重组质粒hTSHR转染低分化甲状腺癌细胞株后对摄碘能力及特异性基因表达的影响

目的 探讨重组真核表达质粒pcDNA3.1/人TSH受体（hTSHR）体外转染TSHR表达下降的低分化滤泡状甲状腺癌细胞株后,细胞摄取放射性碘功能以及甲状腺癌相关基因mRNA表达 的变化.方法 pcDNA3.1/hTSHR转化DH5a感受态菌,进行扩增、酶切,再以核苷酸测序方法鉴定.体外转染pcDNA3.1/hTSHR,通过免疫荧光检测TSHR表达产物,井型γ计数仪检测摄碘率,相对定量实时荧光PCR验证其表达的TSHR蛋白功能和特性.采用SPSS 13.0软件,对计量资料行t检验.结果pcDNA3.1/hTSHR经PCR扩增hTSHR-cDNA片段约113 kb,Kpn Ⅰ和Xha Ⅰ双酶切：hTSHR-cDNA的片段约2.3 kb,pcDNA3.1（＋）的片段约5.5 kb,均同预期片段大小相符;核苷酸测序方法鉴定测序结果与GenBank中收录的hTSHR全长序列一致,表明真核表达质粒构建正确.在hTSH刺激下,转染pcDNA3.1/hTSHR细胞与转染pcDNA3.1（＋）细胞比较：（1）在甲状腺肿瘤细胞胞质、胞膜有增强的绿色荧光,（2）前者125 I摄取率是后者的2.9倍（t=28.63,P＜0.01）,（3）甲状腺碘摄取相关基因TSHR、钠碘转运体（NIS）、甲状腺过氧化物酶（TPO）、Tg的mRNA的表达分别升高1.74倍（t=5.959,P＜0.01）、7.2倍（t=3.807,P＜0.05）、2.88倍（t=4.769,P＜0.01）和2.67倍（t=6.388,P＜0.01）.结论 pcDNA3.1/hTSHR体外转染甲状腺癌肿瘤细胞后,可有效提高碘的摄取;这可为放射性碘治疗失分化甲状腺癌提供新的实验依据.

The effects of human TSH receptor gene transfection on iodide uptake and thyroid-specific gene expression in poorly differentiated thyroid carcinoma cell line

Objective To investigate the changes of iodide uptake and the expression of thyroidspecific genes in poorly differentiated follicular thyroid carcinoma (FTC) cells after transfection of human TSH receptor (hTSHR) gene in vitro. Methods The recombinant eukaryotic expression plasmid PcDNA3. 1/hTSHR-cDNA was transformed into DH5a bacterial for amplification and then the recombinant plasmid was extracted. The recombinant was identified with PCR amplifying, restriction enzyme digestion analysis and DNA sequencing. The recombinant plasmid pcDNA3.1/hTSHR was transfected into FTC-133 cell line by lipofectin methodin vitro. Immunofluorescence, iodide uptake studies and real time-PCR were applied to detect target protein expression. Statistical analysis was performed with t-test using SPSS 13. 0 software. Results Kpn Ⅰ and Xba Ⅰ restriction enzyme digestion, PCR amplifying and DNA sequencing confirmed that pcDNA3. 1/hTSHR was successfully constructed. After transfection of the recombinant plasmid pcDNA3. 1/hTSHR-cDNA and the stimulation of hTSH, the tumor cells displayed the expression of hTSHR protein at cell surface and cytoplasm. The iodine uptake in pcDNA3. 1/hTSHR transfected cells was 2. 9 times higher than that of control(pcDNA3.1(+) transfected cells) group(t = 28.63, P ＜0. 01). The expression of TSHR,NIS, TPO and Tg (mRNA levels) in pcDNA3. 1/hTSHR transfected cells were also significantly elevated by 1.74 (t =5.959, P＜0.01), 7.2 (t =3.807,P＜0.05), 2.88 (t=4.769,P＜0. 01) and 2.67 times (t=6.388,P ＜0.01) respectively compared to those of the control group. Conclusion The study demonstrates that iodide uptake may be reactivated by hTSHR receptor gene transfection in poorly differentiated FTC cell.