Extracellular signal-regulated kinase/mitogen-activated protein kinase(ERK/MAPK) signaling pathway plays an important role in gastrointestinal tumorigenesis and development.ERK/MAPK signal transduction pathway has three important molecular targets: the small G protein Ras,Raf kinase and MEK1/2 and ERK1/2.At present,there are three kinds of approach which can inhibit ERK / MAPK signal transduction pathway:⑴ destroying the structure and(or) function of the target protein,⑵adopting the deficit strategy,⑶damaging the interaction between proteins.These approaches can provide new ideas for the treatment of gastrointestinal cancer.

Objective To investigate the effects of somatostatin on the apoptosis of colorectal cancer cells. Methods The expression of somatostatin mRNA in colorectal cancer tissues from 79 patients who had been admired to Yijishan Hospital from January 2004 to October 2006 was detected by nested RT-PCR. The apoptotic index of colorectal cancer cells was detected by TUNEL, and the protein expressions of somatostatin, Fas, FasL, caspase-3 and caspase-8 in colorectal cancer tissues were detected by immunohistochemistry. All data were analyzed by chi-square test, q test and Spearman rank correlation coefficient. Results There was a positive correlation between the mRNA and protein expression of somatostatin (r = 0.98, P < 0.05). The mRNA and protein expression of somatostatin in poorly and moderately differentiated colorectal cancers were significantly lower than that in well differentiated colorectal cancers (χ~2 = 10.78, 11.24, 5.27, 5.24, P < 0.05). The positive expression rates of mRNA and protein of somatostatin in papillary adenocarcinoma were significantly higher than those in mucinous adenocarcinoma and signet ring cell carcinoma and undifferentiated carcinoma (χ~2= 6.56, 6.99, 5.44, 7.39, P < 0.05). The mRNA and protein expression of somatostatin in colorectal cancer in Dukes A and B were significantly higher than that in Dukes C and D (χ~2 =5.17, 4.06, P <0.05). The apoptotic index in high or moderate somatostatin expression group was significantly higher than that in low somatostain expression group (q = 5.66, 4.21, P < 0.05), and the positive expression rates of Fas, caspase-8 and caspase-3 in high or moderate somatostatin expression group were significantly higher than those in low somatostafin expression group (χ~2= 5.48, 5.62, 6.89, 4.32, 4.19, 3.91, P <0.05). Conclusion Somatostatin plays an important role in the regulation of cell apoptosis in colorectal cancer, and the mechanism may be related to the aberrant expression of Fas/FasL.

