脂肪来源间充质干细胞外泌体转运Sox9治疗急性肾损伤慢性化的基础研究

Traditional therapies fail to prevent the progression of acute kidney injury (AKI) progression into chronic kidney disease (CKD). Due to the strong immunoregulatory, anti-inflammatory and anti-apoptotic functions, adipose tissue derived mesenchymal stem cells (AD-MSCs) have become a therapeutic option in the prevention of AKI to CKD progression. However, potential health risks such as malignant differentiation and transient immune response severely restrict the use of AD-MSCs in the clinical setting. It has recently been discovered that exosomes secreted by AD-MSCs possess most of the functions of AD-MSCs, and do not induce immune responses or carry the risk of malignant differentiation, making exosomes a promising potential substitute for AD-MSCs. In preliminary studies, we found that inhibition of AKI to CKD progression by AD-MSCs derived exosomes correlated with activation of renal endogenous repair factor Sox9 expression. It was verified that Sox9 knockout aggravated renal injury and renal interstitial fibrosis. Unfortunately, AD-MSCs derived exosomes have a limited potency in the induction of renal tubular Sox9 expression. In this project, we will transduce Sox9 expression vectors into AD-MSCs to produce specific exosomes with high Sox9 protein and mRNA, and observe the effect of the high Sox9 exosomes on AKI and explore its mechanism and safety. To identify the hypothesis that high Sox9 exosomes can transfer Sox9 protein and mRNA into renal tubular epithelial cells, we will induce AKI in renal tubular epithelial Sox9-specific knockout mice and treat them with the high Sox9 exosomes. We hope the high Sox9 exosomes derived from AD-MSCs will be demonstrated as a potential therapeutic approach to the alleviation of AKI to CKD progression.

传统疗法不能有效阻遏急性肾损伤（AKI）向慢性化（CKD）进展。脂肪来源间充质干细胞（AD-MSCs）具有免疫调节、抗炎等功能，一度成为防治AKI向CKD转变的重要选择。然而，AD-MSCs治疗存在恶性分化、免疫反应等隐患。近来发现，AD-MSCs分泌的外泌体具有AD-MSCs的大部分功能，不产生免疫反应、诱导恶性分化倾向小，能够替代AD-MSCs。我们发现，AD-MSCs外泌体确实可以抑制AKI向CKD转归，该过程中伴有肾脏的内源性修复因子Sox9的激活。但是，普通AD-MSCs外泌体诱导肾小管Sox9表达和修复的效能有限，本项目拟改造AD-MSCs细胞，使其外泌体过表达Sox9，观察其对AKI向CKD转归的保护作用，同时构建肾小管上皮细胞Sox9特异性敲除小鼠，验证外泌体过载Sox9能强效向损伤肾小管上皮细胞投递Sox9蛋白和mRNA，促进AKI修复，抑制AKI向CKD转归。

间充质干细胞（Mesenchymal Stem Cells，MSCs）具有极强的自我更新及多向分化潜能，具有强大的抗炎、免疫调节。人脂肪来源MSCs可减轻急性肾损伤（AKI）及慢性肾间质纤维化。AKI后的第一个修复阶段以近端小管上皮细胞的增值为特征。Sox9在AKI早期肾小管上皮细胞中的表达明显增加，参与了肾小管上皮细胞的内源性修复。基因敲除Sox9，肾脏损伤及间质纤维化显著加重。通过提取、培养和鉴定了小鼠脂肪来源间充质干细胞（AD-MSCs），利用病毒转染技术获得过表达SOX9的AD-MSCs，选取最适转染滴度。对缺血再灌注模型小鼠进行处理，观察过表达S0X9的AD-MSCs处理的小鼠和未干预的AD-MSCs处理的小鼠对急性肾损伤慢性化的影响。本项目通过构建过表达S0X9的AD-MSCs，发现过表达S0X9的AD-MSCs比未干预的AD-MSCs对急性肾损伤慢性化有更好的治疗作用。为临床急性肾损伤提供全新的治疗方法，遏制其慢性化及纤维化