弓形虫侵入细胞过程中宿主胞内游离钙和胞外花生四烯酸的变化和来源

Variance and resource of intracellular free calcium and extracellular arachidonic acid of host cells during the invasion of Toxoplasma gondii

目的探讨刚地弓形虫侵入不同类型传代细胞过程中宿主胞内游离Ca^2＋和胞外花生四烯酸（AA）的变化和来源。方法采用核素液闪仪和激光共聚焦显微镜检测刚地弓形虫RH株侵入Veto和J774A.1细胞过程中宿主胞外AA和胞内游离Ca^2＋的变化及来源。对所获数据进行方差分析和t检验。结果弓形虫侵入J774A.1和Vero细胞后，宿主胞内游离Ca^2＋有不同程度升高，其最高荧光强度变化分别为（1219．7±58．4）％（t=4.982，P〈0.01）和（356.3±23．6）％（t=2.593，P〈0.05）。Vero细胞内Ca^2＋升高多来源于胞内Ca^2＋库释放，而J774A.1细胞内Ca^2＋升高来源于胞内Ca^2＋库释放和胞外Ca^2＋内流。弓形虫侵入Vero和J774A.1细胞后，宿主胞外AA均明显升高，分别为感染前的5.02倍和8.44倍（t=3.124，t=3.852，均P〈0.01）。磷脂酶怠抑制剂作用弓形虫后可明显抑制AA升高。结论弓形虫感染吞噬性细胞激活宿主胞膜磷脂酶C和弓形虫磷脂酶A2，引起宿主胞内游离Ca^2＋和胞外AA浓度升高，两者共同作用导致虫体侵入；虫体侵入非吞噬性细胞可能仅激活虫体磷脂酶A2。

Objective To explore variance and resource of intracellular free Ca^＋ and extracellular arachidonic acid （AA） in different types of passage cells during the invasion of T. gondii, Methods The variance and resource of extracellular AA and intracellular free Ca^＋ of Vero and J774A.1 cells during the invasion of T. gondii were detected by multi-purpose scintillation counter and laser scanning confocal microscope. Data were analyzed using Chi-square test and t test. Results The intracellular free Ca^2＋ levels in J774A.1 and Vero cells were both increased after T. gondii infection. The maximal changes of fluorescence intensity were （1219.7±58.4）% （P〈0.01） and （356.3±23.6）%（P〈0.05）, respectively. The increase of intracellular Ca^2＋ level in Vero cell was mostly from the release of intracellular Ca^2＋ store. And the Ca^2＋ increase in J774A.1 cell was from both the release of intracellular Ca^2＋ store and extracellular Ca^2＋ influx. Extracellular AA levels were significantly increased in both Vero and J774A.1 cells after T. gondii infection 5.02 and 8.44 times respectively （t=3.124, t=3.852, P〈0.01）. The APt elevations could be significantly inhibited by phospholipase A2 （PLA2） inhibitor pretreating T. gondii. Conclusions The 15hospholipase C of phagocytic host cell and PLA2 of T. gondii are activated by T. gondii infection, which results in the increase of intracellular free Ca^2＋ and extracellular AA level. Combined actions of Ca^2＋ and AA play a major role in the invasion of T. gondii to host cell. While only PLA2 of T. gondii may be activated in nonphagoeytic host cell.