臺灣博碩士論文加值系統

Baicalein 是從中草藥-黃芩的根部中萃取出來的成分之ㄧ，具有很強的生物活性。許多文獻指出，在肺癌、乳癌的細胞中可以發現Baicalein 具有抗發炎、抑制細胞生長、促進凋亡的作用。故本篇研究的目的是運用細胞及動物模式，測試是否Baicalein 對口腔癌也有治療的效果。
在細胞模式中，我們將不同濃度的Baicalein與口腔癌細胞株HSC-3 共同培養。藉由MTT assay，我們發現Baicalein濃度在14~112 μg/ml，有抑制細胞生長的情況；且在IC50 在藥物作用48小時大約是56 μg/ml。而流式細胞儀的分析結果顯示，隨著藥物濃度的上升，細胞週期顯著的停滯在S期。西方墨點法可以發現Bax、PARP的表現量有逐漸上升的趨勢，Bcl-2表現量則相反。細胞免疫染色法可以看到隨著藥物劑量的加大，ki67、Bcl-2有下降的趨勢； Bax的表現則有上升的趨勢 。綜合以上結果顯示，Baicalein 的確具有抑制癌細胞生長的作用，進而促進走向凋亡的路徑。
在動物實驗中，將所有實驗用倉鼠大致分為兩組進行：單純以0.5%DMBA (7,12 –dimethylbenzanthracene) 誘導的組別，分別於八週、十週、十二週時犧牲。另一組則為0.5% DMBA誘導並同時給予不同濃度的Baicalein，於飼養至十二週後犧牲。DMBA 的給予方式是用2號的水彩筆於每週一、三、五，各塗抹口頰兩側的囊袋，各繞八圈為主。而有Baicalein組別，則以同樣方式於每週二、四、六塗抹不同濃度的Baicalein (7~28 mg/ml)。根據組織蘇木紫-伊紅染色法 (Hematoxylin and Eosin stain；H&E stain) 的結果顯示，大約在12週時，單純塗抹DMBA的組別全部隻數的倉鼠可以完全達到carcinoma的階段。而同時給予不同濃度的Baicalein (7~28 mg/ml)的組別，由組織免疫染色法 (Immunohistochemistry stain；IHC)的檢查發現，其與細胞增生的蛋白質PCNA，及與細胞凋亡的指標蛋白質Bax、Bcl-2的表現量與正常倉鼠口腔上皮的同種蛋白質表現沒有顯著的變化。
綜合以上結果，我們可以推測Baicalein 在HSC-3 cell line 中可以抑制細胞的生長，促進凋亡；但在動物試驗中，至目前使用的最大劑量28 mg/ml，似乎無法看到顯著的抑制腫瘤生長的情況。

Baicalein ( 5,6,7-trihydroxyflavone ) is a bioactive flavone isolated from the root of Scutellaria baicalensis Georgi ( Huang Qin ). It has been used as an anti-inflammatory agent in Chinese herb medicine. Recently, Baicalein was found to have anti-proliferative and apoptotic effects on prostate, lung and breast cancer cells. The objective of our study is to investigate the possible chemopreventive effect of Baicalein on both human oral cancer cells ( in vitro ) and oral carcinogenesis animal model ( in vivo ).
By MTT test, we demonstrated that Baicalein ( ranging from 14 to 112 μg/ml ) inhibited cell proliferation in a does-dependent manner in human oral caner cell line, HSC-3. The IC50 at 48hr of Baicalein treatment was 56μg/ml. Cell cycle analysis showed an increased percentage of cells in S-phase and a decreased percentage of G1 phase after incubation with 28 μg/ml of Baicalein for 48 hrs. These results indicated that Baicalein has antiproliferative effect on human oral cancer cells via arresting cells at S-phase.
We further investigated the antiproliferative effect of Baicalin on the DMBA-induced hamster buccal pouch carcinogenesis model. Male Syrian hamsters were painted with DMBA ( 0.5% in mineral oil ) in both sides of buccal pouch 3 times per week for 12 week, and the hamsters were sacrificed. Various concentrations of Baicalein ( 7, 14 or 28mg/ml ) were also applied to the buccal pouch, 3 times per week during the days when DMBA were painted. At the end of the 12 wks, the buccal pouch was dissected and the numbers and the sizes of the tumors were measured before histological examination by H&E. All the hamsters, with DMBA alone or the combination of DMBA + Baicalein, showed squamous cell carcinoma after 12 wks experimental period. Decreased tumor sizes were only observed in hamsters treated with 28mg/ml
Baicalein . The cell proliferation marker PCNA 、apoptosis marker Bcl-2 were not decreased in the group of the 28mg/ml Baicalein treatment by IHC. Another apoptosis marker Bax was not increased in the group of the 28ug/ml Baicalein treatment. All of these results suggested that Baicalein had antiproliferative effect only in vitro.

