成体神经干细胞培养方法的建立

Extablishment of adult neural stem cell culture

目的 探讨体外建立培养成体神经干细胞（MSC）的方法.方法 从6周龄大鼠脑组织中分离神经干细胞,用神经干细胞培养液[DMEM/F12培养液添加1%N2、2%B27、20 μg/L表皮生长因子（EGF）和20μg/L碱性成纤维细胞生长因子（bFGF）]使其稳定增殖,10%胎牛血清诱导其贴壁分化.倒置显微镜下观察神经干细胞形态学变化;流式细胞仪检测神经干细胞表面标记物巢蛋白（Nestin）、神经元特异性烯醇酶（NSE,成熟神经元的特异性标志）、半乳糖脑苷脂（Galc-C,成熟少突胶质细胞的标记物）的表达.结果 分离所得细胞能在体外传代培养,流式细胞仪检测显示Nestin阳性细胞为97.6%,细胞经胎牛血清诱导分化后能形成NSE、Galc-C阳性细胞.结论 采用无血清的神经干细胞培养液能培养出具有分化潜能的成体神经干细胞.

Objective To investigate the method of adult neural stem cell （NSC） culture in vitro.Methods The neural stem cells were derived from the brain tissue of 6-week-age rats and proliferated steadily under the effect of neural stem cell culture fluid （DMEM/F12 with 1% N2, 2% B27, 20 μg/LEGF and 20 μg/L bFGF）. Neural stem cell ifferentiation was induced by 10% fetal calf serum. The change of cell morphology was observed under an inverted microscope. The rat neural stem cells and the differentiated neural stem cells were identified respectively by flow cytometry including Nestin, neuron specific enolase （NSE） and Galc-C. Results The cells derived from the brain tissue could be subcultured in vitro. The rate of positive Nestin cells was 97.6%. After induction by 10% fetal calf serum, these cells were positive for NSE and Galc-C. Conclusion By using fetal calf serum-free neural stem cell culture fluid, the rat adult neural stem cells having potential of multiple differentiations can successfully be cultured in vitro.