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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 apoptotic activity of recombinant Caspase3-promoting U251 glioma cells constructed by wild-type human Caspase3 subunit. Methods The subunit of Caspase3 gene was constructed by reverse transcription polymerase chain reaction (RT - PCR). The recombinant plasmid was cloned into the green fluorescent protein (GFP) eukaryotic expression vector pcDNA3.1 and transfected into U251 glioma cells. The biological characteristics of glioma cell apoptosis were observed by transmission electron microscopy and flow cytometry. Results The recombinant reverse caspase3 gene was successfully obtained. The 330 and 550bp fragments were identified by restriction enzyme digestion, and the reverse recombinant was identified by PCR. The recombinant Caspase3 gene was constructed and transfected into U251 glioma cells, and the recombinant Caspase3 gene was expressed in the cells Electron microscopy showed that the cells showed the typical morphological characteristics of apoptosis. Flow cytometry showed apoptosis peak. Conclusions Recombinant Caspase3 can promote the apoptosis of U251 glioma cells.