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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 in the lung cancer cell model BERP35T1 induced by α-particle irradiation. Methods The eukaryotic expression plasmid p EGFP-CAT was constructed and identified by PCR, double enzyme digestion and sequencing. The recombinant plasmid and empty vector were transfected into BERP35T1 cells by lipofectamine. After selection by G418, Cell lines. Real-time PCR and Western blot were used to detect the mRNA and protein expression of CAT in BERP35T1 cells. Immunocytochemistry, MTT assay, cell scratch healing assay DNA oxidative damage level (8-OHd G level), proliferation (cell viability), migration ability (cell migration distance) and anchorage independence (soft agar colony formation rate). Results The recombinant plasmid pEGFP-CAT was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The BERP35T1-CAT-7 cell line with stable CAT expression was obtained. The m RNA transcription levels of CAT gene in BERP35T1, BERP35T1-p EGFP and BERP35T1-CAT-7 cells were 0.90 ± 0.23,1.00 ± 0.12 and 2.91 ± 0.41, respectively. The relative expression levels of CAT protein were 0.97 ± 0.11 and 1.00 ± 0.00 And 5.72 ± 1.28,8-OHd G were 1.65 × 10-2,1.60 × 10-2 and 8.95 × 10-3, respectively. The cell survival rates were (95.33 ± 4.26)%, (100.00 ± 8.29)% and (61.63 ± 3.08)%, and the cell migration distances were (150.68 ± 9.98) μm, (115.02 ± 30.73) μm and (44.25 ± 10.53) μm respectively. The rates of soft agar cell colonization were (2.50 ± 0.02) ‰ and ± 0.02) ‰ and (0.70 ± 0.02) ‰. Conclusion CAT overexpression may inhibit malignant phenotypes of BERP35T1 cells, such as proliferation, metastasis and anchorage independent growth, by decreasing the level of DNA oxidative damage.