不同来源人参皂甙免疫佐剂作用的研究
摘要
人参作为一味补益类中药使用在我国已有数千年的历史。人参经过蒸制加工而成红参。最近的研究发现,从人参分离到的皂甙具有免疫佐剂作用,增强疫苗的免疫效果。本论文研究了人参根皂甙、红参皂甙、人参茎叶皂甙、人参皂甙单体Re和人参皂甙纳米颗粒的免疫佐剂作用,为进一步研究开发人参皂甙提供参考。
1.人参根皂甙(GS-N)免疫佐剂作用研究
目的观察人参根皂甙(GS-N)经胃肠道途径给药对皮下注射的口蹄疫病毒(FMDV)亚洲I型和流感病毒H3N2型灭活疫苗的免疫增强作用。方法实验一将小鼠分别灌服250μl含0.5、5或50μg GS-N溶液,对照组灌服250μl蒸馏水,1天后皮下注射100μlFMDV抗原,同样方法免疫2次,间隔3周；实验二：将小鼠连续3天,每天灌服250μl含50、100或200μg GS-N溶液,对照组灌服蒸馏水,然后皮下注射含10 ng HA的流感病毒疫苗,同样方法免疫2次,间隔3周。实验一和实验二小鼠均在二免后2周采血测抗体水平,实验二中小鼠取小肠空肠中段进行免疫组化观察IgA+浆细胞数量变化。结果实验一小鼠灌胃1次50μg GS-N能提高血清抗FMDV抗体IgG水平,但和对照组比较无统计学差异。实验二中每天灌服一次100μg GS-N,连续3天的小鼠血清中抗流感HA特异性抗体IgG效价,IgG1、IgG2b和IgG3水平均显著提高(P<0.05),且肠道IgA+浆细胞明显增多,说明黏膜免疫功能得到增强。结论1次口服GS-N虽然提高了小鼠对皮下注射FMD抗原的免疫反应,但无统计学意义；连续3天口服GS-N(每天剂量为50、100或200μg)可以显著提高小鼠对流感疫苗的免疫反应,以及增加肠道IgA+浆细胞数量。
2.红参皂甙(GS-R)免疫佐剂作用研究
目的观察疫苗中添加红参皂甙(GS-R)或口服GS-R对疫苗接种有无免疫增强作用。方法实验一：将20只ICR小鼠随机分成4组,每组5只。以OVA(每只免疫剂量为10μg)、为模式抗原,分别设含人参皂甙10、50或250μg GS-R组,对照组仅免疫10μg OVA。免疫2次,间隔3周。二次免疫后2周采血,检测血清抗OVA抗体IgG水平。实验二：实验组小鼠分别在实验第1天和第21天灌胃250μl含0.5、5或50μg GS-R的蒸馏水溶液,对照组小鼠同时灌服250μl蒸馏水。在第2天和第22天,每组小鼠均皮下注射100μlFMDV抗原。即实验组小鼠在每次免疫前灌胃一次GS-R,小鼠免疫2次,间隔3周。二免后2周(实验第36天),小鼠采血分离血清,检测血清抗FMDV抗体IgG水平。结果与结论实验一和实验二结果表明,红参皂甙和抗原混合以及在动物对注射OVA或FMDV抗原前口服均不能增强相应的的免疫应答,因而红参皂甙不具有免疫佐剂作用。
3.人参茎叶皂甙(GSLS)免疫佐剂作用研究
目的口服人参茎叶皂甙(GSLS)对皮下注射FMDV抗原或者流感疫苗的免疫增强作用。方法实验一：实验组小鼠在免疫前一天灌服0.5、5或50μgGSLS,对照组灌服250μl蒸馏水。小鼠皮下注射100μlFMDV抗原,同样方法免疫2次,间隔3周。二免后2周采血分离血清,检测血清抗FMDV抗体IgG水平；实验二：实验组小鼠在免疫前连续三天灌服50、100或200μgGSLS,对照组同时灌服蒸馏水,完成3天灌胃后,小鼠皮下注射含10 ng HA抗原的流感疫苗,同样方法免疫2次,间隔3周。在二免后2周采血测血清抗HA特异性抗体IgG滴度及其亚类水平,并取小鼠小肠空肠中段制作组织石蜡切片,免疫组化比较口服GSLS组和对照组IgA+浆细胞数量。结果实验一：小鼠2次免疫前均灌胃一次0.5或50μgGSLS能提高血清抗FMDV抗体IgG水平,但和对照组比较无统计学差异。实验二：连续口服3天,每天灌服50μgGSLS使小鼠抗流感HA特异性抗体IgG效价,IgG1和IgG2b水平均显著提高(P<0.05),小鼠空肠中段组织中IgA+浆细胞明显增多,提示黏膜免疫功能得到增强。总体上,通过抗体及其亚类和肠道IgA+浆细胞变化可知,口服低剂量人参茎叶皂甙提高了机体体液免疫和细胞免疫的同时,还增强胃肠道局部黏膜免疫水平,即口服GSLS对皮下注射FMD抗原和流感H3N2病毒抗原均显示一定的免疫调节作用。结论小鼠灌胃1次GSLS能提高机体对皮下注射FMDV抗原的体液免疫水平,但无统计学意义；连续3天口服GSLS可提高小鼠对流感疫苗的抗体免疫反应,并增加肠道IgA+浆细胞数量。
4.人参皂甙单体Re免疫佐剂作用研究
目的观察人参皂甙Re对流感病毒疫苗的免疫佐剂作用。方法40只ICR小鼠随机分成5组,每组8只。组1：10 ng HA(注射含10 ng HA抗原的流感疫苗,下同)；组2：100 ng HA组；组3：10 ng HA+25μg Re组；组4：10 ng HA+50μg Re；组5：10 ng HA+100μg Re组。小鼠腹部皮下注射200μl含上述组分的疫苗,免疫2次,间隔3周。二免后2周摘眼球采血分离血清,间接ELISA法检测血清抗血凝素HA抗体效价及抗体亚类IgG1、IgG2a、IgG2b和IgG3水平,并测血凝抑制效价HI；无菌分离脾淋巴细胞后,采用MTT法检测淋巴细胞增殖和ELISA法检测Con A刺激培养上清中细胞因子水平。结果在含10 ng HA的流感疫苗中添加50μg Re能显著提高ICR小鼠血清中抗HA特异性IgG效价,IgG1、IgG2a和IgG2b水平,以及HI效价和淋巴细胞转化能力(P<0.05),显著刺激淋巴细胞分泌细胞因子IFN-γ和IL-5(P<0.05)。结论流感疫苗中添加人参皂甙Re能显著提高受免小鼠特异性抗体IgG和抗体亚类水平,增强血清HI效价,显著促进脾淋巴细胞转化能力和分泌细胞因子水平,表明Re对流感疫苗免疫具有佐剂作用。
