臺灣博碩士論文加值系統

人類CD93 (C1qRp)是一個高度醣基化的穿膜蛋白，廣泛的表現在骨髓細胞、內皮細胞與幹細胞上。CD93與凝血酶調節素(Thrombomodulin, TM)結構相似。TM對於細胞貼附扮演重要角色，本實驗室先前的研究也指出穩定表現CD93-GFP重組蛋白的A2058細胞，不論貼附、爬行和侵襲能力皆比控制組A2058-GFP細胞還強。本論文主要研究表現CD93對人類臍靜脈內皮細胞(Human Umbilical Vein Endothelial Cells, HUVEC)的影響，使用RNAi技術降低CD93表現後，觀察HUVEC爬行與侵襲能力的變化，並以免疫螢光染色觀察抑制CD93表現後HUVEC的骨架蛋白F-actin的分布情形。為測試CD93對細胞貼附的影響，也使用anti-CD93的抗體中和A2058-CD93間的貼附能力。結果顯示CD93的shRNA能有效抑制HUVEC的CD93表現。以in vitro wound healing與Boyden chamber assay分析，也發現CD93表現降低的HUVEC之爬行與侵襲能力皆顯著的降低。但以酶譜法偵測MMPs活性，發現MMP-2活性並無受到影響，表示侵襲力下降是由其他因素調控。以免疫螢光染色法使用共軛焦螢光顯微鏡以及共同免疫沉澱實驗觀察，發現CD93會與phospho-moesin與F-actin交互作用，並出現在F-actin microdomain。再以TNF-α刺激細胞後，會使控制組的HUVEC表現更多的F-actin microdomain，而CD93表現降低的HUVEC在TNF-α刺激後，形成F-actin microdomain與F-actin branching points的數量也隨之降低。此結果意味著CD93可能透過其它機制增強細胞F-actin branching points的形成，進而使細胞的爬行能力上升。最後使用anti-CD93的抗體，發現能成功的阻止細胞與細胞間的貼附。從本論文的研究成果顯示CD93可調控細胞間的貼附以及在細胞內的骨架重組中扮演重要角色，CD93是細胞形成F-actin microdomain與F-actin branching points的重要蛋白，而F-actin microdomain的存在或許是細胞爬行時形成F-actin branching points的關鍵，使細胞能快速的爬行。

Human CD93 (known as C1qRp) is a highly glycosylated transmembrane protein expressed on monocytes, neutrophils, endothelial cells (ECs), and stem cells. The structure of CD93 is similar with thrombomodulin (TM). CD93 and TM both have five domains including C-type lectin-like domain, EGF-like domain, mucin-like domain, transmembrane domain and cytoplasmic tail. TM play an important role in mediated cell-cell adhesion. In previously study, we established stably expressed green fluorescent protein tagged CD93 transfectants in A2058 melanoma. We demonstrated that the cell-cell adhesion, migration and invasion of A2058 CD93-GFP cells were increased than that of control A2058 GFP cells. In this study, RNAi technology is used to knockdown CD93 expression in HUVECs, an endothelial cells with endogenous CD93 expression. Cell migration and invasion of CD93-knowndown HUVEC were analyzed. In addition, immunofluorescent staining is utilized to observe the location of F-actin and CD93. For investigating the cell-cell adhesion, antibody against CD93 is used to neutralizing CD93 on cell surface in A2058 CD93-GFP cell. We successfully knockdown CD93 expression by CD93 shRNA in HUVEC. The migration and invasion of CD93-knockdown HUVEC are decreased significantly. However zymography result show that the activity of MMP-2 is not changed in CD93-knockdown HUVEC. Immunofluorescent staining and immunoprecipitation results show that CD93 is involved in the formation of F-actin microdomain by interaction with phospho-moesin in HUVEC. Interestingly, TNF-α induced formation of F-actin microdomain and F-actin branching points are decreased in CD93-knockdown HUVEC. This result suggests that CD93 play an important role in the formation F-actin microdomain and F-actin banching points. Finally, CD93 antibody was successfully blocked cell-cell adhesion. Taken together, these results suggest that CD93 enhances cell-cell adhesion and plays a critical role on formation of F-actin microdomain, which may regulate cytoskeletal reorganization, F-actin banching points formation and then enhance cell migration.

