重组型Caspase3对U251胶质瘤细胞促凋亡作用

Investigation on U251 Glioma cells apoptosis induced by recombinat caspase3

目的探讨由野生型人Caspase3大小亚基颠倒构建的重组型Caspase3促U251胶质瘤细胞的调亡活性。方法运用分子克隆技术,使Caspase3基因大小亚基颠倒构建,并将重组基因克隆入绿色荧光蛋白(GFP)真核表达载体pcDNA3.1中,转染人U251胶质瘤细胞。利用透射电镜和流式细胞仪观察胶质瘤细胞凋亡的生物学特征。结果成功地获得了重组型反向Caspase3基因。经限制酶酶切分析鉴定释放330及550bp片段,PCR法鉴定反向重组成功;构建了重组型Caspase3基因的真核表达载体,转染U251胶质瘤细胞后,重组型Caspase3基因在细胞中表达,电镜显示细胞呈现凋亡的典型形态学特征。流式细胞仪可见细胞凋亡峰。结论重组型Caspase3可促进U251胶质瘤细胞的凋亡。

Objective To investigate the facilitaling action of recombinant caspase 3 onU251 glioma cell apoptosis constructed from reversal of two subunits of wild type caspase3. Methods Human caspase3 gene was transformed into recombinant caspase3 gene by settling the small subunit prior to the large ones by molecular cloning . The new gene was cloned into GFP mammalian expression vector pcDNA3.1-EGFP and transfected into U251 glioma cells. The transfected cells were observed with electron microscopes and Flow cytometer . Results Recombinant caspase3 was cloned successfully. After enzymes digestion,recombinant caspase3 gene released 330bp and 550bp fragments, the recombinant rev-caspase3 were identified by PCR. U251 glioma cells transfected by mammalian expression vectors of recombinant caspase3 and displayed typical features of apoptosis . Conclusion recombinant caspase3 can effectively accelerate U251 glioma cell apoptosis.