Objective To investigate the effects and mechanisms of extracellular-signal regulated protein kinase-motogenactived protein kinase(ERK-MAPK)signaling pathway in gastrin-induced cell proliferation and apoptosis of colorectal cancer cells.Methods HT-29 cells were incubated in different media,and then were divided into the control group,gastrin group,proglumide group and gastrin + proglumide group.No reagent was added in the control group,and other groups were dealed with reagent in different concentrations.The changes of proliferation of the HT-29 cells were detected by MTT assay,and the optimal concentration of gastrin and proglumide were determined.The changes of proliferation index and apoptotic rates of HT-29 cells were detected by cell cytometry.The mRNA expressions of gastrin receptor/cholecystokinin-B receptor(CCK-BR),ERK1/2 and K-ras were detected by RT-PCR.The protein of ERK1/2,K-ras protein and phosphorylation levels were detected by Western blot.All data were analyzed by analysis of variance and SNK-q test.Results The proliferation of HT-29 was stimulated by gastrin when the concentration of the gastrin was 6.25-100.00 mg/L,and the optimal concentration of gastrin was 25.00 mg/L(F =31.36,P ＜ 0.05).Proglumide had no obvious effects on the proliferation of HT-29 cells,while it significantly inhibited the proliferation of HT-29 cells stimulated by gastrin when the concentration of proglumide was 8.00-128.00 mg/L,and the optimal concentration was 32.00 mg/L(F =24.31,P ＜ 0.05).The proliferation index of the gastrin(25.00 mg/L)group was 37.5 ％ ± 5.2％,which was significantly higher than 27.7％ ± 5.0％ of the control group and 27.3％ ± 5.8％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =4.56,4.75,P ＜ 0.05).The apoptotic index of the gastrin(25.00 mg/L)group was 1.9％ ± 0.4％,which was significantly lower than 2.5％ ± 0.4％ of the control group and 2.4％ ± 0.3％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =4.23,4.06,P＜0.05).The mRNA expression of CCK-BR was detected in the HT-29 cells.The levels of phosphorylated ERK1/2 protein and phosphorylated K-ras protein were 0.43％ ± 0.04％ and 0.45％ ± 0.06％,which were significantly higher than 0.32％ ± 0.02％ and 0.31％ ± 0.05 ％ of the control group(q =7.78,4.95,P ＜ 0.05),and they were also higher than 0.36％ ± 0.01％ and 0.35 ％ ± 0.04％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =5.72,4.08,P ＜0.05).There were no significant differences in the mRNA and protein expressions of ERK1/2 and K-ras among the control group,gastrin(25.00 mg/L)group,proglumide(32.00 mg/L)group and gastrin (25.00 mg/L)+ proglumide(32.00 mg/L)group(F =0.52,0.72,0.78,0.28,P ＞0.05).Conclusion Gastrin could stimulate the proliferation of HT-29 cells and inhibit their apoptosis by upregulate the phosphorylation levels of ERK and K-ras through the Ras→Raf→ MEK1/2→ ERK1/2 pathway,while the effect can be restrained by gastrin receptor antagonist proglumide.

Objective To investigate the risk factors for pancreatic fistula after duct-to-mucosa pancreaticojejuuostomy (PD). Methods The clinical data of 101 cases undergoing duct-to-mucosa PD in our hospital from January 1994 to January 2008 were reviewed retrospectively. Results The incidence of pancreatic fistula was 9.9% (10/101). Univariate analysis showed level of preoperative jaundice(χ2=5.814, P= 0.016) , duration of jaundice (χ2= 4.17, P = 0.041 ), texture of the remnant pancreas (χ2=5.286, P = 0.021 ), diameter of pancreatic duct (χ2= 4.165, P = 0.041 ), blood loss during operation (χ2=5.273, P=0.022) were significantly associated with pancreatic fistula after duct-to-mucosa PD. Multivariate analysis regression revealed that texture of the remnant pancreas (OR = 13.355, P = 0.023), level of preoperative jaundice (OR = 12.126, P = 0.006), blood loss during operation (OR = 5.92, P =0.032 ) were independent risk factors. Logistic regression equation was as following: P=1/[<1+e-(-6.378+2.592 texture of the remrant pancress + 2.495 level of preopetative jaundice + 1.778 blood loss during operative)>]. The accuracy of the logistic equation was 92.1%. Conclusion Texture of the remnant pancreas, level of preoperative jaundice, blood loss during operation were the independent risk factors for the occurrence of PD after duct-to-mucosa PD. Improvement of operative technique and reduction of blood loss can decrease the incidence of pancreatic fistula.

AIM:To construct eukaryotic expression vector expressing double shRNA sections targeting Survivin gene.METHODS: Eukaryotic expression vector expressing double shRNA sections targeting Survivin gene were designed and chemically synthesized.They were directionally inserted into plasmid pGenesil-1 with respectively U6 promoter and termination code,the common green fluorescence protein(EGFP) gene and Neo gene. In this way,the vector of pGenesil-1 shRNA containing 2 sections of Survivin shRNA were constructed and they were transfected into the pancreatic cancer cell Bx-PC3.Transfection was detected by fluorescence microscope.The inhibition expression of Survivin mRNA was measured by RT-PCR.RESULTS: HE1 and HE2 plasmids were identified by the biocatalyst cut which confirmed the exactitude and were analyzed by the sequence analysis which verified the perfect clone plasmid inserted by them.CONCLUSION: A eukaryotic expression vector of double short hairpin RNA for Survivin gene is successfully constructed.The pancreatic cancer cells Bx-PC3 succeed to be transfected and expression of Survivin mRNA is inhibited obviously.