中文摘要 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ I
英文摘要 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ III
本文目錄 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ V
圖目錄 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ VIII
附圖目錄 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ IX
縮寫表 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ X
第一章 緒論 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 1
1.1. 文獻回顧 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 1
1.1.1. CD93的發現 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 1
1.1.2. CD93的分佈與結構 ‧‧‧‧‧‧‧‧‧‧ 1
1.1.3. CD93的功能 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 2
1.1.4. CD93與其它結構相似蛋白的功能 ‧‧‧‧ 3
1.1.5. CD93與ERM蛋白家族 ‧‧‧‧‧‧‧‧‧ 4
1.1.6. F-actin microdomain與F-actin branching points和細胞爬行或
型態的關係 ‧‧‧‧‧‧‧‧ 5
1.2. 研究動機 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 6


第二章 材料與方法 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 7
2.1. 儀器 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 7
2.2. 藥品 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 10
2.3. 質體的製備 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧14
2.4. 細胞的培養 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧15
2.5. HUVEC starvation ‧‧‧‧‧‧‧‧‧‧‧‧‧ 16
2.6. 電穿孔轉染 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 16
2.7. 西方墨點法 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 17
2.8. 傷口癒合 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 19
2.9. 細胞侵襲試驗 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 19
2.10. 酶譜法 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 20
2.11. 免疫螢光染色 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 21
2.12. 共同免疫沉澱 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 22
2.13. CD93中和試驗 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 23


第三章 結果 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 24
3.1. 質體的萃取與內生性CD93的降低 ‧‧‧‧‧‧ 24
3.2. 降低 CD93對HUVEC爬行能力的影響 ‧‧‧‧ 24
3.3. 降低 CD93對HUVEC侵襲能力的影響 ‧‧‧‧ 25
3.4. 降低 CD93對HUVEC 的MMPs活性的影響 ‧‧ 25
3.5. 以免疫螢光染色法觀察CD93與F-actin的表現 ‧‧ 26
3.6. 以共軛焦螢光顯微鏡觀察CD93、phospho-moesin
與F-actin ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧26
3.7. 共同免疫沉澱法觀察CD93與phospho-moesin的交互
作用 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 27
3.8. 以西方墨點法偵測phospho-moesin在各種狀態下的表
現量 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 27
3.9. 免疫螢光染色觀察CD93-knockdown HUVEC的F-actin
microdomain與 F-actin branching points表現 28
3.10. 中和CD93對細胞與細胞間的貼附能力的影響 ‧‧ 28
第四章 討論 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 30
第五章 結論 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 35
參考文獻 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 36
圖目錄
圖1. 使用限制酵素切割質體 ‧‧‧‧‧‧‧‧‧‧‧‧‧ 39
圖2. 以西方墨點法觀查電穿孔轉殖shRNA後CD93的表現
量 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 40
圖3. 使用傷口 healing assay觀察降低 CD93對HUVEC爬行的影
響 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 41
圖4. 使用Boyden chember觀察降低 CD93對HUVEC侵襲力的影
響 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 42
圖5. 以酶譜法偵測MMPs活性 ‧‧‧‧‧‧‧‧‧‧‧‧ 43
圖6. 以免疫螢光染色觀察CD93與F-actin ‧‧‧‧‧‧‧ 44
圖7. 共軛焦顯微鏡觀察CD93、phospho-moesin與F-actin的分
布‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 45
圖8. 共同免疫沉澱觀察CD93與phospho-moesin的交互作用 46
圖9. 以西方墨點法偵測phospho-moesin在各種狀態下的表現
量 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 47
圖10. 使用免疫螢光染色觀察CD93-knockdown HUVEC的F-actin
microdomain與F-actin branching points表現 ‧‧ 48
圖11. 利用CD93抗體中和CD93觀察細胞間的貼附作用 ‧‧ 52
附圖目錄
附錄1. Moesin與CD93 cytoplasmic tail結合示意圖 ‧‧‧ 53
附錄2. shRNA質體圖譜 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 54
附錄3. CD93示意圖與五個shRNA針對序列 ‧‧‧‧‧ 55

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