稳定表达HBx基因的HepG2细胞对顺铂敏感性的变化及其机制研究

Sensitivity alteration of HepG2 stably expressing HBx toCDDP and its mechanism

目的 探讨乙肝病毒x基因(HBx)对肝癌细胞株HepG2化疗敏感性的影响及其机制。方法 1.构建HBx的真核表达载体;2.脂质体法转染HepG2,G418筛选得到稳定表达HBx-ORF的阳性细胞克隆;3.Western blot法检测转染前后磷酸化细胞外信号调节激酶1/2(p-ERK1/2)的表达变化;4.MTT法检测转染前后HepG2对顺铂的反应,以及用PD98059特异性阻断ERK1/2激活通路后HBx+HepG2对顺铂敏感性的变化。结果 1.稳定表达HBx-ORF的HepG2中p-ERK1/2的表达明显强于阴性细胞;2.稳定表达HBx-ORF的HepG2在2μg/ml顺铂作用24小时后,顺铂对其抑制率为19.45％±2.50,明显低于阴性组的33.15％±2.85和转染空质粒组的30.79％±2.66(P<0.05);用PD98059阻断阳性细胞中ERK1/2的激活之后,相同剂量,时间作用下,对HBx+HepG2的抑制率上升至32.28％±4.22,明显高于阻断前(P<0.05)。结论 HBx通过ERK通路介导肝癌细胞的抗化疗反应,ERK通路有可能成为乙肝后肝癌辅助治疗的靶点。

Objective To investigate the sensitivity alteration of HepG2 stably expressing HBx to CDDP and its mechanism . Methods 1. Construct the mammalian expression vector containing the HBx-ORF; 2. Transfect HepG2 with the vector by liposome and select the positive cell clones that stably expressing HBx-ORF; 3. Test the level of p-ERK1/2 in the HBx positive and negative cells by Western blot; 4. Test the inhibitory rate of 2 jug/ml CDDP on both positive and negative cells after 24 hours of incubation by MTT; block the activation of ERKl/2 with PD98059 in the positive cells and test the inhibitory rate again. Results 1 . The expression of p-ERK1/2 rose significantly in the HBx+ cells; 2. After 24 hour incubation of 2μg/ ml CDDP, the inhibitory rate on positive cells was 19.45% ±2.50, significantly lower than that on negative cells; when the activation of ERK1/2 in the positive cells was blocked, its inhibitory rate rose to 32.28%±4.22. Conclusion The pathway of ERK plays an important role in the mechanism of HBx mediated anti-chemitheraputic effect and may be a target to improve the chemitheraputic effect on HCC.