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。
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.
作者
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
LUO Huina;LUO Dongzhang;FAN Quanbao;WANG Bingyun;ZHAN Xiaoshu;CHEN Shengfeng;CHEN Zhisheng;BAI Yinshan;LIU Canying;JI Huiqin(College of Life Science and Engineering,Foshan University of Science and Technology,Foshan 528231,China)
出处
《中国畜牧兽医》
CAS
北大核心
2020年第3期676-685,共10页
China Animal Husbandry & Veterinary Medicine
基金
广东省自然科学基金(2017A030313171)
广东省自然科学基金(2018A030313893)。
