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目的:建立人肿瘤细胞NKG2D配体基因(MICA、MICB、ULBP1、ULBP2、ULBP3)表达的实时荧光定量PCR(real-timefluorescence quantitative PCR)检测方法。方法:根据NCBI基因库中NKG2D配体基因序列,设计合成引物。用Trizol法从培养的肿瘤细胞(BEC-7402、HeLa、MDA-MB-435、XWLC-05)中提取总RNA,逆转录成cDNA,建立实时荧光定量PCR检测NKG2D配体基因表达的方法,并检测NKG2D配体在肿瘤细胞株中的表达。结果:经过琼脂糖凝胶电泳、熔解曲线和标准曲线分析,用所设计的引物和SYBR Green I能够特异扩增和定量检测NKG2D配体基因的表达。该方法成功检测4种肿瘤细胞NKG2D配体基因的表达。结论:建立了人NKG2D配体基因表达的实时荧光定量PCR检测方法,为进一步研究人NKG2D配体在肿瘤免疫中的作用提供了有效手段。
Objective: To establish a real-time fluorescence quantitative PCR method for the expression of NKG2D ligand gene (MICA, MICB, ULBP1, ULBP2 and ULBP3) in human tumor cells. Methods: According to the sequence of NKG2D ligand gene in NCBI gene bank, the synthetic primers were designed. Total RNA was extracted from cultured tumor cells (BEC-7402, HeLa, MDA-MB-435 and XWLC-05) by Trizol method and reverse transcribed into cDNA to establish real-time fluorescence quantitative PCR to detect NKG2D ligand gene expression. Detection of NKG2D ligand expression in tumor cell lines. Results: After agarose gel electrophoresis, melting curve analysis and standard curve analysis, the designed primers and SYBR Green I could specifically amplify and quantitatively detect the expression of NKG2D ligand gene. The method successfully detected the expression of NKG2D ligand genes in four kinds of tumor cells. Conclusion: The real-time fluorescent quantitative PCR method of human NKG2D ligand gene expression was established, which provided an effective method for further studying the role of human NKG2D ligand in tumor immunity.