Cao Li-Zhen, Lin Zhi-Bin
Department of Pharmacology, Health Science Center, Peking University, Beijing 100083, China.
Acta Pharmacol Sin. 2003 Apr;24(4):321-6.
To study the regulatory effects of Ganoderma lucidum polysaccharides (Gl-PS) on cytotoxicity and mechanism of specific cytotoxic T-lymphocytes (CTL) induced by dendritic cells (DC) in vitro during the stage of antigen presentation.
Cultured murine bone marrow-derived DC were pulsed with P815 tumor cell lysates and co-incubated with or without various concentrations of Gl-PS (0.8, 3.2, or 12.8 mg/L) at the same time. P815 specific CTL were induced by spleen lymphocytes stimulated with mature DC. Non-adherent cells and culture supernatants were harvested on d 5 for analysis of specific cytotoxicity with lactate dehydrogenase (LDH) activity assay, mRNA expression of IFNgamma, granzyme B with RT-PCR assay, and protein expression of IFNgamma, granzyme B with ELISA or Western blot assay, respectively.
Three concentrations of Gl-PS promoted LDH activities released into culture supernatants (P<0.01). It also increased mRNA expression of IFNgamma in CTL (Gl-PS 12.8 mg/L vs RPMI medium 1640, P<0.05) and granzyme B in CTL (P<0.01). Protein production of IFNgamma in culture supernatants (P<0.05) and protein expression of granzyme B in CTL (Gl-PS 12.8 mg/L vs RPMI medium 1640, P<0.05) were also augmented by Gl-PS.
Gl-PS is shown to promote the cytotoxicity of specific CTL induced by DC which were pulsed with P815 tumor antigen during the stage of antigen presentation, and the mechanism of cytotoxicity is believed to be going through IFNgamma and granzyme B pathways.
研究灵芝多糖（Gl-PS）在体外抗原呈递阶段对树突状细胞（DC）诱导的特异性细胞毒性T淋巴细胞（CTL）细胞毒性的调节作用及其机制。
用P815肿瘤细胞裂解物刺激培养的小鼠骨髓来源的DC，并同时与不同浓度（0.8、3.2或12.8 mg/L）的Gl-PS共同孵育或不与Gl-PS共同孵育。用成熟DC刺激脾淋巴细胞诱导产生P815特异性CTL。在第5天收集非贴壁细胞和培养上清液，分别用乳酸脱氢酶（LDH）活性测定法分析特异性细胞毒性，用RT-PCR法检测IFNγ、颗粒酶B的mRNA表达，用ELISA或Western blot法检测IFNγ、颗粒酶B的蛋白表达。
三种浓度的Gl-PS均促进了释放到培养上清液中的LDH活性（P<0.01）。它还增加了CTL中IFNγ的mRNA表达（Gl-PS 12.8 mg/L与RPMI 1640培养基相比，P<0.05）以及CTL中颗粒酶B的mRNA表达（P<0.01）。Gl-PS还增强了培养上清液中IFNγ的蛋白产生（P<0.05）以及CTL中颗粒酶B的蛋白表达（Gl-PS 12.8 mg/L与RPMI 1640培养基相比，P<0.05）。
结果表明，Gl-PS可促进在抗原呈递阶段用P815肿瘤抗原刺激的DC诱导的特异性CTL的细胞毒性，其细胞毒性机制可能通过IFNγ和颗粒酶B途径。
