CAJ | 학술논문

目的：构建环指蛋白6（RNF6）真核表达载体，并探讨其对胰岛素受体底物1（IRS-1）的泛素化作用。方法以人cDNA为模板，PCR 扩增 RNF6全长编码基因，并将其克隆至载体质粒 pcDNA3．1-CHA 中，将重组质粒转染肝癌细胞株 HepG2，利用实时荧光定量 PCR（RT-PCR）、免疫印迹（Western blot）检测细胞内 IRS-1 mRNA 水平及其蛋白表达水平。结果携带RNF6目的基因的质粒转染 HepG2细胞48 h 后 IRS-1的 mRNA 表达水平降低，为对照组的69％，显著低于对照组，差异有统计学意义（P ＜0．01）。结论RNF6引起 IRS-1的表达下调，这一泛素化过程可能导致胰岛素信号转导障碍。
목적：구건배지단백6（RNF6）진핵표체재체，병탐토기대이도소수체저물1（IRS-1）적범소화작용。방법이인cDNA위모판，PCR 확증 RNF6전장편마기인，병장기극륭지재체질립 pcDNA3．1-CHA 중，장중조질립전염간암세포주 HepG2，이용실시형광정량 PCR（RT-PCR）、면역인적（Western blot）검측세포내 IRS-1 mRNA 수평급기단백표체수평。결과휴대RNF6목적기인적질립전염 HepG2세포48 h 후 IRS-1적 mRNA 표체수평강저，위대조조적69％，현저저우대조조，차이유통계학의의（P ＜0．01）。결론RNF6인기 IRS-1적표체하조，저일범소화과정가능도치이도소신호전도장애。
Objective To construct an eukaryotic expression vetor of ring finger protein 6 (RNF6)and to investigate its ubiqu-itylation on insulin receptor substrate-1(IRS-1).Methods Human RNF6 coding sequence was amplified by polymerase chain reac-tion (PCR)method with human cDNA as template.The pcDNA3.1-CHA-RNF6 was constructed with routine molecular methods and transfected into hepatocarcinoma cell HepG2.Relative quantification of IRS-1 mRNA was preformed by real-time reverse tran-scription PCR.Western blot was applied to detect the expression levels of IRS-1.Results 48 h after the plasmid carring RNF6 gene transfected HepG2 cells,the mRNA level of ISR-1 gene reduced to 69% of control group.The expression of IRS-1 was markedly lower than control group (P <0.01).Conclusion The expression of IRS-1 is markedly down-regulated in HepG2 and enhancement of the ubiquitylation level of IRS-1 contribute to the disorder in insulin signal transferring.