Evaluation on the allergenicity of phytase-transgenic rice (Tainung NO.67) using in vitro and in vivo models

Abstract

稻米(Oryza sativa L.)是亞洲國家重要主食之一，其胚乳中14-16 kDa水/鹽溶性蛋白質，為公認的稻米主要過敏原(rice allergen, RA)，對米過敏者，RA能與其IgE抗體產生交互連結反應(crosslink)，造成食物過敏或誘發氣喘反應。本實驗為探討植酸酶基因轉殖稻米是否增加其胚乳蛋白質之過敏可能性，以2001年 FAO/WHO過敏可能性決定樹為評估架構，對基因轉殖前後稻米樣品進行體外模擬胃液(simulated gastric fluid, SGF)消化的耐受性試驗，並利用過敏之免疫學原理，進一步探討植酸酶基因轉殖前後，稻米對脾臟細胞分泌TH1/TH2細胞激素、巨噬細胞分泌發炎媒介物、血清中RA特異性及非特異性抗體含量之影響，並依免疫指標變化，判斷植酸酶基因轉殖後是否增加其過敏性，評估試驗整體架構分為(一) 植酸酶基因轉殖稻米蛋白模擬胃液消化耐受性實驗; (二) 植酸酶基因轉殖稻米過敏性反應之動物體外實驗(in vitro)評估; (三) 植酸酶基因轉殖稻米過敏性反應之動物體內實驗(in vivo)評估。 模擬胃液消化耐受性評估結果顯示，植酸酶基因轉殖前、後稻米之水萃物、鹽萃物、醇萃物、鹼萃物、酸萃物等五種區分物中所含蛋白質，在本實驗條件下皆能有不同程度之水解現象，尤其水萃物及鹽萃物中14-16 kDa蛋白在SGF作用2小時後可被完全水解，植酸酶基因轉殖後並未增加稻米胚乳蛋白之消化耐受性。 在動物體外過敏性反應評估方面，植酸酶基因轉殖前、後稻米之五種區分物，對BALB/c雌鼠初代脾臟細胞皆不具刺激其增生或具毒性作用。進一步分析發現，植酸酶基因轉殖前、後稻米之各萃取區分物其經消化處理後樣品，並未顯著刺激BALB/c雌鼠初代脾臟細胞之TH2細胞激素(IL-4、IL-5)分泌。 本實驗選擇14-16 kDa稻米主要過敏原蛋白存在之水、鹽萃取區分物進行動物體內過敏性反應評估實驗，經腹腔注射小鼠四次以致敏小鼠，結果發現，植酸酶基因轉殖稻米之水、鹽萃取區分物，並未增加BALB/c雌鼠初代脾臟細胞自發性分泌或抗原刺激分泌TH2細胞激素(IL-4、IL-5)，尤其IL-4在消化處理前、後的轉殖前、後樣品組別間均無顯著差異，血清中非特異性抗體含量，尤其是IgE，抗體含量雖隨致敏次數增加而上升，但比較相同時間點之抗體分泌量，水萃取區分物樣品在消化處理前、後的轉殖前、後樣品組別間均無顯著差異，基因轉殖後鹽萃取物消化後樣品則顯著低於基因轉殖前消化後樣品組別，血清RA特異性抗體含量，抗體含量隨致敏次數增加而上升，但比較犧牲時之抗體分泌量，消化後轉殖前、後各組之間並無顯著差異，植酸酶基因轉殖稻米之水、鹽萃取區分物，並未增加BALB/c雌鼠腹腔巨噬細胞自發性及外來抗原(LPS)刺激分泌發炎相關媒介物(IL-1β、IL-6、TNF-α、NO)。綜合本研究動物體外及體內實驗結果，顯示本研究所選取之植酸酶基改稻米，與其母本台農67號稻米之過敏性比較並無顯著性變化。

Rice (Oryza sativa L.) is an important staple food in Asia. However, the water/salt extracts of 14-16 kDa protein from rice endosperm have been recognized as one of the major allergens in rice. The 14-16 kDa rice allergen (RA) can be crosslink with the IgE antibody in subjects whom allergic to rice and may cause food allergy and asthma. The goal of this study was to evaluate the allergenicity of phytase transgenic rice. Based on the FAO/WHO (2001) decision tree concerning the allergenic possibility of GMOs, this study was divided into three topics including 1) the digestibility resistance assay of protein from phytase transgenic and non-transgenic rice using simulated gastric fluid (SGF), 2) the allergenic assay of phytase transgenic rice (PTR) using primary immune cell culture model (in vitro), 3) the allergenic assessment of PTR using animal model (in vivo). The experimental design concerning allergenicity using immune cell cultures and animal model was based on immunologic theory. Therefore, the effects of phytase transgenic rice on the changes of TH1/TH2 cytokine secretion by spleen cells, the titers of specific or non-specific antibody, and the secretion of inflammatory mediators by peritoneal macrophages were determined. The results from SDS-PAGE showed that five protein fractions sequentially extracted from PTR and non-PTR (TN-67 rice) using water, salt, isopropanol, alkali, and acid could be efficiently digested by SGF. Especially, the 14-16 kDa proteins of water- and salt-extracted fractions from PTR and non-PTR were completely degraded by SGF for 2 hours incubation during the digestibility resistance assay. The results demonstrated that the proteins from phytase-transgenic rice did not increase their tolerance to the digestion of SGF. The primary immune cells assay from female BALB/c mice showed that all of five extracted fractions from PTR and non-PTR (TN-67 rice) could not significantly affect the proliferation or cyto-toxicity of splenocytes in vitro. According to the TH1/TH2 cytokine secretions, the extracts from PTR and non-PTR, whether digested by SGF or not, did not significantly change the Th2 cytokine secretions such as IL-4 and IL-5. To evaluate the allergenicity of the rice, the fractions of water- and salt-extracts from PTR and non-PTR, which consist of 14-16 kDa protein, were selected and administrated to female BALB/c mice. The mice were injected intraperitoneally with the extracts to induce the allergenicity bi-weekly for four times. The results demonstrated that PTR and non-PTR extracts, whether digested by SGF or not, did not significantly change the TH2 cytokine (IL-4, IL-5) secretions in vivo, especially IL-4. Titers of serum non-specific antibody (IgE, IgG, IgM, and IgA) increased as the experimental period extended. However, the administration with PTR extracts did not significantly change serum titers of non-specific antibody, especially IgE, compared to those from non-PTR at the same 8th week of experimental period. Administration with PTR extracts digested by SGF also did not significantly change serum titers of RA-specific antibody (IgE, IgG1, IgG2a), especially IgE, compared to those from non-PTR at the same 8th week of experimental period. Furthermore, PTR and non-PTR extracts, whether digested by SGF or not, did not significantly change the secretions of inflammatory mediators by peritoneal macrophages in vivo. In conclusion, the results from both in vitro and in vivo studies suggest that PTR selected in this study did not increase the allergenic possibility.