臺灣博碩士論文加值系統

食道癌(esophageal cancer)在全球癌症死亡人數排名第六，死亡率高達87.8%，目前尚無有效治療方式，主因為食道癌的治療用藥不多和對化療藥劑產生抗藥性及副作用等問題，所以需迫切開發新的治療藥物。吳茱萸次鹼(rutaecarpine, RTP)為一種quinazolinocarboline，是中草藥吳茱萸(Evodia rutaecarpa)主要的生物鹼化合物之一，許多研究發現其它有心血管保護作用、抗發炎(anti-inflammation)及抑制癌細胞增生等作用。然而，RTP是否具有誘發食道癌細胞凋亡的活性，尚未被研究，其相關機制亦不清楚。因此，在本研究中，我們將以細胞及異體移植模式(xenograft model)進行探討。
在細胞模式中，將CE81T/VGH人類食道鱗狀癌細胞與RTP (5、10及20 μM)共同培養，並以常用化療藥物-順鉑(cis-diamminedichloroplatinum (II), CDDP)做為正控制組，進行細胞週(cell cycle)及細胞凋亡(apoptosis)分析，並進一步以西方墨點法分p53、Bax (Bcl-2-associated X)與Bcl-2 (B-cell lymphoma 2)蛋白質表現。在動物模式中，我們將以裸小鼠(BALB/cAnN)進行異體移植試驗，並分別給予低劑量RTP (25 mg/kg body weight /twice weekly/p.o)、高劑量RTP (75 mg/kg body weight/twice weekly/p.o)及CDDP (5 mg/kg body weight/twice weekly/i.p)，六週後犧牲動物並進行各項分析。
細胞試驗發現，RTP可以顯著抑制食道鱗狀癌細胞增生及促進其細胞凋亡，並具有劑量及時間關係(P<0.05)。進一步研究發現RTP可以顯著增加p53蛋白質表現，並誘導促凋亡蛋白Bax和抑制抗凋亡蛋白Bcl-2的表現(P<0.05)，也可以促進Sub-G1生成。推測RTP可能透過增強p53蛋白質的表現並促進Bax及抑制Bcl-2蛋白表現，進而誘發食道鱗狀癌細胞凋亡。
在動物模式研究結果顯示，與腫瘤組相比，RTP高劑量組與CDDP處理組之腫瘤組織體積及重量均顯著降低(P<0.05)。然而RTP低劑量、高劑量及CDDP處理組在p53蛋白質與Bax蛋白質表現均顯著增加，並抑制Bcl-2蛋
白質表現(P<0.05)，其結果與細胞實驗相似。進一步發現RTP低劑量、高劑量及CDDP處理組不但增加caspase-9及caspase-3蛋白質表現(P<0.05)，也能顯著抑制腫瘤組織中PCNA (Proliferating cell nuclear antigen)蛋白質表現(P<0.05)。
我們藉由分析血清尿素氮(urea nitrogen, BUN)與肌肝酸(creatinin, Cre)的濃度及血漿紅血球(red blood cell)、血紅素(hemoglobin)、血球容積比(hematocrit)與平均紅血球血紅素濃度(mean corpuscular hemoglobin concentration)等，探討其RTP是否具有腎毒性(nephrotoxicity)及血液毒性(haematological toxicity)，結果顯示，與控制組相比，RTP高劑量或低劑處理組均無顯著差異(P>0.05)，但CDDP處理組BUN與Cre濃度均顯著上升(P<0.05)。實驗結果說明在不會產生腎毒性及血液毒性的情況下，RTP可以有效抑制食道鱗狀癌細胞生長，且抑制與CDDP相似。
綜合體內外實驗結果發現，RTP可能藉由增加p53蛋白質、促進Bax蛋白質、抑制Bcl-2蛋白質、活化下游caspase-9及caspase-3蛋白質，增進食道鱗狀癌細胞凋亡，進而抑制腫瘤生長，推測其機制可能與RTP增加p53蛋白質表現，經由內在性路徑促進(intrinsic pathway)細胞凋亡有關，但詳細機制尚須進一步探討。

