CAJ | 학술논문

目的观察细胞外信号调节激酶(ERK)信号传导通路在乙肝病毒X基因(HBx)改变肝癌细胞HepG2对顺铂敏感性中的作用.方法采用聚合酶链反应(PCR)法从pCP10质粒中扩增482 bp HBx的开放读码框(ORF),构建表达载体pcDNA3-HBx并进行酶切及测序鉴定.应用脂质体将其转染HepG2,50 mg/L G418筛选阳性克隆,抽提mRNA,逆转录(RT)-PCR鉴定是否稳定表达HBx.Western blot法检测转染前后44×103及42×103磷酸化ERK1/2(p-ERK1/2)的表达.噻唑蓝(MTT)法检测转染空脂质体组、空载体组HepG2、HBx+ HepG2对2 mg/L顺铂敏感性的差异,以及顺铂联合ERK通路特异性抑制剂PD98059对HBx+HepG2抑制率的改变.结果从pCP10中扩增得到482 bp HBx-ORF片段.将其与pcDNA3连接后经酶切及测序鉴定,成功构建HBx-ORF表达载体.从G418抗性细胞克隆中成功扩增出482 bp HBx-ORF,表明转染后的HepG2细胞稳定表达目的片段.经Western blot及灰度扫描鉴定,HBx+细胞中p-ERK蛋白表达是空脂质体组的1.97倍,是空载体组的1.67倍.MTT法检测2 mg/L顺铂作用24 h,对HBx+ HepG2的抑制率为19.56％,明显低于对空脂质体组和空载体组HepG2的抑制率(35.21％和30.18％).而先以100 μmol/L PD98059孵育2h的HBx+ HepG2,顺铂对其抑制率上升至31.20％,与未加PD98059时比较有显著提高(P＜0.05).结论 ERK信号传导通路的激活在抑制HBx+的肝癌细胞的化疗敏感性方面起重要作用,对该通路的特异性抑制有助于提高HBx+肝癌的化疗效果.
목적 관찰세포외신호조절격매(ERK)신호전도통로재을간병독X기인(HBx)개변간암세포HepG2대순박민감성중적작용.방법 채용취합매련반응(PCR)법종pCP10질립중확증482 bp HBx적개방독마광(ORF),구건표체재체pcDNA3-HBx병진행매절급측서감정.응용지질체장기전염HepG2,50 mg/L G418사선양성극륭,추제mRNA,역전록(RT)-PCR감정시부은정표체HBx.Western blot법검측전염전후44×103급42×103린산화ERK1/2(p-ERK1/2)적표체.새서람(MTT)법검측전염공지질체조、공재체조HepG2、HBx+ HepG2대2 mg/L순박민감성적차이,이급순박연합ERK통로특이성억제제PD98059대HBx+HepG2억제솔적개변.결과 종pCP10중확증득도482 bp HBx-ORF편단.장기여pcDNA3련접후경매절급측서감정,성공구건HBx-ORF표체재체.종G418항성세포극륭중성공확증출482 bp HBx-ORF,표명전염후적HepG2세포은정표체목적편단.경Western blot급회도소묘감정,HBx+세포중p-ERK단백표체시공지질체조적1.97배,시공재체조적1.67배.MTT법검측2 mg/L순박작용24 h,대HBx+ HepG2적억제솔위19.56％,명현저우대공지질체조화공재체조HepG2적억제솔(35.21％화30.18％).이선이100 μmol/L PD98059부육2h적HBx+ HepG2,순박대기억제솔상승지31.20％,여미가PD98059시비교유현저제고(P＜0.05).결론 ERK신호전도통로적격활재억제HBx+적간암세포적화료민감성방면기중요작용,대해통로적특이성억제유조우제고HBx+간암적화료효과.
Objective To investigate the role of extracellular signal-regulated kinase (ERK) cascades in the sensitivity alteration to cisplatin (CDDP) of HepG2 expressing hepatitis B virus X gene (HBx).Methods The 482-bp open reading frame (ORF) of HBx was cloned from the plasmid pCP10.Construct HBx-ORF into the expression vector pCDNA3 and check whether it is successful by sequencing and enzyme cutting.Transfect HepG2 with the vetor pCDNA3-HBx by liposome and select G418 50 mg/L resisitant HepG2 clones to identify the existence of HBx-ORF with reverse transcription-polymerase chain reaction(RT-PCR).Compare the expression of 44 × 103 and 42 × 103 p-ERK1/2 protein in the HBx-ORF positive and negative clones by Western blotting.Compare the differences of sensitivity to 2 mg/L CDDP among HBx-ORF positive HepG2,liposome transfected HepG2 and mock vetor transfected HepG2 and changes of sensitivity to CDDP with and without the effect of PD98059 in the HBx + HepG2 with methyl thiazol tetrazolium (MTT) assay.Results A 482 bp fragment of HBx-ORF was cloned from pCP10 plasmid and successfully ligated into the pCDNA3 vector which was identified by sequencing and enzyme cutting.And the 482 bp HBx-ORF existence was also identified in the G418 resistant HepG2 clones after tranfection by reverse transcriptase-polymerase chain reaction (RT-PCR).With Western blotting and density scaning,we found that the expression of p-ERK1/2 protein in the HBx + HepG2was 1.97 and 1.67 times stronger than that in the liposome transfected and mock vetor transfected HepG2.With the MTT assay,the inhibitory rate of CDDP to HBx + HepG2 was 19.56％,significantly lower than that of the liposome transfected and mock vetor transfected HepG2 which was 35.21％ and 30.18％,P ＜0.05.When the ERK pathway was blocked with PD98059 beforehand,the inhibitory rate was improved to 31.20％ in the HBx+ HepG2.Conclusion The ERK cascades activation playes an important role in protection of hepatoma cells from death induced by CDDP and block of it helps to cut off the chemotherapy resistance.