转录调控因子Shh促进胎鼠脊髓神经干细胞体外增殖的效应

Effect on proliferation of neural stem cells in spinal cord of fetal rat in vitro promoted by transcriptional modulator sonic hedgehog

目的:观察体外转录调控因子Shh对脊髓神经干细胞的增殖效应,探讨其在脊髓损伤中可能的应用。方法:以一定含量的Shh处理原代和传代的脊髓神经干细胞,分别在接种细胞后的第2,4,6天计数神经球的形成情况;并用胎牛血清作分化诱导剂,观察Shh中干细胞球的分化情况,8d后,免疫组化双标鉴定并计数神经微丝-200(NF-200)和胶质原纤维酸性蛋白(GFAP)阳性细胞的比例,对比碱性成纤维细胞生长因子(bFGF)作统计学分析。结果:Shh(5～50nmol/L)在体外可以促进胎鼠脊髓原代和传代的神经干细胞增殖,且表现为剂量依赖性,Shh(50nmol/L)的增殖效应几同bFGF(20μg/L);增殖的细胞可分化神经元、星形胶质细胞和少突胶质细胞,其神经元与星形胶质细胞的比例同bFGF组的分别为64%∶36%和63%∶37%。结论:Shh(5～50nmol/L)在体外可以剂量依赖性的促进胎鼠脊髓原代和传代的神经干细胞增殖,且增殖的细胞具有多分化潜能,其分化细胞的趋向类似于bFGF组。

AIM: To observe the proliferative effect of transcriptional modulator sonic hedgehog(Shh) in vitro on the neural stem cells in spinal cord, and explore the possible application of it in spinal cord injury. METHODS: Primary and passage neural stem cells in spinal cord were treated by different concentrations of Shh. Then the formation of neural spheres was observed on the 2nd,4th and 6th day after inoculation. Furthermore, the fetal bovine serum(FBS) was used as differentiated inducer, and the differentiation of neural stem cells in Shh was observed.Eight days later, immunohistochemistry with antibody neural microfilament 200(NF 200) and glial fibrillary acidic protein(GFAP) were taken to identify and calculate the proportion of positive cells.The basic fibroblast growth factor(bFGF) was compared and taken to analyze statistically. RESULTS: The proliferation of primary and passage neural stem cells in spinal cord were promoted by Shh(5-50 nmol/L) with dosage dependence in vitro. The difference between Shh(5-50 nmol/L) and bFGF(20 μg/L) in promoting proliferation of neural stem cells was nearly nothing. Neurons, astrocytes and oligodendrocytes were derived from neural stem cells. The proportion between neurons and astrocytes in Shh and bFGF were 64%∶36%and 63%∶37%respectively. CONCLUSION: Shh(5-50 nmol/L)promoted the proliferation of primary and passage neural stem cells in spinal cord with dosage dependence in vitro. The proliferative cells in Shh were of multipotentiality. The differentiational tendency of cells in Shh was consistent with that in bFGF.