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目的:探索大鼠脑室管膜下区神经干细胞的贴壁培养方法及其体外分化特性。方法:实验于2004-10/12在华中科技大学同济医学院附属同济医院麻醉科实验室进行。取出生24h内的SD大鼠,分离其室管膜下区神经干细胞,应用无血清Neurobasal培养基(含20g/LB27,20μg/L碱性成纤维生长因子、20μg/L表皮生长因子)进行贴壁培养。采用巢蛋白抗体鉴定培养细胞,以5-溴脱氧尿嘧啶核苷掺入法检测细胞增殖能力。体积分数为0.05胎牛血清诱导神经干细胞分化后,用神经元、星形胶质细胞和少突胶质细胞标志物微管相关蛋白、胶质纤维酸性蛋白和2,3-环核苷酸磷酸二酯酶相应抗体检测神经干细胞分化情况。结果:①在细胞培养第4,5天即可见培养基中出现大量的由几十个细胞构成的结构紧密、大小不等的悬浮细胞球,即神经球。②神经球贴壁培养24～48h后形成片状细胞克隆,细胞呈梭形或多角形。③细胞一般七八天传代一次,经七八次传代,培养2个月左右,细胞进入生长停滞状态。④贴壁培养的细胞巢蛋白染色及5-溴脱氧尿嘧啶核苷染色均为阳性。在培养基中加入血清后,免疫细胞化学检测分化后细胞包括微管相关蛋白阳性、胶质纤维酸性蛋白阳性和2,3-环核苷酸磷酸二酯酶阳性细胞,巢蛋白表达均为阴性。结论:贴壁培养的神经干细胞在体外经多次传代培养后仍可保持较强的增殖能力,仍保持了其本身的特性,同时具有多分化潜能的特性,因此可以认为贴壁培养法可作为一种理想的神经干细胞的培养方法。
Objective: To explore the method of adherent culture of neural stem cells in the subventricular zone of rat and its in vitro differentiation. Methods: The experiment was performed at the Anesthesiology Laboratory, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from October to October 2004. SD rats were sacrificed 24h after birth, the subventricular zone of neural stem cells were isolated, the application of serum-free Neurobasal medium (containing 20g / LB27, 20μg / L basic fibroblast growth factor, 20μg / L epidermal growth factor) Wall culture. Nestin antibody was used to identify cultured cells, and cell proliferation ability was detected by 5-bromodeoxyuridine incorporation method. After differentiation of neural stem cells was induced at a volume fraction of 0.05 fetal bovine serum, neurons, astrocytes and oligodendrocyte markers microtubule-associated protein, glial fibrillary acidic protein and 2,3-cyclic nucleotide phosphate Di esterase corresponding antibody detection of neural stem cell differentiation. Results: ①In the fourth and fifth days of cell culture, a large number of suspended cell spheres, consisting of dozens of cells and having different sizes, appeared in the medium, namely, neurospheres. ② neurosphere adherent culture formed after 24 ~ 48h sheet-shaped cell clones, spindle or polygonal cells. ③ cells generally seven or eight days of passage once, after seven or eight passages, cultured for about 2 months, the cells into the stagnant state of growth. ④ adherent culture cells nestin staining and 5 - bromodeoxyuridine staining were positive. After adding serum into culture medium, immunocytochemistry detected the differentiated cells including microtubule-associated protein positive, glial fibrillary acidic protein positive and 2,3-cyclic nucleotide phosphodiesterase positive cells, nestin expression were negative . CONCLUSION: Adherent neural stem cells cultured in vitro can still maintain their strong proliferative capacity after being subcultured for many times, and retain their own characteristics as well as their potential of differentiation. Therefore, adherent culture can be considered as An ideal method for culturing neural stem cells.