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BACKGROUND: Deletion of glial cell-derived neurotrophic factor and endothelin receptor type B gene will induce abnormal development of enteric nervous system. Neural stem cell transplantation can repair nervous system from anatomy and function,and be considered as a vector of gene transfection.OBJECTIVE: To transfect recombinant adenovirus carrying glial cell-derived neurotrophic factor and endothelin receptor type B gene into mouse neural stem cells, and to observe expression of target gene.DESIGN: A cell-gene study.MATERIALS: New-born Kunming mice were provided by the Animal Center of Tongji Medical College, Huazhong University of Science and Technology, China. jetPEI reagent was purchased from PolyPlus Co, France. The pAdTrack-CMV-GE with green fluorescent protein (GFP) was gifted by Doctor Sun Nianfeng and Zhang Jinghui in our laboratory.METHODS: Neonatal mouse brain tissues were sterilely obtained to prepare monoplast suspension. Adenovirus expressing glial cell-derived neurotrophic factor and endothelin receptor type B gene with GFP was dissolved in NaCI to prepare JetPEI/DNA complex. Subcultured neural stem cells in DMEM/F12 were regulated to 5×10~8/L, and 400 μL cell suspension and 100 μL JetPEI/DNA complex were seeded on a 24-well plate at 37 ℃ in 5% CO_2 incubator. Neural stem cells were harvested at 24, 48 and 72 hours following transfection.MAIN OUTCOME MEASURES: The efficiency of transfection was detected using fluorescence microscope and flow cytometry.Target gene expression in neural stem cells was determined using RT-PCR.RESULTS: Bright green fluorescence of the transfected cells could be observed under fluorescence microscope after 24 hours of transfection. The positive rate of GFP was 15.36%, 24.67%, 25.73% at 24, 48 and 72 hours following transfection respectively.Neural stem cells expressed glial cell-derived neurotrophic factor and endothelin receptor type B gene at various time points.Strap brightness was low at 24 hours, and exogenous gene expression was great at 72 hours.CONCLUSION: The target genes were successfully transfected into neural stem cells by using jetPEI reagent. Moreover, glial cell-derived neurotrophic factor and endothelin receptor type B gene effectively transcribed and expressed in target cells.
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Objective:To investigate the expressions of Bag-1 and Bcl-2 in colorectal cancer and evaluate their clinical significance.Methods:Bag-1 and Bcl-2 expressions were studied by immunohistochemical Streptavidin-Biotin-Peroxidase Complex(SABC)method in 64 samples of colorectal cancer tissues,10 normal colorectal tissues.Results:Bag-1 was positively related to the tumor grade,distant metastasis,stages of Dukes and prognosis,but was not related to the pathological cell types,tumor diameter,depth of invasion and lymph node metastasis.Bcl-2 was not correlated to these clinicopathologic factors.There was a positive correlation between Bag-1 and Bcl-2(r=0.475).Conclusion:The over expression of Bag-1 and Bcl-2 protein in colorectal cancer can affect the generation of colorectal cancer by participating in the regulation of apoptosis.
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Objective To summarize the experiences of the treatment of extrahepatic bile duct injury caused by cholecystectomy.Methods A retrospective analysis was made on the treatment of 11 cases of extrahepatic bile duct injuries in our hospital from 1989 to 2001.The position of injuries,types,the time from onset to diagnosis, operation methods and therapeutic effects were analyzed.Results Three of the 11 cases received an emergency operation,while 8 underwent selective operation.Seven cases (62.7%) of extrahepatic bile duct injury were diagnosed during the cholecystectomy,and the remaining 4 cases (37.3%) were diagnosed after operation.Seven cases of extrahepatic bile duct injury diagnosed during operation were treated immediately and 4 cases treated 3-4 days after the first operation.All the patients were cured and discharged.Conclusion The correct principles and methods of handling the cases of extrahepatic bile duct injury caused by cholecystectomy should be mastered;Roux-en-y cholangiojunostomy is the best option for the treatment of extrahepatic bile duct injury.
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Objective To study the inhibitory effect of human antisense high mobility group box 1 (HMGB1) gene on the growth of human pancreatic adenocarcinoma cell line (PANC-1).Methods HMGB1 was cloned and reversely inserted into eukaryotic expression vector pcDNA3.1 to get pcDNA3.1/anti-HMGB1, which was then transfected by liposome method into PANC-1 cells.After G418 selection, HMGB1 expression of PANC-1 cells before and after transfection was detected by RT-PCR and Western blot, the cell proliferating activity in vitro was examined by MTT analysis, and cell cycle and apoptosis were assessed by flow cytometry.Results pcDNA3.1/anti-HMGB1 vector was obtained.After transfection with pcDNA3.1/anti-HMGB1, HMGB1 gene expression of PANC-1 cells was inhibited in both mRNA and protein level, and the proliferation of cultured pancreatic adenocarcinoma cells was suppressed with a suppression rate higher than 40 % on the sixth day.The cells of G1 phase increased obviously while the cell number of S phase decreased, the number of apoptosic cell increased.Conclusion Antisense HMGB1 gene can inhibit the proliferation of human ancreatic adenocarcinoma cells and increase the cell apoptosis rate.