| Objective: By transfecting survivin ASODN into hep-2cells, we explore the effect of survivin ASODN on apoptosisand radiotherapy sensitivity.Methods: Firstly we culture hep-2 cells under theconditions of 37℃,5﹪CO2 and 95﹪humidity. Cells in thelogarithmic phase of cell growth were selected to proceed withthe following experiments.(1)Transfection of survivin ASODNinto hep-2 cells was carried out by lipofectin method.Theexperiment is devided into fourgroups :100,200,400,600nmol/ml. The cells were observedunder the fluorescence inversphase microscope and single cellsuspension was made to be measured with flow cytometry.(2)Measure the inhibition rate of transfection with crystal violetThe cells in the logarithmic phase of cell growth were selectedto be transfected with lipofectin mediated survivingASODN.The experiment is devided into five groups:blankgroup replaced with RPMI-1640 medium, 100nmol/mlsurviving ASODN,200nmol/ml surviving ASODN,400nmol/mlsurviving ASODN,600nmol/ml survivin ASODN. Each groupconsists of three parallel cells.Before transfection the cells werecultured under the conditions of 37℃,5﹪CO2 and 95﹪humidity till the covering rate had been to 85-90﹪.24h aftertransfection ,we remove the medium and add crystalviolet ,vibrate and measure optical density ( OD ) withenzyme-linked analyzer.(3)Observe the changes of survivinmRNA with RT-PCR method As above ,cells in thelogarithmic phase of cell growth are selected .Also theexperiment is devided into five groups:blank group replacedwith RPMI-1640 medium,100nmol/ml survivingASODN,200nmol/ml surviving ASODN,400nmol/ml survivingASODN,600nmol/ml surviving ASODN.24h aftertransfection,we collect 1.7×10~7 cells and extract total RNA withguanidinium thiocyanate method .The same quantity of totalRNA was taken to proceed with RT-PCR in a 20ul reactionsystem according to the manusfacture's instruction ,and thenamplified in a 4ul reaction system.Electrophoresis was used toanalyse the products ,and observe under ultraviolet light.(4)Measure apoptosis and surviving protein with flowcytometry :We use 200nmol/ml survivin ASODN to transfecthep-2 cells ,24h after transfection , cells were irradiated with2,4,8Gy. 24h after irradiation ,we collect cells and make singlecell suspension ,then measure with flow cytometry.Results:(1)Observing with fluorescence inversphasemicroscope,we find changes of cells after transfection,such asreducing of cell volume ,anomaly form ,shrinking , shrinking ofnucleolus ,cytoplast roughness and fewer cell division. Howeverthe untreated cells are in integrity form and well growth. Thecells transfected with survivin ASODN tagged with FITC for 6hare showing green fluorescence ,the green fluorescence mainlydistributes in the nucleolus and less in cytoplasm .we find theefficiency of transfection was 94-98﹪6h after transfection.(2)All concentrations of surviving ASODN can inhibit proliferationand show a dose-and time-dependent manners,the differencewas significant.The inhibition rate of 600nmol/l survivinASODN was the highest.(3)survivin mRNA was expressed ata high level in hep-2 cells .All concentrations of survivinASODN can inhibit the expression of survivin mRNA at 24hafter transfection and present a dose-dependent manner.(4)Apoptosis was detected both in radiotherapy group and inradiotherapy combine with survivin ASODN group .Theapoptosis rate in radiotherapy group was4.74±0.30,6.17±0.49,9.69±0.78(﹪);while the apoptosis rate inradiotherapy combine with survivin ASODN group is12.19±0.76,21.36±1.77,25.69±1.27(﹪). The radiotherapycombine with survivin ASODN group is significantly higherthan the radiotherapy group. survivin protein fluorescence indexin untreated cells was 1; in radiotherapy group was 1.42±0.13,2.27±0.18,2.32±0.17;and in radiotherapy combine withsurvivin ASODN group was 1.26±0.11,1.81±0.13,2.03±0.13. survivin protein in radiotherapy group is significantlyhigher than that of in radiotherapy combine with survivinASODN group(p<0.05). There was a negative correlation...
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