水蛭素对TGF-β_(1)诱导的肺成纤维细胞增殖及转分化的影响研究  

Hirudin inhibits TGF-β_(1) induced proliferation and transdifferentiation of lung fibroblasts

作　　者：王慧 贺彬 黄文杰 刘理静 柯灿 向粤 WANG Hui;HE Bin;HUANG Wenjie;LIU Lijing;KE Can;XIANG Yue(Hunan University of Medicine,Huaihua 418000,Hunan,China;Changsha Social Work College,Changsha 410000,Hunan,China)

机构地区：[1]湖南医药学院,湖南怀化418000 [2]长沙民政职业技术学院,湖南长沙410000

出　　处：《现代中西医结合杂志》2022年第23期3252-3256,共5页Modern Journal of Integrated Traditional Chinese and Western Medicine

基　　金：2019年湖南省教育厅优秀青年资助项目(19B406);2020年国家级大学生创新创业训练计划项目(S202012214009);2022年湖南省自然科学基金青年项目(2022JJ40293)。

摘　　要：目的研究水蛭素对转化生长因子β_(1)(TGF-β_(1))诱导小鼠胚肺成纤维细胞(NIH3T3)向肌成纤维细胞转化的影响及相关机制。方法培养NIH3T3细胞,分别用浓度为0.3 ATU/mL、0.6 ATU/mL、1.2 ATU/mL、2.4 ATU/mL、4.8 ATU/mL、9.6 ATU/mL水蛭素处理NIH3T3细胞24 h,采用CCK-8试剂检测细胞活力,根据CCK-8结果选择浓度为1.2 ATU/mL、2.4 ATU/mL、4.8 ATU/mL水蛭素进行实验。实验分为6组:空白对照组细胞常规培养,水蛭素组细胞加入4.8 ATU/mL水蛭素培养,TGF-β_(1)组细胞加入10 ng/mL TGF-β_(1)培养,TGF-β_(1)+水蛭素1.2 ATU/mL组、TGF-β_(1)+水蛭素2.4 ATU/mL组、TGF-β_(1)+水蛭素4.8 ATU/mL组分别加入相应浓度水蛭素后30 min再加入10 ng/mL TGF-β_(1)培养。采用CCK-8试剂检测各组细胞活力,qRT-PCR和Western blot法分别检测各组细胞中Ⅰ型胶原蛋白(CollagenⅠ)、α-平滑肌肌动蛋白(α-SMA)mRNA及蛋白表达量。结果4.8 ATU/mL水蛭素对细胞无毒性作用,1.2 ATU/mL、2.4 ATU/mL、4.8 ATU/mL水蛭素能抑制TGF-β_(1)诱导所致的NIH3T3细胞活力升高,且2.4 ATU/mL、4.8 ATU/mL水蛭素抑制作用更显著。TGF-β_(1)组细胞中CollagenⅠ、α-SMA mRNA及蛋白表达量均明显高于空白对照组(P均<0.05),TGF-β_(1)+水蛭素2.4 ATU/mL组、TGF-β_(1)+水蛭素4.8 ATU/mL组细胞中CollagenⅠ、α-SMA mRNA及蛋白表达量均明显低于TGF-β_(1)组(P均<0.05)。结论水蛭素可以抑制TGF-β_(1)诱导的NIH3T3细胞增殖和肺成纤维细胞向肌成纤维细胞表型转化。Objective It is to explore the effects of hirudin on the differentiation of NIH3T3 cells into myofibroblasts induced by transform growth factorβ_(1)(TGF-β_(1))and potential mechanism.Methods NIH3T3 cells were cultured and respectively treated with(0.3,0.6,1.2,2.4,4.8,9.6 ATU/mL)hirudin for 24 h,the cell viability was detected by CCK8 assay.According to CCK-8 results,the concentrations of 1.2 ATU/mL,2.4 ATU/mL and 4.8 ATU/mL hirudin were selected for the experiment.The experiment was divided into 6 groups:the cells in the blank control group were cultured conventionally,the cells in the hirudin group were cultured with 4.8 ATU/mL hirudin,the cells in the TGF-β_(1) group were cultured with 10 ng/mL TGF-β_(1),the TGF-β_(1)+hirudin 1.2 ATU/mL group,the TGF-β_(1)+hirudin 2.4 ATU/mL group,the TGF-β_(1)+hirudin 4.8 ATU/mL group were cultured with 10 ng/mL TGF-β_(1)30 min after the addition of the corresponding concentration of hirudin.The cell viability of each group was detected by CCK-8 reagent,the expression of mRNA and protein of collagen type I(Collagen)andα-smooth muscle actin(α-SMA)in the cells of each group were detected by qRT-PCR and Western blot,respectively.Results 4.8 ATU/mL hirudin had no toxic effect on the cells,1.2 ATU/mL 2.4 ATU/mL,4.8 ATU/mL hirudin could inhibit the increase of NIH3T3 cell viability caused by TGF-β_(1) induction,and the inhibitory effect of 2.4 ATU/mL and 4.8 ATU/mL hirudin was more significant.The expression of Collagen I,α-SMA mRNA and protein in the cells of TGF-β_(1) group were significantly higher than those of the blank control group(all P<0.05),the expression of CollagenⅠ,α-SMA mRNA and protein in the cells of the TGF-β_(1)+hirudin 2.4 ATU/mL group and TGF-β_(1)+hirudin 4.8 ATU/mL group were significantly lower than those in the TGF-β_(1) group(all P<0.05).Conclusion Hirudin can inhibit the cell viability of NIH3T3 cells and phenotypic transformation of lung fibroblasts into myofibroblasts induced by TGF-β_(1).

关 键 词：肺纤维化 水蛭素 成纤维细胞 转化生长因子β_(1) 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]