^(19)F磁共振波谱分析CD基因在人结肠癌细胞的表达

ANALYSIS OF EXPRESSION OF CYTOSINE DEAMINASE GENE IN COLON CANCER CELLS WITH ^(19)F MAGNETIC RESONANCE SPECTROSCOPY

为了研究^(19)F-NMR波谱分析胞嘧啶脱氨酶(CD)基因在人结肠癌细胞中表达的可行性,通过逆转录病毒介导方法,将编码大肠杆菌的CD基因转染到人结肠癌细胞系SW1116。通过RT-PCR、MTT方法和^(19)F-NMR波谱分析了 CD基因在人结肠癌细胞中的表达状况。结果显示转基因的肿瘤细胞SWCD_2 中表达CD基因,对前药 5-氟胞嘧啶(5-FC)的敏感性较亲本肿瘤细胞 SW1116明显增高,50％细胞生长抑制率(IC_(50))为 66 μmol/L,而 SW1116细胞IC_(50)为16 mmol/L。^(19)F-NMR波谱分析显示,转导CD基因细胞在774μmol/L 5-FC浓度培养24 h后的样品,在δ-91.7和δ-93.3显示了2个双峰,分别是5-FC和5-氟尿嘧啶(5-FU)的^(19)F-NMR的信号,提示5-FU是 SWCD_2细胞内的 CD酶催化 5-FC的代谢产物。这些结果表明^(19)F-NMR分析是一种能够用于检测CD基因在人结肠癌细胞中表达的新方法,它将有助于对临床基因治疗方案实施监测。

To investigate the feasibility of using ^(19)F-NMR spectroscopy to monitor cyto- sine deaminase (CD) transgene expression in human colon cancer cell line. The SW1116 cell line was transfected with a gene coding for CD from E. coli by the retroviral media- ted method. The assessment of the expression of CD gene in the colon cancer cell line was carried out by RT-PCR, MTT assay of drug sensitivity to 5-FC and ^(19)F-NMR spec- troscopy. The results indicate that the transgenic SWCD, cell line was more sensitive to the prodrug 5-fluorcytosine (5-FC) (IC_(50)= 66μmol/L) as compared to the parent cancer cells SW1116 (IC_(50) = 16 mmol/L). After incubation of the cultured CD^+ cells in 774 μmol/L 5-FC for 24 h, ^(19)F-NMR spectra of the intact cells showed two broad resonances corresponding to 5-FC (taken up into cell chemical shift relative to trifluoroacetate of δ - 91. 7) and 5-FC produced by the action of CD (chemical shif of δ-93. 3), respective- ly. These results demonstrated the feasibility of using ^(19)F-NMR spectroscopy to nonin- vasively monitor CD gene expression in human colon cancer cells. This capability pro- vides a new approach for measuring gene expression that will be useful in clinical gene therapy trials.