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Steroid-refractory (SR) acute graft-versus-host disease (aGVHD) is one of the leading causes of early mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We investigated the efficacy, safety, prognostic factors, and optimal therapeutic protocol for SR-aGVHD patients treated with basiliximab in a real-world setting. Nine hundred and forty SR-aGVHD patients were recruited from 36 hospitals in China, and 3683 doses of basiliximab were administered. Basiliximab was used as monotherapy (n = 642) or in combination with other second-line treatments (n = 298). The cumulative incidence of overall response rate (ORR) at day 28 after basiliximab treatment was 79.4% (95% confidence interval [CI] 76.5%-82.3%). The probabilities of nonrelapse mortality and overall survival at 3 years after basiliximab treatment were 26.8% (95% CI 24.0%-29.6%) and 64.3% (95% CI 61.2%-67.4%), respectively. A 1:1 propensity score matching was performed to compare the efficacy and safety between the monotherapy and combined therapy groups. Combined therapy did not increase the ORR; conversely, it increased the infection rates compared with monotherapy. The multivariate analysis showed that combined therapy, grade III-IV aGVHD, and high-risk refined Minnesota aGVHD risk score before basiliximab treatment were independently associated with the therapeutic response. Hence, we created a prognostic scoring system that could predict the risk of having a decreased likelihood of response after basiliximab treatment. Machine learning was used to develop a protocol that maximized the efficacy of basiliximab while maintaining acceptable levels of infection risk. Thus, real-world data suggest that basiliximab is safe and effective for treating SR-aGVHD.

Relapse to drug seeking can be caused by exposure to drug-associated cues, provoking drug craving even after prolonged abstinence. Recent studies demonstrated that AMP-activated protein kinase (AMPK) regulates neuronal morphology and membrane excitability in neurons. Here, we investigated the role of AMPK activity in the nucleus accumbens (NAc) in relapse to cocaine seeking. We found that exposure to drug-related cues reinstated cocaine-seeking behavior and increased AMPK and p70s6k phosphorylation in the NAc core but not shell. Augmenting AMPK activity by intra-NAc core infusions of the AMPK activator 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) or adenovirus expressing constitutively active subunits of AMPK decreased cue-induced reinstatement of cocaine seeking and inhibited the mammalian target of rapamycin complex 1 (mTORC1) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways. In contrast, inhibition of AMPK activity by intra-NAc core infusions of the AMPK inhibitor compound C or adenovirus expressing dominant-negative subunits of AMPK increased cue-induced reinstatement of cocaine seeking and enhanced mTORC1 and ERK1/2 activity. The regulation of AMPK activity in the NAc shell had no effect on cue-induced cocaine seeking. Altogether, these results indicate that AMPK activity in the NAc core is critical for the cue-induced reinstatement of cocaine seeking, which may be mediated by mTORC1 and ERK1/2 signaling.

To observe the curative efficacy of tyrosine kinase inhibitors (TKIs) in the treatment of e19a2 transcript (P230) CML chronic phase (CML-CP) patients.The clinical data of 11 P230 CML-CP patients were collected from July 2008 to December 2019. Blood routine examination, bone marrow cytology, chromosome, and BCR-ABL qualitative and quantitative tests were performed at initial diagnosis. After TKIs treatment, BCR-ABL (P230)/ABL in peripheral blood was regularly detected to evaluate molecular response by real-time quantitative PCR.There were 11 patients (7 males and 4 females) in chronic phase from 6 domestic hospitals enrolled, their median age was 46 years old (range from 19 to 56 years old). Among 4 patients treated with imatinib (400 mg, qd) firstly, 3 cases switched to nilotinib (400 mg, bid) and 1 case switched to dasatinib (100 mg, qd) due to failure to achieve best molecular response at the landmark time or mutation of ABL kinase. Then major molecular response (MMR) was obtained within 1 year. In addition, 5 patients were treated with nilotinib (300 mg, bid) and 2 patients with dasatinib (100 mg, qd) as first-line treatment, all of them got MMR within 6 months.For intolerance or resistance to imatinib, second-generation TKIs can enable P230 CML patients to achieve deeper molecular response, and MMR in a short time.酪氨酸激酶抑制剂治疗P230慢性髓系白血病临床疗效观察.观察酪氨酸激酶抑制剂（TKI）治疗e19a2转录本（P230）慢性髓系白血病（CML）慢性期患者的疗效。.收集2008年7月至2019年12月11例P230 CML慢性期患者临床资料，初诊时进行血常规、骨髓细胞学、染色体和BCR-ABL定性和定量检测，接受TKI治疗后，定期通过实时定量PCR方法检测外周血BCR-ABL（P230）/ABL评估分子学反应。.来自国内6家医院11例患者中男性7例，女性4例，中位年龄为46（19-56）岁。11例患者均处于慢性期，4例先接受伊马替尼（400 mg，qd）治疗的患者，由于在规定的时间界点未达到最佳分子学反应或出现ABL激酶区突变，其中3例改为尼洛替尼（400 mg，bid）治疗，1例改为达沙替尼（100 mg，qd）治疗，之后均在1年内获得主要分子学反应。另外5例使用尼洛替尼（300 mg，bid）、2例使用达沙替尼（100 mg，qd）作为一线治疗的患者在6个月内获得了主要分子学反应。.对伊马替尼不耐受或疗效不佳的P230 CML患者，二代TKI可使其达到更深层次分子学反应，且能在更短时间达到主要分子学反应。.

Background Hematopoietic stem cell transplantation (HSCT) for haematological disorders. Graft-versus-host disease (GVHD), a cause of morbidity and mortality is treated with corticosteroids. However, patients with steroid-refractory GVHD after HSCT have a poor prognosis. Ruxolitinib, a selective Janus kinase inhibitor, is a novel treatment strategy for steroid-refractory GVHD. Objectives To assess the efficacy of ruxolitinib for the treatment of steroid-refractory GVHD and analyse its adverse effects. Study design Meta-analysis. Search methods Randomised controlled trials (RCTs) and non-RCTs of ruxolitinib-based therapy in patients with steroid-refractory GVHD were found in the Cochrane Central Register of Controlled Trials, EMBASE, PubMed, and Web of Science in March 2021. Outcomes included overall response rate, survival, and adverse effects. The Methodological Index for Non-randomised Studies (MINORS) and the Cochrane collaboration risk-of-bias tool were used to assess methodological quality. Funnel plots, Egger’s test, and the trim and fill method were used to assess publication bias. Results In total, 1470 studies were identified; 19 studies (17 non-RCTs, 2 RCTs) involving 1358 patients met our inclusion criteria. Survival rates at the longest follow-up in non-RCTs, were 57.5% (95% CI 46.9–67.4) and 80.3% (95% CI 69.7–87.9) for acute GVHD (aGVHD) and chronic GVHD (cGVHD), respectively. In non-RCTs, the overall response was 74.9% (95% CI 66.6–81.8, I2 = 49%) in aGVHD and 73.1% (95% CI 62.5–81.6, I2 = 49%) in cGVHD. In aGVHD, the response rates were gastrointestinal, 61.4–90.2%; skin, 52.5–80.6%; and liver, 41.8–71.8%. In cGVHD, the response rates were gastrointestinal, 30.1–70.4%; skin, 30.1–84.4%; lung, 27.0–83.0%; and mouth 3.5–98.1%. In addition, a lower aGVHD grade and moderate cGVHD were associated with a better clinical response. Common adverse events were cytopenia and infectious complications. Conclusions Our systematic review and meta-analysis indicated that ruxolitinib therapy could be a potentially effective and safe treatment for patients with steroid-refractory GVHD.

