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目的:探讨经PTD-HBcAg融合蛋白致敏的树突状细胞(DCs)体外诱导特异性细胞毒T淋巴细胞(CTLs)对HBV的抑制作用。方法:体外分离培养小鼠髓源性DC,加入融合蛋白刺激DC成熟后与T淋巴细胞共培养,ELISA法检测T淋巴细胞上清中IL-2、IL-4、IL-10和INF-γ的分泌水平,流式细胞术检测胞内细胞因子水平,乳酸脱氢酶释放试验检测特异性CTL活性,并对HepG2.2.15细胞上清中HBsAg及HBV DNA水平进行检测。结果:经不同融合蛋白刺激的DCs能有效促进T淋巴细胞的细胞因子分泌,同时融合蛋白PTD-HBcAg组中IL-2(552.7±117.5ng/L)和INF-γ(150.6±7.945ng/L)明显高于HBcAg组中IL-2(420±12.47ng/L)和INF-γ(107.5±12.19ng/L)分泌。流式细胞计数术检测的PTD-HBcAg融合蛋白诱导CTL细胞水平明显高于对照组。经PTD-HBcAg融合蛋白诱导的CTL比HBcAg有明显的特异性杀伤作用(P<0.05),同时对HBsAg及HBV DNA水平有明显的抑制作用。结论:经PTD-HBcAg融合蛋白致敏的DCs能有效刺激T淋巴细胞分泌细胞因子及增加细胞毒T淋巴细胞的表达,并增强特异性CTL活性及对HepG2.2.15细胞上清中HBsAg及HBV DNA水平的抑制。
Objective: To investigate the inhibitory effect of dendritic cells (DCs) primed by PTD-HBcAg fusion protein on HBV induced by specific cytotoxic T lymphocytes (CTLs) in vitro. Methods: Murine myeloid DCs were isolated and cultured in vitro. The fusion proteins were added to stimulate the maturation of DCs and co-cultured with T lymphocytes. The levels of IL-2, IL-4, IL-10 and INF-γ The levels of cytokines were measured by flow cytometry, the specific CTL activity was detected by lactate dehydrogenase release assay, and the levels of HBsAg and HBV DNA in the supernatant of HepG2.2.15 cells were detected. Results: DCs stimulated by different fusion proteins could effectively promote the cytokine secretion of T lymphocytes, and IL-2 (552.7 ± 117.5ng / L) and INF-γ (150.6 ± 7.945ng / L) in the PTD-HBcAg fusion protein group ) Was significantly higher than that of HBcAg group (420 ± 12.47ng / L) and INF-γ (107.5 ± 12.19ng / L). The level of CTL cells induced by PTD-HBcAg fusion protein detected by flow cytometry was significantly higher than that of the control group. CTL induced by PTD-HBcAg fusion protein had a significant specific killing effect on HBcAg (P <0.05), and significantly inhibited the level of HBsAg and HBV DNA. CONCLUSION: DCs primed by PTD-HBcAg fusion protein can effectively stimulate the secretion of cytokines and increase the cytotoxic T lymphocytes in T lymphocytes, enhance the specific CTL activity and inhibit the expression of HBsAg and HBV DNA in the supernatant of HepG2.2.15 cells Horizontal inhibition.