本文關(guān)鍵詞： 黃芪多糖 人參莖葉皂苷 口蹄疫疫苗 腸上皮內(nèi)淋巴細胞 黏膜免疫　出處：《浙江大學》2017年博士論文　論文類型：學位論文

【摘要】：疫苗免疫是養(yǎng)殖業(yè)控制動物疫病的主要措施之一。但現(xiàn)代畜牧業(yè)由于集約化飼養(yǎng)以及應(yīng)激等因素可導致動物免疫功能下降,從而使動物對疫苗免疫的應(yīng)答能力降低,引起免疫失敗。補氣類中藥具有提高動物免疫機能的作用。人參和黃芪是重要的補氣中藥,多糖和皂苷是補氣類中藥的重要化學部位,本文以小鼠為實驗動物,研究口服黃芪多糖、人參莖葉皂苷對口蹄疫(foot-and-mouth diseases,FMD)疫苗免疫的影響。由于大多數(shù)中藥的給藥途徑是口服,給藥后藥物直接作用于消化道,近年來研究發(fā)現(xiàn),腸道黏膜免疫系統(tǒng)對于維持機體消化器官完整性和發(fā)揮局部免疫調(diào)節(jié)作用都至關(guān)重要,因此,口服黃芪多糖、人參莖葉皂苷對腸道黏膜免疫的影響也是本研究的重點。研究結(jié)果可為采用中藥多糖和皂苷提高動物免疫機能提供依據(jù),并為探討它們的免疫增強作用機理提供參考。1 口服黃芪多糖對口蹄疫疫苗免疫的影響為了探究口服黃芪多糖對口蹄疫疫苗免疫效果的影響,將24只小鼠按體重隨機分為4組,每組6只。第1組為生理鹽水對照,第2～4組每只小鼠每天口服含25 mg、50mg、100mg黃芪多糖的水溶液,連續(xù)4天。最后一次給藥后24 h,每只小鼠在腹股溝部皮下注射FMD疫苗(OS/99+OZK/93株)0.2mL,2周后加強免疫一次。二免后1-5周采集血液分離血清,用間接ELISA法檢測FMD特異性IgG及其亞類。結(jié)果表明:二免后3周,與對照組相比口服25 mg黃芪多糖可顯著提高小鼠血清FMD特異性IgG、IgG亞類水平(P＜0.05),說明口服黃芪多糖對FMD疫苗免疫具有增強作用。2 口服黃芪多糖對脾淋巴細胞增殖及腸道黏膜免疫的影響為了探究口服黃芪多糖(astragalus polysaccharides,APS)對脾臟淋巴細胞增殖及腸道黏膜免疫的影響,將40只小鼠隨機分為2組,每組20只。實驗組每只小鼠口服含25 mg黃芪多糖水溶液,連續(xù)4天,對照組口服生理鹽水為對照。用藥后第0、1、2、3周,每組處死5只小鼠,收集脾臟制備脾淋巴細胞懸液進行脾淋巴細胞增殖實驗(MTT法);采集十二指腸組織,采用免疫組化方法檢測腸黏膜上皮內(nèi)淋巴細胞和固有層IgA+細胞。結(jié)果顯示:口服黃芪多糖后1、2周,小鼠脾淋巴細胞對刀豆蛋白A(conconavalin A,ConA)和脂多糖(lipopolysaccharide,LPS)刺激引起的增殖反應(yīng)顯著高于對照組(P0.05);口服黃芪多糖后0、1、2周,小鼠十二指腸黏膜上皮內(nèi)淋巴細胞及固有層IgA+細胞數(shù)顯著高于對照組(P＜0.05),說明口服黃芪多糖能夠促進機體的細胞免疫反應(yīng)和腸道黏膜免疫反應(yīng)。3 口服人參莖葉皂苷對口蹄疫疫苗免疫的影響為了探究人參莖葉皂苷對口蹄疫疫苗免疫的影響,將24只小鼠根據(jù)體重隨機分為4組,每組6只。第1組口服生理鹽水為對照,第2～4組每只小鼠每天口服含0.05 mg、0.5 mg、5 mg人參莖葉皂苷的水溶液,連續(xù)4天。最后一次給藥后24h,每只小鼠在腹股溝部皮下注射FMD疫苗(OS/99+OZK/93株)0.2 mL,2周后加強免疫一次。二免后5周內(nèi)每周采血,分離血清,間接ELISA法檢測FMD特異性IgG及其亞類。