de Mestre A M, Bacon S J, Costa C C, Leadbeater J C, Noronha L E, Stewart F, Antczak D F
Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Placenta. 2008 Feb;29(2):158-69. doi: 10.1016/j.placenta.2007.10.005. Epub 2007 Dec 3.
The chorionic girdle of the equine conceptus is comprised of specialized trophoblast cells which, at day 36-38 of equine pregnancy, gain an invasive phenotype and invade the endometrium to form endometrial cups. Studies of equine endometrial cups remain difficult to perform because of the invasive techniques required to obtain cup tissue and because sampling requires termination of the pregnancy. In this study we developed a system to model trophoblast differentiation and trophoblast-immune interactions in vitro and in vivo. We utilized a method of culturing chorionic girdle pieces in serum-free medium to promote spontaneous formation of vesicle structures enriched for terminally differentiated binucleate cells that secreted equine chorionic gonadotrophin (eCG). Immunohistochemical staining and scanning electron microscopy showed that the cells of the vesicles closely resembled the outer layers of chorionic girdle immediately prior to invasion. Chorionic girdle vesicles were harvested after 72h in culture and ectopically transplanted via injection into the vulvar mucosa of recipient mares. At 7, 14, 21 and 28days after transplantation, biopsies of the injection sites were obtained. Immunohistochemical labeling of cryostat sections of the biopsies with a panel of monoclonal antibodies to horse trophoblast molecules demonstrated survival, differentiation, and presence of trophoblast cells for at least 21days. Serial sections of the biopsies labeled with antibodies to the equine lymphocyte surface markers CD4 and CD8, together with lymphocyte microcytotoxicity assays, revealed that the recipients mounted both cellular and humoral antibody immune responses to the transplanted trophoblast cells. This new method for culturing equine chorionic girdle trophoblast cells, and for transplanting trophoblast vesicles to ectopic sites, should allow identification of key aspects of trophoblast differentiation and the interactions that occur between invasive trophoblast and the maternal immune system.
马妊娠早期胚胎的绒毛带由特殊的滋养层细胞组成，在妊娠36 - 38天时，这些细胞获得侵袭性表型并侵入子宫内膜形成子宫内膜杯。由于获取杯状组织需要采用侵入性技术，且取样需要终止妊娠，因此对马子宫内膜杯的研究仍然难以开展。在本研究中，我们开发了一种系统，用于在体外和体内模拟滋养层细胞分化及滋养层与免疫细胞的相互作用。我们采用在无血清培养基中培养绒毛带组织块的方法，以促进富含分泌马绒毛膜促性腺激素（eCG）的终末分化双核细胞的囊泡结构自发形成。免疫组织化学染色和扫描电子显微镜显示，囊泡中的细胞与侵袭前绒毛带的外层细胞极为相似。培养72小时后收获绒毛带囊泡，并通过注射异位移植到受体母马的外阴黏膜。在移植后的第7、14、21和28天，获取注射部位的活检样本。用一组针对马滋养层分子的单克隆抗体对活检样本的冰冻切片进行免疫组织化学标记，结果表明滋养层细胞至少存活、分化了21天。用针对马淋巴细胞表面标志物CD4和CD8的抗体对活检样本的连续切片进行标记，并结合淋巴细胞微细胞毒性试验，结果显示受体对移植的滋养层细胞产生了细胞免疫和体液免疫反应。这种培养马绒毛带滋养层细胞并将滋养层囊泡移植到异位部位的新方法，应有助于确定滋养层细胞分化的关键方面以及侵袭性滋养层与母体免疫系统之间的相互作用。
