基因修饰的树突状细胞体外免疫功能的初步研究

The preliminary study on the immune function of the gene modified dendritic cells in vitro

目的制备转染乙型肝炎病毒核心抗原(HBcAg)基因、人外周血单个核细胞(PBMC)来源的树突状细胞(DC)疫苗,观察HBcAg的表达效率,检测其在体外诱导的细胞毒性T淋巴细胞(cyto-toxic T lymphocytes,CTL)对HepG2.2.15细胞的杀伤效应。方法体外诱导并培养DC;将HBcAg基因导入DC,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,MTT法检测其对HepG2.2.15细胞的杀伤毒力。结果成功培养了人外周血PBMC来源的DC,并通过脂质体的方法获得表达HBcAg的特异性DC疫苗,脂质体转染后DC的的形态未发生明显变化,CD83表达增加,与对照组比较有显著性差异。HBcAg基因转染DC后72 h的HBcAg表达率为55.8%;MTT法检测特异性细胞毒作用结果显示,试验组效靶比为10∶1、20∶1、40∶1的杀伤率分别为(62.5±4.8)%(、71.8±5.3)%、(81.5±5.0)%,与空转组、未转组比较,均有统计学差异(均P<0.05)。结论应用阳离子脂质体能有效地将HBcAg基因转染到人PBMC来源的DC,转染HBcAg基因的DC具有诱导CTL体外杀伤HepG2.2.15细胞的作用。

Objective To prepare the dendritic cell（DC） vaccine derived from peripheral blood monouclear cells（PBMC） and transfected by the cation liposome bearing the hepatitis B core gene in vitro,and to observe the inductive therapeutic effect of cytotoxic T lymphocyte（CTL） on HepG2.2.15 cells.Methods DC proliferated from human peripheral blood monocytes by adding GM-CSF and IL-4 and PBMC centrifugated by the HES centrifugation were cultured and stimulated into DC by granulocyte-macrophage colony stimulating factor（GM-CSF）,interleukin-4（IL-4）.Their morphology and phenotype were determinated by the inverted microscope and flow cytometry respectively.The hepatitis B core gene was transfected into DC on the fifth day by the liposome.The morphology and phenotype of the transfected DC were observed.The expression efficiency of hepatitis B core gene at 72 hours was tested using the flow cytometry.DC vaccine was used to induce specific CTL and subsequently co-cultured with HepG2.2.15 cells.The killing effect of CTL against HepG2.2.15 was determined using MTT method.Results The morphology of the DC transfected by the liposme did not change significantly.The expressing of CD83 in the transfected DC was higher.The expression efficiency of hepatitis B core gene at 72 hours was 55.8%.The killing effect was higher in transfected DC group than those in untransfecd and the vacancy plasmid group（P0.05）.Conclusion Genetically modified DC vaccine can successfully be prepared by liposome,and it has a significant effect on triggering of specific CTL against HepG2.2.15 cells.