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

发布时间：2018-11-05 09:14

【摘要】：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RAPA[1mg/(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。
[Abstract]:Objective to study the effect of rapamycin (RAPA) on the biological behavior of renal tubular epithelial cells and its molecular mechanism in the treatment of diabetic nephropathy. Methods Renal tubular epithelial cells (HK-2) were cultured in vitro and HK-2 cells were treated with transforming growth factor 尾 1 (TGF- 尾 1) to establish a cell model of transforming (EMT) from renal tubular epithelial cells to myofibroblasts. HK-2 cells were treated with RAPA, and the changes of cell biological behavior such as migration and invasion of HK-2 cells were observed by Transwell migration assay and invasion assay. The expression of EMT molecular markers was observed by RT-PCR,Western blot method. Combined with the kidney tissue of diabetic nephropathy model and the kidney tissue treated by intraperitoneal injection of RAPA [1mg/ (kg d)], the pathological changes of renal tissue were analyzed by HE staining. The expression of EMT molecular markers was detected by immunohistochemistry. Results the results of Transwell assay showed that TGF- 尾 1 induced the migration and invasion of HK-2 cells. After combined application of RAPA, migration and invasion were significantly inhibited. RT-PCR,Western blot and immunohistochemical results showed that TGF- 尾 1 could promote the expression of Vimentin, but the expression of epithelial marker E-cadherin was significantly decreased. Combined use of RAPA could inhibit EMT induced by TGF- 尾 1. HE staining showed that RAPA could significantly improve renal tubular morphology in diabetic nephropathy mice. Conclusion RAPA can inhibit the development of EMT in renal tubular epithelial cells.
【作者单位】：安徽医科大学生物化学与分子生物学教研室;安徽医科大学第二附属医院药剂科;安徽医科大学第二附属医院肾脏内科;
【基金】：国家自然科学基金(编号:81372911)
【分类号】：R587.2;R692.9

【相似文献】