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To investigate the effects of polyvinyl alcohol (PVA) + graphene oxide (GO, weight content 1 wt%) aerogel three-dimensional (3D) scaffolds culture system on the proliferation, phenotype and drug resistance of ALL cell line Jurkat and AML cell line HL-60.Jurkat cells and HL-60 cells were seeded in PVA+GO aerogel scaffolds for culture, and the structure of cells were observed by the scanning electron microscopy. Cell proliferation activity was measured by Cell Counting Kit-8 (CCK-8), cell phenotypes were analyzed by flow cytometry after fluorescent staining, then were compared with 2D cultured cells. Ara-C was used in drug resistance experiment, and CCK8 was used to detected cell proliferation activity.The proliferation activity of Jurkat cells grown in aerogel scaffolds was higher than that by 2D cultured in long-term culture. However, in HL-60 cells, the proliferation activity on 3D scaffold only at the 8th to 20th day was higher than that on the traditional 2D culture. Expression of CD4 in Jurkat cells increased after culture for 30 days, but the cell phenotypes in the 3D aerogel scaffolds were similar to 2D cultured cells. Phenotype of HL-60 cells was certainly changed after culture for 30 days, the cells can be divided into CD13PVA+GO (1 wt%) aerogel scaffolds can improve the proliferation and drug resistance of leukemia cells, and the phenotypes were the same as those in 2D culture, which can be used for cell amplification and biology characteristics studies and drug experiments. However, cell phenotypes should be analyzed before culture, and the effects of phenotypes changes on drug resistance should be eliminated.聚乙烯醇/氧化石墨烯气凝胶三维培养对急性白血病细胞系生物学特性的影响.研讨聚乙烯醇（polyvinyl alcohol，PVA）+氧化石墨烯（graphene oxide，GO，质量百分比1 wt%）气凝胶三维支架培养对急性淋巴细胞白血病细胞系Jurkat及急性髓系白血病细胞系HL-60增殖活性、细胞表型及耐药性的影响.将Jurkat细胞系及 HL-60细胞系植入PVA+GO气凝胶三维支架进行培养，通过扫描电镜观察细胞结构。使用CCK-8法定期检测细胞增殖活性，荧光染色后流式细胞术分析细胞表型，并与传统二维培养的细胞对比。使用阿糖胞苷进行耐药性实验，并使用CCK-8法检测细胞增殖活性.细胞长期培养发现，气凝胶支架中生长的Jurkat细胞增强活性高于传统二维培养的细胞，而在 HL-60细胞中只有d 8-20三维支架组的细胞活性高于传统二维培养的细胞。Jurkat细胞在培养30 d后CD4表达增高，但气凝胶三维支架中的细胞表型和传统二维培养细胞相似；HL-60细胞在培养30 d后表型发生了变化，可分为CD13聚乙烯醇+氧化石墨烯（含量1 wt%）气凝胶三维支架可以提高白血病细胞的增殖活性和耐药性，并且表型与二维培养相同，可以用于细胞扩增及特性的研究以及药物实验，但在使用前应先检测支架是否能够引起表型变化，并注意排除这种变化对耐药性的影响.

Breast cancer (BC) is the most commonly diagnosed cancer in females globally and is more aggressive at later stages. Chromosome region maintenance 1 (CRM1) is involved in the nuclear export of proteins and RNAs and has been associated with a number of malignancies. However, the clinicopathological significance of its expression in BC remains to be elucidated therefore this was investigated in the present study. CRM1 expression in 280 breast cancer tissues and 60 normal tissues was retrospectively analyzed using immunohistochemistry (IHC) and western blotting. IHC investigation demonstrated that CRM1 expression was significantly increased in BC compared with the normal breast epithelium (P

Three dimensional (3D) culture has gradually become a research hotspot in the field of drug screening, stem cell research, and tissue engineering due to its more physiological-like morphology and function. In this study, we compared the differences of cell proliferation, population, protein expression and chemoresistance profiles between two dimensional (2D) and 3D culture of acute lymphoblastic leukemia (ALL) Jurkat cell line. Polycaprolactone (PCL) is used for 3D culture owing to its biochemical properties and compatibility. Culturing of ALL Jurkat cell line in collagen type I coated polycaprolactone scaffold for 168 h increased cell proliferation, attachment, as well as the drug resistance to cytarabine (Ara-C) and daunorubicin (DNR) without changing the original CD2+CD3+CD4+dimCD8−CD34−CD45+dim phenotype, compared to uncoated PCL scaffold and tissue culture plate systems. Molecularly, increased chemoresistance is associated with the upregulation of discoidin domain receptor 1 (DDR1) and transcription factor STAT3. Inhibition of DDR1 activity by DDR1-specific inhibitor DDR-IN-1 accelerated cell death in the presence of Ara-C, DNR or their combination. These results demonstrated that 3D culture enhances chemoresistance of ALL Jurkat cell line by increasing DDR1 expression. Importantly, the cell adhesion-mediated drug resistance induced by DDR1 in the scaffold was similar to the clinical situation, indicating the 3D culture of cancer cells recapitulate the in vivo tumor environment and this platform can be used as a promising pre-clinic drug-screen system.