To explore the effect of soluble CD40 ligand (sCD40L) on the proliferation, apoptosis, and cell cycle of human non-Hodgkin lymphoma (NHL) cells, and analyze its possible mechanism.NHL CA46 cell and Raji cell were treated with different concentrations of sCD40L for 48 h, CCK-8 was used to detect the effect of sCD40L on cell proliferation in vitro, flow cytometry on apoptosis and cycle of NHL cells, and Western blot on the expression of PTEN, BCL-2, and BAX in NHL cells.Compared with the control group, 4 and 8 μg/ml sCD40L could significantly inhibit the proliferation of lymphoma Raji cell and CA46 cell (P0.05). The test results of flow cytometry showed that 4 μg/ml sCD40L could significantly promote the apoptosis of CA46 and Raji cells, and significantly inhibit the S phase proportions (P0.05). Western blot results showed that sCD40L could promote the expression of PTEN and BAX, while inhibit the expression of BCL-2 (P0.05).sCD40L can promote the apoptosis and inhibit the proliferation of NHL cells through the PTEN signaling pathway.可溶性CD40配体对非霍奇金淋巴瘤细胞的影响及相关机制.探讨可溶性CD40配体（sCD40L）对人非霍奇金淋巴瘤细胞增殖、凋亡及细胞周期的影响及其可能机制.用不同浓度的sCD40L处理非霍奇金淋巴瘤CA46细胞和Raji细胞48 h，用CCK-8检测细胞增殖；流式细胞术检测细胞凋亡和周期；通过蛋白免疫印迹检测PTEN、BCL-2和BAX的表达.与对照组比较，4和8 μg/ml的sCD40L均能显著抑制Raji细胞和CA46细胞的增殖（P0.05）。流式细胞术检测凋亡和细胞周期结果显示，4 μg/ml的sCD40L能显著促进CA46和Raji细胞的凋亡和减少S期细胞的比例（P0.05）；蛋白免疫印迹结果显示，sCD40L能显著促进PTEN和BAX的表达，抑制BCL-2的表达（P0.05）.sCD40L抑制淋巴瘤细胞的增殖并促进其凋亡可能是通过调控PTEN信号通路实现.

Acute myeloid leukemia (AML) is a malignant clonal proliferative hematological tumor that originates from hematopoietic stem progenitor cells. Traditional chemotherapy can achieve complete remission in most patients, but so far, only allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only way to cure AML. Recurrence, drug resistance, and transplant-related deaths remain a key issue for AML treatment. Therefore, finding new treatments to improve the prognosis of patients with AML is urgently needed. In recent years, the emergence of new immunotherapy has revolutionized the concept of cancer treatment in the past few decades. Cellular immunotherapy represented by chimeric-antigen receptor T cell (CAR-T) and immunological detection point inhibitor represented by PD-1 blockade have achieved remarkable effects in hematological malignancies. This article mainly reviews the recent research progress of CAR-T and PD-1 blockade in the clinical treatment of AML.CAR-T和PD-1单克隆抗体在急性髓系白血病治疗中的进展和趋势.摘要 急性髓系白血病（AML）是一种起源于造血干祖细胞的恶性克隆增殖性血液系统肿瘤。传统化疗可使大部分患者获得完全缓解，但截至目前，仅有异基因造血干细胞移植（allo-HSCT）是可能治愈AML的唯一方式。复发、耐药及移植相关死亡仍然是AML治疗面临的关键问题。因此，寻找新的治疗方式以改善和提高AML患者的预后在当下显得迫切需要。近年来，新型免疫治疗的出现彻底改变了过去数十年对肿瘤治疗的观念。以嵌合抗原受体T 细胞（CAR-T）为代表的细胞免疫治疗和PD-1单克隆抗体为代表的免疫检测点抑制剂在血液系统恶性肿瘤中取得显著的疗效。本文就近几年CAR-T和PD-1单克隆抗体在AML临床治疗中的最新研究进展作一综述.

Neuronal atrophy and alterations of synaptic structure and function in the medial prefrontal cortex (mPFC) have been implicated in the pathogenesis of depression, but the underlying molecular mechanisms are largely unknown. The protein kinase Mζ (PKMζ), a brain-specific atypical protein kinase C isoform, is important for maintaining long-term potentiation and storing memory. In the present study, we explored the role of PKMζ in mPFC in two rat models of depression, chronic unpredictable stress (CUS) and learned helplessness. The involvement of PKMζ in the antidepressant effects of conventional antidepressants and ketamine were also investigated. We found that chronic stress decreased the expression of PKMζ in the mPFC and hippocampus but not in the orbitofrontal cortex. Overexpression of PKMζ in mPFC prevented the depressive-like and anxiety-like behaviors induced by CUS, and reversed helplessness behaviors. Inhibition of PKMζ in mPFC by expressing a PKMζ dominant-negative mutant induced depressive-like behaviors after subthreshold unpredictable stress and increased learned helplessness behavior. Furthermore, stress-induced deficits in synaptic proteins and decreases in dendritic density and the frequency of miniature excitatory postsynaptic currents in the mPFC were prevented by PKMζ overexpression and potentiated by PKMζ inhibition in subthreshold stress rats. The antidepressants fluoxetine, desipramine and ketamine increased PKMζ expression in mPFC and PKMζ mediated the antidepressant effects of ketamine. These findings identify PKMζ in mPFC as a critical mediator of depressive-like behavior and antidepressant response, providing a potential therapeutic target in developing novel antidepressants.

Cancer-associated fibroblasts (CAF), as one of the most important components of tumor microenvironment, which plays important role in tumorigenesis, development, infiltration and metastasis of cancers. In a variety of solid tumors, CAF can even determine the fate of tumor cells. In view of its pivotal role in promoting tumor progression, CAF has recently become a therapeutic target for a variety of tumors. However, there are a few studies on CAF in hematological malignancies. Recent studies have found that the resistance, relapse of AML, MM, CLL and myelofibrosis of MPN closely relate with CAF, so targeting CAF can effectively enhance the killing effect of chemotherapy drugs on tumor cells, thus improve the efficacy, CAF is expected to become a new target for the treatment of hematological malignancies. This review summarizes recent advances in cancer-associated fibroblasts in hematological malignancies.

Fear extinction forms a new memory but does not erase the original fear memory. Exposure to novelty facilitates transfer of short-term extinction memory to long-lasting memory. However, the underlying cellular and molecular mechanisms are still unclear. Using a classical contextual fear-conditioning model, we investigated the effect of novelty on long-lasting extinction memory in rats. We found that exposure to a novel environment but not familiar environment 1 h before or after extinction enhanced extinction long-term memory (LTM) and reduced fear reinstatement. However, exploring novelty 6 h before or after extinction had no such effect. Infusion of the β-adrenergic receptor (βAR) inhibitor propranolol and glucocorticoid receptor (GR) inhibitor RU486 into the CA1 area of the dorsal hippocampus before novelty exposure blocked the effect of novelty on extinction memory. Propranolol prevented activation of the hippocampal PKA-CREB pathway, and RU486 prevented activation of the hippocampal extracellular signal-regulated kinase 1/2 (Erk1/2)-CREB pathway induced by novelty exposure. These results indicate that the hippocampal βAR-PKA-CREB and GR-Erk1/2-CREB pathways mediate the extinction-enhancing effect of novelty exposure. Infusion of RU486 or the Erk1/2 inhibitor U0126, but not propranolol or the PKA inhibitor Rp-cAMPS, into the CA1 before extinction disrupted the formation of extinction LTM, suggesting that hippocampal GR and Erk1/2 but not βAR or PKA play critical roles in this process. These results indicate that novelty promotes extinction memory via hippocampal βAR- and GR-dependent pathways, and Erk1/2 may serve as a behavioral tag of extinction.

