ADSC对GATA3/T-Bet的转录调控及其在ITP中的作用机制探讨

It is now generally accepted that the Th1 / Th2 cytokines imbalance resulted from the immunologic disorder of T cells play a key role in ITP. Our previous study found that ADSCs could inhibit Th1 cytokine secretion and increase the Th2 cytokines level, and improve the level of platelet in ITP mice. And the applicant found that ADSCs could inhibit the proliferationion of T cell induced by PHA, and promoting the expression of IL–10 and IL - 4 in T cells of ITP paitents reducing IFN-γ and IL - 2 by ADSCs/T cells co-culture. However, it is not clear the specific regulation mechanism of Th1 / Th2 cytokines after ADSCs took part in? especially for Th cell differentiation regulation, all the questions were still to be resolved. In recent years, it confirmed that the differentiation imbalances of CD4 + T in ITP are closely related with GATA3 / T - Bet transcription regulation. Combined with previous work, in this study we would observe the Th1/Th2 differentiation in the ADSCs/CD4+ T co-culture system by detecting Th1 / Th2 factors and GATA - 3 / T - bet, etc. Then to silence GATA - 3 or and T - bet genes in the T cells of mice, and detect its upstream and downstream related genes and cytokines. Finally clarify its mechanism and ensure the ADSCs as new targets to control ITP.

目前认为，ITP的发生发展与机体Th1/Th2细胞因子失衡密切相关。申请者已研究发现，ADSCs可抑制Th1因子分泌，并上调Th2因子，提高ITP小鼠血小板水平。且前期对ADSCs/CD4+T共培养发现，ADSCs可使PHA诱导的CD4+T增殖抑制，促进ITP来源的CD4+T细胞IL-4、IL-10表达并下调IFN-γ、IL-2。但ADSCs对Th1/Th2因子的具体调控机制尚有待进一步阐明？近年研究证实，CD4+T在ITP中分化失衡与GATA3/T-Bet的转录调控密切相关。结合前期工作，本研究继续在共培养体系中观察ADSCs对ITP患者CD4+T的分化影响，检测Th1/Th2因子及GATA-3/T-bet等表达。然后沉默小鼠T细胞GATA-3或和T-bet基因，探究其上下游相关基因及细胞因子变化。进而探明ADSCs对ITP的作用机制，为确立ADSCs作为防治ITP的新靶点提供科学依据。

项目背景：ITP是一种器官特异性自身免疫出血性疾病。目前认为，ITP的发病机制与机体Th1/Th2细胞因子失衡密切相关。我们已研究发现，ADSCs可抑制Th1因子分泌，并上调Th2因子，提高ITP小鼠血小板水平。且前期对ADSCs/CD4+T共培养发现，ADSCs可使PHA诱导的CD4+T增殖抑制，促进ITP来源的CD4+T细胞IL-4、IL-10表达并下调IFN-γ、IL-2。但ADSCs对Th1/Th2因子的具体调控机制尚有待进一步阐明？近年研究证实，CD4+T在ITP中分化失衡与GATA3/T-Bet的转录调控密切相关。据此设想，ADSCs可能通过对GATA-3/T-bet的转录调控进而影响Th1/Th2细胞的分化。.主要研究内容：本项目建立了ADSCs与人CD4+T淋巴细胞的共培养体系，观察ADSCs对ITP患者CD4+T的分化影响，检测Th1/Th2因子及GATA-3/T-bet等表达。然后沉默小鼠T细胞GATA-3或和T-bet基因，探究其上下游相关基因及细胞因子变化。此外还建立ADSCs与小鼠T淋巴细胞的共培养体系，检测实验组及对照组相关基因及蛋白变化。.重要研究结果： ①采用密度梯度离心和贴壁培养相结合方法，成功从人脂肪组织中获得纯度较高的人脂肪干细胞。应用免疫法成功建立了ITP小鼠疾病模型。②CD4+T细胞亚群及细胞中STAT4/6表达变化可能促使机体Th1/Th2细胞因子的失衡，导致B淋巴细胞转化及分泌抗血小板抗体增加，CD8+T血小板坏破增多。STAT4/6的表达异常可能在ITP发病机制中起重要作用。③ADSCs可上调ITP小鼠外周血中CD4+CD25+Foxp3+T细胞比例，且可上调ITP小鼠脾脏、胸腺、淋巴结中的Foxp3、TGFβ1、IL-10mRNA表达水平。 ④通过对GATA-3或和T-bet基因表达干扰，可抑制下游Th1因子分泌，并上调Th2因子，提高ITP小鼠血小板水平。.关键数据及其科学意义：ITP的发病机制与机体Th1/Th2细胞因子失衡密切相关。ADSCs可通过对特异性转录因子GATA-3/T-bet的调控以及信号转导通路中相关基因的影响，进而抑制下游Th1因子分泌，并上调Th2因子，从而提高外周血血小板水平。本项目为确立ADSCs作为防治ITP的新方法提供确凿的科学依据。