供体骨髓间充质干细胞抑制肝移植大鼠免疫排斥的研究

目的 探讨供体骨髓间充质干细胞(MSCs)抑制肝移植大鼠免疫排斥的作用.方法 实验按照随机数字表法分为3组,每组14对雌性大鼠,建立Wistar-SD大鼠原位肝移植模型.A组:肝移植术中输注生理盐水;B组:肝移植术后隔天一次给予他克莫司(0.25 mg/kg)灌胃2周;C组:肝移植术中输注与A组同剂量的雄性Wistar大鼠的MSCs.观察术后第10天肝功能的变化、病理改变、TGF-β1与IL-10的表达、Y染色体定位及受体存活时间.采用完全随机化设计多组比较方差分析AST、ALT值,LSD法分析组间差异;病理分级采用ridit法分析;Kaplan-Meier法计算存活率,Log-rank检验进行生存分析.结果 肝移植术后第10天A、B、C组ALT分别为(756±104)、(197±49)、(103±31)U/L,AST分别为(635±134)、(331±78)、(150±38)U/L,3组ALT和AST比较差异均有统计学意义(F=158,265,P<0.05);两两比较发现,B组及C组均优于A组,且C组优于B组.A组ridit均值为0.8333,为重度排斥,B、C两组ridit均值分别为0.4583、0.2083,排斥反应较A组显著减轻,且C组较B组级别更低.A、B、C组TGF-β1阳性表达率分别为18%±5%、69%±20%及85%±24%,3组比较差异有统计学意义(F=191,P<0.01).A、B、C组IL-10阳性表达率分别为21%±5%、75%±14%及91%±21%,3组比较差异有统计学意义(F=672,P<0.01);B、C两组TGF-β1和IL-10呈强阳性表达且C组比B组更明显,而A组则为弱阳性表达.原位杂交检测发现,C组含有带Y染色体的雄性MSCs,主要在汇管区聚集.A、B、C组大鼠50 d存活率分别为0、10%、90%,3组大鼠存活率比较差异有统计学意义(χ~2=36,P<0.01),C组中位存活时间为63 d,较A组11 d延长,且C组长于B组.结论 肝移植同时向供体输注MSCs能够抑制受体对移植肝的免疫排斥.

Study on the inhibitive effect of mesenchymal stem cells on the immunological rejection in rats after liver transplantation

Objective To investigate the inhibitive effect of mesenchymal stem cells (MSCs) on the immunological rejection in rats after liver transplantation. Methods The recipients and donors were female SD rats and Wistar rats. All rats were randomly divided into 3 groups (28 rats in each group). Rats in group A were infused with normal saline; rats in group B received FK506 (0.25 mg/kg) every 2 days for 2 weeks after liver transplantation; rats in group C were injected with MSCs from male Wistar rats during liver transplantation. The pathological changes, expression of TGF-β1 and IL-10, Y chromosome location, changes of liver function and the survival of the recipients were detected on postoperative day 10. The levels of ALT and AST were analyzed by com-pletely randomised design analysis of variance, and the difference among the 3 groups were analyzed by LSD. Ridit was used to analyze the pathological grading. The survival was analyzed by Log-rank test after the Kaplan-Meier survival curve was drawn. Results The values of ALT and AST were (756±104)U/L and (635±134)U/L in group A, (197±49)U/L and (331±78) U/L in group B, (103±31)U/L and (150±38) U/L in group C, respectively. The difference in the level of ALT and AST among the 3 groups had statistical significance (F = 158, 265, P < 0.05). The liver function of rats in group B and C was better than those in group A (P < 0.01), and the liver function of rats in group C was better than those in group B. The mean values of ridit in group A, B and C were 0.8333, 0.4583 and 0.2083, respectively. The expression rates of TCG-β1 in group A, B and C were 18%±5% , 69%±20% and 85%±24% , with statistical difference among the 3 groups (F=191, P <0.01). There was a significant difference in IL-10 expression among group A (21%±5%), group B (75%±14%) and group C (91%±21%) (F=672, P<0.01). The TCG-β1 and IL-10 had strong positive expression in group B and C, and the expression of TCG-β1 and IL-10 was much stronger in group C than in group B; while the expres-sion of TCG-β1 and IL-10 was weak positive in group A. MSCs cells with Y chromosome were positively stained and were concentrated at the portal area in group C. The 50-day survival rate of rats in group A, B and C were 0, 10% and 90% , respectviely, with significant difference (χ~2=36, P < 0.01). The median survival time of rats in group C was 63 days, which was longer than that in group A and B. Conclusion Simultaneous injection of MSCs from donors during liver transplantation can inhibite the immunological rejection of recipients to the liver graft.