【2017年整理】小鼠大脑皮层星形胶质细胞不同体外培养方法的比较.doc

小鼠大脑皮层星形胶质细胞不同体外培养方法的比较 【关键词】 星形胶质细胞;,离体培养;,反应性星形胶质细胞;bystin 　　［摘 要］ 目的: 比较离体纯化和培养星形胶质细胞的不同方法，旨在获得高纯度的星形胶质细胞，为进一步研究奠定基础。方法: :用原代培养、传代纯化和一次或两次振荡纯化的方法培养星形胶质细胞。 结果: 原代培养的星形胶质细胞纯度不高;经传代纯化培养后纯度达到99%以上，bystin表达量较少，且能经短时间模拟缺血诱导活性明显增高;经振荡纯化后bystin表达量明显增多，GFAP免疫荧光变亮，能耐受长时间模拟缺血，提示经振荡后星形胶质细胞已经处于“活化”阶段。 结论: 经过传代Na3VO4， NaF是磷酸酶抑制剂，如果你作的蛋白是磷酸酶建议不要加。Aprotinin, leupeptin, pepstatin是蛋白酶抑制剂比较昂贵，如果没条件，只加PMSF也可以，但必须严格操作，防止蛋白降解。提取的蛋白放－70保存。纯化的星形胶质细胞纯度最高，可以经模拟缺血诱导成反应性星形胶质细胞，因此能更好地反映其在脑中的功能，是研究星形胶质细胞在脑中的功能和反应性星形胶质细胞较好的培养方法。 　　［关键词］星形胶质细胞; 离体培养; 反应性星形胶质细胞;bystin 　　To establish a cultural method of astrocytes by comparison of different ways of isolation astrocytes from mouse cerebral 　　DU Fang，WU Xiao-mei，ZHU Li 　　　　（Institute of Nautical Medicine， Nantong University， Nantong Jiangsu 226001，China） 　　［Abstract］ Objective: Comparing different ways of isolation and purify astrocytes from mouse cerebral tissue to establish an optimal culture method of astrocytes for future study of their functions in the brain. Methods: To acquire purified astrocytes by four usually used cultural methods: primary culture，subculture for three times， shanken overnight once or twice. Results: The purity of primary culture was the lowest，with the method of being subcultured for three times the purest， appear99% pure. The expression of bys- tin was low and cell ability could be enhanced by short-timed simulated ischemia with the subcultural meth-od. Astrocytes purified by being shanken overnight once or twice highly expressed bystin， immunocytochem-ical staining with anti-mouse GFAP was brighter and could endure simulated ischemia for longer time， which demonstrated that the astrocytes might have been activated by shaking. Conclusion: With the subcultural method we can acquire the purest astrocytes in vitro， which can be induced to reactive astrocytes by simula-ted ischemia， and can best reflect their functions in the brain. It′s an optimal method to acquire the astro-cytes for fut