Relapse to drug seeking can be caused by exposure to drug-associated cues, provoking drug craving even after prolonged abstinence. Recent studies demonstrated that AMP-activated protein kinase (AMPK) regulates neuronal morphology and membrane excitability in neurons. Here, we investigated the role of AMPK activity in the nucleus accumbens (NAc) in relapse to cocaine seeking. We found that exposure to drug-related cues reinstated cocaine-seeking behavior and increased AMPK and p70s6k phosphorylation in the NAc core but not shell. Augmenting AMPK activity by intra-NAc core infusions of the AMPK activator 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) or adenovirus expressing constitutively active subunits of AMPK decreased cue-induced reinstatement of cocaine seeking and inhibited the mammalian target of rapamycin complex 1 (mTORC1) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways. In contrast, inhibition of AMPK activity by intra-NAc core infusions of the AMPK inhibitor compound C or adenovirus expressing dominant-negative subunits of AMPK increased cue-induced reinstatement of cocaine seeking and enhanced mTORC1 and ERK1/2 activity. The regulation of AMPK activity in the NAc shell had no effect on cue-induced cocaine seeking. Altogether, these results indicate that AMPK activity in the NAc core is critical for the cue-induced reinstatement of cocaine seeking, which may be mediated by mTORC1 and ERK1/2 signaling.

Fear extinction has been extensively studied, but little is known about the molecular processes that underlie the persistence of extinction long-term memory (LTM). We found that microinfusion of norepinephrine (NE) into the CA1 area of the dorsal hippocampus during the early phase (0 h) after extinction enhanced extinction LTM at 2 and 14 days after extinction. Intra-CA1 infusion of NE during the late phase (12 h) after extinction selectively promoted extinction LTM at 14 days after extinction that was blocked by the β-receptor antagonist propranolol, protein kinase A (PKA) inhibitor Rp-cAMPS, and protein synthesis inhibitors anisomycin and emetine. The phosphorylation levels of PKA, cyclic adenosine monophosphate response element-binding protein (CREB), GluR1, and the membrane GluR1 level were increased by NE during the late phase after extinction that was also blocked by propranolol and Rp-cAMPS. These results suggest that the enhancement of extinction LTM persistence induced by NE requires the activation of the β-receptor/PKA/CREB signaling pathway and membrane GluR1 trafficking. Moreover, extinction increased the phosphorylation levels of Erk1/2, CREB, and GluR1, and the membrane GluR1 level during the late phase, and anisomycin/emetine alone disrupted the persistence of extinction LTM, indicating that the persistence of extinction LTM requires late-phase protein synthesis in the CA1. Propranolol and Rp-cAMPS did not completely disrupt the persistence of extinction LTM, suggesting that another β-receptor/PKA-independent mechanism underlies the persistence of extinction LTM. Altogether, our results showed that enhancing hippocampal noradrenergic activity during the late phase after extinction selectively promotes the persistence of extinction LTM.

Forty-nine wild oil plants which are abundant in the Tsinling and Zhongtiao Mountains were investigated and screened as raw material for biodiesel production. The oil content was tested and found to be greatly variable, ranging from 10 to 62.8 %. Fatty acid profiles of their vegetable oils were analyzed, and their acid value, saponification number, and iodine value were found to range from 1.6 to 61.3 mg KOH/g, 128.6 to 225.9 mg KOH/g, and 54.3 to 120.9 g I2/100 g, respectively. Cetane number, kinematic viscosity, specific gravity, high heating values, and cold filter plugging point of the 49 vegetable-oil methyl esters (MEs) were empirically calculated and were found to vary from 38.8 to 66.6, 2.9 to 4.2 mm2/s, 841.4 to 883.8 kg/m3, 39.0 to 47.5 kJ/g, and −14.3 to 13.2 °C, respectively. Almost half (25 species) of all the sampled resources were found to be suitable for biodiesel production since the profiles of their vegetable-oil MEs met the major specifications of Chinese and European Union biodiesel standards, GB/T20828 and EN-14214, respectively. Grey relational analysis was used during the optimum-species screening process. The difference between the individual species and the artificial designed ideal species (the best biodiesel plant type in this paper) was calculated and evaluated with the help of grey relational grade, which varied from 0.467 to 0.686. Finally, the selected plants, Prunus salicina Linn. and Amygdalus davidiana (C.) C. de Vos, were considered as the optimal feedstock for biodiesel production.

The intense associative memories that develop between drug-paired contextual cues and rewarding stimuli or the drug withdrawal-associated aversive feeling have been suggested to contribute to the high rate of relapse. Various studies have elucidated the mechanisms underlying the formation and expression of drug-related cue memories, but how this mechanism is maintained is unknown. Protein kinase M ζ (PKMζ) was recently shown to be necessary and sufficient for long-term potentiation maintenance and memory storage. In the present study, we used conditioned place preference (CPP) and aversion (CPA) to examine whether PKMζ maintains both morphine-associated reward memory and morphine withdrawal-associated aversive memory in the basolateral amygdala (BLA). We also investigate the role of PKMζ in the infralimbic cortex in the extinction memory of morphine reward-related cues and morphine withdrawal-related aversive cues. We found that intra-BLA but not central nucleus of the amygdala injection of the selective PKMζ inhibitor ZIP 1 day after CPP and CPA training impaired the expression of CPP and CPA 1 day later, and the effect of ZIP on memory lasted at least 2 weeks. Inhibiting PKMζ activity in the infralimbic cortex, but not prelimbic cortex, disrupted the expression of the extinction memory of CPP and CPA. These results indicate that PKMζ in the BLA is required for the maintenance of associative morphine reward memory and morphine withdrawal-associated aversion memory, and PKMζ in the infralimbic cortex is required for the maintenance of extinction memory of morphine reward-related cues and morphine withdrawal-related aversive cues.

To construct an eukaryotic expression vector containing the aldehyde dehydrogenase-2 (ALDH2) gene, and determine whether transfection with the ALDH2 gene can provide protection against hydrogen peroxide-induced oxidative damage, as well as attenuate apoptosis or cell death in human peripheral blood mononuclear cells (PBMCs). The ALDH2 gene was cloned from human hepatocytes by RT-PCR. The eukaryotic expression vector containing the gene was constructed and then transfected into PBMCs via liposomes. RT-PCR, indirect immunofluorescence assay, and Western blot were used to evaluate the expression of the transgene in target cells. MTT assay and flow cytometry were used to detect the effects of ALDH2 on PBMCs damaged by hydrogen peroxide (H2O2). The level of intracellular reactive oxygen species (ROS) was determined by fluorescence spectrophotometry. The eukaryotic expression vector pcDNA3.1/myc-His-ALDH2 was successfully constructed and transfected into PBMCs. RT-PCR results showed higher mRNA expression of ALDH2 in the gene-transfected group than in the two control groups (empty vector-transfected group and negative control). Indirect immunofluorescence assay and Western blot indicated distinct higher protein expression of ALDH2 in the gene-transfected group. The cell survival rate against H2O2-induced oxidative damage was higher in the ALDH2 gene-transfected group. Moreover, apoptosis rates in gene-transfected PBMCs incubated with 50 and 75 μmol/L H2O2 decreased by 7% and 6%, respectively. The generation of intracellular ROS was also markedly downregulated. ALDH2 gene transfection can protect PBMCs against H2O2-induced damage and attenuate apoptosis, accompanied with a downregulation of intracellular ROS. ALDH2 functions as a protector against oxidative stress.

