【摘要】 目的:探讨二氢嘧啶脱氢酶基因(DPYD)T85C位点突变对乳腺癌细胞5-氟尿嘧啶(5-FU)敏感性的影响。方法:从肝脏组织中提取mRNA,扩增得到野生型DPYD编码序列。利用定点突变技术,获得T85C定点突变的DPYD编码序列。将GFP、野生型和突变型DPYD序列,连入PCDH-CMV-MCS-EF1-Puro载体。转染上述3种质粒到MDA-MB-231细胞中,获得稳定表达的细胞株。MTT法检测不同浓度5-FU(0、1、5、10、50μg/mL)处理24 h后,细胞存活情况。结果:酶切验证3种重组质粒,观察到目的序列。测序验证定点突变质粒,突变位点发生T到C的改变。相比对照组,突变型细胞的存活率显著增高(P<0.01),且突变型细胞存活率高于野生型(P<0.05)。结论:DPYD基因突变影响乳腺癌细胞对5-FU敏感性,T85C突变型DPYD细胞对5-FU的敏感性显著降低。更多还原

【Abstract】 Objective: To explore the relationship between T85 C mutation in dihydropyrimidine dehydrogenase gene(DPYD) and sensitivity of breast cancer cell to 5-fluorouracil(5-FU). Methods: Genomic m RNA was isolated from human liver tissue. DPYD coding sequence was amplified as wild type DPYD. The T was transformed into C by site-directed mutagenesis technique at nucleotide position 85 in DPYD.Then the sequences of GFP, wild type and mutation DPYD were inserted into PCDH-CMV-MCS-EF1-Puro vector respectively. The recombinant plasmids were transfected into MDA-MB-231 to obtain stable cells. After 24 hours of exposure to 5-FU at different concentrations(0, 1, 5, 10, 50 μg/mL), the cell viability was tested using the MTT colorimetric method. Results: The GFP, wild type and mutation DPYD were observed by restricting double enzyme digestion. The nucleotide of mutation DPYD transformed from T to C at 85 in DPYD coding sequence. Compared with control group, the mutation group cell survival rate was significantly increased(P <0.01), and the mutation group cell survival rate was higher than that of wild type group(P <0.05). Conclusion: The mutation of DPYD influences the sensitivity of breast cancer cell to 5-FU, and the sensitivity of T85 C mutation phenotype cell to 5-FU could be significantly reduced.更多还原