雷帕霉素对肾小管上皮细胞生物学行为的影响及其治疗糖尿病肾病分子机制的初步研究

目的 研究雷帕霉素(RAPA)对肾小管上皮细胞生物学行为的影响及其治疗糖尿病肾病分子机制.方法 体外培养肾小管上皮细胞(HK-2),使用转化生长因子 β1(TGF-β1)处理HK-2细胞,以建立肾小管上皮细胞-肌成纤维细胞转化(EMT)的细胞模型,并联合RAPA处理HK-2细胞,通过Transwell迁移实验和侵袭实验观察HK-2细胞的迁移和侵袭等细胞生物学行为变化;通过RT-PCR、Western blot法观察EMT分子标志物表达的变化;结合前期建立的糖尿病肾病小鼠模型的肾组织及通过腹腔注射RAPA1 mg/(kg·d)]处理后的模型小鼠肾组织,利用HE染色分析肾组织的病理变化;利用免疫组化检测EMT分子标志物的表达变化.结果 Transwell实验结果表明,TGF-β1诱导HK-2细胞发生迁移和侵袭能力明显增加;联合应用RAPA后,迁移和侵袭能力明显抑制.RT-PCR、Western blot和免疫组化结果显示TGF-β1可以促进间质细胞标志分子Vimentin的表达,而上皮标志分子E-cadherin的表达明显降低;联合应用RAPA后可以抑制TGF-β1诱发的EMT作用.HE染色显示,RAPA治疗后,能显著改善糖尿病肾病小鼠肾小管形态病变.结论 RAPA具有抑制肾小管上皮细胞发生EMT作用.

On rapamycin regulating biological behaviours of renal tubular epithelial cells and its potiential mechanisms related to treatment for diabetic nephropathy

Objective To investigate biological behaviors of renal tubular epithelial cells treated by rapamycin ( RAPA) and its molecular mechanisms related to treatment for diabetic nephropathy( DN) . Methods Human re-nal tubular epithelial cells (HK-2) were cultured in complete medium with TGF-β1 and (or) RAPA. Transwell migration assay or transwell invasion assay, respectively, was performed to observe HK-2 cells treated by TGF-β1 and ( or) RAPA. RT-PCR and Western blot were used to detect expression of E-cadherin and vimentin in HK-2 cells;meanwhile, the mice models with diabetic nephropathy were established and treated by RAPA. HE staining was used to observe pathological changes in these tissues. Immunohistochemical analysis was performed to detect expression of E-cadherin and vimentin in the kidney tissues. Results The transwell assays showed the abilities of migration and invasion of HK-2 cells treated by TGF-β1 significantly increased,compared to the controls. However, the HK-2 cells treated by RAPA showed weaker migration and invasion abilities. RT-PCR,Western blot and immu-nohistochemistry assay illustrated that E-cadherin expression decreased and vimentin expression increased in HK-2 cells treated by TGF-β1. However, after the combination use of RAPA, EMT was inhibited, which was induced by TGF-β1 . HE staining revealed that RAPA ameliorated the histological tissue damage in the kidney tissues from the DN mice. Conclusion RAPA can reverse epithelial-myofibroblast transdifferentiation of renal tubular epithelial cells.