Recent evidence suggests that cannabinoid CB(1) receptors may represent effective targets for therapeutic agents used to treat cocaine and heroin relapse. However, the role of cannabinoid CB(1) receptors in the potential treatment for other drugs of abuse is still largely unknown. The present study was conducted to determine whether cannabinoid CB(1) receptors play a similar role in relapse to ketamine abuse. To establish a ketamine reinstatement model in the conditioned place preference paradigm, rats were trained to develop place preference conditioned by ketamine, which was subsequently extinguished through daily exposure to the test chambers in the absence of ketamine. On the day following the last extinction session, four groups of rats were injected with ketamine (1, 5, 10 and 15 mg/kg, i.p.) to reinstate previously extinguished conditioned place preference. To investigate the effects of rimonabant, a cannabinoid CB(1) receptor antagonist, on reinstatement of ketamine-induced place preference, different doses of rimonabant (0.1, 0.5 and 3 mg/kg, i.p) were injected 30 min prior to the ketamine (5 and 15 mg/kg, i.p.) priming injection in a separate group of rats. To determine whether rimonabant itself produces conditioned place preference or conditioned place aversion, rats were trained for conditioned place preference or place aversion with rimonabant (0, 0.1, 0.5, 3.0 mg/kg, i.p.). While ketamine priming injections reinstated extinguished place preference, rimonabant administration significantly attenuated the reinstatement of ketamine-induced place preference in a dose-dependent manner. Importantly, rimonabant itself did not produce conditioned place preference or place aversion. Since the reinstatement effects of ketamine administration were inhibited by rimonabant, these findings suggest that a cannabinoid CB(1) receptor antagonist may be useful in preventing relapse to ketamine abuse.

Chronic lymphocytic leukemia (CLL) is infrequent in Chinese people. Conventional cytogenetic analysis underestimates the frequency of chromosome aberrations in CLL due to the low rate of spontaneous mitoses. The aim of this study was to prospectively explore the frequency of chromosomal abnormalities in Chinese patients with CLL using interphase fluorescence in situ hybridisation (FISH) and probes for 12 centromere, 13q14, 14q32, 17p13, 11q22 and 6q23 on 143 patients with CLL. Molecular cytogenetic aberrations were found in 104 patients (72.7%) and 40 patients (28.0%) with more than two abnormalities. The most frequent abnormality was del(13q14) (47.6%), followed by trisomy 12 (21.7%), 14q32 translocation (19.6%), del(17p13) (12.6%), del(11q22) (11.9%) and del(6q23) (4.9%), respectively. The percentages of patients with aberrations by FISH were 75.4%, 72.3% and 67.7% for Binet stages A, B and C, respectively. In early stage (Binet A), del(13q14) aberration was more frequent than in Binet B and C (61.5% vs. 31.9% and 41.9%) (P=0.021). Patients with advanced stage (Binet C) had more frequent del(17p13) aberration than in Binet A and B (32.3% vs. 9.2% and 4.3%) (P=0.008). It was showed that the frequencies of the chromosomal abnormalities in our study population were similar to the frequencies in Western countries.

To explore the effect of decitabine on Dickkopf-1 (DKK1) gene expression level and its downstream Wnt signaling pathway in acute myeloid leukemia (AML) cell line HL-60.Flow cytometry and DNA ladder analysis were performed to detect apoptosis in HL-60 cell treated with different concentration of decitabine. Methylation-specific polymerase chain reaction (MS-PCR) was used to examine the methylation status of DKK1 gene. The expressions of mRNA and protein were determined by qRT-PCR and Western blot, respectively.Flow cytometric detection showed that after treating HL-60 cell line with decitabine of different concentrations for 48 h, the early apoptosis of HL-60 cells increased significantly as compared with control group (P0.05). DNA ladder analysis showed that the DNA ladder and demethylation of DKK1 gene appeared. RT-PCR and Western blot showed that the expressions of mRNA and protein increased. The protein expressions of β-catenin and C-MYC decreased.The decitabine can promote the apoptosis of HL-60 cells throngh demethylation of DDK1 gene and inhibition of Wnt signalling pathway.

A number of studies have demonstrated that the methylation of Dickkopf-1 (DKK1) gene promoter is related with the occurrence and development of many neoplastic diseases. By means of binding with corresponding receptors, DKK1 blocks the transduction pathway of Wnt/β-catenin/TCF and inhibits the proliferation and invasion of tumor cells, inducing apoptosis. Leukemia is a hyperplastic disease of hematopoietic stem cell malignant clone. Its pathogenesis has been confirmed to be closely related with the aberrant activation of Wnt signaling pathway. This pathway is associated with the self-renewal and proliferation of the hematopoietic stem cells, which can regulate growth, differentiation, migration of the cells, angiogenesis and embryonic development. Its expression is regulated by some suppressor genes like Dickkopf 1 (DKK1). Leukemia often accompanied by methylation modification of the DKK1 gene, so as to leads to silencing itself and activation of the Wnt signaling pathway, which cause the occurrence of leukemia. Some therapeutic methods on leukemia aiming at DKK1 gene have been reported, among which DKK1 gene was demethylated. The intensive study on the expression and function of DKK1 should be important for the early diagnosis, treatment and prognosis. This article reviews the current progress in this field.

Both the formation of long-term memory (LTM) and dendritic spine growth that serves as a physical basis for the long-term storage of information require de novo protein synthesis. Memory formation also critically depends on transcription. Adenosine monophosphate-activated protein kinase (AMPK) is a transcriptional regulator that has emerged as a major energy sensor that maintains cellular energy homeostasis. However, still unknown is its role in memory formation. In the present study, we found that AMPK is primarily expressed in neurons in the hippocampus, and then we demonstrated a time-dependent decrease in AMPK activity and increase in mammalian target of rapamycin complex 1 (mTORC1) activity after contextual fear conditioning in the CA1 but not CA3 area of the dorsal hippocampus. Using pharmacological methods and adenovirus gene transfer to bidirectionally regulate AMPK activity, we found that increasing AMPK activity in the CA1 impaired the formation of long-term fear memory, and decreasing AMPK activity enhanced fear memory formation. These findings were associated with changes in the phosphorylation of AMPK and p70s6 kinase (p70s6k) and expression of BDNF and membrane GluR1 and GluR2 in the CA1. Furthermore, the prior administration of an mTORC1 inhibitor blocked the enhancing effect of AMPK inhibition on fear memory formation, suggesting that this negative regulation of contextual fear memory by AMPK in the CA1 depends on the mTORC1 signaling pathway. Finally, we found that AMPK activity regulated hippocampal spine growth associated with memory formation. In summary, our results indicate that AMPK is a key negative regulator of plasticity and fear memory formation.

20-HETE, a cytochrome P-450 4A (CYP4A1)-derived arachidonic acid metabolite, is a major eicosanoid formed in renal and extrarenal microcirculation. 20-HETE inhibits Ca2+-activated K+ channels in vascular smooth muscle cells and thereby may modulate vascular reactivity. We transfected renal interlobar arteries with an expression plasmid containing the cDNA of CYP4A1, the low- K m arachidonic acid ω-hydroxylase, and examined the consequences of increasing 20-HETE synthesis on constrictor responses to phenylephrine. CYP4A1-transfected interlobar arteries demonstrated a twofold increase in CYP4A protein levels and 20-HETE production compared with arteries transfected with the empty plasmid; they also showed increased sensitivity to phenylephrine, as evidenced by a decrease in EC50 from 0.37 ± 0.04 μM in plasmid-transfected arteries to 0.07 ± 0.01 μM in CYP4A1-transfected arteries. The increased sensitivity to phenylephrine was greatly attenuated by N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), a selective inhibitor of 20-HETE synthesis, and by 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid, a specific 20-HETE antagonist. This effect of DDMS was reversed by addition of 20-HETE, further substantiating the notion that increased levels of 20-HETE contribute to the increased sensitivity to phenylephrine in vessels overexpressing CYP4A1. These data suggest that 20-HETE of vascular origin sensitizes renal vascular smooth muscle to phenylephrine.

Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) gene could increase resistance to 1,3-Bis(2- Chloroethyl)-l-Nitrosourea (BCNU). Methods: The eDNA encoding the MGMT was isolated by using RTPCR method from total RNA of fresh human liver, the fragment was cloned into pGEM-T vector and further subcloned into GINa retrovirus vector. Then the G1Na- MGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8×105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMTcDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34 + cells and expressed efficiently. The transgene cord blood CD34 + cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.

To establish a bcr-abl(+) cell line resistance to nilotinib, and to investigate the possible mechanisms of resistance.K562 cells were treated with gradually increasing concentrations of nilotinib to generate resistance cell line K562-RN. The folder of drug-resistance was evaluated by MTT assay. Cells apoptosis rate was detected by flow cytometry, the mRNA level of bcr-abl fusion gene by FISH, and the expression of apoptosis relative gene mRNA and protein (such as bcr-abl, HO-1, mdr1, Bcl-2 and caspase-3) by RQ-PCR and western blot.The resistant cell line K562-RN was successfully established, with 2.01 fold resistant to nilotinib compared with K562 cell line \[the IC(50) value of nilotinib to K562 and K562-RN were (12.320 ± 1.720) µmol/L and (24.742 ± 2.310) µmol/L, respectively\]. It also had the cross resistance to adriamycin, homoharringtonine, etoposide and imatinib. Treated with different concentrations of nilotinib, cell apoptosis rate of K562-RN was significantly lower than that of K562 cells. The rate of bcr-abl gene positive cells was 92% in K562-RN by FISH assay. The mRNA and protein levels of bcr-abl, HO-1 and mdr1 expression up-regulated in K562-RN cells, while those of caspase-3 expression down-regulated, being significantly statistical difference when compared with K562 cells (P0.05).Human leukemic cell line resistance to nilotinib, K562-RN is established successfully by gradually increasing concentrations of drug. The mechanisms of resistance in K562-RN is probably associated with increasing expression of bcr-abl, HO-1, mdr1 and decreasing expression of caspase-3 mRNA and protein levels.

To investigate the effect of retrovirus mediated heme oxygenase (HO)-1 gene on chronic myeloid leukemia (CML) resistance cell apoptosis induced by nilotinib (AMN107).High titer viral particles of pQCXIP-EGFP-HO-1 were prepared, and K562/A02 cells stably transfected with HO-1 gene was established. The expression of HO-1 in K562/A02 cells was detected by RT-PCR. After treated with AMN107 for 24 h, HO-1 mRNA and protein expression by RT-PCR and Western blot, respectively; Cell proliferation by MTT assay; bcr-abl fusion gene by RQ-PCR, and the apoptosis and cell cycle by flow cytometry.Recombinant retrovirus vector was constructed successfully and K562/A02/HO-1 cells were successfully set up. The expression of HO-1 in K562/A02 cells was expressed clearly. After three groups cells treated with AMN107 for 24 h, the expression of HO-1 mRNA and protein was significantly higher in gene-transfected group than in either empty vector or no-transfected group. The difference was statistically significant (P0.05). The cell proliferation ofs was inhibited, but the cell viability was significantly higher in gene-transfected group than in other two groups. The difference was statistically significant(P0.05); After treated with 10 µmol/L AMN107 for 24 h, the CT values of bcr-abl fusion gene were (18.15 ± 0.18) in K562/A02/HO-1 group, being significantly higher than that in K562/A02/LXSN (20.32 ± 0.20) and K562/A02 (20.51 ± 0.21) group, the difference was statistically significant (P0.05); the apoptosis rate were (17.26 ± 0.23)%, (39.47 ± 0.17)%, and (41.84 ± 0.09)%, respectively in three groups, and were (3.74 ± 0.03)%, (5.91 ± 0.08)% in K563/A02/HO-1 untreated with drug and K562/A02 untreated with drug group. The number of G(0)/G(1) phase and S phase cells markedly decreased. The cells were arrested in G(2)/M phase. But cell cycle in gene-transfected group did not change significantly.AMN107 inhibits proliferation of CML resistance cells and induces cell apoptosis. HO-1 gene can protect CML resistance cells to apoptosis. There was a relationship between HO-1 gene and the growth of CML resistance cells.

To investigate the effect of heme oxygenase-1 (HO-1) expression on cell growth and apoptosis in imatinib resistant chronic myeloid leukemia (CML) cells (K562/A02-IM), and explore the relationship between HO-1 gene and CML.The expression of HO-1 in 20 drug-resistant CML patients was detected by RT-PCR. Different concentrations of hemin were used to induce HO-1 expression of K562/A02-IM, HO-1 expression at different time was detected by RT-PCR and Western blot analysis. Cell apoptosis was detected by Annexin V/PI staining, and MTT assay was used to detect viability of K562/A02-IM cells after induction or inhibition of HO-1 gene by hemin and zinc protoporphyrin (ZPP).RT-PCR showed that HO-1 was expressed in the bone marrow mononuclear cells (BMMNCs). When treated with hemin at different concentrations (0, 10, 20, 40 µmol/L) for 16 h, the expression of HO-1 in K562/A02-IM was increased in a dose-dependent manner, and peaked at 20 µmol/L of hemin for 16 h. The apoptosis rates were (17.61 ± 0.01)%, (12.13 ± 0.11)%, (7.94 ± 0.03)% and (4.62 ± 0.15)% at 0,10, 20 and 40 µmol/L of hemin respectively for 16 h and were (14.7 ± 0.05)%, (8.1 ± 0.07)% and (16.3 ± 0.13)% at 20 µmol/L of hemin treatment for 8,16, and 24 h respectively. Hemin induced apoptosis of K562/A02-IM cells in a dose-dependent manner. The expression of HO-1 was induced in K562/A02-IM cells in a dose-dependent manner, and the survival of K562/A02-IM cells was significantly increased as compared to that of control group. When HO-1 was inhibited by ZPP, the cells survival was sharply decreased compared to that of the control group (P0.05).HO-1 was expressed in the BMMNCs. It is a kind of molecules whose expression can be induced and can promote the growth of drug-resistant cells. Inhibition of HO-1 expression probably be used for the treatment of drug-resistant CML.

Exposure to cues previously associated with drug intake leads to relapse by activating previously acquired memories. Based on previous findings, in which cannabinoid CB1 receptors were found to be critically involved in specific aspects of learning and memory, we investigated the role of CB1 receptors in nicotine reward memory using a rat conditioned place preference (CPP) model. In Experiment 1, rats were trained for CPP with alternating injections of nicotine (0.5 mg/kg, s.c.) and saline to acquire the nicotine-conditioned memory. To examine the effects of rimonabant on the reconsolidation of nicotine reward memory, rats were administered rimonabant (0, 0.3, and 3.0 mg/kg, i.p.) immediately after reexposure to the drug-paired context. In Experiment 2, rats were trained for CPP similarly to Experiment 1. To examine the effects of rimonabant on the reinstatement of nicotine reward memory, rimonabant (0, 0.3, and 3.0 mg/kg, i.p.) was administered before the test of nicotine-induced CPP reinstatement. In Experiment 3, to evaluate whether rimonabant itself produces a reward memory, rats were trained for CPP with alternating injections of different doses of rimonabant (0, 0.3, and 3.0 mg/kg) and saline. Rimonabant at a dose of 3.0 mg/kg significantly disrupted the reconsolidation of nicotine memory and significantly blocked the reinstatement of nicotine-induced CPP. However, rimonabant itself did not produce CPP. These findings provide clear evidence that CB1 receptors play a role in nicotine reward memory, suggesting that CB1 receptor antagonists may be a potential target for managing nicotine addiction.

