口服黄芪多糖和人参茎叶皂苷增强口蹄疫疫苗及肠道黏膜免疫的研究

发布时间：2018-01-23 12:27

本文关键词： 黄芪多糖人参茎叶皂苷口蹄疫疫苗肠上皮内淋巴细胞黏膜免疫　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：疫苗免疫是养殖业控制动物疫病的主要措施之一。但现代畜牧业由于集约化饲养以及应激等因素可导致动物免疫功能下降,从而使动物对疫苗免疫的应答能力降低,引起免疫失败。补气类中药具有提高动物免疫机能的作用。人参和黄芪是重要的补气中药,多糖和皂苷是补气类中药的重要化学部位,本文以小鼠为实验动物,研究口服黄芪多糖、人参茎叶皂苷对口蹄疫(foot-and-mouth diseases,FMD)疫苗免疫的影响。由于大多数中药的给药途径是口服,给药后药物直接作用于消化道,近年来研究发现,肠道黏膜免疫系统对于维持机体消化器官完整性和发挥局部免疫调节作用都至关重要,因此,口服黄芪多糖、人参茎叶皂苷对肠道黏膜免疫的影响也是本研究的重点。研究结果可为采用中药多糖和皂苷提高动物免疫机能提供依据,并为探讨它们的免疫增强作用机理提供参考。1 口服黄芪多糖对口蹄疫疫苗免疫的影响为了探究口服黄芪多糖对口蹄疫疫苗免疫效果的影响,将24只小鼠按体重随机分为4组,每组6只。第1组为生理盐水对照,第2～4组每只小鼠每天口服含25 mg、50mg、100mg黄芪多糖的水溶液,连续4天。最后一次给药后24 h,每只小鼠在腹股沟部皮下注射FMD疫苗(OS/99+OZK/93株)0.2mL,2周后加强免疫一次。二免后1-5周采集血液分离血清,用间接ELISA法检测FMD特异性IgG及其亚类。结果表明:二免后3周,与对照组相比口服25 mg黄芪多糖可显著提高小鼠血清FMD特异性IgG、IgG亚类水平(P＜0.05),说明口服黄芪多糖对FMD疫苗免疫具有增强作用。2 口服黄芪多糖对脾淋巴细胞增殖及肠道黏膜免疫的影响为了探究口服黄芪多糖(astragalus polysaccharides,APS)对脾脏淋巴细胞增殖及肠道黏膜免疫的影响,将40只小鼠随机分为2组,每组20只。实验组每只小鼠口服含25 mg黄芪多糖水溶液,连续4天,对照组口服生理盐水为对照。用药后第0、1、2、3周,每组处死5只小鼠,收集脾脏制备脾淋巴细胞悬液进行脾淋巴细胞增殖实验(MTT法);采集十二指肠组织,采用免疫组化方法检测肠黏膜上皮内淋巴细胞和固有层IgA+细胞。结果显示:口服黄芪多糖后1、2周,小鼠脾淋巴细胞对刀豆蛋白A(conconavalin A,ConA)和脂多糖(lipopolysaccharide,LPS)刺激引起的增殖反应显著高于对照组(P0.05);口服黄芪多糖后0、1、2周,小鼠十二指肠黏膜上皮内淋巴细胞及固有层IgA+细胞数显著高于对照组(P＜0.05),说明口服黄芪多糖能够促进机体的细胞免疫反应和肠道黏膜免疫反应。3 口服人参茎叶皂苷对口蹄疫疫苗免疫的影响为了探究人参茎叶皂苷对口蹄疫疫苗免疫的影响,将24只小鼠根据体重随机分为4组,每组6只。第1组口服生理盐水为对照,第2～4组每只小鼠每天口服含0.05 mg、0.5 mg、5 mg人参茎叶皂苷的水溶液,连续4天。最后一次给药后24h,每只小鼠在腹股沟部皮下注射FMD疫苗(OS/99+OZK/93株)0.2 mL,2周后加强免疫一次。二免后5周内每周采血,分离血清,间接ELISA法检测FMD特异性IgG及其亚类。结果表明:二免后3周,与对照组相比口服0.5 mg人参茎叶皂苷可显著提高小鼠血清口蹄疫特异性IgG、IgG亚类水平(P＜0.05),说明口服人参茎叶皂苷能够显著提高小鼠对口蹄疫疫苗的免疫反应。4 口服人参茎叶皂苷对脾淋巴细胞增殖及肠道黏膜免疫的影响为了探究口服人参茎叶皂苷对脾脏淋巴细胞增殖及肠道黏膜免疫的影响,将40只小鼠随机分为2组,每组20只。实验组小鼠口服含0.5 mg人参茎叶皂苷的水溶液,连续4天,对照组口服生理盐水。用药后第0、1、2、3周,每组处死5只小鼠,收集脾脏,制备脾淋巴细胞悬浮液,用于脾淋巴细胞增殖实验(MTT法);同时采集十二指肠肠段,采用免疫组化方法检测肠粘膜上皮内淋巴细胞和固有层IgA+细胞。实验结果表明口服0.5 mg人参茎叶皂苷后1、2周,小鼠脾脏淋巴细胞对Con A和LPS刺激引起的增殖反应显著高于对照组(P0.05);给药后0、1、2周,小鼠十二指肠黏膜上皮内淋巴细胞及固有层IgA+细胞数显著高于对照组(P＜0.05),说明口服人参茎叶皂苷能够促进机体脾脏细胞的增殖功能,增强机体的细胞免疫和体液免疫应答,同时也能增强肠道黏膜免疫。5黄芪多糖和人参茎叶皂苷免疫增强作用的比较研究为了比较口服黄芪多糖和人参茎叶皂苷对口蹄疫疫苗免疫的增强作用,将24只小鼠根据体重随机分为4组,每组6只。第1-3组每只小鼠分别口服生理盐水、含25mg黄芪多糖的水溶液、含0.5mg人参茎叶皂苷的水溶液,连用4天。给药后24小时,第1～3组小鼠每只腹股沟皮下注射O型FMD灭活疫苗(O/My98/XJ/2010株+O/GX/09-7株)200μL,间隔2周后加强免疫一次。第4组不免疫作阴性对照。各组小鼠于二免后1、2、3周采集血液,检测血清特异性IgG、IgG滴度及IFN-y水平。此外,二免后3周处死小鼠,采集十二指肠黏膜组织,采用RT-PCR方法检测肠道黏膜相关免疫基因mRNA表达。结果表明,口服黄芪多糖和人参茎叶皂苷能显著提升血清特异性IgG及IgG滴度水平,上调肠道黏膜组织趋化因子(CCL25、CCL28)、趋化因子受体(CCR9、CCR10)、CD80、CD86、MHC-Ⅱ、TLR4 及 NF-κB p65mRNA 表达水平,提示口服黄芪多糖和人参茎叶皂苷通过上调肠黏膜相关免疫基因mRNA表达,促进肠道上皮内淋巴细胞(IELs)和固有层IgA分泌型浆细胞归巢至肠黏膜上皮及固有层,同时能够促进肠黏膜树突状细胞的成熟,增强机体对抗原的递呈能力和刺激肠黏膜免疫细胞(主要是T、B细胞)活化,从而增强肠道黏膜免疫及获得性免疫应答水平。本研究结果表明口服黄芪多糖和人参茎叶皂苷,能够显著提升口蹄疫疫苗免疫血清特异性IgG、IgG亚类、IgG滴度水平,促进脾脏淋巴细胞经刀豆蛋白A(ConA)和脂多糖(LPS)诱导的增殖反应。免疫组化结果表明,口服黄芪多糖和人参茎叶皂苷显著增加肠道黏膜上皮内淋巴细胞(IELs)和固有层IgA分泌型浆细胞数目,从而提高肠道黏膜免疫。RT-PCR结果提示口服黄芪多糖和人参茎叶皂苷增强肠道黏膜免疫与肠黏膜组织相关免疫基因mRNA表达上调有关。综上所述,黄芪多糖和人参茎叶皂苷具有口服佐剂(oral adjuvant)作用,作为新型中药免疫增强剂改善和提高疫苗免疫效果和预防肠道病原感染都值得进一步深入研究。
