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Objective: To engineer a BCMA mutant resistant to γ-secretase cleavage in order to stabilize wild-type BCMA expression after γ-secretase cleavage and to generate target cells for measuring BCMA CAR-T cell cytotoxicity. Methods: Our study aimed to engineer a mutant BCMA protein (BCMA-CD8α TM) that could resist γ-secretase cleavage by replacing the transmembrane domain of the wild-type BCMA protein with the human CD8α sequence. Four different types of cells in which this mutant gene was expressed excessively were engineered, including U266 (U266BCMA Mut), K562 (K562BCMA Mut), SKOV3 (SKOV3BCMA Mut), and CHO (CHOBCMA Mut) cells. BCMA CAR Jurkat cells, loaded with the NFAT-EGFP reporter gene (BCMA-CAR-Jurkat-Reporter), were engineered and cocultured with U266BCMA Mut cells. The expression level of EGFP was detected by FCM in order to indicate the activation level of NFAT. The cytotoxicity of BCMA CAR-T cells against Luciferase-labeled K562BCMA Mut cells was detected by the luciferase assay. Additionally, real-time cell analysis (RTCA) technique was employed to detect the cytotoxicity of BCMA CAR-T cells against SKOV3BCMA Mut and CHOBCMA Mut cells. Results: Application of γ-secretase inhibitor LY411575 to inhibit γ-secretase activity significantly enhanced the expression level of BCMA on the surface of wild-type U266 cells, and the average fluorescence intensity was increased by more than 10 times. However, the expression level of BCMA gradually decreased after removal of inhibitors (P<0.01). BCMA-CD8α TM mutant could resist the cleavage of γ-secretase and expressed stably on the surface of U266 cells (P>0.05). U266 cells and U266 cells overexpressing BCMA-CD8α TM were co-incubated with BCMA-CAR-Jurkat-Reporter cells, both of which could activate the Reporter system and enhance the expression of EGFP, but the effect was more significant in U266 cells overexpressing BCMA-CD8α TM (P<0.01). BCMA-CD8α TM mutants were successfully overexpressed in 3 BCMA-negative target cells, namely K562, SKOV3 and CHO cells, and the expression level of the mutant was only slightly increased under LY411575 treatment. Luciferase assay results showed that under different target-effect ratios BCMA CAR-T cells could all be specific and efficient in killing K562 cells overexpressing BCMA-CD8α TM. RTCA results showed that under different target ratios BCMA CAR-T cells could all effectively recognize and kill SKOV3 and CHO cells overexpressing BCMA-CD8α TM, but Mock-T cells with the same target ratio had no such effect. Conclusion: The BCMA-CD8α TM mutant engineered in this study can resist γ-secretase cleavage and exhibits stable surface expression on various target cells, thus offering various methods for evaluating the efficacy and specificity of BCMA CAR-T cell cytotoxicity in vitro. [ABSTRACT FROM AUTHOR]

The aim of the experiment was to investigate the effect of VC and bacterial enzyme complex combined with glucose on serum biochemical indexes, immune and antioxidant function of cows with antibiotic poisoning. Twenty cows with antibiotic poisoning, weighing about (250.00±10.23) kg were randomly divided into a control group and an experimental group, with five replicates in each group and two cows in each replicate. The cows in the control group received an intravenous injection of glucose and VC, and the cows in the experimental group received an oral administration of glucose and bacterial enzyme complex for seven days. The results showed that compared with the control group, on the 8th day, the leukocytes, neutrophils, erythrocytes and hemoglobin of cows in the experimental group were significantly decreased ( P<0.05), and the platelets were significantly increased ( P<0.05). The activitiy of ALT and CK, and the concentrations of TBIL, Na, P and BUN in serum significantly decreased ( P<0.05), while the concentrations of ALB and Ca significantly increased ( P<0.05). The concentrations of IgM, IgG, IL-6, IFN-γ, SOD activity and T-AOC in the serum of the cows in the experimental group significantly increased ( P<0.05), while the concentrations of MDA significantly decreased ( P<0.05). On the 8th day of the experiment, there were no significant differences in blood routine, serum biochemistry, antioxidant indexes and immune between the experimental group and healthy cows ( P>0.05). The study indicates that glucose and bacteriase complex treatment can effectively improve the blood routine, serum biochemical, immunity and antioxidant function. [ABSTRACT FROM AUTHOR]

Objective: To investigate the influence of peripheral hemoglobin (Hb)-to-red cell distribution width (RDW) ratio (HRR) on the prognosis of patients with diffuse large B-cell lymphoma (DLBCL)., Methods: Data of 265 patients with diffuse large B-cell lymphoma (DLBCL) at the Affiliated Hospital of Xuzhou Medical University from January 2014 to December 2019 were retrospectively analyzed. 132 healthy people in the same period were used as normal control group. The best cut-off points of HRR was determined by receiver operating characteristics (ROC) curve; the chi-square test was used to analyze the correlation of clinical characteristics with HRR; the Kaplan-Meier method was used to compare the overall survival (OS) and progression-free survival (PFS) of HRR patients in different groups; the Cox proportional risk model was used for univariate and multivariate analysis., Results: The best cut-off value of HRR was 0.936, which was divided into low HRR group and high HRR group. The low HRR group had a higher ECOG score, higher incidence of advanced Ann Arbor stage, higher NCCN-IPI score, and elevated LDH level. K-M survival analysis showed that OS (P<0.001) and PFS (P<0.001) in the low HRR group were significantly shorter than that in the higher HRR group. The multivariate analysis revealed that HRR was an independent predictor of OS（HR＝0.379，95％CI：0.237-0.605，P<0.001） and PFS （HR＝0.384，95％CI：0.241－0.614，P<0.001） in DLBCL patients., Conclusion: Low HRR（<0.936） in patients with DLBCL indicates a poor prognosis, which is an independent prognosis risk factor.

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ -ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ -signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMax TM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.

Objective: To investigate the relationship between the levels of ferritin, C-reactive protein (CRP), lactate dehydrogenase (LDH) and interleukin-6 (IL-6) in peripheral serum and cytokine release syndrome (CRS) in patients with relapse and/or refractory multiple myeloma (R/R MM) after receiving chimeric antigen receptor T cells (CAR-T) immunotherapy., Methods: Twenty-eight patients with R/R MM were treated with 1×10 6 /kg humanized CD19 CAR-T and mouse B cell maturation antigen CAR-T cells after pretreatment chemotherapy based on fludarabine and cyclophosphamide. The concentrations of ferritin, CRP, LDH, and IL-6 in peripheral blood were measured regularly within 30 days after infusion, and the correlation between severity of CRS and above indexes was analyzed., Results: Among the 28 patients, 27 cases (96.4%) developed CRS, 24 cases (85.7%) in 1-2 grade CRS and 3 cases (10.7%) in 3-5 grade. The severity grade of CRS of 27 patients was positively correlated with the peak values of ferritin, CRP, LDH, and IL-6 in peripheral blood (r 1 =0.511, r 2 =0.375, r 3 =0.480, r 4 =0.632). The median peak values of ferritin, CRP, LDH and IL-6 in peripheral serum of patients with grade 3-5 CRS were significantly higher than those in patients with grade 0-2 CRS., Conclusion: After receiving CAR-T cellular immunotherapy, the incidence of CRS in patients with R/R MM is higher, but most of them are in grade 1 or 2. The severity of CRS is positively correlated with the levels of ferritin, CRP, LDH and IL-6 in peripheral blood.

