重组腺病毒介导的ING4对人MCF-7乳腺癌细胞的增殖抑制及化疗增敏作用的实验研究

发布时间：2018-07-07 16:33

本文选题：ING4基因 + 腺病毒载体　；参考：《苏州大学》2015年硕士论文

【摘要】：目的:研究重组腺病毒介导的ING-4(Inhibitor of tumor growth factor 4)基因感染人MCF-7乳腺癌细胞后对后者所起的生长抑制作用及化疗增敏作用。方法:将搭载有ING-4基因的重组腺病毒载体Ad-ING4感染人MCF-7乳腺癌细胞,用荧光显微镜观察感染后的MCF-7细胞形态学变化;RT-PCR和Western-Blot法检测ING-4基因在MCF-7细胞中的转录和表达;CCK法分别测定ADM、5-Fu、CTX对病毒感染前后的MCF-7乳腺癌细胞的半数抑制浓度IC50,以观察其化疗药物增敏现象。用流式细胞技术检测ING-4基因对MCF-7细胞的促凋亡作用;半定量RT-PCR法检测ING-4基因表达对MCF-7细胞中相关凋亡基因表达的影响。结果:搭载有ING-4基因的重组腺病毒载体Ad-ING4成功感染人MCF-7乳腺癌细胞,并且随着感染作用时间的延长,于荧光显微镜下可见Ad-ING4作用下的MCF-7乳腺癌细胞变圆、脱落、皱缩、聚集等现象愈加明显,细胞贴壁数量明显减少;重组腺病毒载体感染MCF-7细胞后ING-4成功转染入细胞内,RT-PCR法检测出其于MCF-7细胞内获得表达;蛋白印迹法表明在感染后的MCF-7细胞内存在ING-4基因相关蛋白的表达;CCK-8试验表明在有ING-4基因表达的情况下,MCF-7细胞对化疗药物的敏感性有所增强;Ad-ING4与化疗药物联合使用时对MCF-7细胞的增殖抑制作用更为明显;RT-PCR检测表明Ad-ING4的转染使MCF-7细胞的Bax表达上升,Bcl-2与Survivin的表达下降,其机制可能与ING4降低Bcl-2/Bax比值和抑制Survivin的表达有关。结论:MCF-7细胞在转染ING4基因后,其细胞增殖受到了明显抑制,且对化疗药物的敏感度更高,更易凋亡,该现象可能是通过改变Bax,Bcl-2及Survivin表达水平来实现的。
[Abstract]:Aim: to study the growth inhibition and chemosensitization of recombinant adenovirus-mediated ING-4 (inhibitor of tumor growth factor 4) gene in human MCF-7 breast cancer cells. Methods: the recombinant adenovirus vector Ad-ING4 was transfected into human MCF-7 breast cancer cells. Morphological changes of MCF-7 cells after infection were observed by fluorescence microscope. RT-PCR and Western-Blot were used to detect the transcription and expression of EM-4 gene in MCF-7 cells. The half inhibitory concentration (IC50) of ADM5-Fu-CTX on MCF-7 breast cancer cells before and after infection was determined by CCK method. The apoptosis of MCF-7 cells was detected by flow cytometry, and the effect of the expression of Ring-4 gene on the expression of apoptotic genes in MCF-7 cells was detected by semi-quantitative RT-PCR. Results: the recombinant adenovirus vector Ad-ING4 was successfully infected with human MCF-7 breast cancer cells. With the extension of infection time, MCF-7 breast cancer cells induced by Ad-ING4 could be seen to become round, shedding and shrinking under fluorescence microscope. The aggregation became more and more obvious, and the number of cell adhesion was decreased, and the expression of the recombinant adenovirus vector in MCF-7 cells was detected by RT-PCR after transfection of the recombinant adenovirus vector into MCF-7 cells. Western blot analysis showed that there was an expression of EM-4 gene related protein in infected MCF-7 cells. CCK-8 test showed that chemosensitivity of MCF-7 cells to chemotherapeutic drugs was enhanced with the presence of EM-4 gene expression, and the combination of Ad-ING4 and chemotherapeutic drugs was also observed. RT-PCR analysis showed that the expression of Bax and survivin in MCF-7 cells increased after transfection of Ad-ING4, and the expression of Bcl-2 and survivin decreased in MCF-7 cells. The mechanism may be related to the decrease of Bcl-2 / Bax ratio and the inhibition of survivin expression by ING4. Conclusion after transfection of ING4 gene, the proliferation of MCF-7 cells was significantly inhibited, and its sensitivity to chemotherapeutic drugs was higher and apoptosis was more likely. This phenomenon may be achieved by changing the expression levels of BaxanBcl-2 and survivin.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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