ATG5通过细胞自噬调控Ⅱ型胶原表达的实验研究

邓琳; 先梦麟; 杨玉有; 耿娜娜; 邝彪; 郭风劲

doi:10.16016/j.2097-0927.202302029

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ATG5通过细胞自噬调控Ⅱ型胶原表达的实验研究

基础医学 | 更新时间：2026-01-09

- ATG5通过细胞自噬调控Ⅱ型胶原表达的实验研究
- ATG5 regulates type Ⅱ collagen expression by modulation of autophagy
- 陆军军医大学学报 2023年45卷第17期页码：1828-1837
- 作者机构：
  
  1.400016 重庆，重庆医科大学基础医学院细胞生物学与遗传学教研室
  2.400010 重庆，重庆医科大学附属第二医院骨科
- 作者简介：
  
  [郭风劲，E-mail: guo.fengjin@cqmu.edu.cnGUO Fengjin, E-mail: guo.fengjin@cqmu.edu.cn
- 基金信息：
  
  国家自然科学基金面上项目(81871769);重庆市博士后研究项目特别资助(2020-379-46)
- DOI：10.16016/j.2097-0927.202302029
  中图分类号： R394-33;R394.3;R684.302
- 收稿：2023-02-07，
  
  修回：2023-03-03，
  
  纸质出版：2023-09-15
- 稿件说明：
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邓琳, 先梦麟, 杨玉有, 等. ATG5通过细胞自噬调控Ⅱ型胶原表达的实验研究[J]. 陆军军医大学学报, 2023,45(17):1828-1837.

Lin DENG, Menglin XIAN, Yuyou YANG, et al. ATG5 regulates type Ⅱ collagen expression by modulation of autophagy[J]. Journal of Army Medical University, 2023, 45(17): 1828-1837.
邓琳, 先梦麟, 杨玉有, 等. ATG5通过细胞自噬调控Ⅱ型胶原表达的实验研究[J]. 陆军军医大学学报, 2023,45(17):1828-1837. DOI： 10.16016/j.2097-0927.202302029.

Lin DENG, Menglin XIAN, Yuyou YANG, et al. ATG5 regulates type Ⅱ collagen expression by modulation of autophagy[J]. Journal of Army Medical University, 2023, 45(17): 1828-1837. DOI： 10.16016/j.2097-0927.202302029.

摘要

目的

探究自噬相关蛋白ATG5通过影响自噬功能调控软骨细胞中Ⅱ型胶原的表达水平。

方法

GEO数据库下载13例人软骨组织样本的转录组数据分析ATG5和COL2A1表达的相关性。雷帕霉素(Rapamycin

Rapa)和巴佛洛霉素A1(Bafilomycin A1

BafA1)处理人软骨细胞C28/I2

Western blot及免疫荧光检测Ⅱ型胶原(CollagenⅡ

COL2)的表达水平。构建人源COL2A1启动子质粒

双荧光素酶报告实验检测过表达ATG5对COL2A1启动子转录活性的影响。mRFP-GFP-LC3自噬双标腺病毒动态监测过表达和敲低ATG5后自噬流的变化。Western blot检测过表达或敲低ATG5后Rapa及BafA1对COL2、LC3Ⅱ/LC3Ⅰ表达水平的影响。

结果

GEO数据库相关性分析结果显示在人软

骨组织中ATG5和COL2A1的mRNA表达呈现正相关; Rapa和BafA1分别显著促进和抑制C28/I2细胞COL2的表达(

0.05);成功构建人源COL2A1启动子质粒

并用双荧光素酶报告实验证实过表达ATG5上调COL2A1启动子转录活性(

0.01);自噬双荧光病毒处理后

过表达ATG5组LC3-GFP荧光淬灭(

0.01)

自噬激活; 敲低ATG5后

LC3-GFP荧光增强(

0.05)

自噬抑制; Rapa促进过表达ATG5上调的COL2和LC3Ⅱ/LC3Ⅰ的表达(

0.05)

BafA1显示与Rapa相反的作用(

0.05)。

结论

过表达ATG5上调Ⅱ型胶原的表达

且ATG5调控Ⅱ型胶原的表达依赖于细胞自噬。

Abstract

Objective

To explore the regulative effect of autophagy-related protein ATG5 on the expression of type Ⅱ collagen in human chondrocytes through affecting autophagy function.

Methods

Transcriptome data of 13 human cartilage tissue samples were downloaded from GEO database to analyze the correlation between ATG5 and COL2A1 expression.Western blotting and immunofluorescence assay were used to detect the expression of type Ⅱ collagen (Collagen Ⅱ

COL2) in human chondrocytes C28/I2 after rapamycin (Rapa) and bafilomycin A1(BafA1) treatment.Human COL2A1 promoter plasmid was constructed

and dual-luciferase reporter assay was applied to detect the effect of overexpression of ATG5 on COL2A1 promoter activity.mRFP-GFP-LC3 autophagy dual fluorescent virus was used to dynamically monitor the changes in autophagic flux of C28/I2 cells with overexpression and knockdown of ATG5.Western blotting was used to detect the effects of Rapa and BafA1 on COL2

LC3Ⅱ/LC3Ⅰ expression levels after overexpression or knockdown of ATG5.

Results

The results of correlation analysis showed a positive correlation between the mRNA expression of ATG5 and COL2A1 in human chondrocytes samples.Rapa and BafA1 significantly promoted and inhibited the expression of COL2 in C28/I2 cells (

0.05)

respectively.The human COL2A1 promoter plasmid was successfully constructed

and

the transcriptional activity of the COL2A1 promoter was up-regulated by the overexpression of ATG5(

0.01).Treatment with autophagy dual-fluorescence virus resulted in LC3-GFP fluorescence quenching after overexpression of ATG5(

0.01)

suggesting autophagy activation; and a greater fluorescence intensity of LC3-GFP was observed after knockdown of ATG5(

0.05)

demonstrating autophagy inhibition.Rapa obviously promoted the expression of COL2 and LC3Ⅱ/LCⅠ

which were up-regulated by ATG5(

0.05)

but BafA1 showed the opposite effect of Rapa (

0.05).

Conclusion

Overexpression of ATG5 up-regulates the expression of type Ⅱ collagen

and the regulation of ATG5 on the expression of type Ⅱ collagen depends on autophagy in chondrocytes.

关键词

Keywords

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