【摘要】 目的:观察差速贴壁技术对星形胶质细胞纯化率的影响,旨在建立一套可靠的大鼠脑皮质星形胶质细胞的取材分离、纯化培养技术。方法:实验于2006-06/08在泰山医学院生命科学研究所完成。实验材料:出生2～3d的Wistar大鼠,雌雄不拘,由泰山医学院生命科学研究所实验动物中心提供。实验方法:选用出生二三天的Wistar大鼠进行脑皮质星形胶质细胞原代培养。实验分两组培养:常规培养组和差速贴壁培养组。差速贴壁培养组分别于15,30min取出,轻轻翻转培养瓶,将上清液移至另一培养瓶中,放入培养箱中继续培养。7～10d后传代,待细胞分层生长后,置于37℃摇床中250r/min振荡18h,倒掉上清液,D-Hank’s液洗3次后,加入0.25%胰酶消化,倒置显微镜下观察,待细胞突起回缩后加入含血清的培养基终止消化,用吸管反复吹打使细胞从瓶壁上脱落,细胞悬液1000r/min离心5min后,弃上清液,加入含体积分数为0.2血清的DMEM培养基混悬沉淀,接种入预先涂有L-多聚赖氨酸的培养瓶中继续培养。采用双重免疫荧光法鉴定星形胶质细胞纯度,测定积分吸光度值判断星形胶质细胞的生长状况。结果:①应用差速贴壁技术培养星形胶质细胞可明显提高星形胶质细胞纯度[常规培养组:(82±3)%,差速贴壁培养组15min:(94±2)%,差速贴壁培养组30min:(95±2)%,P<0.01]。差速贴壁需要充分的时间,15min组和30min组在提高星形胶质细胞纯度方面无明显差别。②差速贴壁培养组星形胶质细胞积分吸光度值高于常规培养组(常规培养组:528±25,差速贴壁培养组15min:972±17,差速贴壁培养组30min:996±35,P<0.05)。结论:①差速贴壁技术可明显提高星形胶质细胞纯化度,并且星形胶质细胞生长状态明显优于常规培养方法。②最佳差速贴壁时间为15min,过长差速贴壁时间对提高星形胶质细胞纯度无明显影响。更多还原

【Abstract】 AIM: To observe the proportion of rat cerebral astrocyte cultured by differential velocity adherent technique, so as to establish a set of reliable technique for the isolation and purification of the astrocytes cerebral cortex. METHODS: The experiment was performed in the Institute of Life Science, Taishan Medical College from June to August 2006. Wistar rats aged 2-3 days after birth of either sex were provided by Experimental Animal Center, Institute of Life Science, Taishan Medical College, and selected to carry out primary culture of cerebral astrocytes. They were divided into two groups: routine culture group and differential velocity adherent group. Astrocytes were removed at minutes 15 and 30 in the latter group, and then culture flask was rotated to remove supernatant into another culture flask, and then kept in incubator for following culture. After 7-10 days, each group was passaged till cells grew into demixing, and then put in swing bed at 37 ℃ and shook at 250 r/min for 18 hours. Supernatant was added, and then the cells were washed with D-Hank’s liquor three times, and treated with 0.25% trypsinization, observed under inverted microscope. Medium containing serum was added to stop digestion when process of cells recovered. The cells were blown and hit by pipette for keeping cells from flask wall, and then centrifuged at 1 000 r/min for 5 minutes. Supernatant was removed before DMEM medium containing 0.2 volume fraction was suspended and precipitated. The cells were incubated in L-polylysine culture flask successively. Double immunofluorescence staining was selected to identify purification of the astrocytes. At the same time, integrated optical density(IOD)was used to measure growth state of astrocytes. RESULTS: ①Purity of astrocytes could be elevated markedly by differential velocity adherent technique [routine culture group:(82±3)%, differential velocity adherent group of 15 minutes:(94±2)%, differential velocity adherent group of 30 minutes:(95±2)%,P < 0.01. Differential velocity adherent technique needed enough time. There was no significant difference in elevating cell purity in the 15 minutes group and 30 minutes group. ②IOD of astrocytes in the differential velocity adherent group was higher than that in the routine culture group (routine culture group:528±25, differential velocity adherent group of 15 minutes:972±17, differential velocity adherent group of 30 minutes:996±35,P < 0.05. CONCLUSION: ①The differential velocity adherent technique can remarkably elevate the purity of astrocytes, and the growth is distinctly better than that by routine culture method. ②The best differential velocity adherent time is 15 minutes, and there is no significant effect of longer adherent time on elevating purity.更多还原