新型体外滋养细胞胎盘屏障模型(PCB-HPT)的建立和鉴定

Establishment and evaluation the neotype placental barrier in vitro with trophoblast cell(PCB-HPT)

目的:建立具有极性和完整性的体外滋养细胞胎盘屏障模型。方法:以永生化的、通过系统鉴定的人早孕滋养细胞株构建胎盘屏障模型(PCB-HPT)。采用细胞生长周期评估细胞屏障构建时间;采用透射电镜评估滋养细胞屏障的极性;采用上皮细胞电阻测量评估滋养细胞屏障的完整性。结果:该滋养细胞胎盘屏障模型采用0.4μm孔径、0.47cm2NUNC插入式细胞培养皿培养,建议小室接种细胞总数为6×104cells,细胞屏障构建完整的培养时间为3d,屏障可维持36～48h。光镜下细胞培养至融合后具有典型的"铺路石"样外观;透射电镜观察显示,屏障具有极性,滋养细胞屏障可形成紧密连接并产生较高的跨内皮阻抗(TER)达600Ω;证实该屏障具有完整性。结论:建立了具有极性和完整性的体外滋养细胞胎盘屏障模型,为研究妊娠期间病原体和物质跨越胎盘屏障机制研究提供了体外研究模型。

Objective：To establish a polar and integrity of trophoblast cells in vitro placental barrier model. Methods：We build placental barrier models（ PCB-HPT） base on immortalized human trophoblast cells （ HPT-8 ） of early pregnancy lines. Using HPT-8 cell cycle to obtain the construction time of cell barrier models. Using transmission electron microscopy to assess the polarization of trophoblast cells barrier. Using epithelial cell resistance measurement to assess the integrity of trophoblast cell barrier. Results：The trophoblast cells barrier model has been cultured in 0. 4μm apertures,0. 47cm2 NUNC insert cell culture dishes. And recommen-ded inoculation cell density is 6 × 104 cells. Moreover, we cultured 3 days in order to build a complete cells barrier and the PCB-HPT barriers can maintain 36~48h. HPT-8 cells cultured in the transwell insert exhibited typical “flagstone” appearance. The PCB-HPT barrier has polarization observed by transmission electron microscopic. The junction between HPT-8 cells is tight and the transmembrane resistance is over 600Ω, which shows the integrity of PCB-HPT barrier. Conclusion：We established the trophoblast cells placental barrier model with polarization and integrity in vitro which provided a model to study the mechanisms that pathogens and substances across the placental barrier during pregnancy.