【摘要】 目的:探讨人早孕蜕膜基质细胞(DSCs)对育龄女性外周血Treg细胞的影响。方法:分离培养育龄期女性外周血淋巴细胞(PBLC);分离培养正常人早孕DSCs并传至第3代或第4代,后分为4组。1对照组:单纯培养PBLC;2共培养组:PBLC+DSCs培养组;3脂多糖(LPS)刺激组:PBLC+DSCs+LPS培养组;4吡咯烷二硫代氨基甲酸盐(PDTC)阻断组:PBLC+DSCs+LPS+PDTC培养组。蛋白质印迹法(Western blotting)检测核因子κB抑制因子α(IκBα)蛋白表达水平;逆转录聚合酶链反应(RT-PCR)检测Fox P3 m RNA表达水平;流式细胞仪检测Treg细胞亚群比例。结果:分离培养的人原代DSCs的纯度大于95%。Western blotting和RT-PCR结果显示,共培养组IκBα蛋白、Fox P3 m RNA表达明显高于其余3组(P<0.05);与共培养组相比,LPS刺激组IκBα蛋白、Fox P3 m RNA表达降低(P<0.05),与LPS刺激组相比,PDTC阻断组IκBα蛋白、Fox P3 m RNA表达升高,但仍低于共培养组(P<0.05)。流式细胞术检测显示,共培养组Treg细胞亚群比例高于其余3组(P<0.05);与共培养组相比,LPS刺激组Treg细胞亚群比例下降;PDTC阻断组Treg细胞亚群比例高于LPS刺激组,但仍低于共培养组(P<0.05)。结论:人早孕DSCs能上调育龄期女性外周血淋巴细胞中Treg细胞亚群比例,加入LPS刺激后Treg细胞亚群比例下降,可能与级联活化IκB激酶,降解IκBα,活化NF-κB信号通路,从而抑制Fox P3表达增高有关。更多还原

【Abstract】 Objective: To investigate the effect of human early decidual stromal cells(DSCs) on the regulatory T cell(Treg cell) subsets in peripheral blood lymphocytes(PBLCs) of women with reproductive age.Methods:Treg cells of peripheral blood were isolated and cultured. DSCs were isolated and cultured from the decidual tissue of normal early pregnancy. When DSCs were passaged to the third or fourth generation, DSCs were randomly divided into 4 groups. 1Control group: PBLC cultured only, 2Co-culture group: PBLC+DSCs cultured together, 3 LPS stimulation group: PBLC +DSCs Co-culture and LPS stimulation, 4 PDTC blockade group: LPS stimulation group plus PDTC blocking. The IκBα protein was detected by Western blotting. The Fox P3 m RNA was detected by RT-PCR. The Treg cell subsets were detected by flow cytometry. Results:The purity of DSCs was more than 95%. The levels of IκBα protein and Fox P3 m RNA in the Co-culture group were significantly higher than those in the other three groups(P<0.05). The levels of IκBα protein and Fox P3 m RNA in the LPS stimulation group were significantly decreased when compared with the Co-culture group, while two parameters in the PDTC block group were significantly increased when compared with the LPS stimulation group although be still less than those in the Co-culture group(P<0.05). Flow cytometry showed that the percentage of Treg cell subset in the Co-culture group was significantly higher than that in the other three groups(P<0.05). Compared to the Co-culture group, this value in the LPS stimulate group was significantly decreased. Interestingly, this parameter in the PDTC blocked group was significantly increased than that in the LPS stimulate group, although be still less than that in the Co-culture group(P <0.05). Conclusions:Human early DSCs can increase the proportion of Treg cell subsets in the peripheral blood lymphocytes from childbearing age women. The potential mechanism of the decreased proportion of Treg cell subsets induced by LPS is related to the activation of IκB kinase and the degradation IκBα, then the activation of the NF-κB signaling pathway and thus inhibition of Fox P3 expression.更多还原