犬UC-MSC-Exo来源miRNA表达分析及其cfa-miR-34a/-143对血管内皮细胞增殖的影响

doi:10.16431/j.cnki.1671-7236.2020.03.004

摘要/Abstract

摘要： 试验旨在分离鉴定犬脐带间充质干细胞分泌的外泌体（UC-MSC-Exo），并研究犬UC-MSC-Exo来源miRNA的表达情况及其对血管内皮细胞（VEC）增殖的影响。采用超高速离心法从犬脐带间充质干细胞培养上清中分离外泌体，采用Western blotting、透射电镜和纳米颗粒跟踪分析法（NTA）进行鉴定。通过对犬UC-MSC-Exo中的miRNA进行测序，筛选出2个目标miRNA（cfa-miR-34a和cfa-miR-143）并合成相应的mimic和inhibitor，转染犬VEC；采用荧光显微镜和实时荧光定量PCR法检测mimic和inhibitor转染情况及其转染效率；CCK-8法检测转染mimic和inhibitor对犬VEC增殖能力的影响，并对其靶基因进行预测。结果显示，犬UC-MSC-Exo表达特异性的外泌体表面蛋白CD9、CD63和CD81，粒径集中在100~200 nm，电镜检测成典型的杯状。免疫荧光结果表明，miRNA mimic和inhibitor均已转染进细胞内，并且聚集于细胞质中；实时荧光定量PCR结果表明，转染cfa-miR-34a mimic和cfa-miR-143 mimic后，细胞内cfa-miR-34a和cfa-miR-143表达量分别升高约400和78倍；而转染cfa-miR-34a inhibitor和cfa-miR-143 inhibitor后，细胞内cfa-miR-34a和cfa-miR-143表达量分别降低了77%和83%；CCK-8检测结果表明，cfa-miR-34a和cfa-miR-143在体外可极显著促进血管内皮细胞的增殖（P<0.01）。cfa-miR-34a靶基因预测结果发现，共有195个保守靶位点，与miRDB预测结果共有69个交集靶基因；cfa-miR-143则有448个保守靶基因，其与miRDB预测结果有128个交集靶基因。本试验成功分离得到犬UC-MSC-Exo，且证实目标miRNA cfa-miR-34a和cfa-miR-143在体外可极显著促进VEC增殖。

关键词: 脐带间充质干细胞(UC-MSC); 外泌体; miRNA; 血管内皮细胞; 增殖

Abstract: This study was aimed to isolate and identify exosomes secreted by canine umbilical cord mesenchymal stem cells(UC-MSC-Exos) and study the expression of miRNAs derived from UC-MSC-Exo and its effect on proliferation of vascular endothelial cells (VECs).Exosomes were isolated from the culture supernatant of canine umbilical cord mesenchymal stem cells by ultra-high speed centrifugation and identified by Western blotting,transmission electron microscopy and nanoparticle tracking analysis (NTA).Two miRNAs (cfa-miR-34a/-143) were screened by sequencing miRNAs in canine UC-MSC-Exo,and the corresponding mimic and inhibitor were synthesized and then transfected into canine VECs.The mimic and inhibitor transfections and their transfection efficiency were detected by fluorescence microscopy and Real-time PCR,and its effect on the proliferation of canine VECs was detected by CCK-8 method.Finally,the target genes were predicted.Canine UC-MSC-Exo expressed specific exosomal surface proteins CD9,CD63 and CD81 with a particle size of 100-200 nm and a typical cup shape.The results of immunofluorescence assay showed that both miRNA mimic and inhibitor were transfected into cells and accumulated in the cytoplasm;Real-time PCR results showed that the expression of cfa-miR-34a in cells increased about 400-fold after transfection of cfa-miR-34a mimic,while cfa-miR-143 increased about 78-fold;Whereas transfection of cfa-miR-34a inhibitor,the expression of cfa-miR-34a in cells decreased by 77%,while that of cfa-miR-143 decreased by 83%,and the cfa-miR-34a and cfa-miR-143 could extremely significantly promote the proliferation of VECs in vitro by CCK-8.The target genes of cfa-miR-34a predicted that there were 195 conserved target sites,and 69 cross-target genes were shared with miRDB prediction results,while cfa-miR-143 had 447 conserved target genes,128 cross-target genes were shared with miRDB prediction results.Canine UC-MSC-Exo was successfully isolated,and it was confirmed that the target miRNAs cfa-miR-34a and cfa-miR-143 could extremely significantly promote VECs proliferation in vitro.

Key words: umbilical cord mesenchymal stem cells (UC-MSCs); exosomes; miRNA; vascular endothelial cells; proliferation

中图分类号:

S829.2

罗惠娜, 罗冬章, 樊全宝, 王丙云, 詹小舒, 陈胜锋, 陈志胜, 白银山, 刘璨颖, 计慧琴. 犬UC-MSC-Exo来源miRNA表达分析及其cfa-miR-34a/-143对血管内皮细胞增殖的影响[J]. 中国畜牧兽医, 2020, 47(3): 676-685.

LUO Huina, LUO Dongzhang, FAN Quanbao, WANG Bingyun, ZHAN Xiaoshu, CHEN Shengfeng, CHEN Zhisheng, BAI Yinshan, LIU Canying, JI Huiqin. Expression of miRNA from Canine UC-MSC-Exo and the Effect of cfa-miR-34a/-143 on Proliferation of Vascular Endothelial Cells[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 676-685.

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