Objective To investigate the ambiguity results distribution of HLA-A,B and DRB1 gene sequence-base typing in Guangxi population and to propose the way to resolve.Methods HLA-A,B and DRB1 genes of 1 000 donors in the Guangxi branch bank of China'bone marrow bank were genotyped by PCR-SBT,and then the ambiguity results distribution of the three loci was analyzed.The typing ambiguities resultswere resolved by high-resolution polymerase chain reaction-sequence-specific primers(PCR-SSP) and group specific sequencing primer(GSSP) methods,respectively.Results Among 1 000 samples,at least 1 locus in HLA-A,B and DRB1 genes in 96.7％ samples appeared the ambiguity results,in which the proportions of HLA-A,B and DRB1 loci appearing ambiguity results were 65.7 ％,58.8 ％ and 77.2 ％ respectively.For the samples of detected ambiguity results,single using the GSSP method could resolve the ambiguity typing results of 87.37％ HLA-A,93.54％ HLA-B and 60.49％ HLA-DRB1,using high-resolution PCR-SSP could resolve the ambiguity typing results of 12.63 ％ HLA-A,4.76 ％ HLA-B and 15.29 ％ HLA-DRB1,and the rest 1.70 ％ HLA-B and 24.22 ％ HLA-DRB1 ambiguity results were resolved by both GSSP and high-resolution PCR-SSPs method.Conclusion GSSP and high-resolution PCR-SSPs methods have high abilities to solve HLA ambiguity results both locate inside and outside the sequencing region,respectively.GSSP and high-resolution PCR-SSPs methods are supplement for each other,which can effectively resolve the problem of ambiguity results in high resolution HLA typing.

Introduction:Transcatheter arterial chemoembolization (TACE) plus thermal ablation has been widely used recently in the treatment of hepatocellular carcinoma (HCC). In this study, we aimed to compare results of the combination of TACE and percutaneous thermal ablation with those of hepatectomy in patients with HCC. Methods:The clinical data of 137 HCC patients who sequentially received TACE and computed tomography (CT)-guided percutaneous thermal ablation as an initial curative treatment (combination group) and 148 matched HCC patients who received hepatectomy (surgery group) between 2004 and 2011 were collected and analyzed. After TACE, multiphase contrast-enhanced CT was performed to identify the total number of tumors as well as lipiodol deposition in the liver. Survival was calculated by using the Kaplan-Meier method and compared by using the log-rank test. The prognostic factors were assessed with multivariate Cox proportional hazards regression analysis. Results:Of all 285 patients, 225 (79.0%) had cancerous lesions≤5 cm in diameter. In preoperative contrast-enhanced CT or magnetic resonance imaging, the number of tumors was 1–4 for each patient. The 1-, 3-, and 5-year overal survival rates were 95, 74%, and 67%in the combination group and 88, 66, and 47%in the surgery group, respectively (P=0.004);the corresponding recurrence-free survival rates for the two groups were 92, 69, and 61%and 75, 58, and 44%, respectively (P=0.001). In the multivariate analysis, treatment al ocation was an independent prognostic factor for survival. Only 60 patients in the combination group had sufficient imaging data, and 135 new lesions with lipiodol deposition were diagnosed as malignancies in 22 of 60 patients, whereas 20 new lesions were found in 11 of 148 patients in the surgery group. Conclusion:The combination of TACE and CT-guided percutaneous thermal ablation for HCC improves survival of HCC patients compared with hepatectomy.