The aim of the present study was to examine the post-infarct acute effect of adenosine-5'-triphosphate (ATP) on myocardial infarction (MI) size as well as its precise molecular mechanism. Sixty New Zealand white male rabbits were exposed to 40 min of ischemia followed by 180 min of reperfusion. The rabbits were intravenously administered 3 mg/kg of ATP (ATP group) or saline (control group) immediately after reperfusion and maintained throughout the first 30 min. The wortmannin+ATP, PD-98059+ATP, and 5-hydroxydecanoic acid (5-HD) sodium salt+ATP groups were separately injected with wortmannin (0.6 mg/kg), PD-98059 (0.3 mg/kg), and 5-HD (5 mg/kg) 5 min prior to ATP administration. MI size was calculated as the percentage of the risk area in the left ventricle. Myocardial apoptosis was determined using a TUNEL assay. Western blot analysis was performed to examine the levels of protein kinase B (Akt)/p-Akt and extracellular signal-regulated kinase (ERK)/p-ERK in the ischemic myocardium, 180 min after reperfusion. The infarct size was significantly smaller in the ATP group than in the control group (p

Vasculogenic mimicry (VM) refers to the unique ability of highly aggressive tumor cells to mimic the pattern of embryonic vasculogenesis, which was associated with invasion and metastasis. The grape seed proanthocyanidins (GSPs) had attracted much attention as a potential bioactive anti-carcinogenic agent. However, GSPs regulation of VM and its possible mechanisms in a triple-negative breast cancer cells (TNBCs) remain not clear. Therefore, we examined the effect of GSPs on VM information in HCC1937 cell model. In this study, we identified the VM structure via the three-dimensional (3D) matrix in vitro. Cell viability was measured using the CCK8 assay. The effects of GSPs on human triple-negative breast cancer cells (TNBCs) HCC1937 in terms of related proteins of VM information were determined using western blot analysis. In vitro, the tubular networks were found in highly invasive HCC1937 cells but not in the non-invasive MCF-7 cells when plated on matrigel. The number of vascular channels was significantly reduced when cells were exposed in GSPs (100 μg/ml) and GSPs (200 μg/ml) groups (all p

Purpose.Using rat conjunctival bleb model, we correlated changes morphology and histology in the bleb with changes in MMP-2 and TIMP-2 levels.Methods.Filtering surgeries were performed on rats. Dynamic changes in morphology and histopathology were observed using HE staining. Expression of MMP-2 and TIMP-2 was determined by immunofluorescence microscopy and western blotting.Results. Well-elevated filtering blebs formed and persisted for an average of 12 days. Histological examination showed that inflammatory was dominant in postoperative days 1–3, and proliferating manifestation became the main sign 5 days later. Western blot showed that MMP-2 was downregulated 1 day after surgery, upregulated at 3 days, and observed with a peak at 7 days; then it persisted until 28 days. The difference was statistically significant (F= 280.18,p<0.01).TIMP-2 was upregulated 1 day after surgery and observed with a peak at 5 days; then it persisted until 28 days. The difference was statistically significant (F= 145.34,p<0.01).Conclusions.During the processes of conjunctival filtering bleb and scar formation in rats, the changes in MMP-2 and TIMP-2 levels in the filtering area, together with a corresponding proliferation of fibroblasts and the accumulation of collagen fibres, resulted in scarring of filtering blebs.

