大鼠脑室膜管下区神经干细胞的贴壁培养及其分化特征

Adherent culture and differentiation character of neural stem cells at subependymal zone in rats

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。

AIM： To explore the adherent culture method and differentiation character of neural stem cells （NSC） at subependymal zone in rats in vitro. METHODS： The experiment was done in laboratory of Department of Anesthesiology, Tongji Affiliated Hospital, Tongji Medical College, Huazhong University of Science ＆ Technology between October and December 2004. The NSCs from subependymal zone of postnatal 24-hours SD rats were isolated and cultured adherently with serum-free Neurobasal medium [containing 20 g/L B27, 20μg/L basal fibroblast growth factor （bFGF） and 20μg/L epidermal growth factor （EGF）]. Nidogen antibodies were used to identify the cultured cells and the capability of cell proliferation was measured by BrdU method. After the differentiation of NSCs with volume fraction of 0.05 fetal bovine serum, neuron, astrocyte and oligodendrocyte marked with microtubule-associated protein-2 （MAP-2） antibody, glial fibrillary acidic protein （GFAP） antibody and 2, 3-cyclic nucleotide phosphodiesterase （CNP） antibody were used to study the differentiation condition of NSC. RESULTS：①Four to five days after primary culture, a large number of suspending cell balls, i.e., neural balls which contained several decades of cells with tight structure and inequality of size. After the neural balls cultured adherently for 24-48 hours, sheet-shaped cell clone was formed, and the cells were showed fusiform or muhi-angle.③ The cells were passaged once every 7-8 days commonly. After seven time or eight times passages, the culture could maintain for two months before growth stopped.④ Primary cultured cells adherently were nidogen positive cell and also stained positive for BrdU. After adding serum to medium and differentiation of immunocytochemical detection, the cells included positive MAP-2, positive GFAP and positive 2,3- CNP, while the expression of the nidogen was negative. CONCLUSION： Primary cultured NSCs with adherent method can retain the fairly strong capability of proliferation after many times of subcuhuring in vitro, still keep itself character, meanwhile, have the characteristics of multi-differentiation potential. Therefore, the adherent culture can be used as an ideal culture method of NSC.