臺灣博碩士論文加值系統

金線連免疫調節蛋白 (IPAF) 純化自台灣金線連 (Anoectochilus formosanus Hayata)，可透過細胞表面受器 TLR4 活化小鼠巨噬細胞，以及提高 B 淋巴細胞的增生，然而，IPAF 活化 B 淋巴細胞之機制及其活化作用尚未明瞭。因此，本研究探討 IPAF 對 B 淋巴細胞之免疫調節作用，以及其活化 B 淋巴細胞與 TLR2/TLR4 訊息傳導路徑之關係，進一步分離 IPAF 中的有效成分，並建立 IPAF 酵素連結免疫分析定量平台。結果發現，IPAF 可提高小鼠 B 淋巴細胞表面的 CD69 及 MHC class II 表現量，也可促進 B 淋巴細胞之細胞增生。同時發現，IPAF 對於 B 淋巴細胞分泌 IgM 抗體具有時間及濃度依存效應，但 IgG 抗體並無明顯增加，推測 IPAF 為胸腺非依賴性第一型抗原，不需要 T 淋巴細胞即能直接活化 B 淋巴細胞。另一方面，IPAF 同樣可活化 TLR2-/- 及 TLR4-/- 小鼠之 B 淋巴細胞，顯示 TLR2 及 TLR4 並非 IPAF 活化 B 淋巴細胞之主要訊息傳導路徑。進一步利用逆相高效能液相層析技術分析 IPAF，可將其分離出 4 個蛋白質樣品，發現其中之 IPAFP3 (14.0 kDa，pI = 8.4) 具有與 IPAF 相似之活性，可促進 B 淋巴細胞的增生及抗體 IgM 的產生。
而在IPAF 酵素連結免疫分析平台之建立方面，利用多株抗體 (J0) 作為捕捉抗體，單株抗體 (I) 作為偵測抗體，進行 IPAF 濃度與吸光值的簡單迴歸分析，可得吸光值與濃度校正曲線 (y = 0.0041 x + 0.6516，r2 = 0.9894，p<0.05)，定量濃度範圍在 6.25 - 400 μg/mL，最低偵測極限為 0.57 μg/mL，其回收率則為 97.8%。實際定量市售金線連茶包與新鮮金線連植株根、莖及葉中 IPAF 之含量，結果發現，其 IPAF 濃度分別為 9.5 mg/g、1323.5 μg/g、87.4 μg/g 及 314.7 μg/g。因此，此方法有助於進一步定量金線連樣品及相關產品中免疫調節蛋白 IPAF 之含量。

IPAF, the immunomodulatory protein complex purified previously from Anoectochilus formosanus Hayata, was capable to activate murine macrophages through TLR4 pathway and to stimulate B lymphocytes proliferation. However, the activation mechanism of IPAF on B lymphocytes remained unclear. The objectives of this study aimed to evaluate the immunoregulatory effects of IPAF on murine B lymphocytes, to investigate the activation of B lymphocyte by TLR2/TLR4 signaling pathways, to further identify the effective component of IPAF proteins and to establish an ELISA assay system to determine IPAF. Our results demonstrated that IPAF could activate murine B lymphocytes. It could up-regulated the expression of surface marker CD69 and MHC class II, stimulated the cell proliferation. Additionally, IPAF increased IgM but not IgG antibody secretion of murine B lymphocytes in a time and dose dependent manner, suggesting IPAF a type-1 thymus-independent antigen to activate B lymphocytes without the assistance of T lymphocytes. On the other hand, IPAF was capable to activate the B lymphocytes from TLR2- and TLR4-deficient mice, which implied that IPAF did not activate through TLR4 and TLR2 signaling pathway in murine B lymphocytes. Furthermore, IPAF could be separated into 4 proteins by RP-HPLC. Among these 4 proteins, IPAFP3 (14.0 kDa, pI = 8.4) showed similar activity with IPAF to increase B lymphocytes proliferation and IgM secretion. Finally, we established a sandwich ELISA platform for IPAF determination by using a polyclonal Ab (J0) as the capture antibody and a biotinylated mAb (I) as the detection antibody. The calibration curve gave a linear line (y = 0.0041 x + 0.6516, r2 = 0.9894, p<0.05) and a dynamic range with IPAF concentrations between 6.25 - 400 μg/mL. Its detection limit was 0.57 μg/mL and the recovery was 97.8%. This method was also used to measure the IPAF content in commercial tea bags and fresh leaves, stem and roots of A. formosanus and the results showed their IPAF amounts were 9.5 mg/g, 1323.5 μg/g, 87.4 μg/g and 314.7 μg/g, respectively. This analytical method could be highly helpful for further determination of IPAF in A. formosanus Hayata samples and its relative products.

