【作者】 李瑞丽；

【导师】 胡松华；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 疫苗免疫接种是防治动物传染病的一个重要手段,但在很多情况下动物注射疫苗后不能产生有效的免疫应答。因此,如何提高动物疫苗的免疫效果是畜牧业生产上的一个重要问题。研究发现一些中药可以增强机体的免疫机能,从而提高疫苗的免疫效果。浙江省出产的道地药材“浙八味”久负盛名,它们是白术、白芍、浙贝母、杭白菊、延胡索、玄参、麦冬、温郁金八味中药材。本论文系统研究浙八味中药以及含浙八味中药的玉屏风散和增液汤的口服免疫佐剂作用,为进一步开发浙八味中药提供参考。1.浙八味中药对小鼠注射模式抗原卵清白蛋白(OVA)的口服佐剂作用比较目的筛选浙八味中具有口服免疫佐剂作用的中药。方法以雌性ICR小鼠为实验动物,将浙八味设为高、中、低三个剂量水平,每只小鼠分别灌胃0.25 mL不同水平的中药,每日一次,连续给药4 d,然后分别在第5 d和第25 d进行皮下注射抗原OVA,并在二免后2周进行眼眶采血。结果与对照组相比,浙八味中的白术能显著提高OVA受免小鼠血清中OVA特异性IgG总抗体及抗体亚类IgG2a、IgG2b和IgG3抗体水平(P＜0.05)。麦冬、玄参可以显著增强小鼠血清中OVA特异性IgG总抗体水平(P＜0.05)。此外,在试验过程中发现浙贝母的毒性较大,灌胃1 g/只即可导致小鼠死亡。2.玉屏风散煎剂对口蹄疫疫苗免疫的口服佐剂作用目的探讨中药复方玉屏风散及其麦冬加味对O型口蹄疫疫苗的免疫佐剂作用。方法将21只ICR小鼠随机分为3组,每组7只,分别为对照组、玉屏风散组和玉屏风散加麦冬组。将玉屏风散及其加味制成生药浓度为2 g/mL的药液,每只小鼠灌胃给药1次(0.25 mL),24 h后经腹股沟皮下注射FMD疫苗(0.2mL/只),2周后同样方法加强免疫一次。二免后3周采血和取脾脏,用间接ELISA检测血清中特异性IgG抗体及其亚类,用MTT法检测淋巴细胞增殖情况。结果二免后各给药组小鼠血清中FMDV特异性IgG抗体及其IgG抗体亚类水平和淋巴细胞刺激指数均显著高于对照组(P＜0.05),而玉屏风散和玉屏风散加麦冬两组间的抗体水平以及淋巴细胞刺激指数均未见显著性差异(P＞0.05)。3.增液汤煎剂对口蹄疫疫苗免疫的口服佐剂作用目的探讨中药复方增液汤(由玄参、麦冬和生地组成)及其单味中药对O型口蹄疫疫苗的免疫调节作用。方法将40只ICR小鼠随机分为5组,每组8只,分别为对照组、单味中药组和增液汤组。每只小鼠于免疫注射前24h灌胃给药一次,剂量为0.25 mL/只,然后对所有小鼠经腹股沟皮下注射O型FMD疫苗免疫,2周后加强免疫一次,二免后3周眼眶采血。结果与对照组相比,口服麦冬和生地均能显著提高免疫O型FMD商品苗的小鼠血清中FMDV特异性IgG总抗体水平以及FMDV特异性IgG1和IgG2a抗体亚类水平(P＜0.05)；但是增液汤组无论在调节IgG总抗体水平方面,还是其对抗体亚类的影响方面,与对照组均未见显著性差异(P＞0.05)。4.白术水煎剂对小鼠注射模式抗原卵清白蛋白(OVA)的口服佐剂作用目的探讨白术对模式抗原卵清白蛋白(Ovalbumin, OVA)均口服佐剂作用。方法选取48只ICR小鼠,随机分为6组,每组8只,雌雄各半,采用单因素多水平实验设计,各组小鼠分别灌服0.05 g、0.1 g、0.2 g、0.4 g或0.8 g/只剂量的白术水煎剂或者生理盐水,连续给药4 d后,分别于试验的第5 d和第25 d分别进行皮下注射免疫,二免后2周眼眶采血并取脾脏。结果与对照组相比,白术水煎剂(0.1 g、0.2 g、0.4 g和0.8 g)能够显著的提高小鼠血清中IgG总抗体滴度(P＜0.05),0.2 g、0.4 g和0.8 g的白术水煎剂均可显著地提高小鼠血清中OVA特异性IgG1、IgG2a、IgG2b和IgG3的水平(P＜0.05)。灌胃0.1 g、0.2 g、0.4 g及0.8 g白术水煎剂也可以显著增强小鼠的脾脏淋巴细胞受刺激物刺激后的刺激指数(P＜0.05),其中0.2 g白术水煎剂灌胃后,小鼠脾脏淋巴细胞受ConA刺激后分泌的IL-5和IFN-γ均显著地高于对照组(P＜0.05)。5.白术水煎剂对口蹄疫疫苗免疫的口服佐剂作用目的探讨白术对O型口蹄疫疫苗免疫的口服佐剂作用。方法将35只ICR雌性小鼠随机分为5组,每组7只,采用单因素多水平实验设计,分别设0.0625g、0.125 g、0.25 g及0.5 g白术水煎剂组和生理盐水组。按上述剂量分别灌胃小鼠,连续给药4 d,在试验的第5 d、第25 d分别免疫O型FMD疫苗,3周后眼眶采血并取脾脏。结果0.25 g和0.5 g剂量的白术水煎剂,可以显著提高免疫O型FMD疫苗小鼠血清中FMDV特异性抗体滴度水平和FMDV特异性IgG抗体亚类水平(P＜0.05)。与对照组相比,不同剂量的白术水煎剂均能显著增强脾脏淋巴细胞在刺激物作用下的刺激指数(P＜0.05),其中,灌胃0.25 g白术水煎剂可以提高脾脏淋巴细胞在ConA刺激下分泌IL-5和IFN-y的能力(P＜0.05),因此0.25 g或0.5g/只·d可以作为提高FMD疫苗佐剂效果的参考剂量。在此基础上,我们研究了不同的灌胃天数,即1 d、2 d和4 d,对O型FMD疫苗的免疫佐剂作用效果的影响,结果发现2 d和4 d的效果相对较好。6.日粮添加白术对口蹄疫疫苗免疫的口服佐剂作用试验一目的用不同的加工方法(粉碎或煎煮)对白术进行处理后,按1%的生药量比例添加到饲料中,探讨其对O型口蹄疫疫苗免疫的佐剂作用。方法试验设免疫前饲喂一周和全程饲喂(共饲喂6周)两个不同的时间,按照两因素三水平析因实验设计方法,将试验分为：对照组、粉碎-1周组、粉碎-6周组、煎煮-1周组和煎煮-6周组。试验用O型FMD疫苗免疫小鼠两次,间隔2周,二免后3周眼眶采血并取脾脏。结果与对照组相比,饲喂添加1%白术水煎剂的日粮6周的处理组小鼠血清中FMDV特异性IgG抗体水平和抗体亚类水平(P＜0.05)显著提高,对ConA和LPS刺激的脾脏淋巴细胞增殖有明显促进作用(P＜0.05),并能显著地增强脾脏淋巴细胞受ConA刺激后分泌IL-5和IFN-y的能力,表明该实验组对O型FMD疫苗具有有较好的免疫增强作用。