Transfer Of The HTPO And HNIS Into Gliomas For Ioiodine Theraphy

Objective:To construct the recombinant hTPO gene with adenvirus and transfer hTPO and hNIS genes into U251 cell line.Stably expressing hTPO and hNIS gene cell line (AdTPO-hNIS-U251) and stably expressing hNIS gene cell line(hNIS-U251) were produced.To study the iodide uptake ablibities and effective half lives of iodide in the above cell lines.Methods:Through cloning,recombination,packaging and amplifying,the recombinant adenosine virus AdTPO was constructed.After purification,the viral titers were calculated.Then by using Western-Blotting,the protein expression of AdTPO was tested.We accessed the recombinant plasmids pcDNA3.1- hNIS.After transecting hNIS gene into human glioma cell line U251 through liposome,stably expressing hNIS gene cell line(hNIS-U251) selected by G418 antibiotics was determined as the negative control groups.By using adenosine virus,hTPO was transducted into hNIS-U251,which was used as the testing group (AdTPO-hNIS-U251).U251 cell without any plasmids was applied as blank control group(U251).Then,we investigated biologic functions of the above cells,including ¹²⁵I uptake assay,¹²⁵I influx-course test,¹²⁵I effiux-course test,perchlorate suppressive assay,¹²⁵I organification degree assay and cell clonogenic assay.Results:1.The uptake ability of ¹²⁵I was 4 fold higher in hNIS-U251 cells than in blank control U251 cells(P＜0.01).2.The uptake ability of ¹²⁵I was 147 fold higher in AdTPO-hNIS-U251 cells than in blank control U251 cells(P＜0.01),and 110 fold higher in hNIS-U251 cells than in blank control U251 cells(P＜0.01).3.¹²⁵I influx test showed:¹²⁵I accumulated quickly in negative control group and in testing group,and reached the steady state with 90-120min after iodide was added. The blank control group couldn't accumulate ¹²⁵I.4.¹²⁵I effiux test showed:The effiux of ¹²⁵I was rapid in negative control group,its effective half life was about 7 min,the effective half life was prolonged to 13 min in testing group. 5.Perchlorate suppressive assay showed:Uptake was inhibited by NaClO₄ in AdTPO-hNIS-U251 cells and hNIS-U251 cells.6.¹²⁵I organification degree assay:In testing group,the ¹²⁵I organifi- cation degree was higher than the groups without AdTPO.7.Cell clonogenic assays demonstrated the clonal forming efficiency of testing group after incubating with ¹³¹I was 13 fold lower than the group which was not incubated with ¹³¹I(P＜0.01).And the clonal forming efficiency of negative control group after incubating was 10 fold lower than the group which was not incubated with ¹³¹I(P＜0.01).The clonal forming efficiecies of all three groups without incubation with ¹³¹I showed no significant difference.Conclusion:High titer AdTPO was constructed by using convenient Adeasier Systerm. Co-transfection with hNIS and hTPO genes could increase iodide uptake and radioiodide organification,which could prolong T_1/2 effiux to 13min and increase retention of radioiodide in the cell.