5.人参皂甙的纳米颗粒(Ginsomes)制备及其佐剂作用研究
目的研究人参皂甙纳米颗粒(Ginsomes)的制备方法、颗粒物理形态及其对模式抗原OVA的免疫佐剂作用。方法分别选取三种人参皂甙：人参根皂甙、人参茎叶皂甙和红参皂甙,与胆固醇、卵磷脂(胆固醇和卵磷脂预先溶于Mega-10)形成不同配比方案,将混合溶液置于不同温度(10、20、30、40和50℃)孵育2 h,此后对PBS透析48 h以移除去污剂Mega-10,此时,皂甙、胆固醇和卵磷脂形成的纳米颗粒产物采用2%磷钨酸溶液负染后进行透射电镜鉴定。电镜确认纳米颗粒形成后,设计ICR小鼠试验检验纳米颗粒的免疫佐剂作用。在实验一中选取不同人参皂甙浓度的红参皂甙和Ginsomesl：含人参皂甙分别为10、50或250μg)与无内毒素污染的卵清白蛋白(OVA)混合后免疫小鼠,间隔3周进行一次加强免疫,二免后2周取血清进行抗OVA特异性抗体水平测定；实验二：将32只ICR小鼠随机分成4组,每组8只。以OVA(每只免疫剂量为10μg)为模式抗原,分别设含人参皂甙50μg的Ginsomes组,含人参皂甙50μg的人参皂甙GS组和50μg Quil A组,对照组仅免疫OVA。抗原和佐剂混合均匀后,腹部皮下注射疫苗,在第1天和第21天分别进行2次免疫。第14天从小鼠眼静脉窦采血,第35天摘眼球采血并分离脾脏淋巴细胞。间接ELISA法检测血清抗OVA抗体效价及抗体亚类IgG1、IgG2a、IgG2b和IgG3水平；取脾淋巴细胞,采用MTT法检测淋巴细胞增殖和ELISA法检测OVA刺激培养上清中细胞因子水平。结果透射电子显微镜观察后发现,三种不同来源人参皂甙中只有红参皂甙能制备形成Ginsomes;形成纳米颗粒的最佳条件为红参皂甙：胆固醇：卵磷脂以质量比20：1：1混合,纳米颗粒平均粒径为：87.8±9.4nm。小鼠免疫试验结果表明,实验一中各个浓度颗粒均诱导出显著高于人参总皂甙免疫小鼠血清抗体水平的特异性IgG(P<0.05),实验二中含50μg人参皂甙的纳米颗粒能显著提高ICR小鼠血清中抗OVA特异性IgG效价,IgG1、IgG2a、IgG2b和IgG3抗体水平,以及受特异性抗原OVA刺激的淋巴细胞转化能力和细胞因子IFN-γ和IL-5的分泌(P<0.05),说明人参皂甙纳米颗粒作为免疫佐剂能同时上调Th1和Th2型免疫应答反应。结论红参皂甙制备的纳米颗粒Ginsomes粒径约为90 nm,OVA与纳米颗粒混合免疫可显著提高血清抗体IgG及其亚类水平,并显著增强小鼠脾淋巴细胞受OVA刺激的增殖能力和分泌IFN-γ和IL-5水平。
综上所述,(1)小鼠皮下注射FMD抗原前灌胃1次GS-N提高了体液免疫水平,但无统计学意义；注射流感疫苗前连续3天口服GS-N可显著提高小鼠抗流感抗原的免疫反应,并增加肠道IgA+浆细胞数量。(2)OVA抗原和GS-R混合或皮下注射口蹄疫疫苗前灌服1次GS-R均未显著增强机体对抗原特异性免疫反应,因而红参皂甙不具有免疫佐剂作用。(3)小鼠口服1次GSLS能提高机体对皮下注射FMD抗原的体液免疫水平,但无显著性差异；连续3天口服GSLS可提高小鼠对流感疫苗的体液反应水平,增加肠道,IgA+浆细胞数量。(4)流感疫苗中添加人参皂甙Re显著促进小鼠抗体IgG和抗体亚类水平,提高血清HI效价,并显著增强脾淋巴细胞转化能力和促进IFN-γ和IL-5的分泌,表明Re对流感疫苗免疫具有佐剂作用。(5)红参皂甙与胆固醇、卵磷脂在质量比20：1：1配比条件下可制备Ginsomes,在OVA溶液中添加纳米颗粒可显著提高小鼠血清OVA特异性IgG及其亚类水平,显著增强小鼠脾淋巴细胞增殖能力和细胞因子水平,表明Ginsomes对OVA具有免疫佐剂作用。
As a traditional medicine, ginseng has been used for thousands of years in China. According to the latest investigation, saponins extracted from Panax ginseng show adjuvant effects against various antigens. However, almost all of the reported ginseng adjuvants were originated from ginseng roots. In the present thesis, normal ginseng saponins (extracted from ginseng root) (GS-N), red ginseng (steam-treated ginseng root) saponins (GS-R) and ginseng stem-leaf saponins (GSLS), as well as ginsenoside Re and ginsenoside-based nanoparticles were studied in order to further evaluate the adjuvant proterties of saponins originated from different ginsengs.
