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研究生：	陳佳政 Chen, Chia-Cheng
論文名稱：	口腔癌中Endothelin-1 對調控VEGF 所扮演的角色 Endothelin-1 Modulate the Expression of VEGF in Oral Cancer Cells
指導教授：	黃振勳 Huang, Jehn-Shyun
學位類別：	碩士 Master
系所名稱：	醫學院 - 口腔醫學研究所 Institute of Oral Medicine
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	44
中文關鍵詞：	口腔癌、內皮素、血管內皮生長因子
外文關鍵詞：	Oral squamous cell carcinoma (OSCC), endothelin-1 (ET-1), vascular endothelial growth factor (VEGF)
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口腔鱗狀上皮細胞癌（OSCC）是一種具侵犯性的口腔黏膜惡性病變，根據衛生署2010年的統計資料，口腔鱗狀上皮細胞癌高居國人頭頸部癌症發生率的第一名，並在癌症死亡率中排名第五。而在惡性腫瘤生長的過程中由於需要大量的養分，所以會分泌許多訊息分子來促使腫瘤周圍的血管新生以供給腫瘤營養，其中以血管內皮生長因子(VEGF)為最主要的訊息分子，其在頭頸部鱗狀上皮細胞癌中有高度表現的情形。在口腔癌的研究指出VEGF的表現量和血管密度具高度正相關，同時也和患者的存活率呈現負相關的情形。此外也有文獻指出由21個胺基酸構成的內皮素(ET-1)，和惡性腫瘤細胞的生長、轉移、甚至腫瘤血管新生有關，而進一步也發現在卵巢癌中，腫瘤所產生的ET-1會藉由autocrine的方式活化癌細胞本身的ETAR受器，使癌細胞的VEGF表現量上升。另外在前人的研究中也發現，比起正常的口腔角質細胞，口腔癌細胞株中的ET-1表現量有明顯的上升的情形，然而在口腔癌中ET-1和VEGF之間的關係尚未被研究過。因此本實驗的目標是想要探討在口腔癌中ET-1是否會透過活化ETAR來調控VEGF的表現，同時希望能在臨床檢體中獲得驗證。首先利用免疫組織化學染色發現，在臨床口腔癌患者的檢體中ET-1和VEGF的表現量有顯著的相關性，而在存活分析方面，VEGF的表現量高的患者其存活率確實有較低的趨勢，並到達統計上的意義，但是ET-1的表現量則否。此外在細胞株實驗中發現口腔癌細胞在給予ET-1刺激之後VEGF的表現量確實會上升，而在給予ETAR的拮抗劑之後VEGF的表現便會明顯受到抑制，顯示在口腔癌細胞中ET-1確實會透過活化ETAR促使VEGF表現量的上升，而此一現象會受到細胞株中的ET-1以及VEGF的基礎表現量的影響。我們同時也發現到和單純給予ETAR拮抗劑的組別比較起來，同時給予ET-1以及ETAR拮抗劑的組別其VEGF的表現量仍然比較高，推論可能是由於ET-1為一強烈的致效劑，也可能因為ET-1的另一個受器ETBR也扮演著同樣功能所導致，但由於VEGF的表現受到許多的因素的調控，因此這部分仍然需要進一步研究。最後在九株不同的口腔癌細胞株的mRNA基礎表現中，發現雖然各個細胞株彼此之間的表現量不一，但是在移除OEC-M1以及OC-2這二株VEGF表現量極高的細胞株之後，ET-1和VEGF彼此之間的表現量成現一正相關的趨勢，符合臨床檢體的發現。綜合上述實驗的結果，在口腔癌中ETAR拮抗劑可以有效的抑制VEGF的表現量，然而是否可以做為對抗癌症的藥物仍需進一步的研究。

