大鼠星形胶质细胞无血清培养方法的研究  

Primary and passage cultures of astrocytes from rat brain cortex by using serum-free medium

作　　者：徐立新[1] 贾梅 师忠芳[1] 董丽萍[1] 闫旭[1] 王玉娇 陈烨[1] 袁芳[1] Xu Lixin;Jia Mei;Shi Zhongorang;Dong Liping;Yan Xu;Wang Yufiao;Chen Ye;Yuan Fang(Belting Neurosurgical Institute, Beifing Tiantan Hospital, Capital Medical University, Belling 100050, China)

机构地区：[1]首都医科大学、北京市神经外科研究所、首都医科大学附属北京天坛医院,100050

出　　处：《中华神经外科杂志》2018年第6期633-636,共4页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金（81271286,51707012）;北京市自然科学基金（7152027）;北京市神经外科研究所创新探索基金（2017-5）

摘　　要：目的 探索应用无血清培养基进行大鼠星形胶质细胞原代培养及传代培养的方法.方法 取出生24 h内Wistar大鼠大脑皮质组织,机械吹打分散为单细胞后在无血清培养基中进行原代培养.细胞传代时分别使用乙二胺四乙酸（EDTA）-胰酶或胰酶作为消化液,浓度为0.2 mg/ml或1 mg/ml的大豆胰蛋白酶抑制剂（SBTI）终止消化作用,按照1∶2或1∶3的比例传代.显微镜下观察细胞生长状态,胶质纤维酸性蛋白（GFAP）细胞免疫荧光染色方法鉴定细胞表型,MTF法检测传代细胞增殖情况.结果 细胞悬液静置15 min后更换新的无血清培养基培养,7d后显微镜下可见细胞生长良好,折光性强,胞体小而突起长;细胞免疫荧光染色鉴定显示＞95％的细胞为GFAP阳性细胞.与应用SBTI终止胰酶反应时未进行离心比较,低温、低速离心（4℃、3 000 ×g）3 min后,传代细胞可在无血清培养基中生长正常并增殖.MTT检测结果显示,EDTA-胰酶消化效果优于胰酶（A值分别为0.290±0.007、0.270±0.006,P＜0.010）;使用浓度为0.2 mg/ml或1 mg/ml的SBTI终止消化（A值分别为0.290 ±0.013、0.290±0.017,P=0.820）及以1∶2或1∶3比例传代（A值分别为0.340±0.070、0.320 ±0.030,P =0.770）,传代星形胶质细胞数量的差异均无统计学意义.结论 本研究建立无血清培养基进行大鼠星形胶质细胞原代及传代培养的方法,为避免血清干扰更好地开展体外星形胶质细胞功能的研究提供了新的细胞模型.Objective To explore the methods of primary and passage cultures of astrocytes by using serum-free medium.Methods Cells from cerebral cortices of Wistar rats within 24 h of birth were mechanically dispersed and cultured in serum-free medium.Reaching the confluence of 80%-90%,cells were digested by EDTA （ethylenediaminetetraacetic acid）-trypsin or trypsin,followed by 0.2 mg/ml or 1 mg/ml soybean trypsin inhibitor （SBTI） treatment and centrifugation for 3 min （3 000 × g,4 ℃） to stop digestion,then cells were subcultured with the ratio of 1 ∶2 or 1 ∶3.The cell phenotype was identified by immunofluorescence staining for glial fibrillary acidic protein （GFAP）.The cell proliferation was detected by MTT assay.Results Under the phase-contrast microscopy,primary cells in serum-free medium were prosperous,showed high refractivity and demonstrated small cell body and nucleus with extensive processes.About 95% of the primary cells were positively stained for GFAP.The passage cells grew and proliferated well in the serum-free medium.The MTT results showed that the cell number in EDTA-trypsin group was higher than that in trypsin group （0.290 ± 0.007 vs.0.270 ± 0.006,P 〈 0.010）.There was no difference in cell count either between 0.2 mg/ml or 1 mg/ml SBTI groups （0.290 ± 0.013 vs.0.290 ± 0.017,P =0.820） or between 1∶2 and 1 ∶ 3 groups （0.340 ± 0.070 vs.0.320 ± 0.030,P =0.770）.Conclusion The results showed that we have established the method of primary and passage cultures of rat astrocytes by using serum.

关 键 词：星形细胞 大鼠 培养基 无血清 原代细胞培养 传代细胞培养 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]