CTLA4基因修饰骨髓间充质干细胞向肝细胞诱导分化及其免疫抑制功能研究

用携带CTLA4Ig基因的重组腺病毒感染大鼠骨髓间充质干细胞(bone marrow mesenchymal stern cels，BMMSCs),体外向肝细胞诱导分化，并检测其免疫抑制功能.用含有HGF等细胞因子培养液诱导重组腺病毒Ad-CTLA4Ig感染大鼠BMMSCs向肝细胞分化.诱导后的细胞可表达AFP、Alb和CK18等肝细胞标志,同时还具有储存糖原和摄取、排泌靛青绿等肝细胞功能.转基因BMMSCs在未经诱导和诱导后均可表达CTLA4Ig,诱导14天时表达量有所减弱.单向混合淋巴细胞反应证实,诱导7天的转基因BMMSCs具有明显的抑制淋巴细胞反应的作用,其抑制作用明显高于未转基因BMMSCs,且CTLA4Ig基因修饰BMMSCs输注还可以明显延长肝移植大鼠的存活时间.用重组腺病毒Ad-CTLA4Ig对BMMSCs进行基因修饰,一方面不会影响BMMSCs的肝细胞分化潜能，另一方面使BMMSCs的免疫抑制特性得到进一步强化.

The Function Identification of Hepatocyte Diffirentiated From CTLA4-Gene Modified Bone Marrow Mesenchymal Stem Cells

Besides differentiating into multiple mesenchymal tissues, bone marrow mesenchymal stem cells(BMMSCs) can also develop into hepatocyte-like cells in vitro and in vivo. CTLA4Ig-gene modified BMMSCs have a strong immunosuppressive function cultured in both normal and hepatic differentiation medium and this gene modification improves the biological function of BMMSCs. Recombinant adenovirus containing CTLA4Ig gene was constructed, and transferred into rat BMMSCs in vitro. The transfected BMMSCs, cultured in hepatic differentiation medium containing HGF for 14 days, could express hepatocyte-specific markers including AFP, Alb and CK18, and these differentiated cells from BMMSCs also had the capabilities of glycogen deposition and ICG uptake and excretion, which are hepatocyte-specific functions. Expression of CTLA4Ig in the transfected BMMSCs cultured in the normal growth medium or the differentiation medium was confirmed by RT-PCR, Western blot and fluorescent immunocytochemistry, and the expression at 14 d was weaker than that at 7 d. In the model of MLR, the transfected BMMSCs could suppress immune response, and the suppression was statistically stronger than that of BMMSCs at the same number. Even if cultured in hepatic differentiation medium, the transfected BMMSCs also had the same immunosuppression function. In rat liver transplantation model, the CTLA4Ig-gene modified BMMSCs infused into liver graft after operation could prolong the survival time of the recipients. Adenovirus-mediated CTLA4Ig gene transfer into BMMSCs can not change the potential of BMMSCs to differentiate into hepatocytes, and on the other hand, this gene transfer can improve the immunosuppressive function of BMMSCs, which can broaden the applying space of BMMSCs in the clinical therapy for liver diseases.