Heat shock protein 70 (HSP70) is known to be downstream of human epidermal growth factor receptor-2 (ERBB2), but little is known regarding the relationship between HSP70 and drug resistance mediated by ERBB2 in breast cancer.After infecting breast cancer cells with lentivirus-mediated Lenti-ShHSP70 and Lenti-ShERBB2, we examined the expression of HSP70 and ERBB2 by real-time polymerase chain reaction and western blotting.Compared to the control groups, mRNA expression of HSP70 was decreased in lentivirus-infected, and western blotting indicated a concordant reduction of HSP70 protein. On the other hand, ERBB2 was significantly down-regulated by HSP70 silencing in SK-BR-3 cells at both the mRNA and protein levels. Expression of HSP70 in transfected cells was also reduced by Lenti-ShERBB2. CCK8 viability assay indicated that inhibition of HSP70 increased the sensitivity of SK-BR-3 cells to fluorouracil treatment.HSP70 affects ERBB2 and ERBB2-mediated drug-resistance in breast cancer cells.

OBJECTIVE To investigate whether polymorphisms in ERC1, XPD, XPG, XRCC1 genes are associated with clinical outcomes in advanced gastric cancer (AGC) patients treated with oxaliplatin-based chemotherapy. METHODS The genetic polymorphisms in ERCC1, XPD, XPG, XRCC1 were determined in 94 advanced gastric cancer patients treated with oxaliplatin-based chemotherapy, using TaqMan-MGB probes. The clinical response of 60 patients with stage IV disease, time to progression (TTP) and overall survival (OS) of 94 patients were evaluated.RESULTS The overall disease control rate (CR + PR + SD) of the 60 patients in stage IV was 70% (42/60). Patients with XRCC1 399 G/G, XPG 46 C/C genotypes showed enhanced response to the oxaliplatin-based chemotherapy compared to those with other genotypes (P < 0.05). The median OS and TTP of the patients were 5.5 months and 9.0 months, respectively. Among the 4 types of polymorphisms in the study, XRCC1 399 G/A + A/A, XPG 46 C/T + T/T genotypes were regarded to be associated with chemoresistance and poor survival ( P < 0.05). Combination analysis of the 2 polymorphisms using the Kaplan–Meier method revealed that the TTP and OS of the patients with a number of risk genotypes were signi fi cantly shortened ( P < 0.05). No significant association was found between the genotypes of the XPD codon 751, the ERCC1 codon 118 and the clinical outcome (P > 0.05). CONCLUSION Testing for XRCC1 399, XPG 46 polymorphisms may allow identification of the gastric cancer patients who will benefit from oxaliplatin-based chemotherapy. Specific polymorphisms may influence clinical outcomes of AGC patients. Selecting specific chemotherapy based on pretreatment genotyping represents an innovative strategy that warrants prospective studies.

Previously we reported that a polypeptide from Chlamys farreri (PCF) was a potent photoprotective agent against ultraviolet (UV) irradiation in vitro. To understand the mechanism by which PCF protects cells from irradiation, we studied anti-apoptotic effects of PCF against UV irradiation on the murine thymocytes in vitro. MTT and flow cytometric analysis assays showed that 2h pretreatment with PCF completely abolished UV induced cell death. TEM examination showed that PCF fully protected the ultrastructure of thymocytes exposed to UV irradiation. Lipid peroxidation and intracellular reactive oxygen species assays indicated that PCF efficiently blocked production of reactive oxygen intermediates induced by UV irradiation. Further, PCF protected UV-irradiated thymocytes from losing mitochondrial transmembrane potential and DNA fragmentation. Based on these observations we propose that PCF is a potent anti-apoptotic factor, which protects cells from irradiation at multiple steps.

At present, the expression of MOR1 and its function in gastric cancer remains unclear with evidence suggesting that it is to be involved in tumor progression and metastasis. The study was to assess the clinicopathologic relevance and prognostic value of MOR1 expression in gastric cancer.Real-time quantitative RT-PCR and immunohistochemical staining were used to detect MOR1 expression in primary gastric cancerous surgical specimens and adjacent nontumorous tissues.High MOR1 expression was detected in cancerous tumor compared with their adjacent nontumorous tissues. In addition, the chi-square test revealed that high MOR1 expression was significantly correlated with depth of invasion (p = 0.006), lymph node metastasis (p = 0.001), distant metastasis (p = 0.017), and TNM staging (p = 0.027). Moreover, Kaplan-Meier analysis revealed a significant association between MOR1 expression and overall survival. High expression of MOR1 was identified as an independent and significant predictor gene of reduced postoperative survival.We conclude that MOR1 expression may be a useful biomarker for better prediction of the clinical outcome and management of gastric cancer patients.

