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.
