间充质干细胞通过旁分泌HGF减轻大鼠肾小管上皮细胞缺氧复氧损伤的作用

Mesenchymal stem cells protect against hypoxia-reoxygenation injury of rat renal tubular epithelial cells via paracrine HGF in vitro

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HGFsiRNA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-α mRNA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HGFsiRNA的效果较好，转染率在70％以上；转染HGFsiRNA后MSC的HGFmRNA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32h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HMGBl、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组问两两比较，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。

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-reoxygenatiom （3） the wild MSCs group, coculture of hypoxia-reoxygenation-treated NRK cells （upper chamber） ＋ wild type MSCs （lower chamber） ; （4） MSCs transfection group, eoeulture of hypoxia-reoxygenation-NRK 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 blottingrespectively. 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 TNFa 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.