| Objective:The purpose of this study was to investigate the apoptotic effect on human cervical cancer cell lines(Hela cells)by transfection of antiapoptotic gene bcl-2 antisense oligonuleutides(ASODN)combined with oncogene c-myc ASODN or protoapoptotic gene bax following irradiation in vitro.And the impact of bcl-2 ASODN and bax combining transfection on the growth and radiotherapy effect of the subcutaneously transplanted Hela cells was also investigated in nude mice.The relationship between combined gene transfection and radiosensitivity of Hela cells was thus explored.Methods:1.ASODN of bcl-2 and c-myc were synthesized and transfected into Hela cells mediated by antionic liposomes.The cells were divided into four groups:bcl-2 ASODN treated group,c-myc ASODN treated group,(bcl-2+c-myc)ASODN treated g roup and control group.And each group was also divided into two subgroups according to whether irradiation was performed on or not.Cellular clone formation analysis,MTT and flow cytometry were used to investigate the proliferation and apoptosis of Hela cells in each group.The expression of bcl-2 and c-myc was analyzed by immunohistochemistry and flow cytometry. 2.We cloned the Bax-αgene from Hela cells with RT-PCR technique and inserted the Bax-αgene into the retroviral vector pLXSN.The transfection of bax in retroviral granules and bcl-2 ASODN were mediated by cationic liposomes.The Hela cells were divided into four groups:bcl-2 ASODN treated group,bax treated group, the bcl-2ASODN+bax treated group,and control group.And each group was then divided into two subgroups according to whether irradiation was accepted or not.The proliferation and apoptosis of Hela cells were measured by cellular morphology analysis under light microscope,MTT,and flow cytometry.RT-PCR was employed to analyze the expression of bcl-2 and bax.3.Hela cells were implanted subcutaneously into 25 nude mice.After tumor formation,the mice were immediately assigned into five groups:bcl-2 ASODN treated group,bax treated group,bcl-2 ASODN+bax treated group,bcl-2 SODN treated group and control group.After transfection,tumor volumes were measured and growth inhibition rates were calculated periodically.Twenty one days later, single-dose irradiation by 6MV-X was performed and terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL)was used to detect the apoptosis.Results:1.(1):The survival rates of the Hela cells after irradiation measured by cellular clone formation analysis were significantly lower(p<0.05)in bcl-2 ASODN treated group,c-myc ASODN treated group and(bcl-2+c-myc)ASODN treated group than that in control group.(2):The apoptotic rates of Hela cells measured by flow cytometry in bcl-2 ASODN treated group,c-myc ASODN treated group and(bcl-2+ c-myc)ASODN treated group were(6.60±0.70)%,(10.29±0.66)%,(11.83±0.57) %,respectively.They were significantly higher than that in control group(p<0.05). (3).The sensitivity enhancement ratio(SER)of bcl-2 ASODN treated group,c-myc ASODN treated group and(bcl-2+c-myc)ASODN treated group calculated by MTT was 1.20,1.53 and 2.18 respectively at a cell survival fraction of 0.1.(4).The expression of bcl-2 and c-myc assessed by immunohistochemistry and flow cytometry in the transfection groups were decreased greatly,compared with the control group.2.(1)We successfully inserted the Bax-αgene to the retroviral vector pLXSN at the restriction nuclease site EcoRI and XhoI orientately and obtained the recombinant retroviral expressive vector pLXSN-Bax.(2)Significant apoptosis was observed under light microscope in bcl-2 ASODN+bax treated group with irradiation.(3)The proliferation inhibitory rate of the Hela cells,which was measured by MTT,in bcl-2 ASODN+bax treated group with irradiation was(63.4±4.6)%,while that in bax and bcl-2 ASODN treated groups with irradiation were(32.7±1.8)%,(37.8±2.0)%, respectively.The inhibitory rate of Hela cells in double-gene transfection groups with irradiation was significantly higher than those in single-gene transfection groups with irradiation and other groups(P<0.05).(4)The apoptotic rate of HeLa cells,which was measured by flow cytometry in bcl-2 ASODN+bax treated group without irradiation was(26.13±2.50)%,and the rates in bcl-2 ASODN and the bax treated groups with irradiation were(33.04±2.71)%,(38.22±2.94)%,respectively.The apoptotic rate in bcl-2 ASODN+bax treated group with irradiation was(58.16±3.91)%,and significant difference was observed when compared with previous three groups(P<0.05).(5) According to the results of RT-PCR,the expression of bax was upregulated and the expression of bcl-2 was downregulated in groups following transfection.3.(1)Tumor formed in all the mice 14 days after implantation.The inhibitory rates of implanted tumor in bcl-2 ASODN treated group,bax treated group and bcl-2 ASODN+bax treated group were significantly higher than those in bcl-2 SODN treated group and control group(P<0.05).(2)Four days after irradiation,the inhibitory rate of tumor in the bcl-2 ASODN+bax treated group was 40.0%and significant difference was observed when compared with other groups(P<0.05).(3) The number of apoptotic cells in bcl-2 ASODN+bax treated group was 40.5±4.9 per high power field(HPF),which was also significantly higher than other groups (P<0.05).Conclusion:1.We successfully decreased the expression of bcl-2 and c-myc and increased the expression of bax through antisense technology and retroviral vector system in Hela cells.2.In vitro,compared with the control group,single-gene and double-gene transfections of bcl-2 ASODN and c-myc ASODN could significantly enhance the apoptosis of Hela cells induced by irridiation in vitro.Double gene transfection had more obvious effect.Bcl-2 ASODN+bax double-gene transfections,compared with single-gene transfection groups and control group,could significantly enhance the apoptosis of Hela cells induced by irridiation.3.In vivo,combining transfection of bcl-2 ASODN and bax could inhibit the tumor growth and induce the apoptosis after irradiation more effectively than single gene transfections in cervical cancer xenograft.4.Combining gene transfection aiming at enhancing radiosensitivity may become a new strategy to fight against malignancy.
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