To construct an eukaryotic expression vector containing the aldehyde dehydrogenase-2 (ALDH2) gene, and determine whether transfection with the ALDH2 gene can provide protection against hydrogen peroxide-induced oxidative damage, as well as attenuate apoptosis or cell death in human peripheral blood mononuclear cells (PBMCs).The ALDH2 gene was cloned from human hepatocytes by RT-PCR. The eukaryotic expression vector containing the gene was constructed and then transfected into PBMCs via liposomes. RT-PCR, indirect immunofluorescence assay, and Western blot were used to evaluate the expression of the transgene in target cells. MTT assay and flow cytometry were used to detect the effects of ALDH2 on PBMCs damaged by hydrogen peroxide (H₂O₂). The level of intracellular reactive oxygen species (ROS) was determined by fluorescence spectrophotometry.The eukaryotic expression vector pcDNA3.1/myc-His-ALDH2 was successfully constructed and transfected into PBMCs. RT-PCR results showed higher mRNA expression of ALDH2 in the gene-transfected group than in the two control groups (empty vector-transfected group and negative control). Indirect immunofluorescence assay and Western blot indicated distinct higher protein expression of ALDH2 in the gene-transfected group. The cell survival rate against H₂O₂-induced oxidative damage was higher in the ALDH2 gene-transfected group. Moreover, apoptosis rates in gene-transfected PBMCs incubated with 50 and 75 μmol/L H₂O₂ decreased by 7% and 6%, respectively. The generation of intracellular ROS was also markedly downregulated.ALDH2 gene transfection can protect PBMCs against H₂O₂-induced damage and attenuate apoptosis, accompanied with a downregulation of intracellular ROS. ALDH2 functions as a protector against oxidative stress.

To construct a eukaryotic expression vector containing aldehyde dehydrogenase-2 (ALDH2) gene and investigate the effects and its possible mechanisms of ALDH2 gene on cell proliferation and anti-oxidative damage in the K562 cells.An eukaryotic expression vector containing the ALDH2 gene cloned from human hepatocytes was constructed and transfected into K562 cells by liposome. RT-PCR and Western blot were used to evaluate the expression of ALDH2. MTT assay was used to check the cell proliferation and trypan blue exclusion to check K562 cells damage induced by hydrogen peroxide (H2O2). RT-PCR and fluorescence spectrophotometry were used to determine the expression of heme oxygenase-1 (HO-1) and the generation of intracellular reactive oxygen species (ROS) respectively.RT-PCR and Western blot analysis showed distinct higher ALDH2 protein expression in gene transfected group. The latter group had a higher cell proliferation (P0.05) and survival rate against H2O2 induced-oxidative damage, being increased by 7.8 times (IC(50) was 12.3 µmol/L and 1.4 µmol/L for K562-pcDNA3.1-ALDH2 and control cells, respectively, P0.01). The HO-1 mRNA expression and the generation of intracellular ROS were downregulated at a specific concentration of H2O2 in the ALDH2 gene transfected group.ALDH2 gene transfection can protect K562 cells against oxidative damage, and the downregulation of HO-1 expression and intracellular ROS may be involved in this process.

To construct the human cytochrome P-450 CYP2E1 (CYP2E1) recombinant adenovirus vector and detect its anti-tumor effects in combination with chemotherapeutic drugs so as to provide rationales for gene directed enzyme prodrug therapy (GDEPT).CYP2E1 cDNA was cloned from human liver and a recombinant adenovirus vector constructed at a titer of 1 × 10(12) pfu/ml. Human bone marrow-derived mesenchymal stem cells (BMSCs) were separated, cultured, purified and detected. The tropism of BMSCs for cancer cells was detected by Transwell technique. These recombinant vectors were transferred into BMSCs and A375 cells and the expressions of EGFP and CYP2E1 detected by fluorescence microscope, RT-PCR and Western blot respectively. Inverted microscope, MTT and Annexin V-FITC/PI were employed to detect the anti-tumor effect of CYP2E1 recombinant adenovirus vectors in combination with chemotherapeutic prodrug dacarbazine (DTIC).The recombinant adenovirus vectors pAd5CMV-NpA-CYP2E1 and pAd5CMV-NpA-EGFP were constructed successfully. BMSCs were successfully separated and they could migrate in vitro through a polycarbonate filter toward K562 and A375 cells in the lower chamber. Fluorescence microscope was used to detect the expression of EGFP while both RT-PCR and Western blot revealed a high expression of CYP2E1 in gene-transfected group cells. The dead cell counts of co-culture group of gene-transfected BMSCs and wild type A375 cells were significantly higher than those of the control under inverted microscope. The results of MTT showed that the growth inhibition of gene-transfected group cells was increased with DTIC concentration in a concentration dependent manner. IC(50) of group BMSCs-CYP2E1 + A375 was (0.17 ± 0.13) mmol/L, and that of the control group was (0.65 ± 0.20) mmol/L (P0.01). The DTIC concentration at which BMSCs were relatively safe might be selected at 0.05 mmol/L. Annexin V-FITC/PI confirmed that the apoptosis rate of BMSCs-CYP2E1 + A375 group was significantly higher than that of the control group after a 48-hour treatment of 0.05 mmol/L DTIC (26.8 ± 2.0 vs 8.7 ± 1.3, P0.01).With an in vitro tropism for cancer cells, BMSCs transferred with CYP2E1 recombinant adenovirus vectors have anti-tumor effects probably synergistically with chemotherapeutic drugs.

Cocaine use and relapse involves learned associations between cocaine-associated environmental contexts and discrete stimuli and cocaine effects. Initially, these contextual and discrete cues undergo memory consolidation after being paired with cocaine exposure. During abstinence, cocaine cue memories can undergo memory reconsolidation after cue exposure without the drug. We used a conditioned place preference (CPP) procedure in rats to study the role of neuronal protein kinase cyclin-dependent kinase 5 (Cdk5) in consolidation and reconsolidation of cocaine cue memories. We found that the expression of cocaine CPP in drug-free tests 1 d after CPP training (four pairings of 10 mg/kg cocaine with one context and four pairings of saline with a different context) increased Cdk5 activity, and levels of the Cdk5 activator p35 in basolateral but not central amygdala. We also found that basolateral (but not central) amygdala injections of the Cdk5 inhibitor β-butyrolactone (100 ng/side) immediately (but not 6 h) after cocaine–context pairings during training prevented subsequent cocaine CPP expression. After training, acute basolateral (but not central) amygdala β-butyrolactone injections immediately before testing prevented the expression of cocaine CPP; this effect was also observed on a second test performed 1 d later, suggesting an effect on reconsolidation of cocaine cue memories. In support, basolateral β-butyrolactone injections, given immediately (but not 6 h) after a single exposure to the cocaine-paired context, prevented cocaine CPP expression 1 and 14 d after the injections. Results indicate that basolateral amygdala Cdk5 activity is critical for consolidation and reconsolidation of the memories of cocaine-associated environmental cues.

This study was aimed to investigate 6 kinds of human cytokines (IL-2, IL-4, IL-12, IL-13, IFN-gamma and TNF-alpha) in patients with acute lymphocytic leukemia (ALL), so as to find their relationship with the disease. The pure monoclonal antibodies were spotted on the prepared NC membrane glass slides under certain conditions to make human cytokine protein microarray. The serum samples were collected from peripheral blood of 28 patients and 25 normal controls, the serum concentration of cytokines was determined by using the protein microarray and ELISA. The results showed that the expression levels of IL-4, IL-12, IL-13, IFN-gamma and TNF-alpha in ALL patients were lower than those in the normal controls, but ELISA indicated that the expression of TNF-alpha in ALL patients and normal controls was negative. In conclusion, both cellular and humoral immunity are all inhibited in patients with ALL. Microarray is more sensitive than ELISA.

