Biological Alteration Of Hepatoma Cells Transfected By XPD In Vitro

Objective To construct the recombinant plasmid pEGFP-N2-XPD and transfect the plasmid into the human hepatoma carcinoma cell line Hep3B. To investigate the role of wild-type XPD in cell cycle and apoptosis of hepatoma cells and its relationship with HBx and Cdk7.Methods The human full length XPD was cloned by RT-PCR using the total RNA extracted from HeLa, and inserted it into the pEGFP-N2 plasmid vector which expression the green fluorescence protein (GFP). And then pEGFP-N2 and pEGFP-N2-XPD were transfected into Hep3B cell line by LipofectAMINE 2000 and selected in medium containing G418,400μg. ml^-1 for Hep3B-pEGFP-N2 and Hep3B-pEGFP-N2-XPD; Cell lines for comparison were matched on the same genetic background and passage . The expression of wild-type XPD, HBxand Cdk7 were detected by RT-PCR and Western blot, cell growth was detected by MTT, TUNEL and FCM were employed for examining the cell cycle and apoptosis of the transfected Hep3B cells.Results 1. We have built the combinational plasmid pEGFP-N2-XPD . 2. Hep3B-pEGFP-N2 and Hep3B-pEGFP-N2/XPD express the green fluorescence protein(GFP) which could be detected under the immunofluorescence microscope. 3. The relative expressin of HBx mRNA in Hep3B, Hep3B-pEGFP-N2 and Hep3B-pEGFP-N2/XPD were : 0.361 ±0.095, 0.368 ± 0.098, 0.113 ± 0.041; The relative expression of HBx mRNA in Hep3B-pEGFP-N2/XPD was significantly lower than the control Hep3B, Hep3B-pEGFP-N2. 4. The relative expression of XPD...