Objective: To investigate the effect of electroacupuncture (EA) on the p38 mitogen-activated protein kinase (p38 MAPK) and signal transducer and activator of transcription 3 (STAT3) pathway in hippocampus and frontal cortex of diabetic rats with cognitive impairment (CI), as well as the mechanism of EA in protection against CI in diabetic rats., Methods: Thirty SD rats were divided into normal, model and EA groups ( n =10 rats/group). The diabetic model was established by i.p.injection of Streptozotocin solution(25 mg/kg), followed by high-fat diet raising for 1 month, and the CI rats was confirmed by Morris water maze tasks. The rats in the EA group were given acupuncture at "Zusanli" (ST36) "Neiting" (ST44) and "Yishu" (EX-B3) 20 min/d, among which ST36 and ST44 were treated with EA. The treatment was conducted 6 times a week for 4 weeks. The fasting blood glucose (FBG) contents were assayed by glucometer before and after treatment. The rats' learning-memory ability was detected by Morris water maze tasks. The expression levels of IL-6、IL-1β、TNF-α、p38 MAPK、p-p38 MAPK、STAT3 and p-STAT3 in hippocampus and frontal cortex were detected by Western blot and quantitative real-time PCR, separately. The mean fluorescence intensity of p38 MAPK and STAT3 was observed by immunofluorescence histochemistry., Results: After modeling, FBG and the escape latency of Morris water maze tasks were significantly increased in the model group compared with the normal group ( P <0.001, P <0.01). Following EA treatment, the increased FBG and average escape latency were markedly reversed in the EA group relevant to the model group ( P <0.05). Compared with the normal group, the proteins and mRNAs expression of IL-6, IL-1β, TNF-α, p38 MAPK, p-p38 MAPK, STAT3 and p-STAT3 in hippocampus and frontal cortex were significantly increased in the model group ( P <0.001), as well as the mean fluorescence intensity of p38 MAPK and STAT3 in hippocampus and frontal cortex ( P <0.001). Following EA intervention, the proteins and mRNAs expression of IL-6, IL-1β, TNF-α, p38 MAPK, p-p38 MAPK, STAT3 and p-STAT3, and the mean fluorescence intensity of p38 MAPK and STAT3 in hippocampus and frontal cortex were down-regulated( P <0.001, P <0.05)., Conclusion: EA can inhibit the over production of pro-inflammatory cytokines in diabetic rats with CI, possibly by regulating the expression of p38 MAPK and STAT3 pathway.

Drugs targeting immune checkpoint are used for cancer treatment, but resistance to single drug may occur. Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome. Therefore, we designed a recombinant protein rPC to block multiple targets, which consists of extracellular domains of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The coding sequence was inserted into expression vector and stably transfected into HEK293 cells. The culture supernatant was collected and rPC was affinity-purified. Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines. The binding of rPC with cancer cells was examined by immunofluorescence cell staining, the influence of rPC on cancer cell growth was assayed by CCK-8. The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells, the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4, no direct impact on cancer cell growth could be observed by rPC treatment. The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.

Objective: To study the level and clinical value of Th17 cell subset in the peripheral blood of the patients with aplastic anemia (AA)., Methods: Eighty-five patients with aplastic anemia in our hospital from May 2017 to February 2018 were selected. Among them, 51 patients with poor curative effect were enrolled in group A. and the 34 patients with good curative effect were enrolled in group B, the 35 healthy persons were selected as controls. The levels of serum IL-17, IL-6, IL-4 and IFN-β were analyzed by ELISA; the levels of peripheral blood Th17 cell subset were analyzed by flow cytometry; the expression levels of RORγt and STAT3 mRNA in lymphocytes were analyzed by RT-PCR; the expression levels of RORγt and STAT3 protein in lymphocytes were analyzed by Western blot., Results: There was no significant difference in sex, age, WBC count and complications between group A and group B (P＞0.05). Non-severe aplastic anemia (NSAA) in group B accounted for 23 cases, and the NSAA ratio (23/34) was significantly higher than that in group A (20/51) (P＜0.05). The Hb level (86.25±7.9 g/L) and Plt count (54.7 ± 6.3×10 9 /L) in group B were significantly higher than those in group A (P＜0.05). ELISA showed that the levels of IL-17 and IL-6 in group A were significantly higher than those in control group (P＜0.05); the levels of IL-17 and IL-6 in group B were significantly lower than those in group A (P＜0.05); the levels of IL-4 and IFN-β in group A were significantly lower than those in control group (P＜0.05); the levels of IL-4 and IFN-βin group B were significantly higher than those in group A (P＜0.05). Flow cytometry showed that the proportion of Th17 cells in peripheral blood of group A was higher than that of group B (P＜0.05). RT-PCR showed that the levels of RORγt and STAT3 mRNA in group A were significantly higher than those in control group (P＜0.05), and the mRNA levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). Western blot showed that the protein levels of RORγt and STAT3 in group A were significantly higher than those in control group (P＜0.05), and the protein levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05)., Conclusion: The Th17 cells in peripheral blood of AA patients increase, and the inflammatory reaction in the patients are enhanced. It may be related with the rise of RORγt and STAT3 gene expression, which provides a research direction for clinical treatment of AA.

Objective: To study the safety and effectiveness of humanized CD19-targeted CAR-T cells (hCART19s) for treatment of patients with refractory/relapsed (R/R) B-ALL., Methods: The analyzed patients were 15 children and adults with relapsed/refractory B-ALL who not received treatment with murine CD19 CAR-T cells. The patients received a single dose (1×10 6 /kg) of autologous hCART19 infusion after lymphodepletion chemotherapy based on cyclophosphamide and fludarabine., Results: Among the 15 patients, 13/14 (92.9%) evaluable patients achieved complete remission (CR) or CR with incomplete recovery of blood cells (CRi) on day 30 after hCART19s infusion. At day 180 after the infusion, the overall survival rate was 73.3%, and the leukemia-free survival rate was 69.2%. The cumulative incidence of relapse was 24.5% and non-relapse mortality rate was 7.7%. During treatment,12/15 patients (80%) developed cytokine release syndrome (CRS) of grade 1-2, and 3 patients (20.0%) developed CRS of grade 3-5. Only one patient (6.7%) suffered from the reversible neurotoxicity., Conclusion: hCART19s can effectively treat refractory/relapsed (R/R) adult and children with B-ALL, and the incidence of treatment-related CRS and neurotoxicity is low.

Objective: To investigatc the curative efficacy of low dose rituximab for glucocorticoid ineffective on dependent ITP patients and its relation with sensitivity to glucocorticoid so as to provide reference basis for rational use of drugs in clinical treatmant., Methods: Seventy-ninth ITP patients enrolled in this study included the glucocorticoid-ineffective patients (19 cases) and glucocorticoid-dependent patients (60 cases). All ITP patients were treated with regimen consisted of high dose dexamethasone plus low dose rituximab (dexal-methasone 40 mg/d for 4 days per os, ritaximab 100 mg by intravenous infusion at D7, 14, 21 and 28 respectively). The patients after treatment were followed-up for 12 month, and the relation of patients sensitivity to glucocorticoid with therapentic response of rituximab was analyzed. The changes of Treg cell ratio and BAFF, IL-2 and sCD40L levels before and after treatment were detected by flow cytometry and ELISA respectively., Results: The overall response rate (ORR) of patients treated with above- mentioned regemen at 1, 3, 6 and 12 months after treatment was 79.7% (63/79), 69.6% (55/79), 63.3% (50/79) and 60.8% (48/79) respectivcly, out of which the ORR of glucocorticoid ineffective and glucocorticoid-dependent ITP patients treated with above-mentioned regimen at 1, 3, 6 and 12 months after treatment was 47.4% (9/19) vs 90.0% (54/60), 36.8% (7/19) vs 80.0% (48/60), 21.1% (4/19) vs 76.7% (46/60), 21.1% (4/19) vs 73.3% (44/60), and the difference between 2 groups was statistically significant. The detection of T reg cell showed that the T reg cell ratio in glucocorticoid- ineffective and dependent patients at 1, 3, 6 and 12 months after treatment was (1.70±0.43)% vs (3.47±0.72)%, (1.66±0.33)% vs (4.29±0.91)%, (1.71±0.37)% vs (4.44±0.97)%, (3.36±0.54)% vs (4.29±1.04)%, respectively. The detection of cytokines showed that the levels of BAFF, IL-2 and sCD40L in plasma of glucocorticoid-dependent patients at 1 month after treatment significanlly decreased (P＜0.05), the levels of BAFF, IL-2 and sCD40L in plasma of glucocorticoid-ineffective patients although decreased at 1 mouth after treatment, but there was no statistical difference as compared with glucocosticoid-depenment patients., Conclusion: The treatment of glucocorticoid-dependent ITP patients with rituximab is more effective. The regulatory effect of rituximab on the T-reg cells, BAFF, IL-2 and sCD40L may be one of its mechanisms.

