CAJ | 학술논문

目的探讨转染hSOD1-G93A对NSC-34细胞内铁代谢相关蛋白表达的影响.方法常规体外培养未转染、转染pcDNA3.1(-)、hSOD 1-pcDNA3.1(-)和hSOD 1-G93A-pcDNA3.1(-)质粒的NSC-34细胞系;分为正常组、空转组、野生组和突变组.利用免疫组化染色法观察各组细胞的形态变化;通过检测丙二醛(MDA)含量观察各组细胞氧化应激水平;用逆转录聚合酶链式反应(RT-PCR)和Western blotting分别检测各组细胞内铁代谢相关蛋白的基因和蛋白表达水平.结果突变组的细胞胞体变圆,突起减少、变短,细胞内MDA含量明显高于其他3组细胞,差异有统计学意义(P＜0.05);各组细胞内的转铁蛋白受体(TfR)、二价金属离子转运体1(DMT1)、铁蛋白等基因在基因水平上表达差异无统计学意义(P＞0.05);各组细胞内的TfR、DMT1、铁蛋白等在蛋白水平上表达差异无统计学意义(P＞0.05),但是突变组细胞铁蛋白表达较正常组细胞明显升高,差异有统计学意义(P＜0.05).结论转染hSOD1-G93A对NSC-34细胞(突变组)内TfR、DMT1表达无明显影响,但可以使细胞内铁蛋白在蛋白表达水平明显升高.
목적 탐토전염hSOD1-G93A대NSC-34세포내철대사상관단백표체적영향.방법 상규체외배양미전염、전염pcDNA3.1(-)、hSOD 1-pcDNA3.1(-)화hSOD 1-G93A-pcDNA3.1(-)질립적NSC-34세포계;분위정상조、공전조、야생조화돌변조.이용면역조화염색법관찰각조세포적형태변화;통과검측병이철(MDA)함량관찰각조세포양화응격수평;용역전록취합매련식반응(RT-PCR)화Western blotting분별검측각조세포내철대사상관단백적기인화단백표체수평.결과 돌변조적세포포체변원,돌기감소、변단,세포내MDA함량명현고우기타3조세포,차이유통계학의의(P＜0.05);각조세포내적전철단백수체(TfR)、이개금속리자전운체1(DMT1)、철단백등기인재기인수평상표체차이무통계학의의(P＞0.05);각조세포내적TfR、DMT1、철단백등재단백수평상표체차이무통계학의의(P＞0.05),단시돌변조세포철단백표체교정상조세포명현승고,차이유통계학의의(P＜0.05).결론 전염hSOD1-G93A대NSC-34세포(돌변조)내TfR、DMT1표체무명현영향,단가이사세포내철단백재단백표체수평명현승고.
Objective To investigate the effect of transfection of hSOD1-G93A on expression of iron-related proteins in NSC-34 cells.Methods The NSC-34 cell lines from four groups (without transfection or stably transfected with pcDNA3.1 (-),hSOD 1-pcDNA3.1 (-) and hSOD 1-G93A-pcDNA3.1 (-) plasmids) were cultured at routine ways.Morphological changes of the transfected cells were observed by immuno-histochemical method.The cell oxidative damage degrees were observed by detecting malondialdehyde content in cells.Real time-PCR was used to determine the mRNA expression levels of iron-related genes;Western blotting was used to determine the changes of protein levels of iron-related genes.Results The soma became round and the number of neurite decreased in the NSC-34 cells transfected with hSOD1-G93A-pcDNA3.1 (-) plasmid,the neurites were shorter and oxidative stress was increased as compared with those in the other three cell lines (P＜0.05).The mRNA expressions of transferrin receptor (TfR),divalent metal ion 1 (DMT1),and ferritin were not significantly different among the four groups (P＞0.05);the protein expressions of TfR and DMT1 were not significantly different among the four groups (P＞0.05),while that of ferritin in the NSC-34 cells transfected with hSOD1-G93A-pcDNA3.1 (-) plasmid was significantly higher than that in cells without transfection (P＞0.05).Conclusion The expressions of TfR and DMT1 are not influenced by hSOD1-G93A but the expression of ferritin in the mutant cells are increased by the influence of hSOD 1-G93A.