摘要………………………………………………………………….........I
Abstract…………………………………………………………….....…III
目錄………………………………………………………………….…..V
圖表目錄……………………………………………………………...VIII
壹、 前人研究………………………………………………….………...1
一、 金線連簡介………………………………….………………....1
（一） 金線連的分類與特性………………………………...1
（二） 金線連的保健功能研究………………………….…..2
二、 金線連免疫調節蛋白 IPAF 的前期研究…………................9
三、 B 淋巴細胞的生長與分化……………………………….….11
（一） B 淋巴細胞的活化………………………………....12
（二） Toll-like receptors 與 B 淋巴細胞之關係………...17
四、 研究動機與目的……………………………………………...21
貳、 材料與方法………………………………………………………..24
一、 金線連免疫調節蛋白樣本製備……………………………...24
二、 金線連免疫調節蛋白生化特性分析………………………...26
（一） 蛋白質濃度測定…………………………………….26
（二） 變性膠體電泳分析………………………………….30
（三） 逆相高效能液相層析分析 (RP-HPLC analysis)…..34
（四） 二維電泳分析……………………………………….36
（五） 西方轉漬分析……………………………………….40
三、 金線連免疫調節蛋白對 B 淋巴細胞的活化效應………....42
（一） 小鼠脾臟細胞之取得……………………………….43
（二） 利用 MACS 磁珠分選 B 淋巴細胞……………...45
（三） 細胞增生分析 (Brd-U method)…………………….49
（四） 利用流式細胞儀分析 B 淋巴細胞表面標記……..52
（五） 酵素連結免疫分析法分析 B 淋巴細胞抗體表現..55
四、 金線連免疫調節蛋白對 B 淋巴細胞作用路徑分析……....59
（一） C57BL/10ScN 小鼠試驗…………………………...59
（二） B6.129-TLR2tmlkir/J 小鼠試驗 ..…………………....60
五、 金線連免疫調節蛋白濃度標準曲線………………………...60
（一） IPAF 抗體生物素化(biotinylation)…………………60
（二） IPAF 捕捉及偵測抗體配對之 ELISA 分析………63
（三） IPAF 濃度標準曲線建立…………………………...66
（四） 利用 IPAF 之 ELISA 定量平台分析金線連樣品..68
六、 統計分析……………………………………………………...70
參、 結果………………………………………………………………..72
一、 金線連免疫調節蛋白之製備及生化特性分析……………...72
二、 金線連免疫調節蛋白 IPAF 的進一步分離………………..73
三、 金線連免疫調節蛋白對 B 淋巴細胞的活化作用………....76
四、 建立金線連免疫調節蛋白之 ELISA 分析方法…………....86
肆、 討論………………………………………………………………..91
一、 IPAF 中的 IPAFP3 可能才是真正活性物質………………91
二、 IPAF 屬於 TI-1 抗原能活化 B 淋巴細胞………………...93
三、 IPAF 活化 B 淋巴細胞與 TLR4 及 TLR2 之關係……...96
四、 IPAF 與 B、T 淋巴細胞之關係…………………………...101
五、 酵素連結免疫分析法應用於 IPAF 之定量………………103
伍、 結論………………………………………………………………107
參考文獻………………………………………………………………108
圖與表…………………………………………………………………114

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