慢病毒介导的SPARC基因沉默对人瘢痕疙瘩成纤维细胞增殖调亡的影响  

Effect of lentivirus-mediated SPARC gene silencing on proliferation and apoptosis of human keloid fibroblasts

作　　者：李茜 李心怡[1] 李小静[1] 朱晓璇 丁以春 Li Xi;Li Xinyi;Li Xiaojing;Zhu Xiaoxuan;Ding Yichun(Dept of Plastic and Cosmetic Surgery,The First Affiliated Hospital of Anhui Medical University,Hefei 230022)

机构地区：[1]安徽医科大学第一附属医院整形外科,合肥230022

出　　处：《安徽医科大学学报》2022年第6期944-948,共5页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金(编号:9021548201)。

摘　　要：目的 观察慢病毒介导的SPARC基因沉默对人瘢痕疙瘩成纤维细胞增殖、凋亡及细胞周期的影响并初步探讨其影响机制。方法 将携带SPARC shRNA的慢病毒载体(LV2N-shRNA-SPARC,sh-SPARC组)及空载体(LV2-NC,sh-NC组)转染人瘢痕疙瘩成纤维细胞(HKF),以未作任何处理的成纤维细胞为空白对照组,应用实时荧光定量PCR(qRT-PCR)和Western blot分别检测SPARC mRNA和蛋白的表达。四甲基偶氮噻唑蓝比色法(MTT)检测各组细胞的增殖情况;流式细胞仪法测定HKF细胞周期及细胞凋亡;Western blot检测各组细胞中TGF-β1及TβRⅠ的表达。结果 ①SPARC-shRNA慢病毒载体感染HKF后,sh-SPARC组细胞中SPARC mRNA和蛋白表达量明显低于sh-NC组和空白对照组,差异有统计学意义(P<0.05);②MTT结果显示,与对照组相比,sh-SPARC组细胞增殖减慢(P<0.05);③流式细胞术结果显示,sh-SPARC组成纤维细胞进入S期的比例低于sh-NC组和空白对照组(P<0.05);细胞凋亡率较对照组增加(P<0.05);④Western blot结果显示,sh-SPARC组TGF-β1及TβRⅠ的蛋白表达水平明显降低。结论 SPARC基因沉默能抑制瘢痕疙瘩成纤维细胞增殖,阻滞细胞周期进程,促进凋亡,其机制可能与TGF-β信号通路的下调有关。Objective To investigate the effect of secreted protien acidic and rich in cysteine(SPARC)gene onthe proliferation and apoptosis of human keloid fibroblasts and its mechanism in vitro.Methods The lentiviral vector carrying SPARC shRNA(LV2N-shRNA-SPARC,sh-SPARC group)and empty plasmid(LV2-NC,sh-NC group)were transfected into human keloid fibroblasts(HKF)respectively.Real-time PCR(qRT-PCR)and Western blot were performed to examine the expression of SPARC mRNA and protein in HKF.MTT assay was used to estimate the cell proliferation.The cell cycle and apoptosis of HKF were detected by flow cytometry.The expression of TGF-β1 and TβRⅠproteins in HKF were detected by Western blot.Results①After transfection of SPARC shRNA lentiviral vector,the expression of SPARC mRNA and protein in HKF were significantly down-regulated compared with those in the control groups(P<0.05).②MTT assay showed that the proliferation of sh-SPARC groups decreased compared with the control groups(P<0.05).③Flow cytometry results showed that the S phase cell ratios of sh-SPARC groups were reduced while the apoptosis rates increased compared with the control groups(P<0.05).④Western blot showed that the protein expression levels of TGF-β1 and TβRⅠsignificantly decreased in sh-SPARC groups.Conclusion SPARC gene silencing inhibits the proliferation blocks cell cycle progression and promotes cell apoptosis in HKF,which may be related to the down-regulation of the TGF-βsignaling pathway.

关 键 词：慢病毒 富含半胱氨酸的酸性分泌蛋白 瘢痕疙瘩 成纤维细胞 转化生长因子-Β 

分 类 号：R619.6[医药卫生—外科学]