关键词: 存活素, 流式细胞术, 细胞凋亡

Abstract: Abstract:Objective To construct the vector of RNA interference(RNAi) targeting survivin gene and observe its effect combined with X-rays radiation on lung adenocarcinoma A549 cell apoptosis. Methods One pair of RNAi sequence targeting survivin gene were designed according to its cDNA sequence reported in GenBank,the recombinant RNAi plasmid pGenesil2-survivin was constructed. After identified by enzyme digestion and sequencing,the pGenesil2-survivin plasmid was trasfeced into A549 cells. In the experiment,normal group,pGenesil2 group,pGenesil2-survivin group,5 Gy irradiation group and pGenesil2-survivin + 5 Gy irradiation group were set up. The apoptosis of A549 cells was measured by flow cytometry with PI/Annexin Ⅴ and TUNEL,the survivin and caspase-3 expressions were measured by Western blotting.Results Two fragments about 389 bp and 4 206 bp were gotten by KpnⅠand EcoRⅠenzyme digestion,they are the same to expected result,the sequencing result was compared to oligonucleotide chain with DNAssist 2.0,they were equal,these indicated the identification of pGenesil2-survivin vector was right; pGenesil2-survivin was transfected into A549 cells for 48 h,the apoptotic percentage in pGenesil2-survivin and 5 Gy X-rays groups increased obviously (P< 0.05),when the both were combined,the effect was more obvious; the Western blotting results appeared that the survivin gray scale/β-actin gray scale in pGenesil2-survivin group was lower than that in normal group (P< 0.01),and the caspase-3 gray scale/β-actin gray scale was higher than that in normal group,and that ratio in pGenesil2-survivin+5 Gy irradiation group was more high (P< 0.01).Conclusion RNAi targeting surviving gene could inhibit survivin protein expression,but enhance caspase-3 protein expression,and promote apoptosis.When it is combined with 5 Gy X-rays irradiation,the promotion of apoptosis is enhanced.

Key words: survivin, flow cytometry, apoptosis