| 摘要: | Cathelicidin 和 Defensin是兩種主要的宿主防禦胜肽 (Host defence peptides, HDPs) 族群 。Cathelicidin的族群成員的序列長度約在十二到八十個氨基酸之間 ， 因此具有不同的分子量大小。 Indolicidin (Indo) 是Cathelicidin家族的成員之一，其序列中含有十三個氨基酸，序列為：ILPWKWPWWPWRR。從序列中可以得知， Indo本身富含了脯氨酸和色氨酸兩種氨基酸。如同其它的Cathelicidin 成員，Indo同樣也具有對抗微生物和免疫調節的功能。Indo之所以讓我感到興趣並成為我的研究主題的原因，是因為它可以有效的刺激動物體內的免疫細胞，產生和發炎反應相關的一些細胞素，例如：TNF-α，以進一步地活化免疫反應。
Tripalmitoyl-cysteinyl-seryl-(lysyl)3-lysine, Pam3CysSerLys4 (P3CSK4)， 是第二型類鐸受器的配體，由一段胜肽序列(CSK4)接上三個棕櫚酸分子(Pamitic acid, Pam)所組成。從這樣的想法開始，或許利用棕櫚酸化的方式，將棕櫚酸接到 Indo 上，也能夠依循相同的機制，刺激第二型類鐸受器，以產生發炎相關的細胞素，最後活化免疫系統。
關於這類型的研究，目前還沒有被研究人員發表相關報導至文獻中。在我的研究當中，我成功的將一個棕櫚酸分子接到了Indo上，然後再透過「高效液相色譜儀」(High- performance liquid chromatography, HPLC) 和「液相層析質譜儀」 (Liquid chromatography-mass spectrometry, LC-MS) 將 Indo 純化並定性。在動物實驗當中，福馬林去活化後的禽流感病毒 (Formalin-inactivated H5N1, FI-H5N1) 被當作是實驗中的測試抗原，並和利用化學方式合成好的 Indo 和 Pam-Indo 混合後，以肌肉注射的方式打入BALB/c (H-2d) 老鼠當中以評估胜肽如何去引響測試抗原 (antigen, FI-H5N1) 所產生的免疫效應。
老鼠以不同的接種配方免疫之後，FI-H5N1-coated Enzyme Linked Immunosorbant Assay (FI-H5N1-coated ELISA) 被用來測量老鼠血清中，針對 FI-H5N1 的抗體濃度。實驗結果顯示，在打入 FI-H5N1 和 Pam-Indo 接種配方的老鼠組別當中，老鼠血清之中針對 FI-H5N1 的抗體表現量顯著地降低；然而，在打入了 FI-H5N1 和 Pam 或 Indo 的老鼠組別中，卻看不到同樣的抑制現象。
B 細胞要活化以防禦抗原，例如：H5N1，是需要「CD4+ 的輔助型T 細胞」的幫助才能達成。而CD4+的輔助型T細胞的活化，則需要和「抗原呈現細胞」 (Antigen presenting cells,APCs) 產生交互作用才能達成。因此，了解 Pam-Indo如何影響 APCs 就成為了下一個要被探討的重要問題。為了驗證這個概念，我將老鼠大腿骨中的骨髓細胞取出，在含有「巨噬細胞集落刺激因子」 (Granulocyte macrophage colony stimulating factor, GM-CSF) 的培養液當中培養，約八到十天後，即可產生未成熟的骨髓樹突細胞。接下來將產生的骨髓樹突細胞加入含有脂多醣 (lipopolysaccharide, LPS) 和 Pam, Indo, Pam + Indo 或 Pam-Indo 的培養基中培養。實驗結果顯示，Pam-Indo 可以有效的抑制，被脂多醣刺激後的樹突細胞產生 CD40 和 CD86 分子。而這樣子的抑制現象，在含有 Pam, Indo 或 Pam + Indo 的組別當中並沒有看到。
綜合以上的實驗結果可以證明，Pam-Indo 可以有效促進耐受性未成熟樹突細胞的生成，進而抑制老鼠免疫系統針對FI-H5N1的抗體反應。
Cathelicidins and Defensins are the two main classes of host defence peptides (HDFs). Cathelicidins contain peptides of different molecular sizes, which can range from 12 amino acids to around 80 amino acids long. Indolicidin (abbreviated as Indo from here on) is a 13 amino acid member of the cathelicidins with the sequence, ILPWKWPWWPWRR. From the sequence, it can be seen that the peptide is rich in proline and tryptophan. Like many other cathelicidins, Indo also posesses anti-microbial as well as immunomodulatory properties. The special feature of the immunodulatory property of Indolicidin is that it has been shown to stimulate cells to produce inflammatory cytokines.
Tripalmitoyl-cysteinyl-seryl-(lysyl)3-lysine, P3CSK4 (Pam3CysSerLys4), is known to be a toll-like receptor-2 (TLR-2) ligand. So, pamilation of Indo may allow it to target TLRs to elicit inflammatory immune responses. These studies have not been reported in the literature. In this research project, I have prepared Pam-Indo by linking monopalmitic acid to Indo, purified it, and characterized it using mass spectrometry.
The potential immunomodulatory property of Pam-Indo was evaluated by testing how it could affect the generation of immune responses against formalin-inactivated H5N1 influenza virus (FI-H5N1) in BALB/c (H-2d) mice. The results of the studies I have carried out showed that mice immunized with Pam-Indo generated much lower level of antibodies against FI-H5N1 as compared to mice of the same age administered with FI-H5N1, or FI-H5N1 formulated in Pam (monopalmitic acid), Indo, or Pam + Indo.
FI-H5N1-coated Enzyme Linked Immunosorbant Assay (ELISA) was used to detect the serum anti-FI-H5N1 antibodies in these mice. B cell response to an antigen (FI-H5N1) requires immunological help from CD4+ T cells, and how effective FI-H5N1-specific CD4+ T cells are generated in turn relies on how well antigen-presenting cells (APCs) present the H5N1 antigen to them. I, therefore, investigated how Pam-Indo affects APCs. To do this, I had cultured bone marrow monocytes in the presence of mouse (granulocyte macrophage colony stimulation factor, GM-CSF) to convert them to become immature myeloid dendritic cells (imDCs). Pam-Indo was found to suppress maturation of DC following stimulation with lipopolysaccharide (LPS). Expression of the CD86 and CD40 costimulatory molecules are downregulated in LPS-treated imDCs in the presence of Pam-Indo.
This effect was not seen in LPS-stimulated DCs exposed to Indo or, Pam. The results suggest Pam-Indo mediates the generation of tolerogenic imDCs to suppress the induction of antibody responses against FI-H5N1. |
