人脐带间充质干细胞来源的外泌体对盆底脱垂患者阴道壁成纤维细胞的调节作用

Regulation of human umbilical cord mesenchymal stem cells derived exosomes on vaginal wall fibroblasts in patients with pelvic floor prolapsed

目的:探究人脐带间充质干细胞来源的外泌体(MSC-exosomes)对盆腔器官脱垂(POP)患者阴道壁成纤维细胞的调节作用。方法:采用超速离心法提取MSC-exosomes,应用透射电镜观察外泌体形态,Western blot法检测特异分子HSP90、CD63和TSG101表达,对外泌体进行综合鉴定。采用组织爬块和胶原酶消化法从POP患者阴道壁组织中分离培养成纤维细胞。采用PKH-67绿色荧光染料标记外泌体,添加PKH-67标记的MSC-exosomes,观察成纤维细胞对外泌体的摄取情况。分别将终浓度为50μg/mL、100μg/mL的MSC-exosomes加入成纤维细胞中,对照组加等体积PBS。MTS法检测3组细胞在12、24、36、48、60h和72h的增殖情况。收集成纤维细胞蛋白,Western blot法检测成纤维细胞胞外基质代谢相关基因CollagenⅠ、CollagenⅢ、LOXL-2、Fibronectin和MMP-2等表达,并进行相对定量统计分析。结果:分离到的外泌体特异表达标志蛋白HSP90和CD63,不表达内质网蛋白TSG101,透射电镜观察到具有双层脂膜结构的圆形囊泡小体,表明分离得到了外泌体。从脱垂患者阴道壁组织中成功培养得到成纤维细胞,其形态呈长梭型或纺锤形,细胞排列成簇状。PKH-67标记的MSC-exosomes添加24h后,细胞内可见丰富的绿色荧光信号,表明MSC-exosomes可被阴道壁成纤维细胞有效大量摄取。50、100μg/mL MSC-exosomes均可促进阴道壁成纤维细胞的增殖。50、100μg/mL exosome作用24h后,CollagenⅠ、CollagenⅢ、LOXL-2、Fibronectin表达增加,其中Fibronectin表达升高最显著,100μg/mL exosome组较50μg/mL exosomes组促进作用更明显,Collagen I在48h时较24h升高作用更明显,而MMP-2在MSC-exosomes添加后均出现蛋白表达下降,表明细胞外基质分解降低。结论:MSC-exosomes可有效促进POP阴道壁成纤维细胞的增殖,并促进成纤维细胞胞外基质的合成,减少细胞外基质的降解。

Objective:To investigate the regulatory effect of human umbilical cord mesenchymal stem cell derived exosomes(MSC-exosomes)on vaginal fibroblasts in patients with pelvic floor prolapse.Methods:MSC-exosomes were extracted by supercentrifugation,and the morphology of exosomes were observed by transmission electron microscopy.The expressions of specific molecules HSP90,CD63 and TSG101 were detected by Western blot,thus exosomes were comprehensively identified.Fibroblasts were isolated and cultured from vaginal wall tissues of patients with pelvic floor prolapse by tissue crawling and collagenase digestion methods.Exosomes were labeled with PKH-67 green fluorescent dye and were added to fibroblasts to observe the uptake developemtn.MSC-exosomes with a final concentration of 50μg/mL and 100μg/mL were added into fibroblasts,respectively,and PBS was added into the control group.MTS method was used to detect the proliferation of cells in the three groups at 12h,24h,36h,48h,60h and 72h.Fibroblast proteins were collected,and the expressions of Collagen I,CollagenⅢ,LOXL-2,Fibronectin and MMP-2 related genes of fibroblast extracellular matrix metabolism were detected by Western blot,and relative quantitative statistical analysis was performed.Results:Extraction and identification of exosomes:the isolated exosomes expressed specific marker proteins HSP90 and CD63,but did not express endoplasmic reticulum protein TSG101.Transmission electron microscopy(TEM)observed circular vesicle bodies with double lipid membrane structure,indicating that exosomes were isolated.Fibroblasts were successfully cultured from vaginal wall tissue,which were in the shape of long spindle or spindle and arranged in clusters.After 24h addition of PKH-67 labeled MSC-Exosomes,abundant green fluorescence signal could be seen in cells,suggesting that MSC-Exosomes could be effectively and abundantly absorbed by vaginal wall fibroblasts.MSC-Exosomes with final concentration of 50μg/mL and 100μg/mL promoted proliferation of vaginal wall fibroblasts.CollagenⅠ,CollagenⅢ,Collagen LOX-2 and Fibronectin expressions were increased after exosome treated with 50μg/mL and 100μg/mL for 24h,and the expression of Fibronectin was the most significant.Collagen I increased more significantly at 48h than at 24h,while protein expression of MMP-2 decreased after MSC-exosomes addition,indicating decreased decomposition of extracellular matrix.Conclusions:Exosomes derived from human umbilical cord mesenchymal stem cells can effectively promote the proliferation of vaginal fibroblasts in patients with pelvic floor prolapse,and promote the synthesis of fibroblasts extracellular matrix,and reduce the degradation of extracellular matrix.