| Objective:To establish and cultivate the L02/HBx cytes.Testing the quantity of mRNA and protein of HBX gene in order to insure the establishing cytes is succeeful. And then, to investigate the influence of Hepatitis B virus X protein to PDCD5 expression.Methods:(1) Revive and cultivate the L02 cytes in order to obtain the minimal concentration of G418 that kill the L02 cytes.(2) The Liposomes transfect the HBx gene into the L02 cells.Then we could obtain the positive clones of L02 cytes by he lowerest concentration of G418 after two weeks.Last. The HBx mRNA was detected by RT-PCR and the protein of HBx was detected western-blot in order to assuring that construction of the cell model of L02/HBx is successful.(3)The levels of mRNA and protein expression of PDCD5 were detected by Real-time PCR and Western Blot in control cell line L02/ pcDNA3.1 and stable transfected cell line L02/HBX.Results:(1) Revive and cultivate the L02 cytes. Using the G418 dosage-reaction test, we can know that the L02 cytes were kill when the concentration of G418 is above 500ug/ml and on the contrary, the L02 cytes still live when the concentration of G418 is under 500ug/ml. So we can consider the 500 ug/ml as the lowest concentration.(2) The Liposomes transfect the HBx gene into the L02 cytes. Then we could obtain the positive clones of L02 cytes by he lowerest concentration of G418 after two weeks. Folloming RT-PCR and western-blot detected mRNA and protein of the HBx. Construction of the cell model of L02/HBx is successful. (3)After HBx gene transfection, the expression of PDCD5 mRNA level was increased(P<0.05),but its protein level was decreased(P<0.05).[Conclusion]:HBx that increases the expression of apoptosis protein PDCD5 further indication of its relationship with apoptosis is very complex and may have a dual regulation of apoptosis. |
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