RESUMO
Objective To establish human HBV/HBx-knockin-p53 rat embryonic stem (ES) cell line, so as to lay a foundation for establishment of animal models. Methods First we constructed the targeting vector pKO-gHBV or pKO-X with HBV whole genome or X and p53 homologue arms by PCR and connection with common gene targeted vector pKO; then after linearized with Sal I the vector was transferred into rat ES cells through electroporation. And 2i ES culture medium containing puromycin was used for a three-cycle selection of puromycin resistant clones. Then PCR was used to screen the obtained positive clones; mycoplasma examination and karyotypic analysis were used to confirm targeted ES cell clones. Results We successfully obtained two targeted vectors: pKO-gHBV and pKO-X; then after three-cycle drug-selection we obtained 2 pKO-X clones and 1 pKO-gHBV clone, which were confirmed by PCR, mycoplasma examination and karyotypic analysis. Conclusion We have successfully established human HBV/HBx-knockin-p53 rat ES cells, paving a way for future establishment of HBV/HBx-knockin-p53 rat model.
RESUMO
Objective To study the effects of the HBV x gene (HBx) on the biological characteristics and the expression of DNA repair enzyme hMTH1 mRNA of the L02/HBx transgene cell model. Methods Light microscopy was used to observe the morphologic characteristics of gene-transfected cell strain Lff2/HBx that stably expressed the HBx protein and the control groups of L02 and L02/PcDNA3.1. The changes of L02/HBx on the proliferation, cell cycle and apoptosis were observed by MTT assays and flow cytometry analysis respectively. Moreover, the malignant transformation of L02/HBxwas assayed by colony formation in soft agar and the expression of DNA repair enzyme hMTH1 mRNA was assayed in each group by real-time qPCR. Results Inversion phase contrast microscope showed that the morphologic characteristics of L02/HBx had changed obviously compared with control groups. The MTT showed that L02/HBx proliferated more quickly and flow cytometry analysis indicated that HBx could accelerate the progression of cell cycle and inhibit apoptosis. Colony formation in soft agar demonstrated that the rate of colony formation of L02/HBx was remarkably higher than the L02 and the L02/peDNA3. 1 cells (P<0. 05). The real-time qPCR detection showed that the expression of hMTH1 mRNA in L02/HBx was significantly higher than that in the control groups ( P < 0. 05 ). Conclusion HBx could play an important role in the malignant transformation of L02/HBx and the over expression of hMTH1 mRNA.
RESUMO
Objective:To construct the retroviral vector containing HBx gene so as to investigate the effects of HBx gene in the pathogenesis of HBV-dependent hepatic cell carcinoma(HCC)and further construct animal models of hepatic cell carcinoma.Methods:HBx gene was amplified from the HBx adenovirus Plasmid by polymerase chain reaction(PCR)technique whose primer was designed according to the announced sequences of HBx gene in gene bank,then cut by two endonucleases and directionally cloned into the pseb-hus vector and analyzed by gene sequencing analyzer,the correct constructed vectors were transfected into L02 cell line.At last,HBx protein was detected by Western Blotting.Results:Through PCR,endonuclease cutting and gene sequencing,the target gene was verified into be correctly cloned to retroviral plasmid pSEB-HUS and the high expression of green fluorescence protein(GFP)in L02 cell line was observed under fluorescent microscope.The expression of HBx protein was also observed by Western Blotting.Conclusion:Recombinant retroviral vector expressing HBx protein was successfully constructed.