人脐带间质干细胞外泌体对肾间质纤维化的修复及机制研究

发布时间：2018-06-04 02:10

本文选题：人脐带间质干细胞 + 外泌体　；参考：《江苏大学》2017年硕士论文

【摘要】：目的课题组前期研究已发现人脐带间质干细胞(human umbilical cord mesenchy stem cell,huc MSC)及其分泌的外泌体(huc MSC-exosome)能缓解由顺铂诱导的急慢性肾损伤,micro RNA(mi RNA)在其中发挥重要作用。在此基础上,本课题建立了大鼠单侧输尿管结扎(unilateral ureteral obstruction,UUO)肾间质纤维化模型和转化生长因子β1(TGF-β1)细胞诱导模型,通过体内外实验评价huc MSC-exosome缓解肾间质纤维化中的作用,并初步探讨其中的作用机制。方法体内实验分3组,假手术组:SD大鼠经左侧腹部切口打开腹腔寻到输尿管后缝合;UUO组:SD大鼠开腹进行单侧输尿管结扎;huc MSC-exosome干预组:SD大鼠单侧输尿管结扎24h后,经尾静脉注射huc MSC-exosome。小动物活体成像观察huc MSC-exosome在大鼠模型体内的分布。术后不同时间点(12h,24h,48h,72h,7d,14d)采集大鼠血清,检测血清肌酐(Cr)、尿素氮(BUN)水平;单侧输尿管结扎14d后处死大鼠,获取肾组织,HE染色观察不同组别的肾脏组织结构,Masson染色分析胶原沉积情况;免疫组织化学、免疫荧光、Western blot检测肾组织中上皮间质转化(epithelia-mesenchymal transition,EMT)相关分子,上皮钙黏着蛋白(E-cadherin,E-cad)、神经钙黏着蛋白(N-cadherin,N-cad)、赖氨酸氧化酶相关蛋白2(LOXL2)及纤维化相关蛋白α-平滑肌肌动蛋白(α-SMA)、TGF-β1、α1-I型胶原(COL1A1)的表达水平。体外实验:大鼠肾小管上皮细胞(NRK-52E)种植于6孔板上,细胞不做任何处理的为对照组。诱导组采用40ng剂量的TGF-β1诱导NRK-52E细胞24h使细胞发生EMT改变,干预组在TGF-β1诱导后加入huc MSC-exosome共培养48h。倒置显微镜观察各组别的细胞形态和细胞密度。Western blot检测各组细胞中E-cadherin、N-cadherin、LOXL2及α-SMA、TGF-β1、COL1A1的表达,q RT-PCR检测各组NRK-52E细胞mi RNA(mi R-199a、mi R-146b)的表达。结果小动物活体成像结果显示经尾静脉注射的荧光标记huc MSCexosome大部分到达损伤左肾,右肾、脾脏、肺中略有存在,假手术组各脏器均没有荧光表达,显示huc MSC-exosome具有向损伤部位趋化的能力;huc MSC-exosome干预组中血清肌酐尿素氮较UUO组显著下降,提示肾脏功能有所恢复;HE染色结果表明huc MSC-exosome干预组较UUO组,肾小管扩张减缓,炎性细胞浸润减少,肾小管结构完整;Masson染色结果显示,huc MSC-exosome干预组肾间质中胶原沉积减少;与UUO组相比,huc MSC-exosome干预组E-cadherin表达增加,N-cadherin减少,提示间质上皮转化,α-SMA、TGF-β1、COL1A1等纤维化相关蛋白表达下降,表明huc MSC-exosome干预后肾间质纤维化改善。免疫荧光、免疫组化和Western blot检测EMT进程中关键蛋白LOXL2,发现UUO组LOXL2表达上调,而huc MSC-exosome干预后LOXL2表达下降。TGF-β1诱导NRK-52E细胞形态出现显著变化,由原来的光滑卵圆形变为不规则长梭形,细胞密度明显下降,E-cadherin表达下降,N-cadherin增加。Huc MSC-exosome干预后,Western blot检测显示E-cadherin表达增加,N-cadherin、α-SMA、COL1A1、TGF-β1下调,EMT进程得到缓解。q RT-PCR检测三组细胞mi R-199a表达发现,TGF-β1诱导后mi R-199a表达低于对照组,而huc MSC-exosome干预后mi R-199a较诱导组含量明显增加,分析后具有统计学意义。制与LOXL2蛋白和huc MSC-exosome中包裹的mi RNAs有关。结论huc MSC-exosome在体内外均可逆转纤维化并缓解EMT,其作用机制与LOXL2蛋白和hucMSC-exosome中包裹的mi RNAs有关。
[Abstract]:Human umbilical cord mesenchy stem cell (HUC MSC) and its secreted exocrine (HUC MSC-exosome) can alleviate acute and chronic renal injury induced by cisplatin, and micro RNA (micro RNA) plays an important role in the preliminary study. On this basis, the unilateral ureteral junction of rats was established. Unilateral ureteral obstruction (UUO) renal interstitial fibrosis model and transforming growth factor beta 1 (TGF- beta 1) induced model were used to evaluate the role of HUC MSC-exosome in alleviating renal interstitial fibrosis in vivo and in vitro. The mechanism of action was preliminarily discussed in 3 groups and sham operation group: SD rats were cut through the left abdomen. Open abdominal cavity and open the abdominal cavity for posterior ureteral suture; group UUO: SD rats were treated with unilateral ureteral ligation; HUC MSC-exosome intervention group: the distribution of HUC MSC-exosome in the rat model body was observed after the unilateral ureteral ligation in SD rats, and the distribution of HUC MSC-exosome in the rat model body by the tail vein injection of HUC MSC-exosome. small animals. 