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目的对人胚胎神经干细胞(NSCs)进行分离、培养,研究纹状体组织提取液对神经干细胞分化方向的影响。方法从临床因故引产的人胚胎(胎龄8~16周)海马组织中分离、培养人胚胎神经干细胞,将其分离纯化并进行抗Nestin染色鉴定后分为两组进行诱导分化实验。A组:以基础培养基作为对照组;B组:基础培养基+人胚胎纹状体组织提取液(50ml/L)。分化的第7d取出细胞爬片,分别进行抗神经特异性烯醇化酶(NSE)免疫细胞化学染色,抗5-羟色氨(TH)免疫荧光化学染色和RT-PCR方法检测TH-mRNA的表达。结果培养的海马细胞经抗Nestin染色后呈Nestin阳性,证明为神经干细胞。诱导分化后对照组与实验组的NSE阳性细胞率分别为(21.89±2.17)%和(23.50±1.60)%,两组比较结果无统计学差异(P>0.05)。TH阳性细胞率分别为(0.53±0.17)%和(7.38±0.84)%,两组比较有统计学差异(P<0.05)。RT-PCR检测结果表明,TH-mRNA在对照组中无明显表达,纹状体组织提取液组神经干细胞分化后,TH-mRNA表达明显,两组比较有统计学差异(P<0.05)。结论在体外培养中,人胚胎纹状体组织提取液对神经干细胞向神经元分化无明显促进作用,但能促进人胚胎神经干细胞向多巴胺能神经元分化。
Objective To isolate and culture human embryonic neural stem cells (NSCs) and to study the effect of striatal tissue extract on the differentiation of neural stem cells. Methods Human embryonic neural stem cells were isolated and cultured from human embryos (gestational age: 8-16 weeks). The neural stem cells were isolated, purified and identified by anti-Nestin staining. The neural stem cells were divided into two groups to induce differentiation. Group A: basic medium as control group; Group B: basal medium + human embryonic striatum tissue extract (50ml / L). On the 7th day after differentiation, the cells were removed and immunostaining for NSE was performed. TH-mRNA expression was detected by immunofluorescence staining and RT-PCR. . Results The cultured hippocampal cells were Nestin positive after anti-Nestin staining and proved to be neural stem cells. The NSE positive rate of the control group and the experimental group were (21.89 ± 2.17)% and (23.50 ± 1.60)%, respectively. There was no significant difference between the two groups (P> 0.05). The positive rate of TH was (0.53 ± 0.17)% and (7.38 ± 0.84)%, respectively, with statistical significance (P <0.05). The results of RT-PCR showed that TH-mRNA was not expressed in the control group. The expression of TH-mRNA in the striatum tissue extract group was significantly lower than that in the control group (P <0.05). Conclusion In vitro culture, human embryonic striatum extract has no obvious effect on differentiation of neural stem cells into neurons, but it can promote the differentiation of human embryonic neural stem cells into dopaminergic neurons.