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以石蓮花與諾麗萃取物開發疫苗佐劑 = Development of va...

國立高雄大學生物科技研究所

以石蓮花與諾麗萃取物開發疫苗佐劑 = Development of vaccine adjuvant using Houseleek and Noni extracts

紀錄類型:	書目-語言資料,印刷品 : 單行本
並列題名:	Development of vaccine adjuvant using Houseleek and Noni extracts
作者:	簡子齡,
其他團體作者:	國立高雄大學
出版地:	[高雄市]
出版者:	撰者;
出版年:	2009[民98]
面頁冊數:	88面圖、表 : 30公分;
標題:	ApxIA
標題:	Actinobacillus pleuropneumoniae
電子資源:	http://handle.ncl.edu.tw/11296/ndltd/73033268001829841342
附註:	指導教授：楊文仁
附註:	參考書目：面
摘要註:	佐劑(adjuvant)是一種能增加疫苗免疫原性的免疫促進劑，其不具有抗原性，主要的功用是延長抗原的作用時間、刺激巨噬細胞(macrophage)吞噬抗原以及非特異性地誘發免疫反應，以達到增強抗原免疫原性、改變免疫反應類型與維持長時間的疫苗保護效力等目的。本實驗室先前的研究結果顯示，以豬胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae, AP)外毒素結構蛋白ApxIA製成的pcDNA-IA DNA疫苗，免疫小鼠後能使其對抗第1血清型菌株感染的保護力達70 %。為了提升此疫苗的效力，擬藉由添加佐劑來達成此目的。文獻指出，石蓮花萃取物能刺激Th細胞和B細胞的增生、增強巨噬細胞吞噬能力且對癌細胞有抑制效果；而諾麗果實具有抗氧化、抗腫瘤並且能提升TNF-α、IL-1β、IL-10、IFN-γ和NO的表現，有助於免疫系統的調節。因此，本研究以石蓮花(H)、諾麗熟果(NI)與諾麗青果(NG)三種材料，評估其水萃(W)、煮萃(B)與粗萃多醣(P)之萃取物發展為佐劑之可行性。首先利用水萃、煮萃與粗萃多醣方法獲得共12種萃取物，利用phenol-sulfuric acid和Bradford protein assay法分析萃取物總糖與蛋白質含量。將萃取物添加進培養基中刺激J774A.1巨噬細胞，分析各萃取物誘發的TNF-α、IL-6以及IL-12細胞激素的表現量，評估萃取物誘導的免疫途徑。從中挑選出六個能有效誘發Th1與(或)Th2免疫途徑的萃取物，包含H-WP、H-B、NI-W、NI-WP、NG-W與NG-B，做為pcDNA-IA DNA疫苗佐劑免疫小鼠，以ELISA檢測專一性抗原的抗體效價，其中添加萃取物的六個免疫組可達51200的抗體效價。進一步分析其抗體亞型IgG1與IgG2a含量，結果顯示H-B和NI-W能提升IgG1含量，誘導Th2免疫途徑；NI-WP與NG-W則能提升IgG2a的含量，誘導Th1免疫途徑。再使用MTT試驗進行萃取物分析，發現H-B具有細胞毒性，較不適合用來發展為佐劑。最後以三次免疫後小鼠的脾臟細胞，進行脾臟細胞增生試驗與TNF-α、IL-6以及IL-12細胞激素表現量分析。結果顯示，NI-W和NG-B能有效誘發Th2免疫途徑，H-WP、NI-WP與NG-W則能夠有效同時誘發Th1和Th2的免疫途徑，具有開發為佐劑之潛能。未來可進一步測試萃取物與疫苗的搭配比例，尋找最佳的使用劑量，進一步應用於不同的疫苗類型中。 Adjuvant is an immune stimulator that can enhance the immunogenicity of vaccine but itself does not have the antigenicity. The major function of adjuvant including prolongs the reaction time of antigen, stimulates macrophage engulfing antigen as well as non-specifically induces immune responses to achieve many goals including that enhances antigen immunogenicity, changes the type of immune response, and maintains the long term potency of vaccine. Previous studies in our laboratory showed that a DNA vaccine, pcDNA-IA, containing Actinobacillus pleuropneumoniae ApxIA provided 70% protective efficacy against serotype 1 challenge in mice. In this study, adjuvant administration was used to improve the vaccine efficacy. Several studies showed that the extracts of houseleek could stimulate the proliferation of T helper cells and B cells, enhance the engulfing activity of macrophage, and inhibit the growth of cancer cells. The fruit of Noni has anti-oxidation and anti-tumor activities and enhance the expression of TNF-, IL-1, IL-10, IFN- and nitrogen oxide. It is useful for the regulation of immune system. Therefore, this study using houseleek (H), Noni mature fruit (NI) and Noni green unmature fruit (NG) to evaluate the potential of that used as vaccine adjuvant from their water extracts, boiling extracts and crude polysaccharide extracts. Twelve different extracts were obtained and the total sugar and protein content of extracts were assayed by phenol-sulfuric acid method and Bradford protein analysis method, respectively. The macrophage J774A.1 incubated with different extracts and then measured the expression levels of cytokines TNF-α, IL-6 and IL-12 to evaluate the immune pathway that these extracts induced. Six extracts were chosen which could induce Th1 and/or Th2 immune pathway, including H-WP, H-B, NI-W, NI-WP, NG-W and NG-B. These extracts were used as adjuvant in pcDNA-IA DNA vaccine for mice immunization. The antibody titers specific to ApxIA of extracts addition groups could reach 51200 by ELISA analysis. The results of antibody IgG subclass analysis showed that H-B and NI-W could enhance IgG1 and induce Th2 pathway; NI-WP and NG-W could increase IgG2a content and toward Th1 pathway. Cytotoxicity analysis of the extracts by MTT assay showed that the H-B extract has cytotoxicity and less suitable to use as adjuvant. The splenocytes of mice which immunized three times with DNA vaccine plus extracts were used for splenocyte proliferation assay and cytokines TNF-α, IL-6 and IL-12 analyses. The results showed that NI-W and NG-B could effectively induce Th2 immune pathway; H-WP, NI-WP and NG-W could effectively induce both of Th1 and Th2 immune responses. It indicated that these extracts have the potential for adjuvant development. It could be further test the most suitable ratio of extracts to vaccine, the best dosage of administration, and their application in different types of vaccines.

以石蓮花與諾麗萃取物開發疫苗佐劑 = Development of vaccine adjuvant using Houseleek and Noni extracts
簡, 子齡

以石蓮花與諾麗萃取物開發疫苗佐劑 = Development of vaccine adjuvant using Houseleek and Noni extracts / 簡子齡撰 - [高雄市] : 撰者, 2009[民98]. - 88面 ; 圖、表 ; 30公分.
指導教授：楊文仁參考書目：面.
ApxIAActinobacillus pleuropneumoniae

以石蓮花與諾麗萃取物開發疫苗佐劑 = Development of vaccine adjuvant using Houseleek and Noni extracts
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