问号钩端螺旋体诱导J774A．1细胞凋亡及caspase-3、-6活化对凋亡的影响

J774A.1 cell apoptosis induced by Leptospira interrogans and effects of caspase-3,-6 activation on apoptosis

目的:了解问号钩端螺旋体(钩体)诱导小鼠单核-巨噬样细胞(J774A.1)凋亡的作用,以及caspase-3、-6活化对凋亡的影响。方法:建立问号钩体黄疸出血群赖型56601株J774A.1细胞感染模型。采用FITC-Annexin V/PI荧光标记流式细胞术检测细胞凋亡或坏死情况。分别采用荧光比色法和WesternBlot,检测感染细胞caspase-3、-6活性及其底物(PARA和LaminA/C)剪切情况。结果:多种不同浓度的问号钩体赖株感染均可使J774A.1细胞发生凋亡,其中以问号钩体∶细胞=100∶1的比例感染时凋亡率最高(48.81%±5.95%)。感染细胞caspase-3和-6最大活性分别为(1453.41±36.07)FU和(618.65±39.82)FU,是未感染细胞的16.38和9.98倍。感染细胞的PARP和Lamin A/C均被剪切。Caspase-3、-6抑制剂对问号钩体赖株诱导的J774A.1细胞凋亡有明显的阻断作用。结论:问号钩体可诱导J774A.1细胞凋亡,caspase-3和-6与该细胞凋亡密切相关。

Objective： To investigate the apoptosis of J774A. 1 interrogan and the effect of caspase-3,-6 activation on the apoptosis. cells induced by Leptospira Methods： Mouse monocytemacrophage like cell line J774A. 1 was infected by L. interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae Lai strain 56601. The apoptosis or necrosis of infected cells was examined by flow cytometry using fluorescein labeling FITC-Annexin V/PI. The activity of caspase-3,-6,and their cleaved substrates PARP and Lamin A/C were measured by fluorometry and Western Blotting,respectively. Results. L. interrogan strain Lai was able to induce apoptosis of J774A. 1 cells and the maximal apoptotic rate was （48.81±5.95）% when microbe ： cell ratio was 100 ： 1. The maximal activities of caspase-3 and -6 in the infected J774A. 1 cells were （1453.41±36.07） and （618.65±39.82） FU,respectively,which were 16.38- and 9.98-fold of those uninfected.cells. PARP and Lamin A/C in the infected cells were detected. Caspase-3 and -6 inhibitors remarkably blocked the L. interrogans-induced apoptosis in J774A. 1 cells. Conclusion. L. interrogan is able to induce the apoptosis of J774A. 1 cells and intracellular caspase-3 and -6 are closely associated with the apoptosis.