碱性成纤维细胞生长因子诱导大鼠表皮干细胞分化为神经干细胞的作用

Role of basic fibroblast growth factor in epidermal stem cells differentiating into neural stem cells in rats

目的探讨碱性成纤维细胞生长因子(bFGF)诱导大鼠表皮干细胞(ESCs)分化为神经细胞的作用。方法分离获得饲养1~3<1的新生SD大鼠表皮基底层组织,利用快速贴壁法消化、分离获得ESCs,于倒置显微镜下观察ESCs的形态。ESCs的培养基为角质细胞无血清培养基(K-SFM),然后按照不同密度ESCs进行分组处理(A组:0.1x10^7/ml,B组;0.3x10^7/ml,C组:0.5x10^7/ml,D组:0.1x10^6/ml),每组加入含20ng/ml的bFGF培养基进行培养。观察细胞形态的变化并计数,连续观察7d;培养7d后利用细胞免疫组织化学法检测细胞标志物巢蛋白和神经元特异性烯醇化酶(NSE)的变化。结果成功分离得到SD大鼠ESCs。bFGF诱导后,A组和B组细胞形态发生变化的数量第1~6天均较C组和D组多(P<0.05);第7天A组较其他三组多(P<0.05);C组第3天组内比较细胞数量最多(P<0.05);D组未出现细胞形态变化。免疫组织化学法检测显示巢蛋白和NSE检测均呈阳性。结论bFGF有助于诱导ESCs向神经细胞的分化,而且该诱导作用与细胞的培养密度存在一定关系。

Objective To investigate the effect of basic fibroblast growth factor(bFGF)on the differentiation of epidermal stem cells(ESCs)into nerve cells in rats.Methods The epidermal basal layer tissue of newborn SD rats(1-3 days)were isolated and obtained.ESCs were digested and isolated by rapid attachment to a substrate,and the morphology of ESCs was observed under an inverted microscope.ESCs were cultured with Keratinocytes serum-free medium(K-SFM).The ESCs were grouped and treated according to the density including Group A:0.1 x 10^7/ml,Group B:0.3 X 10^7/ml,Group C:0.5 x 10^7/ml,Group D:0.1 x 10^6/ml,and each group was added bFGF(20 ng/ml).The changes of cell morphology were observed and counted for seven days.The changes of cell markers Nestin and NSE were detected by immunohistochemistry.Results The ESCs of SD rat were isolated successfully.After bFGF induction,the numbers of cells with morphological changes in Groups A and B were larger than those in other two groups in the first 6 days(P<0.05),and the number in Group A was the largest on the seventh day(P<0.05).On the third day,Group C had the largest number of cells with changes based on the comparison within the group.There was no change in cell morphology in Group D.Immunohistochemistry showed positive Nestin and NSE.Conclusion The bFGF helps induce the differentiation of ESCs into nerve cells,which is associated with the cell density.