This study was aimed to investigate the correlation of 12th exon mutations in the npm1 gene with prognosis of adult AML patients and to explore the relationship of 12th exon mutation with other gene mutations. The specimen of bone marrow and peripheral blood from AML patients, the informations of medical history, symptoms, related image examinations, blood routine examination, NAP, oxygen saturation level in artery blood and EPO level in serum were collected; the bcr/abl fusion gene was detected by routine examination of bone marrow + biopsy + chromosome mapping + FISH. The patients were typed according to WHO classification. The DNA in cells was extracted, the npm1 gene mutation was detected by allele specific PCR combined were the sequencing. The results indicated that the npm1 heterozygote gene mutation was found in 72 out of 150 AML patients with normal cytogenetics (48%, 72/150). 48% patients showed a frameshift mutation in the C-terminal region of the NPM1 protein. The AML patients with npm1 gene mutation had specific clinical, phenotypic and genetic characteristics. The statistical analysis demonstrated the relationship between npm1 and flt3 ITDs. The patients with npm1 mutation showed a better response to induction therapy, furthermore, the overall survival (OS) rate of patients without flt3 ITD mutation was enhanced. The multivariate analysis demonstrated that the npm1 gene mutation and cebpa mutation positively correlated to the OS rate, and the correlation of flt3 mutation to OS rate showed negative. It is concluded that npm1 mutation is a favorable independent prognostic factor for adult AML patients with normal cytogenetics under conditions without FIT3 gene mutation.

To explore bone marrow-derived mesenchymal stem cells (BMSC) mediated gene directed enzyme prodrug targeting anti-tumor therapy (GDEPT).CYP1A2 gene was cloned from human hepatocytes by RT-PCR, and the eukaryote expression vector was constructed and transferred into Raji cells and human BMSCs via liposome. The targeted anti-tumor effect of BMSC-CYP1A2 cooperated with dacarbazine (DTIC) was measured. RT-PCR and Western blot were used to detect the expression of CYP1A2. Migration assay was detected with Transwell Plates. MTT was used to evaluate the growth inhibitory effect. Cell apoptosis was determined with Annexin V-FITC/PI staining by flow cytometry(FCM).The results of FCM and differentiation induction were in line with the characteristics of BMSC. The expression of CYP1A2 was confirmed by RT-PCR and Western blot. Growth inhibition of Raji-CYP1A2 cells was increased with DTIC concentration in a dose-dependent manner (IC(50) was 1.67 mmol/L). However, BMSC was less sensitive to the cytotoxic effects of DTIC (IC(50) of 9.26 mmol/L and 7.53 mmol/L for BMSC-pcDNA3.1 and BMSC-CYP1A2 cells, respectively) than Raji cells did (IC(50) of 5.62 mmol/L and 1.67 mmol/L for Raji-pcDNA3.1 and Raji-CYP1A2 cells, respectively). BMSC migrated toward Raji cells in Transwell plate. BMSC-CYP1A2 cells mediated a bystander killing effect for CYP1A2-negative Raji cells when they were co-cultured with BMSC-CYP1A2 cells.DTIC can be catalyzed by CYP1A2 in vitro. BMSC-based enzyme prodrug system of CYP1A2 and DTIC can induce lymphoma cell apoptosis targetedly via bystander killing effect.

This study was aimed to investigate the frequency of FMS-like tyrosine kinase 3 (FLT3) mutations including internal tandem duplication (ITD) mutation of juxtamembrane region and point mutation of the second tyrosine kinase domain (TKD) in acute myeloid leukemia (AML) patients and its clinical significance. The ITD mutation in FLT3 exon 14, 15 of bone marrow mononuclear cells was detected by genomic DNA-PCR, the TKD point mutation in FLT3 exon 20 was detected by genomic DNA-PCR combined with restriction endonuclease digest. The results indicated that among 131 newly diagnosed AML patients, 21 patients (16.0%) showed FLT3-ITD positive, 3 patients (2.3%) showed FLT3-TKD positive. None was found harboring both mutations. The WBC and bone marrow blast counts in FLT3-ITD positive patients seemed both higher than those in patients with wild-type FLT3 (FLT3-wt), but there was significant difference only in WBC count (p0.05). The complete remission (CR) rate in FLT3-ITD positive patients was 47.6%, which was significantly lower than that in FLT3-wt patients (88.1%, p0.05). There was no statistical difference in CR rate between FLT3-ITD positive and negative patients in 20 cases of M3; the CR rate in FLT3-ITD positive patients with non M(3) was 37.5 (6/16) which was obviously lower than that in FLT3-wt patients with non M3 (90.6%, 48/53) (p0.05). 3 FLT3-ITD positive patients with CR relapsed after CR for 14 (2-20) months with relapse rate 50% (3/6) which was higher than that in FLT3-wt patients (29.2%, 14/48). It is concluded that FLT3 mutation is common in AML patients, while FLT3-ITD mutation is more frequent than FLT3-TKD mutation. The AML patients with FLT3-ITD mutation have a poor prognosis, while FLT3-TKD point mutation does not significantly influences prognosis of the patients. Therefore early detection of FLT3 mutation may be important for targeting therapy and evaluating clinical prognosis of AML patients.

This study was aimed to investigate the status of c-KIT, Fms-like tyrosine kinase 3 (FLT3) and Janus kinase 2 (JAK2) mutations in acute myeloid leukemia (AML) patients with t (8; 21) and to analyze their relation to clinical feature and prognosis. PCR, AS-PCR, restriction and sequencing methods were used respectively to detect the FLT3, JAK 2 and c-KIT mutations in 8 cases of de novo AML with t (8; 21) and 6 cases of relapsed AML with t (8; 21). The results showed that the c-KIT mutation was found in 2 cases out of 14 AML patients with t (8; 21) (14.3%), among them 1 case had c-KIT D816V mutation, the other had c-KIT D816Y mutation. The FLT3-ITD mutation was detect in 1 out of 14 patients (7.1%), but JAK2 mutation could not be detected in all 14 cases. In conclusion, tyrosine kinase mutation relates to AML with t (8; 21), patients with tyrosine kinase mutation may have higher relapse, extramedullary infiltration and poor prognosis. The screening c-KIT, FLT3 mutations may play an important role in evaluating prognosis and guiding treatment of t (8; 21) AML.

Cytokine interferon-alpha (IFN-alpha) is an immunomodulator and neuromodulator, which modulates central nervous system function partially by activating opioid receptors. However, the role that IFN-alpha plays in relapse to drug abuse is still largely unknown. Thus, we studied whether human recombinant INF-alpha (rIFN-alpha) would reinstate morphine-conditioned place preference (CPP) in rats. In Experiment 1, rats were trained for morphine-CPP with 8-day alternate subcutaneous (s.c.) injections of morphine and saline, and the effect of human rIFN-alpha (20 000 IU/5 microl, intracerebroventricularly) on CPP reinstatement was examined after extinction. In Experiment 2, rats underwent morphine (5 mg/kg, s.c.) unconditioned training with 8 daily alternate injections of morphine (5 mg/kg, s.c.) and saline. Then, the effect of human rIFN-alpha (20 000 IU/5 microl, intracerebroventricularly) on reinstatement of CPP was examined after extinction. In Experiment 3, the effect of opioid receptor antagonist naloxone (1 mg/kg, intraperitoneally) on human rIFN-alpha-induced reinstatement of morphine-CPP was investigated. We found that human rIFN-alpha reinstated morphine-CPP in rats trained under morphine conditioning after extinction, but did not affect CPP in rats that underwent unconditioned training. Naloxone significantly inhibited human rIFN-alpha-induced reinstatement of morphine-CPP. These results indicate that IFN-alpha is a stimulus for reinstatement of morphine-CPP by activation of opioid receptors, which extends our understanding on the high comorbidity of heroin relapse and viral infection.

