化學藥物治療與放射線治療引發黏膜炎動物模式之建立與新穎化合物AzP之抗黏膜炎作用與機制探討

Establishment of animal model for chemotherapy- and radiotherapy-induced mucositis and study of anti-mucositis effect and mechanism of novel compound AzP

黃維光(Wei-Kuang Huang)

指導教授 ： 鄭幼文

臺北醫學大學/藥學院/藥學系(碩博士班)/碩士(2013年)

摘要

口腔與小腸潰瘍性黏膜炎是一種在放射線治療與化學藥物治療癌症時常見的毒性反應 (toxicity)。其對於健康、生活品質與經濟支出，甚至是死亡率皆有顯著的影響。近年來的研究發現黏膜炎的病理機轉極為複雜且尚有許多未知的地方，但可大致區分為五個階段：起始期、初期傷害期、訊號擴大期、潰瘍期與癒合期。目前臨床上針對黏膜炎雖有許多輔助療法，像是給予止痛藥、營養針、抗生素或是止瀉劑等，但是針對黏膜炎所研發之藥物除了唯一一種且價格昂貴的人類合成 Keratinocyte Growth Factor (KGF) 蛋白藥物 Palifermin 之外，全世界尚無其他有效降低或防止黏膜炎發生的藥物，更遑論小分子化合物。AzP (Aza-PBHA) 為台北醫學大學藥學系 王惠珀 教授，針對由癌症治療所引起之口腔與小腸潰瘍性黏膜炎研發合成的新穎性化合物。在本文中，首先將會建立口腔與腸道黏膜炎之動物模型，再藉由動物模型來研究 AzP 對於口腔與腸道黏膜炎之效果。另外再觀察 AzP 在大鼠小腸上皮裂隙細胞中存活率與增生的表現。
活體動物模型分成兩部分：一、使用皮膚採樣器在敘利亞倉鼠口中製造傷口，再腹腔給予 5-Fluorouracil (5-FU) 模擬化療造成傷口潰瘍以模擬口腔黏膜炎。傷害造成之後，連續由腹腔給予 AzP 5天並每天觀察傷口並拍照；二、C57BL/6 小鼠給予 Cisplatin 處理、放射線處理或是兩者共同處理，分別模擬化療、放療、以及化療配合放療等臨床常見的癌症治療方式所造成的小腸黏膜炎。由體重、小腸絨毛長度觀察所受傷害嚴重程度，並使用免疫組織染色法與酵素免疫吸附法觀察血中IL-6與腸道黏膜組織中 COX-2 蛋白質表現。由實驗結果發現，倉鼠口腔黏膜炎實驗中，給予 AzP 後傷口大小顯著提早癒合，潰瘍情況也明顯改善；小鼠小腸黏膜炎實驗中，cisplatin 所引起的體重掉落、小腸絨毛斷裂情況以及血中IL-6與腸道黏膜組織中 COX-2 的過度表現也在給予 AzP 後明顯改善。另外使用大鼠的小腸上皮裂隙細胞 (IEC-6) 進行細胞實驗，以觀察 AzP 對於小腸上皮裂隙細胞的存活率與增生情況。給予 AzP 之後小腸上皮裂隙細胞沒有造成死亡甚至在 10 ?嵱 濃度以上會促進細胞的增生。
綜合以上實驗結果顯示，從動物模型可觀察到 AzP 對於由化療藥物所引起的黏膜炎有治療的效果而且效果甚至比 palifermin 還好。這樣的結果可能是因為 AzP 對於細胞保護作用、細胞激素調控與促進細胞再生都有一定的功效。因此我們認為 AzP 具有發展成抗黏膜炎藥物的潛力。

關鍵字

黏膜炎 動物模式 化療 放療

並列摘要

Oral and gastrointestinal ulcerative mucositis is a common toxicity in many forms of radiotherapy and chemotherapy. It has a significant impact on health, quality of life and economic outcomes, as well as mortality. Recent research had indicated that pathological mechanism of mucositis is extremely complex and still so many remain unknown. Mucositis can be broadly divided into five stages: Initiation, Primary damage, Signal amplification, Ulceration and Healing. Many complementary therapies for mucositis, including oral pain control, nutritional support, infection treatment and control of diarrhea. There is no effective drug to reduce or prevent mucositis except Palifermin, a recombinant human keratinocyt
infection treatment and control of diarrhea. There is no effective drug to reduce or prevent mucositis except Palifermin, a recombinant human keratinocyte growth factor (rhKGF), which is extremely expensive. AzP, a novel small molecule compound which is made and developed to against cancer treatment-induced mucositis by Dr. Hui-Po Wang’s lab in Taipei Medical University. In this study, the animal models of intestinal mucositis which induced by cemotherapy, radiotherapy, radio combined cemotherapy were established for studied the efficacy of AzP against intestinal mucositis.
Two parts divided for in vivo study: For imitation of oral mucositis, skin puncher was used to form a 6mm wound in right pouch of Syrian hamster and 5-Fluorouracil (5-FU) was given intraperitoneally. Wound of pouch was observed and photographed for 5 days consecutively. For imitation of intestinal mucositis induced by chemotherapy and/or radiotherapy, cisplatin, radiation and cisplatin combined radiation were given respectively to C57BL/6 mice. Severity of mucositis was observed by body weight change of everyday and villi length after sacrifice. The expression of COX-2 in mucosa and inflammatory cytokine IL-6 of serum were also observed using immunohistochemistry and ELISA. The result showed that the wounds of pouches were healed earlier and less severe of ulcer in hamster model after AzP was given. In C57BL/6 mice, AzP ameliorated the body weight dropping, villi cleavage and over expression of inflammatory related protein which induced by cisplatin. Rat intestinal epithelial crypt cell (IEC-6) was used to observe cell viability and the efficacy of proliferation after treating AzP. Result showed that AzP promotes proliferation in IEC-6 cell above the concentration of 10 ?嵱.
In summary, AzP has the efficacy and yet, better than palifernin, against mucositis which induced by chemotherapy. The possibly reason in that AzP has pleiotropic effect in cytoprotection, cytokine regulation and cell proliferation. So we consider that AzP is a potential compound against chemotherapy-induced mucosuitis.

並列關鍵字

mucositis animal model chemotherapy radiotherapy AzP

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