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背景:神经干细胞的临床应用还尚待时日,现阶段需要解决如何诱导神经干细胞分化为特定表型的神经元以替代丢失、变性的神经元细胞。目的:探讨大鼠海马组织83ku蛋白对神经干细胞向乙酰胆碱酯酶阳性神经元分化的作用。设计、时间及地点:细胞学体外对照观察,于2003-10/2008-04在南通大学医学院完成。材料:清洁级SD大鼠12只,17d龄SD胎鼠多只,均由南通大学实验动物中心提供。方法:取6只正常大鼠及6只切割海马伞后14d大鼠的海马组织制成匀浆,进行非变性聚丙烯酰胺凝胶电泳,根据染色结果切取含有83ku差异蛋白条带进行电洗脱,定量后调整蛋白浓度为300mg/L。取胎鼠前脑组织,体外分离培养神经干细胞,设立3组:空白对照组加入单纯DMEM/F12无血清培养基;83ku蛋白正常组、83ku蛋白切割组分别加入含10mg/L来自正常/割海马伞大鼠海马组织83ku蛋白的DMEM/F12无血清培养基,诱导12d。主要观察指标:用乙酰胆碱酯酶组织化学染色检测神经干细胞分化为乙酰胆碱酯酶阳性神经元的情况。结果:诱导12d后,83ku蛋白切割组乙酰胆碱酯酶阳性神经元较多,胞体大且分化较好,突起粗且长;83ku蛋白正常组乙酰胆碱酯酶阳性神经元较少,胞体小,突起短;空白对照组仅见少量乙酰胆碱酯酶阳性神经元。组间乙酰胆碱酯酶阳性神经元数比较差异有显著性意义(P<0.05),83ku蛋白切割组>83ku蛋白正常组>空白对照组。结论:大鼠海马组织中83ku蛋白可成功诱导神经干细胞定向分化为乙酰胆碱酯酶阳性神经元。
BACKGROUND: The clinical application of neural stem cells is yet to be solved. At this stage, it is necessary to solve how to induce neural stem cells to differentiate into neurons of specific phenotype in order to replace lost and degenerated neurons. Objective: To investigate the effect of 83 ku protein in rat hippocampus on the differentiation of neural stem cells into acetylcholinesterase-positive neurons. DESIGN, TIME AND SETTING: The cytology in vitro control study was performed at Nantong Medical College from October 2003 to April 2008. MATERIALS: Twelve clean-grade SD rats and 17-day-old SD fetal rats were all provided by Experimental Animal Center of Nantong University. METHODS: Six normal rats and six hippocampus of hippocampus were harvested from the hippocampus of rats for 14 days, and then subjected to non-denaturing polyacrylamide gel electrophoresis. The bands containing 83 ku protein were cut out and electroeluted , After adjustment quantitative protein concentration 300mg / L. Cultured neural stem cells were isolated and cultured in vitro. Three groups were established: blank control group was added DMEM / F12 serum-free medium; 83 ku protein normal group and 83 ku protein cutting group were added with 10mg / The rat hippocampus 83ku protein in DMEM / F12 serum-free medium was induced for 12 days. MAIN OUTCOME MEASURES: Acetylcholinesterase histochemical staining was used to detect the differentiation of neural stem cells into acetylcholinesterase-positive neurons. Results: After induced for 12 days, 83 ku protein cleaved more acetylcholinesterase - positive neurons, large cell body and well differentiated, thick and long protuberances; 83 ku protein normal group less acetylcholinesterase neurons, small cell body, short process; A small amount of acetylcholinesterase-positive neurons were found in the blank control group. There were significant differences in the number of acetylcholinesterase positive neurons among the groups (P <0.05), 83ku protein cleavage group> 83ku protein normal group> blank control group. CONCLUSION: 83 ku protein in hippocampus of rats can successfully induce the neural stem cells to differentiate into acetylcholinesterase-positive neurons.