本文選題：人臍帶間質(zhì)干細(xì)胞 + 外泌體�。� 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的課題組前期研究已發(fā)現(xiàn)人臍帶間質(zhì)干細(xì)胞(human umbilical cord mesenchy stem cell,huc MSC)及其分泌的外泌體(huc MSC-exosome)能緩解由順鉑誘導(dǎo)的急慢性腎損傷,micro RNA(mi RNA)在其中發(fā)揮重要作用。在此基礎(chǔ)上,本課題建立了大鼠單側(cè)輸尿管結(jié)扎(unilateral ureteral obstruction,UUO)腎間質(zhì)纖維化模型和轉(zhuǎn)化生長(zhǎng)因子β1(TGF-β1)細(xì)胞誘導(dǎo)模型,通過(guò)體內(nèi)外實(shí)驗(yàn)評(píng)價(jià)huc MSC-exosome緩解腎間質(zhì)纖維化中的作用,并初步探討其中的作用機(jī)制。方法體內(nèi)實(shí)驗(yàn)分3組,假手術(shù)組:SD大鼠經(jīng)左側(cè)腹部切口打開(kāi)腹腔尋到輸尿管后縫合;UUO組:SD大鼠開(kāi)腹進(jìn)行單側(cè)輸尿管結(jié)扎;huc MSC-exosome干預(yù)組:SD大鼠單側(cè)輸尿管結(jié)扎24h后,經(jīng)尾靜脈注射huc MSC-exosome。小動(dòng)物活體成像觀察huc MSC-exosome在大鼠模型體內(nèi)的分布。術(shù)后不同時(shí)間點(diǎn)(12h,24h,48h,72h,7d,14d)采集大鼠血清,檢測(cè)血清肌酐(Cr)、尿素氮(BUN)水平;單側(cè)輸尿管結(jié)扎14d后處死大鼠,獲取腎組織,HE染色觀察不同組別的腎臟組織結(jié)構(gòu),Masson染色分析膠原沉積情況;免疫組織化學(xué)、免疫熒光、Western blot檢測(cè)腎組織中上皮間質(zhì)轉(zhuǎn)化(epithelia-mesenchymal transition,EMT)相關(guān)分子,上皮鈣黏著蛋白(E-cadherin,E-cad)、神經(jīng)鈣黏著蛋白(N-cadherin,N-cad)、賴氨酸氧化酶相關(guān)蛋白2(LOXL2)及纖維化相關(guān)蛋白α-平滑肌肌動(dòng)蛋白(α-SMA)、TGF-β1、α1-I型膠原(COL1A1)的表達(dá)水平。體外實(shí)驗(yàn):大鼠腎小管上皮細(xì)胞(NRK-52E)種植于6孔板上,細(xì)胞不做任何處理的為對(duì)照組。誘導(dǎo)組采用40ng劑量的TGF-β1誘導(dǎo)NRK-52E細(xì)胞24h使細(xì)胞發(fā)生EMT改變,干預(yù)組在TGF-β1誘導(dǎo)后加入huc MSC-exosome共培養(yǎng)48h。倒置顯微鏡觀察各組別的細(xì)胞形態(tài)和細(xì)胞密度。Western blot檢測(cè)各組細(xì)胞中E-cadherin、N-cadherin、LOXL2及α-SMA、TGF-β1、COL1A1的表達(dá),q RT-PCR檢測(cè)各組NRK-52E細(xì)胞mi RNA(mi R-199a、mi R-146b)的表達(dá)。結(jié)果小動(dòng)物活體成像結(jié)果顯示經(jīng)尾靜脈注射的熒光標(biāo)記huc MSCexosome大部分到達(dá)損傷左腎,右腎、脾臟、肺中略有存在,假手術(shù)組各臟器均沒(méi)有熒光表達(dá),顯示huc MSC-exosome具有向損傷部位趨化的能力;huc MSC-exosome干預(yù)組中血清肌酐尿素氮較UUO組顯著下降,提示腎臟功能有所恢復(fù);HE染色結(jié)果表明huc MSC-exosome干預(yù)組較UUO組,腎小管擴(kuò)張減緩,炎性細(xì)胞浸潤(rùn)減少,腎小管結(jié)構(gòu)完整;Masson染色結(jié)果顯示,huc MSC-exosome干預(yù)組腎間質(zhì)中膠原沉積減少;與UUO組相比,huc MSC-exosome干預(yù)組E-cadherin表達(dá)增加,N-cadherin減少,提示間質(zhì)上皮轉(zhuǎn)化,α-SMA、TGF-β1、COL1A1等纖維化相關(guān)蛋白表達(dá)下降,表明huc MSC-exosome干預(yù)后腎間質(zhì)纖維化改善。免疫熒光、免疫組化和Western blot檢測(cè)EMT進(jìn)程中關(guān)鍵蛋白LOXL2,發(fā)現(xiàn)UUO組LOXL2表達(dá)上調(diào),而huc MSC-exosome干預(yù)后LOXL2表達(dá)下降。TGF-β1誘導(dǎo)NRK-52E細(xì)胞形態(tài)出現(xiàn)顯著變化,由原來(lái)的光滑卵圓形變?yōu)椴灰?guī)則長(zhǎng)梭形,細(xì)胞密度明顯下降,E-cadherin表達(dá)下降,N-cadherin增加。Huc MSC-exosome干預(yù)后,Western blot檢測(cè)顯示E-cadherin表達(dá)增加,N-cadherin、α-SMA、COL1A1、TGF-β1下調(diào),EMT進(jìn)程得到緩解。q RT-PCR檢測(cè)三組細(xì)胞mi R-199a表達(dá)發(fā)現(xiàn),TGF-β1誘導(dǎo)后mi R-199a表達(dá)低于對(duì)照組,而huc MSC-exosome干預(yù)后mi R-199a較誘導(dǎo)組含量明顯增加,分析后具有統(tǒng)計(jì)學(xué)意義。制與LOXL2蛋白和huc MSC-exosome中包裹的mi RNAs有關(guān)。結(jié)論huc MSC-exosome在體內(nèi)外均可逆轉(zhuǎn)纖維化并緩解EMT,其作用機(jī)制與LOXL2蛋白和hucMSC-exosome中包裹的mi RNAs有關(guān)。
[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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692

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本文編號(hào)：1975350