四种大鼠星形胶质细胞原代纯化培养方法的比较

目的:寻求一种简单、经济、高效的大鼠大脑皮质星形胶质细胞原代纯化培养方法。方法:40只新生24小时Wistar大鼠,随机分成4组,每组10只。A组:酶消化、差速离心、振荡法;B组:酶消化、差速贴壁、恒温摇床法;C组:机械吹打、差速贴壁、恒温摇床法;D组:D1机械处理、差速贴壁、恒温摇床法D2酶消化、差速离心、振荡法。结果:四组纯化培养出的细胞都具有典型胶质细胞形态学星形特征。经GFAP-FITC免疫荧光结合DAPI染色鉴定纯度A:97.8%B:95.5%C:96.3%D组:(D1:95.7%;D2:97.3%);细胞数量D组>A组>B组>C组。结论:四组方法提取的细胞纯度均达95%以上,均能达到细胞实验的要求,C组方法提取所得细胞纯度最高,操作简单,细胞数也较多,值得推广。D组方法提取得到的细胞最多,虽过程相对繁琐,但也是一种值得推广的星形胶质细胞提取方法。

Objective: To seek a simple, economical and efficient primary purification and culture method for rat cerebral cortical astrocytes. Methods: Forty newborn Wistar rats(in 24 hours) were randomly divided into 4 groups, 10 in each group. Group A:trypsin digesting explants, differential centrifugation,shaking; group B: trypsin digesting explants,differential attachment, Constant temperature shaking table; group C: mechanical blowing,differential attachment, Constant temperature shaking table; group D:( D1:mechanical method,differential attachment, Constant temperature shaking tabl D2:trypsin digesting explants,differential centrifugation,shaking). Results: Cells of the four groups all had typical morphology characteristics of astrocyte. The purities were identified by immunofluorescence staining with anti-GFAP body and DAPI. The purities, A: 97.8% B: 95.5% C: 96.3% D group:(D1:95.7%; D2: 97.3%); cell number, D group > A group > B group > C group. Conclusion: The purities of the cells extracted by the four methods were all more than 95%, which can meet the requirements of cell experiments. Method C have more cells, simple operation and the highest purity, which is worthy of promotion. Method D extracts the most cells, although the process is relatively cumbersome, but it is also worth promoting.