慢病毒介导的DKK3过表达对增生性瘢痕成纤维细胞凋亡的影响  

Effect of lentivirus-mediated DKK3 overexpression on apoptosis of hypertrophic scar fibroblasts

作　　者：柯晓苹[1] 张启国[1] 林维嘉[1] 蔡良奇[1] KE Xiao-ping;ZHANG Qi-guo;LIN Wei-jia;CAI Liang-qi(Department of Dermatology, First Affiliated Hospital of Xiamen University, Xiamen 361000, China)

机构地区：[1]厦门大学附属第一医院皮肤科

出　　处：《中国病理生理杂志》2019年第8期1508-1513,共6页Chinese Journal of Pathophysiology

基　　金：厦门市2017科技惠民项目(No.3502Z20174061)

摘　　要：目的:探讨慢病毒介导的DKK3过表达对增生性瘢痕成纤维细胞凋亡的影响。方法:体外分离培养人增生性瘢痕成纤维细胞,将细胞分成对照(control)组、阴性对照慢病毒感染(vector)组和pcDNA3.1-DKK3慢病毒感染(DKK3)组,RT-qPCR和Western blot检测过表达效果,MTT检测细胞活力,流式细胞术检测细胞凋亡,Western blot检测细胞中激活型caspase-9(cleaved caspase-9)、Ⅱ型胶原(COL II)、Ⅰ型胶原(COL I)和激活型caspase-3(cleaved caspase-3)及胞质、线粒体中细胞色素C(cytochrome C)蛋白表达变化。结果:pcDNA3.1-DKK3感染后增生性瘢痕成纤维细胞中DKK3 mRNA和蛋白表达水平升高(P<0.05)。DKK3组增生性瘢痕成纤维细胞活力降低,细胞凋亡率升高,细胞中cleaved caspase-9和cleaved caspase-3蛋白水平升高,COL II和COL I蛋白水平降低,与vector细胞比较,胞质中cytochrome C蛋白水平升高,线粒体中cytochrome C蛋白水平降低(P<0.05)。结论:慢病毒介导的DKK3过表达能够诱导增生性瘢痕成纤维细胞凋亡,并减少细胞合成胶原蛋白。AIM: To investigate the effect of lentivirus-mediated DKK3 overexpression on the apoptosis of hypertrophic scar fibroblasts. METHODS: Human hypertrophic scar fibroblasts were isolated and cultured in vitro. The cells were divided into control group, vector(negative control lentivirus infection) group and DKK3(pcDNA3.1-DKK3 lentivirus infection) group. The overexpression effect was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein levels of cleaved caspase-9, collagen type II(COL II), COL I and cleaved caspase-3 in the cells, and cytochrome C in the cytoplasm and mitochondrion were detected by Western blot. RESULTS: After transfection with pcDNA3.1-DKK3, the expression of DKK3 at mRNA and protein levels was increased in the hypertrophic scar fibroblasts(P<0.05). The viability of hypertrophic scar fibroblasts in DKK3 group was decreased, and the apoptotic rate was increased. The protein levels of cleaved caspase-9 and cleaved caspase-3 were increased in the cells, and the protein levels of COL II and COL I were decreased. The protein level of cytochrome C was increased in the cytoplasm, while the protein level of cytochrome C in the mitochondrion decreased. Compared with vector group, these differences were statistically significant(P<0.05). CONCLUSION: Lentivirus-mediated DKK3 overexpression induces apoptosis and reduces collagen synthesis in the fibroblasts from hypertrophic scars.

关 键 词：DKK3基因 增生性瘢痕成纤维细胞 细胞凋亡 胶原蛋白 细胞色素C 

分 类 号：R751[医药卫生—皮肤病学与性病学] R363.2[医药卫生—临床医学]