应用质谱技术检测结肠癌细胞DPYD和MTHFR基因单核苷酸多态性

Detection of single nucleotide polymorphisms of DPYD and MTHFR genes in colon cancer cell lines

目的:检测结肠癌细胞株中两种代谢相关基因DPYD和MTHFR的单核苷酸多态性(single nucleotide polymorphisms,SNPs)。方法:培养HT-29及Lovo两株结肠癌细胞,分别提取基因组DNA,PCR扩增DPYD和MTHFR基因相应目的片段,采用基于基质辅助激光解吸电离飞行时间质谱技术进行DPYD基因rs1801159(A/G)、rs1801160(G/A)、rs17376848(A/G),MTHFR基因rs1801131(A/C)、rs1801133(C/T)、rs2274976(G/A)的SNPs位点检测。结果:两种细胞的DPYD基因3个位点rs1801159、rs1801160及rs17376848基因型均为野生纯合型,即A/A、G/G及A/A型。HT-29细胞MTHFR基因rs1801131和rs1801133、rs2274976位点基因型分别为A/C、C/T杂合子和G/G纯合子,而Lovo细胞此3个位点依次为A/A、T/T纯合子和G/A杂合子。结论:本实验中HT-29及Lovo两种细胞株的DPYD基因所测3个SNP位点基因型均相同,而这两种结肠癌细胞株MTHFR基因所测的各SNP位点基因型均不相同。

OBJECTIVE：This study was purposed to identify single nucleotide polymorphisms（SNPs） of two pharmacokinetics-related genes in two colon cancer cell lines.METHODS：HT-29 and Lovo cell lines were cultured. The genomic DNA of the two cell lines were isolated by QIAamp DNA Blood Mini kit.Then we designed the primers and amplified the related DNA fragments by PCR.SNP genotyping of DPYD gene rsl801159（A/G）,rs1801160 （G/A）,rs17376848（A/G） and MTHFR gene rs2274976（G/A）,rs1801131（A/C） and rs1801133（C/T） was performed by means of matrix assisted laser desorption ionisation-time of flight mass spectrometry method（MALDI-TOFMS）. RESULTS：The genotypes of DPYD gene locus rs1801159,rs1801160,rs17376848 were A/A,G/G and A/A, respectively,in the two cell lines.The genotype of MTHFR gene locus rs1801131,rs1801133 and rs2274976 were A/C,C/T and G/G,respectively,in HT-29 cell line.While those were A/A,T/T and G/A,respectively,in Lovo cell line.CONCLUSION：The above-mentioned loci of DPYD were the same in HT-29 and Lovo cell lines.However the detected loci of MTHFR gene in the two cell lines were expressed differently.