刚地弓形虫RH株速殖子对小鼠结肠癌细胞ct26体外增殖和细胞周期的影响  

Effects of Toxoplasma gondii tachyzoites on proliferation and cell cycle of mouse colon cancer cells ct26

作　　者：高剑[1] 叶彬[1] 武卫华[1] 孔璟[1] 

机构地区：[1]重庆医科大学病原生物学教研室,分子医学与肿瘤研究中心,重庆400016

出　　处：《中国人兽共患病学报》2010年第12期1088-1092,共5页Chinese Journal of Zoonoses

摘　　要：目的研究刚地弓形虫RH株速殖子对小鼠结肠癌ct26细胞体外增殖和细胞周期的影响,并探讨其作用的分子机制。方法取对数生长期的小鼠结肠癌ct26细胞,建立弓形虫与ct26复合感染度(moi)分别为1∶1、2∶1、4∶1、8∶1、16∶1的共培养试验模型,台盼兰排斥试验连续7d绘制细胞生长曲线;流式细胞仪检测8∶1共培养模型组6h、12h、24h、48h细胞周期改变;采用半定量RT-PCR方法分别检测ct26细胞cyclinB1、cdc2基因转录水平变化;Westernblot方法检测细胞周期蛋白cyclinB1蛋白水平变化。结果台盼兰排斥试验发现各比例组弓形虫均能有效抑制小鼠ct26细胞的体外增殖,且呈现前4d缓慢抑制,4d后细胞大量死亡的现象,以8∶1模型组的增殖抑制作用最明显,感染7d后细胞抑制率达80.77%(P<0.01);流式细胞仪检测发现8∶1模型组弓形虫可在各作用时间明显改变ct26细胞周期分布,使G0/G1期细胞百分比下降,G2/M期百分比显著升高(P<0.01),24h达到作用高峰,使G2/M期百分比升高12.77%,48h则细胞G2/M期百分比下降;弓形虫感染ct26细胞3～24h,细胞cdc2转录水平在各个时间点未见明显变化,细胞cyclinB1从转录水平和蛋白水平均呈现为感染3h时表达增强,随后各时间点均明显降低的现象,cyclinB1基因转录水平在感染后24h只达到对照组的16.55%。结论弓形虫RH株速殖子可明显抑制小鼠结肠癌ct26细胞体外增殖,感染前期以诱导细胞发生G2/M期阻滞为主要机制,细胞周期蛋白cyclinB1活性下降在G2/M期阻滞中起主要作用。The objective was to investigate the changes of proliferation and cell cycle of mouse colon cancer cell ct26 infected with Toxoplasma gondii RH strain tachyzoites,and to elucidate possible molecular mechanism of G2/M arrest induced by T.gondii tachyzoites.The comitant culture models of ct26 cells with different amounts of tachyzoites of T.gondii RH strain（the proportions between tachyzoites and cells are 1∶1,2∶1,4∶1,8∶1 and 16∶1,respectively）were established,the growth curves of ct26 cells were delineated by trypan blue coloring method,and flow cytometry（FCM）was adopted to analyze the cell cycle progression in ct26 infected with the T.gondii tachyzoites（the proportion between tachyzoites and ct26cells is 8∶1）.Semi-quantitative RT-PCR was used to detect cyclinB1,cdc2 mRNA levels,and Western blot was used to detect cyclinB1 protein level.All different proportions of T.gondii tachyzoites could inhibit the proliferation of ct26 cells in vitro,the inhibition rates of proliferation were increased rapidly after 4 days,and the group of which proportion between tachyzoites and cells was 8∶1 changed more significantly comparing with the control group.The inhibition rates of proliferation increased to 80.77% on day 7（P0.01）.In the flow cytometric analysis,the percentage of G0/Gl phase of the ct26 cells was decreased and that of G2/M cells was increased significantly in the groups infected with T.gondii（P0.01）.The maximum change happened in 24h after treatment,and the percentage G2/M phase of the cells increased by 12.77%,then the percentage G2/M phase of the cells decreased in 48h after treatment.The degrade of cyclinB1 could be observed on both mRNA level and protein level,and there are no significant change in cdc2 mRNA level.T.gondii tachyzoites could inhibit the proliferation of ct26 cells actively in vitro.The blockage of ct26 cells in G2/M phase induced by T.gondii tachyzoites is the key mechanism at the initial stage,and cyclinB1 rather than cdc2 seems to play an important role in G2

关 键 词：刚地弓形虫 小鼠结肠癌ct26细胞 周期阻滞 细胞周期蛋白 

分 类 号：R382.5[医药卫生—医学寄生虫学]