【摘要】：目的：本課題通過檢測傳染性單核細(xì)胞增多癥患兒急性期外周血T淋巴細(xì)胞亞群及調(diào)節(jié)性T細(xì)胞的變化情況,探討CD4+CD25+CD127-調(diào)節(jié)性T細(xì)胞在傳染性單核細(xì)胞增多癥發(fā)病中的可能機(jī)制,同時檢測T淋巴細(xì)胞亞群及調(diào)節(jié)性T細(xì)胞與EBV-DNA濃度是否有相關(guān)性。 方法：1.實驗對象：47例初發(fā)住院確診為傳染性單核細(xì)胞增多癥且EBV-DNA陽性的患兒作為觀察組,32例為同期保健門診的健康兒童作為對照組。各抽取外周靜脈血兩紫管,1個取1ml,1個取2ml,EDTA試劑抗凝。2.檢測指標(biāo)：A流式細(xì)胞術(shù)檢測：a加抗體：于試管中分別加入CD4-PC5、CD25-FITC、CD127-PE單克隆抗體,CD4-FITC/CD8-FE/CD3-PC5三標(biāo)記單克隆抗體各5ul,再在每個試管中加抗凝全血20ul,充分混勻,室溫避光,孵育20分鐘。b溶血：加入紅細(xì)胞裂解液(溶血素)90ul,混勻,15分鐘后以1000轉(zhuǎn)/分離心5分鐘,棄上清。c洗滌：在上述剩余液中加生理鹽水,上機(jī)待檢。每個樣本獲取10000個細(xì)胞,用流式細(xì)胞CELLQUGST軟件檢測CD3+、CD4+、CD8+、CD4+CD25+調(diào)節(jié)性T細(xì)胞、CD4+CD25+CD127-調(diào)節(jié)性T細(xì)胞的表達(dá)值。BEBV-DNA載量檢測：采集清晨空腹外周靜脈血2m1,采用ABI-7300FQ-PCR儀進(jìn)行檢測,檢測步驟、結(jié)果判斷以及質(zhì)量控制方法按其公司試劑操作說明進(jìn)行。檢測極限為5*102copies/ul,大于或等于該值為陽性。 結(jié)果：與正常對照組相比,急性期IM患兒,CD3+、CD8+明顯高于對照組(P均0.05),CD4+、CD4+/CD8+比值、CD4+CD25+、CD4+CD25+CD127-調(diào)節(jié)性T細(xì)胞的表達(dá)值明顯下降,差異有統(tǒng)計學(xué)意義(P均0.05)。相關(guān)性結(jié)果示：在急性期EB病毒載量與CD3+CD8+T細(xì)胞均呈正相關(guān),與CD4+、CD4+/CD8+、CD4+CD25+、CD4+CD25+CD127-調(diào)節(jié)性T細(xì)胞呈負(fù)相關(guān)。 結(jié)論：傳染性單核細(xì)胞增多癥患兒急性期存在細(xì)胞免疫及免疫調(diào)節(jié)功能的紊亂,其免疫功能紊亂的程度與EB病毒感染量有關(guān),為臨床IM患兒的治療提供理論依據(jù)。調(diào)節(jié)性T細(xì)胞在IM患兒急性期減少,說明調(diào)節(jié)性T細(xì)胞可能參與了IM的發(fā)病和病情的發(fā)展。
[Abstract]:Objective: To explore the possible mechanism of CD4+CD25+CD127-regulatory T cells in the pathogenesis of infectious mononucleosis by detecting the changes of T lymphocyte subsets and regulatory T cells and EBV-DNA levels in peripheral blood of children with infectious mononucleosis at acute stage. Is there any correlation?
Methods: 1. Experimental subjects: 47 children with infectious mononucleosis and positive EBV-DNA were selected as observation group and 32 healthy children as control group. Two purple tubes of peripheral venous blood were taken from each group, one was taken from 1 ml, one was taken from 2 ml, and EDTA reagent was used for anticoagulation. Antibody: Add CD4-PC5, CD25-FITC, CD127-PE monoclonal antibody, CD4-FITC/CD8-FE/CD3-PC5 tri-labeled monoclonal antibody 5 UL in test tube, then add 20 UL of anticoagulant whole blood in each test tube, mix well, avoid light at room temperature, incubate for 20 minutes. B hemolysis: Add erythrocyte lysate (hemolysin) 90 ul, mix well, 15 minutes later, 1000 revolutions / heart separation. 5 minutes, discard supernatant. C washing: add normal saline to the remaining solution, go on the machine to be checked. Each sample obtained 10,000 cells, CD3 +, CD4 +, CD8 +, CD4 + CD25 + regulatory T cells, CD4 + CD25 + CD127 - regulatory T cells expression value. BEBV - DNA load detection: collection of fasting peripheral venous blood 2m1, using flow cytometry CELLQUGST software to detect CD3 +, CD4 +, CD8 +, CD4 + CD25 + regulatory T cells, CD4 + CD127 - regulatory T cells. BI-7300FQ-PCR is used to detect, detect, judge the results and control the quality according to the company's reagent instructions. The detection limit is 5*102 copies/ul, greater than or equal to the positive value.
Results: Compared with the normal control group, the expression of CD3 +, CD8 +, CD4 +, CD4 +, CD4 + / CD8 +, CD4 + CD25 +, CD4 + CD25 + CD127 - regulatory T cells in the acute phase of IM patients were significantly higher than those in the control group (P 0.05). The correlation results showed that the load of EB virus and CD3 + CD8 + T cells were positively correlated in the acute phase, and CD4 + CD25 + CD127 - regulatory T cells were significantly lower than those in the control group (P 0.05). CD4+, CD4+/CD8+, CD4+CD25+ and CD4+CD25+CD127- showed negative correlation with regulatory T cells.
CONCLUSION: There are disorders of cellular immunity and immune regulation in children with infectious mononucleosis in the acute phase. The degree of immune dysfunction is related to the amount of EBV infection, which provides theoretical basis for the treatment of IM. The development of the disease.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R725.1

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