ISOLATION OF PURE VILLOUS CYTOTROPHOBLAST FROM TERM HUMAN-PLACENTA USING IMMUNOMAGNETIC MICROSPHERES
ISOLATION OF PURE VILLOUS CYTOTROPHOBLAST FROM TERM HUMAN-PLACENTA USING IMMUNOMAGNETIC MICROSPHERES
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DOI：
10.1016/0022-1759(89)90405-5
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发表时间：
1989-05-12
影响因子：
2.2
通讯作者：
KING, BF
中科院分区：
文献类型：
--
作者：
DOUGLAS, GC;KING, BF
一个程序已经开发，产生一个纯人口绒毛细胞滋养层从足月人胎盘。作为第一步，使用Kliman等人（1986）的方法的改进获得高度富集细胞滋养层的细胞制剂（通过阳性细胞角蛋白染色鉴定）。然后通过用抗I类和II类主要组织相容性抗原的小鼠单克隆抗体处理，然后用山羊抗小鼠IgG包被的磁性微球处理，去除剩余的污染细胞（通过阳性波形蛋白染色鉴定）。这一步骤的基本原理是基于绒毛滋养层不能表达HLA抗原，而绒毛间充质细胞表达这些表面抗原的事实。使用磁体固定玫瑰花状细胞，允许通过移液管容易地取出非玫瑰花状细胞。当将非玫瑰花状细胞置于原代培养物中时，通过免疫磁性分离程序没有HLA阳性去除这些组分。细胞对细胞角蛋白呈阳性，24小时后，对妊娠特异性β 1-糖蛋白（SP1）和人绒毛膜促性腺激素呈阳性染色。细胞滋养层细胞的恢复率大于92%，仅轻微丧失活力。
A procedure has been developed which yields a pure population of villous cytotrophoblast from term human placenta. As a first step, a cell preparation highly enriched for cytotrophoblast (identified by positive cytokeratin staining) was obtained using a modification of the method of Kliman et al. (1986). The remaining contaminating cells (identified by positive vimentin staining) were then removed by treatment with mouse monoclonal antibodies against class I and class II major histocompatibility antigens followed by magnetic microspheres coated with goat anti-mouse IgG. The rationale for this step was based on the fact that villous trophoblast fails to express HLA antigens whereas cells from the villous mesenchyme do express these surface antigens. Rosetted cells were immobilized using a magnet allowing the non-rosetted cells to be easily withdrawn by pipette. When the non-rosetted cells were placed in primary culture, no HLA-positive removal of these components by the immunomagnetic separation procedure. The cells were positive for cytokeratin and, after 24 h, showed positive staining for pregnancy-specific .beta.1-glycoprotein (SP1) and human chorionic gonadotropin. Recovery of cytotrophoblast was greater than 92% with only a slight loss of viability.