摘要
目的 研究特定大小聚苯乙烯微粒作为抗原载体介导树突状细胞 (DC)摄取和提呈抗原 ,诱导体液和细胞免疫应答的能力 ,并与DC 微粒 卵清蛋白 (OVA)及DC E7抗原表位的诱导能力进行比较。方法 用粒 巨噬细胞集落刺激因子 (GM CSF)联合白细胞介素 (IL) 3从小鼠骨髓黏附细胞培养制备DC ,流式细胞术分析表型 ,细胞经荧光素异硫氰酸酯 (FITC)标记的微粒 OVA或微粒 E7饲育 ,或用OVA抗原表位 (SIIFEKL多肽 )或E7抗原表位 (RAHYNIVTF多肽 )负载 ,流式细胞术分析DC对微粒 OVA和微粒 E7的摄取能力 ,B3Z细胞活化试验检测DC提呈微粒 OVA和OVA抗原表位的能力。分别用DC 微粒 OVA、DC 微粒 E7、DC OVA抗原表位、DC E7抗原表位经双侧足掌免疫小鼠 ,36h后取髂部引流淋巴结 ,流式细胞仪分析微粒阳性细胞的表型 ,10d后用酶联免疫吸附实验 (ELISA)法测定小鼠血清的抗体水平 ,酶联免疫斑点法 (ELISPOT法 )检测小鼠脾细胞干扰素γ(IFNγ)形成单位。结果 小鼠骨髓黏附细胞在GM CSF和IL 3条件下培养 5d后大部分细胞呈树突状形态和表型 ,经微粒 OVA或微粒 E7饲育后 ,能高效地摄取和提呈抗原 ,用DC 微粒 OVA或DC 微粒 E7免疫小鼠后引流淋巴结微粒阳性细胞 ,主要组织相容 (抗原 )复合物 (MHC)
Objective To investigate the feasibility of using dendritic cell (DC) beads antigen (Ag) as novel cancer vaccine form with E7 as the target antigen Methods C57BL/6 mouse was killed and the femora were taken out Marrow cells were isolated and cultured with IL 3 and granulocyte macrophage growth stimulating factor (GM CSF) so as to prepare DCs Flow cytometry was conducted to analyze the phenotype FITC labeled polystyrene beads ovalbumin (OVA) or polystyrene beads human papilomavirus (HPV) E7 protein were added into the culture fluid to be co cultured with the DCs for 3 hours Flow cytometry was conducted to analyze the up taking rates of polystyrene beads OVA and of polystyrene beads HPV E7 protein by the DCs and B3Z T cell hybridoma cells were co cultured and then beads OVA or beads SIIFEKL polypeptide was added into the culture fluid The absorbance was read Twelve mice were injected with DC beads OVA, DC beads E7, DC OVA antigen epitope (SIIFEKL), or DC E7 epitope (RAHYNIVTF) into the plantae and killed in 36 hours to take out the iliac lymph nodes Flow cytometry was conducted to analyze the phenotypes of the bead positive cells Mice were killed 10 days after immunization and their heart blood was collected Enzyme linked immunosorbent assay was used to detect the levels of antibodies Immunized and non immunized mice were killed and their spleens were taken out ELISPOT was used to detect the number of cells secreting interferon (IFN)γ Results DCs were seen in the culture fluid of mice marrow cells 5 days after culture with IL 3 and GM CSF The bead positive rates of bead E7 and bead OVA were 64%±18% and 58%±16% respectively ( P >0 05) in the IL 3DCs The CD40 expression rate in the bead positive cells in the graining iliac lymph nodes was significantly higher after feeding by beads E7 in comparison with that before the feeding ( P <0 05), the NLDC145, and MHC II expression rates were increased to a certain degree, however the F4/80 expression rate was decreased The DCs fed with bead OVA or with OVA antigen epitope SIIFEKL, especially the former, significantly activated the B3Z cells The serum IgG level in the mice immunized by beads E7 was significantly increases, the IgM level was increased slightly, however, the IgA level almost remained unchanged The numbers of IFNγ SFU in the splenic cells of the mice immunized by DC bead OVA and DC bead E7 were significantly higher than that in the unimmunized mice, especially those immunized by DC bead OVA Conclusion DCs fed with beads E7 can migrate to the draining lymph nodes and induce high level humeral and cellular immunity DC beads Ag seems better than DC epitope according to the strength of induced immunity DC beads Ag may be an ideal vaccine form and can be used to develop other cancer vaccine
出处
《中华医学杂志》
CAS
CSCD
北大核心
2004年第11期932-936,共5页
National Medical Journal of China
基金
国家自然科学基金资助项目 ( 3 0 3 715 63 )
