Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology · 2013
[Establishment of hepatic stem cell line stably expressing HBx protein and the effect of HBx on cell proliferation].
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Lu Yongliang, Shen Lihong, Li Hongli, Hu Daixi, Zhang Xifeng, Hu Bin, Feng Tao, Huang Jiayi
Аннотация
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).
CONCLUSION: 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.
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).
CONCLUSION: 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.