细胞周期及其调控基因对问号钩端螺旋体诱导单核-巨噬细胞凋亡的影响

目的 了解不同细胞周期及其调控基因对问号钩端螺旋体(简称钩体)诱导人或鼠单核-巨噬细胞凋亡的影响.方法 采用细胞周期染色试剂盒及流式细胞仪检测问号钩体黄疸出血群赖型赖株感染前后小鼠单核-巨噬样细胞株J774A.1和人单核细胞株THP-1的细胞周期及其变化.采用细胞周期阻滞剂及流式细胞仪建立细胞周期同步化的J774A.1和THP-1细胞并进行鉴定.采用AnnexinV/PI凋亡检测试剂盒及流式细胞仪检测问号钩体赖株感染后细胞周期同步化与非同步化J774A.1和THP-1细胞早期凋亡、晚期凋亡/坏死率.采用实时荧光定量RT-PCR检测问号钩体赖株感染前后J774A.1和THP-1细胞的细胞周期及凋亡基因p21、p27、p53、c-myc和cycA mRNAs水平变化.结果 未感染钩体的正常J774A.1和THP-1细胞均分别处于G1、S和G2/M期,感染后均以G1期细胞为主,但S期THP-1细胞有所增加,J774A.1细胞则否(P＜0.05).J774A.1和THP-1细胞可分别被不同细胞周期阻滞剂阻滞在G1、S、G2/M或M期.G1期J774A.1和THP-1细胞感染后均无明显的早期凋亡现象,M期细胞早期凋亡、晚期凋亡/坏死率均明显升高(P＜0.05),G1期THP-1细胞晚期凋亡/坏死率明显升高(P＜0.05),J774A.1细胞则否.J774A.1和THP-1细胞感染后,p21 mR-NA水平均明显高于未感染细胞(P＜0.05),J774A.1细胞c-myc和p27 mRNAs水平、THP-1细胞cycAmRNA水平也高于未感染细胞(P＜0.05).结论 不同细胞周期及其调控基因对问号钩体诱导人或鼠单核-巨噬细胞凋亡有明显影响,但存在细胞种类差异性.

Effects of cell cycles and their regulating genes on apoptosis of mononuclear-macrophages induced by Leptospira interrogans

Objective To investigate the effects of different cell cycles and their regulating genes on apoptosis of mononuclear-macrophages induced by Leptospira interrogans. Methods The diversity and alteration of cell cycles of murine mononuclear-macrophage line(J774A. 1 ) and human monocyte line(THP-1 ) before and after infected with L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai were detected using Cell Cycle Stain Kit plus flow cytometer. The cell cycle synchronized J774A. 1 and THP-1 cells were generated and then identified by using different cell cycle blocking agents and flow cytometer. By using Annexin V/PI Detection Kit combined with flow cytometer, the rates of early-apoptosis and late-apoptosis/necrosis in the synchronized and non-synchronized J774A. 1 and THP-1 cells after infection with L. interrogans strain Lai were determined. Several real-time fluorescence quantitative RT-PCRs were performed to the changes of mRNAs levels of p21, p27, p53, c-myc and cycA genes that associated with cell cycle and apoptosis in J774A. 1 and THP-1 cells before and after infected with L. interrogans strain Lai. Results There were G1, S and G2/M phases in both the non-infected normal J774A. 1 and THP-1 cells. On the contrast,the majority of infected J774A. 1 and THP-1 cells were stagnated at G1 phase, but the amount of S phase THP-1 cells was elevated while that of S phase J774A. 1 cells was not(P ＜0.05). No remarkable early-apoptosis in both the infected G1 phase J774A. 1 and THP-1 cells was found, whereas the rates of early-apoptosis and late-apoptosis/necrosis in the infected M phase J774A. 1 and THP-1 cells were significantly increased (P ＜0.05 ). Additionally, late-apoptosis / necrosis rate in the infected G1 phase THP-1 cells (P ＜ 0.05 )that not found in the infected G1 phase J774A. 1 cells. Compared to the non-infected cells, the p21 mRNA levels in the infected J774A. 1 and THP-1 cells were significantly elevated(P ＜0.05), and the c-myc and p27 mRNA levels in the infected J774A. 1 cells and the cycA mRNA level in the infected THP-1 cells were also higher than those in both the non-infected cells ( P ＜ 0.05 ). Conclusion Different cell cycles and their regulating genes have a role to affect the apoptosis of human and murine mononuclear-macrophages caused by L. interrogans with a diversity of cell line origins.