人早孕胎盘绒毛膜滋养层细胞体外培养模型的建立

ESTABLISHMENT OF THE IN VITRO CULTURE MODEL OF TROPHOBLASTIC CELLS FORM FIRST-TRIMESTER HUMAN PLACENTA

目的通过对早孕胎盘绒毛膜滋养层细胞的分离,纯化和培养,寻找一种稳定、简便可获得较高纯度滋养层细胞的培养方法。方法通过胰酶/DNA酶联合消化法对妊娠6-10周绒毛组织进行消化,获得单细胞悬液,比较Per-coll密度梯度离心和淋巴细胞分离液对滋养层细胞的分离纯化效果。含10%FBS的DMEM/F12培养基培养,并比较是否应用鼠尾胶原对细胞贴壁和生长的影响。通过免疫荧光方法对滋养层细胞进行鉴定。结果经简化Percoll密度梯度离心分离纯化的滋养层细胞纯度高,明显优于淋巴细胞分离液的分离效果(P<0.001);细胞生长表面预先经鼠尾胶原处理后,细胞贴壁良好,分裂生长旺盛。结论利用简化Percoll密度梯度离心法分离细胞,并在应用鼠尾胶原的条件下进行培养,可以获得满意的人绒毛膜滋养层细胞的体外培养模型。

Objective To establish a stable and convenient method of purification and culture of human placenta trophoblastic cells. Methods Single cell suspension was obtained from healthy placental tissues between 6 and 10 weeks of gestation. Trypsin and Dnase I were used for digestion. The separating resuits of the trophoblastic cells by Percoll gradient centrifugation were compared with those by the lymphocyte separating solution. DMEM/F12 culture medium containing 10% FBS was used for cell culture. Cell adherence and reproduction with or without collagen I treatment of the culture surface was compared. These cells were identified by immunofluorescent staining. Results More trophoblastic cells with higher purity were obtained by simplified Percoll gradient centrifugation than by lymphoeytes separating solution （P〈0. 001）. The culture surface treated with collagen beforehand was better in cells adherence and proliferation. Conclusion Purifying cells with simplified Percoll gradient centrifugation and culturing them under suitable conditions has established a satisfactory in vitro culture model of trophoblastic cells.