试验二目的试验采用煎煮法加工的白术,按5%的比例添加到饲料中,探讨其对O型口蹄疫疫苗免疫的口服佐剂作用。方法采用单因素多水平实验设计,分别饲喂2 d、4 d和6 d,用O型FMD疫苗免疫小鼠两次,间隔2周,二免后3周眼眶采血并取脾脏和小肠。结果用添加5%白术的饲料饲喂小鼠2 d、4 d和6 d后,与对照组相比,各组小鼠血清中FMDV特异性IgG抗体水平和抗体亚类水平均显著升高,刺激物作用后的脾脏淋巴细胞的刺激指数和ConA刺激后脾脏淋巴细胞中IL-5和IFN-y的分泌量也显著上升(P＜0.05)。用添加5%白术的饲料饲喂小鼠2 d后,在第一次免疫FMD疫苗后1周和二免后3周的小鼠小肠IgA分泌细胞的阳性率高于对照组,说明口服白术具有增强机体黏膜免疫反应的作用。综上所述,浙八味中的麦冬、玄参、白术具有口服免疫佐剂作用,可以提高ICR小鼠对于OVA免疫的体液免疫反应水平。进一步的研究表明,口服一定剂量的白术能提高ICR小鼠对于模式抗原OVA和FMD疫苗免疫的特异性体液免疫和细胞免疫水平,能同时上调Th1/Th2型免疫应答；日粮添加白术饲喂小鼠,能提高FMD疫苗的免疫效果,提高动物机体的体液免疫、细胞免疫和黏膜免疫水平。另外,口服含浙八味中药的复方玉屏风散可以增强ICR小鼠免疫FMD疫苗的免疫效果,但是增液汤的效果则不明显。更多还原

【Abstract】 Vaccination is a common practice against infectious disease in the world. However, failure to elicit effective immune responses by vaccination has been frequently reported. Therefore, there is a need to improve currently available vaccines in order to effectively protect humans and animals from infection. There is growing evidence that medicinal herbs and the ingredients enhance the immune response to vaccines direct against infectious agents. The "Eight Zhe Herbs" (Zhe Ba Wei [Z-8]) is well known because the medicinal quality of the 8 herbs produced in Zhe Jiang Province is believed to be better than in other parts of China. They include Ophiopogon japonicus Ker-Gawl., Scrophularia ningpoensis Hemsl., Curcuma wenyujin, Chrysanthemum morifolium Ramat., Paeonia lactiflora Pall., Atractylodes macrocephala Koidz., Fritillaria thunbergii Miq., and Eorydalis yanhusuo. The thesis was to study the effects of oral administration of herbal soups made from Zhe Ba Wei and the related compound recipe of Yu Ping Feng San and Zeng Ye Tang on the immunity adjuvant effect in mice.1. Screening of Zhe Ba Wei for their immunity adjuvant activityObjective Zhe Ba Wei is a collective term for 8 well known traditional Chinese medicinal plants, including Ophiopogon japonicus Ker-Gawl., Scrophularia ningpoensis Hemsl., Curcuma wenyujin, Chrysanthemum morifolium Ramat., Paeonia lactiflora Pall., Atractylodes macrocephala Koidz., Fritillaria thunbergii Miq., and Eorydalis yanhusuo.The purpose of this study was to evaluate their immunity adjuvant activity in mice immunized with OVA. Methods Zhe Ba Wei were cooked as herbal soups. Groups of five ICR mice were s.c. immunized twice as a two-week interval with OVA (10μg). One day before each immunization, the mice had already been daily orally administered with a high, medium or low dose of herbal soups daily for 4 days. Blood was collected 2 weeks after the second immunization for measurement of IgG and the IgG subclasses. Results Oral administration of a soup made from Ophiopogon japonicus Ker-Gawl, Scrophularia ningpoensis Hemsl or