ABSTRACT
Objective To over-express the P53 upregulated modulator of apoptosis(PUMA) in ovarian cancer COC1 cells for playing the role to restrain the cell growth .Methods Adenovirus vector (Ad)-human telomerase reverse transcriptase promoter (hTERT)-PUMA was transfected into ovarian cancer COC1 cells .Then the growth of COC1 cells was studied by CCK-8 and the apoptosis kit and the level of PUMA was analyzed .Results After transfection of Ad-hTERT-PUMA ,PUMA was over-expressed in COC1 cells ,which restrained the cells growth and promoted the cell apoptosis .Conclusion Ad-hTERT-PUMA is successfully transfected into ovarian cancer COC1 cells and PUMA is over-expressed to play the role for restraining the cells growth .
ABSTRACT
Objective To construt the adenovirus vectors of Ad-hTERT-PUMA-EGFP containing human telomerase reverse transcriptase(hTERT) ,PUMA and enhanced green fluorescent protein (EGFP) .Methods By a series of molecular biology tech-niques ,PUMA gene was inserted into downstream of the hTERT promoter .And then EGFP was inserted into downstream of the PUMA in adenovirus vectors .Finally ,recombinant adenovirus vector ,Ad-hTERT-PUMA-EGFP was assembled into the adenovi-rus .Results Correct adenovirus laoding vectors was verified by viral genome PCR technology .After 12 h of transfection ,HEK293 cells showed green fluorescence in 72 h .Conclusion The correct structure of the adenovirus vectors containing PUMA gene and EGFP gene was identified by restriction enzyme analysis .
ABSTRACT
Objective:To investigate the effect of P53 up-regulate modulator of apoptosis(PUMA) on the apoptosis of pancreatic carcinoma BxPC-3 cells and the possible mechanism.Methods: BxPC-3 cells were infected with recombinant adenovirus containing PUMA gene(Ad-PUMA) at 100 MOI for 0-96 h.Apoptosis of BxPC-3 cells was examined by FCM.Expressions of PUMA,Bcl-2,Bax,Cytochrome C and Caspase-3 proteins in BxPC-3 cells were detected by Western blotting.Bax expression in the cytoplasm and mitochondrion and Bax oligomer expression expression in BxPC-3 cells were determined by Western blotting.Results: Apoptosis rates of BxPC-3 cells were significantly increased with the time of Ad-PUMA infection,and peaked after 48 h.Ad-PUMA infection increased the expressions of PUMA,Cytochrome C and Caspase-3 proteins in BxPC-3 cells,and decreased the expression of Bcl-2 protein.Apoptosis rate of BxPC-3 cells after Ad-PUMA infection was correlated with PUMA expression.Ad-PUMA did not affect the expression of total Bax protein in BxPC-3 cells,but Bax expression in cytoplasm was dramatically decreased after infection,and Bax expression in mitochondrion was markedly increased.Furthermore,Ad-PUMA infection induced Bax oligomerization in BxPC-3 cells.Conclusion: PUMA can promote apoptosis of pancreatic carcinoma cells through mitochondrion pathway.