Objective: To establish the reference intervals of the hematological parameters in normal adult people of Xuzhou erea., Methods: The red blood cells (RBC), platelets, white blood cells (WBC) and related parameters were detected by hematoanalyzers in 82514 healthy people including 41257 males and 41257 females in the Medical Center of the Affiliated Hospital of Xuzhou Medical University., Results: The range of RBC count: (4.33-5.51) ×10 12 /L in male and（3.82-4.85）×10 12 /L in female, the range of Hb level: (132-172) g/L in male and（107-145）g/L in female, the range of HCT: 40 %-50 % in male and 34 %-44 % in female, the range of platelet count: (113-268) ×10 9 /L in male and (126-289) ×10 9 /L in female, the range of WBC count: (4.00-9.40) ×10 9 /L in male and （3.54-9.30）×10 9 /L in female, the range of NEUT count: (1.91-5.76) ×10 9 /L in male and（1.67-5.30）×10 9 /L in female, the range of MONO count: (0.18-0.58) ×10 9 /L in male and（0.16-0.52）×10 9 /L in female, the range of LYM is（1.3-3.4）×10 9 /L in male and（1.2-3.1）×10 9 /L in female, etc.Conclusion: There is significant difference in the blood cell parameters between Xuzhou and other areas. Among them, the lower limit of Hb reference interval for adult women in Xuzhou area is obviously lower, and the upper and lower limits of adult Plt reference interval are significantly lower than other areas in China and abroad. The upper and lower limits of the WBC reference interval are close to the domestic areas, but lower than that in some foreign regions. Through this survey, the reference intervals of different hematologic parameters of healthy people in Xuzhou area primarily has been established, Some of the indexes, such as RBC, Hb, HCT, and Plt have significant sex differences. The reference intervals for them have been estublisted respectively.

Objective: To analyze the risk factors and prognosis of hepatic chronic GVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT)., Methods: The clinical data of 147 patients undergoing allo-HSCT from January 2013 to December 2016 were analyzed, the correlation between recipient age and sex, disease state, matched degree of HLA, donor sex, stem cell sources, ATG in GVHD prophylaxis, liver dysfunction during conditioning period, pre-transplant HBsAg, prior aGVHD and hepatic cGVHD were studied, and the correlation between hepatic cGVHD and prognosis were analysed., Results: Thirty-two patients had hepatic cGVHD, cumulative incidence of 26.4%. In univariate analysis, pre-transplant HBsAg + and liver dysfunction during conditioning period were not significantly related with hepatic cGVHD (P>0.05). In multivariate analysis, prior acute GVHD (HR=2.087, P=0.045) was the independent risk factor for hepatic cGVHD, ATG (HR=0.231, P=0.000) was significantly related with a lower incidence of hepatic cGVHD. In univariate analysis, patients with hepatic cGVHD had a lower 2 years relapse rate (P=0.038)., Conclusion: Prior acute GVHD is the independent risk factor for hepatic cGVHD, the ATG can significantly reduce the incidence of hepatic cGVHD. Hepatic cGVHD has been found to relate with a lower 2 years relapse rate.

Objective: To evaluate biological effects of OCT4A gene on K562 cells and explore the molecular mechanism of K562 cell apoptosis., Methods: Two recombinant lentiviral vectors were constructed, which could stablely up- regulate and down- regulate OCT4A protein. Recombinant lentivirus was generated by co-transfection of three-plasmids and transfec-ted into K562 cells. The experiments were divided into 5 groups: normal, pLVX-OCT4A-ZsGreen1, pLVX vector control, PLB-OCT4A shRNA and non-specific shRNA groups. Western blot was applied to detect the expression of OCT4A protein, the cell counting kit-8 was applied to evaluate the effect of OCT4A on proliferation of K562 cells. The apoptosis and differentiation of K562 cells were detected by flow cytometry with AnnexinV/7-AAD double staining. The mRNA expressions of caspase-3,BIM,BCL-xL,BAX in K562 cells were determined by real time PCR., Results: The OCT4A fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR), the 2 lentiviral vectors were successfully constructed. In comparson with those in the control group, the expression of OCT4A protein of pLVX-OCT4A-ZsGreen1 group was significantly increased, but decreased in PLB-OCT4A shRNA group. CCK-8 assay showed that the higher the content of OCT4A protein, the faster the cell proliferation. The apoptosis rate was (3.48±0.52)% of pLVX-OCT4A-ZsGreen1 group, which was lower than that of control group, while the apoptosis rate PLB-OCT4A shRNA group was (7.25±0.57)%, which was higher than that of control group (P<0.05), however, the K562 cells differentiation was not influenced(P>0.05). Compared with control group, the gene expression of Caspase-3,BIM and BAX was down-regulated(P>0.05), but a significant up-regulation of BCL-xL gene expression was observed(P<0.05)., Conclusion: Two lentiviral vectors have been successfully constructed, which can stably up- and down- regulate the expression of OCT4A in K562 cells respectively. OCT4A can promote the K562 cell proliferation and inhibit the apoptosis, the mechanism may be related with up-regulation of BCL-xl expression.

Objective: To evaluate the clinical significance of thrombopoietin (TPO) level in diagnosis of pateints with aplastic anaemia (AA)., Methods: The TPO levels in sera from 54 AA patients and 119 healthy controls were examined. A total of 92 samples were collected from AA patients including 43 samples harvested at diagnosis, 23 samples in the cytopenic period after treatment, and 26 samples of patients in partial (n=10) or complete remission (n=16) following immunosuppressive treatment. Serum TPO levels were assessed by a sandwich-antibody ELISA that utilized a polyclonal rabbit antiserum for both capture and signal., Results: Serum samples from normal donors revealed a mean TPO level of 95.3±54.0 pg/ml, the mean TPO levels in AA sera collected at diagnosis and before onset of treatment were 2728±1074 pg/ml (P<0.001) compared with normal controls; mean platelet count at that time: 27×10 9 /L). Serum TPO levels of AA patients in partial or complete remission after immunosuppressive treatment were significantly lower than TPO levels at diagnosis (P<0.001). However, despite normal platelet counts (mean 167×10 9 /L), TPO levels remained significantly elevated in complete remission (mean TPO 1009±6590 pg/ml (P<0.001) compared with normal controls. There was a significant negative correlation between serum TPO levels and platelet counts in AA patients (coefficient of correlation r=-0.70, P<0.001)., Conclusion: TPO levels are highly elevated in sera of patients with AA. Thrombopoietin did not return to normal levels in remission, indicating normal platelet count in remission of AA needs sustained high level of TPO.