The vimentin gene is a hallmark of epithelial-to-mesenchymal transition and has been observed to be overexpressed in various types of tumor cell line and tissue. Previous studies have reported correlations between vimentin DNA methylation levels and subsequent vimentin expression levels in solid tumors, including breast and colorectal cancer; however, to the best of our knowledge, such a correlation has not been reported for gastric cancer (GC) using Lauren classification. Therefore, the present study aimed to quantify DNA methylation levels of the vimentin gene using quantitative (q) methylation-specific polymerase chain reaction (PCR) in intestinal-type GC cell lines (MKN-28, AGS and MKN-1), diffuse-type GC cell lines (SGC-7901, SNU-5 and KATO III), the GES-1 immortalized human non-neoplastic gastric epithelial cell line, as well as in tumor and paratumor normal tissue samples. Furthermore, the present study analyzed the messenger RNA expression of the vimentin gene in these cell lines and tissues by reverse transcription-qPCR. A comparison of the clinicopathological features was conducted between patients, grouped according to the Lauren classification. The present study identified that the vimentin promoter region was hypermethylated in all GC cell lines and tumor tissue samples when compared with immortalized normal gastric epithelial cells and paratumor normal tissues. In addition, vimentin promoter methylation levels were observed to be higher in intestinal-type cell lines when compared with those of diffuse-type lines and tissues. Correspondingly, vimentin expression levels were lower in intestinal-type gastric cell lines compared with those of diffuse-type cell lines and tissues, and were lowest in the non-neoplastic gastric cell line and paratumor normal tissues. Patients with diffuse-type GC were on average younger (P=0.023), and exhibited higher tumor (P=0.020), node (P=0.032) and TNM classification of malignant tumor stage (P=0.039) than those with intestinal-type GC. Following treatment of AGS cells (which demonstrated the highest methylation level of the vimentin gene) with 5-aza-2′-deoxycytidine, vimentin expression was restored significantly. Thus, the present study revealed that vimentin promoter methylation levels are inversely correlated with vimentin expression levels in GC (according to Lauren classification). High levels of methylation in the vimentin gene promoter region may be involved in carcinogenesis and the development of GC, and may provide a novel molecular classification for GC.

To assess the association between matrix metalloproteinase-3 (MM-3) gene polymorphisms and subtypes of ischemic stroke (IS) in northern Han Chinese population.A total of 289 patients with acute IS (within 3 days after the onset, including 185 with large artery atherosclerosis (LAA) and 104 for small artery occlusion (SAO)) and 175 matched healthy controls were recruited for this case-control study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or sequenc-based typing (SBT) was carried out to analyze 3 SNPs of the MMP-3 gene.An incomplete linkage disequilibrium (LD) block was constructed with the 3 SNPs, and the distribution of genotypes of the 3 SNPs differed between the LAA group and controls in a dominant model: Carriers of 5A allele (5A5A+5A6A) of the rs3025058 locus were 1.72 times more susceptible to LAA stroke compared with carriers of 6A6A alleles (P=0.017, OR=1.72, 95% CI: 1.10-2.69), carriers of G alleles (GG+AG) of the rs522616 locus were 0.52 times more susceptible to LAA stroke compared with carriers of AA alleles (P=0.005, OR=0.52, 95% CI: 0.33-0.82), whilst carriers of A allele of the rs679620 locus were 1.55 times more susceptible to LAA stroke compared with carriers of GG alleles (P=0.042, OR=1.55, 95% CI: 1.01-2.37). However, no significant difference has been found between particular genotypes of such SNPs between SAO patients and controls (P0.05). Furthermore, 5A-A-A and 6A-A-A haplotypes were significantly more common in LAA group than the controls (P0.05), whilst 6A-G-G haplotype has been the opposite (P0.01).Our study has demonstrated that serum MMP-3 level is significantly increased at acute stage of LAA as well as SAO type strokes. There may be an association of rs3025058, rs522616 and rs679620 of MMP-3 gene with susceptibility to LAA stoke in northern Han Chinese population.

