CAJ | 학술논문

目的通过体外实验研究骨髓间充质干细胞(MSC)旁分泌肝细胞生长因子(HGF)对大鼠肾小管上皮细胞缺氧复氧损伤的修复作用及其相关机理.方法获取和培养大鼠骨髓MSC;构建HGF siRNA质粒对MSC进行转染,将大鼠MSC进行相应的基因沉默;建立大鼠大鼠肾小管上皮细胞系NRK-52E(简称:NRK)缺氧复氧模型.在前述基础上,实验共分4组建立上下共培养体系,(1)对照组:将未进行缺氧复氧处理的NRK单独培养;(2)缺氧复氧组:将进行了缺氧复氧处理的NRK单独培养;(3)野生MSC组:上室中加入缺氧复氧后的NRK,下室中加入野生型MSC;(4)转染MSC组:上室中加入缺氧复氧后的NRK,下室中加入转染了HGF siRNA的MSC.采用MTT法检测NRK增殖情况,采用实时荧光定量聚合酶链反应试剂盒检测各组HGF、HMGB1、IL-1β及TNF-αmRNA的相对表达量,采用蛋白质印迹法检测HGF、HMGB1、IL-1β及TNF-α蛋白的表达.结果 MSC转染HGF siRNA的效果较好,转染率在70％以上;转染HGF siRNA后MSC的HGF mRNA表达水平显著降低.随培养时间的延长各组NRK均有增殖,培养32 h时野生型MSC组NRK的增殖率最高,转染MSC组次之,缺氧复氧组最低(P＜0.01).缺氧复氧组HMGB1、IL-1β及TNF-αmRNA的相对表达量均为最高,转染MSC组次之,野生MSC组最低,3组间两两比较,差异均有统计学意义(P＜0.05).缺氧复氧组HMGB1、IL-1β及TNF-α蛋白的相对表达量均为最高,转染MSC组次之,野生MSC组最低,3组间两两比较,差异均有统计学意义(P＜0.01).结论 MSC通过旁分泌HGF对大鼠肾小管上皮细胞缺氧复氧损伤具有明显的修复作用.
목적 통과체외실험연구골수간충질간세포(MSC)방분비간세포생장인자(HGF)대대서신소관상피세포결양복양손상적수복작용급기상관궤리.방법 획취화배양대서골수MSC;구건HGF siRNA질립대MSC진행전염,장대서MSC진행상응적기인침묵;건립대서대서신소관상피세포계NRK-52E(간칭:NRK)결양복양모형.재전술기출상,실험공분4조건립상하공배양체계,(1)대조조:장미진행결양복양처리적NRK단독배양;(2)결양복양조:장진행료결양복양처리적NRK단독배양;(3)야생MSC조:상실중가입결양복양후적NRK,하실중가입야생형MSC;(4)전염MSC조:상실중가입결양복양후적NRK,하실중가입전염료HGF siRNA적MSC.채용MTT법검측NRK증식정황,채용실시형광정량취합매련반응시제합검측각조HGF、HMGB1、IL-1β급TNF-αmRNA적상대표체량,채용단백질인적법검측HGF、HMGB1、IL-1β급TNF-α단백적표체.결과 MSC전염HGF siRNA적효과교호,전염솔재70％이상;전염HGF siRNA후MSC적HGF mRNA표체수평현저강저.수배양시간적연장각조NRK균유증식,배양32 h시야생형MSC조NRK적증식솔최고,전염MSC조차지,결양복양조최저(P＜0.01).결양복양조HMGB1、IL-1β급TNF-αmRNA적상대표체량균위최고,전염MSC조차지,야생MSC조최저,3조간량량비교,차이균유통계학의의(P＜0.05).결양복양조HMGB1、IL-1β급TNF-α단백적상대표체량균위최고,전염MSC조차지,야생MSC조최저,3조간량량비교,차이균유통계학의의(P＜0.01).결론 MSC통과방분비HGF대대서신소관상피세포결양복양손상구유명현적수복작용.
Objective To preliminarily explore the effect of paracrine hepatocyte growth factor (HGF) from mesenchymal stem cells (MSCs) on the repair of hypoxia-reoxygenation injury in renal tubular epithelial cells of rats and the relevant mechanism.Method The bone marrow MSCs of rats were harvested and cultured,and transfected with HGF-siRNA plasmid.The corresponding genes of rat MSCs were silenced to build a hypoxia-reoxygenation model of the renal tubular epithelial cells (NRK-52E) in rats.On the basis of the foregoing,the experiment was divided into four groups to establish co-culture system:(1) the control group,simple culture of NRK cells not treated with hypoxia-reoxygenation; (2) hypoxia-reoxygenation group,simple culture of NRK cells treated with hypoxia-reoxygenation; (3) the wild MSCs group,coculture of hypoxia-reoxygenation-treated NRK cells (upper chamber) + wild type MSCs (lower chamber) ; (4) MSCs transfection group,coculture of hypoxia-reoxygenatiowNRK cells (upper chamber) + HGF siRNA-MSCs (lower chamber).The proliferation of NRK cells was assayed by MTT method.The mRNA and protein relative expression of HGF,HMGB1,IL-1β and TNFα was detected by real-time PCR,and by Western blotting respectively.Result The effect of HGF siRNA transfection was satisfactory and transfection efficiency was above 70％.The HGF mRNA relative expression in MSCs transfection group was decreased significantly.The proliferation of NRK cells was detectable in each group along with the extension of incubation time.The proliferation of NRK cells in wild MSCs group was highest,followed by MSCs transfection group,and lowest in hypoxia-reoxygenation group (P＜0.01) at 32 h after culture.The relative expression of HMGB1,IL-1β and TNFα mRNA and protein was highest,follwed by hypoxia-reoxygenation group,and lowest in MSCs transfection group,and there was significant difference between two groups among the three groups (P＜0.05).Conclusion MSCs can promote the repair of hypoxia-reoxygenation injury on renal tubular epithelial cells in rats by the paracrine of HGF.