Oral squamous cell carcinoma (OSCC) is one kind of invasive malignancy arising from oral epithelial cells. According to the statistical data of the Department of Health in Taiwan, OSCC is most common cancer in head and neck and also the fifth leading cause of cancer mortality in 2010. In order to promote their own growth and dispersion, malignant tumors will secrete variant signaling molecules to recruit host blood vessels to grow into the vicinity of the tumor (so-called tumor angiogenesis). Among these signaling molecules, vascular endothelial growth factor (VEGF) is the most important and usually overexpresses in head and neck squamous cell carcinoma. In OSCC, VEGF highly correlates with microvessel density (MVD) of oral cancer micro-environment, and negatively correlates with the survival rates of OSCC patients. Besides, endothelin-1 (ET-1) composed of 21 amino acids plays an important role in tumor growth, metastases, and angiogenesis. Furthermore, it is proved that ET-1 released by tumors would activate endothelin A receptor (ETAR) of the tumor cells and induce VEGF expression in ovarian. Moreover, studies indicated that the expression of ET-1 is increased in OSCC cell lines. However, the relationship between ET-1 and VEGF in OSCC is still not clear. Therefore, in this study we want to testify that whether ET-1 will modulate the expression of VEGF in OSCC via ETAR, and also confirm this phenomenon in clinical samples at the same time. Firstly, we find the significant correlation between ET-1 and VEGF expressions with immunohistochemical stain in clinical OSCC samples. In survival analysis, the patients with high VEGF expressions, instead of ET-1, have lower survival rates, and there is a statistical significance. In the studies of OSCC cell lines, after ET-1 stimulated, the expression of VEGF was increased in OSCC cells, and was inhibited by ETAR antagonist (BQ-123). This phenomenon indicated that ET-1 would induce the expression of VEGF via ETAR in OSCC, and the basal expressions of ET-1 and VEGF would regulate this result. On the other hand, VEGF expression did not inhibit completely by ETAR antagonist when compared with the control group or the group with BQ-123 only. We speculated that ET-1 might be a strong agonist, or the other receptor of ET-1, endothelin B receptor (ETBR), had the similar function as ETAR did. However, the expression of VEGF is regulated by many factors, so therefore we need to study the underlying mechanism of carcinogenesis. Finally, the basal expressions of mRNA of ET-1 and VEGF were varied between selected nine OSCC cell lines. There was a positive correlation between ET-1 and VEGF when we omitted OEC-M1 and OC-2 which expressed great levels of VEGF mRNA. This result matched the finding of clinical samples. According to the experimental results, ETAR antagonist will inhibit the expression of VEGF in OSCC; however, it still further studies whether ETAR antagonist can become a candidate of the anti-cancer drug.

中文摘要 ............................................................................... i
English abstract ..................................................................... iii
致謝 ...................................................................................... v
Contents ................................................................................ vi
Figure contents .................................................................... viii
Table contents ....................................................................... ix
Abbreviations ......................................................................... x
Introduction ........................................................................... 1
1. Oral squamous cell carcinoma ........................................... 1
2. Endothelin-1 ...................................................................... 2
3. Vessel endothelial growth factor ....................................... 4
4. The mechanism of ET-1induces tumor anginogenesis ....... 5
5. Study purpose .................................................................... 6
Materials and methods .......................................................... 7
Materials ................................................................................ 7
1. Cell lines ............................................................................. 7
2. Primary antibodies ............................................................. 8
3. Secondary antibodies ......................................................... 8
4. PCR Primers ........................................................................ 8
5. Labwares and reagents ....................................................... 9
6. OSCC specimen collection and data establishment .......... 10
Methods ............................................................................... 10
1. Cell culture conditions ...................................................... 10
2. Labeled streptavidin biotin (LSAB) stain ........................... 11
3. RNA extraction .................................................................. 11
4. RNA reverse transcription ................................................. 12
5. Real-time PCR .................................................................... 12
6. Protein extraction and quantification ................................ 13
7. Western blot analysis ......................................................... 13
8. Immunohistochemistry (IHC) stain assays .......................... 14
9. Statistical methods .............................................................. 15
Results ..................................................................................... 16
1. Baseline expression of ET-1 and VEGF in OSCC cells ........... 16
2. ET-1 Increased VEGF productions ....................................... 16
3. ETAR antagonists inhibited VEGF expressions ..................... 17
4. ET-1-induced VEGF production was inhibited by ETAR
 antagonist .............................................................................. 18
5. The correlation between ET-1 and VEGF ............................. 19
6. Positive correlation between ET-1 and VEGF in human
 OSCC ..................................................................................... 19
Discussion ............................................................................... 21
Conclusion .............................................................................. 24
Reference ................................................................................ 25
Figures ..................................................................................... 30
Tables ....................................................................................... 38

                                    

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校內：2014-08-04公開
校外：2014-08-04公開

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