荧光定量-逆转录-聚合酶链反应测定IL-2mRNA和IL-4mRNA方法的建立及其应用  

The FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA and its application

作　　者：朱晴晖 陆青 章谷生 

机构地区：[1]上海纺一医院,长江免疫基因检验中心,200060

出　　处：《中华微生物学和免疫学杂志》2006年第2期185-188,共4页Chinese Journal of Microbiology and Immunology

摘　　要：目的建立荧光定量-逆转录-聚合酶链反应（FQ-RT-PCR）测定TH细胞中IL-2 mRNA和IL-4 mRNA方法，并作临床初步应用。方法制备IL-2 cDNA和IL-4 cDNA，分别构建含有人IL-2基因和叫基因片段的pMD18-T质粒，克隆后作为定量阳性模板；设计和制备用于FQ-RT-PCR的引物和FAM、TAMRA标记的探针，优化实验条件，建立FQ-RT-PCR方法；用固相单抗方法从健康人、妇科良性疾患和恶性肿瘤患者以及慢性肾功能衰竭（cRF）患者PBMC中富集CD4（TH）细胞，经PMA和calcium ionophore诱导，提取总RNA，继用所建FQ-RT-PCR方法，作IL-2 mRNA和IL-4 mRNA定量测定。实验设阻actin为内控基因以监测RNA的质量。结果根据系列模板浓度的对数与CT值作直线回归建立标准曲线，线性范围为10^2～10^7copies/μl；不精密度试验显示，高值样品的批内、批间CV分别为7．8％和12．5％，低值样品的批内、批间CV分别为10．8％和19．5％；妇科恶性肿瘤患者与健康对照组和妇科良性疾患组比较，CRF患者与健康对照组比较，IL-2 mRNA表达均显著降低，IL-4 mRNA表达均显著升高（P〈0．001）。结论用所建立的FQ-RT-PCR法可对TH细胞内IL-2 mRNA和IL-4 mRNA作定量测定，方法简便、敏感、准确；妇科恶性肿瘤患者和CRF患者TH细胞内IL-2mRNA和IL-4 mRNA表达有明显的极化现象，呈TH2反应，提示恶性肿瘤患者和CRF患者TH细胞功能处于失衡状态。可通过改善和调整TH细胞的失衡提高恶性肿瘤患者的免疫监视功能和纠正CRF患者免疫紊乱。Objective To establish a FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA of TH cells and apply it to the patients with gynaecological tumors and chronic renal failure（CRF）. Methods IL-2 cDNA and IL-4 cDNA were prepared, the plasmid pMD18 carried IL-2 cDNA or IL-4 cDNA fragment were constructed and cloned as a templet for quantitative determination, the primers and probes labbed with 6-carboxy-fluoreseein（FAM） and 6-carbexy-tetramethylrhedamine （TAMRA） were prepared. The IL-2 mRNA and IL-4 mRNA of TH. cells enriched from PBMCs of 20 healthy volunteers（HVs）, 16 gyrmecological benign（GB） cases, 18 gynaecological malignant（GM） cases and 16 CRF patients were detected by FQ-RT-PCR, the housekeeping gene β-actin was used as the internal control gene of the experiment. Results The standard curve for Log concentration of series of quantitative templet vs threshold cycle（CT） was established by linear regression, the linear range is 102-107 copies/μl; the imprecision test shows the CV of inter-assay and intra-assay of a high content sample by FQ-RT-PCR were 7.8% and 12.5% respectively, the CV of inter-assay and intra-assay of a low content sample were 10.8% and 19.5% respectively; the IL-2 mRNA expression in TH. of the patients with gynaecological malignant tumor（compared with the HVs and the patients with gynaecological benign disease） and of the CRF patients （compared with the HVs） significantly declined and the IL-4 mRNA expression significantly increased （P 〈 0.001）. Condusion A simple, sensitive and accurate FQ-RT-PCR method for quantitatively detecting IL-2 mRNA and IL-4 mRNA has been established; IL-2 mRNA and IL-4 mRNA expression in TH. cells of the patients with gynaecological malignant tumor and the CRF patients was polarized and suggested that the function of the TH. cells from the patients with gynaecological malignant tumor and CRF were out of balance.

关 键 词：荧光定量-逆转录-聚合酶链反应 白细胞介素2 白细胞介素4 mRNA TH1 TH2 

分 类 号：R450[医药卫生—治疗学]