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目的:建立可用于测定淋巴细胞免疫功能的SYBRGreen I实时荧光定量PCR技术。方法:根据NCBI基因库中4种基因(NKG2D、穿孔素、颗粒酶B和内参照GAPDH)的序列,设计合成相应的引物,扩增上述基因。建立SYBRGreen I实时荧光定量PCR方法,检测肿瘤患者外周血淋巴细胞和诱导培养的患者CIK细胞(cytokine induced killer,CIK)中NKG2D、穿孔素和颗粒酶B mRNA的含量。结果:应用设计的引物扩增NKG2D、穿孔素和颗粒酶B基因后,经琼脂糖凝胶电泳和溶解曲线分析表明具有特异性。SYBR Green I实时荧光定量PCR检测结果表明,肿瘤患者的淋巴细胞中颗粒酶B基因的表达降低,而经细胞因子和单克隆抗体诱导培养的肿瘤患者CIK细胞与其淋巴细胞相比,细胞中穿孔素和颗粒酶B基因的表达明显增加(P<0.01)。结论:该SYBRGreen I实时荧光定量PCR方法可用于检测淋巴细胞中NKG2D、穿孔素和颗粒酶B的mRNA的表达、作为研究淋巴细胞免疫功能的有力手段。
Objective: To establish SYBR Green Ⅰ real-time fluorescence quantitative PCR technique which can be used to determine the immune function of lymphocytes. Methods: According to the sequences of four genes (NKG2D, perforin, granzyme B and internal reference GAPDH) in NCBI gene bank, corresponding primers were designed and synthesized to amplify these genes. SYBR Green Ⅰ real-time quantitative PCR method was established to detect the contents of NKG2D, perforin and granzyme B mRNA in CIK cells from peripheral blood lymphocytes of patients with cancer and in induced cytokine induced killer (CIK). Results: The NKG2D, perforin and granzyme B genes were amplified by using designed primers and showed specificity by agarose gel electrophoresis and lysis curve analysis. SYBR Green I real-time quantitative PCR results showed that the expression of granzyme B gene in lymphocytes of tumor patients was decreased, whereas CIK cells in tumor patients induced by cytokines and monoclonal antibodies had higher levels of perforin And granzyme B gene expression was significantly increased (P <0.01). Conclusion: The SYBRGreen I real-time PCR method can be used to detect the mRNA expression of NKG2D, perforin and granzyme B in lymphocytes, which can be used as a powerful means to study lymphocyte immune function.