Objective To investigate the prevention of HBV reinfection in the perioperative period of liver transplantation on HBV-related diseases． Methods Published papers were collected and reviewed． Results HBV-related diseases were the main indications of liver transplantation．The prevention for HBV reinfection affects the survivals remarkably. Nowadays, a lot of medication have been used in the prevention of HBV reinfection， and the therapeutic regimens were different from each other. Conclusion Liver transplantation is an effective treatment for HBV-related disease． Appropriate prevention of HBV reinfection in the perioperative period of liver transplantation is important for the survivals of patients．
Objective To determine the value of detection of micrometastasis in peripheral blood to hepatocellular carcinoma （HCC） metastasis or recurrence． Methods Reviewed the related literatures, the methods and significances of the detection of HCC micrometastasis in peripheral blood were analyzed. Results Currently, there are mainly two methods, hematogenous dissemination cell detection and HCC specific mRNA biomarker detection, for detection of HCC micrometastasis in peripheral blood. Theoretically, although they are considered as early detections of HCC metastasis or recurrence, researches still not have a abroad agreeable conclusion from different studies. After adjusting and improving the methods and detection time, different studies also have not gotten a quite consistent conclusion. Conclusion There is a great significance in detection of HCC micrometastasis in peripheral blood to understanding the mechanisms of HCC metatasis and recurrence, and also to improving the clinical therapy. Theoretically and practically, the method should be improved for facilitating the mechanism research of HCC metastasis and recurrence, and the application of detection.
ObjectiveTo investigate the expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in human pancreatic adenocarcinoma and their correlation with clinicobiological behavior.MethodsThe expression of COX-2 and VEGF in 51 cases of human pancreatic ductal adenocarcinoma were detected with immunohistochemistry of Envision.ResultsExpression of COX-2 and VEGF in pancreatic ductal adenocarcinoma were 74.5% and 68.6%, respectively; no expression of COX-2 and VEGF in adjacent normal tissue was detected. Both COX-2 and VEGF expression in clinical stage Ⅲ-Ⅳ were much higher than those in clinical stage Ⅰ-Ⅱ, and also higher in positive group of lymph node metastasis than in negative group as well (Plt;0.05). None of them had relation with histological grades, age, sex, tumor size and location. The expression of COX-2 was closely correlated with VEGF (r=0.411, Plt;0.01).ConclusionCOX-2 and VEGF may play a pivotal role in tumorigenesis and tumor progression in pancreatic cancer, they may provide new targets for therapy of pancreatic cancer.
Objective To examine the relationship between ratio of proliferating cell nuclear antigen (PCNA)/apoptosis and biology of hepatocellular carcinoma. Methods Thirty five cases of hepatocellular carcinoma were studied with TUNEL and immunohistochemistry. Results Positive rate of apoptosis in grade Ⅰ, Ⅱ, Ⅲand Ⅳ was 1.37%,0.70%, 0.67% and 0.25% respectively. Positive rate of PCNA in grade Ⅰ, Ⅱ, Ⅲ and Ⅳ was 17%, 19%, 75% and 80%. Ratio of PCNA/apoptosis in grade Ⅰ,Ⅱ,Ⅲ and Ⅳ was 19.04, 75.51, 138.01 and 345.52. Conclusion Ratio of PCNA/apoptosis is correlated with histological classification.
Objective To construct a green fluorescent protein expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28, containing anti-TAG72 single chain variable fragment (scFv) fused into the transmembrane and intracellular domain of the signal-transducing chain of CD28 gene, and to transfect it into peripheral blood mononuclear cells． Methods Recombinant transmembrane and intracellular domain of CD28 cDNA and anti-TAG72 scFv cDNA fragment was subcloned into pEGFP-C3 vector. Recombinant clones were selected by Kanamyein， and then identified by PCR， enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into peripheral blood mononuclear cells by means of lipofection. The recombinant protein expression was confirmed by immunocytochemistry, laser scanning confocal microscope, PCR and Western blot analysis. Results The fused gene fragment of anti-TAG72 scFv-CD28 was successfully inserted into pEGFP-C3 plasmid, and it was confirmed by enzyme digestion and DNA sequencing. The fused anti-TAG72 scFv-CD28 gene and its protein was identified in peripheral blood mononuclear cells． Conclusion The eukaryotic expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28 was successfully constructed and transiently expressed in peripheral blood mononuclear cells， which would lay a foundation for further studies on the role of it to activate tumor-associated antigen-specific T lymphocyte, for generating of modified T lymphocytes targeting gastrointestinal tumors.