總目錄
中文摘要 VI
英文摘要 IX
第一章、 前言 1
第一節、 口腔癌 1
第二節、 口腔癌的研究模式: in vitro 4
第三節、 口腔癌的研究模式: in vivo 17
第四節、 中國傳統藥物在癌症治療的應用 18
第五節、 研究目的 21
第二章、 材料與方法 22
第一節、 實驗材料 22
A. 儀器 22
B. 材料 23
C. 試劑 23
第二節、 實驗方法 26
1. 動物飼養 26
2. 組織冷凍切片 26
3. 組織染色(Hematoxylin & Eosin Stain ; H&E stain ) 27
4. 免疫組織染色(Immunohistochemistry stain；IHC) 27
5. 免疫細胞染色(Immunocytochemistry stain；ICC) 28
6. 細胞培養 29
7. 細胞存活率試驗 (MTT assay) 29
8. 細胞週期試驗(Cell cycle analysis) 30
9. 西方墨點法(Western blot)偵測相關蛋白質變化 32
第三節、 統計方法 33
第三章、 實驗設計 35
第四章、 實驗結果 37
?? In vivo 37
1. Baicalein 對DMBA誘導的倉鼠之飲食及體重的影響 37
2. Baicalein 對DMBA誘導的倉鼠之腫瘤大小及癌化階段之影響 37
3. Baicalein 對DMBA誘導的倉鼠口腔癌細胞增生及細胞凋亡等相關的蛋白質之影響 39
?? In vitro 39
1. Baicalein對HSC-3細胞存活率的影響 39
2. Baicalein 對HSC-3細胞週期之影響 40
3. Baicalein 誘導HSC-3細胞凋亡與相關蛋白之表現 41
4. Baicalein 對HSC-3細胞之細胞免疫染色結果 42
第五章、 結論 43
第六章、討論 45
第七章、 未來發展方向 50
第八章、結果圖表目錄 51
第九章、 Appendix 69
第十章、 參考文獻 71

結果表目錄
Table 1、在DMBA誘導的倉鼠動物模式中Baicalein 對於腫瘤大小 53
Table 2、以DMBA誘導的倉鼠動物模式中其癌化分級結果 54
Table 3、Baicalein 對於以DMBA誘導的倉鼠動物模式中其癌化階段 55

結果圖目錄
圖一、Baicalein 對於體重及飲食量的影響 52
圖二、以DMBA誘導倉鼠口腔上皮細胞癌化過程，不同組別的組織 56
圖三、以DMBA誘導倉鼠口腔上皮細胞癌化過程(a)及Baicalein對 57
圖四、DMBA誘導及 Baicalein對 (a)PCNA、(b)Bax、(c)Bcl-2 60
圖五、不同濃度的Baicalein 對於口腔癌細胞株HSC-3在48小時的存活率 61
圖六Baicalein 對於口腔癌細胞株HSC-3在24、48、72小時的存活率 62
圖七、不同濃度的Baicalein 對於口腔癌細胞株HSC-3在48小時的細胞週期影響 63
圖八、不同濃度的Baicalein 對於口腔癌細胞株HSC-3在48及72 64
圖九、不同濃度的Baicalein 48小時的作用在HSC-3與細胞凋 65
圖十、Baicalein 作用在HSC-3細胞48小時後，細胞增生的指標 68

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