臺灣博碩士論文加值系統

Slit2是一個胞外基質醣蛋白，過去文獻指出Slit2蛋白在神經軸突導向上扮演重要的角色，有趣的是，近年來發現Slit2也參與血管內皮細胞的血管新生，但眾說紛紜，本研究想探討這些不一致的結果是否與不同的剪接變異型的Slit2有關。我們發現slit2在exon15的位置有兩個剪接變異型的表現: Slit2-△E15 (exon15缺失)及Slit2-WT (具有exon15)。Slit2-△E15過度表現會抑制CL1-5肺癌細胞的生長及侵襲能力，而Slit2-WT只會抑制細胞的侵襲能力。因此我們分析了Slit2-△E15及Slit2-WT對臍靜脈內皮細胞的移動能力、體外血管的形成及通透性的影響。結果顯示處理Slit2-△E15條件培養液的內皮細胞可在體外形成較完整也較粗的血管，而且在細胞遷徙試驗中處理Slit2-△E15條件培養液可以抑制肺癌細胞所誘導的絲狀結構。CL1-5肺癌細胞的條件培養液會誘導HUVEC的通透性，表現Slit2-WT的條件培養液可以部份的抑制內皮細胞被誘導的通透性，而Slit2-△E15可以完全抑制肺癌細胞的條件培養液對HUVEC細胞所誘導的通透性。綜合以上這些in vitro血管新生的實驗結果我們推測處理了 Slit2-△E15能完全抑制條件培養液對HUVEC細胞所誘導的血管通透性，而形成完整的新生血管。我們利用酵母菌雙雜合試驗偵測到包含或不包含Exon15的Slit leucine-rich repeat 2 (LRR2/D2)都會與Robo4 Ig1-2有很強的交互作用，所以未來我們可以更進一步的探討Robos與Slit2在內皮細胞的血管新生中扮演了什麼樣的角色。

Slit2 is a 200 kDa extracellular matrix (ECM) glycoprotein. Previous studies indicated that Slit2 plays an important role in neural axon guidance. Interestingly, recent studies pointed that Slit2 has effect on angiogenesis, however, with great discrepancy; some studies stated that Slit2 inhibits angiogenesis, while others supported its role in enhancing angiogenesis. Therefore, we want to investigate whether slit2 splicing variants may cause these inconsistent results. We discovered two slit2 splicing variants at exon15: Slit2-△E15 (lacking exon15) and Slit2-WT (containing exon15). Overexpression of Slit2-△E15 inhibited growth and invasion of CL1-5 lung cancer cells, but overexpression of Slit2-WT only inhibited invasion but not growth of CL1-5. For this reason, we analyzed the effect of Slit2-WT or Slit2-△E15 expressed conditioned medium on migration, in vitro tube formation and permeability of HUVECs. Our results showed that HUVECs treated with Slit2-△E15 expressed conditioned medium formed thicker blood vessel in vitro with better integrity compared with the cells treated with conditioned medium collected from CL1-5 cells expressing empty vector and Slit2-WT. In migration assay, Slit2-△E15 conditioned medium inhibited cancer cells’ conditioned medium induced filamentous morphology on migrated HUVECs. Furthermore, conditioned medium collected from CL1-5 cells harboring empty vector enhances HUVEC permeability, Slit2-△E15 expressed conditioned medium can completely inhibited the permeability induced by cancer cells’ conditioned medium, but Slit2-WT expressed conditioned medium only partially inhibited endothelial cell permeability. Baed on these data, we hypothesized that Slit2-△E15 may inhibit permeability of HUVECs and thus increasing the integrity of newly synthesized blood vessel. Although Robo4 plays important role in angiogenesis, however the physical interaction between Slit2 and Robo4 has not been clarified. We tried to examine the possible interaction between Slit2-LRR2/D2 and Robo4-Ig1,2 domains in yeast two hybrid system, and found that Slit2-LRR2/D2 domain strongly interacts with Robo4 Ig1-2 domain. Our future studies will be focused on how exon15 modulates permeability and integrity of newly synthesized blood vessel.