Background We studied the relationships among Cx43, CD105, and VEGF in specimens of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) with different serum AFP levels with respect to recurrence and metastasis. Methods Expressions of Cx43, CD105, and VEGF in 234 HCC tissue specimens were examined using tissue microarray and immunohistochemistry. Cx43 mRNA expression was examined in 38 frozen HCC specimens using RT-PCR. Correlations between these expressions and tumor recurrence, metastasis, and prognosis were analyzed using Kaplan–Meier and Cox regression analyses. Results Cx43 expression correlated with early tumor recurrence (P = 0.001), disease-free survival (P = 0.026), and overall survival (P = 0.000) in patients with serum AFP P = 0.018 and 0.023, respectively), histological differentiation (P = 0.002), vessel tumor embolism (P = 0.029), and focal number (P = 0.017). Immunohistochemistry showed that Cx43 expression in patients with low AFP was lower in patients with distant metastases than in those with no metastasis or those with liver metastasis. Patients with early recurrence expressed less Cx43 mRNA than did those with no recurrence (χ2 = 9.827, P = 0.002). Conclusions Cx43 expression can delay early HCC recurrence, metastasis, and poor prognosis after radical hepatectomy in patients with HBV-related HCC and low AFP.

To investigate the expression of CK19 in HBV-related hepatocellular carcinoma (HCC) tissues in patients with low serum AFP concentration and the relationship between them and the recurrence and prognosis of HCC after R0 radical hepatectomy.The expressions of CK19 and Ki67 in HCC tissues of 235 cases were examined using tissue microarray and two-step methods of PV-6000 immunohistochemistry. The expression of CK19 mRNA in 20 frozen HCC specimens was examined by RT-PCR. The correlation between gene expressions and tumor recurrence and prognosis was analyzed.Among the 235 HBV-related HCC patients after R0 radical hepatectomy, the median disease-free survival (DFS) was 31.2 months in the patients with serum AFP400 µg/L and 13.8 months in the patients with serum AFP ≥ 400 µg/L (P = 0.041), the overall survival (OS) was 84.0 and 58.6 months in the two subgroups (P = 0.125), and the tumor recurrence within one year was in 43 cases (27%) and 37 cases (49.3%), respectively, (P = 0.001). The DFS was 11.6 months in the CK19-positive cases and 27.0 months in the CK19-negative cases (P0.05). The OS was significantly lower in the CK19-positive cases than that in the CK19-negative cases (P = 0.023). Both DFS and OS in the CK19-positive cases with AFP400 µg/L were significantly lower than those in the CK19-negative cases with AFP400 µg/L (both P0.05). The CK19 expression was significantly correlated with histological differentiation (P = 0.023), number of tumor foci (P = 0.044), vascular tumor embolism (P = 0.005), regional lymph node metastasis (P = 0.023), and 1-year recurrence (P = 0.006). Among the patients with AFP400 µg/L, the 1-year recurrence was 53% in the CK19-positive cases and 23% in the CK19-negative cases (P0.001), the median DFS was 11.3 months in CK19-positive cases and 34.0 months in CK19-negative cases (P = 0.010), and the median OS was 19.5 months in the CK19-positive cases, significantly lower than 84.0 months in the CK19-negative cases (P = 0.001). Cox regression analysis showed that CK19-positive expression was an independent factor affecting early HCC recurrence and prognosis.In HBV-related HCC patients after radical hepatectomy with AFP400 µg/L, positive expression of CK19 indicates a higher proliferation and invasiveness of HCC, and is an important factor of early recurrence and poor prognosis.

To explore the associations between vitamin D receptor (VDR) gene Tru9I polymorphism and myasthenia gravis (MG).A total of 302 MG patients, diagnosed and treated at Affiliated Hospital of Medical College, Qingdao University and Beijing Friendship Hospital from December 2006 to July 2010, were recruited. And 283 normal subjects were selected as the controls. Tru9I polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The frequencies of genotypes and alleles were compared among different MG subgroups and control group. The relationship between the genotype and susceptibility or severity of MG and immediate efficacies of glucocorticoid were explored. The SPSS17.0 was applied to statistical analysis.In the MG patients whose age of onset was above 15 years, the frequency of TT and tt genotypes in females was lower than that of the control group. However the frequency of Tt genotype was higher than that of the control group. And there were significant differences (χ(2) = 8.847, P = 0.012). The frequency of Tt + tt genotype in females (58/139, 41.7%) was higher than that of the control group (56/189, 29.6%). And there were also significant differences (OR = 1.70, 95% CI 1.07 - 3.41, χ(2) = 5.169, P = 0.023). Although the frequency of t alleles in females (61/278, 21.9%) was higher than that of the control group (65/378, 17.2%), no significant differences existed (P0.05). There were no differences in frequencies of genotypes and alleles between the patients with varying severity and different immediate efficacies of glucocorticoid (P0.05).VDR gene Tru9I polymorphism may be related to the risk of MG in females aged above 15 years.