1. Adjuvant effects of normal ginseng saponins (GS-N)
Objective To measure the immunomodulatory activity of orally-administrated normal ginseng saponins (GS-N) against subcutaneously-injected FMDV (serotype Asia 1) and influenza virus strain H3N2 antigens. Methods Two experiments were designed and described as follows. In experiment A, ICR mice were orally-administrated GS-N (0.5,5 or 50μg in 250μl sterilized water) in GS-N groups one day before subcutaneous immunization of FMDV antigen. The control mice received 250μl water at the same time. Mice were immunized twice with 3-week's interval. Two weeks after the second immunization, blood samples were collected for detection of antigen-specific antibody response. In experiment B, Mice received three dose (1 dose/day) of orally-administrated GS-N (50,100 or 200μg in 250μl sterilized water) in GS-N groups before subcutaneous injection of influenza virus antigen. Mice administrated 250μl water were served as control. All of the mice were immunized twice with 3-week's interval. Two weeks after the boost, blood samples were collected for detection of antigen-specific antibody response. Intestine jejunum of mice were seperated and tissue paraffin sections were prepared for immunohistochemical analysis of IgA+ plasma cells. Results Results in experiment A showed that oral administration of GS-N (50μg) tended to enhance serum sepcific IgG response against FMDV immunization. In experiment B, orally-administrated GS-N (100 p.g/day for 3 days) significantly improved serum anti-influenza hemagglutinin (HA) IgG titer as well as IgG1, IgG2b and IgG3 levels (P< 0.05). The tissue paraffin section immunohistochemistry revealed increased numbers of IgA+ plasma cells in mouse jejunum in 100μg GS-N group when compared with those in control. Conclusions Oral administration of GS-N could improve serum antigen-specific antibody responses to immunization of FMDV serotype Asia 1 and influenza virus strain H3N2 antigens and increase jejunum tissure IgA+ plasma cells, suggesting that both systemic immune response and local mucosal immune response were enhanced when GS-N was administrated orally before subcutaneous vaccination.
2. Adjuvant effects of red ginseng saponins (GS-R)
Objective To investigate adjuvant effects of subcutaneously-injected and orally-administrated red ginseng saponins (GS-R) to immunization of ovalbumin (OVA) and FMDV serotype Asia 1 antigens, respectively. Methods In experiment A, 20 ICR mice were randomly divided into 4 groups with 5 mice each. Mice were immunized with OVA alone as control, or in combination with 10,50 or 250μg GS-R. Three weeks after the first immunization, mice were boosted with the same route. Blood samples were then collected two weeks after the boost for detection of serum OVA-specific antibody responses. In experiment B,35 mice were randomly seperated into 7 groups with 5 each. Mice in groups 2-7 received intragastric administration of GS-R (0,5,5 or 50μg in 250μl sterilized water) on days 1 and 21. Mice in group 1 administrated 250μl water were served as control. On days 2 and 22, all of the mice were injected with 100μl FMDV antigen. Mice were bleeded and serum samples were colleced for measurement of FMDV-specific IgG levels. Results and
Conclusions Results from experiments A and B revealed that there was no immunomodulatory activity recorded when GS-R was administrated orally or subcutaneously. Hence, GS-R showed no adjuvant effects against OVA and FMDV antigens.
3. Adjuvant effects of ginseng stem-leaf saponins (GSLS)
Objective To evaluate the immunomodulatory properties of orally-administrated GSLS against immunization of FMDV Asia 1 and influenza virus H3N2 antigens.
