胞质转导肽-HBcAg18-27-Tapasin体外诱导小鼠髓源性树突状细胞成熟及在T淋巴细胞增殖中的作用

Effects of cytoplasmic transduction peptide-HBcAg18-27-Tapasin induced murine bone marrow derived dendritic cell maturation on T lymphocyte proliferation in vitro

目的观察融合蛋白胞质转导肽（CTP）-HBcAg18-27-Tapasin诱导体外培养的小鼠髓源性树突状细胞（Dc）成熟和对T淋巴细胞增殖的作用。方法体外分离、培养近交系BALB／c小鼠髓源性DC，加入重组粒细胞一巨噬细胞集落刺激因子和IL-4培养5d，再加入脂多糖诱导成熟。10,ug／LCTP—HBcAg18-27-Tapasin、50,ag／LCTP—HBcAg18-27-Tapasin、10p．g／LCTP-HBcAg18-27及RPMI-1640培养液加入培养介质。流式细胞术测定DC表面分子表达，EusA法测定DC培养上清液中的IL-12p70的水平，细胞计数试剂盒检测T淋巴细胞增殖反应，流式细胞术检测增殖的T淋巴细胞内的细胞因子。多个样本均数间的比较采用单因素方差分析，组间两两比较采用LSD法。结果成功体外诱导小鼠髓源性DC；CTP—HBcAg18-27-Tapasin能明显上调DC表面分子CD80、CD86及主要组织相容性复合体-1分子的表达；509g／LCTP—HBcAg18-27-Tapasin组诱导DC分泌的IL-12p70水平为（61．12±10．25）pg／mL，依次高于10／xg／LCTP-HBcAg18-27-Tapasin组的（50．43±10．42）pg／mL、10〉g／LCTP-HBcAgl8m组的（40．17±8．54）pg／mL和空白组的（30．51±8．03）pg／mL（F=15．85，P=0．030和P=0．037）；CTP—HBcAg18-27-Tapasin诱导DC刺激T淋巴细胞增值能力明显高于对照组；流式细胞仪检测融合蛋白诱导的CTL水平50μg／LCTP-HBcAg18-27-Tapasin组为（2．05±0．41）％、10μg／LCTP-HBcAg18-27-Tapasin组为（1．06±0．10）％，高于10〉g／LCTP-HBcAg18-27组的（O．45±0．11）％和空白组的（O．09±0．02）％（F=60．22，P=0．003）。结论CTP-HBcAg18-27-Tapasin可以促进DC的分化、成熟，增强DC刺激T淋巴细胞增殖能力并能增加CTL的表达。

Objective To observe the effects of cytoplasmic transduction peptide （CTP）- HBcAg18-27-Tapasin induced murine bone marrow-derived dendritic cell （DC） maturation on T lymphocyte proliferation in vitro. Methods Bone marrow derived DC isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleutin （IL）-4 for 5 days followed by lipopolysaccharide added to induce DC maturation. 10 μg/L CTP-HBcAg18-27- Tapasin, 50μg/L CTP-HBcAg18-27-Tapasin, 10 μg/L CTP-HBcAg1827 or RPMI-1640 were added into culture medium to induce DC maturation. IX; phenotypes were analyzed by flow cytometry. The level of IL-12p70 in the supernatant was detected by enzyme linked immunosorbent assay. The proliferation ofT lymphocytes was performed by using cell counting kit-8 and intracellular cytokine of proliferative T cells were analyzed by flow cytometry. The means among groups were compared using one-way ANOVA and those between two groups were compared by least significant difference test. Results DC were cultured and induced successfully. The molecules on DC surface, such as CDS0, CD86 and major histocompatibility antigen-I were upregulated by CTP-HBcAg18-27-Tapasin. IL-12p70 level induced by 50μg/L CTP- HBcAg18-27-Tapasin was （61.12±10.25） pg/mL, which was higher than those induced by 10 μg/L CTP- HBcAg1827-Tapasin （50. 43±10.42） pg/mL, 10 μg/L CTP-HBcAg18-27 （40.17±8.54） pg/mL and medium control （30.51 ± 8.03） pg/mL （F= 15.85, P = 0. 030 and 0. 037）. The proliferation of T lymphocytes induced by CTP-HBcAg18-27-Tapasin was higher than control groups. The amounts of cytotoxic T lymphocyte （CTL） induced by 50μg/L CTP-HBcAg18-27-Tapasin [（2. 05±0.41）%] and 10 μg/L CTP-HBcAg18-27- Tapasin [-（1. 06±0.10）%] were both significantly higher than the 10 μg/L CTP-HBcAg1827 group [（0. 45±0.11）%] and medium group [（0. 09±0.02）%, F=60.22, P=0. 003]. Conclusions CTP- HBcAg1827-Tapasin could promote the differentiation and maturation of DC, and enhance the ability of DC stimulating T lymphocytes proliferation and increase CTL expression effectively.