Objective To study the prokaryotic expression of the anti-tumor associated glycoprotein-72 (TAG-72) singlechain fragment variable (scFv) antibody and its specific affinity to hepatocellular carcinoma cell lines and tissues. Methods The cDNA of anti-TAG-72 scFv antibody was inserted into pCANTAB5E to obtain phage vector anti-TAG-72 -scFv-pCANTAB5E. Isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of anti-TAG-72 scFv antibody. SDS-PAGE and Western blot were used to identify the anti-TAG-72 scFv antibody. Human hepatocellular carcinoma cells were cultured, and TAG-72 was determined with the obtained scFv by immunohistochemistry in the cells and paraffin-embedded hepatocellular carcinoma tissues. Results SDS-PAGE and Western blot showed that the anti-TAG-72-scFv antibody was successfully expressed. Anti-TAG-72 scFv could bind to hepatocellular carcinoma cell lines SMMC7721, HepG2, and HHCC, but not to BEL7402, suggesting that SMMC7721, HepG2, and HHCC cells expressed TAG-72. For the 40 cases of hepatocellular carcinoma tissues, the positive rate of TAG-72 in stage Ⅰ and Ⅱ-Ⅲ was 23.08% (3/13) and 62.96%(17/27), respectively. While no TAG-72 expression was found in the 10 normal cases. TAG-72 expression was significantly different between hepatocellular carcinomas and normal tissues (Plt;0.05). Conclusions The prokaryotic expression of anti-TAG-72 scFv antibody is successfully achieved, and can be used to identify TAG-72 antigen. TAG-72 is highly expressed in hepatocellular carcinoma, but not in normal liver tissue, which may be suggested as cancer marker of hepatocellular carcinoma.
ObjectiveTo construct the human small interfering RNA (siRNA) lentiviral vector who targeting inhibitor of differentiation-1 (Id1) gene, and to detect its efficiency of gene silence for the HepG2 cells. MethodsThe most effective RNA interference sequences was screened from 4 kinds of siRNA vectors targeting Id1 gene (included pCGSIL-GFP-Id1-1, pCGSIL-GFP-Id1-2, pCGSIL-GFP-Id1-3, and pCGSIL-GFP-Id1-4), who was transfected to 293T cells. The selected siRNA vector was used to build lentiviral vector (Id1-RNAi-LV) and then infected human HepG2 cells. Then the expression levels of Id1 mRNA and its protein were detected by the real time PCR and Western blot method respectively. ResultsExpression level of Id1 protein in pCGSIL-GFP-Id1-4 group was lower than those of pCGSIL-GFP-Id1-1 group, pCGSIL-GFP-Id1-2 group, and pCGSIL-GFP-Id1-3 group (P < 0.05), who had the best efficiency of gene silence. The Id1-siRNA lentiviral vector (Id1-RNAi-LV) was successfully constructed by using pCGSIL-GFP-Id1-4. The titer of lentiviral was 2.0×109 TU/mL.results of real time-PCR and Western blot showed that, the expression levels of Id1 mRNA and its protein in HepG2 cells of Id1-RNAi-LV group were lower than those of blank control group and negative control group (P < 0.05). ConclusionsThe specific lentiviral can constantly down-regulate the expression of Id1 gene.
Objective To construct a mammalian vector encoding angiostatin kringle 5 (K5) under the control of αfetoprotein （AFP） enhancer and albumin promoter, and to observe the expression of angiostatin by introducting angiostatin gene into hepatocellular carcinoma cells through gene transfection. Methods Angiostatin cDNA was amplified from normal human eukaryotic cells by using RTPCR. Meanwhile, AFP enhancer and albumin promoter sequences were directed cloned and were inserted into vector pcDNA3.1. The recombinant vector of pcDNA3.1AFABangiostatin K5His was constructed, which contained the angiostatin K5 cDNA sequence that was under the control of the AFP enhancer and promoter. Angiostatin K5 cDNA was introduced into human AFP positive hepatocellular carcinoma cell lines with the transfected cultured cells that were mediated with Lipofectamine 2000. The expression of angiostatin K5 was analyzed by Western blot and the protein was dectected with antiHis antibody. Results The 500base pair of angiostatin K5 was in accordance with the expected sequence and the recombinant vector of pcDNA3.1AFABangiostatin K5His was also confirmed as the anticipated sequence. The expression of angiostatin K5 in AFP positive hepatocellular carcinoma cells was detected both by SDSPAGE and Western blot. Conclusion Efficient construction and expression of angiostatin K5 to AFP positive cells make it possible for antiangiogenesis therapy of human hepatocellular carcinomas, which may provide a promising approach.