过氧化氢酶过表达对α粒子致肺癌细胞模型DNA氧化损伤和表型的影响  

Effect of catalase overexpression on DNA oxidative damages and phenotype of lung cancer cell model induced by radiation with α particles

作　　者：佟鹏[1] 魏志权[1] 张伟[1] 邵帅[1] 王春燕[1] 齐雪松[1] 苟巧[1] 

机构地区：[1]中国疾病预防控制中心辐射防护与核安全医学所辐射防护与核应急中国疾病预防控制中心重点实验室毒理学研究室,北京100088

出　　处：《中国生物制品学杂志》2015年第10期1011-1017,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(81000862)

摘　　要：目的探讨过氧化氢酶(catalase,CAT)过表达对α粒子辐射致肺癌细胞模型BERP35T1内DNA氧化损伤水平和恶性表型的影响。方法构建p EGFP-CAT真核表达质粒,经PCR、双酶切和测序鉴定后,采用脂质体法将该重组表达质粒及空载体转染至BERP35T1细胞中,经G418筛选出具有抗性的细胞株。Real-time PCR和Western blot法检测BERP35T1细胞中CAT基因m RNA转录和CAT蛋白表达水平;免疫细胞化学法、MTT法、细胞划痕愈合试验和锚着独立生长试验分别检测CAT过表达对BERP35T1细胞DNA氧化损伤水平(8-OHd G水平)、增殖(细胞存活率)、迁移能力(细胞迁移距离)和锚着独立性(软琼脂克隆形成率)的影响。结果重组表达质粒p EGFP-CAT经PCR、酶切鉴定和DNA测序证实构建正确,获得了CAT稳定表达的BERP35T1细胞株BERP35T1-CAT-7。空白对照BERP35T1、BERP35T1-p EGFP和BERP35T1-CAT-7细胞中CAT基因m RNA转录水平分别为0.90±0.23、1.00±0.12和2.91±0.41,CAT蛋白相对表达水平分别为0.97±0.11、1.00±0.00和5.72±1.28,8-OHd G表达水平分别为1.65×10-2、1.60×10-2和8.95×10-3,细胞存活率分别为(95.33±4.26)%、(100.00±8.29)%和(61.63±3.08)%,细胞迁移距离分别为(150.68±9.98)μm、(115.02±30.73)μm和(44.25±10.53)μm,细胞软琼脂克隆形成率分别为(2.50±0.02)‰、(2.10±0.02)‰和(0.70±0.02)‰。结论 CAT过表达可能通过降低DNA氧化损伤水平,抑制BERP35T1细胞的增殖、转移和锚着独立生长能力等恶性表型。Objective To investigate the effect of catalase（CAT） overexpression on DNA oxidative damage and malignant phenotype of lung cancer cell model BERP35T1 induced by radiation with α particles. Methods Eukaryotic expression vector p EGFP-CAT was constructed and identified by PCR, restriction analysis and sequencing, then transfected to BERP35T1 cells in mediation of liposome, using empty vector as control. G418-resistant cell strains were screened, in which the m RNA transcription and protein expression of CAT were identified by real-time PCR and Western blot. The effect of CAT overexpression on DNA oxidative damage（8-OHd G level）, proliferation（cell surviva）, migration ability（distance of cell migration）and anchoring independence（formation rate of soft agar colonies）in BERP35T1 cells were evaluated by immunocytochemical method, MTT assay, wound healing test and anchorage independent growth test respectively.Results PCR, restriction analysis and sequencing proved that recombinant plasmid p EGFP-CAT was constructed correctly. BERP35T1-CAT-7 cell strain for stable expression of CAT was obtained. The transcription levels of CAT m RNA in BERP35T1, BERP35T1-p EGFP and BERP35T1-CAT-7 cells were 0. 90 ± 0. 23, 1. 00 ± 0. 12 and 2. 91 ± 0. 41, while the relative expression levels of CAT protein were 0. 97 ± 0. 11, 1. 00 ± 0. 00 and 5. 72 ± 1. 28, and the expression levels of 8-OHd G were 1. 65 × 10-2, 1. 60 × 10-2and 8. 95 × 10-2, respectively. However, survival rates of BERP35T1,BERP35T1-p EGFP and BERP35T1-CAT-7 cells were（95. 33 ± 4. 26）%,（100. 00 ± 8. 29）% and（61. 63 ± 3. 08）%,while the migration distances were（150. 68 ± 9. 98）,（115. 02 ± 30. 73） and（44. 25 ± 10. 53） μm, and the formation rates of soft agar colonies were（2. 50 ± 0. 02）‰,（2. 10 ± 0. 02）‰ and（0. 70 ± 0. 02） ‰, respectively. Conclusion CAT overexpression may inhibit the proliferation, migration and anchoring independence of BERP35T1 cells by decreasing the level of DNA oxidati

关 键 词：过氧化氢酶 过表达 Α粒子 肺癌 DNA氧化损伤 

分 类 号：R734.2[医药卫生—肿瘤] R73-36[医药卫生—临床医学]