Atractylodes macrocephala Koidz for 4 days significantly increased the IgG, IgG2a, IgG2b and IgG3 response in mice immunised with OVA. Oral administration of a high dose of Fritillaria thunbergii Miq may results in a complete inhibition of respiration, eventuating in death.2. Effects of Yu Ping Feng San and Yu Ping Feng San plus on the Immune Responses to Vaccination against Foot-and-mouth Virus in MiceObjective A compound recipe (Yu Ping Feng San) is effective therapy clinically in Traditional Chinese Medicine, so the study investigated the immunity adjuvant effects of the compound recipe. Methods Twenty one ICR mice were randomly divided into 3 groups with 7 animals in each group. The animals received daily oral administration of 0.25 mL of herbal liquid equivalent to 2 g of Yu Ping Feng San or Yu Ping Feng San plus per mL or saline solution. Twenty-four hours later, each mouse was s.c. immunized twice with commercial FMD vaccine at two week intervals. Three weeks after boosting, blood samples were collected for measurement of FMDV-specific IgG level and the IgG subclasses, and splenocytes were prepared for lymphocyte proliferation test. Results Serum FMDV-specific IgG and IgG subclass responses and splenocyte stimulation indexes were significantly enhanced in mice orally administered Yu Ping Feng San and Yu Ping Feng San plus compared with the control group (P< 0.05). No significant difference was found between Yu Ping Feng San and Yu Ping Feng San plus groups for serum FMDV-specific IgG and IgG subclass responses and splenocyte stimulation indexes.3. Effects of Zeng Ye Tang on the Immune Responses to Vaccination against FMDV in MiceObjective This study investigated the immunity adjuvant effects of Zeng Ye Tang recipe. Methods Forty ICR mice were randomly divided into 5 groups with 8 animals in each group. The animals received daily oral administration of 0.25 mL of herbal soup equivalent to 2 g of Zeng Ye Tang, Scrophularia ningpoensis Hemsl. including Ophiopogon japonicus Ker-Gawl., Rehmannia glutinosa (Gaerin) Libosch per mL or saline solution. Twenty-four hours later, each mouse was s.c. immunized twice with FMD vaccine at two week intervals. Three weeks after boosting, blood samples were collected for measurement of FMDV-specific IgG level and subclasses.Results The serum FMDV-specific IgG and IgG subclass responses were significantly enhanced in mice orally administered Scrophularia ningpoensis Hemsl. and Ophiopogon japonicus Ker-Gawl (P<0.05). No significant difference was found between Zeng Ye Tang for serum FMDV-specific IgG and IgG subclass responses (P>0.05).4. Adjuvant effects of Oral administration of water