TIMP-3缺失对子宫免疫微环境及母胎界面髓系细胞分化的调控研究

The immunocytic infiltration occurs during the several stages of pregnancy, such as decidualization of uterine endometrium, implantation of blastocyte, and pregnancy maintenance. The aberrant expression of the immunocytes is one of the crucial factors of pathological pregnancy, pregnancy complications and infertility, and this abnormity is associated with many factors at the maternal-fetal interface. TIMP-3 has a unique expression profile in the uterine microenvironment. The deficiency or low-expression of TIMP-3 is associated with the high risk of pre-eclampsia and endometriosis, suggesting its importance in the uterine immuno-microenvironment. However, the specific effects and regulation mechanism are still not fully understood. The myeloid-derived immunocytes are essential components of the uterine immuno-microenvironment, and are involved in the regulation of many physiological processes during pregnancy, including immuno-response and angiogenesis. Exploring the regulation mechanism of their differentiation is of great significance in the field of reproductive immunology. There are clear evidences that the expression of TIMP-3 was closely associated with the differentiation of myeloid-derived immunocytes, and the down-regulation of TIMP-3 affected DCs differentiation and Th1 polarization. Therefore, it could prove to be of great value to explore the mechanisms of the influences that TIMP-3 deficiency has on uterine immuno-microenvironment and differentiation of myeloid immunocytes at different pregnancy phases. In this study, Cre-LoxP system would be used to establish the Timp-3 conditional knockout mice model with triple mutant luciferase reporter genes. Moreover, fluorescent tracer, flow cytometry and genomic techniques would be employed to analyze the effects of TIMP-3 deficiency on immuno-microenvironment and differentiation of myeloid immunocytes at the maternal-fetal interface. This study would provide important evidences for the treatment of endometrial diseases and pregnancy complications.

子宫内膜蜕膜化、胚泡植入及妊娠维持均伴有免疫细胞浸润，免疫细胞群体异常是病理性妊娠等发生的关键因素之一，局部许多因子影响这一过程。TIMP-3在子宫微环境表达独特， TIMP-3表达缺失与先兆子痫及子宫内膜异位症发生有关，提示该分子对子宫免疫微环境有重要影响。髓系免疫细胞是子宫免疫微环境中的重要群体，参与调控局部免疫应答类型、血管生成等多个过程。已证实TIMP-3调控髓系细胞分化，下调TIMP-3影响DCs分化程度并优势诱导Th1。因此，揭示TIMP-3缺失对子宫免疫微环境及局部髓系免疫细胞分化的影响及机制具有重要意义。本项目组拟利用Cre-LoxP系统，制备条件敲除子宫内膜组织Timp-3及三突变荧光报告小鼠模型，流式细胞术、荧光示踪及组学研究技术揭示TIMP-3缺失对子宫免疫微环境及髓系细胞分化的影响。本研究结果将为治疗子宫内膜疾病及病理性妊娠提供重要依据。

TIMP3是TIMPs家族中最为独特的成员，对髓源性细胞的分化有重要的调控作用。TIMP3在胎盘中高表达，但其对子宫微环境影响的研究甚少，目前关于世界上已报道的TIMP3敲除小鼠的模型均为全身敲除模型，本研究首次成功的构建出了条件敲除子宫内膜组织TIMP3的模型小鼠，获得了PR受体高表达组织（子宫内膜组织）TIMP3纯合及杂合缺失的模型小鼠。该模型小鼠将对研究TIMP3在特定组织缺失对其结构、特定时期微环境的重构改变，阐明TIMP3对子宫内膜及妊娠的影响具有重要的意义。然而后续的研究发现TIMP3条件缺失小鼠的繁殖能力与野生型对照鼠的繁育能力没有显著区别，平均每窝的胎仔数无统计学差异。对子宫内膜的组织学及组学的分析也未发现组织微环境的显著异常，推测在小鼠子宫内膜中TIMP3的功能可能存在冗余机制。我们在该研究发现，人与小鼠的TIMP3在子宫内膜的分布有显著的差异，小鼠的TIMP3主要分布在间质细胞，而人的TIMP3主要表达在腺上皮细胞且呈现高度的表达异质性，提示TIMP3在人与小鼠子宫中的作用可能有较大差异。我们进而对妊娠早期蜕膜细胞、重复性流产、内膜异位症及腺肌瘤等不同状态的子宫内膜组织进行了TIMP3表达及全基因组、转录组、微生物组及microRNA表达的差异研究，并基于该研究的发现参与了2018国家重点研发计划“孕早期重复性流产的免疫机制【项目编号2018YFC1002803】”的项目研究。我们对TIMP3条件敲除小鼠模型的研究提示，在评价TIMP3的医学意义时由于该分子在人鼠表达的差异等因素，需要慎重评价小鼠研究结果可能对人生殖的影响。我们将基于该项目对人不同病理状态下子宫内膜组学数据予以分析，继续研究阐释子宫内膜微环境免疫对人类生殖健康影响的关键机制。

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