胎盘滋养层合体化必需分子syncytin单核苷酸多态性对基因转录和蛋白质功能的影响及与妊娠结局相关性的研究

Placental syncytiotrophoblast, which is formed by cell-cell fusion of cytotrophoblast cells and secretes human chorionic gonadotropin and progesterone, is indispensable for the establishment and maintenance of normal pregnancy. Dysregulation in syncytialization leads to gestational diseases such as pre-eclampsia and habitual abortion. To date, syncytin-1 and -2 are the only genuine placenta-specific fusogens that have been discovered, whose expression can induce fusion of nonfusogenic cells. In the promoter and coding sequences of syncytin-1 and -2, dozens of single nucleotide polymorphisms (SNPs) have been released from public SNP database, and some of these SNPs are located within transcription factor binding sites of the promoter or important functional motifs of syncytins. However, whether these SNPs affect gene transcription/fusogenic function of syncytins and placental syncytialization remains unelucidated. Utilizing pregnancy-related clinical samples, multiple placental syncytialization models and research platform of SNP identification, transcriptional regulation and in vitro fusion assays, we sets out to investigate the linkage of SNPs, which affect gene transcription and fusogenic function of syncytins, to pregnancy outcome. We aim to elucidate the molecular mechanism of syncytialization and pathological mechanism of dysregulated placentation in gestational diseases, and gain insights into diagnostic markers and therapeutic strategies of these complications.

胎盘绒毛合体滋养层由细胞滋养层细胞融合形成，分泌人绒毛膜促性腺激素和孕酮等激素，对于正常妊娠的建立和维持至关重要。合体化异常能引起子痫前期和习惯性流产等妊娠疾病。Syncytin是迄今发现的唯一的胎盘滋养层合体化的必需分子(fusogen)，其表达直接介导细胞融合。目前已发现syncytin-1和-2启动子和编码区的数十种单核苷酸多态性(SNP)，其中很多SNP位于启动子转录因子结合元件或蛋白质重要功能结构域中。然而，迄今没有报道表明这些SNP是否影响syncytin的基因转录、fusogen功能及胎盘合体化。本项目拟利用妊娠标本库和多种滋养层合体化模型，以搭建好的SNP鉴定、转录调控分析、体外蛋白质功能检测平台，研究影响syncytin转录和蛋白质功能的SNP与妊娠结局的相关性，以阐明胎盘滋养层合体化的分子机制，为揭示妊娠疾病胎盘发育障碍的机制及临床早期诊断和干预治疗提供重要的理论依据。

胎盘绒毛合体滋养层由细胞滋养层细胞融合形成，分泌人绒毛膜促性腺激素和孕酮等激素，对于正常妊娠的建立和维持至关重要。合体化异常能引起子痫前期和习惯性流产等妊娠疾病。人类内源性逆转录病毒囊膜蛋白syncytins是目前发现的唯一的直接介导滋养层细胞合体化的融合蛋白（fusogen）。我们通过构建syncytin-2野生型和21个单核苷酸多态性（SNP）的表达载体（包括10个N-糖基化位点缺失突变，N118S引入N-糖基化位点和10个位于重要功能结构域的非同义SNP），观察其介导293T细胞的融合能力，进而研究syncytin-2结构和功能的联系。在这21个突变体中，N133Q、N312Q、N443Q、C46R（CXXC结构域）和R417H（HRP和ISD结构域）完全失去了诱导细胞融合的功能，而N332Q、N118S、T367M（融合肽）、V483I（跨膜结构域）和T522M（胞内域）5个突变体的融合功能显著增强。同时，我们也证明了在293T细胞中，syncytin-2的N133、N146、N177、N220、N241、N247、N312、N332和N443会发生糖基化。免疫共沉淀结果分析表明受体MFSD2A与syncytin-2突变体N443Q、C46R、R417H和T367M的相互作用较弱，而与N118S有较强的结合能力。我们对125例重度子痫前期患者和272例正常妊娠妇女syncytin-2编码区序列进行了测序，发现一个非同义杂合子T522M SNP频率有异（分别为0.0320和0.0618）。本项目的研究结果表明，在融合蛋白syncytin-2结构中，糖基化位点133、312、332和443对其融合功能极其重要，而SNP C46R、N118S、T367M、R417H、V483I和T522M可以改变syncytin-2的融合功能。这些研究解释了胎盘滋养层合体化的分子机制及syncytin SNP与妊娠疾病的相关性。