extracts of Rhizoma Atractylodis Macrocephalae (RAM) on the immune responses to OVA in miceObjective In this study, the immunomodulation activity of oral administration in different doses of Rhizoma Atractylodis Macrocephalae (RAM) in OVA-immunized mice has been evaluated. Methods ICR mice were daily oral administration of water extracts of RAM (0.05 g,0.1 g,0.2 g,0.4 g or 0.8 g) or saline for 4 days and intramuscularly injected with OVA (10μg) in saline on days 5. The OVA-specific IgG titer, IgGl, IgG2a, IgG2b and IgG3 antibody levels and mitogen-stimulated splenocytes proliferation were measured two weeks after the booster. Results The serum IgG titer in mice immunized with OVA was significantly enhanced by water extracts of RAM (0.1 g,0.2 g,0.4 g and 0.8 g) as compared with control group (P< 0.05). Water extracts of RAM (0.2 g,0.4 g,0.8 g) significantly enhanced the total IgGl, IgG2a, IgG2b and IgG3 (P< 0.05). Mitogen-stimulated splenocyte proliferation in the mice immunized with OVA (10μg) and oral administration of water extract of RAM (0.1 g,0.2 g,0.4 g and 0.8 g) were significantly higher than control group (P< 0.05).Water extracts of RAM (0.2 g) significantly increased the cytokine IFN-y (Thl cytokine) and IL-5 (Th2 cytokine) by splenocytes (P< 0.05). Above all, RAM has the potential adjuvant effects to enhance the immune responses to OVA in mice. Hence RAM deserve for further studies as an adjuvant.5. Enhancement of the immune responses to vaccination against FMDV in mice by oral administration of an extract made from Rhizoma Atractylodis Macrocephalae (RAM)Objective Present study was designed to evaluate the effects of oral administration of a water extract made from RAM on the immune responses in mice immunized with FMDV type O vaccine. Methods Thirty five ICR mice were randomly divided into 5 groups with 7 animals in each group, and orally administered for 4 days at a daily dose of RAM of 0,0.0625 g,0.125 g,0.25 g or 0.5 g, respectively. After that, the animals were s.c. immunized twice with FMD vaccine at two week intervals. Blood samples were collected 3 weeks after boosting for measurement of FMDV-specific IgG titers and the IgG subclasses, lymphocyte proliferation as well as production IL-5 and IFN-y. Results The serum FMDV-specific IgG titers and the IgG subclass responses were significantly enhanced in mice orally administered RAM at the dose of 0.25 or 0.5 g compared with the control group (P< 0.05). Splenocyte proliferation in response to Con A and LPS and production of IL-5 and IFN-y by splenocytes were also significantly enhanced (P<0.05). Together, the results demonstrate that oral administration of RAM