热休克蛋白72调节成纤维细胞增殖的体外实验研究  

In vitro experimental study of Heat shock protein 72 to regulate fibroblast proliferation

作　　者：方雪 孙商益 朱庆贤 刘航航 王正 张旺 谭微 沈超[3] 王欣[1] Fang Xue;Sun Shangyi;Zhu Qingxian;Liu Hanghang;Wang Zheng;Zhang Wang;Tan Wei;Shen Chao;Wang Xin(Department of Orthopedics Trauma and Microsurgery,Zhongnan Hospital of Wuhan University,Wuhan 430071,China;Wuhan University Second Clinical College,Wuhan 430071,China;College of Life Sciences,Wuhan University,Wuhan 430072,China)

机构地区：[1]武汉大学中南医院创伤与显微骨科,武汉430071 [2]武汉大学第二临床学院,武汉430071 [3]武汉大学生命科学学院,武汉430072

出　　处：《中华实验外科杂志》2024年第11期2460-2463,共4页Chinese Journal of Experimental Surgery

基　　金：湖北省骨外科学科能力建设项目(YYXKNL2022009)。

摘　　要：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) mRNA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�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 fibroblast cells overexpressing HSP72 plasmid).Mouse primary fibroblasts(L929)and human primary fibroblasts(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 expression 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.ResultsFollowing transfection with the HSP72 overexpression 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 transfection 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-transfection(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 t

关 键 词：热休克蛋白72 信号转导和转录激活因子3 信号通路 成纤维细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]