hMSC对异体DC-CIK细胞分泌细胞因子的影响

Effects of human bone marrow mesenchymal stem cells on cytokines secretion from allogeneic dendritic cell activated cytokine-induced killer cells

目的:通过检测人骨髓间充质干细胞(hMSCs)对共培养的异体树突状细胞活化的细胞因子激活的杀伤细胞(DC-CIK细胞)分泌细胞因子IFN-γ、TNF-α、IL-10、IL-6、IL-4和IL-2的影响,探讨hMSCs的免疫调控机制。方法:从健康人骨髓液中分离、培养hMSCs,通过观察细胞形态,检测其分化为神经元样细胞的神经元烯醇化酶(NSE),脂肪样细胞的油红O染色以及CD29和CD44的表达鉴定hMSCs。从健康人的外周血分离、培养DC,CIK细胞。用流式细胞术(FCM)检测CD1α,HLA-DR的表达来鉴定DC;检测CIK细胞CD3+CD56+的表达。将hMSCs与DC-CIK细胞以1:10的比例共培养4d,用FCM检测培养上清中IFN-γ、TNF-α、IL-10、IL-6、IL-4和IL-2的含量。结果:hMSCs呈长梭形,表达CD29和CD44的阳性细胞数分别为96.6%,94.6%,分化为神经元样细胞的NSE阳性;脂肪样细胞的油红O染色为阳性。DC细胞表达CD1α,HLA-DR的阳性细胞数分别为(91.9±10.04)%,(88.80±8.92)%。DC与CIK细胞共培养第4天表达CD3+CD56+的细胞数为29.23±12.23%。hMSCs与DC-CIK细胞共培养第4天的培养上清中IFN-γ为(135.05±48.19)ng/L;TNF-α为(11.33±1.42)ng/L;IL-10为(10.15±2.25)ng/L;IL-6为(494.63±235.222)ng/L;IL-4为(7.07±2.30)ng/L;IL-2为(1074.63±303.74)ng/L。对照组(DC-CIK细胞)的IFN-γ为(717.60±248.15)ng/L;TNF-α为(17.78±7.52)ng/L;IL-10为(29.95±12.76)ng/L;IL-6为(8.03±0.21)ng/L;IL-4为(9.08±3.07)ng/L;IL-2高于1250ng/L。结论:DC-CIK细胞与hMSCs共培养后,IFN-γ分泌减少,IL-10轻微下调。表明hMSCs可能通过干扰DC-CIK细胞分泌细胞因子来发挥免疫调节作用。

AIM：To study the effect of human bone marrow derived mesenchymal stem cells （hMSCs） on cytokines secretion （IFN-γ,TNF-α,IL-10,IL-6,IL-4 and IL-2） of allogeneic DC-CIK cells （in co-culture of CIK cells with DC）,which investigate the mechanism of immunoregulation induced by hMSCs.METHODS：The hMSCs from bone marrow were isolated,expanded and identified by cell morphology,differentiation into neuron-like cells with NSE,fat-like cells with red-oil stain,and expression of CD29,CD44.The DC and CIK cells from peripheral blood were isolated,expanded and identified by CD1α,HLA-DR or CD3＋CD56＋.The hMSCs were co-cultured with DC-CIK cells according to ratio 1：10.The expression of the six cytokines in supernatant was evaluated by flow cytometry after 4 days of DC-activated CIK cells in co-culture with hMSCs.RESULTS：The hMSCs displayed a fibroblast-like morphology and the positive cells of CD29 and CD44 were 96.6%,94.6%,which have the capacity of differentiation into neuron-like cells with expressed NSE as well as fat-like cells with red-oil stain positive.The expression of CD1α,HLA-DR in DC was （91.9±10.04）% and （88.8±8.92）%.The CD3＋CD56＋ double positive cells in DC-CIK cells was （29.23±12.23）% compared to CIK cells with （15.98±2.49）%.The cytokines secretion of DC-CIK cells in co-culture with hMSCs was IFN-γ （135.05±48.19） ng/L;TNF-α （11.33±1.42） ng/L;IL-10 （10.15±2.25） ng/L;IL-6 （494.63 ±235.222） ng/L;IL-4 （7.07±2.30） ng/L and IL-2 （1074.63±303.74） ng/L.In control group （DC-CIK cells） the secretion of IFN-γ,TNF-α,IL-10,IL-6,IL-4 and IL-2 was （717.6±248.15） ng/L;（17.78±7.52） ng/L;（29.95±12.76） ng/L;（8.03±0.21） ng/L,（9.08±3.07） ng/L as well as IL-2 1 250 ng/L.CONCLUSION：The secretion of IFN-γ and IL-10 were down-regulated.It probably implied that hMSCs had the effect of immunoregulation on DC-CIK cells.