胎鼠脊髓神经干细胞分离培养及诱导分化

目的 探讨机械分离法与酶消化法在脊髓源性神经干细胞（NSCs）体外培养的优劣,并观察脊髓源性NSCs体外培养时增殖和分化的特点.方法 取妊娠第15天（E15 d）的胎鼠神经管组织,分别采用酶消化法和机械吹打分离法离散细胞,进行无血清培养,并以上述两种方法传代培养.借助免疫细胞化学法对NSCs及分化结果进行鉴定,细胞球计数法检测成球率,测量神经球直径分析细胞增殖能力,MTT法监测细胞生长情况.结果 ①培养的细胞具有增殖成球生长的特性并且抗巢蛋白免疫荧光阳性、血清可诱导分化出胶质细胞酸性蛋白（GFAP）、神经元特异性希醇化酶（NSE）阳性表达的细胞.②细胞直径：酶消化组（137.74±11.62）μm,机械分离组（143.69±12.20） μm（P ＞0.05）.③生长曲线结果显示酶消化组NSCs综合生长增殖能力较高,OD值：酶消化组（0.39 ±0.15）,机械分离组为（0.36±0.13）.结论 胎鼠脊髓组织中存在具有自我增殖和多分化潜能的NSCs,原代无血清成球培养时采用酶消化法更有利于获得较多NSCs.

The Isolation Culture and Differentiation of Neural Stem Cells from Rat Fetal Cord

Objective To compare the livability of isolation and culture methods of neural stem cells （NSCs） by mechanical separation and enzymatic digestion methods,and to observe the proliferation and differentiation characteristics in the cell culture process.Methods The NSCs were isolated from neural tube tissue of E15 d rat fetal with micro-anatomise method.And they were cultured in serum-free culture medium,and they were used mechanical separation and enzymatic digestion methods to discrete cells and passaging.The characterizations of NSCs and their differentiation were identified by immunocytochemical strining.Results ①The cultured cells could proliferate into a ball growth characteristics,and anti-nestin immunofluorescence was positive.serum could induce differentiation of GFAP,NSE positive cells.② The cell diameter in enzymatic digestion group was （137.74 ± 11.62） while it was （143.69 ± 12.20） in mechanical separation group（P ＞0.05）.③Growth curve showed enzymatic digestion group possessed more proliferation ability.OD value in enzymatic digestion group was （0.39-± 0.15） while OD value in mechanical separation group was （0.36 ± 0.13）.Conclusion NSCs present in fetal spinal cord tissue.It has self-proliferation and pluripotent characterizations.Primary culture in serum into a ball with enzyme digestion is more conducive to obtain more neural stem cells.