調控巨噬細胞的表現型態減緩類風濕性關節炎造成的疼痛

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研究生：	江澔 Hoa Jiang
論文名稱：	調控巨噬細胞的表現型態減緩類風濕性關節炎造成的疼痛 Modulation of phenotypic diversity of macrophages attenuates rheumatoid arthritis induced mechanical hyperalgesia
指導教授：	孫維欣 Wei-Hsin Sun
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2018
畢業學年度：	106
語文別：	中文
論文頁數：	79
中文關鍵詞：	巨噬細胞、類風濕性關節炎
外文關鍵詞：	macrophages, rheumatoid arthritis
相關次數：	點閱：11 下載：0
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類風濕性關節炎(RA)是一種自體免疫疾病，其特徵在於慢性關節炎症導致骨頭侵蝕和軟骨損傷。RA 患者經常伴隨著持續性疼痛，並將疼痛改善作為首要任務。目前 RA 患者的治療著重於抑制炎症，在減緩慢性疼痛方面並沒有顯著的效果。治療 RA 引發的疼痛仍然是臨床實踐中的主要挑戰。我用發炎性疼痛模式動物篩選了小分子化合物和胜肽類藥物用於止痛效果。初步篩選後，我選擇小分子藥物NSC745885和Peptide X 用於 RA 模式小鼠做進一步測試。在 RA 模式小鼠中，腹腔注射 NSC745885 可以透過促發炎表型巨噬細胞(M1)的數量下降和抗發炎表型巨噬細胞(M2)的數量增加而減少 RA 誘導的機械性痛覺過敏。口服低劑量的 Peptide X 可以減少 RA 誘導的機械性痛覺過敏，推測可能是通過減少 M1 巨噬細胞和血液中 IL-6 的濃度。

Rheumatoid arthritis (RA), an autoimmuedisease, is characterized by chronic joint inflammation leading to bone erosion and cartilage damage. RA patients often have persistent pain and declare pain as the greatest problem. Current treatments focusing on suppression of inflammation may produce an inadequate response in relieving chronic pain in many RA patients. The treatment of RA pain continues to be a major management challenge in clinical practice. I have screened small molecule compounds and peptides for analgesic effects using inflammatory pain animal models. After pre-screening, I have identified compound NSC745885 and Peptide X for further tests on RA models. In RA model, intraperitoneal administration of NSC745885 reduced RA-induced mechanical hyperalgesia, probably through a decline in the number of pro-inflammatory phenotype macrophage (M1) and an increase in the number of anti-inflammatory phenotype macrophage (M2). Oral delivery of low dose of Peptide X reduced RA-induced mechanical hyperalgesia, probably by the reduction of the number of M1 macrophage and IL-6 level.

