GRIM-19调控Treg/Th17免疫平衡参与原因不明复发性流产的分子机制研究

GRIM-19 is a functional component of mitochondrial complex I and is essential for the maintenance of pregnancy. Our study found that the expression of GRIM-19 decreased in the decidual lymphocytes of unexplained recurrent spontaneous abortion (URSA) patients. And, the percentage of Th17 cells increased and the percentage of Treg cells decreased in peripheral blood mononuclear cells of GRIM-19+/- mouse, which lead to the higher embryo absorption rate in the GRIM-19 +/- pregnant mouse. Therefore, we presume that GRIM-19 may participate in the occurrence of URSA by affecting the balance of Treg/Th17. ROS are mainly produced by mitochondrial complex I, and can regulate the balance of T cells by activating mTOR. In our previous study, we found that the levels of ROS and p-mTOR were increased in the decidual lymphocytes of URSA patients. The phenomenon indicates that the balance of Treg/Th17 is possibly regulated by GRIM-19 through ROS and mTOR. In this study, we will investigate the roles of GRIM-19 in the progress of ROS generation, mTOR activation and Treg/Th17 balance. And we are going to focus on the GRIM-19-ROS-mTOR-Treg/Th17 axis in the occurrence of URSA and its involved mechanism. This study may provide foundation for the elucidation of the molecular mechanism of URSA, and provide basis for its early prediction and targeted reversion of URSA.

GRIM-19是线粒体呼吸链复合物I的基本功能单位，在妊娠维持中发挥重要作用。我们前期研究发现，URSA患者蜕膜淋巴细胞GRIM-19表达下调，GRIM-19+/-小鼠外周血Treg/Th17平衡向Th17偏移，其孕鼠的胚胎吸收率增高，提示GRIM-19可能通过调节Treg/Th17免疫平衡参与URSA发生。线粒体复合物I是ROS产生的主要部位，ROS可通过激活mTOR调节T细胞平衡。我们发现URSA患者蜕膜淋巴细胞中ROS和p-mTOR水平升高，推测GRIM-19可能通过ROS、mTOR调节Treg/Th17平衡。本课题拟深入探讨GRIM-19对ROS产生、mTOR激活和Treg/Th17平衡的调控作用，并进一步验证GRIM-19-ROS-mTOR-Treg/Th17轴在URSA发生中的作用效应和机制。本研究将为阐明URSA发生的分子机制并对其进行早期预测和靶向逆转提供新思路和实验依据。

深入研究URSA的发病机制并寻找早期预测和治疗的靶点是生殖医学领域亟需解决的难题。GRIM-19是线粒体呼吸链复合物I的基本功能单位，在妊娠维持中发挥重要作用。本项目从临床标本、动物模型以及体外细胞实验三个方面开展研究，证实GRIM-19异常表达通过ROS-mTOR途径调控Treg/Th17细胞免疫平衡参与URSA的发生。临床标本中的研究发现，URSA患者外周血单个核细胞中发挥免疫耐受作用的Treg细胞数量减少，介导炎性反应的Th17细胞数量增多。项目组分离URSA患者外周血淋巴细胞，发现其GRIM-19表达显著降低，ROS水平明显提高，AKT/mTOR信号通路激活。动物模型方面，项目组构建GRIM19+/+和GRIM-19+/-孕鼠模型，发现GRIM-19+/-孕鼠的胚胎吸收率显著提高，提示GRIM-19表达降低与流产发生相关。分离GRIM-19+/-小鼠脾脏淋巴细胞，体内实验证实GRIM-19低表达时，淋巴细胞ROS水平提高，AKT/mTOR信号通路激活。体外细胞实验中，项目组通过转染siRNA下调人T淋巴瘤Jurkat细胞中GRIM-19表达水平，证实下调GRIM-19表达，Jurkat细胞ROS水平提高，且AKT/mTOR信号通路激活。为研究GRIM-19是否通过ROS调控AKT/mTOR信号通路激活，我们进一步采用N-乙酰半胱氨酸（NAC）处理细胞，发现下调细胞ROS水平后，AKT/mTOR信号通路激活被抑制。以上研究证实GRIM-19通过ROS调控AKT/mTOR信号通路的激活，进而影响Treg/Th17细胞免疫平衡。该项目的研究阐明了线粒体蛋白GRIM-19表达异常在原因不明复发性流产发生中的作用及机制，为原因不明复发性流产的早期预测和靶向治疗提供新的思路和实验依据。