Cholangiocarcinoma, a type of malignant tumor, originates from epithelial cells of the bile duct. Perineural invasion is common path for cholangiocarcinoma metastasis, and it is highly correlated with postoperative recurrence and poor prognosis. It has been reported that muscarinic acetylcholine receptor M3 (mAChR M3) is widely expressed in digestive tract cancer, and may play an important role in the proliferation, differentiation, transformation and carcinogenesis of tumors. This study was to explore the effect of mAChR M3 on the growth of cholangiocarcinoma cells in vitro and provide a new approach to the pathogenesis and treatment of cholangiocarcinoma.Streptavidin-biotin complex immunohistochemistry was carried out to assess the expression of mAChR M3 in surgical specimens of cholangiocarcinomas (40 cases) and normal bile duct tissues (9), as well as to investigate nerve infiltration. The cholangiocarcinoma cells were treated with different concentrations of selective M-receptor agonist pilocarpine and M-receptor blocker atropine sulfate to induce changes in cell proliferation. The experimental data were analyzed by the Chi-square test.The strongly-positive expression rate of mAChR M3 was much higher in poorly-differentiated (69%, 9/13) than in well- and moderately-differentiated cholangiocarcinomas (30%, 8/27) (X2=5.631, P0.05). The strongly-positive mAChR M3 expression rate in hilar cholangiocarcinoma (50%, 14/28) was higher than that in cholangiocarcinomas from the middle and lower common bile duct (25%, 3/12) (X2=2.148, P0.05). Cholangiocarcinomas with distant metastasis had a strongly-positive expression rate (75%, 9/12), which was much higher than those without distant metastasis (29%, 8/28) (X2=7.410, P0.01). The absorbance value in the pilocarpine+atropine group was significantly higher than the corresponding value in the pilocarpine group.The expression of mAChR M3 is influenced by the extent of differentiation, distant metastasis and the site of cholangiocarcinoma. It also plays a key role in the proliferation and metastasis of cholangiocarcinoma.

The formation and growth of tumors are related to the synthesis of the DNA. The enzyme ribonucleotide reductase (RR) is an enzyme that regulates the total rate of DNA synthesis and thus plays a pivotal role in cell growth. Catalytic subunit M2 (RRM2) is the main unit modulating the ribonucleotide reductase enzymatic activity. This study aimed to investigate the expression of RRM2 mRNA and protein in patients with ovarian cancer and its relevance to diagnosis and clinical outcome of the patients.RRM2 mRNA levels and protein expression were detected in 98 ovarian specimens with immunohistochemistry and real-time quantitative polymerase chain reaction (PCR). Expression of the RRM2 protein and correlation of the RRM2 gene expression with clinical pathological features were analyzed. The Kaplan-Meier test was used for evaluating RRM2 expression and time to progression and survival. The Cox proportional model was used to analyze the risk factors in prognosis of patients.Positive RRM2 immunostaining was found in 43 of 62 (69.4%) patients with epithelial ovarian cancer, 10 of 15 (66.7%) patients with borderline neoplasm, 4 of 15 (26.7%) patients with benign growths, and none of the normal group. The RRM2 mRNA levels were significantly over expressed in epithelial ovarian cancer (1.722 ± 0.639) and borderline ovarian neoplasms (1.365 ± 0.615), compared to the normal group (0.678 ± 0.446) and benign group (0.828 ± 0.545). Patients with ovarian caner in clinical FIGO-stages III-IV presented higher RRM2 gene expression than those in clinical FIGO-stages I-II. Furthermore, the survival of patients with low RRM2 mRNA level was significantly better than patients with high levels (P0.05). By Cox proportional risk model analysis, the risk of mortality of patients with high level expression of RRM2 mRNA was 2.553 times greater than those with low expression.RRM2 expression closely correlates with the development of ovarian tumor and may serve as a novel predictive marker for diagnosis and prognosis of the disease.

