盆底功能障碍性疾病患者阴道壁成纤维细胞受力后生物学功能的变化

Biological function alternations with stretch in vaginal wall fibroblasts from patients with pelvic floor dysfunction

目的:研究盆底功能障碍性疾病(PFD)阴道壁成纤维细胞在受力情况下,Ⅰ型胶原(colⅠ)、Ⅲ型胶原(colⅢ)、转化生长因子β1(TGF-β1)和基质金属蛋白酶-1(MMP-1)基因表达水平的变化,探讨其与PFD发病的关系。方法:选取2009年10月至2010年5月在河北医科大学第二医院妇产科接受手术治疗的12例PFD患者的阴道壁组织,用组织块法对阴道壁成纤维细胞进行原代培养,取2～5代稳定的传代阴道壁成纤维细胞,采用Flexcell-4000细胞柔性基底拉伸系统进行0.3Hz、12%、正弦波的拉伸应变加载实验,以未加载力的静态细胞作为对照组。用RT-PCR反应检测对照组及受力后4h、15h、24h colⅠ、colⅢ、TGF-β1、MMP-1基因的表达水平。结果:对照组colⅠmRNA的表达水平为1.178±0.071,受力后4h、15h、24h表达分别为1.092±0.038、1.198±0.031、1.120±0.098,不同时间colⅠmRNA水平比较,差异有统计学意义(P=0.035);对照组colⅢ表达为0.673±0.029,受力后4h、15h、24h分别为0.313±0.019、0.504±0.033、0.853±0.210,不同时间colⅢmRNA水平比较,差异有统计学意义(P=0.015);对照组TGF-β1表达为0.852±0.039,受力后4h、15h、24h分别为0.673±0.052、0.897±0.051、0.895±0.015,不同时间TGF-β1 mRNA水平比较,差异有统计学意义(P=0.0001)。对照组MMP-1表达为1.167±0.036,受力后4h、15h、24h表达分别为0.669±0.109、1.085±0.176、1.325±0.048,不同时间MMP-1 mRNA水平比较,差异有统计学意义(P=0.017)。随着加力时间的延长,目的基因的表达水平呈现先下降,再上升的趋势。结论:阴道壁成纤维细胞受到机械拉伸刺激后,其细胞外基质的合成代谢和分解代谢均呈旺盛状态,细胞外基质发生了重塑,这可能是导致PFD发生的重要原因。

Objective：To investigate the expression of typeⅠcollagen（colⅠ）,type Ⅲ collagen（colⅢ）,transforming growth factorβ1（TGF-β1）and Matrix metalloproteinase-1（MMP-1） in vaginal wall fibroblasts obtained from patients with pelvic floor dysfunction（PFD） with stretch.Method：Twelve patients with PFD who underwent pelvic reconstructive surgery in the second hospital of Hebei Medical University from Oct 2009 to May 2010 were recruited in the study.2~5 stable passages from primary cultured vaginal wall fibroblasts were cultured in monolayer on flexible substrates and were subjected to stretch 12% with sinusoidal waveforms at 0.3HZ by using Flexcell-4000TM unit,with vaginal wall fibroblasts in static conditions as a control.Total RNA was extracted from fibroblasts and gene expression of colⅠ,colⅢ,TGF-β1 and MMP-1 were measured before stretch applied and after 4 hours,15 hours and 24 hours with stretch.Results：The expression of colⅠ,colⅢ,TGF-β1 and MMP-1 were 1.178±0.071,0.673±0.029,0.852±0.039 and 1.167±0.036 in control.The expression of colⅠwere 1.092±0.038,1.198±0.031 and 1.120±0.098 after 4 hours,15 hours and 24 hours with stretch.The expression of col Ⅲ were 0.313±0.019,0.504±0.033 and 0.853±0.210 after 4 hours,15 hours and 24 hours with stretch.The expression of TGF-β1 were 0.673±0.052,0.897±0.051 and 0.895±0.015 after 4 hours,15 hours and 24 hours with stretch.The expression of MMP-1 were 0.669±0.109,1.085±0.176 and 1.325±0.048 after 4 hours,15 hours and 24 hours with stretch.Significant difference was observed between them.With time increasing,gene expression levels decreased first and then showed upward trend.Conclusion：Vaginal wall fibroblasts from PFD patient were stimulated by mechanical stretch,which may lead to extracellular matrix remodeling and the occurrence of PFD.