中国地方病学杂志
中國地方病學雜誌
중국지방병학잡지
CHINESE JOURNAL OF ENDEMIOLOGY
2010年
3期
258-261
,共4页
杨丽%谢菁%仙玲玲%张金莉%徐文静
楊麗%謝菁%仙玲玲%張金莉%徐文靜
양려%사정%선령령%장금리%서문정
三磷酸腺苷结合盒转运子A1%质粒%转染%真核细胞%抗砷性
三燐痠腺苷結閤盒轉運子A1%質粒%轉染%真覈細胞%抗砷性
삼린산선감결합합전운자A1%질립%전염%진핵세포%항신성
ATP-binding cassette transporter A1 gene%Plasmids%Transfection%Eukaryotic cells%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 × 103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.
目的 研究含有三燐痠腺苷結閤盒轉運子A1(ABCA1)基因的真覈錶達載體pcDNA3.1/ABCA1轉染真覈細胞後,在細胞中的錶達及對細胞抗砷性的影響.方法 由脂質體介導將pcDNA3.1/ABCA1重組質粒轉染入人HeLa細胞中(轉染重組質粒組),同時設轉染空質粒組及未轉染組作為對照,用實時定量PCR法檢測各組細胞ABCA1 mRNA錶達水平,用蛋白免疫印記法(Western blot)檢測ABCA1蛋白的錶達.轉染48 h後,以不同劑量[0(對照)、4、8、16、32、64、128 μmol/L]亞砷痠鈉(NaAsO2)染毒,用四甲基偶氮噻唑藍(MTT)法檢測細胞生存率.結果 轉染重組質粒組、轉染空質粒組及未轉染組ABCA1 mRNA錶達量分彆為(2.09±0.08)×10-4、(0.09±0.02)×10-4、(0.08±0.02)×10-4,組間比較差異有統計學意義(F=1499.23,P<0.01),其中轉染重組質粒組ABCA1 mRNA錶達量高于轉染空質粒組和未轉染組(P均<0.01).轉染重組質粒組、轉染空質粒組及未轉染組在相對分子質量(Mr)為254 × 103處均有特異性蛋白條帶,其大小與ABCA1蛋白大小相符,且轉染重組質粒組蛋白錶達量明顯高于轉染空質粒組及未轉染組.在NaAsO2劑量為4、8、16、32、64、128μmol/L時,轉染重組質粒組細胞生存率[(94.8±0.9)%、(86.5±2.6)%、(77.8±2.0)%、(56.0±2.0)%、(23.8±1.7)%、(18.6±0.6)%]均高于轉染空質粒組[(85.3±1.1)%、(78.7±0.6)%、(67.8±2.4)%、(43.2±1.5)%、(14.5±1.3)%、(8.0±0.4)%],二者比較差異有統計學意義(t值分彆為18.985、6.689、5.922、9.504、9.481、32.634,P均<0.01).結論 ABCA1基因經轉染後在HeLa細胞中呈現高錶達狀態,併使HeLa細胞對砷的耐受性提高.
목적 연구함유삼린산선감결합합전운자A1(ABCA1)기인적진핵표체재체pcDNA3.1/ABCA1전염진핵세포후,재세포중적표체급대세포항신성적영향.방법 유지질체개도장pcDNA3.1/ABCA1중조질립전염입인HeLa세포중(전염중조질립조),동시설전염공질립조급미전염조작위대조,용실시정량PCR법검측각조세포ABCA1 mRNA표체수평,용단백면역인기법(Western blot)검측ABCA1단백적표체.전염48 h후,이불동제량[0(대조)、4、8、16、32、64、128 μmol/L]아신산납(NaAsO2)염독,용사갑기우담새서람(MTT)법검측세포생존솔.결과 전염중조질립조、전염공질립조급미전염조ABCA1 mRNA표체량분별위(2.09±0.08)×10-4、(0.09±0.02)×10-4、(0.08±0.02)×10-4,조간비교차이유통계학의의(F=1499.23,P<0.01),기중전염중조질립조ABCA1 mRNA표체량고우전염공질립조화미전염조(P균<0.01).전염중조질립조、전염공질립조급미전염조재상대분자질량(Mr)위254 × 103처균유특이성단백조대,기대소여ABCA1단백대소상부,차전염중조질립조단백표체량명현고우전염공질립조급미전염조.재NaAsO2제량위4、8、16、32、64、128μmol/L시,전염중조질립조세포생존솔[(94.8±0.9)%、(86.5±2.6)%、(77.8±2.0)%、(56.0±2.0)%、(23.8±1.7)%、(18.6±0.6)%]균고우전염공질립조[(85.3±1.1)%、(78.7±0.6)%、(67.8±2.4)%、(43.2±1.5)%、(14.5±1.3)%、(8.0±0.4)%],이자비교차이유통계학의의(t치분별위18.985、6.689、5.922、9.504、9.481、32.634,P균<0.01).결론 ABCA1기인경전염후재HeLa세포중정현고표체상태,병사HeLa세포대신적내수성제고.
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.
