人早孕绒毛组织的体外原代培养和人滋养细胞株的建立

目的:建立快速、有效的绒毛滋养细胞体外原代培养的方法以及建立人滋养细胞株和初步评价。方法:采用胰酶和胶原酶I混合消化法消化绒毛组织,用胰蛋白酶消化传代体外培养,通过光镜、透射电镜和免疫荧光激光共聚焦方法对培养出的细胞进行形态和骨架评价。结果:显微镜下可见细胞呈多边形,细胞平铺生长,有细胞融合后形成的合体滋养层细胞;透射电镜下可见细胞表面的微绒毛丰富;胞质丰富,内质网扩张明显,有丰富的糖原颗粒、髓样小体和明显的脂滴,以及细胞间紧密的桥粒样结构连接;直接免疫荧光双标记染色检测到角蛋白18和波形蛋白均为阳性。结论:采用胰酶和胶原酶I混合消化法可获得较纯的滋养细胞,通过光镜、透射电镜和免疫荧光激光共聚焦检测细胞外形和骨架,确定该体外传至56代的细胞株为滋养细胞株。

A primary cell culture system for the first trimester human placental villus in vitro and establishment of a human trophoblastic cell line

Objective:To establish the quick and valid primary culture method of human placental villus in vitro and set up a human trophoblastic cell line with preliminary assessment.Methods:The placental villus were digested by pancreatic enzyme and collagenase I.Primary culture of human trophoblast cells was carried out by combination digestion,and the subsequent serial passage of the cells performed by trypsin digestion,identified and assayed by light microscope,transmission electron microscope and immunofluorescence laser confocal microscopy.Results:Under the microscope,the polygon cell grew monolayer slice and there were some syncytiotrophoblast by cell fusion.Abundance kytoplasm,glycogen granule,myelin body and lipid droplet were observed under the transmission electron microscope.Endocytoplasmic reticulum was distended markedly.There was desmosome structure between the trophoblast cells.Keratin 18 and vimentin specifically bound to the cytoplasm of trophoblastic cells with direct immunofluorescence double marker.Conclusion:Pure trophoblastic cell is gained by combination digestion with pancreatic enzyme and collagenase I.The results of light microscope,transmission electron microscope and immunofluorescence laser confocal microscopy detection confirmed that the human trophoblast cell line with 56 passage numbers in vitro was established from appearance and cystoskeleton.