人脐带间质干细胞来源的exosome下调LOXL2缓解小鼠肝纤维化的作用机制研究
发布时间：2019-01-18 13:11
【摘要】：目的肝纤维化是肝硬化的必经中间环节,其主要病理过程是肝星状细胞(hepatic stellate cell,HSC)活化为肌成纤维细胞、细胞外基质(extracellular matrix,ECM)过量沉积及肝组织结构破坏。前期研究已表明人脐带间质干细胞(human umbilical cord mesenchymal stem cell,huc MSC)来源的exosome(huc MSC derived-exosome,huc MSC-Ex)可缓解小鼠肝纤维化,但其具体机制仍不清楚。本研究在此基础上,进一步探讨huc MSC-Ex抗纤维化的作用机制。方法分离培养huc MSC,蔗糖/重水超速离心法提取huc MSC-Ex,透射电镜观察huc MSC-Ex形态,Nano Sight纳米颗粒跟踪分析仪检测huc MSC-Ex直径分布,Western blot检测huc MSC-Ex表面标志物CD9、CD63。体内注射四氯化碳(carbontetrachloride,CCl4)建立BALB/c小鼠肝纤维化模型,并应用huc MSC-Ex进行治疗,分组如下:PBS组(n=10),huc MSC-Ex组(n=10);动物活体成像检测huc MSC-Ex在小鼠体内分布,通过肝脏大体观、HE染色、Masson染色等技术观察肝纤维化组织结构及胶原沉积,免疫组化和Western blot检测赖氨酰氧化酶样蛋白2(lysyl oxidase-like 2,LOXL2)、HSC活化指标成纤维细胞活化蛋白(Fibroblast Activation Protein,FAP)及α-平滑肌肌动蛋白(α-Smooth Muscle Actin,α-SMA)的表达。体外诱导肝星状细胞(LX-2)细胞活化,与huc MSC-Ex共培养,RT-PCR、Western blot和免疫荧光检测LOXL2、一型胶原(collagenⅠ,COL1)及FAP表达。免疫组化及Western blot检测肝组织及LX-2细胞中Yes相关蛋白(Yes-associated protein,YAP)表达;利用重组腺病毒或质粒过表达或敲除YAP,Western blot检测LOXL2蛋白表达;荧光素酶报告基因检测YAP对LOXL2的转录活性,分析YAP对LOXL2的转录调控作用。Western blot检测huc MSC-Ex中14-3-3ζ表达;利用重组腺病毒转染huc MSC过表达14-3-3ζ,收集提取并鉴定14-3-3ζ过表达exosome(Ad-14-3-3ζ-Ex);Ad-14-3-3ζ-Ex及对照组exosome(Ad-GFP-Ex)与活化的LX-2细胞共培养,Western blot、免疫荧光检测LX-2细胞14-3-3ζ、YAP表达,分析14-3-3ζ与YAP核定位的相关性。结果Huc MSC-Ex呈球形膜性囊状结构,直径30~100nm左右,表达CD9、CD63等exosome表面标志物。动物活体成像显示荧光染料标记的huc MSC-Ex主要集中于肝纤维化小鼠肝脏组织,并且在huc MSC-Ex处理的肝脏中检测到exosome标志物CD63表达。与PBS组相比,huc MSC-Ex组小鼠肝纤维化减轻,肝组织中坏死结构减少,胶原沉积减弱,同时LOXL2表达下调、HSC活化指标FAP及α-SMA表达降低;huc MSC-Ex与诱导活化的LX-2细胞共培养后,LOXL2、COL1及FAP表达下降。在肝纤维化小鼠肝脏及活化的LX-2细胞中YAP核定位及LOXL2表达相关;在293T、LX-2、HL7702细胞中过表达YAP导致LOXL2蛋白表达增加,敲减YAP后,LOXL2蛋白表达下降;荧光素酶报告基因检测表明YAP对LOXL2具有转录调控作用。在活化的LX-2细胞中,Ad-14-3-3ζ-Ex具有比Ad-GFP-Ex更强的LOXL2表达抑制及YAP核定位抑制作用。结论HucMSC-Ex通过下调LOXL2表达抑制HSC的活化,缓解肝纤维化,其机制可能通过转运14-3-3ζ抑制YAP核定位相关。
[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 伪 -smooth muscle actin (伪 -Smooth Muscle Actin, 伪 -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，
本文编号：2410753
[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 伪 -smooth muscle actin (伪 -Smooth Muscle Actin, 伪 -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，
本文编号：2410753
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