线粒体融合素基因2对人骶韧带成纤维细胞增殖及功能影响.docVIP

线粒体融合素基因2对人骶韧带成纤维细胞增殖及功能影响 　　【第一作者简介】陈华云（1979-），男，主治医师、博士，主要从事女性盆底疾病相关的临床诊治与研究工作。 　　【摘要】目的:研究外源性线粒体融合素基因-2（Mfn2）表达对骶韧带成纤维细胞的增殖及其合成前胶原蛋白功能的影响。方法:取新鲜骶韧带组织，用组织块法在体外培养出成纤维细胞。将第2~4代细胞分组：转染LV-Mfn2-GFP组（Mfn2+组），转染LV-GFP组（Mfn2-组）和未转染组（对照组）。流式细胞仪检测细胞绿色荧光蛋白（GFP）表达，RT-PCR和Western blot检测Mfn2表达。细胞计数和cck-8法检测Mfn2对细胞增殖的影响，流式细胞术检测细胞周期分布。RT-PCR和Western blot检测细胞前胶原1A1/1A2/3A1的表达。结果:转染慢病毒的成纤维细胞GFP蛋白表达率73??2%~79??1%，转染Mfn2+组成纤维细胞稳定高表达Mfn2蛋白，显著高于Mfn2-组和对照组（P＜0??05，both）。cck-8实验提示，转染表达Mfn2基因96h后，成纤维细胞增殖明显受到抑制，Mfn2+组细胞数显著低于Mfn2-组和未转染组（P＜0??05 both）；DNA直方图分析法测定显示转染Mfn2基因的细胞大部分停滞于G0/G1期，Mfn2+组G0/G1期细胞比例(60??7%±3??26%)，显著高于Mfn2-组（43??2%±3??32%）和对照组（40??8%±2??28%），（P＜0??05，both）。半定量RT-PCR和Western blot示Mfn2+组成纤维细胞合成表达前胶原1A1/1A2/3A1显著低于Mfn2-组和对照组（P＜0??05，all）。结论:Mfn2基因在体外转染成纤维细胞后，抑制细胞增殖并使成纤维细胞前胶原蛋白表达下降，可能是导致骶韧带细胞外基质减少，影响韧带纤维数量和质量的重要原因。 　　【关键词】线粒体融合素基因-2；成纤维细胞；前胶原蛋白；增殖；细胞周期 　　Effects of mitofusin gene-2 on proliferation and function of human uterosacral ligament fibroblastCHEN Huayun, LU Ye, LIAO Qinping△. Department of Obstetrics and Gynecology, Peking University First Hospital, Beijing 100034, China 　　【Abstract】Objectives: To investigate the effect of exogenous mitofusin gene -2 (Mfn2) expression on the proliferation and procollagen of human uterosacral ligament fibroblast. Methods: Taking fresh uterosacral ligament tissue from the removal womb, the method of explant culture was used to get fibroblast cell. The 2nd to 4th generations of fibroblasts were divided into transfected LV-Mfn2-GFP, transfected LV-GFP group and non-transfected group. The green fluorescent protein (GFP) was observed by flow cytometry (FCM). The mRNA and protein expressions of Mfn2 and procollagen 1A1/1A2/3A1 in cells were detected by RT-PCR and western blot assay. Cell proliferation was measured by cck-8 assay. Cell cycle of fibroblasts was observed by FCM. Results: The GFP protein expression rate of fibroblasts tansfected with LV-Mfn2-GFP or LV-GFP was 73??2-79??1%, and Mfn2 gene was stably expressed in transfect