嵌合重组caspase-3诱导表达促进肿瘤细胞凋亡  

Inducible Expression of Chimeric Recombinant Caspase-3 Promotes Apoptosis in Tumor Cells

作　　者：贾林涛[1] 张立红[1] 于翠娟[1] 纪宗玲[1] 曹云新[2] 王成济[1] 杨安钢[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,西安710032 [2]第四军医大学基础部免疫学教研室,西安710032

出　　处：《生物化学与生物物理进展》2003年第2期272-277,共6页Progress In Biochemistry and Biophysics

基　　金：国家高技术(863 )计划资助项目 (2 0 0 1AA2 1710 1);国家杰出青年科学基金 (3 992 5 0 3 6);军队杰出青年科学基金 (98J0 0 9)资助项目~~

摘　　要：通过稳定转染人宫颈癌HeLa细胞 ,建立了野生型caspase 3(wt casp3) ,大小亚基序列颠倒的重组caspase 3(r casp3) ,和N端融合绿脓杆菌外毒素 (PE)转膜肽段的嵌合重组caspase 3(cr casp3)的诱导表达细胞系 .蜕皮素诱导后细胞中检测到目的基因的表达 ,MTT检测和细胞计数结果表明 ,r casp3和cr casp3诱导表达后有效地导致HeLa细胞死亡 ,通过测定细胞中caspase 3活性 ,以及细胞周期检测、DNA梯状电泳条带检测(DNAladder)、电镜观察等证实r casp3和cr casp3诱导表达后细胞发生了凋亡 ,且二者的促凋亡活性相当 ,而wt casp3诱导表达细胞并未出现上述效应 .结果表明 ,与野生型caspase 3活化需要上游分子的切割不同 ,重组caspase 3具有自发的促凋亡活性 ,而N端PE肽段的融合不影响这种活性 ,因此PE转膜结构域和重组caspase 3有望参与构建能转膜进入细胞内部 。Human cervix HeLa cells were stably transfected to establish cell lines that inducibly expressed 3 types of caspase-3 constructs, respectively. These constructs involved wild-type caspase-3 (wt-casp3), recombinant caspase-3 (r-casp3) in which the order of the small and large subunits was reversed in contrast to the original protein, and chimeric recombinant (cr-casp3) in which a Pseudomonas exotoxin A (PE) -derived peptide was fused to N-terminus of r-casp3. The expression of the interest genes was detected upon induction with ponasterone. The genes of r-casp3 and cr-casp3 were demonstrated to effectively cause cell death by MTT assay and cell counting. Cells that expressed r-casp3 or cr-casp3, but not wt-casp3, underwent apoptosis in a comparable level as determined by cell cycle analysis, genomic DNA ladders, and electronic microscopy. These results prove that unlike wild-type caspase-3 which is inactive unless proteolytically processed by upstream caspase, both recombinant caspase-3s are naturally active, and the N-terminal fusion of PE translocation domain does not interfere with the natural caspase-3 activity, suggesting their applications on the construction of novel tumor therapeutics that efficiently translocate to the cytosol of tumor cells and cause cell death.

关 键 词：嵌合重组 CASPASE-3 诱导 肿瘤 细胞凋亡 绿脓杆菌外毒素 转膜肽段 

分 类 号：R730[医药卫生—肿瘤] Q255[医药卫生—临床医学]