培养雪旺细胞NGF、CNTF和GDNF共表达和共定位实验研究

Experimental study of co-expression and co-localization of NGF, CNTF and GDNF of cultured Schwann cells

目的探讨将雪旺细胞和背根节神经元共同培养后去除背根节神经元后雪旺细胞神经生长因子(nervegrowthfactor,NGF)、睫状神经营养因子(ciliangneurotrophicfactor,CNTF)和胶质细胞源性神经营养因子(glialcellline-derivedneurotrophicfactor,GDNF)共表达变化特点及其细胞共定位。方法运用多彩色荧光原位杂交和计算机图像处理技术实现雪旺细胞NGF、CNTF和GDNFmRNA共表达变化的定量分析和细胞共定位,并通过免疫组织化学方法验证有关结果。结果NGF、CNTF和GDNFmRNA表达变化可同时定位于单个雪旺细胞;通过mRNA和蛋白表达水平的图像分析发现,去除背根节神经元后NGF和GDNF表达在迅速下降后随即逐渐增高,但2周时仍低于共培养表达水平(P<0.05),CNTF表达则持续下降(P<0.01)。结论雪旺细胞和背根节神经元共同培养后建立相互营养支持的体系,当失去神经元营养支持后,需要分泌神经生长因子和多种神经营养因子以维持自身环境的稳定;同时上述研究提供的方法能对雪旺细胞中多种神经营养因子的共表达定位进行可视化研究。

Objective To investigate the co-expression characteristics of NGF, CNTF and GDNF and the co- localization of these factors in Schwann cells initially co-cultured with dorsal root ganglion neurons and then deprived of these neurons. Methods The co-localization of NGF, CNTF and GDNF mRNA and quantitative analysis of their coexpression characteristics were performed by means of multicolor fluorescent in situ hybridization and computer image processing teehnique. Immunohistochemistry staining was also used to confirm above results. Results The coexpression of NGF, CNTF and GDNF could be localized on a cellular level. Image quantitative analysis results showed that the expression of NGF and GDNF mRNA decreased rapidly then followed with a gradual increase after the neurons were removed.The expression, however, was still lower till 2 weeks as compared with that of the co-cultured one （ P 〈 0.05） . Expression of CNTF mRNA decreased successively （ P 〈 0.01 ） . Conclusion Sehwann cells secret nerve growth factor and several kinds of neurotrophic factors to keep the stability of self environment without the support of dorsal ganglion neurons. Co-expression and co-localization could be visually ascertained by the method presented in this paper o