- 學位論文
建立原位膀胱癌模式並使用中藥桂枝茯苓丸治療泌尿上皮腫瘤並探討其抑制機制
Investigation of the Potential Usage of GFW in the Treatment of Urothelial Carcinoma Using the Orthotopic Mouse Bladder Model
摘要
泌尿移形上皮細胞癌在泌尿系統中佔90%的發生率，目前臨床治療方式大多為經過顯微手術去除部分癌細胞之後使用卡介苗或是絲裂霉素清除殘留癌細胞。由於之前實驗室所使用皮下注射方式建立膀胱癌並沒有辦法完整模擬臨床的治療方式，因此我們建立了小鼠原位膀胱癌動物模型，並且現階段臨床使用的絲裂霉素或是卡介苗其副作用較高，我們在本研究即選擇了在中醫中有去瘀血療效的桂枝茯苓丸做為治療的藥物。我們在膀胱植入小鼠膀胱MB-49細胞株的隔天灌注180 mg/ml 濃度的桂枝茯苓丸，之後每隔一天灌注，總共治療十次，結果可以有效減小膀胱腫瘤約80%的大小。另外在細胞實驗中我們也證實了桂枝茯苓丸使用1.5 mg/ml 以上的高濃度會降低小鼠膀胱癌細胞株MB-49的存活率，接著在探討藥物對於癌細胞造成的死亡機制中看到了當藥物的濃度給得越高，代表自噬作用的蛋白LC3-II表現量越高，抗凋亡蛋白Bcl-2會減少，在細胞週期的實驗中看到當給予高於1.5 mg/ml 濃度的桂枝茯苓丸藥物，會使細胞DNA的碘化丙啶 (Propidium Iodide) 螢光訊號升高，表示細胞給予高濃度的藥物時可能會直接造成細胞的壞死。我們也測試了給予藥物後細胞培養基裡釋放出來的乳酸脫氫酶 (Lactate dehydrognase ; LDH) 濃度，也可以看到相符的結果; 綜合以上的實驗結果我們認為高濃度的桂枝茯苓丸的確可以有效地減輕膀胱癌腫瘤大小，並且藥物對癌細胞造成的影響為細胞產生壓力發生自噬作用之後走向壞死。
並列摘要
Urothelial carcinoma is the most common tumor type in the urinary system, it has high recurrent rate after surgery. After transurethral resection of bladder tumor, it will combine treatment with Bacillus Calmette-Guerin (BCG) or mitomycin C to prevent the recurrence. However, the BCG treatment caused severe side effects such as hepatitis, abscess or pneumonitis. Thus, in this study we want to use the traditional chinese medicine Guei-Chih-Fu-Ling-Wan (桂枝茯苓丸, GFW) to investigate whether it will improve the treatment of bladder tumor. First, we established the orthotopic bladder cancer model to investigate the efficiency of GFW treatment and compared the efficacy with BCG, and mitomycin C in vivo. We found that GFW could inhibit the tumor growth in mouse bladder, and the efficacy was similar with BCG and mitomycin C, but did not show sever side effect such as hematuria. Next, we investigated the inhibitory mechanisms of GFW toward urothelial carcinoma. In vitro, cell survival were gradually decreased after GFW treatment and with dose dependent under 1.5mg/ml, however, most cell died when GFW dose were greater than 1.5mg/ml. And, the underlying mechanism of cell death after GFW treatment is through autophagy and necrosis pathway at the low and high dosage of GFW, respectively. Summarized our above data, in vivo, we established the orthotopic bladder cancer model and found that GFW inhibited the mouse bladder tumor growth orthotopically and with minor side effect compared with BCG and mitomycin C. After treatment with GFW, the cell performed cytotoxcicty through autophagy in lower dose but cell necrosis at higher dose.