内源性过表达Nestin对NIH3T3细胞增殖、迁移及MAPK信号传导通路的影响

Effects of endogenous nestin overexpression on proliferation, migration and MAPK signal transduction pathway in NIH3T3 cells

目的通过在成纤维细胞NIH3T3中内源性过表达巢蛋白(Nestin),研究其对细胞增殖、迁移及转化生长因子(TGF-β1)作用下细胞胶原蛋白表达与MAPK相关通路的影响。方法通过CRISPR/SAM技术构建内源性过表达Nestin的细胞株,用MTS实验、Western blot检测细胞增殖能力,细胞划痕实验检测细胞迁移能力,经qPCR、Western blot与免疫荧光染色明确TGF-β1对其胶原蛋白与MAPK通路的影响。结果 CRISPR/SAM技术能使内源性Nestin表达显著提高(P<0.05),而内源性过表达Nestin对NIH3T3细胞的生长、迁移有明显促进作用(P<0.05),对TGF-β1诱导的胶原蛋白表达及MAPK的激活也有明显的抑制效应(P<0.01)。结论用CRISPR/SAM技术内源性过表达Nestin可以明显促进NIH3T3细胞的生长和迁移,但却抑制TGF-β1诱导下胶原蛋白的表达及MAPK的激活。

Objective To observe the effects of endogenous nestin overexpression on the proliferation, migration and transforming growth factor β(TGF-β1)-induced expression of collagen and MAPK-related pathway in NIH3T3 fibroblasts. Methods CRISPR/SAM technique was used to construct a nestin-overexpressing cell line in NIH3T3 fibroblasts. The effects of nestin overexpression on cell proliferation and migration were evaluated using MTS assay, immunoblotting and scratch-wound assay. Quantitative PCR, immunoblotting and immunofluorescence staining were used to determine the effect of nestin overexpression on TGF-β-induced expression of collagen and activation of MAPK pathway in the fibroblasts. Results CRISPR/SAM significantly increased the expression of endogenous nestin in NIH3T3 fibroblasts(P<0.05), and nestin overexpression significantly promoted the growth and migration of NIH3T3 cells(P<0.05). Nestin overexpression obviously inhibited the expression of collagen and the activation of MAPK in TGF-β1-induced cells(P<0.01). Conclusion Endogenous overexpression of nestin achieved by CRISPR/SAM technique significantly promotes the growth and migration of NIH3T3 cells and inhibits TGF-β1-induced expression of collagen and activation of MAPK.