Methods A total of 2 experiments were carried out and described as follows. In experiment A, ICR mice were orally-administrated GSLS (0.5,5 or 50μg in 250μl sterilized water) in GSLS groups one day before subcutaneous immunization of FMDV antigen. The control mice received 250μl water at the same time. Mice were immunized twice with 3-week's interval. Two weeks after the second immunization, blood samples were collected for detection of antigen-specific antibody response. In experiment C, Mice received three dose (1 dose/day) of orally-administrated GSLS (50,100 or 200μg in 250μl sterilized water) in GSLS groups before subcutaneous injection of influenza virus antigen. Mice administrated 250μl water were served as control. All of the mice were immunized twice with 3-week's interval. Two weeks after the boost, blood samples were collected for detection of antigen-specific antibody response. Intestine jejunum of mice were seperated and tissue paraffin sections were prepared for immunohistochemical analysis of IgA+ plasma cells.
Results Results in experiment A revealed that oral administration of GSLS (0.5 or 50μg) tended to enhance serum FMDV-specific IgG level. In experiment B, oral administration of GSLS (50 jig/day for 3 days) significantly improved serum anti-influenza HA IgG titer as well as IgGl and IgG2b levels (P< 0.05). The tissue paraffin section immunohistochemistry showed increased numbers of IgA+ plasma cells in mouse jejunum in 50μg GSLS group when compared with those in control.
Conclusions GSLS administrated orally showed adjuvant effects to immunization of FMDV and influenza antigens. Considering its ralatively lower price than saponins extracted from ginseng root, the adjuvant effects of GSLS deserve further study in different animals against various pathogens.
4. Adjuvant effects of ginsenoside Re
Objective The present investigation was designed to evaluate the adjuvant effect of ginsenoside Re from the root of Panax ginseng on the immune responses in mice against hemagglutination (HA) protein of influenza virus. Methods Forty ICR mice were randomly distributed into five groups with 8 mice each. All animals were subcutaneously (s.c.) immunized twice with 100 ng HA, or with 10 ng of HA with or without Re (25,50 and 100μg) at 3 week intervals. Two weeks after the boost, blood samples were collected for measurement of serum HI titers using chicken red blood cells, and HA-specific IgG titers and isotype levels. Splenocytes were separated for the detection of lymphocyte proliferation responses and production of IFN-y and IL-5 in vitro. Results Re (50μg) augmented significantly higher responses of serum specific IgG, IgG1, IgG2a and IgG2b responses, HI titers, lymphocyte proliferation responses as well as IFN-y and IL-5 secretions to immunization of HA protein of influenza virus A/H3N2 in mice(P< 0.05). Conclusions Considering the adjuvant effect demonstrated in this study, Re deserve further studies for improving the quality of vaccines where mixed Thl/Th2 immune responses are needed.
5. Preparation of ginsenoside-based nanoparticles and evaluation of its adjuvant effects
Objective To prepare ginsenoside-based nanoparticles (ginsomes) and investigate its activity for up-regulation of immune response in mice. Methods Three ginseng extracts were used for screening out an optimal extract to make nanoparticles. They were GS-N, GSLS and GS-R. Different weight ratios of ginseng saponin: cholesterol:phospholipid by weight and the reaction temperature for the formation of ginsomes were selected to prepare nanoparticles. After dialysis against PBS for 48 h, solutions were applied onto carbon-coated grids, stained with 2% of phosphotungstic acid, and then envisaged under a transmission electron microscope. After confirmation by electron microscopy, two experiments were designed and described as follows. In the first experiment,35 mice were randomly divided into 7 groups of 5 animals. The mice were subcutaneously immunized twice at a 3-week interval with OVA (10μg) in saline alone or with OVA in saline containing GS-R (10,50,250μg) or ginsomes (10,50,250μg). Sera were collected 2 weeks after the booster immunization for measurement of OVA-specific IgG levels. In the second experiment, 32 mice were randomly divided into 4 groups of 8 animals each. Mice were subcutaneously immunized twice at weeks 0 and 3 with OVA (10μg) alone as a control, or in combination with GS-R (50μg), or Quil A (50μg), or ginsomes (50μg). Blood samples were collected from the orbital venous sinus at weeks 0,2 and 5 for measurement of serum OVA-specific IgG titers and the isotype responses. Splenocytes were harvested at week 5 for determination of lymphocyte proliferation and production of IFN-y and IL-5. Results Ginsomes were assembled during removal of the detergent by dialysis in presence of GS-R, cholesterol and phosphatidyl choline. The nanoparticles were spherical with diameters ranging from 50 to 100 nm, and contained ginsenosides Rb2, Rc, Rbl and Rd. When co-administered with a model antigen ovalbumin (OVA) in ICR mice, ginsomes at a dose range from 10 to 250μg promoted significantly higher IgG responses than OVA alone (P< 0.05). Co-administration of ginsomes with OVA also significantly increased the levels of specific IgGl, IgG2a, IgG2b and IgG3, as well as T and B lymphocyte proliferation in response to Con A, LPS and OVA than when OVA was used alone (P< 0.05). The enhanced IgG and the subclasse titers paralleled the increased production of IFN-y (Th1 cytokine) and IL-5 (Th2 cytokine). Conclusions Ginsomes as an adjuvant have up-regulated both Thl and Th2 immune responses.
All together, it could be concluded that supplement of GSLS, ginsenoside Re and ginsomes enhanced antigen-specific humoral and cell-mediated immunity to vaccination of various antigens. Both Thl and Th2 immune responses were up-regulated in mice. Besides, orally-administrated GS-N and GSLS tended to improve antigen-specific humoral immune response as well as local mucosal immunity. No adjuvant activity of GS-R was detected in the studies carried out here, whereas, it was the only kind of crude extracts out of three which was able to form nanoparticles (ginsomes) with cholesterol and phosphatidyl choline. The adjuvant properties of ginsomes were also confirmed in this work.