[Abstract]:Vaccination is one of the main measures of aquaculture animal disease control. But modern animal husbandry due to intensive feeding and stress and other factors can lead to animal immune function decline, thereby reduce animal response on the vaccine, causing the immune failure. Buqi medicine could improve immune function of animal. The effect of ginseng and Astragalus is an important traditional Chinese medicine the polysaccharide and saponin chemistry is an important part of traditional Chinese medicine of Buqi, taking the mouse as the experimental animal study of Astragalus polysaccharide, ginsenoside of foot-and-mouth disease (foot-and-mouth diseases, FMD) of vaccine. Because most of the traditional Chinese medicine oral route of administration is, to a direct effect of the drug on the digestive medicine in recent years, the study found that the intestinal mucosal immune system for maintaining the integrity of the digestive organs and play local immune regulation are crucial, therefore, oral Astragalus polysaccharide, effects of ginsenosides on intestinal mucosal immunity is the focus of this study. The research results can provide a basis for the use of Chinese medicine polysaccharides and saponins to improve animal immune function, and to explore the effect of their immune enhancement mechanism of reference.1 oral astragalus polysaccharide to FMD vaccine in order to explore oral Huangqi effect of Polysaccharide on the immune effect of foot-and-mouth disease vaccine, 24 mice were randomly divided into 4 groups, 6 rats in each group. The first groups for the saline control, second to 4 mice per group per day orally with 25 mg, 50mg, 100mg aqueous solution of Astragalus polysaccharide, 4 consecutive days last time. After Administration of 24 h, each mouse in the groin subcutaneous injection of FMD vaccine (OS/99+OZK/93 strain) 0.2mL, 2 weeks after the booster immunization. Two free 1-5 weeks after collection of blood serum were detected by indirect ELISA FMD specific IgG and its subclasses. The results are as follows: two 3 weeks after oral administration of 25 mg free, compared with the control group of Astragalus polysaccharide can significantly improve serum FMD specific IgG and IgG subclass levels (P < 0.05), indicating astragalus polysaccharide can increase the effect of.2 oral administration of Astragalus Polysaccharide on the proliferation of spleen lymphocytes and intestinal mucosal immune to explore oral Huangqi sugar of FMD vaccine (Astragalus polysaccharides, APS) on the proliferation of spleen lymphocytes and intestinal mucosal immunity, 40 mice were randomly divided into 2 groups, 20 rats in each group. The experimental group containing 25 mice per oral mg astragalus polysaccharide aqueous solution, for 4 consecutive days, the control group was treated with normal saline as control after treatment. In the 0,1,2,3 weeks, 5 mice of each group were killed by splenic lymphocyte proliferation of spleen lymphocyte suspension experiments were collected for the spleen (MTT); acquisition of duodenal tissue was detected by immunohistochemical method in intestinal intraepithelial lymphocytes and