In vitro experimental study of Heat shock protein 72 to regulate fibroblast proliferation
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目的 探究热休克蛋白72(HSP72)调节成纤维细胞增殖的机制.方法 本实验共分为3组,空白对照组(未经任何处理的原代成纤维细胞组),转染HSP72空白质粒组(转染HSP72空白质粒原代成纤维细胞组),过表达HSP72质粒组(过表达HSP72质粒原代成纤维细胞组).将小鼠原代成纤维细胞(L929)以及人原代成纤维细胞(MRC5)(均购自武汉大学保藏中心)转染HSP72质粒后,通过实时荧光定量聚合酶链反应(RT-qPCR)分析过表达HSP72(OE-HSP72)组,转染空白质粒(OE-NC)组和空白对照(Control)组中纤连蛋白(Fibronectin)和Ⅰ型胶原蛋白(Collagen Ⅰ)mR-NA表达情况.采用细胞计数试剂盒(CCK-8)以及5-乙炔基-2'-脱氧尿苷(EdU)染色检测过表达HSP72的L929细胞增殖能力.采用蛋白质印迹法(Western blot)分析过表达HSP72的L929细胞中信号转导和转录激活因子3(STAT3)以及STAT3磷酸化的表达情况.采用单因素方差分析多组间的差异性.结果 L929转染过表达HSP72质粒后,OE-HSP72组中细胞外基质相关基因Collagen Ⅰ表达量(0.692±0.063)低于 Control 组和 OE-NC 组(1.000±0.157、1.000±0.029,F=9.658,P＜0.05),Fibronectin 表达量(0.627±0.066)低于 Control 组和 OE-NC 组(1.000±0.047、1.000±0.051,F=45.100,P＜0.05).CCK-8 实验结果显示,L929 转染 HSP72 质粒48 h 后,OE-HSP72 组中细胞生长活力[(71.729±1.303)％]低于 Control 组和 OE-NC 组[(100.000±6.443)％、(101.189±7.660)％,F=24.560,P＜0.01].EdU染色结果显示,L929转染过表达HSP72质粒48 h后,OE-HSP72 组细胞增殖(0.489±0.022)低于 Control 组和 OE-NC 组(0.586±0.048、0.600±0.054,F=9.479,P＜0.05).Western blot 结果显示在 L929 转染过表达 HSP72 质粒后,OE-HSP72 组STAT3 蛋白表达(1.431±0.046)高于 Control 组和 OE-NC 组(0.979±0.047、0.960±0.045,F=101.700,P＜0.01),OE-HSP72 组 p-STAT3 蛋白表达(0.616±0.024)低于 Control 组和 OE-NC 组(0.848±0.045、0.847±0.030,F=46.160,P＜0.01).结论 上调 HSP72 的表达能够抑制成纤维细胞增殖以及细胞外基质基因的表达.
Objective To explore whether heat shock protein 72(HSP72)can regulate fibroblast proliferation.Methods The experiment was divided into three groups:the blank control group(primary fibroblast cells without any treatment),the HSP72 empty plasmid transfection group(primary fibroblast cells transfected with HSP72 empty plasmid),and the HSP72 overexpression plasmid group(primary fibro-blast cells overexpressing HSP72 plasmid).Mouse primary fibroblasts(L929)and human primary fibro-blasts(MRC5),both purchased from Wuhan University's preservation center,were transfected with HSP72 plasmid.The expression of fibronectin and collagen Ⅰ mRNA in the overexpressed HSP72 group(OE-HSP72),empty plasmid control group(OE-NC),and blank control group(Control)was analyzed using real-time quantitative PCR(RT-qPCR).Cell proliferation in HSP72-overexpressing L929 cells was assessed using the cell counting kit-8(CCK-8)and 5-ethynyl-2'-deoxyuridine(EdU)staining.The ex-pression of signal transducer and activator of transcription 3(STAT3)and its phosphorylated form(p-STAT3)in HSP72-overexpressing L929 cells was analyzed through Western blotting.One-way ANOVA was used for statistical analysis among multiple groups.Results Following transfection with the HSP72 over-expression plasmid,the expression level of Collagen Ⅰ in the OE-HSP72 group(0.692±0.063)was lower than that in the Control and OE-NC groups(1.000±0.157,1.000±0.029,F=9.658,P＜0.05),and the expression level of Fibronectin(0.627±0.066)was lower than that in the Control and OE-NC groups(1.000±0.047,1.000±0.051,F=45.100,P＜0.05).CCK-8 results showed that 48 hours after trans-fection with the HSP72 plasmid,the cell viability in the OE-HSP72 group[(71.729±1.303)％]was lower than in the Control and OE-NC groups[(100.000±6.443)％,(101.189±7.660)％,F=24.560,P＜0.01].EdU staining indicated lower cell proliferation in the OE-HSP72 group 48 hours post-transfec-tion(0.489±0.022)compared to the Control and OE-NC groups(0.586±0.048,0.600±0.054,F=9.479,P＜0.05).Western blotting results revealed that STAT3 expression in the OE-HSP72 group(1.431±0.046)was higher than in the Control and OE-NC groups(0.979±0.047,0.960±0.045,F=101.700,P＜0.01),whereas p-STAT3 expression(0.616±0.024)was lower than in the Control and OE-NC groups(0.848±0.045,0.847±0.030,F=46.160,P＜0.01).Conclusion Upregulation of HSP72 expression can inhibit fibroblast proliferation and extracellular matrix gene expression.
Heat shock protein 72Signal transduction and transcriptional activator 3Signa-ling pathwayFibroblast
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