1.人参根皂甙(GS-N)免疫佐剂作用研究
目的观察人参根皂甙(GS-N)经胃肠道途径给药对皮下注射的口蹄疫病毒(FMDV)亚洲I型和流感病毒H3N2型灭活疫苗的免疫增强作用。方法实验一将小鼠分别灌服250μl含0.5、5或50μg GS-N溶液,对照组灌服250μl蒸馏水,1天后皮下注射100μlFMDV抗原,同样方法免疫2次,间隔3周；实验二：将小鼠连续3天,每天灌服250μl含50、100或200μg GS-N溶液,对照组灌服蒸馏水,然后皮下注射含10 ng HA的流感病毒疫苗,同样方法免疫2次,间隔3周。实验一和实验二小鼠均在二免后2周采血测抗体水平,实验二中小鼠取小肠空肠中段进行免疫组化观察IgA+浆细胞数量变化。结果实验一小鼠灌胃1次50μg GS-N能提高血清抗FMDV抗体IgG水平,但和对照组比较无统计学差异。实验二中每天灌服一次100μg GS-N,连续3天的小鼠血清中抗流感HA特异性抗体IgG效价,IgG1、IgG2b和IgG3水平均显著提高(P<0.05),且肠道IgA+浆细胞明显增多,说明黏膜免疫功能得到增强。结论1次口服GS-N虽然提高了小鼠对皮下注射FMD抗原的免疫反应,但无统计学意义；连续3天口服GS-N(每天剂量为50、100或200μg)可以显著提高小鼠对流感疫苗的免疫反应,以及增加肠道IgA+浆细胞数量。
2.红参皂甙(GS-R)免疫佐剂作用研究
目的观察疫苗中添加红参皂甙(GS-R)或口服GS-R对疫苗接种有无免疫增强作用。方法实验一：将20只ICR小鼠随机分成4组,每组5只。以OVA(每只免疫剂量为10μg)、为模式抗原,分别设含人参皂甙10、50或250μg GS-R组,对照组仅免疫10μg OVA。免疫2次,间隔3周。二次免疫后2周采血,检测血清抗OVA抗体IgG水平。实验二：实验组小鼠分别在实验第1天和第21天灌胃250μl含0.5、5或50μg GS-R的蒸馏水溶液,对照组小鼠同时灌服250μl蒸馏水。在第2天和第22天,每组小鼠均皮下注射100μlFMDV抗原。即实验组小鼠在每次免疫前灌胃一次GS-R,小鼠免疫2次,间隔3周。二免后2周(实验第36天),小鼠采血分离血清,检测血清抗FMDV抗体IgG水平。结果与结论实验一和实验二结果表明,红参皂甙和抗原混合以及在动物对注射OVA或FMDV抗原前口服均不能增强相应的的免疫应答,因而红参皂甙不具有免疫佐剂作用。
3.人参茎叶皂甙(GSLS)免疫佐剂作用研究
目的口服人参茎叶皂甙(GSLS)对皮下注射FMDV抗原或者流感疫苗的免疫增强作用。方法实验一：实验组小鼠在免疫前一天灌服0.5、5或50μgGSLS,对照组灌服250μl蒸馏水。小鼠皮下注射100μlFMDV抗原,同样方法免疫2次,间隔3周。二免后2周采血分离血清,检测血清抗FMDV抗体IgG水平；实验二：实验组小鼠在免疫前连续三天灌服50、100或200μgGSLS,对照组同时灌服蒸馏水,完成3天灌胃后,小鼠皮下注射含10 ng HA抗原的流感疫苗,同样方法免疫2次,间隔3周。在二免后2周采血测血清抗HA特异性抗体IgG滴度及其亚类水平,并取小鼠小肠空肠中段制作组织石蜡切片,免疫组化比较口服GSLS组和对照组IgA+浆细胞数量。结果实验一：小鼠2次免疫前均灌胃一次0.5或50μgGSLS能提高血清抗FMDV抗体IgG水平,但和对照组比较无统计学差异。实验二：连续口服3天,每天灌服50μgGSLS使小鼠抗流感HA特异性抗体IgG效价,IgG1和IgG2b水平均显著提高(P<0.05),小鼠空肠中段组织中IgA+浆细胞明显增多,提示黏膜免疫功能得到增强。总体上,通过抗体及其亚类和肠道IgA+浆细胞变化可知,口服低剂量人参茎叶皂甙提高了机体体液免疫和细胞免疫的同时,还增强胃肠道局部黏膜免疫水平,即口服GSLS对皮下注射FMD抗原和流感H3N2病毒抗原均显示一定的免疫调节作用。结论小鼠灌胃1次GSLS能提高机体对皮下注射FMDV抗原的体液免疫水平,但无统计学意义；连续3天口服GSLS可提高小鼠对流感疫苗的抗体免疫反应,并增加肠道IgA+浆细胞数量。
4.人参皂甙单体Re免疫佐剂作用研究
目的观察人参皂甙Re对流感病毒疫苗的免疫佐剂作用。方法40只ICR小鼠随机分成5组,每组8只。组1：10 ng HA(注射含10 ng HA抗原的流感疫苗,下同)；组2：100 ng HA组；组3：10 ng HA+25μg Re组；组4：10 ng HA+50μg Re；组5：10 ng HA+100μg Re组。小鼠腹部皮下注射200μl含上述组分的疫苗,免疫2次,间隔3周。二免后2周摘眼球采血分离血清,间接ELISA法检测血清抗血凝素HA抗体效价及抗体亚类IgG1、IgG2a、IgG2b和IgG3水平,并测血凝抑制效价HI；无菌分离脾淋巴细胞后,采用MTT法检测淋巴细胞增殖和ELISA法检测Con A刺激培养上清中细胞因子水平。结果在含10 ng HA的流感疫苗中添加50μg Re能显著提高ICR小鼠血清中抗HA特异性IgG效价,IgG1、IgG2a和IgG2b水平,以及HI效价和淋巴细胞转化能力(P<0.05),显著刺激淋巴细胞分泌细胞因子IFN-γ和IL-5(P<0.05)。结论流感疫苗中添加人参皂甙Re能显著提高受免小鼠特异性抗体IgG和抗体亚类水平,增强血清HI效价,显著促进脾淋巴细胞转化能力和分泌细胞因子水平,表明Re对流感疫苗免疫具有佐剂作用。
