Objective: To investigate the role of bone marrow mesenchymal stem cells (BMSCs) with CTLA4Ig and CD40LIg gene modification in rejection reaction after liver transplantation in rats and possible mechanisms. Methods: The modified Kamada's two-cuff technique was used to establish a Lewis-BN rat model of orthotopic liver transplantation, and a total of 75 rats were randomly divided into groups A, B, C, D, and E, with 15 rats in each group. The rats in group A (control group) were given infusion of isotonic saline via the portal vein during liver transplantation, those in group B (BMSC group) were given infusion of BMSCs via the portal vein during liver transplantation, those in group C (BMSCs with CTLA4Ig gene modification) were given infusion of BMSCs carrying the CTLA4Ig gene via the portal vein during liver transplantation, those in group D (BMSCs with CD40LIg gene modification) were given infusion of BMSCs carrying the CD40LIg gene via the portal vein during liver transplantation, and those in group E (BMSCs with CTLA4Ig and CD40LIg gene modification) were given infusion of BMSCs carrying CTLA4Ig and CD40LIg gene modification via the portal vein during liver transplantation. Postoperative survival and change in liver function were observed. HE staining was used to observe the pathomorphological changes of the graft liver, and ELISA was used to measure the levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), and interferon-γ (IFN-γ) in peripheral blood. A one-way analysis of variance was used for comparison of means of multiple samples, and the Kaplan-Meier survival curve analysis was used for comparison of survival rates between multiple groups. Results: Group E had a significantly longer survival time after surgery than groups A, B, C, and D (P < 0.05), groups C and D had a significantly longer survival time than groups A and B (P < 0.05), and there was no significant difference between groups C and D (P > 0.05). On day 10 after surgery, group A had significantly higher levels of alanine aminotransferase and total bilirubin than the other four groups (P < 0.05). HE staining showed severe rejection reaction in group A, moderate rejection reaction in group B, and mild rejection reaction in groups C and D; pathological examination showed no marked rejection reaction in group E. Group A had significant increases in the levels of IL-2 and IFN-γ and significant reductions in the levels of IL-4 and IL-10 after surgery compared with the other four groups (all P < 0.05). Conclusion: Infusion of BMSCs with modification of both CTLA4Ig and CD40LIg genes can significantly inhibit acute rejection reaction after liver transplantation in rats and effectively prolong the survival time of the graft liver, with a better effect than infusion of BMSCs alone or BMSCs with modification of CTLA4Ig or CD40LIg gene.
目的： 探讨细胞毒性T淋巴细胞相关抗原4免疫球蛋白（CTLA4Ig）和白细胞分化抗原40配体免疫球蛋白（CD40LIg）基因修饰的骨髓间充质干细胞（BMSCs）在大鼠肝移植排斥反应中的作用及其可能的机制。 方法： 采用改良"Kamada"双袖套法建立Lewis-BN大鼠原位肝移植模型75只，随机分为5组，每组15只。A组（对照组）：肝移植时门静脉输注等渗盐水；B组（BMSCs组）：肝移植时门静脉输注BMSCs；C组（CTLA4Ig转基因BMSCs组）：肝移植时门静脉输注携带CTLA4Ig基因的BMSCs；D组（CD40LIg转基因BMSCs组）：肝移植时门静脉输注携带CD40LIg基因的BMSCs；E组（CTLA4Ig和CD40LIg转基因BMSCs组）：肝移植时门静脉输注CTLA4Ig和CD40LIg转基因的BMSCs。观察大鼠术后存活时间和肝功能变化情况，HE染色观察移植肝脏病理形态学改变，酶联免疫吸附法检测大鼠外周血中白细胞介素2（IL-2）、IL-4、IL-10、干扰素（IFN）γ水平。多组样本均数的比较采用单因素方差分析，多组生存率比较采用Kaplan-Meier生存曲线分析。 结果： 术后E组大鼠存活时间显著长于A、B、C组和D组（F = 18.634，P = 0.018），C组和D组又较A、B组显著延长（P < 0.05），C组和D组的差异无统计学意义（P > 0.05）。术后第10天A组丙氨酸氨基转移酶、总胆红素水平明显高于4个治疗组（P < 0.05）。A组术后移植肝HE染色显示重度排斥反应；B组显示中度排斥反应；C组和D组显示轻度排斥反应；E组病理学检查未显示明显排斥反应。术后与4个治疗组相比，A组IL-2、IFNγ水平明显升高，而IL-4、IL-10水平显著降低（P值均< 0.05）。 结论： CTLA4Ig转基因BMSCs联合CD40LIg转基因BMSCs输注能显著抑制肝移植大鼠急性排斥反应，有效延长移植肝存活时间，其效果要优于单纯BMSCs组和单基因修饰BMSCs组。.
Keywords: Bone mesenchymal stem cells; Liver transplantation; Rejection.