壹、 中文摘要 1
貳、 英文摘要 2
參、 縮寫表 4
肆、 前言 6
一、 血管新生(Angiogenesis) 6
1.血管的組成 6
1.1血管內皮細胞(Vascular endothelial cells)
1.1.1血管內皮細胞的功能 7
2.血管新生的過程 8
(A) Formation of immature vasculature： 8
(B) Stabilization of immature vasculature： 8
(C) Branching, remodeling and pruning of vasculature : 9
(D) Vessel specialization 10
3.血管內皮生長因子(vascular endothelial growth factor，VEGF) 11
3.1 血管內皮生長因子的家族 (VEGF family) 11
3.2 血管內皮生長因子的接受器 (VEGF receptor) 12
二、 癌症與血管新生 13
三、 Slits 蛋白 14
1.Slit 家族成員 15
2.Slit 的結構 15
2.1 Slit2 15
四、 Robos 16
1.Robo的家族成員 16
2.Robo 的結構 16
2.1 Robo1/DUTT1(Deleted in U Twenty Twenty)的結構與其結合蛋白 17
2.2 Robo4/Magic Roundabout的結構與其其結合蛋白及下游訊息傳遞 17
五、 Slit2-Robo 訊號傳遞 18
1.Slit2與神經軸突導向的關係 18
2.Slit2與腫瘤的關係 18
3.Slit2與血管新生(Angiogenesis)的關係 19
3.1 Slit2與premeability的關係 20
4.Slit2 leucine rich repeats (LRRs)的晶體結構 20
5.Heparan sulphate binding 21
六、 先前本實驗室研究Slit2的結果 21
七、 研究動機 22
伍、 材料與儀器 24
一、 酵素 24
二、 商業套組 24
三、 試藥 24
四、 質體 25
五、 細胞株 25
六、 抗體 26
七、 儀器 26
陸、 實驗方法 28
一、 質體DNA的萃取（Plasmid extract） 28
(1)微量質體DNA萃取 28
(2)中量質體萃取(Midi-prep) 29
2-1 Invitrogen 29
2-2 VIOGENE 29
二、 細胞RNA的萃取 31
三、 利用去氧核糖核酸酶除去total RNA裡的染色體DNA 31
四、 反轉錄反應（Reverse Transcription；RT） 32
五、 定量PCR（Real time PCR） 32
六、 構築穩定表現Slit2基因載體 33
（1） 限制酶處理（Enzyme Digestion） 33
（2） 從電泳凝膠中純化DNA 33
（3） 利用CIP（Calf Intestinal）將載體酵素切位點去磷酸化 34
（4） 接合反應（Ligation） 35
（5） 利用化學法製備勝任細胞（Competent cell） 35
（6） 轉型作用（Transformation） 36
（7） 菌落聚合酵素連鎖反應（Colony Polymerase Chain Reaction） 36
（8） 自動定序的製備（Autosequence） 37
（9） 菌種保存 38
七、 內皮細胞株培養 38
（1） 解凍細胞（Thaw cell cline） 38
（2） 內皮細胞培養 39
（3） 細胞繼代 39
（4） 冷凍細胞（Frozen cell） 40
八、 西方墨點法（Western blot） 40
（1） 製備細胞均質液（Cell extract） 40
（2） 製備條件培養液（Conditioned medium） 41
（3） 蛋白定量分析 41
（4） SDS PAGE的製備 42
（5） SDS PAGE電泳操作及轉漬（Transfer blot） 43
（6） 抗體作用及偵測方法 43
九、 細胞計數生長試驗（Growth curve and Dobuling time） 44
十、 細胞遷徙能力試驗（Migration assay） 45
十一、 血管新生試驗（In Vitro Angiogenesis） 45
(1) 人類臍帶內皮細胞(HUVECs)血管生成分析(Tube formation assay) 45
