Stro-1^+和Stro-1^-间充质干细胞免疫抑制作用机制的探讨

Studies on the molecular mechanism involved in different immunosuppressive effects between Stro-1^+ and Stro-1^- MSC

目的探讨细胞因子(TGF-β1、IL-10)与Stro-1+、Stro-1-间充质干细胞(MSC)免疫抑制效应的相关性。方法采用实时定量RT-PCR比较TGF-β1、IL-10在MSC两种细胞亚群中的表达,以磷酸甘油醛脱氢酶(GAPDH)作为内参照。以Pri mer Expre-ssTM软件进行引物设计,以Ct表示PCR的相对产量,ΔCt代表靶基因与GAPDH之间的Ct之差,以2-ΔΔCt表示ΔCt(Stro-1+MSC)与ΔCt(Stro-1-MSC)之间的比值;在3种不同条件下[①MSC单独培养;②MSC与混合淋巴反应体系(MLR)直接接触;③MSC通过Transwell与MLR间接接触],用ELISA法检测Stro-1+与Stro-1-细胞培养上清中TGF-β1的表达。结果IL-10在Stro-1+与Stro-1-MSC中均低表达,而TGF-β1在Stro-1-MSC中显著高表达(P<0.05);ELISA结果显示在3种培养条件下,TGF-β1在Stro-1-MSC中的表达均显著高于Stro-1+MSC(P<0.05)。结论Stro-1+MSC较Stro-1-MSC免疫抑制作用更显著,其与TGF-β1和IL-10的表达无关,表明TGF-β1、IL-10在MSC不同亚群免疫抑制效应中不起主要作用。

Objective To evaluate whether TGF-β and IL-10, which are two well-known inhibitors of T-cell activation and proliferation, are involved in the difference of immunosuppressive effects between Stro-1 and Stro-1 mesenchymal stem cells （MSC）. Methods Quantitative real time RT-PCR was performed to compare the expression levels of TGF-β1 and IL-10 genes between these two MSC subsets. Glyceraldehyde phosphate dehydrogenase （GAPDH） was used as internal reference. The sequences of the forward and reverse primers were designed with Primer ExpressTM software. Total RNA from Stro-1^＋ and Stro-1^- cells was extracted after co-cultured with mixed lymphocyte reaction （MLR） system for 3 days. 1μg of total RNA was reversely transcribed into cDNA, and all reactions were performed in the ABI Prism 7700 Sequence Detection System. The relative quantity of PCR product was evaluated by Ct value. ACt represented the difference between the means of Ct （target gene） and Ct （GAPDH）, while 2 , represented the ratio of ACt（Stro-1^＋ MSC）/ACt（Stro-1 MSC）. Furthermore, the protein content of TGF-β1 in the supernatants of 3 different settings （OMSC alone; （2）MSC plus MLR; （3）MSC plus MLR in Transwell system） were determined by enzyme-linked immunoassays （ELISA）. Results The present study showed that low expression of IL-10 was detected in both subsets （Ct〉35）, while the expression level of TGF-β1 in Stro-1 MSC was significant higher than that detected in Stro-1 MSC （2 = 0. 39, P（0. 05）. In all the 3 different culture conditions, significantly higher expression of TGF-β1 was further detected in Stro-1 MSCs supernatant by ELISA assays （P（0. 05）. Conclusion It is demonstrated that Stro-1 MSC has the more significantly immunosuppressive effect than Stro-1 MSC, whilst TGF-β1 and IL-10 do not, at least not dominant, thus contributes to the difference of the immunosuppressive effects between the MSC subsets.