脂质体介导乙型肝炎病毒核心抗原基因转染人树突状细胞的效率  

Protein expression rate of liposome-mediated hepatitis B core gene transfected into dentritic cell

作　　者：孔练花[1] 李军[1] 韩亚萍[1] 陈念[1] 黄祖瑚[1] 

机构地区：[1]南京医科大学第一附属医院感染病科,210029

出　　处：《江苏医药》2007年第4期341-343,I0004,共4页Jiangsu Medical Journal

基　　金：江苏省医学重点学科工程资助(135-07);江苏省卫生厅医学科技发展基金(H200311);江苏省"333工程"培养资金资助项目(2002)

摘　　要：目的观察脂质体介导HBcAg基因转染来源于人外周血单个核细胞(PBMC)树突状细胞(DC)的转染效率。方法用HES离心沉淀法分离人外周血PBMC,经粒-巨细胞集落刺激因子(GM-CSF)、白细胞介素-4(IL-4)诱导培养DC,培养第5天以阳离子脂质体为载体将报告基因GFP(DNA:脂质体比例为1∶3、1∶4、1∶5、1∶6)及HBcAg基因(DNA∶脂质体1∶5)导入DC,于72h经流式细胞仪检测绿色荧光蛋白(GFP)的表达率,于48、72、96、120h流式细胞仪检测HBcAg的表达率。将基因转染后的DC与自体淋巴细胞混合培养,检测其细胞内γ干扰素(IFN-γ)、IL-4表达水平。结果倒置显微镜下观察脂质体转染后DC的的形态无明显变化。报告基因GFP与脂质体比例不同,其转染效率有差异,72h的表达率为37.12%(1∶3)、48.55%(1∶4)、52.13%(1∶5)、50.75%(1∶6)。HBcAg基因转染DC后48、72、96、120h的HBcAg表达率分别为31.58%、55.80%、54.18%、56.99%。转染后DC与自体淋巴细胞混合培养后,淋巴细胞高表达IFN-γ,较少表达IL-4,呈现Th1为优势的免疫应答。结论应用阳离子脂质体能有效的将HBcAg基因转染到人PBMC来源的DC,转染72h后抗原表达稳定;转染HBcAg基因的DC仍以诱导Th1免疫应答反应为主。Objective To evaluate the hepatitis B core antigen （HBcAg） expression efficiency in the dentritic cell （DC） derived from peripheral blood monouclear cells （PBMCs） transfected by the cation liposome bearing the hepatitis B core gene. Methods PBMCs centrifugated by the HES centrifugation were cultured and tronsfected into DC by granulocyte-macrophage colony stimulating factor （GM-CSF） and interleukin-4 （IL-4）, and their morphology and phenotype were determinated by the inverted microscope and flow cytometry, respectively. The report gene GFP （ratios of DNA to liposome are 1 ： 3, 1 ： 4, 1 ： 5 and 1 ： 6 ） and hepatitis B core gene （ ratio of DNA to liposome is 1 ： 5 ） was respectively transfected into DC on the fifth day. The expression efficiency of GFP at 72 hours and that of hepatitis B core gene at 48, 72, 96 and 120 hours were tested by flow cytometry. The levels of IFN-γ and IL-4 in cell after mixed culture of the transfected DC and lymphocytes were detected. Results The morphology of the DC transfected by the liposme was not changed significantly. The expression efficiency of GFP at 72 hours of four different DNA to liposome rates mentioned above were 37.12%, 48. 55%, 52.13% and 50. 75%,respectively. The expression rates of hepatitis B core gene at 48, 72, 96 and 120 hours were 31.58%, 55.80%, 54. 18% and 56.99%,respectively. The mixed culture of transfected DC and the self lymphocytes stimulated the high level of IFN-γ and low IL-4,and mainly Th1 response. Conclusion The hepatitis B core gene could be efficiently transfected into DC derived from PBMC by the cation liposome with stable protein expression at 72 hours after transfection. The main function of the transfected DC is to induce Type-1 immune response.

关 键 词：树突状细胞 乙型肝炎 脂质体 转染 

分 类 号：R392[医药卫生—免疫学]