Objective: To explore the effect of Caspase 1 inhibitor Ac-YVAD-CMK on acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation(allo-HSCT) and its mechanism., Methods: Experiments were divided randomly into 3 groups: allogeneic hematopoietic stem cell transplantation combined with splenic cell infusion group (TS group, n=12), allogeneic hematopoietic stem cell transplantation combined with splenic cell infusion plus injection of low dose Caspase 1 inhibitor group (TS+low dose of C group, n=16) and plus high dose Caspase1 inhibitor (TS+high dose of C group, n=19). The body weight of mice in each group was dynamically detected, and the clinical manifestation of GVHD and score of aGVHD were determined, and the chimerism rate of mice was detected after transplantation for 14 days. Th1, Th2 and Th17 cells in peripheral blood were examined by flow cytometry. Peripheral proinflammatory cytokines IL-1β, IFN-γ, IL-1α and IL-18 were examined by enzyme-linked immunosorbent assay(ELISA). The tissues sections of GVHD target organs (liver, lung, colon and skin) were stained with HE for histopathologic examination and histopathologic score., Results: Ac-YVAD-CMK could alleviate murine aGVHD and pathological injury, decreare the incidence and severity of aGVHD in recipient mice. The detection of Th cell subsets in peripheral blood by flow cytometry showed that compared with TS group, the Th1 cell ratio in TS+low dose of C and TS+high dose of C groups was significantly reduced (P<0.05), while the Th2 and Th17 cell ratio was significantly enhanced (P<0.05) in TS+low dose of high dose of C groups. The detection of peripheral inflamematory cytokines by ELISA demonstrated that the inflammatory cytokines including IL-1β,IFN-γ,IL-18 and IL-1α were reduced significantly (P<0.05)., Conclusion: Ac-YVAD-CMK can improve aGVHD by inhibiting Caspase 1 and reducing the release of some inflammatory cytokines, thereby alleviated the aGVHD pathological damage.

Objective: To establish the human colorectal cancer cell model SW620/PAR4D with inducible suppression of proteinase activated receptor 4 (PAR4) expression, and investigate the role PAR4 plays in the proliferation and migration of cancer cells., Methods: A human colorectal cancer cell line with tetracycline-inducible expression regulatory system, namely SW620/Tet-on, was established; inducible expression lentiviral vector with artificial microRNA targeting PAR4, pLVX-Tight-Puro-PAR4-miR, was constructed and transfected into SW620/Tet-on to make an inducible PAR4-suppressed cell model SW620/PAR4D. Western blotting was used to confirm the suppression of PAR4 expression after the doxycycline (DOX) treatment. CCK-8 assay was used to evaluate the impact of suppressed PAR4 expression on cell proliferation, and wound-healing assay was used to analyze the migration of the cells., Results: The SW620/PAR4D cell model was established successfully. Suppression of PAR4 expression by DOX treatment had no significant impact on the growth/proliferation of SW620/PAR4D cells, but markedly inhibited the cell migration., Conclusion: Suppression of PAR4 expression has no significant effect on the proliferation of SW620 cells, but can inhibit the migration of the cells.

Objective: To determine the role of phosphatidylinositol 3-kinase--protein kinase B (PI3K-Akt) signaling pathway in the pro- tective effect of aerobic endurance training on the skeletal muscular mitochondria., Methods: Thirty-six rats were randomly divided into three groups( n = 12): control group, aerobic endurance training group and one-time exhaustive group. After the intervention, the quadriceps femoris muscle sample was obtained to detect the mitochondrial membrane potential( MMP), the activities of succinate dehydrogenase (SDH) and cy- tochrome coxidase (COX), and the protein levels of p-PI3K and p-Akt., Results: Compared with the control group, the levels of mitochondrial membrane potential, the activities of succinate dehydrogenase and cytochrome coxidase, and the protein levels of p-PI3K and p-Akt were all significantly decreased in the one-time exhaustive group (P < 0.05). However, all the above was partially reversed in the endurance training group (P < 0.05), and there was no obvious difference with the control group (P > 0.05)., Conclusion: Aerobic endurance training plays an important role in the protective effect on the skeletal muscular mitochondria, the mechanism may be related to activation PI3K-Akt signaling pathway.

Objective: To investigate the effect of ADAM10 inhibitor GI254023X on the proliferation and apoptosis of acute T-lymphoblastic leukemia Jurkat cells and its mechanisms., Methods: Jurkat cells were treated with different concentrations of GI254023X, the proliferation-inhibition curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis was detected by flow cytometry with Annexin V and 7-AAD staining, the cleavage of Notch1 protein was determined by Western blot, the transcripts of anti-apoptotic genes BCL-2, MCL-1, BCL-xl and Notch1 target gene Hes-1 were detected by real-time PCR., Results: The GI254023X obviously inhibited the proliferation of Jurkat cells in concentration-dependent manner. As compared with the control group, the apoptosis of cells increased along with increment of GI254023X concentration. Compared with control group, the expression of Cleaved Notch1 was down-regulated while the expression of Notch1 was up-regulated in a time-dependent manner after the treatment with GI254023X. The levels of MCL-1 and Hes-1 mRNA transcripts in Jurkat cells were reduced in GI254023X treated group, but did not show obvious effect on the level of BCL-2 and BCL-xl mRNA transcripts., Conclusion: GI254023X can remarkably inhibit proliferation and induce apoptosis of Jurkat cells. The inhibition of Notch1 activation and the down-regulation of apoptosis-related gene MCL-1 may be involved in the process of apoptosis.

Objective: To investigate the effects of bromodomain-containing protein 4 (BRD4) inhibitor GSK525762A on the proliferation and apoptosis of primary common B-cell acute lymphoblastic leukemia (common B-ALL) cells from adult patients, then to further explore the possible mechanisms., Methods: Purified leukemia cells from 14 common B-ALL adult patients (4 Ph⁺ and 10 Ph⁻ cases) were obtained by flow cytometry sorting, and maintained in a mimic bone marrow microenvironment culture system for short-term culture. Leukemia cells were treated with various concentrations of GSK525762A. The inhibitory effects of BRD4 inhibitor on common B-ALL leukemia cells were measured by CCK-8 assay and the apoptosis of those cells was determined by AnnexinⅤ/7-AAD staining using flow cytometry. The transcripts of c-MYC, CDK6 and Bcl-2 were detected by quantitative RT-PCR, and the expression of c-MYC, CDK6 and Bcl-2 proteins were detected via Western blot., Results: GSK525762A could inhibit the proliferation of leukemia cells from all 14 common B-ALL patients in a dose-dependent manner, the median value of IC50 was 256.25 (90.64-1 378.39)nmol/L. GSK525762A could promote cells apoptosis of B-ALL leukemia cells in a dose-dependent manner, the median apoptosis rates respectively were 45.17%(9.38%-70.91%), 66.02% (24.36%-96.34%) and 89.29% (39.29%-99.37%) after treated by 500, 1 000 and 2 500 nmol/L GSK525762A. GSK525762A has a similar effect on Ph⁺ ALL and Ph⁻ B-ALL, but the effect of proliferation inhibition and apoptosis enhancement on Ph+ B-ALL is weaker than that on Ph⁻ B-ALL. Compared with vehicle control group, the levels of c-MYC, Bcl-2 and CDK6 transcripts in leukemic cells were reduced after treatment for 24 h and 48 h by 1 000 nmol/L GSK525762A, and there are no significant differences in the downregulation of c-MYC and CDK6 mRNA between Ph⁺ and Ph⁻ B-ALL; however, the inhibitory effect on Bcl-2 transcription was weaker in Ph⁺ B-ALL cells than that in Ph⁻ B-ALL cells. Moreover, c-MYC, Bcl-2 and CDK6 protein levels decreased in GSK525762A treated group., Conclusion: GSK525762A could strongly inhibit the proliferation of common B-ALL and trigger apoptosis; meanwhile it has certain effects against Ph⁺ ALL in vitro. The effect may be achieved by down-regulation of c-MYC, CDK6 and Bcl-2 expression.

