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| Experimental study on the construction of hepatic oval cell strain stably expressing HBx and M icroRNA-21 gene |
| ZHANG Hong-hui XIE Bin-hui▲ |
| Departmentof Emergency Surgery,the First Affiliated Hospital of Gannan Medical College in Jiangxi Province,Ganzhou 341000,China |
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Abstract ObjectiveTo study the constructionmethod of stably expressing HBx and MicroRNA-21 gene of hepatic oval cell strain.MethodsOval cell strain (LE/6)in rat was cultured using LE culture medium,and HBVx sequence in plasmid of PcDNA3.1-HBVx was used as the template,and target gene fragment A was obtained by PCR method amplification.HBx target gene fragment and plasmid vector pEGFP-Nl were conducted double enzyme digestion,and the recombinant plasmid obtained by linearization was named as pEGFP-HBx.Competence of Escherichia coli DH5a was transformed by recombinant plasmid to conduct screening and culture.The plasmid was transfected into oval cells by liposome method (the pEGFP-HBx group)while plasmid vector pEGFP-Nl was transfected by liposome method(the pEGFP-NIplasmid group).Finally,Pre-MicroRNA-21 and HBx-LE/6were given co-culture,and after 24 h culture,fluorescence expression of transfected cellwas observed by fluorescencemicroscopy.ResultsThe expression volume of GFP in pEGFP-HBx determined by fluorescence quantitative PCR instrumentwas 92.28%,with strong expression degree.After the co-culture of Pre-MicroRNA-21 and HBx-LE/6 was completed,the transcription level of MicroRNA-21 in pEGFPHBx groupmeasured by instrumentwas(1.77±0.24),and itwassignificantly higher than thatin the pEGFP-NIplasmid group (0.36±0.21),and differences between the two groupswas statistically significant(P＜0.05).ConclusionThe stable expression of HBx and MicroRNA-21 gene in hepatic oval cell strain is successfully constructed,which provides the experimental basis for further study on the relationship between HBx protein,miRNA-21,hepatic oval cells and liver cancer.
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