Experimental Study On The Anti-tumor Effect And The Mechanism Of Adenovirus-mediated PTEN Gene Against Cutaneous T Cell Lymphoma Cell Line

Objective: To investigate the anti-proliferative and apoptosis effect and mechanism of adenovirus-mediated PTEN gene in the cutaneous T cell lymphoma cell line Hut78 cells.In order to explore the pathogenesis of cutaneous T cell lymphoma and the mechanism of inhibiting effect of PTEN gene on the cells growth in cutaneous T cell lymphoma.Methods:1 Objects of study: The cutaneous T cell lymphoma cell line Hut78 cells was purchased from Beijing Beina Bio-Link Biotechnology Research Institute.Human embryonic kidney cell line HEK293 cells were preserved by Hebei Institute of Oncology,adenovirus-pten?ad-pten?and adenovirus-gfp?ad-gfp?plasmid were purchased from Abm Corporation of Canada.2 Cell culture: Cutaneous T cell lymphoma cell line Hut78 cell was cultivated in the RPMI1640 incubate medium containing 10% volume fraction of fetal bovine serum,HEK293 cell line was cultivated in the DMEM incubate medium containing 10% volume fraction of fetal bovine serum,and putted in 37?,saturated humidity and 5% CO₂ environment,all of our experiments were performed with the cells among the logarithmic growth phase.3 Preparation of adenoviruses: The adenovirus plasmid carrying PTEN and GFP gene was transfected into HEK293 cells for adenovirus amplification,purification and titer determination.4 Analysis of adenovirus infection rate: Fluorescence microscopy was used to observe the efficiency of ad-GFP which was carrying green fluorescent protein infected Hut78 cells.The hut78 cells among the logarithmic growth phase with a concentration of 2*10⁶/ml were inoculated into a six-well plate?1ml/well?.The cells were infected with ad-GFP with different infection numbers?MOI =1,10,50?.The expression of green fluorescent protein was observed under fluorescence microscope at 24 h and 48 h.5 The cell morphological changes after infection with adenovirus: After ad-PTEN and ad-GFP were infected into Hut78 cells for 24 hours and 48 hours with different infection numbers?MOI=1,10,50?.The morphology and growth condition of cells was observed by optical microscope.6 Analysis of cell proliferation inhibition after infection with adenovirus: CCK-8 method was used to determine the inhibitory rate of proliferation of the Hut78 cell lines which infected with ad-PTEN and ad-GFP on different infection numbers and different infection time.The Hut78 cells among logarithmic growth phase has been diluted into 2*10²cell/?l and inoculated in the 96-well plate,each well of 50?l.Then the cells were infected with ad-PTEN or ad-GFP at different infection numbers?MOI=1,10,50?,50?l per well.We also set up blank group?medium without cell?and control group?cells without infected ad-PTEN or ad-GFP?.24 or 48 hours later,CCK-8 was added to each well,the plates were incubated in 37?,5%CO₂ environmental,then we test the optical density?OD?in each group at 1h,2h,3h,4h measured by Microplate Reader,and the corresponding proliferation inhibition rate was calculated.Results are expressed as the mean±standard deviation.7 Analysis of cell apoptosis after infection with adenovirus: Flow cytometry was used to measure the apoptotic rate of cells.The Hut78 cells in logarithmic growth phase have been diluted into 5*10⁶cell/ml and inoculated in the 6–well plate,1ml per well.Infected the cells with ad-PTEN with different infection number?MOI=1,10?were the control group.After infection for 24 h,the flow cytometry was employed to analyze the apoptosis condition.8 Expression of PTEN and downstream signaling pathway proteins: The expression of PTEN and its downstream signaling pathways protein PI3 k,Akt and Caspase-3 was detected by Western Blot.The Hut78 cells in logarithmic growth phase have been diluted into 5*10⁶cell/ml and inoculated in the 6–well plate,1 ml per well.Then the cells were infected with ad-PTEN with different infection number?MOI=1,10,50?.After 48 hours of infection,total protein was extracted from the Hut78 cells for Western Blot.Results:1 Adenovirus preparation results: After amplification,purification and titer determination of the virus,the final titer of the virus was 1*10⁹pfu/ml;2 The efficiency of adenovirus infection: The efficiency of infection of ad-GFP in Hut78 cells was observed by fluorescence microscopy.The efficiency of infection was increased with the growth of the infection number and the infection times.The rank of infection efficiency was 50?MOI?>10?MOI?>1?MOI?,48h>24h;3 The cell morphological changes after infection with adenovirus: The number of vacuoles and particles in the Hut78 cells infected with ad-PTEN was increased with the growth of the number of infection and the time of infection.The nuclear condensation,cell membrane fragmentation,apoptosis and death was observed in part of the cells.While the growth condition and morphology of the blank control group and ad-GFP infected group was changed limited;4 Analysis of cell proliferation inhibition after infection with adenovirus: The proliferation of Hut78 cells after infection with PTEN gene was inhibited,and the inhibition rate increased with the augment of infection number and the prolongation of infection time.After infected 24 h the proliferation inhibition rates of ad-PTEN groups?MOI=0,1,10,50?were 0,?35.87±12.95?%,?58.53±16.71?%,?73.04±5.07?%,The difference was statistically significant?P<0.01?.The MOI = 10 group compared with the MOI = 50 group,the difference was not statistically significant?P>0.05?,the other groups within the P value was less than 0.05,the difference was statistically significant.The proliferation inhibition rates of ad-GFP groups?MOI = 0,1,10,50?were 0,?4.83 ± 0.84?%,?16.93 ± 4.35?% and?21.87 ± 5.72?%,P = 0.39,and the P values > 0.05 in each group,the difference was not statistically significant.The proliferation inhibition rates of Hut78 cells after infected with ad-PTEN?MOI=0,1,10,50?for 48 h were 0,?46.43 ± 12.63?%,?64.28±13.40?%,?91.96±3.76?%,P value was less than 0.01,the difference was statistically significant.Compared in each groups,there was significantly different between MOI=1group and MOI=50 group.The proliferation inhibition rates of ad-GFP group?MOI = 0,1,10,50?were 0,?18.98 ± 5.28?%,?22.11 ± 8.39?%,?20.31 ± 1.56?%,P = 0.20,the difference was not statistically significant;5 Analysis of cell apoptosis after infection with adenovirus: The early apoptosis rate of Hut78 cells in the blank control group and the ad-PTEN infection groups?MOI = 1,10?were 6.5%,12.8% and 18.2% respectively.With the growth of the infection numbers,the early apoptosis of cells increased gradually;6 Expression of PTEN and downstream signaling pathway proteins: In the blank control group and the ad-PTEN?MOI=1,10,50?groups the PI3 k and Akt protein expression were gradually decreased.The expression of PTEN protein and Caspase-3 protein in the blank control group,MOI=1 group and 10 group increased gradually,in the MOI=50 group were declined than the MOI=10group.Conclusion:1 The Cutaneous T-cell lymphoma cells could be infected by the recombinant adenovirus.The efficiency was increased with the growth of the infection number and the infection times.2 The normal PTEN protein inducing apoptosis causes significant morphological changes in the cutaneous T-cell lymphoma cells.3 The normal PTEN protein inhibits the growth and proliferation of cutaneous T-cell lymphoma.4 The normal PTEN protein promotes the early apoptosis of cutaneous T-cell lymphoma cells.5 The normal PTEN protein inhibits the phosphorylation of PI3/Akt pathway in cutaneous T-cell lymphoma cells,and promotes the early apoptosis,suggesting that PTEN gene may play a role in inducing apoptosis by this pathway.