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目的:建立重组腺病毒介导肝细胞生长因子HGF促ADSCs定向分化肝细胞的方法,并对其参与肝损伤修复能力进行验证,为作为治疗肝损伤细胞来源提供参考。方法:采用消化培养的方法,分离SD大鼠腹股沟脂肪组织ADSCs细胞,连续传代3次对其进行纯化培养,利用形态学鉴定、流式细胞术检测ADSCs表面标志物方法对其间充质干细胞样特征进行鉴定,加入成脂肪细胞诱导液观察其分化成脂肪细胞的能力;构建腺病毒表达HGF载体Adeno-HGF-EGFP,并转染ADSCs细胞,利用免疫细胞化学染色方法检测肝细胞标志分子表达水平;最后建立大鼠肝损伤动物模型,观察Adeno-HGF-EGFP转染的ADSCs细胞参与肝损伤修复能力情况。结果:分离的ADSCs细胞形态较为一致,绝大多数呈梭形,排列不规则。流式细胞术结果显示,该细胞表达CD29、CD90、CD106等间充质干细胞细胞表面标记物,低表达造血干细胞细胞表面标记物CD34、CD45,同时,分离的ADSCs细胞具有诱导分化成脂肪细胞能力;Adeno-HGF-EGFP转染ADSCs后,AFP、ALB、CK18等肝细胞特异性分子表达水平升高;经尾静脉注射ADSCs细胞后,肝损伤大鼠的AST、ALT、TBIL等分子表达水平恢复正常。结论:建立了重组腺病毒介导肝细胞生长因子HGF促ADSCs定向分化肝细胞的方法,并且表达HGF的ADSCs细胞具有修复大鼠肝损伤模型能力,这为通过细胞治疗肝损伤提供了新的细胞来源。
OBJECTIVE: To establish a method of adenovirus-mediated hepatocyte growth factor-HGF-mediated differentiation of hepatocytes into ADSCs, and to verify its ability to participate in the repair of liver injury, so as to provide reference for the treatment of liver injury cells. Methods: ADSCs cells were isolated from the inguinal adipose tissue of SD rats by digestion culture. The cells were cultured and purified three times in succession. Morphological identification and flow cytometry were used to detect ADSCs surface markers for their characteristics of mesenchymal stem cells HGF-EGFP was constructed and transfected into ADSCs. Immunocytochemical staining was used to detect the expression level of hepatocyte marker molecules. Finally, an animal model of hepatic injury in rats was established to observe the ability of ADSCs transfected with Adeno-HGF-EGFP to repair hepatic injury. Results: The morphology of the isolated ADSCs was more consistent, the vast majority of spindle cells were arranged irregularly. Flow cytometry results showed that the cells expressed CD29, CD90, CD106 and other mesenchymal stem cell surface markers, low expression of hematopoietic stem cell surface markers CD34, CD45, at the same time, isolated ADSCs cells have the ability to differentiate into adipocytes After Adeno-HGF-EGFP was transfected into ADSCs, the expressions of AFP, ALB, CK18 and other hepatocyte-specific molecules were increased. After ADSCs were injected into tail vein, the expression of AST, ALT and TBIL in liver injury rats recovered normal. CONCLUSIONS: A recombinant adenovirus-mediated hepatocyte growth factor-HGF-directed differentiation of hepatocytes into ADSCs was established and ADSCs expressing HGF had the ability to repair rat liver injury models, which provided new cells for the treatment of liver injury by cells source.