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Fluorogenic quantitative PCR method for detecting perforin gene expression in peripheral-blood under physiological state

A gene expression and perforin technology, which is applied in the fields of sports training and sports medicine, can solve the problems of lack of accurate and effective detection methods for the determination of immunity level.

Inactive Publication Date: 2005-06-22
SHANGHAI UNIV OF SPORT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] To sum up, at present, there is a lack of accurate and effective detection methods for the determination of the level of immunity under physiological conditions, resulting in the inability to effectively avoid the decline in the body's immune function caused by excessive exercise load training in physical training

Method used

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  • Fluorogenic quantitative PCR method for detecting perforin gene expression in peripheral-blood under physiological state
  • Fluorogenic quantitative PCR method for detecting perforin gene expression in peripheral-blood under physiological state
  • Fluorogenic quantitative PCR method for detecting perforin gene expression in peripheral-blood under physiological state

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Embodiment 1: primer design and quantitative PCR product detection method

[0126] 1.1 Materials

[0127] Lymphocytes come from healthy people. Fetal bovine serum and DMEM culture medium are all products of GIBCO Company. The classical RNA extraction kit was provided by Shanghai Shenergy Biotechnology Co., Ltd. Revert Aid TMThe first-strand cDNA synthesis kit was purchased from MBI Fermentas, Lithuania. The PCR primers and fluorescein-labeled probes were synthesized by Shanghai Shenyou Biotechnology Co., Ltd. 10mM dNTP, Pfu high-fidelity DNA polymerase (5Unit / μl) was purchased from Shanghai Shenergy Bocai Company. PCR product sequencing was completed by Shanghai Shenyou Company.

[0128] 1.2 Method

[0129] According to the gene sequences of IL-2 (v00564), IL-4 (M13982), IL-10 (M57627), IFN-γ (X13274), PFR (NM_005041) and GAPDH (M33197) published in the Genbank database, using The primer design software provided by Parkson Company designed 6 sets of primers re...

Embodiment 2

[0152] Example 2: Expression of IFN-γ, IL-2, IL-4, IL-10 and PFR genes in healthy human peripheral blood leukocytes under physiological conditions

[0153] 2.1 Materials

[0154] 25 healthy volunteers come from the 2003 freshmen of Shanghai Institute of Physical Education. TriBlue RNA extraction kit and glycogen were purchased from Shanghai Shenneng Bocai Biotechnology Co., Ltd. Revert Aid TM The first-strand cDNA synthesis kit was purchased from MBI Fermentas, Lithuania. Taq DNA polymerase was purchased from Shanghai Shenyou Company.

[0155] 2.2 Method

[0156] RNA extraction and reverse transcription into cDNA

[0157] Take 2ml of heparin-anticoagulated whole blood, dissolve the red blood cells in the whole blood, prepare white blood cell pellet and store at -20°C. Extract the RNA according to the requirements of the kit.

[0158] Take 2 μg RNA to synthesize cDNA according to the requirements of the kit, and the reaction volume is 20 μl.

[0159] Quantitative PCR de...

Embodiment 3

[0171] Example 3: Expression of IFN-γ, IL-2, IL-4, IL-10 and PFR Genes in Peripheral Blood Leukocytes of Patients with Pulmonary Diseases

[0172] 3.1 Materials

[0173] Twenty-five patients with lung diseases were inpatients from Shanghai Pulmonary Hospital. TriBlue RNA extraction kit and glycogen were purchased from Shanghai Shenneng Bocai Biotechnology Co., Ltd. Revert Aid TM The first-strand cDNA synthesis kit was purchased from MBIFermentas, Lithuania. Taq DNA polymerase was purchased from Shanghai Shenyou Company.

[0174] 3.2 Method (same as the method in Example 2)

[0175] RNA extraction and reverse transcription into cDNA

[0176] Take 2ml of heparin-anticoagulated whole blood, dissolve the red blood cells in the whole blood, prepare white blood cell pellet and store at -20°C. Extract the RNA according to the requirements of the kit. Then take 2 μg RNA to synthesize cDNA according to the requirements of the kit, and the reaction volume is 20 μl.

[0177] Quan...

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Abstract

The invention provides a method for performing quantitative determination to immunity-relating cytokine in peripheral blood, the method can be applied for specifically detect the cytokine gene expression (PFR) from 2ml of peripheral blood, and no any stimulation is needed before detection. The method can detect the cytokine gene expression under physiologic status, thus can be used for determining early stage fatigue, preventing decrease of body immunity function caused by exercises of excess sport load. The invention also provides a corresponding detection kit.

Description

technical field [0001] The present invention relates to the fields of physical training and sports medicine. Specifically, the present invention relates to a method for quantitatively detecting immune-related cytokines in peripheral blood. This method can specifically detect the gene expression of cytokines (perforin, referred to as PFR) directly from 2ml of peripheral blood, and without any stimulation before the detection, the expression of the above cytokine genes can be directly detected under the physiological state, thereby using It can be used to judge early fatigue and avoid the decline of immune function caused by excessive exercise load training. Background technique [0002] Human helper T cells (Th) contain two subgroups that mediate cellular immunity and humoral immunity, namely Th1 and Th2. Th1 cells mainly secrete IFN-γ, IL-2 and TNF-α to regulate cellular immune function; Th2 cells mainly secrete IL-4, IL-5 and IL-10 to regulate humoral immune function. TH...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12Q1/68G01N33/53
Inventor 陈佩杰董强刚王茹
Owner SHANGHAI UNIV OF SPORT
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