5.人参皂甙的纳米颗粒(Ginsomes)制备及其佐剂作用研究
目的研究人参皂甙纳米颗粒(Ginsomes)的制备方法、颗粒物理形态及其对模式抗原OVA的免疫佐剂作用。方法分别选取三种人参皂甙：人参根皂甙、人参茎叶皂甙和红参皂甙,与胆固醇、卵磷脂(胆固醇和卵磷脂预先溶于Mega-10)形成不同配比方案,将混合溶液置于不同温度(10、20、30、40和50℃)孵育2 h,此后对PBS透析48 h以移除去污剂Mega-10,此时,皂甙、胆固醇和卵磷脂形成的纳米颗粒产物采用2%磷钨酸溶液负染后进行透射电镜鉴定。电镜确认纳米颗粒形成后,设计ICR小鼠试验检验纳米颗粒的免疫佐剂作用。在实验一中选取不同人参皂甙浓度的红参皂甙和Ginsomesl：含人参皂甙分别为10、50或250μg)与无内毒素污染的卵清白蛋白(OVA)混合后免疫小鼠,间隔3周进行一次加强免疫,二免后2周取血清进行抗OVA特异性抗体水平测定；实验二：将32只ICR小鼠随机分成4组,每组8只。以OVA(每只免疫剂量为10μg)为模式抗原,分别设含人参皂甙50μg的Ginsomes组,含人参皂甙50μg的人参皂甙GS组和50μg Quil A组,对照组仅免疫OVA。抗原和佐剂混合均匀后,腹部皮下注射疫苗,在第1天和第21天分别进行2次免疫。第14天从小鼠眼静脉窦采血,第35天摘眼球采血并分离脾脏淋巴细胞。间接ELISA法检测血清抗OVA抗体效价及抗体亚类IgG1、IgG2a、IgG2b和IgG3水平；取脾淋巴细胞,采用MTT法检测淋巴细胞增殖和ELISA法检测OVA刺激培养上清中细胞因子水平。结果透射电子显微镜观察后发现,三种不同来源人参皂甙中只有红参皂甙能制备形成Ginsomes;形成纳米颗粒的最佳条件为红参皂甙：胆固醇：卵磷脂以质量比20：1：1混合,纳米颗粒平均粒径为：87.8±9.4nm。小鼠免疫试验结果表明,实验一中各个浓度颗粒均诱导出显著高于人参总皂甙免疫小鼠血清抗体水平的特异性IgG(P<0.05),实验二中含50μg人参皂甙的纳米颗粒能显著提高ICR小鼠血清中抗OVA特异性IgG效价,IgG1、IgG2a、IgG2b和IgG3抗体水平,以及受特异性抗原OVA刺激的淋巴细胞转化能力和细胞因子IFN-γ和IL-5的分泌(P<0.05),说明人参皂甙纳米颗粒作为免疫佐剂能同时上调Th1和Th2型免疫应答反应。结论红参皂甙制备的纳米颗粒Ginsomes粒径约为90 nm,OVA与纳米颗粒混合免疫可显著提高血清抗体IgG及其亚类水平,并显著增强小鼠脾淋巴细胞受OVA刺激的增殖能力和分泌IFN-γ和IL-5水平。
综上所述,(1)小鼠皮下注射FMD抗原前灌胃1次GS-N提高了体液免疫水平,但无统计学意义；注射流感疫苗前连续3天口服GS-N可显著提高小鼠抗流感抗原的免疫反应,并增加肠道IgA+浆细胞数量。(2)OVA抗原和GS-R混合或皮下注射口蹄疫疫苗前灌服1次GS-R均未显著增强机体对抗原特异性免疫反应,因而红参皂甙不具有免疫佐剂作用。(3)小鼠口服1次GSLS能提高机体对皮下注射FMD抗原的体液免疫水平,但无显著性差异；连续3天口服GSLS可提高小鼠对流感疫苗的体液反应水平,增加肠道,IgA+浆细胞数量。(4)流感疫苗中添加人参皂甙Re显著促进小鼠抗体IgG和抗体亚类水平,提高血清HI效价,并显著增强脾淋巴细胞转化能力和促进IFN-γ和IL-5的分泌,表明Re对流感疫苗免疫具有佐剂作用。(5)红参皂甙与胆固醇、卵磷脂在质量比20：1：1配比条件下可制备Ginsomes,在OVA溶液中添加纳米颗粒可显著提高小鼠血清OVA特异性IgG及其亚类水平,显著增强小鼠脾淋巴细胞增殖能力和细胞因子水平,表明Ginsomes对OVA具有免疫佐剂作用。
As a traditional medicine, ginseng has been used for thousands of years in China. According to the latest investigation, saponins extracted from Panax ginseng show adjuvant effects against various antigens. However, almost all of the reported ginseng adjuvants were originated from ginseng roots. In the present thesis, normal ginseng saponins (extracted from ginseng root) (GS-N), red ginseng (steam-treated ginseng root) saponins (GS-R) and ginseng stem-leaf saponins (GSLS), as well as ginsenoside Re and ginsenoside-based nanoparticles were studied in order to further evaluate the adjuvant proterties of saponins originated from different ginsengs.