Objective: To investigate the effect of metformin on proliferation, differentiation and apoptosis of THP-1 cells and explore its possible mechanism., Mehods: THP-1 cells were cultured with different concentrations of metformin for 24 h and 48 h. The cell proliferation was evaluated by CCK-8, the cell apoptosis was analyzed by Annexin V/7-AAD double labeling, the expression of CD14 and CD11b (surface differentiation antigens on THP-1 cells) was evaluated by flow cytometry, the BCL-XL, BAX, BIM and caspase-3 mRNA expressions of THP-1 cells were detected by real time quantitative PCR., Results: Metformin could significantly inhibit the growth of THP-1 cells in a time- and dose- dependent manner. After treated with 20 mmol/L metformin for 24 h, the expressions of CD14 and CD11b in THP-1 cells didn't change much (P>0.05), the early apoptosis rates in exprimental and control groups were (2.02±0.85)% and (4.46±1.33)% respectively, the late apoptosis rates in experimental and control groups were (1.43±0.83)% and (3.31±0.59)% respectively. In process of inducing effect of 20 mmol/L metformin on THP-1 cells, the expressions of BCL-XL and BIM did not significantly changed, while the expressions of BAX and caspase-3 significantly increased (P<0.01)., Conclusion: Metformin can effectively inhibit proliferation and induce apoptosis of THP-1 cells. However, it has no significant effect on differentiation of THP-1 cells, its mechanism inducing apoptosis maybe related with up-regulating BAX and caspase-3.

Objective: To observe the efficacy of high-dose dexamethasone in combination with low-dose rituximab as a second-line treatment for patients with immune thrombocytopenia (ITP)., Methods: 65 patients with ITP, previously by conventional dose of glucocorticoids, received high-dose dexamethasone in combination with low-dose rituximab (dexamethasone 40 mg/d for 4 days, rituximab 100 mg, d 7, 14, 21, 28 intravenous infusion). Treatment response, regulatory T cells (Treg), cytokines levels and treatment-related adverse effects were observed., Results: Total response rate 1 month after treatment was achieved in 81.5% (53/65) of patients, and complete response at 3,6 and 12 months was 72.3% (47/65), 66.2%(43/65), 63.1%(41/65). The higher efficiency and complete response rate was achieved in preexisting glucocorticoid-dependent patients. For patients with complete response, Treg cells continued to show a high level state [(3.01 ± 0.95)% vs (1.69 ± 0.35)%, P=0.032], cytokines of BAFF [(648.03 ± 79.63) ng/L vs (972.35 ± 93.64) ng/L, P=0.001], IL-2 [(2.84 ± 0.32) ng/L vs (4.18 ± 0.46) ng/L, P=0.012], sCD40L[(4.55 ± 0.66) ng/L vs (7.73 ± 1.04) ng/L, P=0.006] significantly lower than that before treatment. The level of IL-10 was increased, but without significance compared with that before treatment (P=0.136). All patients completed the protocol with no serious adverse reactions., Conclusion: The data show high-dose dexamethasone in combination with low-dose rituximab still has a satisfactory outcomes for patients previously with conventional dose of glucocorticoid.

This study was purposed to construct a lentiviral vector carrying human OCT4A gene and green fluorescent protein (GFP) , and infect the leukemic cell line K562, observe the expression of OCT4A in K562 cells. According to the sequence of OCT4A mRNA which was found in GenBank, the special primer sequences were synthesized. The OCT4A gene was amplified by RT-PCR, and then cloned into the pCR-Blunt vector. The OCT4A DNA fragment was subcloned into the lentiviral vector pLVX-IRES-ZsGreen1 which was restricted by EcoR1 to generate a lentiviral vector pLVX-OCT4A-ZsGreen1. The sequence of the recombinant plasmid was identified by DNA sequencing. Recombinant lentivirus was generated by co-transfection of three-plasmids into 293FT cells using lipofectamine 2000 and transfected into K562 cells. Real-time PCR and Western blot were applied to detect the expression of OCT4A mRNA and protein, CCK-8 and colony formation assay were performed to evaluate the effects of OCT4A on proliferation of K562 cells. The results showed that the recombinant lentiviral vector pLVX-OCT4A-ZsGreen1 was successfully constructed. The virus titers were (1.43 ± 0.25) × 10(8) U/ml. After infection of K562 cells with the lentivirus, the recombinant plasmid could stably up-regulate the expression of OCT4A gene and protein according the real-time PCR and Western blot detection results. CCK-8 and colony formation assay showed that exogenous OCT4A gene could significantly promote cell growth and the colony formation of K562 cells. It is concluded that the recombinant lentiviral vector pLVX-OCT4A- ZsGreen1 carrying human OCT4A gene is successfully constructed; K562 cells which stably up-regulates the expression of OCT4A mRNA are obtained, the results of this study provide fundamental basis for further study on mechanism of OCT4A in human leukemia development.

Objective: To explore gene expression of NANOG in T-cell acute lymphoblastic leukemia (T-ALL) cell lines and the effects of NANOG gene down-regulation on apoptosis of leukemia cells., Methods: Real-time PCR (RT-PCR) and Western blot were used to detect the expression level of NANOG gene and protein in MOLT-4, CCRF-HSB2 and Jurkat cells. To test the efficiency of RNA interference, MOLT-4 cells were firstly infected by lentiviral vectors, which were successfully constructed with NANOG specific shRNA. NANOG expression levels were subsequently re-evaluated by RT-PCR and Western blot. The percentages of early apoptotic cells (Annexin V⁺/7-AAD⁻) and late apoptotic cells (Annexin V⁺/7-AAD⁺) were analyzed by flow cytometry. The expression of apoptosis-related genes was also detected., Results: Both NANOG gene and protein expression was positive in MOLT-4 and CCRF-HSB2 cells. The lentiviral vectors pLB-shNANOG-1, pLB-shNANOG-2, and pLB-sh control were successfully constructed, as evidenced by the viral titers (1.83-3.12)× 10⁸ IU/ml. The experimental data on infection of MOLT-4 cells with such lentiviral vectors revealed that both shRNA interfering sequences (shNANOG-1 and shNANOG-2) could stably down-regulate NANOG gene and protein expressions. The percentages of early apoptotic cells in groups of shNANOG-1[(8.06 ± 1.61)%]and shNANOG-2[(5.67 ± 1.59)%]were significantly increased as compared to that of MOLT-4 group[(1.13 ± 0.40)%]or sh-control [(1.15±0.49)%](P<0.05). However, no statistical difference among them was observed for late apoptotic cells (P>0.05). The gene expression of TP53, PMAIP1, and CASP9 of either shNANOG-1 or shNANOG-2 group was augmented as compared to that of MOLT-4 group or sh-control (P<0.05). Reversely, a significant down-regulation of Bcl-2 gene expression was observed (P<0.05)., Conclusion: NANOG can be expressed in various human T-ALL cell lines. Down-regulation of NANOG can trigger leukemia cellular apoptosis through mitochondria-dependent apoptosis pathway.

