目的:研究IL-4mRNA和IL-4蛋白在哮喘大鼠CD34+细胞中的转录表达及孟鲁司特(montelukast,MK)对其表达的影响。方法:将SD大鼠随机分为3组:哮喘组、MK组和正常对照组。用卵白蛋白制备大鼠哮喘模型。应用双抗体夹心酶联免疫吸附试验测定血浆...目的:研究IL-4mRNA和IL-4蛋白在哮喘大鼠CD34+细胞中的转录表达及孟鲁司特(montelukast,MK)对其表达的影响。方法:将SD大鼠随机分为3组:哮喘组、MK组和正常对照组。用卵白蛋白制备大鼠哮喘模型。应用双抗体夹心酶联免疫吸附试验测定血浆中IL-4和γ-干扰素(IFN-γ)浓度;用MiniMACS磁珠分选系统分离骨髓CD34+细胞;采用SYBR GREEN I荧光实时定量PCR法测定CD34+细胞中IL-4 mRNA的相对表达量。采用免疫组化技术测定CD34+细胞中IL-4蛋白的表达量。结果:哮喘组骨髓CD34+细胞中IL-4mRNA和IL-4蛋白的表达量高于其它各组(P<0.01);哮喘组除了IFN-γ水平低外,IL-4浓度和嗜酸性粒细胞(Eos)绝对值都是三组中最高的(P<0.01);除哮喘组外,其余组的各项指标相近(P>0.05)。IL-4 mRNA表达量与IL-4浓度、Eos绝对值呈正相关(P<0.01),与IFN-γ浓度呈负相关(P<0.01)。结论:哮喘大鼠骨髓CD34+细胞中IL-4mRNA的表达增强;孟鲁司特可以下调IL-4mRNA的表达,可能成为其抑制哮喘气道炎症形成的重要机制之一。展开更多
The purpose of the study is to establish a fluorescence quantitative reverse transcription polymerase chain response （FQ-RT-PCR） method for the quantitative determination of IL-2 mRNA and IL-4 mRNA in Th cells, with...The purpose of the study is to establish a fluorescence quantitative reverse transcription polymerase chain response （FQ-RT-PCR） method for the quantitative determination of IL-2 mRNA and IL-4 mRNA in Th cells, with which the Th cells status of the patients with gynaecological tumors and chronic renal failure （CRF） can be analyzed. IL-2 eDNA and IL-4 eDNA were prepared, and the plasmid pMD18 carrying IL-2 eDNA or IL-4 eDNA fragment was constructed and cloned as the template for quantitative determination. The primers and probes labelled with 6-carbexy-fluorescein （FAM） and 6-carboxy- tetramethylrhodamine （TAMRA） were prepared, and the experimental conditions were optimized to set up the FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA. Th cells enriched from peripheral blood mononuclear cells （PBMCs） of 20 healthy volunteers （HVs）, 16 gynaecological benign （GB） cases, 18 gynaecological malignant （GM） tumor cases and 16 chronic renal failure （CRF） patients were tested for IL-2 mRNA and IL-4 mRNA by FQ-RT-PCR. The house-keeping gene β-actin was used as the internal control gene of the experiment. The standard curve for log concentration of series of quantitative templates vs threshold cycle （CT） was established by linear regression, and the linear range was 102-107 copies/μl. The imprecision test showed the CV of inter-assay and intra-assay of a high cont- ent 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 expressions in Th of the patients with gynaecological malignant tumor （compared with the HVs and the patients with gynaecological benign disease） and in Th of the CRF patients （compared with the HVs） were declined significantly and at the same time the IL-4 mRNA expression increased significantly （ P 〈 0. 001 ）. A simple, sensitive and accurate FQ-RT-PCR method for the quantitative detection of IL-2 mRNA and IL-4 mRNA has been established. The IL-2 mRNA and IL-4 mRNA expressions in Th cells of the patients with gynaecological malignant tumor and the CRF patients were polarized and displayed Th2 response. It suggests that the function of the Th cells of the patients with gynaecological malignant tumor or CRF is at unbalance.展开更多
文摘目的:研究IL-4mRNA和IL-4蛋白在哮喘大鼠CD34+细胞中的转录表达及孟鲁司特(montelukast,MK)对其表达的影响。方法:将SD大鼠随机分为3组:哮喘组、MK组和正常对照组。用卵白蛋白制备大鼠哮喘模型。应用双抗体夹心酶联免疫吸附试验测定血浆中IL-4和γ-干扰素(IFN-γ)浓度;用MiniMACS磁珠分选系统分离骨髓CD34+细胞;采用SYBR GREEN I荧光实时定量PCR法测定CD34+细胞中IL-4 mRNA的相对表达量。采用免疫组化技术测定CD34+细胞中IL-4蛋白的表达量。结果:哮喘组骨髓CD34+细胞中IL-4mRNA和IL-4蛋白的表达量高于其它各组(P<0.01);哮喘组除了IFN-γ水平低外,IL-4浓度和嗜酸性粒细胞(Eos)绝对值都是三组中最高的(P<0.01);除哮喘组外,其余组的各项指标相近(P>0.05)。IL-4 mRNA表达量与IL-4浓度、Eos绝对值呈正相关(P<0.01),与IFN-γ浓度呈负相关(P<0.01)。结论:哮喘大鼠骨髓CD34+细胞中IL-4mRNA的表达增强;孟鲁司特可以下调IL-4mRNA的表达,可能成为其抑制哮喘气道炎症形成的重要机制之一。
文摘The purpose of the study is to establish a fluorescence quantitative reverse transcription polymerase chain response （FQ-RT-PCR） method for the quantitative determination of IL-2 mRNA and IL-4 mRNA in Th cells, with which the Th cells status of the patients with gynaecological tumors and chronic renal failure （CRF） can be analyzed. IL-2 eDNA and IL-4 eDNA were prepared, and the plasmid pMD18 carrying IL-2 eDNA or IL-4 eDNA fragment was constructed and cloned as the template for quantitative determination. The primers and probes labelled with 6-carbexy-fluorescein （FAM） and 6-carboxy- tetramethylrhodamine （TAMRA） were prepared, and the experimental conditions were optimized to set up the FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA. Th cells enriched from peripheral blood mononuclear cells （PBMCs） of 20 healthy volunteers （HVs）, 16 gynaecological benign （GB） cases, 18 gynaecological malignant （GM） tumor cases and 16 chronic renal failure （CRF） patients were tested for IL-2 mRNA and IL-4 mRNA by FQ-RT-PCR. The house-keeping gene β-actin was used as the internal control gene of the experiment. The standard curve for log concentration of series of quantitative templates vs threshold cycle （CT） was established by linear regression, and the linear range was 102-107 copies/μl. The imprecision test showed the CV of inter-assay and intra-assay of a high cont- ent 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 expressions in Th of the patients with gynaecological malignant tumor （compared with the HVs and the patients with gynaecological benign disease） and in Th of the CRF patients （compared with the HVs） were declined significantly and at the same time the IL-4 mRNA expression increased significantly （ P 〈 0. 001 ）. A simple, sensitive and accurate FQ-RT-PCR method for the quantitative detection of IL-2 mRNA and IL-4 mRNA has been established. The IL-2 mRNA and IL-4 mRNA expressions in Th cells of the patients with gynaecological malignant tumor and the CRF patients were polarized and displayed Th2 response. It suggests that the function of the Th cells of the patients with gynaecological malignant tumor or CRF is at unbalance.
