HBxAg对胎盘滋养细胞凋亡的影响及其作用机制
Effect of hepatitis B virus X on the apoptosis of placental trophoblastic cells and its potential mechanism

摘要：目的 探讨乙型肝炎病毒X(HBx)蛋白对胎盘滋养层细胞凋亡水平的影响及可能的作用机制。方法 将已构建好的HBx基因的真核表达载体pcDNA3.1(+)-HBx瞬时转染人绒毛膜癌细胞株JEG-3及HTR-8，分别以转染空载体pcDNA3.1(+)和未转染作为阴性对照组和空白对照组，转染后48h，以逆转录-聚合酶链式反应(RT-PCR)鉴定HBx-mRNA，间接免疫荧光法和蛋白印迹法(Western blot)鉴定HBxAg的表达；流式细胞仪AnnexinⅤ+PI双染方法检测细胞凋亡状态；间接免疫荧光法、流式细胞仪及Western blot检测PI3K、pAKT的表达。结果 JEG-3及HTR-8转染组中均有HBx蛋白表达，而在对照组未被检测到；转染组JEG-3、HTR-8细胞早期凋亡率小于阴性对照组(P<0.05)，PI3K和pAKT1的表达水平高于阴性对照组(P<0.05)，然而AKT1蛋白表达水平在转染组、阴性对照组和空白对照组间无显著性差别。结论 HBx基因可以成功地转入JEG-3及HTR-8细胞，HBx转染后可抑制滋养细胞凋亡，这可能与HBx激活PI3K/pAKT信号通路有关。
ABSTRACT: Objective To investigate the effect of hepatitis B virus X (HBx) protein on the apoptosis of placental trophoblastic cells and its potential mechanism. Methods A pcDNA3.1 expression vector of HBx gene was constructed and transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines, respectively. After transfection for 48h, RT-PCR and immunofluorescence analyses were made to detect HBx mRNA and protein expressions. Flow cytometry was used to detect the early apoptosis status of JEG-3 and HTR-8 cells. The expressions of PI3K and p-Akt were detected by immunofluorescence and Western blotting. Results After transfection for 48h, RT-PCR and immunofluorescence analyses showed that HBx mRNA and protein expressions were detected in JEG-3 and HTR-8 cells. Flow cytometry revealed that early apoptosis of JEG-3 and HTR-8 cells was reduced by pcDNA-HBx transfection (P<0.05). Immunofluorescence and Western blotting showed that PI3K and p-Akt were significantly upregulated in HTR-8 cells (P<0.05). Conclusion HBx gene can be transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines, respectively. After the transfection, the early apoptosis of JEG-3 and HTR-8 cells is reduced. Its inhibition on apoptosis is related to the activation of the PI3K/Akt signaling pathway