胎儿基因MICA遗传变异及其与环境内分泌干扰物的交互作用对胎儿生长及早产的作用及机制研究

Intrauterine growth restriction (IUGR) and preterm birth (PTB) are the main cause of neonatal morbidity and mortality. It is known that both genetic and environmental factors contribute to their etiology. ..Development of the placenta is a highly regulated process that is essential for normal fetal growth and maintenance of a healthy pregnancy. Maternal immune tolerance towards the fetus and placenta is important for the implantation and development of placenta, which has been associated with suppression of cell-mediated immune responses of the mother. Abnormal implantation and development of placenta can lead to reduced nutrient transport and uteroplacental blood flow to the fetus and placenta, which will result in IUGR and PTB. ..Recently, MHC class I chain-related sequence A (MICA) was found to be expressed in human syncytiotrophoblast and released to the serum. The MICA molecules in pregnancy serum can down-regulate the activating receptor NKG2D on natural killer (NK) and T cells, with the consequent inhibition of NKG2D-dependent cytotoxic response. These findings suggest a novel mechanism of silencing the maternal immune system that promotes fetal immune escape. Three functional variants in MICA have been reported that can affect the function or transcription of MICA. In our previous study, we found that two genetic variants in MICA can affect the serum level of MICA protein, one of which is in perfect linkage disequilibrium (LD) with the functional variant that affects the transcriptional activity of MICA. Given the importance of MICA in the fetal evasion of maternal immune attack and the development of placenta in fetal growth and maintenance of a healthy pregnancy, we hypothesize that functional variants in MICA gene that affect the function or the serum level of MICA protein may impact cytotoxic response of NK and T cells against fetus during early pregnancy and in turn affect the implantation and development of placenta, which could result in different susceptibility to IUGR and PTB. Meanwhile, environmental endocrine disruptors (EEDs) may interact with MICA in the etiology of IUGR and PTB. To test this hypothesis, we will investigate the association of functional variants in MICA with IUGR and PTB as well as the role of serum level of MICA protein, number of NKG2D-expression NK and T cells and placenta morphology in IUGR and PTB, and the impact of MICA variants on the level of serum MICA, number of NKG2D-expression NK and T cells and placenta morphology (weight and size) in a prospective cohort including 3000 pregnant women. To investigate the interaction between EEDs and MICA, we will examine the impact of EEDs on the methylation of MICA promoter, MICA expression and the cell-mediated cytotoxicity. This study will provide evidence on the role of MICA and its interaction with EEDs in the etiology of IUGR and PTB, greatly advance our understanding of the pathogenesis and open new routes to novel treatments and predictive measures.

胎儿宫内生长受限和早产是新生儿死亡的主要原因。胎盘合体表达MICA蛋白，将其释放到血液中，通过下调母体免疫细胞表面NKG2D的表达削弱其毒性反应，使胎儿逃逸母体的免疫排斥。我们前期研究发现了MICA基因上影响血清中MICA蛋白水平的遗传变异。我们推测孕早期MICA基因的功能位点可能通过影响MICA蛋白的功能或水平来调节母体NKG2D阳性免疫细胞数，破坏母-胎免疫耐受，影响胎盘的植入和发育，导致胎儿生长受限和早产。为了验证这一假说，我们将通过前瞻性队列研究和细胞分子生物学实验，探讨胎儿MICA基因上的功能位点对母体孕早期血清中MICA蛋白水平、NKG2D阳性免疫细胞数、胎盘大小和胎儿生长及早产的影响，以及环境内分泌干扰物与MICA间的交互作用，从胎儿、环境、母体多层面深入研究MICA对胎儿生长和早产的作用和分子机制，明确胎儿生长受限和早产的分子靶标，为防治胎儿生长受限和早产提供理论依据。

早产和低出生体重是导致新生儿发病和死亡的主要原因，也是影响新生儿健康发育和生长的重要危险因素。胎盘合体滋养细胞组成型表达MICA蛋白，并将其释放到细胞外。游离型MICA蛋白可以下调NK、 T细胞表面NKG2D的水平，削弱它们对胎盘的细胞毒性反应，是胎儿免疫逃逸的新机制。鉴于母胎免疫耐受以及胎盘发育对维持正常妊娠和胎儿生长的关键性作用，我们推测MICA基因上的遗传变异可能会通过影响MICA蛋白的功能或水平调节母体NKG2D阳性NK和T细胞数，干扰母-胎免疫耐受，从而影响胎盘的发育，导致胎儿低出生体重和早产。我们在127例早产儿和634例足月儿中对MICA基因上的19个单核苷酸位点进行等位基因分型，研究发现MICA基因SNP位点rs2256318 A/A基因型与胎盘重量减低（β=-25.331; P=0.033）及早产风险增加（OR=6.97, P=0.001）显著相关，提示rs2256318 A/A基因型可能通过影响胎盘的发育导致早产。另一方面， SNP位点rs117246140 A等位基因与胎儿低出生体重显著相关（β=-98.22, P=0.027）。此外，我们发现rs2596538, rs2256318及rs3869132与胎儿性别存在交互作用（P=0.024, P=0.040, 0.045）；而rs2596538及rs2256318与孕周存在交互作用（P=0.008, 0.007）。在细胞水平，用不同浓度的全氟化合物PFOS（0um、75um、150um）处理细胞24h，CCK8检测发现PFOS能够抑制HTR8细胞增殖，流式细胞术及TUNEL检测发现PFOS能够促进细胞凋亡；RT-PCR检测发现PFOS能够促进P53、Bim、Bid、caspase3的mRNA表达，而抑制抗凋亡蛋白bcl-2的mRNA表达水平且其表达水平呈现剂量依赖性。用不同浓度的全氟化合物PFOS处理HTR8细胞24h，RT-PCR检测发现PFOS能够抑制MICA的mRNA表达水平。本研究为揭示早产和低出生体重的发病机制提供了线索，对早产和低出生体重的早期干预具有重要意义。