Objective: To establish a high efficient human coagulation factor VIII (FVIII) eukaryotic stable expression system using lentiviral vector, and determine its biosafety., Methods: Lentiviral transfer plasmid carrying human B-domain-deleted FVIII(BDDhFVIII)-IRES-GFP(BDDhFVIII/pXZ9)or IRES-GFP(pXZ9) was constructed. Lentivirus particles were produced by transiently co-transfected 3-plasmids into 293FT cells and further concentrated via ultracentrifugation. CHO cells were infected, 72h later, the FVIII antigen (FVIII:Ag) concentration in the medium was examined by ELISA, the activity was detected via one stage coagulation,and the transcription of FVIII in the infected CHO cells was determined by RT-PCR.Virus infection ability in the medium and the gag gene in CHO cells were determined to evaluate the model's biosafety., Results: Lentiviral transfer plasmid BDDhFVIII-IRES-GFP(BDDhFVIII/pXZ9)carrying human B-domain-deleted FVIII or IRES-GFP (pXZ9) was successfully constructed, and high titer lentiviruses has been prepared. The lentivirus could infect CHO cells efficiently, after an additional 72 h, the FVIII:Ag concentration had up to (1724.9±283.7) mU/ml, the FVIII:C level increased to (10.58±1.55)%, and transcripts of BDDhFVIII mRNA could be measured by RT-PCR. Neither the gag gene nor the virus in the supernatant was detected., Conclusion: Lentivirus-mediated human coagulation factor VIII could be expressed efficiently in CHO cells. The system couldn't produce offspring virus, proving a good biosafety.

Objective: To investigate the change of CD4⁺CD25(high)CD127(low) regulatory T cells (Tregs) percentage in patients with primary immune thrombocytopenia (ITP) treated by different methods., Methods: One hundred and thirty-eight newly diagnosed adult ITP patients (57 male, median age 40 years, range 18-70 years) were enrolled in this study, who were randomly separated into three regiment groups, namely prednisolone (PSL, 1.5 mg/kg for 2-4 weeks and subsequently stepwise reduction) group enrolled 49 patients, dexamethasone [(one course of high-dose dexamethasone (HDD) 40 mg/day, d1-4] 45 patients, and rituximab plus HDD (rituximab 100 mg on days 7, 14, 21, 28 and HDD) group 44 patients. Peripheral blood was taken in ITP patients of each group before treatment, 14 d and 28 d after treatment. The percentages of CD4⁺CD25(high)CD127(low) Tregs in 30 healthy controls and 138 patients were analyzed by flow cytometry., Results: Overall response (OR) rates of PSL, HDD and R+HDD groups at day 28 were 69.4%, 66.7% and 79.5% respectively with no difference. After the following 12 months, sustained response (SR) was more pronounced in R+HDD group compared to the other two groups (R+HDD vs PSL: 66.7% vs 37.8%, P<0.05; R+HDD vs HDD: 66.7% vs 22.7%, P<0.05). The percentage of CD4⁺CD25(high)CD127(low) Tregs in peripheral blood of ITP patients [(1.67±0.70)%] was significantly lower than in healthy control group; After treatment, the percentages of Tregs in peripheral blood of patients both at day 14 and 28 in R+HDD group remarkably decreased compared with before treatment [(4.28±1.09)% vs (1.68±0.68)%, P<0.05; (4.44±0.63)% vs (1.68±0.68)%]. The percentages of Tregs at day 14 in both other two groups decreased notably compared with before treatment. But the Tregs levels measured at day 28 in PSL and HDD groups were similar with before treatment., Conclusion: The percentage of CD4⁺CD25(high)CD127(low) Tregs in peripheral blood of ITP patients was lower than healthy individual. The higher SR of patients treated by R+HDD was related to its ability to up-regulate the percentage of CD4⁺CD25(high)CD127(low) Tregs.

Objective: To investigate the sensitivity of imatinib mesylate (IM) on Sup-B15 Ph⁺ acute lymphoblastic leukemia (ALL) cells knockdown of VE-cadherin (CD144), and to further explore its mechanism., Methods: CD144 in Sup-B15 leukemia cells was stably knock downed via lentivirus-mediated RNA interference (named as Sup-B15/shVEC). The inhibitory effects of IM on Sup-B15/shVEC and Sup-B15 leukemia cells were measured by CCK-8 test, and the apoptosis of those cells was determined by AnnexinV/7-AAD dyeing using flow cytometry, the percentage of CD34⁺CD38⁻ leukemia cells also by flow cytometry. ALDH1 mRNA levels were detected by real-time RT-PCR, and protein levels of CD144, CD133, Bcr-abl and β-catenin by Western blot., Results: IM treatment presented inhibitory effects on Sup-B15/shVEC and Sup-B15 leukemia cells at multiple concentrations of IM. The IC50 of IM on Sup-B15/shVEC and Sup-B15 leukemia cells were 25.1μmol/L and 18.7μmol/L, respectively (P<0.05). After 48h of 20 μmol/L IM treatment, the percentages of apoptosis cell in Sup-B15/shVEC cells and Sup-B15 cell were (13.52±2.06)% and (3.03±0.72) %, respectively (P<0.05). The percentage of CD34⁺CD38⁻ cells in Sup-B15 cells was significantly higher than in Sup-B15/shVEC cells [(2.39±0.28)% vs (0.96±0.07)%, P<0.05). As compared to Sup-B15 cells, the transcription of ALDH1 in Sup-B15/shVEC was remarkably downregulated, and the CD133 protein level was also downregulated in Sup-B15/shVEC cells. Both cytoplasmic and nucleic β-catenin protein levels (but not for Bcr-abl levels) decreased in Sup-B15/shVEC cells as compare to Sup-B15 cells., Conclusion: Knockdown of CD144 sensitized Sup-B15 Ph+ ALL cells to IM. The possible mechanisms underlying this phenomenon might be via inhibiting β-catenin nucleic translocation and facilitating β-catenin degradation.

Objective: To construct self-inactivating (SIN) lentiviral vector carrying human soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) gene and to observe the effects of sTRAIL gene on apoptosis in SGC-7901 cells, so as to assess the value of sTRAIL in gene therapy for gastric cancer., Methods: The bicistronic SIN lentiviral transfer plasmid containing sTRAIL gene and internal ribosomal entry site-green fluorescent protein gene (IRES-GFP) was constructed. Recombinant lentivirus containing sTRAIL were packaged using liposome by lentiviral packing system. Several cell lines including HL-7702, HLF-1 and SGC-7901 cells were infected with the viral supernatant. Flow cytometry was used to measure apoptosis of cells and recombinant sTRAIL. protein was assayed by ELISA at 24 h after infection., Results: The lentiviral transfer plasmid pXZ208-sTRAIL was constructed, and the virus titres were above 10(6) IU/mL in the supernatant. SGC-7901 cells were efficiently infected by recombinant virus. The apoptosis rate of SGC-7901 cells was increased with the virus multiplicity of infection (MOI) increasing. When the MOI was > or = 0.5, a dose-dependent apoptosis rate was observed. At MOI of 6.0, the highest apoptosis rate (29.12 +/- 2.87)% was observed, while the expression of sTRAIL in the supernatant was only (34.08 +/- 3.43) ng/mL. However, no significant apoptosis was observed in HL-7702 cells and HLF-1 cells., Conclusion: Recombinant lentivirus carrying human sTRAIL gene can efficiently infected SGC-7901 cells, which induced apoptosis in SGC-7901 cells in vitro by secreting bioactive sTRAIL protein. However, this effect is not seen in normal cells.