lamina propria I GA+ cells. The results showed that oral administration of Astragalus polysaccharide after 1,2 weeks, mice spleen lymphocyte to concanavalin A (conconavalin A ConA) and lipopolysaccharide (lipopolysaccharide, LPS) proliferation responses was significantly higher than the control group (P0.05); astragalus polysaccharide after 0,1,2 weeks, epithelial duodenal mucosa and lamina propria lymphocytes in mice the number of IgA+ cells was significantly higher than the control group (P < 0.05), indicating astragalus polysaccharide can promote cellular immune response and intestinal mucosal immune response.3 oral Ginseng Stem on Ye Zaogan's immune foot-and-mouth disease vaccine in order to explore the influence of ginsenoside effects on immune foot-and-mouth disease vaccine, 24 mice were divided randomly according to the body weight into 4 groups, 6 rats in each group. The first group was treated with normal saline as control group, second to 4 mice per day orally with 0.05 mg, 0.5 mg, 5 mg aqueous solution of ginsenoside, last for 4 days. After administration of 24h, each mouse in the groin subcutaneous injection of FMD vaccine (OS/99+OZK/93 strain) 0.2 mL, 2 weeks after immunization. A weekly blood sampling, 5 weeks after two free serum separation, indirect ELISA for the detection of FMD specific IgG and its subtypes. The results showed that 3 weeks after two free, compared and the control group was treated with 0.5 mg ginsenoside can significantly improve the mouse serum FMDV specific IgG and IgG subclass levels (P < 0.05), that oral ginsenoside can significantly improve the effect of mice to FMD vaccine immune response.4 oral ginseng stem leaf saponin on proliferation of spleen lymphocytes and intestinal mucosal immunity in order to explore the effect of oral administration of ginsenosides on proliferation and intestinal mucosal immune spleen lymphocytes, 40 mice were randomly divided into 2 groups, 20 rats in each group. The experimental group of mice oral aqueous solution containing 0.5 mg of ginsenoside, for 4 consecutive days, the control group was treated with normal Saline. After the administration of 0,1,2,3 weeks, 5 mice of each group were killed to collect the spleen, preparation of spleen lymphocyte suspension for spleen lymphocyte proliferation assay (MTT method); at the same time collecting duodenum section, immunohistochemical method was used to detect intestinal intraepithelial lymphocytes and lamina propria of IgA+ cells. The experimental results show that 1,2 oral 0.5 weeks mg ginsenoside, Con and A on mice spleen lymphocyte proliferation responses of LPS was significantly higher than the control group (P0.05); 0,1,2 weeks after injection, mice in duodenal mucosa epithelial cells and lamina propria IgA+ cell number was significantly higher than the control group (P < 0.05), that oral ginsenoside can promote the spleen cell proliferation, enhance cellular immunity and humoral immune response, also can enhance intestinal mucosal