To explore the effect and mechanism of tagalsin on hepatoma cells.The animal models were established by transplanting H(22) mouse hepatoma cells to mouse liver, and ten days later the mice were randomly divided into five groups: blank group, carmofur positive group and tagalsin groups, including low-dose, middle-dose and high-dose groups. Then medicine or oil was given to the mice by gastric gavage in consecutive 5 days with a 2-days interval as a course of treatment, two courses in all. All mice were killed at 24 hours after medication, and the survival period, ascites conditions, aggressive conditions intra- or extra-liver, weight changes, tumor volume and spleen index of the tumor-bearing mice were observed. Pathological changes of the tumors were examined. Apoptotic factors p53 and Bcl-2 protien and mRNA were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).tagalsin inhibited the hepatoma growth effectively without influencing spleen index to some extent. The tumor inhibition rate of tagalsin low, middle and high dose groups were 17.9%, 63.1% and 71.8%, respectively. Immunohistochemical results showed that the p53 and Bcl-2 protein positive cell counts of the positive control and experimental groups were significantly lower than those of the blank group (P0.01). RT-PCR results showed that the p53 mRNA expression was significantly enhanced and Bcl-2 mRNA expression was decreased in the positive control groups and tagalsin treatment groups, especially in the high dose group, compared with those of the blank group (P0.05).tagalsin can inhibit the growth of mouse hepatoma cells significantly. The mechanism of its anti-tumor effect may work via up-regulating the wild type p53 gene expression and down-regulating Bcl-2 gene expression and thus regulating tumor cell apoptosis.

The purpose of this study was to investigate the somatic mutations of human mitochondria succinate dehydrogenase subunit B (SDHB) in sporadic paragangliomas.Eight exons of SDHB gene in 8 sporadic paragangliomas cases were amplified by PCR and sequenced, respectively. The sequences were analyzed to find mutations compared with human homology sequence in Genebank and SNP database.Nine sequence variations were found in 8 cases, in which one mutation was found in one case (1/8, 12.5%). The mutation was identified as the sixty four base pair in exon 2 of SDHB(c.136CT), resulting in a change from a arginine to a stop codon (p.Arg90X). The left 8 variations were polymorphisms.The mutation of SDHB exists in sporadic paragangliomas patients and it might play a significant role in paragangliomas tumorigenesis.

To evaluate the association between the polymorphisms of excision repair cross complementation group 1 (ERCC1), X-ray repair cross complementing 1 (XRCC1), glutathione S-transferase Pi 1 (GSTP1) and the survival of advanced gastric cancer patients treated with oxaliplatin-based combination chemotherapy.Eighty five patients with advanced gastric cancer accepted oxaliplatin/5-FU-based chemotherapy as first-line chemotherapy were investigated. Peripheral venous blood was taken before chemotherapy. DNA was extracted from peripheral venous blood. The genetic polymorphisms were detected by real-time PCR assay. The association between time to progression, overall survival and the polymorphisms was analyzed.The median time to progression of the 85 cases was 5.3 months, and the median overall survival was 8.0 months. ERCC1-118 C/C, XRCC1-399 G/G and GSTP1-105 A/G + G/G were favorable genotypes and the number of the favorable genotypes was associated with survival of the patients. The median overall survival was 12.5 months, 10.0 months, 6.5 months and 4.5 months for patients with 3 favorable genotypes, 2 favorable genotypes, 1 favorable genotype and none favorable genotype, respectively, with a significant difference (χ(2) = 35.54, P0.01).Genetic polymorphisms of ERCC1-118, XRCC1-399 and GSTP1-105 are associated with TTP and OS of advanced gastric cancer patients treated with oxaliplatin/5-Fu-based combination chemotherapy as the first-line chemotherapy.

This study was to investigate the proliferative inhibition and apoptosis of human leukemia HL-60 cells induced by crocin and their possible mechanisms. The cell viability was tested by cell counting. The morphology of HL-60 cells was observed by fluorescence microscopy. The MTT assay was used to evaluate the inhibitory effect of crocin on the growth of HL-60 cells. Flow cytometry was used to measure the cell cycle. RT-PCR was used to detect bcl-2 and bax expression. The results indicated that the growth of HL-60 cells was inhibited remarkably in the dose and time dependent way. When the crocin concentration was higher than 5 mg/ml, the percentage of apoptotic HL-60 cells was not increased, on the contrary this percentage decreased, the cells manifested necrosis. Flow cytometry profiles revealed that cells were blocked in G₀/G₁ phase, the cell proliferation was inhibited obviously at 5 mg/ml. RT-PCR detection revealed that the expression of bcl-2 was down-regulated strikingly and bax was up-regulated. It is concluded that the crocin can inhibit the proliferation of HL-60 cells effectively, and block cells in G₀/G₁ phase. The mechanisms by which crocin induced apoptosis in HL-60 cells may be related to the inhibition of bcl-2 and activation of bax.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, yet effective therapeutic options for advanced HCC are limited. Kringle 1 domain of HGF (HGFK1) has been demonstrated as a potent anti-tumor molecule and p53 is a well established tumor suppressor. Recently we developed AAV transducing HGFK1 (AAV-HGFK1) as a gene therapy for HCC. Here we investigated the possibility of enhancing the effect of AAV-HGFK1 by combining it with Adv transducing p53 (Adv-p53). In vitro expression experiments suggested a small amount of Adv-p53 could increase the expression of AAV transgenes. AAV-HGFK1+Adv-p53 cocktail strongly inhibited the proliferation of microvascular endothelial cell (MEC) and two HCC cell lines, Hepa1-6 and McA-RH7777. In two orthotopic mice and rat HCC models the cocktail gene therapy also significantly reduced the tumor burdens and prolonged the survival time by inhibiting tumor angiogenesis and inducing tumor cell death. Significantly, tumor metastasis was completely prevented. AAV-HGFK1+Adv-p53 viral cocktail may be a promising cancer therapy for the treatment of HCC.

