黄芪多糖诱导成熟的树突状细胞肿瘤疫苗体外抗肿瘤作用的实验研究

In vitro Anti-tumor Effect of Human Dendritic Cells Vaccine Induced by Astragalus Polysacharin:an Experimental Study

目的探讨经黄芪多糖(astragalus polysacharin,APS)诱导成熟的树突状细胞(dendritic cell,DC)肿瘤疫苗体外抗肿瘤作用及其机制。方法分离人外周血单个核细胞(peripheral blood mononuclear cell,PBMC),制备DC细胞,加入营养液体外诱导为未成熟的DC细胞,培养第5天,黄芪多糖组加入终浓度为100μg/mL的黄芪多糖,细胞因子组加入终浓度为20 ng/mL重组人肿瘤坏死因子(rhTNF)-α诱导DC成熟,观察树突状细胞形态及表型变化;成熟DC以SGC-7901肿瘤抗原致敏,与同种异体T细胞共培养,采用MTT法检测T淋巴细胞增殖功能,E LISA法检测共培养上清IL-12、IFN-γ水平;经DC激活的细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)细胞与SGC-7901肿瘤细胞共培养,采用LDH释放法检测CLT对靶细胞特异性杀伤能力。结果黄芪多糖诱导的DC经形态学观察和表型鉴定,符合成熟DC的特征。黄芪多糖诱导成熟的DC能刺激同种异体T淋巴细胞增殖,T细胞增殖指数随刺激细胞与效应细胞比值的增加而增加(P<0.05);DC与T细胞共培养上清IL-12、IFN-γ的水平显著增加且呈时间依赖性(P<0.05);经致敏DC激活的CTL能显著杀伤肿瘤细胞,随着效靶比的增加,杀伤作用增强。结论黄芪多糖可在体外诱导DC成熟,并促进其抗原递呈能力,有效活化CTL,增强机体抗肿瘤功能。

Objective To explore the in vitro anti-tumor effect and mechanism of dendritic cell（DC） tumor vaccine induced by astragalus polysacharin（APS）.Methods Peripheral blood mononuclear cells（PBMCs） isolated from human peripheral blood.DCs obtained from human peripheral blood were cultivated and added with culture solution for in vitro inducing them to immature DCs.On the 5th day of culture,100 μg/mL（as the final concentration） APS was added to cells in the APS group.DCs were induced to mature in the cytokine groups by adding 20 ng/mL rhTNF-α（as the final concentration）.Changes of morphology and phenotype of DCs were observed.Mature DCs were sensitized with tumor antigen SGC-7901 and co-cultured with allogeneic T cells.The proliferative function of T lymphocytes was detected by MTT assay.Levels of IL-12 and IFN-7 in co-cultured supernatant were detected by ELISA.Cytotoxic lymphocytes（CTL） activated by DC were co-cultured with tumor cell SGC-7901.The specific killing capacity of CTL to target cells was detected by LDH release assay.Results The morphological observation and phenotypic identification of APS induced DCs were in accordance with the characteristics of mature DCs.APS induced mature DCs could stimulate the proliferation of allogeneic T lymphocytes.The proliferation index of T cells increased with increased ratio of stimulator cells to effector cells（P 0.05）.Levels of IL-12 and IFN-γ in co-culture supernatant significantly increased in a time-dependent manner（P〈0.05）.CTL cells activated by sensitization of DCs could significantly kill tumor cells,and the killing effect increased along with increased effector-to-target ratio.Conclusion APS could in vitro induce DCs to mature,promote its antigen-presenting capacity,effectively activate CTLs,and enhance anti-tumor function of the organism.