(2) Permeability assay 46
十二、 Yeast two hybrid system 47
1. 酵母菌勝任細胞的製備 47
2. 酵母菌轉型作用 48
3. 酵母菌的PCR 48
4. 酵母菌雜合 49
柒、 實驗結果 50
一、 過度表現Slit2-WT以及Slit2-△E15蛋白對臍靜脈血管內皮細胞(HUVEC)受到VEGF165刺激移動能力的影響 50
二、 不同剪接型的Slit2對臍靜脈血管內皮細胞在體外形成血管的影響 52
三、 不同剪接型的Slit2對臍靜脈血管內皮細胞受到VEGF165刺激通透性的影響 52
四、 利用real-time PCR分析在HUVEC與CL1-5細胞株中，其Robo 以及SDC基因的相對表現量 53
五、 構築表現Slit2-D2以及Slit2-D2/ΔE15 在pACT2 54
六、 構築表現Robo1-Ig1-2以及Robo4-Ig1-2 在pGBKT7 55
七、 構築表現Robo1-Ig1-2 domain以及Robo4-Ig1-2 domain 在pET-28a和pGEX-4T-1 56
八、 利用酵母菌雙雜合系統(Yeast two hybrid system)偵測Slit2-D2, Slit2-D2/ΔE15與Robo1-, Robo4-Ig1-2 的交互作用 57
捌、 討論 59
一、 Robos對不同剪接型的Slit2所調控臍帶靜脈血管內皮細胞的遷徙能力、血管生成及通透性的影響 59
二、Slit2-D2(LRR2)、D2/Exon15與Robo1-和Robo4-Ig 1-2 domains的交互作用
62
玖、 圖目錄 65
圖一、 含有 Slit2-WT以及Slit2-ΔE15蛋白的CL1-5的細胞條件培養液對臍靜脈血管內皮細胞(HUVEC) 細胞移動能力的影響 65
圖二、 過度表現Slit2-WT以及Slit2-ΔE15蛋白對HUVEC細胞移動能力的量化圖 66
圖三、 不同剪接型的Slit2對臍靜脈血管內皮細胞(HUVEC)在體外形成血管的影響 67
圖四、 表現不同剪接型的Slit2的條件培養液對HUVEC細胞受到VEGF165刺激通透性的影響 68
圖五、 利用real-time PCR分析在臍靜脈血管內皮細胞(HUVEC)與肺癌細胞(CL1-5)中，其Robo1、Robo4 以及Syndecan (SDC)基因的相對表現量 70
圖六、 構築Slit2 D2、D2/Exon15 在pACT2 clone過程圖 71
圖七、 構築Robo1-、Robo4-Ig1-2 在pGBKT7 clone過程圖 72
圖八、 構築Robo1-、Robo4-Ig1-2 在pET-28a clone過程圖 73
圖九、 構築Robo1-、Robo4-Ig1-2 在pGEX-4T-1 clone過程圖 74
圖十、 篩選酵母菌 75
圖十一、 利用酵母菌的雜合偵測Slit2-D2, Slit2-D2/ΔE15與Robo1-, Robo4-Ig1-2的交互作用 76
拾、 附圖 78
附圖一 血管新生的過程 78
附圖二、 成熟血管的組成成分 79
附圖三、 成熟血管網的形成 80
附圖四、 圖解人類VEGF isoform 81
附圖五、 Slit2 Wild-type (Slit2-WT) 81
附圖六、 Structure of Slit2-D4 and the Decorin dimer in similar orientations 82
附圖七、 Robo1與Robo4蛋白結構示意圖 83
附圖八、 Robo與Slit訊息傳遞在內皮細胞移動的可能機制 84
附圖九、 由Microarray分析Slit2基因在肺癌病人組織的相對表現量 85
附圖十、 Slit2基因在Exon15剪接變異型 85
附圖十一、 pcDNA3.1(-)/myc-His-A 示意圖(Invitrogen) 86
附圖十二、 yT&A vector之示意圖 (Yeastern) 87
附圖十三、 Robos對Slit2所抑制CL1-5侵犯作用的影響 88
附圖十四、 Schematic drawing of Drosophila Slit, Robo and syndecan 90
拾壹、 參考文獻 91

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