本文選題：人臍帶間充質干細胞來源的外泌體　切入點：預防　出處：《江蘇大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:前期的研究結果表明,人臍帶間充質干細胞來源的外泌體(exosome secreted by human umbilical cord mesenchymal stem cells,hucMSC-ex)能有效的預防和減緩順鉑對大鼠腎臟的損傷,但其作用機制尚不明確。本研究構建了hucMSC-ex體外預防順鉑對人腎小管上皮細胞(HK-2)的損傷模型,揭示了hucMSC-ex預防順鉑引起HK-2細胞的分子機制,為臨床上hucMSC-ex預防順鉑誘導腎毒性提供了實驗依據(jù)。方法:常規(guī)培養(yǎng)的HK-2細胞為control組;cis組為16μmol/L順鉑處理16h;ex+cis組為用200μg/ml的hucMSC-ex與HK-2細胞孵育24h后,加入16μmol/L的順鉑繼續(xù)培養(yǎng)16h;3-MA+ex+cis組為100μmol/L的3-MA和200μg/ml的hucMSC-ex同時預孵育HK-2細胞24h后,加入16μmol/L的順鉑繼續(xù)處理16h。透射電子顯微鏡觀察hucMSC-ex的形態(tài),成像流式細胞儀和Western blot檢測hucMSC-ex表面標記CD9、CD63的表達。過表達和敲減功能研究用于探索14-3-3ζ在hucMSC-ex預防順鉑腎毒性中的作用。TUNEL實驗和凋亡雙染流式分析檢測HK-2細胞的凋亡。免疫熒光分析了核增殖抗原(PCNA)的表達。超高分辨率熒光顯微鏡觀察細胞自噬小體形成的數(shù)量和形態(tài)大小。Western blot技術檢測了14-3-3ζ、LC3B、P62、PCNA、Bax等蛋白的表達水平。結果:人臍帶間充質干細胞來源的外泌體(hucMSC-ex)呈圓形或橢圓形,表面表達特異性分子CD9、CD63。HucMSC-ex與HK-2細胞孵育后,可通過減少細胞凋亡數(shù)量、增加PCNA的表達來預防順鉑誘導的細胞損傷。在此過程中,自噬相關指標LC3BⅡ/LC3BⅠ比值增加,自噬底物P62表達水平下降,提示自噬活化。自噬抑制劑3-甲基腺嘌呤(3-MA)則可逆轉hucMSC-ex的順鉑損傷保護作用。HucMSC-ex預處理后,14-3-3ζ表達水平增加。敲減hucMSC-ex中的14-3-3ζ蛋白后,14-3-3ζ表達水平下調,hucMSC-ex誘導細胞自噬的作用減弱,同時預防順鉑的損傷作用也弱化。過表達HK-2細胞中的14-3-3ζ可以增強細胞自噬水平而緩解細胞的順鉑損害。敲減HK-2細胞中的14-3-3ζ可以下調細胞自噬水平,同時增加了順鉑的細胞損傷。而通過hucMSC-ex攜帶14-3-3ζ回補了14-3-3ζ表達后,被增強的順鉑損傷又得以逆轉。結論:HucMSC-ex攜帶的14-3-3ζ蛋白可以預防順鉑對人腎小管上皮細胞(HK-2)的損傷作用。其作用機制可能是通過上調HK-2細胞的自噬水平而保護細胞免受順鉑的損傷。這一研究為hucMSC-ex預防順鉑誘導的腎損傷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，

本文編號：1575910