竹节参醇提物免疫佐剂作用的实验研究

The adjuvant effect of Panax japonicus ethanol extract

目的采用高效液相色谱法(HPLC)筛选竹节参醇提物中最佳免疫佐剂活性部位,进而探讨此有效部位作为HBs Ag佐剂的作用及可能机制。方法通过HPLC初步分离和分析竹节参醇提物,MTT法检测不同浓度乙醇洗脱的竹节参皂苷中最佳佐剂活性部位,将此部位与HBs Ag共同注入到雌性Balb/c小鼠体内进行免疫,小鼠随机分为正常对照组,HBs Ag模型组和(HBs Ag+竹节参醇提物)低(300μg)、高剂量(600μg)组,末次免疫7 d后收集小鼠淋巴细胞并采集血清,CCK-8检测脾淋巴细胞增殖情况;间接ELISA法检测小鼠血清中抗体滴度;RT-PCR法检测脾淋巴细胞中相关细胞因子的表达;流式细胞仪检测细胞表面蛋白的表达。结果竹节参醇提物显著促进淋巴细胞的增殖,60%浓度的醇过大孔树脂所得竹节参皂苷对淋巴细胞的增殖最显著(P<0.01);显著增强小鼠血清中抗HBs Ag的Ig G、Ig G1和Ig G2b亚型抗体滴度;上调细胞因子IFN-γ、IL-4和IL-10的表达;增强TLR4受体的表达。结论竹节参醇提物中60%乙醇提取物具有较强的免疫佐剂作用,作为HBs Ag抗原佐剂是通过活化TLR4受体来发挥作用的。

Saponins has been developed for use as vaccine adjuvants and immunomodulators because of their ability to stimulate innate and adaptive immune responses. The main effective component of Panax japonicus is saponins, and total saponins of Panax japonicus were reported to enhance the body＇s immune function. Here, we aimed to investigate the effect and the mechanism of Panax japonicus ethanol extract as an adjuvant of HBsAg vaccine. Female Balb/c mice were immunized with saline or HBsAg antigen with or without Panax japonicus ethanol extract, respectively. On 7 days and 21 days after the immunization, the lymphocyte proliferation was determined with CCK-8 method, antibody titer in serum was detected with ELISA, the expression of cytokines were detected with RT-PCR, and cell surface receptor was evaluated by FCM. Our data showed that Panax japonicus ethanol extract enhanced lymphocyte proliferation and elevated the antibody production of specific IgG, IgG1 and IgG2b significantly. In addition, the expression of cytokines IFN-γ, IL-4 and IL-10 and surface receptor TLR4 were upregulated by Panax japonicus ethanol extract. The result suggested that Panax japonicus 60% ethanol extract has strong immune adjuvant effect, and the mechanism relates with TLR4 signal pathway.