机械应力对人子宫旁韧带成纤维细胞增殖及胶原代谢的影响

Effects of mechanical stress on proliferation and collagen metabolism of female parauterine ligament fibroblasts

目的探讨人子宫旁韧带成纤维细胞增殖及胶原代谢受到机械应力的影响。方法选取2020年2月-2020年5月于上海市第一妇婴保健院医院妇科因良性肿瘤行子宫全切手术的5例患者,年龄43~49岁,均无盆底功能障碍疾病,均未绝经,使用酶消化法获得人子宫旁韧带成纤维原代细胞并鉴定。对子宫旁韧带成纤维细胞进行不同时间(12 h及24 h)及不同大小(6%、12%及18%)周期性拉伸应力加载,检测成纤维细胞的增殖能力及细胞胶原代谢相关基因MMP-2、TIMP-1、CollagenⅠ、CollagenⅢ及LOXL1的RNA水平的表达变化,以未加力细胞组作为对照组,进行对照分析。结果与对照组相比,6%、12%及18%的拉伸应力都可以抑制成纤维细胞的增殖能力,且在一定范围内随着应力增加(6%~12%)及作用时间延长(12~24 h)其抑制作用增加。与对照组相比,6%的拉伸应力使成纤维细胞MMP-2、TIMP-1、CollagenⅠ、CollagenⅢ及LOXL1的RNA水平出现轻度上调趋势,但差异不显著(P>0.05)。12%及18%的拉伸应力使成纤维细胞MMP-2、TIMP-1、CollagenⅠ、CollagenⅢ及LOXL1的RNA水平下调。且随作用力加大(12%~18%),MMP-2、TIMP-1、CollagenⅠ、CollagenⅢ及LOXL1 mRNA表达都有进一步下降的趋势,其中LOXL1 mRNA表达有明显的下降(P<0.05);随着作用时间延长(12~24 h),MMP-2、TIMP-1、CollagenⅠmRNA的表达明显下降(P<0.05),而CollagenⅢ及LOXL1 mRNA的表达差异无统计学意义。在各基因中,CollagenⅢ和MMP-2 mRNA表达水平的最大下调幅度为21.1%和24.8%,TIMP-1、CollagenⅠ及LOXL1 mRNA分别达到43.6%、40.1%及47.9%。结论较短时间的轻度的拉伸应力似乎具有促进子宫骶韧带成纤维细胞的增殖及上调胶原相关基因表达的趋势,但仍需进一步实验验证;较大及较长时间的拉伸应力可以抑制成纤维细胞的增殖并使胶原相关基因表达下调,但各基因的下调具有不一致性。

Objective To explore the effects of mechanical stress on proliferation and collagen metabolism of female parauterine ligament fibroblasts.Methods Five patients aged 43~49 years who underwent total hysterectomy for benign tumors in the gynecology department of Shanghai First Maternity and Infant Hospital were selected during February 2020 to May 2020.All patients had no pelvic floor dysfunction disease and were not postmenopausal.The primary fibroblasts of human uterosacral ligament were obtained and identified by enzyme digestion.Parauterine fibroblasts were subjected to cyclic tensile stress loading at different times(12 and 24 h)and different sizes(6%,12%and 18%).The proliferation ability of fibroblasts and the mRNA expression changes of Collagen metabolism-related genes MMP-2,TIMP-1,Collagen I,Collagen III and LOXL1 were detected.The non stressed cell group was used as the control group in the comparision analysis.Results Compared with the control group,tensile stress in 6%,12%and 18%group could inhibit the proliferation of fibroblasts respectively,and the effect of inhibition increased with the increase of stress(from 6%to12%)and the extension of treatment time(from 12h to 24 h)in a certain range.Compared with the control group,the RNA levels of MMP-2,TIMP-1,Collagen I,Collagen III and LOXL1 in fibroblasts were slightly up-regulated at group of tensile stress(6%),but there was no significant difference(P>0.05).The RNA levels of MMP-2,TIMP-1,Collagen I,Collagen III and LOXL1 in fibroblasts were down-regulated at group of tensile stress(12%and 18%).The mRNA expressions of MMP-2,TIMP-1,Collagen I,Collagen III and LOXL1 were decreased with increasing force(from 12%to18%),and the mRNA expressions of LOXL1 were decreased significantly(P<0.05);The mRNA expressions of MMP-2,Collagen I and TIMP-1 decreased significantly with the extension of treatment time(from 12h to24 h)(P<0.05),while there was no significant difference in CollagenⅢand LOXL1 mRNA expression(P<0.05).Among the genes,the maximum downregulation of the expression levels of mRNA levels of Collagen III and MMP-2 were 21.1%and 24.8%,while the mRNA levels of TIMP-1,Collagen I and LOXL1 were 43.6%,40.1%and 47.9%,respectively.Conclusion Mild tensile stress in a relatively short duration of seems to promote the proliferation of uterosacral ligament fibroblasts and up-regulate the expression of collagens related genes,however,further experimental verification is still needed.The proliferation of fibroblasts and the expression of collagen-related genes were inhibited by higher or longer tensile stress,but the downregulation of each gene was inconsistent.