人早孕期蜕膜存在CD34+成血管干细胞及其与胚胎停育相关性的研究

The etiology is still not clear for about 50% embryonic diapause patients. It is difficult to prevent and treat the disease in that situation. We found the fact in previous study the micro- vessel density (MVD) in these patients were much lower than that in the normal pregnant women in the same period. We demonstrated that the decidual CD34+ cells (dCD34+ cells) decreased in the embryonic diapause patients. Colonies developed from dCD34+ cells were proved. These dCD34+ cells were separated by CD34 magnetic bead method. These dCD34+ cells did not express VEGFR-2, VE-cadherin and CD31 which were characteristic expressed by endothelial cells. But these cells performed partly as the endothelial cells after cultured in differentiation medium. Therefore we guess the decidual tissues of early pregnant patients had the CD34+ vasculogenesis stem cells. The abnormal number and biological characters of dCD34+ cells may cause the decrease of MVD and lead to the abnormal blood supply which will induce the embryonic diapause. We will study the dCD34+ cell difference between the early normal pregnant women and the embryonic diapause patients in the same period. We will focus on the cell number, cell specific markers, the capacity to generate colonies, the endothelial specific gene and protein expression after cultured in the differentiation medium, the ability to induce angiogenesis by paracrine secretion, the influence of progesterone and the in vivo vasculogenesis ability. We will demonstrate for the first time if there are CD34+ vasculogenesis stem cells in the decidua tissues of early pregnant women. To explore the biological characters of the dCD34+ vasculogenesis stem cell and to study its correlation with embryonic diapause will help us to understand the mechanism of embryonic diapause better and provide the new target for the treatment.

胚胎停育约50%原因不明且防治困难，此类蜕膜微血管密度（MVD）比正常妊娠降低。我们在前期研究中发现：蜕膜CD34+细胞（dCD34+）在胚胎停育患者减少；磁珠法提取的dCD34+细胞有成克隆性，内皮细胞特征性VEGFR-2、VE-cadherin及CD31阴性，诱导分化后有部分内皮细胞活性。故推测早孕蜕膜存在CD34+成血管干细胞，其数量、生物学特性异常引起蜕膜MVD下降，供血异常，导致胚胎停育。本课题中我们拟磁珠法分离dCD34+细胞，离体及在体研究其成克隆性、分化及成血管能力，并对比其在正常早孕及胚胎停育患者间的差异：细胞数量、标志物、成克隆性，诱导分化后内皮细胞特征性基因及蛋白表达，在体成血管能力，旁分泌促血管形成能力，孕激素对其影响。本研究将首次证实人类早孕蜕膜是否存在CD34+成血管干细胞，并深入研究其生物学特性及其与胚胎停育的相关性，阐明胚胎停育相关机制，为治疗提供新的靶点。

本研究利用免疫荧光染色及免疫组化连续切片染色分析早孕期蜕膜组织CD34（dCD34+细胞）表达及分布情况，发现CD34在蜕膜内呈阳性表达，多数表达于血管内皮细胞（呈CD34、CD31双阳性表达），同时在非血管部位存在少量CD34+/CD31-细胞。通过流式细胞技术分析蜕膜组织CD34、VEGF-R2、PDGF-βR等表达情况，采用单染、双染、三染法分析干细胞标志物的表达情况及其相互关系，并对比正常早孕及胚胎停育蜕膜组织的差别。结果显示正常早孕期蜕膜组织中存在CD34阳性/VEGF-R2阴性的非内皮细胞，并且在这一细胞群中干细胞标志物PDGF-βR呈一定比率的阳性表达。正常早孕及胚胎停育的蜕膜组织上述标志物表达存在一定差别。通过磁珠法分离正常早孕蜕膜组织dCD34+细胞并纯化、培养，利用PCR技术证实为非内皮细胞（内皮细胞特异性基因CD31、VE-cadherin呈阴性表达），体外培养研究证实其具有成克隆性及诱导后的分化能力、在体研究证实其成血管能力，并证实能被孕激素放大在体成血管能力。对比分析正常早孕蜕膜组织及胚胎停育蜕膜组织的dCD34+细胞，其在正常早孕蜕膜组织中的细胞数量多于胚胎停育蜕膜组织，且其成克隆能力强于胚胎停育蜕膜组织。本研究首次证实人类早孕期蜕膜组织中存在CD34+成血管干细胞，正常早孕及胚胎停育的蜕膜组织dCD34+细胞存在差异，胚胎停育的发生与CD34+成血管干细胞异常存在着相关性，为治疗和预防早孕期胚胎停育的提供新靶点奠定基础。项目资助发表国内核心期刊论文5 篇，待发表1篇，培养硕士生9 名，其中5名已经取得硕士学位，4名在读。项目经费共23万元，剩余经费2.7957万元，剩余经费计划用于本项目研究后续支出。