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

doi:10.16016/j.1000-5404.201908194-

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

更新时间：2021-09-22

- Effects of endogenous nestin overexpression on proliferation， migration and MAPK signal transduction pathway in NIH3T3 cells
- Journal of Third Military Medical University Pages: 1-9(2022)
- 作者机构：
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81472596)
- DOI：10.16016/j.1000-5404.201908194-
  CLC：
- Received：27 August 2019，
  
  Revised：20 November 2019，
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Effects of endogenous nestin overexpression on proliferation， migration and MAPK signal transduction pathway in NIH3T3 cells. [J/OL]. Journal of Third Military Medical University 1-9(2022)
DOI：

Effects of endogenous nestin overexpression on proliferation， migration and MAPK signal transduction pathway in NIH3T3 cells. [J/OL]. Journal of Third Military Medical University 1-9(2022) DOI： 10.16016/j.1000-5404.201908194-.

摘要

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（

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（

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（

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。

Abstract

Objective

To observe the effects of endogenous nestin overexpression on the proliferation and migration and transforming growth factor β （TGF-β1）-induced expressions 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 （

0.05）， and nestin overexpression significantly promoted the growth and migration of NIH3T3 cells （

0.05）. Nestin overexpression obviously inhibited the expression of collagen and the activation of MAPK in TGF-β1-induced cells （

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.

关键词

NestinNIH3T3CRISPR/SAM增殖迁移MAPK

Keywords

nestinNIH3T3CRISPR/SAMproliferationmigrationmitogen-activated protein kinase

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