Esophageal cancer is the sixth leading cause of cancer deaths worldwide with a mortality rate of 87.8%. There is currently no effective treatment for esophageal cancer due to a lack of appropriate medications and drug resistance and side effects resulting from chemotherapy drugs; thus, there is an urgent need for the development of new medications. Rutaecarpine (RTP) is a type of quinazolinocarboline and an important alkaloid from the Chinese medicinal herb Evodia rutaecarpa. Many studies have shown that RTP has cardiovascular protective, anti-inflammatory, anti-proliferative. However, no research has been conducted on the ability of RTP to induce apoptosis in esophageal cancer cells and its mechanism remains unclear. Therefore, this study investigated the issue using cells and xenograft models.
In the cell model, CE81T/VGH human esophageal squamous-cell carcinoma cells were treated with various concentrations of RTP (5, 10, and 20 μM). Cisplatin (cis-diamminedichloroplatinum (II), CDDP), a chemotherapy drug, was treated as the positive control group. The cell cycle and cell apoptosis were analyzed. The expression of proteins p53, Bax (Bcl-2-associated X), and Bcl-2 (B-cell lymphoma 2) was identified using Western blot. In the animal model, a xenograft test was conducted on nude mice (BALB/cAnN) treated with low dose RTP (25 mg/kg body weight /twice weekly/p.o), high dose RTP (75 mg/kg body weight/twice weekly/p.o), or CDDP (5 mg/kg body weight/twice weekly/i.p). The animals were sacrificed and analyzed after six weeks.
The cell model results indicated that RTP significantly inhibited the proliferation of esophageal squamous-cell carcinoma cells and induced apoptosis, which was dose- and time-dependent (P<0.05). Further investigation showed that RTP significantly increased p53 protein expression, induced Bax, and inhibited Bcl-2 expression (P<0.05). It is supposed that RTP can induce the apoptosis of esophageal squamous-cell carcinoma cells by increasing the expression of p53 and Bax proteins and inhibiting Bcl-2 protein.
The animal model results indicated that tumor tissue volume and weight were significantly reduced in the groups with high dosage RTP and CDDP treatments (P<0.05). The expression of p53 and Bax was significantly increased and the expression of Bcl-2 was reduced in the groups treated with low dosage RTP, high dosage RTP, and CDDP (P<0.05). These results are similar to those in the cell experiment. Further analysis showed that low and high dosage RTP and CDDP treatment increased the expression of caspase-9 and caspase-3 proteins (P<0.05). Moreover, RTP significantly inhibited the expression of proliferating cell nuclear antigen (PCNA) in tumor tissue (P<0.05). As such, these findings confirmed the results obtained in the cell experiment.
Furthermore, this study also analyzed the concentrations of blood urea nitrogen (BUN) and creatinine (Cre) in blood serum, red blood cells, hemoglobin, hematocrit, and mean corpuscular hemoglobin to examine whether RTP had nephrotoxicity and haematological toxicity. The results indicated that there were no significant differences between the control group and the groups treated with high and low dosage RTP (P>0.05), whereas in the group treated with CDDP, BUN and Cre concentrations significantly increased (P<0.05). With regard to the blood test, there were no significant differences between the control group and the groups treated with high and low dosage RTP (P>0.05). This shows that the RTP dosages used in this study did not cause nephrotoxicity or haematological toxicity. However, its inhibiting effect on esophageal squamous-cell carcinoma cells was similar to that of CDDP.
Taken together results showed that RTP increases p53 and Bax proteins, inhibits Bcl-2 protein, and activates caspase-9 and caspase-3 proteins, inducing the apoptosis of esophageal squamous-cell carcinoma cells and inhibiting tumor growth. These findings suggest that the RTP increases the expression of protein p53 and induces apoptosis via an intrinsic pathway. However, further research is needed to determine its exact mechanism.

中文摘要……………………………………………Ⅰ
英文摘要……………………………………………Ⅲ
目錄………………………………………………………Ⅴ
表目次…………………………………………………Ⅷ
圖目次…………………………………………………Ⅸ
縮寫表…………………………………………………ⅩⅠ
實驗架構……………………………………………ⅩⅢ
1. 文獻探討………………………………………1
2. 研究動機與目的……………………………6
3. 材料與方法…………………………………7
4. 結果………………………………………………20
5. 討論………………………………………………25
6. 結論………………………………………………28
7. 表目次…………………………………………29
8. 圖目次…………………………………………36
9. 參考文獻……………………………………54
10. 附錄……………………………………………61

衛生福利部統計處。民國104年主要死因分析。網址: http://www.mohw.gov.tw
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