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目的建立绒毛外细胞滋养层细胞(EVCT)体外侵袭模型,为探讨滋养层细胞侵袭调节机制提供实验平台。方法取健康早孕妇女(孕7~9周)人工流产绒毛组织,利用Percoll梯度离心法及差速贴壁法,将纯化的绒毛外滋养层细胞,按所需浓度接种于涂有Matrigel培养板或24孔Transwell侵入系统中培养。用细胞免疫荧光法检测TGFβ2、cytokeration7、integrinα1β1。用CCK-8评价细胞生长状况。用Transwell细胞侵入系统检测EVCT的体外侵袭作用。结果 EVCT在Matrigel包被的Petri皿培养4h,用细胞免疫荧光法显示有95%以上的细胞表达TGFβ2、cytokeration7,integrinα1β1。EVCT在Matrigel培养不同时间后,其生长指数未有明显变化,提示Matrigel对EVCT的生长未见明显影响。EVCT在Transwell侵袭系统中培养不同时间后,根据侵袭细胞的OD值,提示48h细胞侵袭作用达到平台期。结论本实验成功地建立了EVCT体外侵袭模型,可利用此模型研究滋养层细胞的侵袭机制。从而为研究妊娠生理、妊娠病理、妊娠免疫耐受及正常细胞到肿瘤细胞之间的转化提供了实验平台。
Objective To establish an in vitro invasion model of villous extracellular trophoblast cells (EVCT) and provide an experimental platform for exploring the regulatory mechanism of trophoblast invasion. Methods Induced abortion villi in healthy pregnant women (7-9 weeks of gestation) were collected. The purified villous trophoblast cells were inoculated into Matrigel culture plates or 24-well Transwell invasion system. TGFβ2, cytokeration7 and integrinα1β1 were detected by immunofluorescence. CCK-8 was used to evaluate cell growth. Transwell invasion system was used to detect the in vitro invasion of EVCT. RESULTS: EVCT was cultured in Matrigel coated Petri dishes for 4 h, and more than 95% of cells expressed TGFβ2, cytokeration7 and integrinα1β1 by immunofluorescence. The growth index of EVCT did not change obviously after Matrigel cultured for different time, which indicated that Matrigel had no obvious effect on the growth of EVCT. After being cultured in Transwell invasion system for different time, EVCT suggested that invasion of 48h cells reached plateau according to OD value of invasive cells. Conclusion This experiment successfully established the EVCT in vitro invasion model, which can be used to study the invasion mechanism of trophoblast cells. This provides an experimental platform for the study of pregnancy physiology, pregnancy pathology, pregnancy immune tolerance and the transformation from normal cells to tumor cells.