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Inhibition of Fas-induced apoptosis in cultured cells infected with Trypanosoma cruzi

抑制 Fas 诱导的克氏锥虫感染培养细胞凋亡

基本信息

批准号：
09670272
负责人：
SHIMADA Junko
金额：
$ 1.73万
依托单位：
Juntendo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

In our laboratory, Trypanosoma cruzi, the parasitic protozoan flagellate that causes Chagas' disease, and infected HeLa cells are utilized as a model system in which to investigate the cell biology and pathology of the host cells. We report here that T.cruzi infection inhibits Fas-mediated apoptosis in HeLa cells. Exponentially growing HeLa cells were inoculated into each well of a 24-well plate, and allowed to grow at 37ﾟC for 2 days. The cells were infected with T.cruzi trypomastigotes and further incubated for 4-5 days. Apoptosis was induced in control and infected HeLa cells, by the addition of anti-Fas monoclonal antibody. The cultures were further incubated, washed with PBS, and stained with Giemsa or Hoechst 33342 for observation under a microscope. To examine the translocation of phospholipids from the inside to the outside of the plasma membrane, Fas-induced HeLa cells were stained with a fluorescein-labeled probe specific for phosphatidylethanolamine and analyzed by flow cytometry. One day after the addition of anti-Fas antibody, most of the control HeLa cells appeared roundish and detached from the well. In contrast, some infected cells showed an apparently normal morphology, adhering to the well. Six hours after the induction of Fas-mediated apoptosis, nuclear staining with Hoechst 33342 showed that 59.5 % of control cells were undergoing apoptosis, whereas 30.0 % of the infected cells were apoptotic, indicating that T.cruzi infection markedly inhibits host-cell apoptosis. The control population of HeLa cells contained more fluorescein-positive cells than the infected cell population. This implies that the translocation of phosphatidylethanolamine from the inner side to the outer side of the plasma membrane takes place in an early stage of apoptosis. Inhibition of Fas-mediated apoptosis may constitute a novel immune escape mechanism for T.cruzi . Potentially critical molecules from parasite and host cells are now under investigation.

本实验室以导致恰加斯病的寄生原生动物鞭毛虫克氏锥虫（Trypanosoma cruzi）和被感染的HeLa细胞为模型系统，研究宿主细胞的细胞生物学和病理学。我们在这里报道了克氏锥虫感染抑制fas介导的HeLa细胞凋亡。将指数生长的HeLa细胞接种到24孔板的每孔中，在37℃的温度下生长2天。用克氏锥虫感染细胞，孵育4-5天。通过添加抗fas单克隆抗体，诱导对照和感染的HeLa细胞凋亡。培养物进一步孵育，PBS洗涤，Giemsa或Hoechst 33342染色，显微镜下观察。为了检测磷脂从质膜内到质膜外的易位，用磷脂酰乙醇胺特异性荧光标记探针对fas诱导的HeLa细胞进行染色，并通过流式细胞术进行分析。添加抗fas抗体1 d后，对照组HeLa细胞大部分呈圆形，与孔分离。相比之下，一些感染细胞表现出明显的正常形态，粘附在孔上。诱导fas介导的细胞凋亡6小时后，Hoechst 33342核染色显示59.5%的对照细胞发生凋亡，而30.0%的感染细胞发生凋亡，表明克氏T.cruzi感染明显抑制宿主细胞凋亡。对照HeLa细胞群比感染细胞群含有更多荧光素阳性细胞。这表明磷脂酰乙醇胺从细胞膜内侧向细胞膜外侧的易位发生在细胞凋亡的早期。抑制fas介导的细胞凋亡可能是一种新的克氏锥虫免疫逃逸机制。目前正在研究来自寄生虫和宿主细胞的潜在关键分子。