灭活仙台病毒Tianjin株诱导人乳腺癌MDA-MB-231细胞和宫颈癌HeLa细胞凋亡机制的研究

发布时间：2018-06-01 13:49

  本文选题：仙台病毒Tianjin株 + MDA-MB-231细胞　； 参考：《天津医科大学》2015年硕士论文

【摘要】：目的:近年来,利用病毒治疗肿瘤取得了巨大进展。仙台病毒Tianjin株是仙台病毒新基因型。本论文拟采用紫外线灭活仙台病毒Tianjin株(以下简称UV-Tianjin)感染人乳腺癌MDA-MB-231细胞和人宫颈癌HeLa细胞,通过检测细胞的凋亡情况及凋亡发生的可能机制来评价UV-Tianjin的抗肿瘤作用,以期为病毒抗肿瘤作用机制研究及开发新的治疗乳腺癌和宫颈癌的生物制剂提供参考依据。方法:用不同滴度UV-Tianjin分别感染人乳腺癌细胞MDA-MB-231和人宫颈癌细胞HeLa,24小时后,(1)MTT法检测不同滴度UV-Tianjin(MOI:10,20,40,80,160,320)对MDA-MB-231细胞和人正常乳腺上皮细胞MCF 10A,或HeLa细胞增殖的影响;(2)Hoechst染色荧光显微镜观察UV-Tianjin(MOI:40,80)感染后MDA-MB-231细胞或HeLa细胞的形态学改变;(3)AnnexinⅤ-FITC/PI标记流式细胞仪检测UV-Tianjin(MOI:20,40,80)诱导MDA-MB-231细胞或HeLa细胞产生凋亡的凋亡率;(4)分光光度法检测UV-Tianjin(MOI:20,40,80)感染后MDA-MB-231细胞或HeLa细胞中Caspase-3、-8和-9的活性变化;(5)JC-10染色流式细胞仪检测UV-Tianjin(MOI:5,20,80)感染后MDA-MB-231细胞或UV-Tianjin(MOI:20,40,80)感染后HeLa细胞线粒体膜电位的变化;(6)Western blot检测UV-Tianjin(MOI:20,80)感染后MDA-MB-231细胞中Bcl-2、Bax、细胞色素c、caspase-9、Fas、FasL、caspase-8和caspase-3蛋白表达水平或UV-Tianjin(MOI:20,40,80)感染后HeLa细胞中细胞色素c、APAF-1、caspase-9、Fas、FasL、FADD、caspase-8和caspase-3蛋白表达水平。结果:(1)MTT检测结果显示UV-Tianjin能够抑制MDA-MB-231细胞或HeLa细胞的增殖,且呈剂量依赖性;而对正常乳腺上皮细胞MCF 10A无明显抑制作用;(2)Hoechst染色结果显示灭活病毒组MDA-MB-231细胞或HeLa细胞均发生了明显的形态学变化:细胞核致密浓染,呈高亮的蓝色荧光,且具有剂量依赖性趋势;(3)AnnexinⅤ-FITC/PI标记流式细胞仪检测结果显示灭活病毒组MDA-MB-231细胞或HeLa细胞的凋亡率呈剂量依赖性升高(P0.01);(4)分光光度法检测结果显示灭活病毒组MDA-MB-231细胞或HeLa细胞中caspase-3,-8和-9的活性增加且呈剂量依赖性(P0.05);(5)JC-10染色流式细胞仪分析显示灭活病毒组MDA-MB-231细胞或HeLa细胞线粒体膜电位呈剂量依赖性降低,且与细胞对照组比较均有统计学差异(P0.01);(6)Western blot结果显示高剂量灭活病毒组MDA-MB-231细胞中Bcl-2、Bax、细胞色素c、caspase-9、Fas、FasL、caspase-8和caspase-3蛋白表达水平或HeLa细胞中细胞色素c、APAF-1、caspase-9、Fas、FasL、FADD、caspase-8和caspase-3蛋白表达水平与对照组比较均有显著差异(P0.05)。结论:灭活仙台病毒Tianjin株能够抑制人乳腺癌MDA-MB-231细胞和人宫颈癌HeLa细胞的增殖,诱导MDA-MB-231细胞和HeLa细胞发生凋亡,且呈剂量依赖性,其凋亡发生的机制可能与线粒体内源性途径和死亡受体外源性途径相关。
[Abstract]:Objective: in recent years, great progress has been made in the treatment of tumor with virus. Sendai virus Tianjin strain is a new genotype of Sendai virus. In this paper, ultraviolet inactivated Sendai virus Tianjin strain (UV-Tianjin) was used to infect human breast cancer MDA-MB-231 cells and human cervical cancer HeLa cells. The anti-tumor effect of UV-Tianjin was evaluated by detecting the apoptosis of the cells and the possible mechanism of apoptosis. The aim of this study was to provide a reference for the study of the antitumor mechanism of the virus and the development of new biological agents for the treatment of breast and cervical cancer. Methods: the effects of different titers of UV-Tianjin on the proliferation of MDA-MB-231 cells and normal breast epithelial cells (MCF 10A or HeLa cells) were detected by MTT assay after 24 hours after infection with human breast cancer cell line MDA-MB-231 and human cervical cancer cell line HeLa. Morphologic changes of MDA-MB-231 cells or HeLa cells after UV-Tianjin MOI: 4080) the apoptosis rate of MDA-MB-231 cells or HeLa cells induced by UV-Tianjin: MOI: 4040 ~ 80) was detected by flow cytometry with annexexin 鈪，

本文编号：1964398