Objective: To compare the efficacy and safety of low-dose rituximab combined with different dosage of glucocorticoids for immune thrombocytopenia (ITP)., Methods: Seventy-four patients (35 male, median age 34 years, range 18-70 years) including 60 newly-diagnosed, 6 persistent, 5 chronic and 3 refractory patients were enrolled in this study, and separated into control (36 cases) and experimental (38 cases) groups according to the dosage of glucocorticoids. Patients in both groups received dexamethasone 40 mg/day on days 1-4, followed by rituximab 100 mg on days 7, 14, 21, 28. The patients in experimental group also received decrements of prednisone 60, 30, 20, 10 mg/day on days 5-7, 8-14, 15-21, 22-28. The initial, long-term efficacy and safety were evaluated., Results: Platelet counts of all patients at day 4 remarkably increased, with the median platelet count from 11(1-26) × 10⁹/L to 84(23-132) × 10⁹/L in control group, and 10(2-20) × 10⁹/L to 80(22-115) × 10⁹/L in experimental group; the platelet counts of patients at day 14 in experimental group [163(19-262) × 10⁹/L] was higher than that of control group [98(18-238) × 10⁹/L] (P<0.05). The overall response (OR) rates at day 28 in experimental group (84.21%) was significantly higher than that of control group (66.67%, P = 0.03). There was no significant difference of sustained response (SR) rates in two groups (63.89%vs 65.79%, 58.33%vs 60.53%, P > 0.05) at six and twelve months follow-up points. Both groups showed similar incidence of adverse events, and no patients discontinued the treatment due to side effects., Conclusion: Low-dose rituximab and glucocorticoids was an effective method for ITP patients, and maintenance treatment with decrements of prednisone contributed to improving earlier CR rate.

Objective: To investigate the effect of AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on proliferation, differentiation and apoptosis of U937 cells and explore its possible mechanism., Methods: U937 cells were cultured with different concentrations of AICAR for 24 h and 48 h. Cell proliferation was evaluated. Cell growth curve was analyzed by CCK-8; cell apoptosis was analyzed by cell morphology, Annexin V/7-AAD double labeling. The differentiation of U937 cells was evaluated by expression of CD11b. The Bcl-xL, Bax, Bim, caspase-3 mRNA expressions of U937 cells were determined by real time PCR., Results: AICAR significantly inhibited the growth of U937 cells in a time-and dose-dependent manner, with a 24 h IC50 value of 1.1 mmol/L and 48 h of 0.9 mmol/L. 1.0 mmol/L AICAR didn't induce differentiation of U937 cells with the increase of CD11b expression for 24 h (P > 0.05). The U937 cells apoptosis was confirmed by cell morphology and Annexin V/7-AAD labeling. AICAR induced apoptosis of U937 cells and the apoptosis rate was (6.81 ± 1.16)% at 1 mmol/L AICAR higher than control group (2.74 ± 0.32)% without AICAR for 24 h treatment (P < 0.05). The real time PCR assay revealed that as compared with control group, the expression of Bim and caspase-3 mRNA were increased, while Bcl-xL and Bax were unchanged on the AICAR treatment., Conclusion: AICAR can effectively inhibit proliferation and induce apoptosis of U937 cells. However, it has no significant effect on differentiation of U937 cells. The mechanism may be related with up-regulating Bim and Caspase-3.

The aim of this study was to detect the expression of interleukin-22 (IL-22) and relative CD4(+) T cell subsets in untreated adult patients with immune thrombocytopenia (ITP), and to explore their roles in the pathogenesis of ITP. Fourty adult active ITP patients were enrolled in this study and 40 healthy subjects were taken as control. Plasma IL-22 level was quantified by ELISA. The percentages of Th1, Th17 and Th22 cells in peripheral blood were determined by flow cytometry. The Pearson correlation test was used to evaluate correlations between IL-22 level and Th1 cells, Th17 cells and Th22 cell percentages. The results showed that the plasma IL-22 levels in untreated ITP patients [(364.12 ± 94.22) pg/ml] were significantly higher than that in healthy controls (P < 0.001). The percentages of both Th1 [(18.92 ± 6.03)%] and Th22 [(2.28 ± 0.51)%] cells in ITP patients were elevated as compared to healthy controls (P < 0.05). Elevated plasma concentration of IL-22 positively correlated to the percentages of Th1 (r = 0.42, P = 0.022) and Th22 (r = 0.40, P = 0.030) cells in untreated ITP patients. The percentage of Th17 cells was not significantly different between untreated patients and normal controls, and there was no statistical correlation between the IL-22 level and the percentage of Th17 cells in active ITP patients. It is concluded that elevated IL-22 level correlates to Th1 and Th22 cells percentage, which may play a synergistic effect in the immunopathogenesis of ITP, while Thl7 cells may not be associated with the occurrence of active ITP.

This study was aimed to clone mouse adam10 gene promoter and construct its dual luciferase report vector, and to investigate its transcriptional activity. Total DNA was extracted from mouse brain and used for amplifying the fragment containing adam10 gene promoter by PCR. The amplified product was inserted into pGL-4.10 vector to construct pGL4.10-adam10. The pGL4.10-adam10 and control plasmid pGL4.74 were co-transfected into HEK293 FT cells by lipofectamine 2000. The activity of adam10 gene promoter was assayed by luciferase system. The results showed that the recombinant plasmid pGL4.10-adam10 containing promoter of mouse adam10 was correctly constructed. The method was optimized by changing ratio of two plasmids. Moreover, the transcriptional activity of pGL4.10-adam10 stimulated by ionomycin increased. It is concluded that the dual luciferase reporter system is successfully established, which is useful in bioluminescence imaging technology in vitro. The effect of ionomycin can enhance the transcriptional activity of adam10 gene promoter.

Objective: To construct eukaryotic expression plasmids containing green fluorescent protein gene and CYP19 wild-type or its variants (W39R, R264C, W39R-R264C) and to observe its expression in MCF-7 and Bcap-37 cells., Methods: The aromatase WT cDNA sequence was obtained by RT-PCR amplification and cloned into the eukaryotic expression vector pcDNA3.1(+). pcDNA3.1(+)-CYP19-GFP plasmid was then used as the template for site-directed mutation to create variant constructs (W39R, R264C, W39R-R264C). pcDNA3.1(+)-CYP19-GFP was transfected and expressed in MCF-7 and Bcap-37 cells., Result: The construction of pcDNA3.1(+)-CYP19-GFP plasmid was confirmed by enzyme digestion and DNA sequencing. pcDNA3.1(+)-CYP19(W39R)-GFP, pcDNA3.1(+)-CYP19(R264C)-GFP, pcDNA3.1(+)- CYP19(W39R-R264C)-GFP plasmids were confirmed by DNA sequencing. The MCF-7 and Bcap-37 cells transfected with the pcDNA3.1(+)-CYP19-GFP plasmid expressed reporter gene of GFP., Conclusion: The eukaryotic expression plasmids have been constructed and expressed in MCF-7 and Bcap-37 cells successfully, which lays the foundation for the research of biological activities of CYP19 variant allozymes.

Objective: To explore the influence of the lentiviral vector mediated mouse genetic engineering regulatory T cells (Treg) infusion on post-allogeneic bone marrow transplantation (allo-BMT) acute graft-versus-host disease (GVHD) in mice., Methods: Lentivirus-mediated Forkhead box P3 (Foxp3) gene was transduced into BALB/c mice CD4(+)CD25(-) T cells (Treg) to construct engineered Tregs in vitro. An allo-BMT model of BALB/c to C57BL/6 mice was established. After irradiation, the recipients were injected with donor cells plus the genetic engineering Tregs. Survival time, clinical GVHD score, histopathological findings, activation of donor T cells or serum levels of inflammatory cytokines were observed after allo-BMT., Results: The mean survival times for radiation alone group (Gp I), transplantation control group (Gp II), engineering Treg infusion group (Gp III) and empty vector control group (Gp IV) were (8.8 ± 0.6) d, (36.7 ± 2.5) d, (51.6 ± 4.0) d and (34.1 ± 2.3) d, respectively. The survival time was significantly longer in Gp III than in other groups (P < 0.05). Histopathological finding in several target organs (skin, liver and small intestine) confirmed the presence of severe GVHD in Gp II and Gp IV, while no histological signs of GVHD were observed in long survival recipients in Gp III, and clinical GVHD scores in Gp III were significantly lower than that in Gps II and IV. The numbers of donor T cells and the percentage of IFN-producing donor T cells in the spleen of recipients in Gp III were significant lower than those in Gps II and IV at days 3 and 4, and at day 3 after transplantation, respectively (P < 0.05). The serum levels of IFN-γ, IL-2 and TNF-α were increased at day 21 to 28 after transplantation in all groups. The peak concentrations of IFN-γ, IL-2 and TNF-α in Gp III were significantly lower than those in Gps II and IV control groups at day 21 (P < 0.05)., Conclusion: Co-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD after allo-BMT in mice by inhibiting the early activation and expansion of donor T cells and reducing the serum levels of inflammatory cytokines.

