细胞外信号调节激酶通路在乙肝病毒X基因改变肝癌细胞化疗敏感性中的作用

The role of extracellular signal-regulated kinase cascades in the sensitivity alteration to cisplatin of HepG2 expressing hepatitis B virus X gene

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.

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.