could enhance the FMDV-specific immune responses.6. Immune responses induced by feeding Rhizoma Atractylodis Macrocephalae (RAM) to vaccination against FMDV in miceIn experiment 1, Objective Present study was designed to evaluate the effects of feeding different treatment of RAM in different days on the immune responses in mice immunized with FMDV type O vaccine. Methods Fifty ICR mice were randomly divided into 5 groups with 10 animals in each group, and the basic diet containing 1%crush of RAM for a 1 week ad libitum feeding period,1%crush of RAM for a 6 week ad libitum feeding period,1%decoction of RAM for a 1 week ad libitum feeding period,1%decoction of RAM for a 6 week ad libitum feeding period, respectively. Blood samples were collected 3 weeks after boosting for measurement of FMDV-specific IgG level and the IgG subclasses, lymphocyte proliferation as well as production IL-5 and IFN-y. In experiment 2, Objective Four groups mice were feed ad libitum a basic diet, and a basic diet containing with 5%decoction of RAM for 2 d,4 d,6 d feeding periods, respectively. Methods Same as experiment 1 and blood samples were collected for detecting IgG and IgG subclasses, splenocytes for lymphocyte proliferation as well as production IL-5 and IFN-y, and small intestine were preserved for determination of secreting IgA (sIgA).Results 1% decoction of RAM for a 1 week ad libitum feeding period group and 5% decoction of RAM for 2 d,4 d and 6 d ad libitum feeding period group enhance the FMDV-specific immune responses significantly. Results demonstrate immune responses against FMDV type O was affected by doses and time to supplemental feeding of RAM.In conclusion, the oral administration for 4 days of a soup made from Ophiopogon japonicus Ker-GawlOphiopogon, Scrophularia ningpoensis Hemsl Scrophularia or Rhizoma Atractylodes macrocephala Koidz. included in Zhe Ba Wei significantly increased the immune response in OVA-immunised ICR mice. Further investigation found oral administration of water extracts of RAM significantly increased the antigen-specific humoral and cell-mediated immunity to immunization of ovalbumin(OVA) and a commercial FMD vaccine, Both Thl and Th2 immune responses were up-regulated in mice. Immune response to the commercially FMD vaccine could be enhanced by diet supplement containing RAM, the antigen-specific humoral, cell-mediated and mucosal immune responses were up-regulated in mice. Moreover, oral administration of Yu Ping Feng San that contain some medicine herb of Zhe Ba Wei is able to enhance the immune response to the commercially FMD vaccine. Whereas Zeng Ye Tang were not effective to the FMD vaccine.更多还原