富含半胱氨酸蛋白61对转化生长因子β1活化肾脏成纤维细胞的抑制作用及机制

Effect and mechanism of cysteine-rich protein 61 on transforming growth factor -β1-activated renal fibroblasts

目的观察富含半胱氨酸蛋白61（Cyr61）在转化生长因子p1（TGF-β1）活化的肾成纤维细胞（NRK-49F）中的表达，探索Cyr61的作用及可能机制。方法（1）0．0、0．5、1．0、2．0、5．0μg／LTGF．B1刺激活化NRK-49F细胞，Western印迹检测细胞Cyr61蛋白表达，CCK-8法检测细胞增殖活性。（2）质粒转染构建过表达及低表达Cyr61的NRK-49F细胞，分为对照组（转染空质粒组）、过表达组和低表达组。CCK-8法检测3组细胞培养24、48、72h的增殖活性。5．0μg／LTGF-β1诱导活化3组细胞，CCK-8法检测细胞增殖活性，流式细胞术分析细胞周期，实时荧光定量PCR检测纤维化标志物（Co11α1、Col3α1、MMP9、MMP13）、细胞衰老信号通路分子（p53、p21、Rb、p16）的mRNA表达，Western印迹检测C013、MMP9蛋白的表达。结果（1）与0．0μg/L TGF-β1组比较，0．5、1．0、2．0、5．0μg／LTGF-β1组NRK-49F细胞的增殖活性均增加（均P〈0．05），1．0μg／L组Cyr61蛋白表达降低，5．0μg／L组Cyr61蛋白表达升高（均P〈0．05）。（2）Cyr61过表达组NRK-49F细胞24、48、72h的增殖活性均低于对照组（均P〈0．05），且呈时间依赖性。（3）TGF-β1刺激后，与对照组比较，Cyr61过表达组NRK-49F细胞增殖活性降低，促纤维化因子Col1α1和Col3α1表达减少，同时抗纤维化因子MMP9和MMP13的表达增加，周期停滞在G1期的细胞比例增加，细胞衰老信号通路基因p53、p21和RbmRNA表达增加（均P〈0．05），而Cyr61低表达组上述效应正好相反（均P〈0．05）；3组细胞p16基因mRNA表达差异无统计学意义（P〉0．05）。结论Cyr61抑制肾成纤维细胞增殖及纤维化表型分泌，从而减缓肾脏纤维化进程，p53／p21／Rb细胞衰老信号通路可能参与Cyr61的抑纤维化作用。

Objective To observe the expression of cysteine- rich protein 61 （Cyr61） in transforming growth factor -β1 （TGF- β1）-activated renal fibroblasts （NRK-49F）, and to explore its effect and mechanism. Methods （1） NRK- 49F ceils were activated by TGF- β1 with different concentrations （0.0, 0.5, 1.0, 2.0, 5.0 μg/L）. Western blotting was used to detect the expression of Cyr61 protein, and CCK-8 assay was used to test the proliferative activity of NRK-49F cells. （2） NRK- 49F cells with low expression and over expression of Cyr61 were established by plasmid transfection. The cells were divided into control group （null vector transfection）, over-expression group and low- expression group. The proliferation was discovered by CCK-8 assay after 24, 48 and 72 h. Further, 5.0 μg/L TGF-β1 activated these three groups. The proliferation was also discovered by CCK-8 assay and the cell cycle was analyzed by flow cytometry. The mRNA expressions of fibrosis markers （Col1α1, Col3α1, MMPg, MMP13） and factors of cell senescence signal pathway （p53, p21, Rb, p16） were ascertained by real time PCR, and the protein expressions of Co13 and MMP9 were detected by Western blotting. Results （1） Compared with 0.0 Ixg/L TGF- β1 group, the proliferation of NRK- 49F cells was enhanced in 0.5, 1.0, 2.0 and 5.0 μg/L TGF- β1 groups （all P〈 0.05）, while the expression of Cyr61 protein was decreased in 1.0 μg/L group and increased in 5.0 μg/L group （all P 〈 0.05）. （2） The proliferation of over-expression group was lower than that of control group after 24, 48 and 72 h （all P 〈 0.05）, which was in a time- dependent manner. （3） Compared with control group activated by TGF-β1, the over-expression group expressed less fibrosis factors （Col1α1 and Col3α1） and more anti- fibrosis factors （MMP9 and MMP13） with decreased proliferation （all P〈 0.05）. Simultaneously, the proportion of cells bogged down in G1 phases, as well as the expressions of p53, p21 and Rb mRNA increased （all P 〈 0.05）. The above effects of low- expression group were just opposite to over-expression group. Moreover, there was no significant difference in the expression of p16 gene among the three groups （P 〉 0.05）. Conclusions Cyr61 can curb the proliferation and fibrotic phenotypes of fibroblasts, thereafter slowing down the process of renal fibrosis. The p53/p21/Rb interrelated cell senescence signal pathway may be involved in the anti-fibrosis process.