放射诱导辣根过氧化物酶/吲哚乙酸自杀基因表达对肺癌细胞系的生物效应研究

Radiobiology effects of radiation-induced horseradish peroxidase/indole-3-acetic suicide gene expression in lung cancer cells

目的 检测放射诱导的肿瘤特异性嵌合启动子介导的辣根过氧化物酶/吲哚乙酸自杀基因系统对肺癌细胞A549、SPC-A1的生物效应.方法 构建含有6个CArG元件的人端粒酶逆转录酶基因嵌合启动子调控的辣根过氧化物酶表达的质粒载体,及单个端粒酶逆转录酶启动子质粒和对照质粒,分别为pE6-hTERT-HRP、phTERT-HRP、pControl-HRP、pControl-luc.分别用细胞计数法、Annexin V-FITC染色检测此嵌合启动子质粒系统对肺癌细胞A549、SPC-A1和正常人胚肺细胞（hEL）的生长抑制及凋亡效应.利用成克隆分析法检测此系统对肺癌细胞A549、SPC-A1放射敏感性的影响.结果 在6 Gy放射线诱导下质粒pE6-hTERT-HRP、phTERT-HRP、pControl-HRP、pControl-luc介导的自杀基因系统对A549细胞的平均生长抑制率分别为72.92%、40.60%、51.00%、25.19%（F=67.31,P〈0.01）,对SPC-A1细胞的平均抑制率分别为64.63%、30.02%、48.23%、23.16%（F=64.94,P〈0.01）,而对正常hEL细胞则分别为20.81%、18.05%、44.20%、18.32%（F=52.19,P〈0.01）.同样四种质粒对A549细胞的平均早期凋亡率分别为36.63%、22.30%、24.33%、12.53%（F=50.99,P〈0.01）,对SPC-A1细胞的平均早期凋亡率分别为33.73%、17.37%、22.43%、11.20%（F=20.76,P〈0.01）,而对正常hEL细胞则分别为13.53%、12.5%、21.93%、12.16%（F=15.08,P〈0.01）.同样四种质粒对A549细胞放射增敏比分别为3.45、2.29、3.05、1.21,对SPC-A1细胞的放射增敏比则分别为2.68、2.15、2.28、1.15.结论 放射诱导的肿瘤特异性嵌合启动子介导的辣根过氧化物酶/吲哚乙酸自杀基因系统对肺癌细胞A549、SPC-A1具有特异杀伤作用,具有很好的应用前景.

Objective To detect specific cell killing effect of radiation combined with horseradish peroxidase （HRP）/indole-3-acetic （IAA） suicide gene therapy controlled by a novel radio-inducible and cancer-specific chimeric gene promoter in lung cancer. Methods We constructed a plasmid expressing HRP enzyme under the control of chimeric human telomerase reverse transcriptase （hTERT） promoter carrying 6 CArG elements, a plasmid expressing HRP enzyme under the control of hTERT promoter carrying single CArG element, and two control plasmids, which named pE6-hTERT-HRP, phTERT-HRP, pControl-HRP, and pControl-luc, respectively. After radiation, the proliferation inhibition and apoptosis induction effect of each type of plasmid in lung cancer cells （A549, SPC-A1） and normal lung cells （hEL） was detected by cell counting and Annexin V-FITC staining. The change of radiosensitivity of lung cancer cells with plasmid system was also detected by clonogenic assays. Results After a single dose radiation of 6 Gy,the average proliferation inhibition rates of pE6-hTERT-HRP, phTERT-HRP, pControl-HRP, and pControl-luc systems were 72. 92% ,40.60% , 51.00% and 25.19% （F= 67.31 , P〈 0.01） in A549 cells ,64.63%,30.02%,48.23% and 23.16% （F=64.94, P〈 0.01） in SPC-A1 cells, and 20.81%,18.05%, 44.20% and 18.32% （F=52. 19,P〈0.01） in normal hEL cells, respectively. The average early apoptosis rates of these four plasmid systems were 36. 63%, 22. 30%, 24. 33% and 12. 53% （F =50. 99,P 〈0. 01） in A549 cells, 33.73%, 17. 37%, 22. 43% and 11.20% （F = 20. 76, P 〈 0. 01） in SPC-A1 cells, and 13.53 %, 12. 5%, 21.93% and 12. 16% （F = 15. 08, P 〈 0. 01） in normal hEL cells,respectively. The sensitizing enhancement ratios of the four plasmid systems were 3.45, 2. 29, 3.05 and 1.21 in A549 cells, while 2. 68, 2. 15, 3.05 and 1.21 in SPC-A1 cells, respectively. Conclusions The new suicide gene system controlled by chimeric promoter may provide a novel therapeutic modality for lung cancer.