携带TAT-Apoptin重组减毒鼠伤寒沙门菌对肿瘤细胞的作用
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- 论文作者：田文静 ; 郁川 ; 陈桂华 ; 廖成水 ; 翟崇凯 ; 张春杰 ; 程相朝
- 年：2016
- 作者机构：河南科技大学动物疫病与公共安全重点实验室;洛阳职业技术学院;
- 论文关键词：减毒鼠伤寒沙门菌 ; TAT-Apoptin ; 表达 ; 信号通路 ; 抗肿瘤
- 英文论文关键词：attenuated Salmonella typhimurium ; TAT-Apoptin ; expression ; signal pathway ; anti-tumor
- 会议召开时间：2016-11-04
- 会议录名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会论文集
- 语种：中文
- 分类号：S857.4
- 学会代码：ZGXJ
- 会议名称：中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会
- 会议地点：中国山东青岛
- 主办单位：中国畜牧兽医学会动物微生态学分会
- 学会名称：中国畜牧兽医学会
- 页数：1
- 文件大小：94k
- 原文格式：O
- 会议级别：全国
摘要
为研究携带TAT-Apoptin重组减毒鼠伤寒沙门菌对肿瘤细胞的作用,将构建好的能稳定携带TAT-Apoptin的重组减毒沙门菌体外感染黑色素瘤细胞B16F10和肺癌细胞LL/2,Western-blot检测TAT-Apoptin在不同肿瘤细胞内的表达情况,CCK-8法检测不同MOI及感染时间条件下细胞增殖情况,TUNEL法检测细胞凋亡情况,并分析Caspase-1,3,6,8,9和细胞色素C等信号蛋白的表达情况。Western-b1ot结果显示,重组菌感染两细胞后,均可检测到约20KD的TAT-Apoptin蛋白条带;CCK-8法实验结果表明,MOI浓度越大,抑制率越高,并且随着感染时间的增加,抑制率逐渐下降,说明重组菌抑制B16F10和LL/2细胞增殖具有浓度依赖性和时间依赖性;重组菌感染B16F10、LL/2细胞24h后,荧光显微镜下可观察到TUNEL阳性细胞;这表明重组菌能够诱导B16F10和LL/2细胞凋亡;进一步对凋亡信号蛋白分析发现,重组菌感染肿瘤细胞B16F10和LL/2后,能诱导Caspase-3,Caspase-6,Caspase-8,Caspase-9和细胞色素C的表达,互补菌对照组和细胞培养空白组均不能诱导上述信号蛋白的表达,此外Caspase-1在LL/2细胞中表达显著升高,以上结果表明,重组减毒鼠伤寒沙门菌能够有效递呈TAT-Apoptin基因在黑色素瘤细胞B16F10和肺癌细胞LL/2内表达,且能够诱导这两种肿瘤细胞凋亡,其可能通过外源性死亡受体途径和内源性线粒体途径诱导肿瘤细胞凋亡,并且诱导肺癌细胞LL/2凋亡还与炎症相关因子Caspase-1的介导有关。
In order to study the Anti-tumor cells effect of combinant attenuated Salmonella Typhimurium Harbouring TAT-Apoptin Gene,B16F10 melanoma cells and lung cancer cells LL/2 were infected by recombinant attenuated Salmonella in vitro.Western-blot were used to detect the expression of TAT-Apoptin in different tumor cells.The proliferation of cells under different MOI and infection time conditions were detected by CCK-8,The apoptosis was detected by flow cytometry and TUNEL method.Furthermore,the expression of Caspase-1,3,6,8,9 and cytochrome C and other signaling proteins were analyzed.Western blot assay displayed that TAT-Apoptin protein bands of about 20 KD can be detected in cells after infection.The results of CCK-8 showed that the higher the concentration of MOI,the higher the inhibition rate,And with the increase of the infection time,the inhibition rate decreased gradually,The results indicated that the recombinant attenuated inhibited B16F10 and LL/2 cells proliferation in a dose-dependent and time-dependent manner;Recombinant attenuated infected B16F10,LL/2 cells for 24 h,TUNEL positive cells were observed by fluorescence microscope,the results indicated that the recombinant attenuated could induce B16F10 and LL/2 cells apoptosis;After infected tumor cells B16F10 and LL/2,the apoptosis signaling proteins analysis showed that the recombinant strain could induce the expression of Caspase-3,Caspase-6,Caspase-8,Caspase-9 and cytochrome C,in contrast,cell control group and Complementary control group did not.In addition,the expression of Caspase-1 was significantly increased in LL/2 cells,The results indicated that recombinant strain could effectively present TAT-Apoptin gene to express in melanoma cells B16F10 and lung cancer cell LL/2,and induce the apoptosis of them,the signal pathway of recombinant attenuated Salmonella in vitro anti-cancer cells of melanoma cells and lung cancer might through the death receptor-mediated extrinsic pathway and the mitochondria-mediated intrinsic pathway,and the process of lung cancer cells apoptosis might also be related to the inflammation related factors Caspase-1.
In order to study the Anti-tumor cells effect of combinant attenuated Salmonella Typhimurium Harbouring TAT-Apoptin Gene,B16F10 melanoma cells and lung cancer cells LL/2 were infected by recombinant attenuated Salmonella in vitro.Western-blot were used to detect the expression of TAT-Apoptin in different tumor cells.The proliferation of cells under different MOI and infection time conditions were detected by CCK-8,The apoptosis was detected by flow cytometry and TUNEL method.Furthermore,the expression of Caspase-1,3,6,8,9 and cytochrome C and other signaling proteins were analyzed.Western blot assay displayed that TAT-Apoptin protein bands of about 20 KD can be detected in cells after infection.The results of CCK-8 showed that the higher the concentration of MOI,the higher the inhibition rate,And with the increase of the infection time,the inhibition rate decreased gradually,The results indicated that the recombinant attenuated inhibited B16F10 and LL/2 cells proliferation in a dose-dependent and time-dependent manner;Recombinant attenuated infected B16F10,LL/2 cells for 24 h,TUNEL positive cells were observed by fluorescence microscope,the results indicated that the recombinant attenuated could induce B16F10 and LL/2 cells apoptosis;After infected tumor cells B16F10 and LL/2,the apoptosis signaling proteins analysis showed that the recombinant strain could induce the expression of Caspase-3,Caspase-6,Caspase-8,Caspase-9 and cytochrome C,in contrast,cell control group and Complementary control group did not.In addition,the expression of Caspase-1 was significantly increased in LL/2 cells,The results indicated that recombinant strain could effectively present TAT-Apoptin gene to express in melanoma cells B16F10 and lung cancer cell LL/2,and induce the apoptosis of them,the signal pathway of recombinant attenuated Salmonella in vitro anti-cancer cells of melanoma cells and lung cancer might through the death receptor-mediated extrinsic pathway and the mitochondria-mediated intrinsic pathway,and the process of lung cancer cells apoptosis might also be related to the inflammation related factors Caspase-1.
引文
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