慢性乙型肝炎患者外周血树突细胞的体外扩增及鉴定  

Proliferation and Identification of Peripheral Blood Dendritic Cells in Patients with Chronic Hepatitis B in vitro

作　　者：周雨迁[1] 詹俊[1] 邓庆丽[1] 黄志清[1] 邵静[1] 智慧[1] 黄志明[1] 

机构地区：[1]中山医科大学孙逸仙纪念医院医学中心,广州510120

出　　处：《胃肠病学和肝病学杂志》2001年第3期256-260,共5页Chinese Journal of Gastroenterology and Hepatology

基　　金：97 3分课题资助项目 (G1 9990 541 0 5)

摘　　要：目的　尝试从慢性乙型肝炎患者外周血扩增培养出大量树突细胞 ,为进一步临床应用树突细胞治疗慢性乙型肝炎提供物质基础。方法　分别取健康人和患者外周血分离培养单核细胞 ,加入rhGM CSF、rhIL 4,经 7天培养后收获即为不完全成熟树突细胞 (DC)。以倒置显微镜观察摄影、透射电镜摄影、流式细胞仪测定表面特异性分子的表达、混合淋巴细胞反应测定功能 ,对DC进行鉴定。结果　第七天记数有明显树突状突起的大个细胞或细胞团 ,产率为 0 2～ 4 5× 10 5/6ml血 ;透射电镜可见细胞表面伸展的突起 ;70 %以上细胞有DC特异性标志CD1a高表达 ,而单核细胞特异性标志CD14表达小于 4% ;能刺激同种异体淋巴细胞强烈增殖 ,随刺激细胞数目的增加 ,其促增殖作用增强 ,P <0 0 1。结论　在本实验条件下 ,从慢性乙型肝炎患者外周血单核细胞可诱导扩增出大量DC。Aim To proliferate and identify dendritic cells from the peripheral blood of patients with chronic hepatitis B.Methods Peripheral blood mononuclear cells (PBMC) were isolated from normal donors and patients by centrifugation in Ficoll Hypaque.PBMC were cultured in 24 holes plate for two hours to separate monocytes and lymphocytes.The adherent monocytes were cultured in RPMI 1640 medium supplemented with rhGM CSF and rhIL 4 After 7 days culture harvest the DC.Taking photographs with invert microscope and electron microscope,analysing the phenotype of DC by flow cytometry,assaying the function of DC with mixed lymphocyte reaction(MLR) Results After 7 days culture,a lot of big cells with long process were seen under invert microscope,and many mitochondrion in cytoplasm under electricity scope.This method proliferated DC 0 2～4.5×10 5/6 ml blood and held 2%～9% of PBMC.More than 85% cells highly expressed MHC Ⅰ?MHC Ⅱ?CD40 and CD80 on the surface,and CD1a, the specific symbol of DC,expressed highly more than 70%,but CD14 less than 4%,which is the specific symbol of monocyte.The cells can stimulate vigorous proliferation of allogenic lymphocytes.With the DC number increased,proliferation of lymphocytes enhanced correlatively Conclusion Under this condition,a lot of DC s can be proliferated from the peripheral blood mononuclear cells of patients with chronic hepatitis B.

关 键 词：树突细胞 乙型肝炎细胞 体外扩增 鉴定 

分 类 号：R512.62[医药卫生—内科学]