银杏多糖对小鼠淋巴细胞免疫调节作用的研究

Effects of Ginkgo biloba Polysaccharides on Immune Regulation of Lymphocyte in Mice

为研究银杏多糖对小鼠淋巴细胞的免疫调节作用,无菌分离小鼠脾淋巴细胞,制备淋巴细胞悬液,实验设空白组、阳性组(终浓度为5μg/mL的左旋咪唑)、银杏多糖处理组(终浓度为25、50、100、200、400μg/mL)。采用MTT法检测银杏多糖对淋巴细胞增殖的影响,流式细胞术检测银杏多糖对淋巴细胞周期的影响,ELISA法检测银杏多糖对淋巴细胞白细胞介素-4(IL-4)、干扰素-γ(IFN-γ)分泌量的影响,qRT-PCR法检测银杏多糖对淋巴细胞IL-4、IFN-γmRNA表达的影响。结果显示,与空白组相比,银杏多糖处理组和阳性组的淋巴细胞增殖指数均极显著高于空白组(P<0.01);与阳性组相比,200μg/mL银杏多糖处理组的淋巴细胞增殖指数低于阳性组,但无统计学意义(P>0.05)。与空白组相比,25~400μg/mL银杏多糖处理组G 0/G 1期占比极显著降低、S期和G 2/M期细胞总占比极显著增加(P<0.01);与阳性组相比,200μg/mL时G 0/G 1期占比降低、S期和G 2/M期细胞总占比极显著升高(P<0.01)。与空白组相比,阳性组、25、100、200μg/mL银杏多糖处理组的IFN-γ/IL-4比值均极显著低于空白组(P<0.01),400μg/mL银杏多糖处理组的IFN-γ/IL-4比值极显著高于空白组(P<0.01);与阳性组相比,100、200μg/mL银杏多糖处理组IFN-γ/IL-4比值低于阳性组,但无统计学意义(P>0.05)。与空白组相比,银杏多糖处理组和阳性组的细胞因子IFN-γ、IL-4分泌量及其mRNA相对表达量均极显著高于空白组(P<0.01);与阳性组相比,200μg/mL银杏多糖处理组的细胞因子IFN-γ、IL-4分泌量及其mRNA相对表达量低于阳性组,但无统计学意义(P>0.05)。由此可见,银杏多糖可以通过增加小鼠脾淋巴细胞增殖、减少G 0/G 1期细胞的阻滞、促进DNA合成、促进细胞因子IFN-γ、IL-4的分泌及其mRNA的表达、维持IFN-γ/IL-4(Th1/Th2)动态平衡,来提高机体免疫功能。

In order to study the immunomodulative effect of ginkgo biloba polysaccharide(GBP)on mouse lymphocytes,lymphocyte suspension was prepared by sterile isolation of mouse spleen lymphocytes.Blank group,positive group(levamisole with final concentration of 5μg/mL)and ginkgo biloba polysaccharide treatment group(final concentration of 25,50,100,200,400μg/mL)were set up for the experiment.The effects of the GBP on the lymphocyte proliferation were detected by MTT assay,the cell cycle were detected by flow cytometry,the secretion of cytokines IL-4 and TNF-γwere detected by ELISA,and the mRNA relative expression levels of IL-4 and TNF-γwere detected by qRT-PCR.The results showed that the proliferation index of lymphocytes in GBP treatment group and the positive group was significantly higher than that in the blank group(P<0.01).Compared with the positive group,lymphocyte proliferation index in the 200μg/mL GBP treatment group was lower than that in the positive group,but there was no statistical significance(P>0.05).Compared with the blank group,the proportion of G 0/G 1 in the 25~400μg/mL GBP group decreased significantly,while the proportion of S and G 2/M cells increased significantly(P<0.01).The proportion of G 0/G 1 in 200μg/mL GBP group was lower than that in the positive group,while the proportion of S and G 2/M cells was higher than that in the positive group,with statistical significance(P<0.01).Compared with the blank group,the IFN-γ/IL-4 ratio of the 25、100 and 200μg/mL GBP treatment group was significantly lower than that of the blank group(P<0.01),and the IFN-γ/IL-4 ratio of the 400μg/mL GBP treatment group was significantly higher than that of the blank group(P<0.01).Compared with the positive group,the IFN-γ/IL-4 ratio of the 100 and 200μg/mL GBP treatment group was lower than the positive group,but there was no statistical significance(P>0.05).Compared with the blank group,the secretion of cytokines IFN-γ、IL-4 and its mRNA expression in the GBP treatment group and the positive group were significantly higher than those in the blank group(P<0.01).Compared with the positive group,the secretion of cytokines IFN-γ、IL-4 and its mRNA expression in the 200μg/mL GBP treatment group were lower than those in the positive group,but there was no statistical significance(P>0.05).Therefore,ginkgo biloba polysaccharides can improve the immune function of mice by increasing the proliferation of spleen lymphocytes,reducing G 0/G 1 cell block,promoting DNA synthesis,promoting the secretion of cytokines IFN-γ、IL-4 and its mRNA expression,and maintaining the dynamic balance of IFN-γ/IL-4(Th1/Th2).