摘要
为了进一步探讨端粒酶RNA(hTR)的反义cDNA对乳腺癌MCF 7细胞凋亡可诱导性的影响 ,构建了能将外源基因整合至细胞基因组的整合型腺病毒载体vAd AAV ,并将hTR全长cDNA反向连接至此载体上 ,获得反义重组腺病毒vAdT AAV .vAd AAV和vAdT AAV分别感染MCF 7细胞后 ,获得两个细胞株MCF 7 vAd AAV和MCF 7 vAdT AAV ,其中MCF 7 vAdT AAV细胞基因组内整合有hTR反义cDNA并能稳定表达 .利用生存曲线、细胞形态学观察、DNA片段分析和流式细胞分析来测定NaBu和无血清DMEM诱导后细胞凋亡的反应性 .通过生存曲线 ,发现NaBu诱导的MCF 7 vAdT AAV细胞比对照组MCF 7和MCF 7 vAd AAV细胞更早出现凋亡 .电镜下 ,NaBu或去血清诱导的MCF 7 vAdT AAV细胞更早出现凋亡形态学指标 .流式细胞分析和DNA片段凝胶电泳实验均显示MCF 7 vAdT AAV细胞对凋亡的抵抗力下降 .研究结果表明 ,端粒酶RNA的反义cDNA使乳腺癌MCF
To examine the effects of antisense human telomerase RNA (hTR) cDNA on tumor cells after induction of apoptosis, the antisense of hTR cDNA was introduced into human breast cancer cell line MCF 7.A hybrid adenovirus/adeno associated virus vector vAd AAV which can integrate foreign gene (antisense hTR cDNA) into cell genome and express constantly was used. The control recombinant virus vAd AAV and the antisense recombinant virus vAdT AAV were constructed. The whole hTR cDNA was integrated inversely into MCF 7 cells after infected with the antisense recombinant virus vAdT AAV. Two cell lines were obtained.One was MCF 7 vAd AAV which had been infected with the control virus vAd AAV and another was MCF 7 vAdT AAV which had been infected with the antisense recombinant virus vAdT AAV and could express constantly antisense hTR cDNA.The apoptosis reaction was measured by viability curve,morphology of cells, DNA fragmentation assays and cell flow cytometry analysis after cells were induced by NaBu and serum deprivation.Through viability curve, it was found that apoptosis was observed earlier in MCF 7 vAdT AAV cells than in MCF 7 vAd AAV and MCF 7 cells after introducing of NaBu. MCF 7 vAdT AAV cells showed earlier and deeper apoptotic phenotype under electron microscope after treatment of NaBu. Both flow cytometry analysis and DNA fragmentation electrophoresis indicated that MCF 7 vAdT AAV had less resistance to apoptosis. The results suggested that the potential induction of MCF 7 apoptosis was enhanced after antisense hTR cDNA was introduced into the cancer cells.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2001年第4期447-452,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家重点基础研究发展规划 (No .G2 0 0 0 0 5 70 0 1与G19990 5 390 6 )
国家自然科学基金重点项目 (No .39930 170 )资助课题&&