目錄
中文摘要................................................................................................................................ I Abstract ................................................................................................................................. II 致謝 ..................................................................................................................................... III 第一章 緒論 ......................................................................................................................... 1
1.1 類風濕性關節炎藥物的市場價值.......................................................................................... 2 1.2 類風濕性關節炎的發炎機制 ................................................................................................. 2
1.2.1 類風濕性關節炎中免疫系統扮演的角色......................................................................................3 1.3 類風濕性關節炎的疼痛機制 ................................................................................................. 4
1.3.1 類風濕性關節炎中與疼痛相關的基因..........................................................................................6
1.4 研究目的................................................................................................................................7 1.4.1 小分子藥物 NSC745885...............................................................................................................8 1.4.2 胜肽類藥物 Peptide X ...................................................................................................................8
第二章 材料與方法 ............................................................................................................. 9
2.1 實驗材料 ............................................................................................................................... 10 2.1.1 實驗動物 (Experimental animals)................................................................................................10 2.1.2 實驗藥品 (Experimental drugs)...................................................................................................10 2.1.3 實驗器具(Experimental tools).................................................................................................11
2.2 實驗方法 ............................................................................................................................... 11 2.2.1 注射酸引發傷害性疼痛模式 (Acid model).................................................................................11 2.2.2 注射CFA引發發炎性疼痛模式 (CFAmodel)............................................................................11 2.2.3 小鼠關節炎模式 (RAmodel)......................................................................................................12 2.2.4 關節炎評估 (Arthritis score) ....................................................................................................... 12 2.2.5 機械性痛覺行為實驗(Mechanical behavior test)...................................................................... 12 2.2.6 蘇木素-伊紅染色(Hematoxylin and eosin stain , H&E stain)........................................................13 2.2.7 免疫組織化學染色法( Immunohistochemistry)............................................................................14 2.2.8 血液中細胞因子定量分析 ELISA(The enzyme-linked immunosorbent assay) .............................. 15
2.3 統計分析 (Statistics) ..................................................................................................... 15 第三章 結果 ....................................................................................................................... 16
IV
3.1 NSC745885 及其衍生物 B1-C1、RV13 與胜肽類藥物 Peptide X 降低完全弗氏佐劑引發 的機械性痛覺過敏感.................................................................................................................. 17
3.2 重複注射完全弗氏佐劑(CFA)於小鼠關節誘發長期關節疼痛........................................18 3.3 M1 巨噬細胞數目在關節炎後期 (> 8W)大量增加;M2 巨噬細胞數目在關節炎中期
(4W-8W)增加 ............................................................................................................................. 19
3.4 NSC745885 有效抑制關節炎模式小鼠誘發的機械性痛覺過敏感 ................................... 20
3.5 長期注射NSC745885減緩關節炎引發滑膜增生、骨頭侵蝕與軟骨破壞.......................20
3.6 NSC745885 降低關節炎模式小鼠中 M1 巨噬細胞數量，增加 M2 巨噬細胞數量 ......... 21
3.7 關節炎模式小鼠中，NSC745885對血液中TNF-α和IL-6的影響................................22
3.8 Peptide X 抑制關節炎模式小鼠誘發的機械性痛覺過敏感 .............................................. 22
3.9 Peptide X 有效減緩關節炎引發滑膜增生、骨頭侵蝕與軟骨破壞................................... 22
3.10 長期給予PeptideX降低關節炎模式小鼠中M1巨噬細胞數目....................................23 3.11 Peptide X 有效降低關節炎模式小鼠血液中 IL-6 的濃度............................................... 23
第四章 討論 ....................................................................................................................... 24 4.1 模擬類風溼性關節炎模式小鼠..........................................................................................25
4.2 NSC745885 在發炎性疼痛的止痛效果 ........................................................................... 26
4.3 NSC745885 在關節炎中調控巨噬細胞的數量................................................................ 27
4.4 Peptide X 在發炎性疼痛的止痛效果 .............................................................................. 28
4.5 Peptide X 在關節炎疼痛的止痛效果 .............................................................................. 28
4.6 Peptide X 在關節炎中調控巨噬細胞的數量................................................................... 29
第五章 參考文獻 ............................................................................................................... 30


圖目錄
Figure 3.1. Screening analgesic compounds and peptides using acid model and sub-chronic inflammatory model. ........................................................................................................... 37
Figure 3.2. Repeat injection of CFA induced chronic arthritic pain. .................................. 39
Figure 3.3. The number of CD68+ macrophages is increased with time in RA mice. ...... 40
Figure 3.4. The number of CD80+ macrophages is increased with time in RA mice. ...... 42
Figure 3.5. The number of CD163+ macrophages is increased with time in RA mice. .... 44
Figure 3.6. NS745885 treatment induces mechanical hyperalgesia in RA mice. ............. 46
Figure 3.7. Histology of ipsilateral side joints in arthritic ICR mice................................ 47
Figure 3.8. The number of CD68+ macrophages effects with compound treatment in RA mice..................................................................................................................................... 48
Figure 3.9. The number of CD80+ macrophages effects with compound treatment in RA mice..................................................................................................................................... 50
Figure 3.10. The number of CD163+ macrophages effects with compound treatment in RA mice............................................................................................................................... 52
Figure 3.11. Levels of serum IL-6 and TNF-α effect by NSC745885 in chronic arthritis pain. .................................................................................................................................... 54
Figure 3.12. Peptide X treatment induces mechanical hyperalgesia in RA mice. ............ 55 Figure 3.13. Histology of ipsilateral side joints in arthritic ICR mice.............................. 56
Figure 3.14. The number of CD68+ macrophages effects with compound treatment in RA mice..................................................................................................................................... 57
Figure 3.15. The number of CD80+ macrophages effects with compound treatment in RA mice..................................................................................................................................... 59
Figure 3.16. The number of CD163+ macrophages effects with compound treatment in RA mice............................................................................................................................... 61
Figure 3.17. Levels of serum IL-6 and TNF-α effect by Peptide X in chronic arthritis pain. .................................................................................................................................... 63
 
表目錄
Table 3.1 Pre-screening of compounds and peptides........................................................... 64 Table 3.2 The number of macrophages in RA mice............................................................65 Table 3.3 The number of macrophages effects with NSC745885 treatment in RA mice .. 66 Table 3.4 The number of macrophages effects with Peptide X treatment in RA mice ...... 67
                                

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