發(fā)布時(shí)間：2019-01-18 13:11

【摘要】：目的肝纖維化是肝硬化的必經(jīng)中間環(huán)節(jié),其主要病理過(guò)程是肝星狀細(xì)胞(hepatic stellate cell,HSC)活化為肌成纖維細(xì)胞、細(xì)胞外基質(zhì)(extracellular matrix,ECM)過(guò)量沉積及肝組織結(jié)構(gòu)破壞。前期研究已表明人臍帶間質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cell,huc MSC)來(lái)源的exosome(huc MSC derived-exosome,huc MSC-Ex)可緩解小鼠肝纖維化,但其具體機(jī)制仍不清楚。本研究在此基礎(chǔ)上,進(jìn)一步探討huc MSC-Ex抗纖維化的作用機(jī)制。方法分離培養(yǎng)huc MSC,蔗糖/重水超速離心法提取huc MSC-Ex,透射電鏡觀察huc MSC-Ex形態(tài),Nano Sight納米顆粒跟蹤分析儀檢測(cè)huc MSC-Ex直徑分布,Western blot檢測(cè)huc MSC-Ex表面標(biāo)志物CD9、CD63。體內(nèi)注射四氯化碳(carbontetrachloride,CCl4)建立BALB/c小鼠肝纖維化模型,并應(yīng)用huc MSC-Ex進(jìn)行治療,分組如下:PBS組(n=10),huc MSC-Ex組(n=10);動(dòng)物活體成像檢測(cè)huc MSC-Ex在小鼠體內(nèi)分布,通過(guò)肝臟大體觀、HE染色、Masson染色等技術(shù)觀察肝纖維化組織結(jié)構(gòu)及膠原沉積,免疫組化和Western blot檢測(cè)賴氨酰氧化酶樣蛋白2(lysyl oxidase-like 2,LOXL2)、HSC活化指標(biāo)成纖維細(xì)胞活化蛋白(Fibroblast Activation Protein,FAP)及α-平滑肌肌動(dòng)蛋白(α-Smooth Muscle Actin,α-SMA)的表達(dá)。體外誘導(dǎo)肝星狀細(xì)胞(LX-2)細(xì)胞活化,與huc MSC-Ex共培養(yǎng),RT-PCR、Western blot和免疫熒光檢測(cè)LOXL2、一型膠原(collagenⅠ,COL1)及FAP表達(dá)。免疫組化及Western blot檢測(cè)肝組織及LX-2細(xì)胞中Yes相關(guān)蛋白(Yes-associated protein,YAP)表達(dá);利用重組腺病毒或質(zhì)粒過(guò)表達(dá)或敲除YAP,Western blot檢測(cè)LOXL2蛋白表達(dá);熒光素酶報(bào)告基因檢測(cè)YAP對(duì)LOXL2的轉(zhuǎn)錄活性,分析YAP對(duì)LOXL2的轉(zhuǎn)錄調(diào)控作用。Western blot檢測(cè)huc MSC-Ex中14-3-3ζ表達(dá);利用重組腺病毒轉(zhuǎn)染huc MSC過(guò)表達(dá)14-3-3ζ,收集提取并鑒定14-3-3ζ過(guò)表達(dá)exosome(Ad-14-3-3ζ-Ex);Ad-14-3-3ζ-Ex及對(duì)照組exosome(Ad-GFP-Ex)與活化的LX-2細(xì)胞共培養(yǎng),Western blot、免疫熒光檢測(cè)LX-2細(xì)胞14-3-3ζ、YAP表達(dá),分析14-3-3ζ與YAP核定位的相關(guān)性。結(jié)果Huc MSC-Ex呈球形膜性囊狀結(jié)構(gòu),直徑30~100nm左右,表達(dá)CD9、CD63等exosome表面標(biāo)志物。動(dòng)物活體成像顯示熒光染料標(biāo)記的huc MSC-Ex主要集中于肝纖維化小鼠肝臟組織,并且在huc MSC-Ex處理的肝臟中檢測(cè)到exosome標(biāo)志物CD63表達(dá)。與PBS組相比,huc MSC-Ex組小鼠肝纖維化減輕,肝組織中壞死結(jié)構(gòu)減少,膠原沉積減弱,同時(shí)LOXL2表達(dá)下調(diào)、HSC活化指標(biāo)FAP及α-SMA表達(dá)降低;huc MSC-Ex與誘導(dǎo)活化的LX-2細(xì)胞共培養(yǎng)后,LOXL2、COL1及FAP表達(dá)下降。在肝纖維化小鼠肝臟及活化的LX-2細(xì)胞中YAP核定位及LOXL2表達(dá)相關(guān);在293T、LX-2、HL7702細(xì)胞中過(guò)表達(dá)YAP導(dǎo)致LOXL2蛋白表達(dá)增加,敲減YAP后,LOXL2蛋白表達(dá)下降;熒光素酶報(bào)告基因檢測(cè)表明YAP對(duì)LOXL2具有轉(zhuǎn)錄調(diào)控作用。在活化的LX-2細(xì)胞中,Ad-14-3-3ζ-Ex具有比Ad-GFP-Ex更強(qiáng)的LOXL2表達(dá)抑制及YAP核定位抑制作用。結(jié)論HucMSC-Ex通過(guò)下調(diào)LOXL2表達(dá)抑制HSC的活化,緩解肝纖維化,其機(jī)制可能通過(guò)轉(zhuǎn)運(yùn)14-3-3ζ抑制YAP核定位相關(guān)。
[Abstract]:Objective Hepatic fibrosis is a necessary intermediate link in liver cirrhosis. The main pathological process is the activation of hepatic stellate cells (hepatic stellate cell,HSC) to myofibroblasts, excessive deposition of extracellular matrix (extracellular matrix,ECM) and destruction of liver tissue structure. Previous studies have shown that exosome (huc MSC derived-exosome,huc MSC-Ex derived from human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cell,huc MSC) can alleviate liver fibrosis in mice, but its mechanism is still unclear. On the basis of this study, the mechanism of anti-fibrosis of huc MSC-Ex was further explored. Methods huc MSC, was isolated and cultured by sucrose / heavy water ultracentrifugation method. Huc MSC-Ex, was extracted by transmission electron microscope. Huc MSC-Ex morphology was observed by, Nano Sight nanoparticle tracking analyzer, huc MSC-Ex diameter distribution was detected by, Western blot, huc MSC-Ex surface marker CD9, was detected by, Western blot CD63. Liver fibrosis model of BALB/c mice was established by intravivo injection of carbon tetrachloride (carbontetrachloride,CCl4) and treated with huc MSC-Ex as follows: PBS group (n + 10), huc MSC-Ex group); Animal in vivo imaging was used to detect the distribution of huc MSC-Ex in mice. Liver fibrosis tissue structure and collagen deposition were observed by gross view of liver, HE staining and Masson staining. The expression of lanyl oxidase like protein 2 (lysyl oxidase-like 2) and 偽 -smooth muscle actin (偽 -Smooth Muscle Actin, 偽 -SMA) was detected by immunohistochemistry and Western blot. Hepatic stellate cells (LX-2) were induced to activate in vitro and co-cultured with huc MSC-Ex. The expression of collagen 鈪，

本文編號(hào)：2410753