传染性单核细胞增多症患儿EB病毒载量与T细胞亚群的相关性分析
发布时间：2018-08-27 06:37
【摘要】：目的：本课题通过检测传染性单核细胞增多症患儿急性期外周血T淋巴细胞亚群及调节性T细胞的变化情况,探讨CD4+CD25+CD127-调节性T细胞在传染性单核细胞增多症发病中的可能机制,同时检测T淋巴细胞亚群及调节性T细胞与EBV-DNA浓度是否有相关性。 方法：1.实验对象：47例初发住院确诊为传染性单核细胞增多症且EBV-DNA阳性的患儿作为观察组,32例为同期保健门诊的健康儿童作为对照组。各抽取外周静脉血两紫管,1个取1ml,1个取2ml,EDTA试剂抗凝。2.检测指标：A流式细胞术检测：a加抗体：于试管中分别加入CD4-PC5、CD25-FITC、CD127-PE单克隆抗体,CD4-FITC/CD8-FE/CD3-PC5三标记单克隆抗体各5ul,再在每个试管中加抗凝全血20ul,充分混匀,室温避光,孵育20分钟。b溶血：加入红细胞裂解液(溶血素)90ul,混匀,15分钟后以1000转/分离心5分钟,弃上清。c洗涤：在上述剩余液中加生理盐水,上机待检。每个样本获取10000个细胞,用流式细胞CELLQUGST软件检测CD3+、CD4+、CD8+、CD4+CD25+调节性T细胞、CD4+CD25+CD127-调节性T细胞的表达值。BEBV-DNA载量检测：采集清晨空腹外周静脉血2m1,采用ABI-7300FQ-PCR仪进行检测,检测步骤、结果判断以及质量控制方法按其公司试剂操作说明进行。检测极限为5*102copies/ul,大于或等于该值为阳性。 结果：与正常对照组相比,急性期IM患儿,CD3+、CD8+明显高于对照组(P均0.05),CD4+、CD4+/CD8+比值、CD4+CD25+、CD4+CD25+CD127-调节性T细胞的表达值明显下降,差异有统计学意义(P均0.05)。相关性结果示：在急性期EB病毒载量与CD3+CD8+T细胞均呈正相关,与CD4+、CD4+/CD8+、CD4+CD25+、CD4+CD25+CD127-调节性T细胞呈负相关。 结论：传染性单核细胞增多症患儿急性期存在细胞免疫及免疫调节功能的紊乱,其免疫功能紊乱的程度与EB病毒感染量有关,为临床IM患儿的治疗提供理论依据。调节性T细胞在IM患儿急性期减少,说明调节性T细胞可能参与了IM的发病和病情的发展。
[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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.1
本文编号：2206432
[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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.1
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