Effects of Ginkgo biloba Polysaccharides on Immune Regulation of Lymphocyte in Mice
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摘要: 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5 μg/mL的左旋咪唑）、银杏多糖处理组（终浓度为25、50、100、200、400 μg/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-γ mRNA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，200 μg/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，25~400 μg/mL银杏多糖处理组G0/G1期占比极显著降低、S期和G2/M期细胞总占比极显著增加（P<0.01）；与阳性组相比，200 μg/mL时G0/G1期占比降低、S期和G2/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、200 μg/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），400 μg/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、200 μg/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，200 μg/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G0/G1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。Abstract: 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 5 μg/mL)and ginkgo biloba polysaccharide treatment group(final concentration of 25,50,100,200,400 μg/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-γ were detected by ELISA,and the mRNA relative expression levels of IL-4 and TNF-γ were 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 200 μg/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 G0/G1 in the 25~400 μg/mL GBP group decreased significantly,while the proportion of S and G2/M cells increased significantly(P<0.01). The proportion of G0/G1 in 200 μg/mL GBP group was lower than that in the positive group,while the proportion of S and G2/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 200 μg/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 400 μg/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 200 μg/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 200 μg/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 G0/G1 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).
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