间充质干细胞和Stro-1^+亚群免疫抑制的比较及IDO1、IDO2在其中的作用

Comparison of immunosuppression between MSC and Stro-1^+ MSC and the role of IDO1 and IDO2 in the effect

目的:比较间充质干细胞(MSC)和经Stro-1抗体分选的Stro-1+MSC对淋巴细胞增殖反应的影响;探讨吲哚胺2,3二氧化酶1(IDO1)和IDO2在上述两种间充质干细胞免疫调节功能中的作用。方法:从全髋关节置换术患者的近端股骨粉碎物中得到细胞悬液,培养MSC,并筛选Stro-1+MSC。体外设立单向混合淋巴细胞反应(MLR)体系,分别加入1×103～5×104个经照射的Stro-1+MSC或MSC,检测对T淋巴细胞增殖反应的影响。再分别加入IDO1和IDO2的特异性抑制剂L-1MT和D-1MT,检测T淋巴细胞增殖反应的变化;以实时定量PCR法比较IDO1、IDO2在Stro-1+MSC和MSC中的表达,以GAPDH作为内参照基因。结果:(1)不同剂量组,Stro-1+MSC对MLR的抑制效应均显著强于相同剂量的未经分选的MSC(P<0.05);(2)加入L-1MT后,Stro-1+MSC和MSC对MLR的抑制作用均显著减弱(P<0.05),而加入D-1MT后Stro-1+MSC和MSC对MLR的抑制作用与未加入抑制剂的对照组相比无显著变化(P>0.05);(3)加入L-1MT后,Stro-1+MSC和MSC对MLR抑制的显著差异不复存在(P>0.05),加入D-1MT后Stro-1+MSC对MLR的抑制作用仍显著强于未经分选的MSC(P<0.05);(4)实时定量PCR检测发现IDO1在Stro-1+MSC中的表达显著高于MSC(P<0.05),而IDO2的表达在两者间无差异(P>0.05)。结论:IDO1在MSC的免疫调节和Stro-1+MSC的强势抑制功能中发挥重要作用,该作用可被IDO1的特异性抑制剂L-1MT所抑制,而IDO2的特异性抑制剂D-1MT无此作用。

Objective： To compare the immunosuppressive effect between mesenchymal stem cells （MSC） and Stro-1 positive subset and detect the role of indoleamine 2,3-dioxygenasel （IDOl） and IDO2 in their effects. Methods： MSC were isolated and cultured from proxi- mal femur marrow of patients undergoing hip replacement, Stro-1 positive cells were sorted out. 1×103^-5×10^4 irradiated Stro-1 ＋ MSC or MSC were added into mixed lymphocyte reaction （MLR） , the proliferation of T lymphocytes was measured. L-1MT and D-1MT were added into the reaction systems, respectively, changes of T lymphocytes proliferation were detected. Real time PCR was preformed to compare the expression levels of IDOl and IDO2 genes between MSC and Stro-1 ＋ MSC. GAPDH was used as internal reference. Re- sults： （ 1 ）In various dose of cells, Stro-1 ＋MSC elicited significant inhibition of lymphocyte proliferation in MLR than unselected MSC （P〈 0.05）; （2）The inhibitory effects of both Stro-1 ＋MSC and MSC decreased significantly when L-1MT was added （P〈0.05）, but they did not change when D-1MT was added （P〉0.05）; （3）The significant difference of the suppressive effect between Stro-1 ＋MSC and MSC was disappeared when L-1MT was added （P〉0.05）, but it still existed when D-1MT was added （P〈0.05）; （4）Real time PC R indicated that the expression level of IDO 1 gene in Stro-1＋ MSC was significantly higher than that in unselected MSC （P〈0.05）, but IDO2 gene expression lev- el had no significant difference between the two groups （P〉0.05）. Conclusion： It is IDOl, not IDO2, that play a major role in the immuno- suppressive effect of MSC and the profound inhibition of Stro-1＋MSC. Such effects can be abrogated by L-1MT, D-1MT is inefficient.