不同分离纯化法建立人早孕滋养细胞模型的效果比较

Isolation and culture of trophoblast cells from human placenta in first trimester with different methods

目的对4种建立滋养细胞的方法进行比较,以期寻求一种简便高效的人早孕滋养细胞的体外培养方法,为相关研究提供基础。方法早孕绒毛通过胰酶联合胶原酶消化分离后,分别采用直接接种、差速贴壁联合差速消化法、人外周血淋巴细胞分离液分离纯化法、完整绒毛消化联合简化的percoll密度梯度分离法4种方法纯化培养,倒置显微镜观察细胞形态,应用HE染色、免疫组化和免疫细胞化学法检测细胞纯度。结果直接接种、差速贴壁联合差速消化法、人外周血淋巴细胞分离液分离纯化法得到的细胞细胞角蛋白7(CK-7)表达阳性率不足60%;简化的percoll密度梯度分离法得到的细胞CK-7表达阳性率达90%以上。结论完整绒毛消化联合简化的percoll密度梯度分离纯化可以得到大量较高纯度的滋养细胞以供后期实验。

Objective To study the methodology for purification of human first trimester trophoblast cells from placentas with different methods , thus to seek a simple method for purification of human first trimester trophoblast cells in vitro.Methods Target cells isolated from first trimester human placenta with trypsin and collagenase Ⅰ were incubated directly; purified by differential time attachment and digestion ; purified by lymphocyte separation medium ; and dissociated in trypsin /collagenase Ⅰ with purification by simplified percoll gradient centrifugation , respectively.Morphologic observa-tion was applied with inverted phase contrast microscope , with purity was assessed by HE staining and immunocytochemis -try.Results Cytokeratin -7 positive cells were more than 90% only with the last method, while cytokeratin -7 positive cells were less than 60% with the other methods.Conclusion First trimester human trophoblast cells can be obtained through whole first trimester human placenta digestion and followed by simplified percoll gradient centrifugation purification .