【作者】 龙雷；

【导师】 谭建；

【作者基本信息】 天津医科大学 ， 影像医学与核医学， 2009， 硕士

【摘要】 目的:构建含有人甲状腺过氧化物酶基因（hTPO）的重组腺病毒AdTPO,并将AdTPO和人钠碘转运体基因（hNIS）分别转入胶质瘤细胞系U251中,获得稳定表达hTPO和hNIS基因的细胞系hNIS-AdTPO-U251和hNIS-U251,在（125）^I的作用下研究细胞的摄碘能力及摄碘时间。方法:克隆,重组,包装并扩增纯化得到重组腺病毒（AdTPO）,并且测定病毒滴度,Western-Blotting检测重组腺病毒的表达。鉴定已构建好的重组质粒（pcDNA3.1-hNIS）。使用脂质体转染法将hNIS基因转染入人胶质瘤细胞系U251中,经过G418抗生素筛选获得稳定表达hNIS的细胞系,为阴性对照组（hNIS-U251）;使用重组腺病毒将hTPO基因转导入U251中,使胶质瘤细胞获得hTPO基因,为实验组（AdTPO—hNIS-U251）。未转入hTPO和hNIS的细胞为空白对照组（U251）。进行感染后稳定表达细胞系的体外摄（125）^I实验,体外（125）^I外流实验,体外（125）^I内流实验,过氯酸盐抑制实验,（125）^I有机化测定试验,细胞克隆形成实验等。结果:1.hNIS在U251细胞中有瞬时表达,转染了hNIS的U251细胞比未转染hNIS的空白组摄（125）^I能力增高约4倍左右（P＜0.01）。2.通过重组腺病毒感染已经稳定表达hNIS基因的U251细胞系,将hTPO基因转导至U251细胞后,hNIS联合AdTPO共转染组与空白对照组的摄（125）^I能力相比增高约147倍左右（P＜0.01）,hNIS单独转染组与空白对照组的摄碘能力相比增高约110倍（P＜0.01）。3.（125）^I内流的动态演变实验证实（125）^I在实验组和阴性对照组稳定表达细胞系中均快速聚集,约90-120min时达到稳定阶段。4.（125）^I外流的动态演变实验证实阴性对照组中（125）^I的有效半衰期为7min,实验组的（125）^I外流减慢,其有效半衰期延长至13min。5.NaClO₄抑制试验表明加入NaClO₄的实验组和阴性对照组的摄（125）^I能力明显受到抑制,摄碘能力均降低30倍左右（P＜0.001）。6.（125）^I有机化测定试验证实hNIS和hTPO共转染U251细胞的有机化程度高于单独hNIS转染U251细胞。7.细胞克隆实验表明经（131）^I孵育后实验组比未经（131）^I孵育的空白对照组的克滦纬陕视兴档?P＜0.01),约降低13倍左右;经（131）^I孵育后阴性对照组比未经（131）^I孵育的空白对照组的克隆形成率有所降低（P＜0.01）,约降低10倍左右;而未经（131）^I孵育的实验组与阴性对照组及空白对照组的细胞克隆形成率相比未见明显差异。结论:成功获得高滴度的重组腺病毒AdTPO。将hTPO基因和hNIS基因共转染至胶质瘤细胞系U251后,实验组摄碘能力有明显增高,可以部分有机化碘,有效半衰期延长至13min,可以延长放射性碘在细胞中的停留时间。更多还原

【Abstract】 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.更多还原