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背景与目的细胞凋亡是一种进化上高度保守的细胞死亡方式,将高活性的促凋亡分子靶向性地导入肿瘤细胞而诱导肿瘤细胞凋亡是肿瘤基因治疗的潜在策略。在证实了大、小亚基次序颠倒的重构型人caspase-8具有持续的诱导宫颈癌细胞HeLa凋亡活性的基础上,本研究旨在分析3种重构型人caspase-8(Casp8CD、Rev8和Rev8L)促HeLa细胞凋亡的效率,探讨重构型人caspase-8作为诱导肿瘤细胞凋亡候选分子的可行性。方法脂质体法将Casp8CD、Rev8和Rev8L基因的pIRES2鄄EGFP真核表达载体转染入人HeLa和MCF鄄7细胞,用荧光显微镜观察目的基因的表达及其促凋亡效应,MTT法和细胞计数法检测3种重构型人caspase鄄8基因表达产物的促凋亡效率。用生物信息学方法分析Rev8和Rev8L分子大、小亚基间连接肽段的柔性。结果荧光显微镜观察结果表明,3种重构型人caspase-8基因在HeLa和MCF-7细胞中得到表达,其中Rev8和Rev8L基因的表达能有效促进被转染细胞的凋亡,细胞表现为凋亡性容积减少(AVD)。MTT结果显示,Rev8和Rev8L基因转染组在转染后20h,A570值即开始明显降低(与对照组相比)。细胞计数结果表明,转染后24h,Casp8CD、Rev8和Rev8L基因转染组的细胞死亡率分别为16.9%、52.3%和47.7%,而转染后48h各组相应的细胞死亡率分别为12.9%、51.6%和61.2%?
BACKGROUND & OBJECTIVE: Apoptosis is a highly conserved cell death mode. Targeting the introduction of highly active pro-apoptotic molecules into tumor cells and inducing tumor cell apoptosis is a potential strategy for gene therapy of tumors. On the basis of confirming that the reconstructed human caspase-8 with large and small subunit inverted has a continuous activity of inducing HeLa apoptosis in cervical cancer cells, the aim of this study was to analyze the expression of three reconstructed human caspase-8 (Casp8CD, Rev8 and Rev8L) to promote apoptosis of HeLa cells and to explore the feasibility of reconstituting human caspase-8 as a candidate molecule for inducing tumor cell apoptosis. Methods The eukaryotic expression vector pIRES2-EGFP of Casp8CD, Rev8 and Rev8L was transfected into HeLa and MCF-7 cells by lipofectamine. The expression of target gene and its pro-apoptotic effect were observed by fluorescence microscopy. MTT assay and cell proliferation assay Counting method was used to detect the pro-apoptotic efficiency of three recombinant human caspase-8 gene expression products. Bioinformatics methods were used to analyze the flexibility of linker between the large and small subunits of Rev8 and Rev8L. Results The results of fluorescence microscopy showed that the three reconstructed human caspase-8 genes were expressed in HeLa and MCF-7 cells. The expression of Rev8 and Rev8L genes could effectively promote the apoptosis of transfected cells. Decreased fatal volume (AVD). The results of MTT showed that the A570 values of Rev8 and Rev8L gene transfected groups began to decrease significantly (compared with the control group) 20 h after transfection. The results of cell counting showed that the cell death rates of Casp8CD, Rev8 and Rev8L gene transfected groups were 16.9%, 52.3% and 47.7% respectively at 24 hours after transfection, while the corresponding cell death rates of all groups at 48 hours after transfection were 12.9% , 51.6% and 61.2%?