臺灣博碩士論文加值系統

氯化鋁是牙科治療過程中常用來控制牙齦出血的收斂劑。但是這類藥品接觸牙齦的時間越久，對口腔角化上皮以及纖維母細胞的毒性就越強，結果可能導致牙齦萎縮、牙根敏感、甚至影響美觀。牙齦組織的血管豐富，治療過程中如果造成牙齦損傷出血，收斂劑可能經由傷口吸收而影響血管內皮細胞的生理特性，連帶影響牙齦組織傷口癒合之血管新生作用。因此，本篇研究論文主要在探討氯化鋁對牙齦血管內皮細胞生物特性的影響，以分離自人類牙齦組織的血管內皮細胞與人類臍靜脈內皮細胞為模式細胞，探討臨床使用之氯化鋁依照不同的處理時間，對兩種血管內皮細胞的毒性作用，及其對人類血管內皮細胞生長因子(VEGF)所誘發兩種血管內皮細胞血管新生作用的生物效應。實驗結果顯示，臍靜脈血管內皮細胞和牙齦血管內皮細胞在25%氯化鋁處理5分鐘的情況下，48小時的細胞存活率僅剩30%及27% 。利用Hoechst 33258的細胞核染色觀察到25%氯化鋁會誘發凋亡小體的形成並伴隨著細胞質內與細胞凋亡機制相關酵素caspase 3的活性提高。細胞內p53蛋白質會隨著細胞接觸25%氯化鋁的時間增加而增加，然而預先處理antisense p53 oligonucleotides以抑制由25%氯化鋁所誘發p53蛋白質的表現，可以降低其誘發血管內皮細胞凋亡的情況。由以上結果顯示25%氯化鋁在處理5分鐘的情況下即能對血管內皮細胞產生毒性作用，主要是引發p53/ caspase 3相關途徑之細胞凋亡。在探討短時間的暴露是否會影響VEGF誘發內皮細胞血管新生作用的實驗結果顯示，臍靜脈血管內皮細胞和牙齦血管內皮細胞經過25%氯化鋁3分鐘的處理之後，對24小時的細胞存活率沒有明顯的毒性影響，然而25%氯化鋁會抑制VEGF調控血管內皮細胞通透性的能力、抑制VEGF誘發細胞移動/侵襲的作用、以及抑制VEGF誘發血管內皮細胞在Matrigel上形成管狀結構的功能。在鷄胚尿囊膜活體試驗的結果顯示，經過25%氯化鋁溶液2分鐘的處理會明顯抑制VEGF所誘發的微小血管新生。機轉研究上，我們發現25%氯化鋁會抑制VEGF誘發血管內皮細胞VEGF receptor 2的磷酸化。由以上的實驗結果顯示，氯化鋁作用時間對血管內皮細胞的耐受度有很大的關係，可能引發細胞凋亡或抑制血管新生作用。

Aluminum chloride acts as an astringent agent with excellent haemostatic properties during gingival retraction. However, the chemical may result in the recession of gingival tissue in prolonged exposure time. The gingiva possesses rich terminal blood supplies, which are mainly composed of vascular endothelial cells. When gingival tissue undergoes trauma or iatrogenic laceration, capillaries rupture facilitates more absorption of retraction solution in gingival tissue. In this study, we investigated the cytotoxic effects of aluminum chloride on human gingival vascular endothelial cells (HGECs) and human umbilical vascular endothelial cells (HUVECs). The results of MTT assay revealed 20% and 25% aluminum chloride significantly lessened survival rate of both HUVECs and HGECs. The 25% aluminum chloride could induce HUVECs and HGECs apoptotic body formation and high caspase 3 activity significantly in 5-minute treatment. However, the p53-dependent apoptosis induced by 25% aluminum chloride could be rescued by antisense p53 oligonucleotides. 25% aluminum chloride showed the anti-angiogenic effects in the shorter exposure time, including the inhibition of endothelial cells tube formation on Matrigel and the inhibition of endothelial cells migration/ invasion in 3-minute treatment, the inhibition of VEGF-induced monolayer cell permeability, and the inhibition of VEGF-induced angiogenesis on chicken chorioallantoic membrane in 2-minute treatment. 25% aluminum chloride also inhibited VEGF-induced VEGF receptor 2 tyrosine phosphorylation. Therefore, we concluded that 25% aluminum chloride could induce human gingival vascular endothelial cells p53-dependent apoptosis or inhibit VEGF modulated angiogenesis, depending on the length of exposure time.

中文摘要.....................................01
Abstract.....................................02
Introduction.................................03
Specific Aims................................20
Materials and Methods........................21
Result.......................................31
Discussion...................................38
Conclusion...................................44
Future Perspective ...........................45
Figures......................................47
Reference ....................................63
Appendix.....................................70

List of Figures and Tables:
Figure 1. The histological photograph of human gingival tissue.......................................47
Figure 2. Characterization of isolated HGECs and HUVECs.......................................48
Figure 3. Identification of HUVECs and HGECs with immunohistochemical staining.................49
Figure 4. Cytotoxic effects of AlCl3 on HUVECs and HGECs........................................50
Figure 5. 25% AlCl3 induced HUVECs and HGECs apoptosis....................................51
Figure 6. 25% AlCl3 induced HGECs p53 expression...................................53
Figure 7. 25% AlCl3 induced caspase 3 activation in HUVECs.......................................54
Figure 8. 25% AlCl3 induced HGECs p53-dependent apoptosis....................................55
Figure 9. Effects of 25% AlCl3 on VEGF-induced HUVECs and HGECs monolayer permeability.................56
Figure 10. Effects of 25% AlCl3 on VEGF-induced HUVECs and HGECs tube formation on Matrigel..57
Figure 11. Effects of 25% AlCl3 on VEGF-induced HUVECs and HGECs migration..................58
Figure 12. Effects of 25% AlCl3 on VEGF-induced HUVECs and HGECs invasion capability.........59
Figure 13. Effects of 25% AlCl3 on VEGF-induced angiogenesis on CAM..........................60
Figure 14. Effects of 25% AlCl3 on VEGF-induced VEGF receptor 2 tyrosine phosphorylation.....61
Figure 15. Schematic mechanism of AlCl3 inhibits VEGF-modulated HGECs angiogenesis............62

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