摘要
目的观察重构型caspase-7的表达对肿瘤细胞的凋亡诱导作用。方法用反转录PCR方法获取野生型人caspase-7基因,用重组PCR构建了两种重构型caspase-7基因,即大、小亚基序列次序颠倒的rev-ca2spase-7以及N-端带有绿脓杆菌外毒素转膜结构域部分肽段的融合蛋白(PEⅡ=rev-caspase-7)的基因。将两种重构型caspase-7基因克隆入表达载体pIRES2-EGFP,脂质体法转染肿瘤细胞,通过荧光显微镜观察、电镜观察、MTT检测等方法检测重组基因的表达及其对肿瘤细胞的促凋亡活性。结果 成功获得了caspase-7基因,构建了rev-caspase-7基因和rev-caspase-7与绿脓杆菌外毒素(PE)转膜肽段的融合蛋白(PEⅡ-rev-caspase-7)基因及其表达载体。将两种重构型caspase-7转染肿瘤细胞后,证实细胞发生凋亡。结论两种重构型caspase-7均可以有效地诱导肿瘤细胞发生凋亡。
Objective To investigate the induction of tumor cells to apoptosis by the expression of reconstructed caspase-7. Methods RT-PCR and recombinant PCR were used to amplify wild-type human caspase-7 gene, followed by the generation of the genes of two reconstructed caspase-7, i. e. a rev-caspase-7, in which the sequences encoding the large and the small subunits were reversed in comparison with the wild-type caspase-7, and a rev-caspase-7 fusion protein, in which a translocation domain of Psuedomonas exotoxin A (PE) was fused N-terminally to rev-caspase-7. After cloning into expression vector pIRES2-EGFP and lipofectin-mediated transfection of tumor cells, we evaluated the growth status and apoptosis of these cells by electron microscopy, MTT assay, etc. Results We successfully obtained the genes of wild-type caspase-7 and rev-caspase-7, as well as that of a fusion protein of PE translocation domain and rev-caspase-7. After transfected with tumor cells, the expression of both reconstructed caspase-7s could induce the cells to apoptosis. Conclusion The expression of reconstructed human caspase-7 genes effictively contributes to apoptosis of tumor cells.
出处
《医学分子生物学杂志》
CAS
CSCD
2004年第1期15-20,共6页
Journal of Medical Molecular Biology
基金
国家杰出青年科学基金(39925036)
国家高技术研究发展计划
(863计划)(2001AA217101)
