间质干细胞来源的microvesicles对顺铂肾毒性的保护作用及机制研究

Cisplatin is a major antineoplastic drug used for the treatment of solid tumors. Its chief dose-limiting side effect is nephrotoxicity. The current measures of nephroprotection used in patients receiving cisplatin are not satisfactory, and studies have focused on the investigation of new possible protective strategies. Our previous studies have showed that mesenchymal stem cells (MSCs) are particularly attractive cells for cellular and gene therapy in acute kidney injury (AKI). Furthermore, we have successfully isolated microvesicles, which are active components secreted by MSCs. Therefore, MSC-MVs may play a critical role in cisplatin-induced nephrotoxicity. In present study, we will determine the preventative and therapeutical effect of human bone marrow MSCs-derived microvesicles(BM-MSC-MVs) and umbilical cord-derived MSCs(uc-MSC-MVs),investigate the role of autophagy in models of cisplatin induced nephrotoxicity in vivo and in vitro. The different microRNAs from MSC-MVs and human fibroblast cell line (HFL1) derived microvesicles(HFL1-MVs) will be detected via microRNA microassay. Then，we will verify the key microRNAs in MSC-MVs mediated nephroprotection. The function of key microRNAs in cisplatin induced nephrotoxicity models will be confirmed. On this basis, we will also bring to light sites of action in key microRNAs corresponding to target gene, clarity the key signaling pathway. Based on these evidences, we will explore the effects of MSC-MVs on enhanced recovery of nephrotoxicity by cisplatin. Enhancing autophagy may provide a novel therapeutic option to minimize cisplatin-induced nephrotoxicity. Microvesicles isolated from MSCs may be an effective component of preventation and therapy in nephrotoxicity by cisplatin via a horizontal transfer of microRNAs. These results will provide basis for a new therapeutic approach of nephrotoxicity by cisplatin. Thus, it will lead to improve curative effect of MSC-MVs in tumor therapy.

顺铂(cisplatin)是当前联合化疗中最常用的药物之一，但是肾毒性影响了其临床疗效，目前仍缺乏有效的应对方案, 因此寻找新的保护策略并探讨其作用机制显得尤为重要。本课题组前期进行了间质干细胞（MSC）分离纯化和移植治疗急性肾损伤（AKI）模型的研究，并成功从MSC的培养上清中提取了微囊泡（MVs）。在此基础上，本研究通过体内和体外MSC-MVs保护顺铂引发的肾毒性模型的建立，揭示人 MSC-MVs对顺铂肾毒性的预防和修复作用，阐明细胞自噬在MSC-MVs保护顺铂肾毒性中的作用，筛选MSC-MVs中调节自噬的关键microRNAs分子并寻找其调控的靶基因及相关信号通路，从而揭示MSC-MVs对顺铂肾毒性的分子保护机制。本课题不仅为MSC-MVs的临床应用提供了可靠的实验依据，而且为提高化疗药物顺铂的抗癌疗效提供了新方案，拓展了MSC-MVs在组织损伤中的应用。

顺铂(cisplatin)的肾毒性影响了其化疗疗效，目前仍缺乏有效的应对策略，本课题旨在寻找新的顺铂肾毒性保护策略并探讨其作用机制。课题组前期已成功分离间质干细胞（MSC）及其来源的微囊泡，鉴定为外泌体（exosome）。在此基础上，本研究通过体内和体外间质干细胞外泌体(MSC-Ex)保护顺铂诱发的肾毒性模型的建立，揭示了MSC-Ex对顺铂肾毒性的预防和修复作用。研究发现人脐带间质干细胞来源的exosome（hucMSC-Ex）可以通过活化自噬预防顺铂诱导的肾损伤。通过凋亡分析、ELISA、Western blot、qRT-PCR等技术，发现体外hucMSC-Ex预处理可抑制顺铂诱导的NRK-52E细胞的凋亡及炎症因子分泌。HucMSC-Ex预处理增强了自噬相关蛋白LC3B的表达，自噬相关基因ATG5、ATG7表达也升高，自噬促进剂雷帕霉素和hucMSC-Ex有相似的作用，相反自噬抑制剂3-甲基腺嘌呤可减弱hucMSC-Ex的预防保护作用。体内实验显示hucMSC-Ex经肾被膜预注射，hucMSC-Ex组大鼠肾组织炎细胞浸润和组织结构破坏程度均有所减轻，血清Cr和BUN水平降低，自噬相关蛋白LC3B表达增强，细胞凋亡减弱，炎症因子TNF-α、ILl-β表达降低。自噬促进剂与hucMSC-Ex有相似的作用，以上结果表明hucMSC-Ex可通过活化自噬预防顺铂诱导的肾损伤。我们进一步研究揭示hucMSC-Ex通过转运14-3-3ζ蛋白从而调节自噬，发挥顺铂肾毒性的预防保护作用。本课题阐明了MSC-Ex介导的顺铂肾毒性保护作用中发挥关键作用的自噬通路及关键分子，不仅为MSC-Ex的临床应用提供了可靠的实验依据，而且为提高顺铂的抗癌疗效提供了新思路和方案。