乙型肝炎病毒X基因诱导卵圆细胞恶性转化的研究

目的 研究乙型肝炎病毒X(HBx)基因对卵圆细胞转化和分化的影响.方法 用稳定表达HBx的转基因即圆细胞株pEGFP-HBx卵圆细胞作为实验组,pEGFP卵圆细胞及LE/6卵圆细胞作为对照组,运用倒置相差显微镜和透射电镜、流式细胞术、糖原染色、软琼脂生长、实时定量PCR、细胞免疫化学技术观察和检测pEGFP-HBx卵圆细胞在细胞形态、细胞周期、分化标志物、癌基因c-myc及TGF-α的变化.结果 (1)与对照组相比,pEGFP-HBx卵圆细胞体积增大,核具有明显异型性;(2)流式细胞仪检测显示,与对照组相比,pEGFP-HBx卵圆细胞G0/G1期细胞百分比下降,S期和G2/M期细胞百分比明显升高,而且非整倍体细胞明显增多;(3)糖原染色显示,pEGFP-HBx卵圆细胞胞质内可见大量糖原颗粒;(4)软琼脂中可见pEGFP-HBx卵圆细胞克隆形成;(5)癌基因c-myc及TGF-α表达量明显增高,卵圆细胞分化指标HNF-4α、AFP表达上升,CK-7、CK-19表达下降,未见有cps1 mRNA表达.结论 HBx基因可以引起卵圆细胞异常分化,并直接诱导卵圆细胞发生恶性转化.

The studies of hepatitis B virus X gene induces oval cell malignant transformation

Objective To find out the mechanisms of HBx gene inducing oval cell malignant transformation into hepatoma carcinoma cell. Methods The changes of morphology, cell cycle,differentiated markers,c-myc and TGF-α in pEGFP-HBx oval cells strain,which stably expressed HBx gene, were studied by inversion phase contrast microscope and transmission electron microscopy, flow cytometry, periodic acid-schiff (PAS) staining, soft agar growth assay,real-time PCR,immunlocytochemistry. pEGFP-oval cells and LE/6 oval cells were used as control groups. Results (1) The pEGFP-HBx oval cells showed biger in size with malformed nucleus as compared with control groups. (2) Flow cytometry showed that, in contrast with the control groups,the propotion of pEGFP-HBx oval cells arrested in GO/G1 phase decreased but in S or G2/M phase rose. Moreover, the population of aneuploid cells increased obviously. (3) PAS staining showed that there were many glycogen granules in the cytoplasm of pEGFP-HBx oval cell. (4) The pEGFP-HBx oval cell formed colonies in the soft agar. (5) Compared with the control groups,the expression of HNF-4α,AFP, c-myc and TGF-αt rose obviously ,whereas the expression of CK-7 and CK-19 decreased. And the expression of cps1 mRNA was not in the extent of detection. Conclusions The HBx gene can provoke abnormal differentiation of oval cell and induce oval cell malignant transformation.