To investigate the effect of lipopolysaccharide(LPS) on the expression and activity of Toll-like receptor 4(TLR-4) in mesenchymal stem cells (MSC).MSCs were harvested from adult rats bone marrow cells by density gradient centrifugation and adhesive culture. The purity of MSC were identified with the cell morphology and osteogenic capacity. The phenotypes were assayed by flow cytometry. Cultured MSCs were treated by LPS with various concentration (1 microg/ml, 10 microg/ml or 100 microg/ml) for 24 hours. The relative expression levels of TLR-4 mRNA were detected by semiquantitative RT-PCR, and costimulatory molecules (CD80, CD86 and MHC-II) expressed on MSC were analyzed by flow cytometry. The levels of TNF-alpha in supernatants were determined by double-antibody sandwich ELISA.The expression levels of CD80, CD86, MHC-II, TLR-4 mRNA and TNF-alpha in MSC were (9.56 +/- 0.69)%, (22.03 +/- 2.03)%, (2.51 +/- 0.97)%, relative magnitude (0.61 +/- 0.10), (4.97 +/- 2.98) pg/ml, respectively. After incubation with LPS, MSC expressed higher levels of TLR-4 mRNA and costimulatory molecules, and the levels of TNF-alpha were higher than that in untreated group. Among the various concentration of LPS, 10 microg/ml emerged as the most effective in increasing the levels of TLR-4 mRNA (relative magnitude 1.55 +/- 0.02), costimulatory molecules [CD80 (41.70 +/- 2.92)%, CD86 (59.72 +/- 2.00)%, MHC-II (24.56 +/- 2.19)%] and TNF-alpha [(213.12 +/- 69.08) pg/ml] (P0.01). The levels of TLR-4 mRNA, costimulatory molecules and TNF-alpha began to decrease when MSC exposed to 100 microg/ml LPS (P0.05). Except for the levels of TNF-alpha [(158.05 +/- 28. 05) pg/ml] and MHC-II [(5.62 +/- 2.31)%] (P0.05), the levels of CD80, CD86, MHC-II and TLR-4 mRNA were significantly lower than the 10 microg/ml treatment group (P0.01).MSCs are able to express TLR-4 mRNA. LPS could activate the expression of TLR-4 in MSC obviously, but the activity is dependent on the specific concentration.

The objective of this study was to investigate the relationship between the expressions of breast cancer resistance protein (BCRP) gene and drug resistance as well as prognosis in adult patients with acute lymphocytic leukemia (ALL). Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of BCRP gene in 97 adult patients with acute lymphocytic leukemia (ALL) and 30 normal subjects. Beta(2) microglobulin (beta(2)-MG) was used as an internal reference. BCRP/beta(2)-MG ratioor= 0.48 was defined as BCRP positive. The results showed that the positive ratio of BCRP gene expression in untreated group was 28.7%, in contrast that in refractory and relapsed patients was 51.2%. The first complete remission rates in patients with negative and positive expression were 71.7% and 29.7% respectively. In ALL subtypes BCRP gene expression of L3 type was distinctly higher than that of L(1), L(2) types. In immune types the BCRP gene expression of B-ALL was higher than that of T cell type, especially in mature B cell type with obviously statistical significance (p0.01). It is concluded that the high expression of BCRP gene may induce clinical drug resistance, and may be an unfavorable factor for prognosis in adult patients with acute lymphocytic leukemia.

Background O(6)-methylguanine-DNA-methyltransferase (MGMT) is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells. Methods Mammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells (PBMCs) via liposome, then assayed for gene expression at RNA and protein levels. MTT assay was used to check the drug resistance of cells transfected with MGMT gene. Results MGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively. Results of Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC(50) values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC. Conclusion The alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.

To investigate JAK2V617F mutation and its clinical significance in patients with idiopathic myelofibrosis (IMF), genomic DNA was extracted from peripheral blood cell samples of 12 IMF cases. Allele-specific PCR (AS-PCR) was performed to identify JAK2V617F mutation, and the results were confirmed by sequence analysis. A retrospective study was performed to explore the correlation between JAK2V617F mutation and the clinical, hematologic features. The results showed that in follow-up for 2 to 15 months, the occurrence of the positive point mutation in 12 patients with IMF was 50%, and the half of these positive patients had thrombosis. Patients with JAK2V617F point mutation had a higher counts of platelets and megakaryocytes in bone marrow than those in patients without JAK2V617F point mutation. Out of other 6 IMF patients without JAK2V617F point mutation only 1 patient had thrombosis, and lower counts of platelets in peripheral blood and megakaryocytes in bone marrow. It is concluded that majority of IMF patients with positive JAK2V617F point mutation have typical clinical and hematologic features, higher incidence of thrombosis, and higher counts of platelets in peripheral blood and megakaryocytes in bone marrow.

Recent studies have demonstrated that angiostatin, a newly discovered specific inhibitor of endothelial cells, may significantly suppress the growth of a variety of tumors. We constructed an eukaryotic expression vector containing angiostatin (pAG3 ). To study the effect of pAG3, we intramuscularly injected pAG3 into B16 melanoma bearing C57 mice, we found that pAG3 could obviously inhibit tumor growth and reduce the size of tumors in B16 melanoma bearing C57 mice compared to the untreated control mice. In addition, we also investigated whether pre-treatment of pAG3 can prevent the tumor formation in mice treated with B16 melanoma. Normal C57 mice which received 5 days of treatment of pAG3 prior to implanting tumors resulted in the inhibitory effect when compared to control mice. No promotive effect was observed when pAG3 was combined with DTIC (Dacarbazine). These findings provide a basis for the further development of nonviral delivery of angiogenic gene therapy.

Cytochrome P-450–4A1 (CYP4A1) is an ω-hydroxylase that catalyzes the metabolism of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to determine the vasomotor consequences of vascular overexpression of CYP4A1. Isolated rat gracilis muscle arterioles transfected ex vivo with an expression plasmid containing CYP4A1 cDNA expressed more CYP4A protein than vessels transfected with the control plasmid. In arterioles pressurized to 80 mmHg, the internal diameter of vessels transfected with CYP4A1 cDNA (55 ± 3 μm) was surpassed ( P < 0.05) by that of vessels transfected with control plasmid (97 ± 4 μm). Treatment with a CYP4A inhibitor ( N-methylsulfonyl-12,12-dibromododec-11-enamide; DDMS) or with an antagonist of 20-HETE actions [20-hydroxyeicosa-6( Z),15( Z)-dienoic acid; 20-HEDE] elicited robust dilation of arterioles transfected with CYP4A1 cDNA, whereas the treatment had little or no effect in vessels transfected with control plasmid. Examination of the intraluminal pressure-internal diameter relationship revealed that pressure increments over the range of 40–100 mmHg elicited a more intense ( P < 0.05) myogenic constrictor response in arterioles transfected with CYP4A1 cDNA than in those with control plasmid. Arterioles transfected with CYP4A1 cDNA also displayed enhanced sensitivity to the constrictor action of phenylephrine. Treatment with DDMS or 20-HEDE greatly attenuated the constrictor responsiveness to both constrictor stimuli in vessels overexpressing CYP4A1, whereas the treatment had much less effect in control vessels. These data suggest that CYP4A1 overexpression promotes constriction of gracilis muscle arterioles by intensifying the responsiveness of vascular smooth muscle to constrictor stimuli. This effect of CYP4A1 overexpression appears to be mediated by a CYP4A1 product.