1. Adjuvant effects of normal ginseng saponins (GS-N)
Objective To measure the immunomodulatory activity of orally-administrated normal ginseng saponins (GS-N) against subcutaneously-injected FMDV (serotype Asia 1) and influenza virus strain H3N2 antigens. Methods Two experiments were designed and described as follows. In experiment A, ICR mice were orally-administrated GS-N (0.5,5 or 50μg in 250μl sterilized water) in GS-N groups one day before subcutaneous immunization of FMDV antigen. The control mice received 250μl water at the same time. Mice were immunized twice with 3-week's interval. Two weeks after the second immunization, blood samples were collected for detection of antigen-specific antibody response. In experiment B, Mice received three dose (1 dose/day) of orally-administrated GS-N (50,100 or 200μg in 250μl sterilized water) in GS-N groups before subcutaneous injection of influenza virus antigen. Mice administrated 250μl water were served as control. All of the mice were immunized twice with 3-week's interval. Two weeks after the boost, blood samples were collected for detection of antigen-specific antibody response. Intestine jejunum of mice were seperated and tissue paraffin sections were prepared for immunohistochemical analysis of IgA+ plasma cells. Results Results in experiment A showed that oral administration of GS-N (50μg) tended to enhance serum sepcific IgG response against FMDV immunization. In experiment B, orally-administrated GS-N (100 p.g/day for 3 days) significantly improved serum anti-influenza hemagglutinin (HA) IgG titer as well as IgG1, IgG2b and IgG3 levels (P< 0.05). The tissue paraffin section immunohistochemistry revealed increased numbers of IgA+ plasma cells in mouse jejunum in 100μg GS-N group when compared with those in control. Conclusions Oral administration of GS-N could improve serum antigen-specific antibody responses to immunization of FMDV serotype Asia 1 and influenza virus strain H3N2 antigens and increase jejunum tissure IgA+ plasma cells, suggesting that both systemic immune response and local mucosal immune response were enhanced when GS-N was administrated orally before subcutaneous vaccination.
2. Adjuvant effects of red ginseng saponins (GS-R)
Objective To investigate adjuvant effects of subcutaneously-injected and orally-administrated red ginseng saponins (GS-R) to immunization of ovalbumin (OVA) and FMDV serotype Asia 1 antigens, respectively. Methods In experiment A, 20 ICR mice were randomly divided into 4 groups with 5 mice each. Mice were immunized with OVA alone as control, or in combination with 10,50 or 250μg GS-R. Three weeks after the first immunization, mice were boosted with the same route. Blood samples were then collected two weeks after the boost for detection of serum OVA-specific antibody responses. In experiment B,35 mice were randomly seperated into 7 groups with 5 each. Mice in groups 2-7 received intragastric administration of GS-R (0,5,5 or 50μg in 250μl sterilized water) on days 1 and 21. Mice in group 1 administrated 250μl water were served as control. On days 2 and 22, all of the mice were injected with 100μl FMDV antigen. Mice were bleeded and serum samples were colleced for measurement of FMDV-specific IgG levels. Results and
Conclusions Results from experiments A and B revealed that there was no immunomodulatory activity recorded when GS-R was administrated orally or subcutaneously. Hence, GS-R showed no adjuvant effects against OVA and FMDV antigens.
3. Adjuvant effects of ginseng stem-leaf saponins (GSLS)
Objective To evaluate the immunomodulatory properties of orally-administrated GSLS against immunization of FMDV Asia 1 and influenza virus H3N2 antigens.
Methods A total of 2 experiments were carried out and described as follows. In experiment A, ICR mice were orally-administrated GSLS (0.5,5 or 50μg in 250μl sterilized water) in GSLS groups one day before subcutaneous immunization of FMDV antigen. The control mice received 250μl water at the same time. Mice were immunized twice with 3-week's interval. Two weeks after the second immunization, blood samples were collected for detection of antigen-specific antibody response. In experiment C, Mice received three dose (1 dose/day) of orally-administrated GSLS (50,100 or 200μg in 250μl sterilized water) in GSLS groups before subcutaneous injection of influenza virus antigen. Mice administrated 250μl water were served as control. All of the mice were immunized twice with 3-week's interval. Two weeks after the boost, blood samples were collected for detection of antigen-specific antibody response. Intestine jejunum of mice were seperated and tissue paraffin sections were prepared for immunohistochemical analysis of IgA+ plasma cells.
