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研究生:陳佳靜
研究生(外文):Jia-Jing
論文名稱:穿山龍萃取物抑制黑色素瘤細胞侵入及移動能力之機制探討
論文名稱(外文):Study of the inhibitory effect and its mechanism ofDioscorea nipponic Makino extract on melanoma cancer invasion and migration
指導教授:謝易修謝易修引用關係
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生化暨生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:107
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黑色素癌 (melanoma),為皮膚色素腫瘤的一種,主要好發在皮
膚,但也會在眼睛以及腸中發現。惡性的黑色素癌具有侵襲性並會擴
散至全身淋巴結,較其他皮膚癌更常出現轉移的情況。惡性黑色素癌
的病例,治療效果不佳,約10 ~ 40%的患者只有5 年的存活率。惡性
黑色素癌的死亡率根據國家世界衛生組織報告,全球每年有16 萬的
新病例產生,造成48,000 位以上的患者死亡,平均每70 位美國人就
有一位罹患黑色素瘤,大多發生在白人以及成人身上。其他的癌症相
比,惡性黑色素癌細胞進行細胞凋亡的速率較緩慢,細胞不易死亡,
轉移至其他臟器的機會較高。目前癌症治療上,無法達到癌症的治癒
,化學治療或放射療法對人體也會造成傷害,是許多研究開始找尋藥
性溫和的漢方藥物,對人體無傷害性,可以增強人體抵抗力的藥物活
性成分。我們用實驗室99 種漢方藥物進行癌症相關研究,研究發現
穿山龍 (Dioscorea nipponica Makino) 以50% 酒精萃取物 (DNEs)
對黑色素癌細胞 (B16F10) 抑制侵襲與轉移效果最好,於是做更深入
的研究。穿山龍屬民間中草藥,被中草藥店拿來做治療筋骨痠痛與損
傷的漢方療藥,許多研究發現穿山龍可以降低兔子的血膽固醇酯、治
療各種風濕痛、過敏性皮炎、結膜炎、支氣管炎等病,而在癌症上的
相關研究則是完全沒有的。本實驗以極度惡性的老鼠黑色素癌細胞
(B16F10) 做研究,利用穿山龍以50%酒精萃取來看其侵襲與轉移的
能力以及機制作一系列研究。首先以B16F10 做Wound healing assay
,發現穿山龍萃取物抑制細胞移動效果顯著,在MTT assay 中不影響
細胞存活率。接著利用Boyden chamber assay 觀察到在處理穿山龍萃
3
取物50 μg/ml 時invasion 和 migration 被抑制 (P<0.05) 。用
Zymography assay 看MMP-2 與u-PA 活性,細胞處理穿山龍萃取物
20 μg/ml 時MMP-2 就有明顯下降,而u-PA 在處理穿山龍萃取物10
μg/ml 時有意義 (P<0.05) 。以Western blot 看其路徑,主要透過
PI3K/Akt/IkBα/NF-kappa B 路徑抑制核內轉錄因子NF-kappa B 的結
合能力。另外在C57BL/6J 老鼠中,在腹腔注射腫瘤,並隨機分組 (
對照組、DNE 2g/kg body weight/day、DNE 1g/kg body weight/day),
結果顯示,腫瘤隨著餵藥濃度增加而減少。以尾靜脈注射,同樣隨機
分組,肺部組織切片有因為餵藥而使黑色素瘤轉移的部分明顯減少,
2g 組也回到正常切片的組織狀況。在活體實驗中,穿山龍明顯的抑
制腫瘤生長,及侵襲與轉移能力明顯受到抑制。在實驗中再以人類黑
色素癌細胞(A2058)處理穿山龍萃取物,以MTT assay、Zymography
assay、boyden chamber assay 實驗同樣發現相似結果,可以推測穿山
龍萃取物對黑色素癌細胞都有抑制轉移與侵襲的效果。在未來可以發
現更多穿山龍的其他治療功能,也能對其他的疾病有很好的療效,使
其在研究上可以對人類有更多的福祉貢獻。

Melanoma is a malignant tumor of melanocytes which are found predominantly in skin but also in the bowel and the eye. It is one of the rarer types of skin cancer but causes the majority of skin cancer related deaths with only approximately10-40% of a 5-year survival rate. Around 160,000 new cases of melanoma are diagnosed worldwide each year, and it is more frequent in males and Caucasians. It is more common in Caucasian populations living in sunny climates than in other groups. According to a WHO report about 48,000 melanoma related deaths occur worldwide per year. Malignant melanoma accounts for 75 percent of all deaths associated with skin cancer. The treatment includes surgical removal of the tumor, adjuvant treatment, chemo- and immunotherapy, or radiation therapy. Chinese medicinal herbs have been used for therapeutic purposes in traditional and folk medicine for a long time. In this study, we determined the effect of 99 herb extracts on cell migration; meanwhile, showed that 50% alcohol extracts of Dioscorea nipponica Makino (DNEs) could efficaciously inhibit melanoma cell migration and invasion by wound healing assay and Boyden chamber invasion assay. From this result, DNEs were chosen for the subsequent experiments. Dioscorea nipponica has long been used as a folk medicine for asthma, rheumatoid arthritis, bronchitis, and other diseases. These compounds have also been reported to be able to decrease serum lipids in cholesterol-fed rabbits. However, these studies on functions of Dioscorea nipponica have been mainly focused on the effects of anti-inflammatory, anti-obesity or anti-allergic property, whereas the effect of Dioscorea nipponica Makino on migration and invasion of tumor cells has not been clearly clarified. This study first demonstrates that, in the absence of cytotoxicity, DNEs exerted a dose-dependent inhibitory effect on the invasion, migration, and motility of highly metastatic murin melanoma cells (B16F10) and human melanoma cells (A2058). We examined the effect of DNEs on factors of cancer metastasis. We treated tumor cells with various concentrations of DNEs, for set periods, and then subjected cells to gelatin zymography, casein zymography, and Western blot to investigate the expression of matrix metalloproteinase-2 (MMP-2), urokinase-type plasminogen activator (u-PA), and tissue inhibitor matrix metalloproteinase -2 (TIMP-2). Following treatment with DNEs was found to decrease the expression of MMP-2 and u-PA in a concentration-dependent manner and enhance the expression of TIMP-2. To investigate the possible mechanisms involved in these events, we performed western blot analysis to find that DNEs inhibited phosphorylation of Akt. A treatment with DNEs to B16F10 cells also inhibited the activation of NF- kappa B as shown by Western blot and electrophoretic mobility shift assay (EMSA). Finally, DNEs were evidenced by its inhibition on the metastasis and tumor volume of B16F10 cells in vivo. Taken together, these findings suggested that DNEs could reduce the metastasis of murin melanoma cells, thereby constituting an adjuvant treatment for metastasis control.

目錄
目錄………………………………………………………………..1
壹、 中英文摘要……………………………………………… 2
貳、 縮寫表………………………………………………….... 7
參、 緒論……………………………………………………… 9
肆、 研究動機……………………………………………….. .25
伍、 實驗材料與方法……………………………………….. .27
陸、 實驗結果………………………………………………....45
柒、 討論………………………………………………..……. .55
捌、 參考文獻………………………………………………... .63
玖、 圖表與圖表說明……………………………………….. ..75
拾、 附圖表…………………………………………………….106


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