結(jié)果表明:二免后3周,與對照組相比口服0.5 mg人參莖葉皂苷可顯著提高小鼠血清口蹄疫特異性IgG、IgG亞類水平(P＜0.05),說明口服人參莖葉皂苷能夠顯著提高小鼠對口蹄疫疫苗的免疫反應(yīng)。4 口服人參莖葉皂苷對脾淋巴細胞增殖及腸道黏膜免疫的影響為了探究口服人參莖葉皂苷對脾臟淋巴細胞增殖及腸道黏膜免疫的影響,將40只小鼠隨機分為2組,每組20只。實驗組小鼠口服含0.5 mg人參莖葉皂苷的水溶液,連續(xù)4天,對照組口服生理鹽水。用藥后第0、1、2、3周,每組處死5只小鼠,收集脾臟,制備脾淋巴細胞懸浮液,用于脾淋巴細胞增殖實驗(MTT法);同時采集十二指腸腸段,采用免疫組化方法檢測腸粘膜上皮內(nèi)淋巴細胞和固有層IgA+細胞。實驗結(jié)果表明口服0.5 mg人參莖葉皂苷后1、2周,小鼠脾臟淋巴細胞對Con A和LPS刺激引起的增殖反應(yīng)顯著高于對照組(P0.05);給藥后0、1、2周,小鼠十二指腸黏膜上皮內(nèi)淋巴細胞及固有層IgA+細胞數(shù)顯著高于對照組(P＜0.05),說明口服人參莖葉皂苷能夠促進機體脾臟細胞的增殖功能,增強機體的細胞免疫和體液免疫應(yīng)答,同時也能增強腸道黏膜免疫。5黃芪多糖和人參莖葉皂苷免疫增強作用的比較研究為了比較口服黃芪多糖和人參莖葉皂苷對口蹄疫疫苗免疫的增強作用,將24只小鼠根據(jù)體重隨機分為4組,每組6只。第1-3組每只小鼠分別口服生理鹽水、含25mg黃芪多糖的水溶液、含0.5mg人參莖葉皂苷的水溶液,連用4天。給藥后24小時,第1～3組小鼠每只腹股溝皮下注射O型FMD滅活疫苗(O/My98/XJ/2010株+O/GX/09-7株)200μL,間隔2周后加強免疫一次。第4組不免疫作陰性對照。各組小鼠于二免后1、2、3周采集血液,檢測血清特異性IgG、IgG滴度及IFN-y水平。此外,二免后3周處死小鼠,采集十二指腸黏膜組織,采用RT-PCR方法檢測腸道黏膜相關(guān)免疫基因mRNA表達。結(jié)果表明,口服黃芪多糖和人參莖葉皂苷能顯著提升血清特異性IgG及IgG滴度水平,上調(diào)腸道黏膜組織趨化因子(CCL25、CCL28)、趨化因子受體(CCR9、CCR10)、CD80、CD86、MHC-Ⅱ、TLR4 及 NF-κB p65mRNA 表達水平,提示口服黃芪多糖和人參莖葉皂苷通過上調(diào)腸黏膜相關(guān)免疫基因mRNA表達,促進腸道上皮內(nèi)淋巴細胞(IELs)和固有層IgA分泌型漿細胞歸巢至腸黏膜上皮及固有層,同時能夠促進腸黏膜樹突狀細胞的成熟,增強機體對抗原的遞呈能力和刺激腸黏膜免疫細胞(主要是T、B細胞)活化,從而增強腸道黏膜免疫及獲得性免疫應(yīng)答水平。本研究結(jié)果表明口服黃芪多糖和人參莖葉皂苷,能夠顯著提升口蹄疫疫苗免疫血清特異性IgG、IgG亞類、IgG滴度水平,促進脾臟淋巴細胞經(jīng)刀豆蛋白A(ConA)和脂多糖(LPS)誘導的增殖反應(yīng)。免疫組化結(jié)果表明,口服黃芪多糖和人參莖葉皂苷顯著增加腸道黏膜上皮內(nèi)淋巴細胞(IELs)和固有層IgA分泌型漿細胞數(shù)目,從而提高腸道黏膜免疫。RT-PCR結(jié)果提示口服黃芪多糖和人參莖葉皂苷增強腸道黏膜免疫與腸黏膜組織相關(guān)免疫基因mRNA表達上調(diào)有關(guān)。綜上所述,黃芪多糖和人參莖葉皂苷具有口服佐劑(oral adjuvant)作用,作為新型中藥免疫增強劑改善和提高疫苗免疫效果和預(yù)防腸道病原感染都值得進一步深入研究。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S852.4

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