immune.5 of Astragalus Polysaccharide and ginsenoside immune enhancement effect In order to enhance the research effect of Astragalus Polysaccharide and oral administration of ginsenosides on immune foot-and-mouth disease vaccine, 24 mice were randomly divided into 4 groups, 6 rats in each group. Each group 1-3 mice were treated with normal saline, 25mg solution containing astragalus polysaccharide, 0.5mg solution containing ginseng stem Ye Zaogan the last 4 days. For 24 hours after the drug, first to 3 mice per groin subcutaneous injection of O type FMD inactivated vaccine (O/My98/XJ/2010 strain +O/GX/09-7) 200 L, 2 week interval after a second immunization. The fourth groups are not immune to negative control. The mice in the two free 1,2,3 weeks after acquisition the blood serum specificity IgG, IgG titer and IFN-y level. In addition, the mice were sacrificed 3 weeks after two free, collection of duodenal mucosa, using the RT-PCR method to detect the expression of intestinal mucosal immune related gene mRNA. The results showed that Astragalus Polysaccharide and ginseng stem leaf saponin significantly Increase of serum specific IgG and IgG titers in intestinal mucosa, upregulation of chemokines (CCL25, CCL28) and chemokine receptors (CCR9, CCR10), CD80, CD86, MHC- II, TLR4 and NF- kappa B p65mRNA expression level, suggesting that Astragalus Polysaccharide and ginsenoside by up regulation of intestinal mucosa associated immune gene mRNA expression, promote intestinal intraepithelial lymphocytes (IELs) and lamina propria IgA secretion of mucosal homing to the intestinal epithelium and lamina propria of plasma cells, and can promote intestinal mucosal dendritic cell maturation, enhance the body of the antigen presenting ability of intestinal mucosa and stimulate immune cells (mainly T, B cell activation). In order to enhance intestinal mucosal immunity and acquired immune response. The results of this study show that Astragalus Polysaccharide and ginsenoside, can significantly improve the FMD vaccine immune serum specific IgG, IgG subtype, IgG titers, promote splenic lymphocyte Pakistan cells by concanavalin A (ConA) and lipopolysaccharide (LPS) induced proliferative response. Immunohistochemistry results showed that Astragalus Polysaccharide and ginseng stem leaf saponin significantly increased intestinal intraepithelial lymphocytes (IELs) and lamina propria IgA secreting plasma cell number, so as to improve the intestinal mucosal immune.RT-PCR showed oral Huangqi polysaccharide and ginsenoside enhance intestinal mucosal immunity and intestinal mucosal immune related gene expression of mRNA. In conclusion, astragalus polysaccharide and ginsenoside with oral adjuvant (oral adjuvant), as a new type of Chinese traditional immunopoteniators, improve and enhance the immune effect of vaccine and prevention of intestinal pathogens are worthy of further study.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.4

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