Aim: To construct an inducible artificial microRNA expression vector targeting eIF3g gene and use it to inhibit the expression of eIF3g in K562 cells., Methods: The microRNA targeting human eIF3g was designed and obtained by PCR. After confirmed by sequencing, the microRNA was cloned into the pRevTRE2 plasmid. The Tet-off plasmids were transfected into K562 cells and selected for the stable tetracycline inducible K562/Tet-off transfectants. The pRevTRE2-eIF3g-miRNA plasmid was then transfected into stable K562/Tet-off transfectants and the expression of eIF3g was detected by Western blot., Results: The microRNA targeting eIF3g was confirmed by sequencing. GFP fluorescence assay showed that the stable transfectants of Tet-off plasmids were under tetracycline control. Western blot results showed that the stable transfectants of pRevTRE2-eIF3g-microRNA inhibited eIF3g expression under the regulation of tetracycline., Conclusion: The tetracycline-inducible artificial microRNA expression vector targeting eIF3g gene has been successfully constructed and effectively inhibits eIF3g expression in K562 cells.

Aim: To construct artificial microRNA expression vector targeting PAR4 and suppress the expression of PAR4 in human colorectal cancer SW620 cells with the artificial microRNA., Methods: Artificial microRNA was designed and amplified by two rounds of PCR and cloned into pMD-19T vector. The sequence of the cloned artificial microRNA was verified by DNA sequencing. Eight tandemly-repeated artificial microRNAs were subcloned into mammalian expression vector pcDNA3.1(+) to make the artificial microRNA- expressing vector pcDNA3.1(+)-8xPAR4-microRNA. The vector was transfected into human colorectal cancer SW620 cells, and stable transfectants were selected by G418. The expression of PAR4 was examined by Western blot., Results: DNA sequencing showed that the sequence of the cloned artificial microRNA targeting PAR4 was correct. Western blot result showed that the expression of PAR4 in SW620 cells stably transfected with pcDNA3.1(+)-8xPAR4-microRNA was markedly downregulated when compared to SW620 parental cells., Conclusion: Artificial microRNA expression vector targeting PAR4 is successfully constructed with significant suppression effect on PAR4 expression in SW620 cells. This provides the basis for future studies on the function of PAR4 and potential cancer gene therapy targeting PAR4.

Objective: To evaluate the efficacy and adverse effects of transcatheter closure of perimembranous ventricular septal defect (pmVSD) with modified double-disk occluder device (MDVO)., Methods: Clinical data including clinical examination, electrocardiography daily after the procedure for a week, chest-X-rays and TTE before discharge and at 3-5 days after the procedure were analyzed from 604 patients underwent percutaneous closure of a pmVSD with MDVO at our department between December 2001 and December 2008., Results: Procedure was successful in 576 out of 604 patients (95.4%) and 583 VSD occluders were placed. Endocarditis, thromboembolism, or deaths were not observed after procedure. Conduction block occurred in 81 patients (56 RBBB, 14 LBBB) and transient nonparoxysmal ventricular tachycardia in 31 patients after the procedure. Complete heart block occurred in 11 patients, 9 of them recovered in 3 weeks, permanent pacemaker was implanted in 2 patients (one had transient III degrees AVB before the procedure, the other underwent simultaneous closure of ventricular septal defect and atrial septal defect). Trivial/small residual shunts were found in 69 patients (12.0%). The residual shunts disappeared in 31 patients and remained unchanged in 38 patients (6.6%) 7 days after procedures. Aortic regurgitation developed in 5 patients (2 trivial/small, 3 small/moderate), and tricuspid regurgitation was present in 35 patients (32 trivial/small, 3 moderate). Five patients developed haemolysis (device retrieved via catheter in 1 patient due to persistent haemolysis, the other 4 patients recovered 3-14 days post procedure). Pseudoaneurysm of femoral artery occurred in 1 patient, and disappeared by pressure dressing. Device was successfully replaced in 2 patients with either device embolization (n = 1) or device misplacement (n = 1) after device retrieval by catheter., Conclusion: It is safe and effective to close congenital perimembranous ventricular septal defect with domestic-made occluder device.

Objective: To prepare the genetic engineering regulatory T cells (Treg) via the self-inactivating (SIN) lentiviral vectors carrying Foxp3 gene, and assay the phenotype and abilities of its proliferation and immunosuppression., Methods: The bicistronic SIN lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene (IRES-GFP) was constructed. Human embryonic kidney 293T cells were co-transfected using liposome by lentiviral packing system, which included the packaging plasmid Delta NRF, the transfer plasmid and the envelope plasmid VSVG. The efficiency of gene transduction and the expressions of Foxp3, CD25, GITR, CTLA-4 of CD4(+)CD25(-) T cells, which were isolated by magnetic beads from the spleen, and then co-cultured with 293T cells, were detected by flow cytometry (FCM). The proliferative and suppressive capacities of transduced T cells were estimated by Cell Count Kit-8 (CCK-8) and the cytokine production was performed by ELISA., Results: The lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was successfully constructed, the virus titers were above 10(6) IU/ml in the supernatant. pXZ208-IRES-GFP was used as control group. After cocultured, the CD4(+)CD25(-) T cells expressed significantly higher Foxp3, CD25, GITR and CTLA-4 in experimental group than in control group. Upon stimulation with anti-CD3 epsilon and APCs, the proliferative capacity of Foxp3-transduced T cells and the production of IL-2, IL-4, IL-10, IFN-gamma were significantly lower than those in control group (P < 0.01); Foxp3-transduced T cells also significantly inhibited the proliferation of CD4(+)CD25(-) T cells., Conclusions: The genetic engineering Treg mediated by SIN lentiviral vectors are successfully constructed and their phenotype and function are similar to natural CD4(+)CD25(+) Treg.

Objective: To construct the self-inactivating lentiviral vector containing murine Foxp3 gene and detect its expression in the CD4+ CD25- T cells., Methods: The bicistronic lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene (IRES-GFP) was constructed. Human embryonic kidney 293T cells were co-transfected by lentiviral packing system, which including the packaging plasmid deltaNRF, the transfer plasmid and the envelope plasmid VSVG using liposome. Then manifested the efficiency of gene transduction with Flow Cytometry (FCM) and the expressions of Foxp3 mRNA and protein were assayed by RT-PCR and Western blotting., Results: The lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was constructed, the virus titres were above 10(6) IU/mL in the supernatant. After coculture with thus cells, the CD4+ CD25- T cells in experimental group expressed significantly higher amounts of Foxp3 mRNA and protein than control group, and the efficiency of gene transduction was about 55.95%., Conclusions: The self-inactivating lentiviral vector containing murine Foxp3 gene are successfully constructed. This gene delivery system can transduce Foxp3 gene into CD4+ CD25- T cells with highly efficient expression.