HGF与CTLA-4Ig双基因修饰增强脐带间充质干细胞的免疫抑制和促增殖作用

Enhanced Immunomodulatory and Pro-proliferation Properties of Human Umbilical Cord Mesenchymal Stem Cells Modified by HGF-gene and CTLA-4Ig-gene

【目的】探讨人脐带间充质干细胞(hUCMSC)经肝细胞生长因子(HGF)和细胞毒性T细胞相关抗原4免疫球蛋白(CTLA4-Ig)双基因修饰后免疫调节能力的变化及其促进肝细胞增殖作用的影响。【方法】酶联合消化法提取并鉴定人脐带间充质干细胞;对照组的hUCMSC转染携带绿色荧光蛋白(EGFP)基因的Ad5-EGFP,实验组的hUCMSC转染携带HGF和CTLA-Ig双基因的腺病毒Ad5-HGF/CTLA-4Ig。流式细胞仪检测对照组hUCMSC转染Ad5-EGFP后72 h表达EGFP细胞比率;实验组Ad5-HGF/CTLA-4Ig转染hUCMSC 72 h后,CCK8检测细胞存活率,同时再次检测hUCMSC免疫表型和向成骨细胞分化的能力,ELISA检测细胞上清中HGF含量,Western blot检测细胞中CTLA4-Ig蛋白的表达水平,通过CCK8法检测双基因修饰后hUCMSC对淋巴细胞增殖的影响及其上清对L02细胞增殖的影响。【结果】携带外源基因的腺病毒可有效转染hUCMSC,不影响其干细胞特性和多向分化能力,经HGF/CTLA-4Ig基因修饰hUCMSC可分泌高浓度HGF并高表达CTLA-4Ig,同时能有效地抑制淋巴细胞增殖,其上清能明显促进肝细胞增殖。【结论】hUCMSC经HGF/CTLA-4Ig基因修饰后生物学特性无明显改变,并能高表达HGF和CTLA-4Ig,其免疫调节及促进肝细胞增殖的能力进一步加强,为肝移植术后存在的肝细胞损伤的保护治疗提供了新思路。

[Objective] To determine whether the human umbilical cord mesenchymal stem cells （hUCMSC） transfected with adenovirus carrying hepatocyte growth factor （HGF） and cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin （CTLA-4Ig） genes still preserve immunosuppressive function as well as pro-proliferation activity. [ Methods] hUCMSC were isolated from Wharton＇s Jelly tissue by combined enzymatic digestion and identified by immunophenotype and differentiation capacity analysis. The hUCMSC in experimental group were transfected by adenovirus-5 carrying HGF and CTLA4-Ig genes （AdS-HGF/CTLA-4Ig） and the hUCMSC in control group were transfected by adenovirus-5 containing EGFP gene （AdS-EGFP）. Expression of EGFP and cell survival rate of hUCMSC transfected with AdS-EGFP and AdS-HGF/CTLA-4Ig were tested by Fluorescence Activating Cell Sorter and CCK8. Then the immunophenotye and differentiation capacity of hUCMSC （HGF/CTLA-4Ig） were further tested after adenoviruses transfection. The expression of HGF and CTLA-4Ig was assessed by ELISA and Western blot 72 h after hUCMSC adenoviral transfection. The proliferation ability of L02 cell by the condition medium of hUCMSC （HGF/CTLA-4Ig） and the suppression to T lymphocyte proliferation by hUCMSC （HGF/CTLA-4Ig） were tested by CCK8 analysis. [ Results ] The double genes modified hUCMSC preserved the same immunophenotype and differentiation capacity as un-transfected hUCMSC. The expression of HGF and CTA-4Ig genes in hUCMSC transfected with AdS-HGF/CTLA-4Ig could be detected 72h after transfection. Proliferation test showed that the conditionmedium of hUCMSC （HGF/CTLA-4Ig） can significant improve the proliferation of L02 cell. When co-cultured with hUCMSC （HGF/ CTLA-4Ig）, the T lymphocyte proliferation was inhibited and the inhibition rate was hUCMSC （HGF/CTLA-4Ig） dosage-dependent. [Conclusion] hUCMSC modified by Ad5-HGF/CTLA-4Ig have the same immunophenotye and differentiation capacity as those untransfected cells. The hUCMSC transfect with HGF/CTLA-4Ig genes can efficiently suppress the proliferation of T cells but promote L02 proliferation by HGF and CTA-4Ig expression.