摘要
目的:观察融合蛋白胞质转导肽(cytoplasmic transduction peptide,CTP)-HBcAg18-27-Tapasin体外诱导HBV转基因小鼠髓源性树突状细胞(dendritic cell,DC)成熟和对T淋巴细胞增殖的作用.方法:体外分离、培养HBV转基因小鼠及近交系C57BL/6小鼠髓源性DC,加入重组粒细胞-巨噬细胞集落刺激因子和白介素(interleukin,IL)-4培养5d,再加入实验组10μg/mL CTP-HBcAg18-27-Tapasin、对照组10μg/mL CTP-HBcAg18-27、10μg/mL HBcAg18-27-Tapasin及空白组RPMI1640完全培养液.流式细胞术测定DC表面分子CD80、CD83、MHC-1的表达,ELISA法测定DC培养上清液中的IL-12p70的水平,细胞计数试剂盒(CCK-8)检测T淋巴细胞增殖反应,流式细胞仪检测增殖的T淋巴细胞内的细胞因子.结果:体外成功诱导小鼠髓源性DC;CTP-HBcAg18-27-Tapasin能明显上调DC表面分子CD80、CD83、MHC-1的表达;并且CTP-HBcAg18-27-Tapasin组诱导DC分泌的IL-12p70水平及诱导DC增殖T淋巴细胞增殖能力明显高于对照组及空白组[IL-12p70转基因小鼠(F=205.85,P=0.000);C57BL/6小鼠(F=406.20,P=0.000)];流式细胞仪检测实验组融合蛋白诱导的CTL水平也高于对照组[转基因小鼠(F=155.45,P=0.000);C57BL/6小鼠(F=392.90,P=0.000)],同时HBV转基因小鼠DC表面分子及在T淋巴细胞增殖中的作用要比C57BL/6小鼠低.结论:分子伴侣Tapasin修饰胞内化抗原肽能促进HBV转基因小鼠髓源性DC的分化、成熟,并能增强DC刺激T淋巴细胞增殖能力及诱导CTL的产生.
AIM： To investigate the effects of cytoplasmic transduction peptide （CTP）-HBcAg18-27-Tapasin on the maturation of HBV transgenic mouse bone marrow-derived dendritic cells （DCs） and proliferation of T lymphocytes in vitro. METHODS： DCs derived from bone marrow of HBV transgenic mice or C57BL/6 mice were cultured with recombinant granulocytemacrophage colony-stimulating factor and recombinant interleukin-4 for 5 days. The fusion proteins CTP-HBcAg18-27-Tapasin （10 μg/mL）, CTP-HBcAg18-27 （10 μg/mL）, HBcAg18-27-Tapasin （10 μg/mL） or PRMI 1640 were then added into culture medium to induce DC maturation. DC phenotypes were analyzed by flow cytometry. The level of IL-12p70 in supernatants was detected by enzyme-linked immunosorbent assay. The proliferation of T lymphocytes was assessed using cell counting kit-8, and intracellular cytokines in proliferative T cells were analyzed by flow cytometry.RESULTS： DCs were cultured and identified successfully. DC surface molecules, such as CD80, CD83 and major histocompatibility anti-gen-I, were up-regulated by treatment with CTP- HBcAgzs,27-Tapasin. IL-12p70 level in cells treated with CTP-HBcAg^8_ay-Tapasin was higher than those in cells treated with CTP-HBcAg18.27, HB- cAg^Ry-Tapasin or medium. Treatment with CTP- HBcAgl^2^-Tapasin not only significantly induced T lymphocyte proliferation, but also significantly increased the percentages of IFN-γCD8＋ T cells compared to treatment with CTP-HBcAg18-27, HB- cAg18-27-Tapasin or medium. However, the effects of CTP-HBcAg18-27-Tapasin on DC maturation and T lymphocyte proliferation were more significant when DCs and T lymphocyte were derived from C57BL/6 mice.CONCLUSION： Molecular chaperone tapasinmodified intracellular antigen peptide could ef- fectively promote the differentiation and maturation of dendritic cells derived from bone marrow cells of HBV transgenic mice and enhance the ability of DCs to stimulate T lymphocyte proliferation and induce cytotoxic T lymphocytes.
出处
《世界华人消化杂志》
CAS
北大核心
2013年第2期122-129,共8页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.31000414~~
