GPX1过表达对肺癌BERP35T1细胞DNA氧化损伤和恶性表型的影响

目的: 探讨谷胱甘肽过氧化物酶1(GPX1)过表达对α粒子辐射致肺癌BERP35T1细胞DNA氧化损伤和恶性表型的影响。方法: 构建pEGFP-GPX1真核表达载体,经PCR、双酶切和测序鉴定后,采用脂质体法将重组质粒及其对照空载体分别转染至BERP35T1细胞中,筛选出具有G418抗性的细胞株,经Western blot法测定GPX1蛋白在细胞株中的表达情况;通过免疫细胞化学法、四甲基噻唑蓝(MTT)法、划痕愈合试验和锚着独立生长试验分别检测GPX1过表达对BERP35T1细胞内DNA氧化损伤产物8-羟化脱氧鸟苷(8-OHdG)水平,细胞增殖、迁移以及锚着独立生长能力的影响。结果: pEGFP-GPX1经PCR、双酶切鉴定和DNA测序证实,GPX1片段的序列完全正确;并获得GPX1稳定表达BERP35T1细胞株BERP35T1-GPX1-6,其GPX1蛋白水平是对照空载体转染细胞的4.01倍(P<0.05);与BERP35T1细胞和对照空载体转染细胞相比,BERP35T1-GPX1-6细胞内8-OHdG水平降低,细胞的增殖、迁移和锚着独立生长能力均减弱(P<0.05)。结论: 过表达GPX1可能通过清除细胞内活性氧降低DNA氧化损伤水平,从而抑制BERP35T1细胞增殖、迁移和锚着独立生长能力等恶性表型。

Effects of glutathione peroxidase 1 overexpression on DNA oxidative damage and phenotype of malignant BERP35T1 cells

OBJECTIVE: To study the effects of glutathione peroxidase 1(GPX1) overexpression on DNA oxidative damage level and phenotype of malignant BERP35T1 cells exposed upon α-particles. METHODS: The eukaryotic expression vector pEGFP-GPX1 was constructed. After PCR,enzyme digestion analysis and sequencing pEGFP-GPX1 and control vector were transfected into BERP35T1 cells with lipofectamine 2000 and G418 screening was used to obtain resistant cell lines. The expression of GPX1 was measured by Western blot. MTT,scratching healing test and anchorage independence growth test were used to analyze the effects of GPX1 overexpression on growth rate,migration and colony forming efficiency of BERP35T1 cells. RESULTS: pEGFP-GPX1 was confirmed by PCR,enzyme digestion analysis and sequencing. The sequence of the target gene GPX1 was entirely correct. The protein expression level of GPX1 in pEGFP-GPX1 transfected group BERP35T1-GPX1-6 was 4.01 times higher than in BERP35T1-pEGFP cells(P<0.05). Compared with BERP35T1 and BERP35T1-pEGFP,the level of growth rate,migration and colony forming efficiency in BERP35T1-GPX1-6 decreased significantly(P<0.05). CONCLUSION: GPX1 overexpression may inhibit the proliferation and metastasis of malignant BERP35T1 cells exposed to α-particles through reducing the level of DNA oxidative damage.