三磷酸腺苷结合盒转运子A1在真核细胞中的表达及对细胞抗砷性的影响

Influence of ATP-binding cassette transporter A1 protein in eukaryocyte and its expression on arsenic resistance

目的 研究含有三磷酸腺苷结合盒转运子A1（ABCA1）基因的真核表达载体pcDNA3.1/ABCA1转染真核细胞后,在细胞中的表达及对细胞抗砷性的影响.方法 由脂质体介导将pcDNA3.1/ABCA1重组质粒转染入人HeLa细胞中（转染重组质粒组）,同时设转染空质粒组及未转染组作为对照,用实时定量PCR法检测各组细胞ABCA1 mRNA表达水平,用蛋白免疫印记法（Western blot）检测ABCA1蛋白的表达.转染48 h后,以不同剂量[0（对照）、4、8、16、32、64、128 μmol/L]亚砷酸钠（NaAsO2）染毒,用四甲基偶氮噻唑蓝（MTT）法检测细胞生存率.结果 转染重组质粒组、转染空质粒组及未转染组ABCA1 mRNA表达量分别为（2.09±0.08）×10^-4、（0.09±0.02）×10^-4、（0.08±0.02）×10^-4,组间比较差异有统计学意义（F=1499.23,P〈0.01）,其中转染重组质粒组ABCA1 mRNA表达量高于转染空质粒组和未转染组（P均〈0.01）.转染重组质粒组、转染空质粒组及未转染组在相对分子质量（Mr）为254 × 10^3处均有特异性蛋白条带,其大小与ABCA1蛋白大小相符,且转染重组质粒组蛋白表达量明显高于转染空质粒组及未转染组.在NaAsO2剂量为4、8、16、32、64、128μmol/L时,转染重组质粒组细胞生存率[（94.8±0.9）%、（86.5±2.6）%、（77.8±2.0）%、（56.0±2.0）%、（23.8±1.7）%、（18.6±0.6）%]均高于转染空质粒组[（85.3±1.1）%、（78.7±0.6）%、（67.8±2.4）%、（43.2±1.5）%、（14.5±1.3）%、（8.0±0.4）%],二者比较差异有统计学意义（t值分别为18.985、6.689、5.922、9.504、9.481、32.634,P均〈0.01）.结论 ABCA1基因经转染后在HeLa细胞中呈现高表达状态,并使HeLa细胞对砷的耐受性提高.

Objective To examine the expression of ATP-binding cassette transporter A1（ABCA1）in eukaryotie cells and the effect of arsenic resistance after the transfection of eukaryotic expression vector containing ABCA1 gene.Methods HeLa cells were transfected with the recombinant plasmid by lipofectaonmine 2000 （recombinant plasmid group）,empty plasmid and untransfected HeLa cell as the control group.The level of the mRNA was examined by real-time PCR,and the expression of ABCA1 protein wag examined by Western blot,the change of cell survival rate was examined by methyl thiazolyl tetrazolium（MTT）after exposure in a series of arsenic [0（contro1）,4,8,16,32,64,128 μmol/L]for 48 hours.Results Expression level of ABCA1 mRNA in recombinant plasmid,empty plasmid and untransfeeted groups was（2.09±0.08）×10^-4,（0.09±0.02）×10^-4,（0.08±0.02）×10^-4,there was a significant difference between the groups（F=1499.23,P〈0.01）.The level of ABCA1 mRNA in recombinant plasmid group was higher than empty plasmid and untransfected group（all P〈0.01）.Western blot showed that specific protein straps existed at 254×10^3 in all the three groups,with a similar size to the ABCA1 protein.The amount of the recombinant plasmid group was higher than the other two groups.MTT shows that arsenic concentration at 4,8,16,32,64,128 μmol/L,the survival rates of recombinant plasmid group was（94.8±0.9）%,（86.5 ± 2.6）%, （77.8 ± 2.0）%, （56.0 ± 2.0）%, （23.8 ± 1.7）%, （18.6 ± 0.6）%, higher than that of empty plasmid group[ （85.3 ± 1.1）%, （78.7 ± 0.6）%, （67.8 ± 2.4）%, （43.2 ± 1.5）%, （14.5 ± 1.3）%, （8.0 ± 0.4）%], the difference of survival rate had a statistical signifieance（t = 18.985,6.689,5.922,9.504,9.481,32.634, all P 〈 0.01）. Conclusions ABCA1 protein is over expressed in HeLa cells after transfect ABCA1 gene. ABCA1 protein increases resistance of arsenic in HeLa cells.