4D) collect rat serum, detect serum creatinine (Cr), urea nitrogen (BUN) level; kill rats after unilateral ureteral ligation, kill rats, obtain renal tissue, HE staining to observe the renal tissue structure of different groups, Masson staining analysis of collagen deposition, immunohistochemistry, immunofluorescence, Western blot detection of epithelial mesenchymal transformation in renal tissue (epithelia-m) Esenchymal transition, EMT) related molecules, E-cadherin (E-cad), N-cadherin (N-cad), lysine oxidase related protein 2 (LOXL2) and fibrosis related protein alpha smooth muscle actin (alpha -SMA), TGF- beta 1, alpha 1-I collagen (COL1A1) expression level. In vitro experiment: rat renal tubule epithelium fine The cell (NRK-52E) was planted on the 6 orifice plate, and the cells did not do any treatment as the control group. The induction group used the 40ng dose of TGF- beta 1 to induce NRK-52E cell 24h to make the cell EMT change. The intervention group was added to the HUC MSC-exosome co culture 48h. inversion microscope after TGF- beta 1 to observe the cell morphology and cell density.Western blot test of each group. The expression of E-cadherin, N-cadherin, LOXL2 and alpha -SMA, TGF- beta 1, COL1A1, Q RT-PCR detection of NRK-52E cell mi RNA (MI) expression. Results the small animal living body imaging results showed that most of the fluorescent markers injected into the tail vein reached the injured left kidney, the right kidney, spleen, and lung were slightly existing, sham operation. There was no fluorescence expression in all the organs of the group, indicating that HUC MSC-exosome had the ability to chemotaxis to the injured part, and the serum creatinine nitrogen in the HUC MSC-exosome intervention group was significantly lower than that in the UUO group, suggesting that the renal function was restored. The results of HE staining showed that the HUC MSC-exosome intervention group was more than the UUO group, the renal tubule dilation slowed down, the inflammatory cell infiltration decreased and the kidney was reduced. Masson staining showed that the collagen deposition in the renal interstitium of the HUC MSC-exosome intervention group decreased. Compared with the UUO group, the expression of E-cadherin in the HUC MSC-exosome intervention group increased, the N-cadherin decreased, the interstitial epithelium was transformed, the expression of alpha -SMA, TGF- beta 1, COL1A1 and other fibrosis related proteins decreased. The improvement of interstitial fibrosis. Immunofluorescence, immunohistochemistry and Western blot detection of the key protein LOXL2 in the EMT process, it was found that the LOXL2 expression in the UUO group was up-regulated, while the HUC MSC-exosome dry prognosis of LOXL2 expression decreased.TGF- beta 1 induced significant changes in the morphology of NRK-52E cells, from the original smooth oval shape to irregular spindle shape, the cell density decreased significantly. E The expression of -cadherin was decreased, and the prognosis of.Huc MSC-exosome was increased by N-cadherin. Western blot detection showed that the expression of E-cadherin was increased, N-cadherin, alpha -SMA, COL1A1, TGF- beta 1 were down, and EMT process was relieved. The content of 199a increased significantly compared with the induced group, which was statistically significant. It was related to the MI RNAs wrapped in LOXL2 protein and HUC MSC-exosome. Conclusion HUC MSC-exosome can reverse fibrosis in vivo and in vitro and alleviate EMT, and its mechanism is related to MI LOXL2 in LOXL2 protein and hucMSC-exosome.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

【参考文献】