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目的克隆人 Mucin (MUC1)黏蛋白的启动子序列,并研究其驱动人钠/碘共转运体(hNIS)基因在胰腺癌细胞内的靶向性表达功能。方法利用巢式 PCR 方法从人胰腺癌细胞株CAPAN-Ⅱ细胞中扩增出 MUC1启动子。采用基因重组方法构建质粒 pDC316-MUC1/hNIS。采用脂质体转染的方法把 pDC316-MUC1/hNIS 导入到 MUCI 阳性的人胰腺癌细胞株 CAPAN一Ⅱ、PANC-1和MUC1阴性的人宫颈癌细胞株 HeLa,转染后48h 采用 RT-PCR 方法及免疫组化方法检测转染细胞内的 hNIS mRNA 水平和蛋白表达,转染48h 后检测肿瘤细胞内 NIS 对125碘吸收功能。结果扩增的MUC1启动子核心调控区序列与参考序列一致。重组 pDC316-MUC1/hNIS 转染后48h,hNIS 蛋白在CAPAN-Ⅱ、PANC-1细胞阳性表达,而在 Hela 细胞内不表达。pDC316-MUC1/hNIS 转染后仅在CAPAN-Ⅱ、PANC-1细胞内检测到高水平吸碘,分别是 pDC316-MUC1转染组的7和12倍。结论MUC1启动子能驱动 NIS 基因在 MUC1阳性的肿瘤细胞内靶向功能表达,为进一步在体内运用放射碘靶向治疗胰腺癌奠定了基础。
Objective To clone the promoter sequence of human Mucin (MUC1) mucin and study its target-driven expression of hNIS gene in pancreatic cancer cells. Methods The promoter of MUC1 was amplified from human pancreatic cancer cell line CAPAN-Ⅱ by nested PCR. Plasmid pDC316-MUC1 / hNIS was constructed by gene recombination. PDC316-MUC1 / hNIS was transfected into human pancreatic cancer cell line MUCI-CAPAN-Ⅱ, PANC-1 and MUC1-negative human cervical cancer cell line HeLa by lipofectamine transfection method. RT-PCR Methods The expression of hNIS mRNA and protein in transfected cells were detected by immunohistochemical method. After 48 hours of transfection, the activity of NIS on 125 iodine was detected. Results The amplified MUC1 promoter core regulatory region sequence was consistent with the reference sequence. At 48h after recombinant pDC316-MUC1 / hNIS transfection, hNIS protein was expressed in CAPAN-Ⅱ and PANC-1 cells but not in Hela cells. High levels of iodine uptake were detected only in CAPAN-II and PANC-1 cells after pDC316-MUC1 / hNIS transfection, which were 7 and 12 times higher than that of pDC316-MUC1 transfection group respectively. Conclusion MUC1 promoter can drive NIS gene expression in MUC1-positive tumor cells, which lays the foundation for further targeting of pancreatic cancer with radioiodine in vivo.