真核细胞atp结合盒转运蛋白A1及其表达对砷抗性的影响

Q3 Medicine 中华地方病学杂志 Pub Date : 2010-05-20 DOI:10.3760/CMA.J.ISSN.1000-4955.2010.03.006

Li Yang, Jing Xie, L. Xian, Jin-li Zhang, Wenqin Xu

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Xian, Jin-li Zhang, Wenqin Xu","doi":"10.3760/CMA.J.ISSN.1000-4955.2010.03.006","DOIUrl":null,"url":null,"abstract":"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 254×103 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). 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引用次数: 0

摘要

目的探讨含ABCA1基因的真核表达载体转染真核细胞后，atp结合盒转运体A1(ABCA1)的表达及其对砷抗性的影响。方法将重组质粒lipofectaonmine 2000(重组质粒组)转染HeLa细胞，空质粒和未转染的HeLa细胞作为对照组。采用实时荧光定量PCR检测mRNA水平，Western blot检测ABCA1蛋白表达，甲基噻唑四氮唑(MTT)检测暴露于砷[0(对照1)、4、8、16、32、64、128 μmol/L] 48 h后细胞存活率的变化。结果ABCA1 mRNA在重组质粒、空质粒和未转染组的表达量分别为(2.09±0.08)×10-4、(0.09±0.02)×10-4、(0.08±0.02)×10-4，组间差异有统计学意义(F=1499.23,P<0.01)。重组质粒组ABCA1 mRNA表达水平高于空质粒组和未转染质粒组(均P<0.01)。Western blot结果显示，三组均在254×103处存在特异性蛋白条带，其大小与ABCA1蛋白相似。重组质粒组的表达量高于其他两组。MTT表明砷浓度在4、8、16、32、64128μmol / L,重组质粒组的生存率(94.8±0.9)%,(86.5±2.6)%,(77.8±2.0)%,(56.0±2.0)%,(23.8±1.7)%,(18.6±0.6)%,高于空质粒组[(85.3±1.1)%,(78.7±0.6)%,(67.8±2.4)%,(43.2±1.5)%,(14.5±1.3)%,(8.0±0.4)%],存活率的差异有统计signifieance (t = 18.985, 6.689, 5.922, 9.504, 9.481, 32.634, P < 0.01)。结论转染ABCA1基因后，ABCA1蛋白在HeLa细胞中过表达。ABCA1蛋白增加HeLa细胞对砷的抗性。关键词:atp结合盒转运蛋白A1基因;质粒;转染;真核细胞;砷抗性

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

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 254×103 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. Key words: ATP-binding cassette transporter A1 gene; Plasmids; Transfection; Eukaryotic cells; Arsenic resistance

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来源期刊

中华地方病学杂志我国对人类健康危害特别严重的地方性疾病：克山病、大骨节病、碘缺乏病、地方性氟中毒、地方性砷中毒、鼠疫、布鲁氏菌病、寄生虫、新冠肺炎等疾病，同时还报道多发性自然疫源性疾病。

CiteScore

1.60

自引率

0.00%

发文量

8714

期刊介绍： The Chinese Journal of Endemiology covers predominantly endemic diseases threatening health of the people in the areas affected by the diseases including Keshan disease, Kaschin-Beck Disease, iodine deficiency disorders, endemic fluorosis, endemic arsenism, plague, epidemic hemorrhagic fever, brucellosis, parasite diseases and the diseases related to local natural and socioeconomic conditions; and reports researches in the basic science, etiology, epidemiology, clinical practice, control as well as multidisciplinary studies on the diseases.