刚地弓形虫RH株速殖子协同VP-16体外杀伤小鼠结肠癌细胞ct26的作用  

Tachyzoites of Toxolasma gondii enhances the cytotoxicity of Etooside (V-16) to mouse colon cancer cell ct26 in vitro

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8期720-724,共5页Chinese Journal of Zoonoses

摘　　要：目的探讨刚地弓形虫RH株速殖子对小鼠结肠癌ct26细胞周期的影响及其协同细胞周期特异性抗癌药依托泊甙(VP-16)对ct26细胞的杀伤作用。方法建立弓形虫RH株速殖子与小鼠结肠癌ct26细胞不同比例的共培养模型(虫/细胞比例分别为2:1、4:1、8:1、16:1),流式细胞仪检测24h细胞周期改变;以虫细胞比例2:1预先感染ct26细胞24h,CCK-8比色法观察不同浓度VP-16对细胞增殖抑制的改变;荧光显微镜观察VP-16(40μg/mL)对感染虫体细胞的形态学改变。台盼蓝染色观察不同浓度VP-16分别作用弓形虫RH株速殖子悬液(1×106/mL)2h、4h、12h、24h的虫体存活度改变。琼脂糖凝胶电泳观察VP-16(20μg/mL)对虫体DNA影响。结果流式细胞仪检测发现各浓度弓形虫速殖子均可明显改变ct26细胞24h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)。2×106个速殖子感染细胞24h可使S期和G2期细胞百分比共同增长14.92%。CCK-8比色法检测结果显示虫细胞比例2:1预先感染ct26细胞24h可有效增强各浓度VP-16对ct26细胞的杀伤作用;Hochest33258染色结合荧光显微镜观察到虫体胞质寄生形态及VP-16诱导感染细胞凋亡典型形态;10μg/mL及更高浓度VP-16作用弓形虫4h以上均可使虫体全部死亡;琼脂糖凝胶电泳发现VP-16(20μg/mL)可使弓形虫速殖子DNA呈现典型的云梯状条带。结论弓形虫RH株速殖子可使小鼠结肠癌ct26细胞发生G2/M期,可有效增强细胞周期特异性抗癌药VP-16的抗癌作用,且弓形虫增殖亦明显受抑并发生凋亡。The urose of this study was to investigate the changes of cell cycle of mouse colon cancer cell ct26 infected with Toxolasma gondii（T.gondii） RH strain tachyzoites and test the synergistic action of T.gondii tachyzoites with cell cycle secific anticancerogen Etooside（V-16） for enhancing cytotoxicity to cancer cell ct26.The co-culture models of ct26 cells with different tachyzoites numbers of T.gondii RH strain（the roortions were 2∶1,4：1,8∶1 and 16∶1,resectively） were established,and the changes of cell cycle of ct26 cells were analyzed by flow cytometry.The ct26 cells（1×10^6/mL） infected with T.gondii tachyzoites（2×10^6/mL） had been cultured for 24 hours before being treated with different concentrations of V-16,then the growth inhibition rates of ct26 cells were tested by the CCK-8 stain method.The tachyzoites infected ct26 cells treated with 40μg/mL V-16 were stained with Hochest 33258,then their morhological changes were observed by fluorescence microscoy.The inhibition rates of roliferation of T.gondii tachyzoites treated with different concentrations of V-16 for different hours（2h,4h,12h and 24h resectively） were evaluated by tryan-blue exclusion method.The DNA changes of T.gondii tachyzoites treated with V-16（20μg/mL） were detected by agarose electrohoresis.Results showed that the different concentrations of T.gondii tachyzoites could change the cell cycle of ct26.And comared with the control grou,the ercentage of G0/Gl hase of the ct26 cells was decreased and that of G2/M cells was increased significantly in the grous infected with T.gondii（P〈0.01）.The ct26 cells（1×10^6/mL） infected with tachyzoites（2×10^6/mL） for 24 hours could make the ercentage of S and G2/M hase of the cells increased by 14.92%,and could enhance cytotoxicity of different concentrations of V-16 to the cancer cells.The arasitism of T.gondii tachyzoites in ct26 cells was observed under fluorescence microscoe,and the aotosis of ct26 cell and tachyzoites treated with V-16 were obvious.

关 键 词：弓形虫 小鼠结肠癌ct26细胞 依托泊甙 细胞周期 

分 类 号：R382.5[医药卫生—医学寄生虫学]