N-乙酰半胱氨酸对支气管上皮细胞辐射致癌模型DNA氧化损伤和存活率的影响

目的:探讨N-乙酰半胱氨酸(N-acetyl cysteine,NAC)对人支气管上皮细胞辐射致癌模型BERP35T-1细胞DNA氧化损伤和存活率的影响。方法:运用Western blot法检测人支气管上皮细胞BEP2D,RH22和BERP35T-1,以及用不同浓度NAC(0～2mmol/L)作用BERP35T-1 48 h后,细胞内DNA双链断裂产物γH2AX的表达水平变化;免疫细胞化学法检测1 mmol/L NAC作用BERP35T-1不同时间(0～48 h)后细胞内DNA氧化损伤产物8-OH-dG的表达水平;DCFH-DA荧光探针标记结合流式细胞仪检测1mmol/L NAC作用BERP35T-1 48 h后细胞内活性氧(active oxygen radicals,ROS)水平变化;二苯基溴化四氮唑蓝(MTT)法检测空白对照组、药物组(1 mmol/L NAC作用48 h)、照射组(2 Gyγ射线照射)和联合作用组(1 mmol/L NAC预处理48 h后+2 Gyγ射线照射)BERP35T-1细胞的存活率差异。结果:与BEP2D细胞相比,RH22和BERP35T-1细胞中γH2AX表达增强(P0.01);BERP35T-1较RH22中γH2AX表达增强(P0.01);1～2 mmol/LNAC作用BERP35T-1细胞48 h后,细胞中γH2AX的表达受到显著抑制(P0.01);1 mmol/L NAC作用BERP35T-1细胞24～48 h可显著下调细胞内8-OH-dG的表达(P0.05或P0.01);1 mmo1/L NAC作用BERP35T-1细胞48 h后,细胞内ROS水平显著降低(P0.05);药物组与空白对照组相比,BERP35T-1细胞存活率降低(P0.01);联合作用组与单纯照射组相比,BERP35T-1细胞存活率增高(P0.05)。结论:抗氧化剂NAC可能通过提高恶性转化细胞BERP35T-1的抗氧化能力,降低细胞内ROS和DNA氧化损伤水平,促进基因组稳定性,部分逆转BERP35T-1细胞的恶性表型,并降低其辐射敏感性。

Effects of N-acetyl cysteine on the oxidative DNA damage and cell survival rate of malignant BERP35T-1 cells exposed to a-particles

OBJECTIVE:To study the effects of N-acetyl cysteine(NAC) on the oxidative DNA damage and cell survival rate of malignant BERP35T-1 cells exposed toα-particles.METHODS:Western blot was used for the detection of protein expression ofγH2AX in the human bronchial epithelial cell lines BEP2D,RH22 and BERP35T-1, and BERP35T-1 treated with 0-2 mmol/L NAC for 48 h.Immunocytochemistry was used to compare the different expression levels of 8-OH-dG in BERP35T-1 treated with 1 mmol/L NAC for 0-48 h.Using DCHF-DA,the generation of active oxygen radicals(ROS) in BERP35T-1 treated with 1 mmol/L NAC for 48 h was monitored by flow cytometry.MTT assay was used to examine the cell survival rate of BERP35T-1 cells treated with 1 mmol/L NAC for 48 h and/or 2 Gyγ-rays. RESULTS:Compared to BEP2D cells,increased level ofγH2AX was detected in RH22 and BERP35T-1 cells (P<0.01).The protein expression ofγH2AX in BERP35T-1 was higher than in RH22 cells(P<0.01).After treatment with 1-2 mmol/L NAC for 48 h,the expression level ofγH2AX in BERP35T-1 was significantly decreased(P<0.01). Decreased expression of 8-OH-dG was seen in BERP35T-1 treated with 1 mmol/L NAC for 24-48 h(P<0.05 or P<0.01). After treatment by 1 mmol/L NAC for 48 h,the basal level of ROS in BERP35T-1 decreased(P<0.05).In addition,the cell survival rate of BERP35T-1 treated with NAC was reduced(P<0.01).Meanwhile,compared to BERP35T-1 treatment withγ-rays alone,increased cell survival rate was detected in BERP35T-1 when treated withγ-rays combined with NAC(P<0.05).CONCLUSION:NAC,as an antioxidant,could up-regulate the antioxidant ability of BERP35T-1, resulting in decreased ROS level and oxidative DNA damage and increased genomic stability to partially reverse the malignant phenotype of BERP35T-1,such as inhibiting the malignant proliferation,and reducing its radiosensitivity.