人羊水间充质干细胞对乙型肝炎肝衰竭患者外周血Th1／Th2的体外调节

In vitro effect of human amniotic fluid-derived mesenchymal stem cells on Th1/Th2 ratio in peripheral blood of hepatitis B patients with liver failure

目的探讨人羊水间充质干细胞（human amniotie fluid—derived mesenchymal stem cells，h-AFMsc）对乙型肝炎肝衰竭患者外周血Th1／Th2的体外调节作用。方法选择2011年2—5月中山大学附属第三医院6例乙型肝炎肝衰竭患者和6名健康志愿者，提取外周血单个核细胞（peripheral blood mononuelear cells，PBMC），同时从孕妇中期羊水中提取h-AFMSC。将h-AFMSC分别与乙型肝炎肝衰竭患者和健康捐献者的PBMC共培养72h，乙型肝炎肝衰竭患者和健康捐献者的PBMC单独培养组作为对照。应用流式细胞仪检测共培养前后不同组PBMCIFNγ（Th1）和IL-4（Th2）的变化情况，计算Th1／Th2比值。对共培养前后数据进行Mauchly球形检验，P〉0．1满足球形假设检验则采用重复测量方差分析。结果共培养72h，乙型肝炎肝衰竭患者PBMC与h—AFMSC共培养组的Th1细胞比例与其PBMC单独培养组比较无明显差异（F=0．82，P〉0．05），但Th2细胞比例明显下降（F=10．24，P〈0．05），从而导致Th1／Th2比值显著上升（F=17．28，P〈0．01）。健康捐献者PBMC与h-AFMSC共培养组的Th1、Th2和Th1／Th2比值与其PBMC单独培养组比较差异均不明显（F值分别为0．54、0．68和0．74，P值均〉0．05）。结论h-AFMSC对乙型肝炎肝衰竭患者的Th1／Th2有体外调节作用，具有作为移植种子来源移植治疗乙型肝炎肝衰竭的可能性。

Objective To investigate the effect of human amniotic fluid-derived mesenchymal stem cells （h-AFMSCs） on Th1/Th2 ratio in peripheral blood of hepatitis B patients with liver failure in vitro. Methods Peripheral blood samples were taken from 6 hepatitis B patients with liver failure and 6 healthy subjects from the Third Affiliated Hospital of Sun Yat-sen University during February and May, 2011. Peripheral blood monouuclear cells （PBMCs） were isolated, h-AFMSCs were extracted from amniotic fluid in mid-pregnacy women, h-AFMSCs and PBMCs were co-cultured in vitro for 72 h, and the monocuhure of PBMCs was taken as the control. Flow cytometry was used to detect the IFN-γ, （ Thl ） and IL-4 （ Th2 ） changes in PBMCs in each group, and the ratio of Th1/Th2 was calculated. Mauchly＇ s sphericity test was performed for data before and after the co-culture, and repeated measure ANOVA was used for data with P 〉 0.1. Results In liver failure patients, there was no significant difference in Thl cells between co-culture group and monocuhure group （ F = 0.82, P 〉 0. 05 ） , but Th2 cells in co-culture group were decreased markedly compared with the monocuhure group （ F = 10.24, P 〈 0.05 ） , which resulted in the increase of the ratio of Th1/Th2 in co-culture group （ F = 17.28, P 〈 O. O1 ）. In control group, there was no significant difference on Thl cells, Th2 cells and Th1/Th2 ratio between co-culture and monoculture groups （ F = 0.54, 0.68 and 0. 74, P 〉 0.05）. Conclusion h-AFMSCs regulate Th1/Th2 ratio in hepatitis B patients with liver failure in vitro, which may be used as a source of transplantation for the treatment of liver failure.