- 學位論文
探討不同剪接型的Slit2 蛋白對血管內皮細胞生長、血管生成及通透性的影響
Study The Effect of Slit2 Splicing Variant on Growth, Tube Formation and Permeability of Endothelial Cells
摘要
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。
並列摘要
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.
參考文獻
延伸閱讀
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