摘要
目的脐带间充质干细胞(UCMSC)已被证实通过其外泌体发挥加速损伤子宫内膜修复的作用,但其具体作用机制仍不明确。文中旨在探索UCMSC外泌体所携带的miR-21-5p对于子宫内膜间质细胞增殖的影响。方法增殖期子宫内膜标本取自2018年1月至6月首都医科大学附属北京妇产医院妇科微创中心,因宫颈上皮内瘤变III级、子宫肌瘤等行开腹或腹腔镜下全子宫切除的3例患者。通过胶原酶消化法原代分离培养子宫内膜间质细胞并鉴定。采用差速离心法提取UCMSC外泌体并鉴定。荧光定量PCR检测UCMSC外泌体及其处理后的子宫内膜间质细胞内的miR-21-5p的表达水平后,并通过CCK8和EdU染色检测抑制组(转染miR-21-5p inhibitor)、对照组(转染miR inhibitor NC)、空白组(未转染)子宫内膜间质细胞的增殖活性。此外,采用生物信息学分析预测外泌体miR-21-5p在子宫内膜间质细胞内的潜在靶mRNA及其所参与的主要细胞生物学进程。结果 CCK8结果显示,24、48、72 h,抑制组的吸光度显著低于对照组和空白组(P<0.01)。EdU染色结果显示,转染后48 h后,抑制组的外泌体增殖活性[(7.47±0.44)%]显著低于对照组和空白组[(13.47±0.47)%、(14.60±1.08)%],差异有统计学意义(P<0.01)。通过GO分析和KEGG通路富集分析,发现miR-21-5p的潜在靶基因参与2162种生物学进程(P<0.05)、300种分子生物学功能(P<0.05)和214种细胞学组分(P<0.05),并富集到3条细胞信号通路(P<0.05),推测miR-21-5p可能通过这些途径促进外泌体增殖和损伤子宫内膜损伤修复。结论 UCMSC外泌体通过转运miR-21-5p能够参与调控多种与子宫内膜再生修复相关的基因表达及信号通路,从而有效促进子宫内膜间质细胞增殖,加速损伤子宫内膜修复进程。
Objective Umbilical cord mesenchymal stem cells(UCMSC) have been proven to accelerate repairing of damaged endometrium via their exosomes, whereas the mechanism is still not clear. Thus, our study was aimed toinvestigate the effect of miR-21-5 p carriedby UCMSC-derived exosomes on proliferation of endometrial stromal cells. Methods Proliferative phase endometrial tissues were obtained from premenopausal patients who underwent laparoscopic or laparotomic hysterectomy for early-stage cervical cancer or uterine myomas in Department of Minimally Invasive Gynecologic Center, Beijing Obstetrics and Gynecology Hospital from January to June 2018.Endometrial stromal cells(ESC) were isolated bycollagenase digestionand identified. UCMSC-derived exosomes were isolated by differentially ultracentrifugationand characterized. Quantitative real time RT-PCR was performed to detect the relative expression level of miR-21-5 p in UCMSC-derived exosomes and exosome-treated ESC. CCK8 and EdU incorporation assays were performed to test the cell viability and proliferation of ESC in miR-21-5 p inhibitor group,NCgroupand nil control group, respectively. Furthermore, bioinformatic analysis was adopted to predict the potential targeted mRNAs of miR-21-5 p and the relevantbiological processes in ESC. Results CCK8 assays showed that the absorbance values at 450 nm in miR-21-5 p inhibitor group at 24, 48 and 72 h after transfection were significantly lower than other two control groups(P<0.01).The EdUincorporation assays showed that the EdUpositive rate of ESC in miR-21-5 p inhibitor group[(7.47±0.44)%] at 48 h after transfection was significantly lower than other two control groups [miR inhibitor NC group:(13.47±0.47)%;nil control group:(14.60±1.08)%](P<0.01). GO and KEGG pathway enrichment analysis showed that these genes participated in 2162 Biological Processes(P<0.05), 300 Molecular Functions(P<0.05), 214 Cellular Components(P<0.05)and 3 pathways(P<0.05) were highly enriched, indicating exosomal miR-21-5 p might participate in prolife
作者
吕承晓
段华
汪沙
甘露
徐倩
LV Cheng-xiao;DUAN Hua;WANG Sha;GAN Lu;XU Qian(Department of Minimally Inasive Gynecologic Center,Bejying Obtetrics and Gynecology Hospital,Capital Medical University,Beijing 100006,China)
出处
《医学研究生学报》
CAS
北大核心
2020年第12期1262-1268,共7页
Journal of Medical Postgraduates
基金
国家重点专项研发计划(2018YFC1004803)
国家自然科学基金(81801403)。
