摘要
目的:探讨miR-30c-2-3p对小鼠足细胞Nephrin、Podocin保护作用及雷公藤甲素(TP)干预作用。方法:采用嘌呤霉素(PAN)致小鼠足细胞损伤的体外模型,将足细胞分成5组:空白组、转染阴性质粒组(阴性质粒组)、转染阴性质粒+PAN组(阴性质粒+PAN组)、转染miR-30c-2-3p+PAN组(阳性质粒+PAN组)、转染miR-30c-2-3p+PAN+TP组(阳性质粒+PAN+TP组)。瞬时转染miR-30c-2-3p mimics,质粒终浓度均为50 nmol/L,转染6 h后进行药物干预,PAN干预浓度均为45 mg/L,TP干预浓度均为1 ng/ml,药物干预时间均为48 h。CCK-8法检测足细胞活力;实时荧光定量PCR检测细胞中miR-30c-2-3p表达;Western Blot检测足细胞Nephrin、Podocin蛋白表达。结果:(1)阴性质粒+PAN组足细胞活力明显低于空白组及阴性质粒组(P<0.01),阳性质粒+PAN组较阴性质粒+PAN组足细胞活力提高(P<0.05),使用TP干预后其活力进一步提高(P<0.05)。(2)与空白组、阴性质粒组比较,阴性质粒+PAN组miR-30c-2-3p表达减少,差异有统计学意义(P<0.01);与阴性质粒+PAN组相比,阳性质粒+PAN组、阳性质粒+PAN+TP组miR-30c-2-3p表达均增加(P<0.05);但与阳性质粒+PAN组相比,雷公藤甲素干预组未见统计学意义(P>0.05)。(3)与空白组及阴性质粒组比较,阴性质粒+PAN组Nephrin、Podocin两者表达均减少,差异有统计学意义(P<0.01);与阴性质粒+PAN组相比,阳性质粒+PAN组Nephrin、Podocin蛋白表达增加(P<0.05);在使用了TP干预后两者表达进一步增加(P<0.05)。结论:PAN能降低足细胞活力,降低miR-30c-2-3p表达,而增加其表达可以上调足细胞活力以及Nephrin、Podocin的表达。TP可以减轻PAN诱导的足细胞损伤以及Nephrin、Podocin的表达下降,但其作用机制似乎与miR-30c-2-3p无关。
Objective： This study aimed to investigate the protection of miR- 30c- 2- 3p on podocyte Nephrin,Podocin and the effect of triptolide（ TP） on it. Methods： Used the model of mice podocyte injury induced by puromycin aminonucleoside（ PAN） in vitro. The podocytes divided into five groups： blank group; negative plasmid group; negative plasmid ＋ PAN group; positive plasmids ＋ PAN group; positive plasmids ＋ PAN and TP group. The final concentration of transfected plasmid was 50 n M / L.After 6 hours＇ transfection,drug intervention initiated. PAN concentration was 45 mg / L,TP concentration was 1 ng / ml,the drugs intervention time was 48 hours. Then,Cell Counting Kit- 8 detected the podocytes viability; real- time quantitative PCR（ RT-PCR） detected the expression of miR- 30c- 2- 3p; western blot detected the expression of Nephrin,Podocin. Results：（ 1） Aafter transfection and drug intervention,the podocytes activity in the negative plasmid ＋ PAN group was significantly lower than the blank group and negative plasmid group（ P〈0. 01）. Compared with the negative plasmids ＋ PAN group,the podocytes activity in the positive plasmid ＋ PAN group increased（ P〈0. 05）. And it was further increased in the intervention of TP（ P〈0. 05）.（ 2） The expression of miR- 30c- 2- 3p in the negative plasmid ＋ PAN group was significantly lower than the blank group and negative plasmid group（ P〈0. 01）. Compared with the negative plasmids ＋ PAN group,the expression of miR- 30c- 2- 3p in the positive plasmid ＋ PAN group and positive plasmids ＋ PAN and TP group increased（ P〈0. 05）. But,it showed no significant difference inthe intervention of TP（ P〈0. 05）.（ 3） The expressions of Nephrin and Podocin in the negative plasmid ＋ PAN group were significantly lower than the blank group and negative plasmid group（ P〈0. 01）. Compared with the negative plasmids ＋ PAN group,the expressions of Nephrin and Podocin in the positive plasmid ＋ PAN group and positive plasmids ＋ PAN and TP group increased（ P〈0. 05）. And it was further increased in the intervention of TP（ P〈0. 05）. Conclusion： PAN reduces cell viability and miR-30c- 2- 3p expression,while increasing the expression of miR- 30c- 2- 3p increase the podocyte viability and the Nephrin,Podocin expression. TP increase the expression of podocyte viability and Nephrin,Podocin expression,but this effect seems not to be regulated by miR- 30c- 2- 3p.
出处
《中国中西医结合肾病杂志》
2016年第11期952-955,共4页
Chinese Journal of Integrated Traditional and Western Nephrology
基金
杭州市科技发展计划项目(No.20110733Q17)
浙江省卫生厅基金资助项目(No.2013KYA167)
杭州市卫生局基金资助项目(No.2013A50)
杭州市卫生局重点项目(No.2011Z013)
浙江省自然科学基金资助项目(No.Y2101410)
浙江省中医药科技计划项目(No.2013ZA091)