To study the protective effect of polypeptide isolated from Chlamys farreri (PCF) on Hela cells damaged by ultraviolet A (UVA) in vitro.Cell proliferation was determined by MTT method; intra-cellular free calcium [Ca2+]i and rates of apoptosis and death were measured by flow cytometry (FCM).PCF (0.5 %-2 %) enhanced the activities of glutathione peroxidase (GSH-px), superoxide dismutase (SOD), and catalase (CAT), and stimulated cell proliferation. The concentration of [Ca2+]i was increased while the amounts of MDA and the rates of apoptosis and death of the cells were decreased. The differences between the PCF groups and control group were significant (P0.05, P0.01).PCF protected Hela cells against damage by UVA via its anti-oxidative mechanisms.

To study the protective effect of the polypeptide isolated from Chlamys farreri (PCF) on hairless mice skin damaged by ultraviolet A.Enzymes and malondialdehyde (MDA) were determined by biochemical methods; the expressions of Bcl-2 protein and NOS protein were examined by immunohistochemical technique. The ultra-structure of the skin was observed through electronic microscope.PCF could enhance the activities of glutathione peroxidase (GSH-px), superoxide dismutase (SOD), and total anti-oxidative capacity (T-AOC). Also PCF could reduce the amount of MDA, increase the expression of Bcl-2 protein, and inhibit the expression of NOS protein. The ultra-structure of epidermis and fibroblasts remained normal in 20 % PCF groups; there were vacuoles in smooth endoplasm reticulum in epidermis of mice and the number of rough endoplasm reticulum in fibroblasts was decreased in model group.PCF had the protective effects on hairless mice skin damaged by ultraviolet A via its anti-oxidative mechanisms.

Objective: Adenosine-5'-triphosphate (ATP) has been reported to protect the heart from myocardial ischemia/reperfusion injury when administered at the onset of reperfusion and is as effective as mechanical postconditioning. However, whether the protective effect of postconditioning with ATP is mediated through the same signaling pathways as those of ischemic postconditioning is still unclear. We examined the postinfarct acute effect of ATP on myocardial infarct size and its precise molecular mechanism. Methods: Forty-eight New Zealand white male rabbits were exposed to 40 min of ischemia followed by 180 min of reperfusion. Rabbits were intravenously injected 3mg/kg of ATP (ATP group) or saline (control group) immediately after reperfusion within 30min. The wortmannin+ATP, PD-98059+ATP, and 5-hydroxydecanoic acid sodium salt (5-HD)+ATP groups were respectively injected with wortmannin [a phosphatidyl-inositol 3-kinase (PI3-kinase) inhibitor, 0.6 mg/kg], PD-98059 [an extracellular signal regulated protein kinase (ERK) inhibitor, 0.3 mg/kg], and 5-HD [a mitochondrial ATP-dependent K+ (KATP) channel blocker, 5 mg/kg] 5 min before ATP administration. Myocardial infarct size was calculated as a percentage of the risk area of the left ventricle. Myocardial apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) methods. Western blot analysis was performed to examine the signals such as protein kinase B (Akt) and extracellular signal-regulated protein kinase (ERK) in the ischemic myocardium after 180min of reperfusion. Results: The infarct size was significantly smaller in the ATP group (12.8±1.9%) than in the control group (29.1±2.9%). The infarct size-reducing effect of ATP was completely blocked by wortmannin (26.5±2.7%), PD-98059 (27.9±3.2%), and 5-HD (26.1±4.0%). Compared with control group, ATP group significantly reduced cardiomyocytes apoptosis (apoptotic index: 27.0±5.8% vs 10.3±6.0%, P