慢性乙型肝炎患者树突状细胞培养及功能检测方法.docVIP

慢性乙型肝炎患者树突状细胞培养及功能检测方法 　【摘要】 目的: 建立从人外周血分离培养树突状细胞（dendritic cell，DC）及检测其表型与诱导淋巴细胞增殖能力的方法。方法:从未接受抗病毒治疗的CHB患者外周血中分离单个核细胞，在含GM-CSF、IL-4、TNF-α的完全培养基中诱导培养为DC，流式细胞仪检测细胞表型，混合淋巴细胞反应检测DC刺激淋巴细胞增殖的能力。结果:CHB患者外周血单核细胞，在体外可诱导出具有典型形态及生物学活性的DC。患者DC的HLA-DR、CD80、CD86的表达率分别为（44.77±8.127）%、(36.65±6.867）%、（32.16±6.340）%，CHB患者DC刺激淋巴细胞增殖的SI为1.39±0.375。与正常人DC相比明显降低（Plt;0.01）。结论:CHB患者单个核细胞能被诱导培养为DC；通过检测DC表型和刺激淋巴细胞增殖能力可以了解CHB患者抗HBV免疫功能。 【关键词】 树突状细胞 慢性乙型肝炎 免疫状态 Abstract Objective：To establish the method of culturing DC from human peripheral blood and testing the phenotype and the capacity of stimulating leukocyte. Methods：Selecting CHB without receiving antiviral treatment,PBMCs were separated from peripheral blood，and then cultured in vitro with medium containing GM-CSF IL-4 TNF-αinto DC. The phenotype of DC was identified by flow cytometry. The abilities to stimulate T cell proliferate were detected by mixed leukocytes reaction (MLR). Results: PBMC from CHB patients which were cultured in vitro could be induced into typical DC. The expression of costimulating molecules CD80、CD86、HLA-DR in the surface of DCs from CHB patients before antiviral treatment were (32.16±6.340)%、(36.65±6.867)%、(44.77±8.127)%.The stimulation index (SI) that DC stimulate lymphocytes to proliferate was 1.39±0.375.Conclusion:The PBMC from CHB patients could be induced into DC. Testing the phenotype and the capacity of stimulating leukocyte of DC from CHB can be known the ability of antiviral immunity. Key words Dendritic cell；Chronic hepatitis B；Immunitic situation 机体特异性免疫反应的产生，是由抗原提呈细胞（APC）捕获抗原，在其对抗原处理后将抗原信息提呈T、B淋巴细胞后引发的。树突状细胞（dendritic cell，DC）是功能最强大的专职的抗原提呈细胞，在机体的免疫反应中起着重要作用［１，２］。HBV持续感染与多方面因素有关，其中患者DC功能低下，抗原提呈能力下降，不能正常诱导机体抗HBV免疫反应是最重要因素之一。HBV持续感染患者仅产生弱的HBV特异的细胞毒性淋巴细胞反应（CTLs），导致HBV病毒不能清除和慢性乙型肝炎的迁延不愈。检测和通过治疗改善患者的DC功能在乙肝免疫治疗中有重要意义。本实验拟建立从人外周血分离培养DC及检测其表型与诱导淋巴细胞增殖能力的方法，并对HBV 感染的慢性肝炎患者体内DC功能进行了检测。 1 材料与方法 1.1 病例选择 选择慢性乙型病毒肝炎10例，男7例，女3例，年龄18岁～50岁，血清丙氨酸转氨酶（ALT）在69 U/L～515 U/L，平均值为182 U/L，6个月内均未使用过抗病毒药物，所有病例参照2005年慢