title: [Establishment of hepatic stem cell line stably expressing HBx protein and the effect of HBx on cell proliferation].
abstract: OBJECTIVE
  To establish HP14.5 cell line stably expressing hepatitis B virus X (HBx) and detect the effect of HBx on cell proliferation, cell cycle and the wnt/β-catenin signal pathway of hepatic progenitor cells.
  
  
  METHODS
  The plasmid of pSEB-Flag-HBx and the packaging plasmid pAmpho were co-transfected into HEK 293T cells to construct the recombinant retrovirus carrying HBx gene. The recombinant retrovirus wan then transfected into mouse hepatic progenitor cells HP14.5. The blasticidin-resistant clones of HBx cells (HP14.5/HBx) were selected out and cultured as the experimental group, paralleled with the vector control HP14.5/Rv (HP14.5/pSEEB-Flag) and HP14.5 cell as negative control. The cell proliferation was tested by MTS assay, the cell cycle was measured by flow cytometry, the expression levels of cyclin D1 and c-myc mRNA were tested by real time PCR, and the expression leveIs of HBx, cyclin D1, c-myc, GSK3β, p-GSK3β (ser-9), β-catenin proteins were examined by Western blotting.
  
  
  RESULTS
  The expression of HBx at both mRNA and protein levels was positive in HP14.5/HBx cell line as confirmed by RT-PCR and Western blotting. Compared with the control groups, the proliferation of HP14.5/HBx cells increased significantly (P<0.05), and the expression level of cyclin D1 and c-myc mRNA rose significantly(P<0.05). G1-phase cell proportion was reduced while the proportion in S and G2 phases went up significantly (P<0.05). The expression levels of cyclin D1, c-myc, β-catenin and p-GSK3β (ser-9) proteins increased significantly (P<0.05)except GSK3β (P>0.05).
  
  
  CONCLUSIONS
  The hepatic stem cell line stably expressing HBx has been constructed successfully. HBx can promote the proliferation and malignant transformation of hepatic stem cells via the activation of Wnt/β-catenin signal pathway.
authors: College of Basic Medicine, Chongqing Medical University, Chongqing, China.
authors: Yongliang
authors: Y
authors: Lu
authors: Lihong
authors: L
authors: Shen
authors: Hongli
authors: H
authors: Li
authors: Daixi
authors: D
authors: Hu
authors: Xifeng
authors: X
authors: Zhang
authors: Bin
authors: B
authors: Hu
authors: Tao
authors: T
authors: Feng
authors: Jiayi
authors: J
authors: Huang
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008099:Liver / Q000166:cytology*; D009705:Nucleosides / Q000494:pharmacology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins; D051153:Wnt Proteins / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
chemical_list: D009705:Nucleosides; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; D051153:Wnt Proteins; D051176:beta Catenin; C057628:hepatitis B virus X protein; D019938:Cyclin D1; C004500:blasticidin S