人与鼠神经干细胞体外培养特性的比较

Comparison of the property of human and rat neural stem cells cultured in vitro

目的:比较分析人及鼠神经干细胞体外培养的生长特性。方法:实验于2004-04/12在华中科技大学同济医学院附属同济医院麻醉科实验室完成。孕14d及新生Wistar大鼠由华中科技大学同济医学院实验动物中心提供,孕9周及孕16周水囊引产胎儿经胎儿家属同意捐赠,用1×1010L-1或1×108L-1的起始密度分离培养人和鼠神经干细胞。原代培养出现大量悬浮神经干细胞球后,一部分细胞继续悬浮培养,另一部分细胞种入用0.001%多聚鸟氨酸和0.2%明胶包被的培养瓶中贴壁培养。在培养9代左右的人神经干细胞及4代左右的鼠神经干细胞培养液中加入5-溴脱氧尿嘧啶核苷或5%胎牛血清继续培养1周。应用抗巢蛋白及抗5-溴脱氧尿嘧啶核苷作为人及鼠神经干细胞初步鉴定,应用抗神经胶质酸性蛋白、微管相关蛋白2和2,3-环核苷酸磷酸二脂酶抗体检测其分化,SABC法进行免疫细胞化学染色。结果:①用较高(1×1010L-1)起始密度可有效的分离培养原代人神经干细胞,较低(1×108L-1)的起始密度对鼠神经干细胞分离有利,贴壁培养的人及鼠神经干细胞保持干细胞的特性长期增殖。②人神经干细胞自发聚集能力强、培养维持时间长,相比较而言,鼠神经干细胞单个细胞体积大、分裂增殖快、对起始密度依赖性小、贴壁能力强。③悬浮培养及贴壁培养的人和鼠神经干细胞,经抗巢蛋白及抗5-溴脱氧尿嘧啶核苷免疫细胞化学染色,均显示阳性。血清诱导分化后,免疫细胞化学染色可显示出微管相关蛋白2、神经胶质酸性蛋白染色阳性的细胞,人与鼠神经干细胞分化后2,3-环核苷酸磷酸二脂酶的染色效果不佳。结论:人神经干细胞和鼠神经干细胞体外培养时生长特性有较大差异。表现为前者需要较高的起始培养密度、聚集能力强、培养维持时间长,后者单个细胞体积大、分裂增殖较快、贴壁能力强。

AIM： To compare the property of human and rat neural stem cell cultured in vitro. METHODS： The experiment was conducted in the Laboratory of Anesthesiology in Tongji Hospital of Tongji Medical College, Huazhong University of Science ＆ Technology from April to December 2004. The 14-day pregnant rats and newborn rats were provided by the experimental animal center of Tongji Medical College, Huazhong University of Science ＆ Technology. Human fetus aged 9 or 16 weeks induced by labor with water bag were donated by their parents. Human and rat neural stem cells were isolated and cultured in different densities （1 ×10^10 L^-1 or 1×10^8 L^-1）. After the floating neurosphere was formed in primary culture, some neural stem cells were used for suspension culture whereas part of cells was cultured adhesively to the flask precovered by 0.001% polyomithine and 0.2% gelatin. 5-bromodeoxyundine or 5% fetal bovine serum was added to the medium of human neural stem cells at passage 9 and rat neural stem cells at passage 4. After a week by SABC immunocytochemical staining, the human and rat neural stem cells were identifed primarily by nestin antibody and 5-bromodeoxyundine antibody. The ability of multipotentiality of the neural stem cells we isolated was identified by microtubule-associated protein 2 antibody, glial fibrillary acidic protein antibody and cyclic nucleotide phosphohydrolase antibody. RESULT： （1）We can isolate human neural stem cells in high primary density （1×10^10 L^-1） and rat neural stem cell in low primary density （1×10^8 L^-1）. Human and rat neural stem cells cultured by adhere-wall method could preserve the property of neural stem cell and proliferate stably for a long time. （2）Human neural stem cell was easy to gather and lasted for more time. Compared to human neural stem cell, rat neural stem cell had bigger volume for each cell, more rapid dividing growth speed, less dependent on primary density, and more powerful ability to adhere wall. （3） Nestin or 5-bromodeoxyundine positive staining cells were found in suspension cultured and adhere-wall cultured human and rat neural stem cells. After serum-induced differentiation, microtubule-associated protein 2 and glial fibrillary acidic protein positive staining cells could be found in human and rat neural stem cells. Whereas no satisfied result was found in cyclic nucleotide phosphohydrolase antibody. CONCLUSION： There are significant differences in the properties between human and rat neural stem cell cultured in vitro. Human neural stem cell is easy to gather, lasts for more times, and need higher primary culture density. Rat neural stem cell has bigger volume for each cell, more rapid division grwoth speed, and more powerful ability to adhere wall.