结缔组织生长因子反义寡核苷酸体外对人增生性瘢痕成纤维细胞凋亡的影响

Effects of antisense oligonucleotide of connective tissue growth factor on apoptosis of hypertrophic scar fibroblasts and the related mechanism

目的:观察结缔组织生长因子(CTGF)反义寡核苷酸(ASODN)体外对人增生性瘢痕组织中成纤维细胞凋亡的影响并探讨其可能的作用机制。方法:体外培养的人增生性瘢痕组织中成纤维细胞,分为CTGF-ASODN脂质体治疗组(ASODN treatment,AT)、空白脂质体对照组(liposome control,LC)和空白对照组(control,C),荧光显微镜观察各组CTGF-ASODN时相性分布,流式细胞术检测各组凋亡率随时相的变化,观察Fas受体表达;RT-PCR检测各组CTGF、Fas、bcl-2 mRNA的表达。砖果:AT组细胞转染24 h后胞质内可见有大量黄绿色荧光,呈离散型、点状分布,而LC和C组成纤维细胞内未见荧光。转染24 h后LC组和C组成纤维细胞的凋亡率及Fas受体阳性表达率无显著差异;而AT组凋亡率及Fas受体阳性表达率明显增加,明显高于LC组和C组(P<0.01)。RT-PCR结果表明,转染组CTGF mRNA相对表达量明显低于空白对照组(P<0.05),Fas mRNA表达明显高于空白对照组(P<0.01),转染组bcl-2 mRNA与空白对照组无显著差异。结论:CTGF反义寡核苷酸体外能促进人增生性瘢痕成纤维细胞凋亡,可能与其上调Fas mRNA及其蛋白表达有关。

Objective： To explore the effects of antisense oligonucleotide （ASODN） of connective tissue growth factor （CTGF） on the apoptosis of human hypertrophic scar fibroblasts （HSF）and the related mechanisms. Methods： The hypertrophic scar fibroblasts were cultured in vitro and were divided into the following 3 groups： CTGF-ASODN-treated group, liposome control group and blank control group. The distribution of CTGF-ASODN in HSF of different phases was observed under fluorescent microscope in 3 groups. Flow cytometer was employed to detect the apoptotic rate of HSF and the expression of Fas. The expression of CTGF, Fas and bcl-2 mRNA in HSF was assessed by RT-PCR. Results： 24 h after transfection,large quantity of yellow-green fluorescence was seen in the HSF cytoplasm of CTGF-ASDON group in a scattered or doted manner, but not found in that of the other 2 groups. The apoptosis rate of HSF in CTGF-ASDON group was significantly higher than those in the other 2 groups （P〈0. 01）. RT-PCR result showed that CTGF mRNA expression in transfection group was significantly lower than that in the blank control group（P〈0.05） and the Fas mRNA expression was significantly higher than that in the blank control group（P〈0.01）;the expression of bcl-2 mRNA had no significant difference between the 2 groups. Conclusion： CTGF-ASODN can promote the apoptosis of HSF in vitro, which might be related to the up-regulation Fas gene.