实时荧光定量PCR检测淋巴细胞中NKG2D、穿孔素和颗粒酶B基因的表达

The quantitative detection of NKG2D,perforin and granzyme B gene expressions in lymphocytes by SYBR green Ⅰ real-time fluorescence quantitative PCR

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。

AIM： To develop a SYBR Green I real-time fluorescence quantitative PCR for the detection of lymphocyte immune function.METHODS： The primers of NKG2D,perforin,granzyme B and keeping-home gene GAPDH were designed and synthesized according to NCBI gene sequences,and the real-time fluorescence quantitative PCR was established.The gene expressions of NKG2D,perforin and granzyme B in lymphocytes from cancer patients and CIK induced from the cancer patients lymphocytes in vitro were quantified by real-time fluorescence quantitative PCR.RESULTS： NKG2D,perforin and granzyme B mRNA could be specifically amplified and quantitatively detected by the SYBR Green I real-time fluorescence quantitative PCR according to agarose gel electrophoresis and melt curve analysis.The mRNA expression of granzyme B was reduced in lymphocytes from cancer patients,however the mRNA expressions of perforin and granzyme B were increased in CIK induced by cytokines and monoclone antibody compared with their lymphocytes（P0.01）.CONCLUSION： The SYBR Green I real-time fluorescence quantitative PCR is a useful method for the quantitative detection of NKG2D,perforin and granzyme B mRNA to investigate cellular immune function.