Results Results in experiment A revealed that oral administration of GSLS (0.5 or 50μg) tended to enhance serum FMDV-specific IgG level. In experiment B, oral administration of GSLS (50 jig/day for 3 days) significantly improved serum anti-influenza HA IgG titer as well as IgGl and IgG2b levels (P< 0.05). The tissue paraffin section immunohistochemistry showed increased numbers of IgA+ plasma cells in mouse jejunum in 50μg GSLS group when compared with those in control.
Conclusions GSLS administrated orally showed adjuvant effects to immunization of FMDV and influenza antigens. Considering its ralatively lower price than saponins extracted from ginseng root, the adjuvant effects of GSLS deserve further study in different animals against various pathogens.
4. Adjuvant effects of ginsenoside Re
Objective The present investigation was designed to evaluate the adjuvant effect of ginsenoside Re from the root of Panax ginseng on the immune responses in mice against hemagglutination (HA) protein of influenza virus. Methods Forty ICR mice were randomly distributed into five groups with 8 mice each. All animals were subcutaneously (s.c.) immunized twice with 100 ng HA, or with 10 ng of HA with or without Re (25,50 and 100μg) at 3 week intervals. Two weeks after the boost, blood samples were collected for measurement of serum HI titers using chicken red blood cells, and HA-specific IgG titers and isotype levels. Splenocytes were separated for the detection of lymphocyte proliferation responses and production of IFN-y and IL-5 in vitro. Results Re (50μg) augmented significantly higher responses of serum specific IgG, IgG1, IgG2a and IgG2b responses, HI titers, lymphocyte proliferation responses as well as IFN-y and IL-5 secretions to immunization of HA protein of influenza virus A/H3N2 in mice(P< 0.05). Conclusions Considering the adjuvant effect demonstrated in this study, Re deserve further studies for improving the quality of vaccines where mixed Thl/Th2 immune responses are needed.
5. Preparation of ginsenoside-based nanoparticles and evaluation of its adjuvant effects
Objective To prepare ginsenoside-based nanoparticles (ginsomes) and investigate its activity for up-regulation of immune response in mice. Methods Three ginseng extracts were used for screening out an optimal extract to make nanoparticles. They were GS-N, GSLS and GS-R. Different weight ratios of ginseng saponin: cholesterol:phospholipid by weight and the reaction temperature for the formation of ginsomes were selected to prepare nanoparticles. After dialysis against PBS for 48 h, solutions were applied onto carbon-coated grids, stained with 2% of phosphotungstic acid, and then envisaged under a transmission electron microscope. After confirmation by electron microscopy, two experiments were designed and described as follows. In the first experiment,35 mice were randomly divided into 7 groups of 5 animals. The mice were subcutaneously immunized twice at a 3-week interval with OVA (10μg) in saline alone or with OVA in saline containing GS-R (10,50,250μg) or ginsomes (10,50,250μg). Sera were collected 2 weeks after the booster immunization for measurement of OVA-specific IgG levels. In the second experiment, 32 mice were randomly divided into 4 groups of 8 animals each. Mice were subcutaneously immunized twice at weeks 0 and 3 with OVA (10μg) alone as a control, or in combination with GS-R (50μg), or Quil A (50μg), or ginsomes (50μg). Blood samples were collected from the orbital venous sinus at weeks 0,2 and 5 for measurement of serum OVA-specific IgG titers and the isotype responses. Splenocytes were harvested at week 5 for determination of lymphocyte proliferation and production of IFN-y and IL-5. Results Ginsomes were assembled during removal of the detergent by dialysis in presence of GS-R, cholesterol and phosphatidyl choline. The nanoparticles were spherical with diameters ranging from 50 to 100 nm, and contained ginsenosides Rb2, Rc, Rbl and Rd. When co-administered with a model antigen ovalbumin (OVA) in ICR mice, ginsomes at a dose range from 10 to 250μg promoted significantly higher IgG responses than OVA alone (P< 0.05). Co-administration of ginsomes with OVA also significantly increased the levels of specific IgGl, IgG2a, IgG2b and IgG3, as well as T and B lymphocyte proliferation in response to Con A, LPS and OVA than when OVA was used alone (P< 0.05). The enhanced IgG and the subclasse titers paralleled the increased production of IFN-y (Th1 cytokine) and IL-5 (Th2 cytokine). Conclusions Ginsomes as an adjuvant have up-regulated both Thl and Th2 immune responses.
All together, it could be concluded that supplement of GSLS, ginsenoside Re and ginsomes enhanced antigen-specific humoral and cell-mediated immunity to vaccination of various antigens. Both Thl and Th2 immune responses were up-regulated in mice. Besides, orally-administrated GS-N and GSLS tended to improve antigen-specific humoral immune response as well as local mucosal immunity. No adjuvant activity of GS-R was detected in the studies carried out here, whereas, it was the only kind of crude extracts out of three which was able to form nanoparticles (ginsomes) with cholesterol and phosphatidyl choline. The adjuvant properties of ginsomes were also confirmed in this work.
引文
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