Objective To explore the best ratio of platelet-rich plasma (PRP) to activator and the synergistic action of thrombin and the growth factors in PRP gel on proliferation of human marrow-derived mesenchymal stem cells (hBMSCs). Methods The activator was made up of 1000 U bovine thrombin and 1 mL 10% calcium chloride solution. PRP was mixed with the activator at the ratios of 1:1, 5:1, 10:1, 20:1, 40:1 respectively in groups A, B, C, D and E. A quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was used for examining the amounts of transforming growth factor-β1 (TGF-β1) and platelet derived growth factnr-AB (PDGF-AB) in PRP gel in each group after incubation for 0,1,8,24, and 120 hours. MTT assay was used to evaluate the effect of PRP gel in each group on hBMSCs proliferation. Results When PRP was mixed with thrombin, concentrations of PDGF-AB and TGF-β1 in PRP gel increased immediately and reached the peak value in 1 hour. The PRP gel in groups B, C, and D contained the highest amounts of PDGF-AB and TGF-β1, and accelerated hBMSCs growth re-markably. The cells proliferation in group A was inhibited. Conclusions The effect of PRP on the hBMSCs proliferation depends on the concentrations of PDGF-AB and TGF-β1 in PRP. Thrombin may influ-ence the hBMSCs proliferation by regulating concentrations of growth factors in PRP to certain extent, but too high a level of thrombin may inhibit hBMSCs growth.

AIM: To investigate the effect of sitagliptin on the autopaghy and the expression of extracellular matrix in mesangial cells induced by advanced glycation end products (AGEs).METHODS: The cells were divided into 5 groups: control group, AGE group, and sitagliptin (5, 10 and 20 μmol/L) groups.After 48 h, the cell viability was measured by MTT assay, and the content of collagen (Col) Ⅳ in the supernatant of the cell culture was detected by ELISA.The protein levels of beclin-1, adenosine monophosphate-activated protein kinase (AMPK), p-AMPK, p70S6K and p-p70S6K were determined by Western blot.RESULTS: Compared with control group, the viability and the expression of Col IV induced by AGEs in the cultured mesangial cells were significantly increased (P<0.01).Sitagliptin decreased the viability and the expression of Col IV induced by AGEs in the mesangial cells in a dose-dependent manner.AGEs significantly inhibited the protein levels of beclin-1 and p-AMPK, but significantly increased the protein level of p-p70S6K.Compared with AGE group, sitagliptin significantly reversed the above results in a dose-dependent manner.CONCLUSION: Autophagy may mediate the protective effect of sitagliptin on mesangial cells induced by AGEs.

Objective To evaluate the application of pooling HIV nucleic acid amplification testing (NAAT) among men who had sex with men (MSM) population, and to investigate suitable HIV screening strategy and the feasibility of calculation of HIV incidence using pooling NAAT among MSM population in China.Methods Four thousand eight hundred and fifty-six samples were collected from MSM population from April 2008 to September 2009 among with 4 156 were in Heilongjiang province and 700 were in Beijing in China. After standard testing with an HIV ELISA and WB confirmation testing, HIV antibody-negative samples were pooled and screened for HIV using NAAT.A three-stage pooling strategy was adopted.The HIV positive rate estimated by the four HIV screening strategies was calculated.In addition, 4 156 HIV positive specimens from Heilongjiang province were screened with the BED capture enzyme immunoassay (BED-CEIA).The HIV-1 incidences were estimated by BED-CEIA assay and pooling NAAT individually.ResultsOne hundred and forty-three of 4 856 subjects were HIV infected.130 were 3rd and 4th generation ELISA positive; 13 were antibody-negative but acutely HIV infected.According to the evaluation of four HIV screening strategies, routine HIV screening test together with pooling NAAT was more effective than other strategies for screening out window period generation ELISA+WB+pooling NAAT' were 2.68%(95% confidence interval CI=2.22%-3.14%), 2.82%(95%CI=2.35%-3.29%), 2.94%(95%CI=2.46%-3.42%) and 2.94%(95%CI=2.46%-3.42%), respectively.The differences were not significant (χ2=0.854 3, P=0.836 4).Of the 88 HIV positive samples from Heilongjiang province, 44 participants were tested as recent HIV infections by BED-CEIA assay. The estimated HIV-1 incidence was 2.36% (95%CI=1.63%-3.08%) and 2.92% (95%CI=1.01%-4.83%) based on BED-CEIA assay and pooling NAAT,respectively.Conclusions Pooling NAAT is a effective screening test in HIV negative population to detect window period infection among MSM population in China.