刚地弓形虫细胞培养上清对人结肠癌细胞sw480增殖与凋亡的影响

Inhibition of proliferation and apoptosis of human colon cancer cell sw480 cell line induced by the co-culture supernatant of Toxoplasma gondii

目的观察刚地弓形虫RH株速殖子与人结肠癌sw480细胞系共培养上清对人结肠癌sw480细胞增殖的影响和诱导其凋亡与坏死的情况。方法取对数生长期的人结肠癌sw480细胞(1×106),建立弓形虫RH株速殖子数目分别为2×106、4×106、8×106、16×106的共培养模型,观察弓形虫速殖子在sw480细胞中的寄生,提取共同孵育72h后的培养上清,以新鲜培养基稀释为半量,体外作用人结肠癌sw480细胞12h、24h、48h、72h,CKK-8法检测吸光度(A450值)并计算抑制率;Annexin-v-FITC/PI染色细胞后上流式细胞仪检测细胞凋亡与坏死率;琼脂糖凝胶电泳观察细胞凋亡DNA条带;透射电镜和荧光显微镜观察细胞形态学改变。结果弓形虫RH株速殖子可在人结肠癌sw480细胞内寄生和增殖。CKK-8法检测结果显示上述共培养上清对sw480细胞增殖抑制率随上清作用时间延长均明显增大,48h最大抑制率达44.55%。流式细胞仪检测显示实验组sw480细胞早期凋亡率和晚期凋亡与坏死率随上清作用时间延长均明显增加,48h早期凋亡率达最高值(11.54%),48h以后早期凋亡率下降,晚期凋亡和坏死率显著上升,72h总死亡率可达46.11%,细胞杀伤效果明显;琼脂糖凝胶电泳显示典型的DNA云梯状条带;荧光显微镜和透射电镜观察到细胞凋亡和坏死典型形态。结论弓形虫RH株速殖子与人结肠癌sw480细胞共培养上清对体外培养的sw480细胞增殖有明显的抑制作用,并可诱导sw480细胞凋亡与坏死。

To investigate the inhibitive effect of the co-culture supernatant of Toxoplasma gondii on the proliferation of human colon cancer cell line sw480 and/or its induced apoptosis and necrosis in vitro,the co-culture model of sw480 cells（1x106） was established with different number of Toxoplasma gondii（the concentration of tachyzoites was 2×106,4×106,8×106,16×106 respectively）,and the co-culture supernatants were gathered 72 hours later.The sw480 cells were treated for different hours with different models of the co-culture supernatant.The parasitism and proliferation of Toxoplasma gondii tachyzoites in sw480 cells was observed by Giemsa staining,and the growth inhibition rates of these cells were investigated by the CKK-8 method.Meanwhile,the apoptosis and necrosis of sw480 cells were detected with transmission electron microscopy（TEM） and fluorescence microscopy with Hochest 33258 staining to observe the morphological changes of sw480 cells.Agarose electrophoresis was used to detect the DNA change of sw480 cells,and flow cytometric analysis was used for investigation of rates of apoptosis and necrosis of these cells stained with Annexin-v-FITC/PI.It was found that the parasitism and proliferation of Toxoplasma gondii tachyzoites in sw480 cells could be observed;and as demonstrated by the CKK-8 method,the inhibitive effect of the co-culture supernatant depended upon the treatment time,and a maximum inhibition ratio of 44.55% was detected at 48 hours.The karyopyknosis and karyorrhexis in the treated sw480 cells were observed under the fluorescence microscope;and the cellular necrosis were observed under TEM.In addition,the DNA fragments in the treated cells were revealed by agarose electrophoresis.In the flow cytometric analysis,a maximum prophase apoptosis rate of 11.54% was detected at 48 hours after treatment with the supernatants,while the rate of prophase apoptosis decreased,and the rates of advanced stage/necrosis increased markedly at the treatment time beyond 48 hours.It is concluded that the co-culture supernatant of Toxoplasma gondii and human colon cancer cell sw480 cell line can induce inhibition of proliferation,apoptosis and necrosis of this cell line in vitro.