重组pDC316-MCMV/hNIS真核表达质粒的构建及在肿瘤细胞的功能  

Construction of pDC316-MCMV/hNIS and Its Effects on Non-thyroid Tumor Cell

作　　者：周泉波[1] 陈汝福[1] 李志花[1] 周嘉嘉[1] 唐启彬[1] 陈积圣[1] 

机构地区：[1]中山大学附属第二医院肝胆外科,广东广州510120

出　　处：《中山大学学报（医学科学版）》2007年第4期383-386,407,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家高科技计划(863)基金(2002AA214061);广东省自然科学基金(2003A031700);广东省医学科学研究基金(A2005228);广州市科委基金(2004Z3-D2011)

摘　　要：【目的】克隆人的钠/碘同向转运体(hNIS)基因可编码序列,并研究其在非甲状腺肿瘤细胞内的功能表达。【方法】利用逆转录和聚合酶链反应(RT-PCR)从毒性弥漫性甲状腺肿(又称Graves病)病人的甲状腺组织中扩增得到NIS的可编码区基因,并克隆到T载体,测序后亚克隆到腺病毒载体穿梭质粒pDC316载体上,获得重组真核表达质粒载体pDC316-MCMV/hNIS。采用脂质体转染的方法,把pDC316-MCMV/hNIS重组质粒(实验组)或pDC316空质粒(对照组)分别导入到胰腺癌细胞株CAPAN-Ⅱ细胞、PANC-1细胞和卵巢癌细胞株SK-OV-3细胞,转染后48h采用RT-PCR方法检测转染细胞内的hNIS mRNA水平,转染后第2、3、4天分别检测肿瘤细胞对125I的吸收功能。【结果】序列分析结果证实克隆片段与文献报道的hNIS基因cDNA序列完全一致。实验组转染后48h,在CAPAN-Ⅱ细胞、PANC-1细胞及SK-OV-3细胞内均能检测到hNIS mRNA高水平表达,对照组基本无hNIS mRNA表达。转染后第3天细胞吸碘达到高峰,实验组CAPAN-Ⅱ细胞、PANC-1细胞和SK-OV-3细胞对125I的吸收量分别比对照组高24倍、17倍和13倍。【结论】从Graves病人的甲状腺组织中克隆的hNIS基因转染肿瘤细胞后,能使肿瘤细胞发挥高水平吸碘功能,为进一步运用放射性核素体内靶向治疗非甲状腺肿瘤奠定了基础。[Objective] To clone human sodium / iodide symporter （hNIS） genes, and study the function expression of hNIS in non-thyroid tumor cell. [Methods] Human sodium/iodide symporter genes cDNA were amplified from thyroid tissue of the patients suffering from Graves disease by RT-PCR , and cloned into pGEM-T easy/hNIS, pGEM-T easy/hNIS was sequenced and subcloned into pDC316-MCMV/hNIS. We transfected the recombinant pDC316-MCMV/ hNIS into CAPAN-Ⅱ cells, PANC-1 cells, and SK-OV-3 cells by lipofectamine 2000. 48h after recombinant plasmid transfection, the level of hNIS mRNA was determined. The 2, 3, 4 day after recombinant plasmid transfection , ^125I uptake in the cells was measured by gamma-counting. [Results] The results showed that the sequence data of hNIS gene in recombinant plasmids pGEM-T easy/hNIS and pDC316-MCMV/ hNIS was corresponded to those of reference report. The level of hNIS mRNA could be determined highly expression in the cells transfection with pDC316-MCMV/ hNIS, but the low level was measured in the cells transfected with pDC316. The similar results could be seen in ^125I uptake, a 24-fold increase in 12sI uptake was observed in CAPAN- 2 cells transfected with pDC316-MCMV/ hNIS when compared with the cells transfected with pDC316, and a 17- fold, a 13-fold increase was also been seen in PANC-1 cells and SK-OV-3 cells. [Conclusion] hNIS could be amplified successfully from thyroid tissue of the patient suffering from Graves disease and could be express in tumor cells after transfection with recombinant pDC316-MCMV/ hNIS, it would provide academic evidence for further research.

关 键 词：人钠/碘同向转运体 逆转录聚合酶链反应 基因转染 碘放射性同位素 

分 类 号：R736.1[医药卫生—肿瘤]