27nt-miRNA对人脐带间充质干细胞定向内皮细胞分化和管腔形成的影响及其分子机制  

Effects of 27nt-miRNA on directional endothelial differentiation and tube formation of human umbilical cord mesenchymal stem cells

作　　者：陶晓静[1] 杨鹏[1] 沈凤[1] 颜渊鸳 李丹 罗雪兰 甘娜 覃裕旺[2] 欧和生[1,2] TAO Xiaojing;YANG Peng;SHEN Feng;YAN Yuanyuan;LI Dan;LUO Xuelan;GAN Na;QIN Yuwang;OU Hesheng(College of Pharmacy,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学药学院,南宁530021 [2]广西中医药大学附属广西国际壮医医院

出　　处：《山东医药》2018年第35期23-27,共5页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81373403)

摘　　要：目的探讨27nt-miRNA对人脐带间充质干细胞(h UCMSCs)定向内皮细胞分化和分化后细胞管腔形成能力的影响,并探讨其分子机制。方法构建27nt-miRNA、anti-27nt-miRNA及对照慢病毒载体。将h UCMSCs分为27nt-miRNA组、anti-27nt-miRNA组、对照组,分别转染27nt-miRNA、anti-27nt-miRNA及对照慢病毒载体后观察转染效率,诱导分化并观察细胞形态变化,MTT检测分化后细胞增殖能力,硝酸还原酶法检测分化过程中细胞的一氧化氮(NO)产量,人工基底膜检测分化后细胞的成管能力,RT-PCR、免疫细胞化学和Western blotting分别检测分化后细胞的内皮型一氧化氮合酶(eNOS)mRNA和蛋白。结果与对照组相比,27nt-miRNA组h UCMSCs分化程度低,未见管腔样网络结构;分化第9天,27nt-miRNA组细胞NO产量较对照组降低33.49%(P<0.05)、eNOS mRNA表达量较对照组降低23.81%(P<0.01)、eNOS蛋白表达较对照组降低24.56%(P<0.05);anti-27nt-miRNA组的上述指标较对照组及27nt-miRNA组升高(P<0.05或P<0.01)。结论 27nt-miRNA明显抑制h UCMSCs定向内皮细胞分化及管腔形成能力,其作用机制与27nt-miRNA抑制eNOS蛋白表达及NO合成有关。Objective To investigate the effects of 27nt-miRNA on the differentiation of human umbilical cord mesenchymal stem cells(hUCMSCs)and the ability of tube formation in differentiated endothelial cells,and to explore the underlying mechanism.Methods The 27nt-miRNA,anti-27nt-miRNA and control lentiviral vector were constructed.The hUCMSCs were divided into the 27nt-miRNA group,anti-27nt-miRNA group,and control group,then were transfected with 27nt-miRNA,anti-27nt-miRNA,and control lentiviral vector,respectively,and the transfection efficiency was evaluated.After induction of differentiation,cell morphology was observed,the proliferation capacity of differentiated cells was detected by MTT,the NO production of differentiated cells was detected by nitrate reductase assay,the ability of tube formation of differentiated cells was detected by artificial basement membrane.RT-PCR,immunocytochemistry and Western blotting were used to detect the expression of eNOS mRNA and protein in differentiated cells.Results Compared with the control group,hUCMSCs of 27nt-miRNA group had the lowest differentiation degree,and no complete tube-like network structure was formed;27nt-miRNA inhibited the cell proliferation(P<0.01);on the 9th day of differentiation,NO production in the 27nt-miRNA group was reduced by 33.49%as compared with that of the control group(P<0.05).The expression of eNOS mRNA and protein was decreased by 23.81%(P<0.01)and 24.56%(P<0.05)as compared with that of the control group.In the 27nt-miRNA inhibitor group,the above indexes significantly increased as compared with those of the control and 27nt-miRNA group(P<0.05 or P<0.01).Conclusion 27nt-miRNA significantly inhibits the directional endothelial differentiation and tube formation in hUCMSCs,which is related to the inhibition of eNOS protein expression and NO synthesis by 27nt-miRNA.

关 键 词：27nt-微小RNA 非编码RNA 干细胞分化 间充质干细胞 人脐带间充质干细胞 内皮细胞 一氧化氮合酶 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]