人脐带MSC来源exosome活化自噬对HK-2细胞顺铂损伤的预防及机制研究

发布时间：2018-03-06 18:07

  本文选题：人脐带间充质干细胞来源的外泌体　切入点：预防　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:前期的研究结果表明,人脐带间充质干细胞来源的外泌体(exosome secreted by human umbilical cord mesenchymal stem cells,hucMSC-ex)能有效的预防和减缓顺铂对大鼠肾脏的损伤,但其作用机制尚不明确。本研究构建了hucMSC-ex体外预防顺铂对人肾小管上皮细胞(HK-2)的损伤模型,揭示了hucMSC-ex预防顺铂引起HK-2细胞的分子机制,为临床上hucMSC-ex预防顺铂诱导肾毒性提供了实验依据。方法:常规培养的HK-2细胞为control组;cis组为16μmol/L顺铂处理16h;ex+cis组为用200μg/ml的hucMSC-ex与HK-2细胞孵育24h后,加入16μmol/L的顺铂继续培养16h;3-MA+ex+cis组为100μmol/L的3-MA和200μg/ml的hucMSC-ex同时预孵育HK-2细胞24h后,加入16μmol/L的顺铂继续处理16h。透射电子显微镜观察hucMSC-ex的形态,成像流式细胞仪和Western blot检测hucMSC-ex表面标记CD9、CD63的表达。过表达和敲减功能研究用于探索14-3-3ζ在hucMSC-ex预防顺铂肾毒性中的作用。TUNEL实验和凋亡双染流式分析检测HK-2细胞的凋亡。免疫荧光分析了核增殖抗原(PCNA)的表达。超高分辨率荧光显微镜观察细胞自噬小体形成的数量和形态大小。Western blot技术检测了14-3-3ζ、LC3B、P62、PCNA、Bax等蛋白的表达水平。结果:人脐带间充质干细胞来源的外泌体(hucMSC-ex)呈圆形或椭圆形,表面表达特异性分子CD9、CD63。HucMSC-ex与HK-2细胞孵育后,可通过减少细胞凋亡数量、增加PCNA的表达来预防顺铂诱导的细胞损伤。在此过程中,自噬相关指标LC3BⅡ/LC3BⅠ比值增加,自噬底物P62表达水平下降,提示自噬活化。自噬抑制剂3-甲基腺嘌呤(3-MA)则可逆转hucMSC-ex的顺铂损伤保护作用。HucMSC-ex预处理后,14-3-3ζ表达水平增加。敲减hucMSC-ex中的14-3-3ζ蛋白后,14-3-3ζ表达水平下调,hucMSC-ex诱导细胞自噬的作用减弱,同时预防顺铂的损伤作用也弱化。过表达HK-2细胞中的14-3-3ζ可以增强细胞自噬水平而缓解细胞的顺铂损害。敲减HK-2细胞中的14-3-3ζ可以下调细胞自噬水平,同时增加了顺铂的细胞损伤。而通过hucMSC-ex携带14-3-3ζ回补了14-3-3ζ表达后,被增强的顺铂损伤又得以逆转。结论:HucMSC-ex携带的14-3-3ζ蛋白可以预防顺铂对人肾小管上皮细胞(HK-2)的损伤作用。其作用机制可能是通过上调HK-2细胞的自噬水平而保护细胞免受顺铂的损伤。这一研究为hucMSC-ex预防顺铂诱导的肾损伤作用提供了新的思路和方案。
[Abstract]:Objective: previous studies have shown that exosome secreted by human umbilical cord mesenchymal stem cells MSC-exes derived from human umbilical cord mesenchymal stem cells can effectively prevent and attenuate the renal injury induced by cisplatin in rats. In this study, we constructed a model of hucMSC-ex to prevent the injury of human renal tubular epithelial cells (HK-2) in vitro, which revealed the molecular mechanism of hucMSC-ex preventing cisplatin induced HK-2 cells. Methods: the conventional cultured HK-2 cells were treated with control group and 16 渭 mol/L cisplatin for 16 h after incubation with HK-2 cells with 200 渭 g / ml hucMSC-ex for 24 hours. HK-2 cells were preincubated with cisplatin of 16 渭 mol/L for 16 hours after incubation with 100 渭 mol/L 3-MA and 200 渭 g / ml hucMSC-ex for 24 hours. The morphology of hucMSC-ex was observed by transmission electron microscope. The cells were treated with cisplatin at 16 渭 mol/L for 16 h. Imaging flow cytometry and Western blot were used to detect the expression of CD9hCD63 on hucMSC-ex surface. Overexpression and knockdown function were used to explore the role of 14-3-3 味 in the prevention of cisplatin nephrotoxicity by hucMSC-ex. Tunel assay and apoptosis double staining flow cytometry were used to detect apoptosis of HK-2 cells. Immunofluorescence was used to analyze the expression of nuclear proliferating antigen (PCNA). Ultrahigh resolution fluorescence microscopy was used to observe the number and morphological size of autophagy. Western blot technique was used to detect the expression level of 14-3-3 味 -LC3BP62P62P62PCNA1-Bax protein. Results: human umbilical cord. HucMSC-exes derived from MSCs were round or oval. After incubated with HK-2 cells, CD9mCD63. HucMSC-ex could prevent the cell damage induced by cisplatin by reducing the number of apoptosis and increasing the expression of PCNA. During this process, the ratio of autophagy related index LC3B 鈪，

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