过表达klotho基因的脂肪干细胞对糖尿病肾病大鼠肾脏的修复作用及机制的研究

Diabetic nephropathy(DN) is a microvascular complication of diabetes and has become the leading cause of end-stage renal failure. Currently, DN patients lack effective therapeutic strategies. In the preliminary study, we found that adipose-derived mesenchymal stem cells (ADMSCs) could significantly improve the symptom of DN in rats. However, shorter survival time and aggravating renal fibrosis of ADMSCs limited its application. Improving the efficacy of ADMSCs treating DN need to be solved urgently. Klotho gene is considered as an anti-aging gene,it can control proliferation and apoptosis of mesenchymal stem cells. which is expressed primarily in kidney. It plays an important protective effect on kidneys. Studies showed that there was persistent and irreversible decreased levels of Klotho in chronic kidney diseases,which will lead to kidney inflammation and fibrosis,because the role of protecting kidneys was significantly weakened. Therefore, in the present study, we will upregulate klotho gene in ADMSCs, then transplant into the rats with DN.The proliferation and survival of ADMSCs by upregulating Klotho gene was detected in kidney of DN in rats. We aimed to explore the mechanism of Klotho gene promoting ADMSCs in the improvement and treatment of kidney of DN in rats by these index of renal function, renal pathology and microenvironment(inflammation,oxidative stress, fibrosis). The study will provide a new method for the application of stem cell therapy in DN patients.

糖尿病肾病（DN）是糖尿病微血管并发症，病因复杂，目前尚无有效疗法。本课题组研究发现，脂肪干细胞（ADMSCs）具有改善糖尿病肾病症状的潜能，但其在DN大鼠肾脏内的存活率明显降低，且具有加重肾纤维化的倾向。如何增强ADMSCs对DN的修复治疗效果？尚待探讨。Klotho基因是抗衰老调节基因，参与调控干细胞的增殖与凋亡。Klotho基因在肾脏高表达，对肾脏具有重要保护作用。研究发现当慢性肾病时，Klotho基因表达量出现持续不可逆性降低，保护肾脏作用明显减弱，肾脏炎症及纤维化进程加剧。因此，本研究拟上调ADMSCs的Klotho基因，移植治疗大鼠糖肾病，探讨过表达Klotho基因的ADMSCs在DN肾脏内的增殖及存活情况，并从DN大鼠的肾功、肾组织病理及微环境（炎症、氧化应激、纤维化程度）等方面，分析Klotho基因对ADMSCs修复改善DN大鼠肾脏的效果及机制，为临床DN治疗提供新思路。

本项目应用慢病毒转染的方式上调脂肪间充质干细胞（ADMSCs）的Klotho基因，移植治疗大鼠糖尿病肾病。原代分离培养大鼠ADMSCs，流式术检测细胞周期和表面抗原，发现原代培养的细胞具有较强的增殖能力，并表达间充质干细胞相关抗原。还具有向成骨、成软骨、成脂方向分化的特性。构建含Kl质粒转染ADMSCs，获得Klotho基因过表达的细胞模型ADMSCs（Kl+），检测发现ADMSCs（Kl+）仍具有明确的间充质干细胞特性。制备糖尿病肾病大鼠模型，发现正常肾脏内Klotho的表达量明显高于糖尿病肾病大鼠肾脏的表达量。进一步应用ADMSCs（Kl+）移植入糖尿病肾病大鼠体内后，发现ADMSCs（Kl+）治疗组肾脏内Klotho蛋白表达量有所升高。肾组织内细胞增殖能力增强，大鼠的肾脏功能得到恢复。肾脏病理改变明显减轻，肾组织炎症因子表达量减少。氧化应激产物表达量降低，肾纤维化程度显著减轻。研究结果提示，应用慢病毒转染技术使ADMSCs过表达Klotho基因，有助于上调受损肾脏内Klotho蛋白表达，增强ADMSCs对糖尿病肾病大鼠肾脏的治疗修复作用，改善肾组织微环境，抑制肾纤维化及炎症的进展。本项目的开展对于明确ADMSCs治疗糖尿病肾病中的作用提供数据支持，为临床糖尿病肾病的治疗提供新思路。