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題 名 | Targeting Transforming Growth Factor-Beta1 by RNA Interference Attenuates Melanoma Cell Growth and Inhibits Pulmonary Metastasis in an ex-vivo Animal Model=針對轉型生長因子β1進行核糖核酸干擾抑制作用能縮減黑色素瘤細胞在體內的生長並抑制腫瘤細胞轉移 |
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作 者 | 戴國峰; 曾斯偉; 徐啟仁; 葉宇捷; 楊世頤; 王健興; | 書刊名 | 臺灣整形外科醫學會雜誌 |
卷 期 | 19:1 2010.03[民99.03] |
頁 次 | 頁1-15 |
分類號 | 415.78 |
關鍵詞 | RNA interference; TGF-β1; Melanoma; |
語 文 | 英文(English) |
中文摘要 | 背景: 在罹患惡性黑色素瘤的病患中,轉型生長因子β被視為降低病患對抗腫瘤免疫反應的重要因子。 目的: 本研究之目的是要以核糖核酸干擾抑制技術降低黑色素瘤細胞內轉型生長因子β1的表現,藉此達成抑制黑色素瘤在小黑鼠皮下的生長與轉移。材料與方法:我們將設計好的轉型生長因子β1構築到一個反轉錄病毒載體pSM2(TGF-β1-RNAi/pSM2)並穩定地轉植到小黑鼠黑色素瘤細胞B16F0細胞株(B16F0/TGF-β1-RNAi),同時也將反轉錄病毒載體pSM2穩定地轉植到小黑鼠黑色素瘤細胞(B16F0/vector-control),作為實驗中的載體控制組細胞。之後分別將5×10^6個野生型腫瘤細胞(B16F0),B16F0/vector-control或是B16F0/TGF-β1-RNAi細胞植入六至九週大且具正常免疫力的小黑鼠(C57BL/6)皮下,比較這些腫瘤細胞在小黑鼠皮下的生長情形。這些細胞也藉由尾靜脈注射到C57BL/6老鼠體內,評估這些腫瘤細胞在肺臟的轉移情形。 結果: 與野生型腫瘤細胞(B16F0)或是載體控制組細胞比較,B16F0/TGF-β1-RNAi細胞內轉型生長因子β1的表現明顯減少,這三株細胞在體外培養時生長速率差不多,在植入小鼠皮下後第十四天,植入野生型腫瘤細胞(B16F0)的小黑鼠腫瘤細胞其大小為495.32±77.25立方厘米,植入載體控制組細胞(B16F0/vector-control)的小黑鼠腫瘤細胞其大小為516.65±73.71立方厘米,植入B16F0/TGF-β1-RNAi細胞的小黑鼠腫瘤細胞其大小則只有326.72±97.34立方厘米,明顯降低許多,以one-wayANOVA統計分析p<0.05,在B16F0/TGF-β1-RNAi腫瘤內發現有明顯的CD4(上标 +)及CD8(上标 +)T細胞浸潤,而且B16F0/TGF-β1-RNAi腫瘤內微血管密度明顯降低。以尾靜脈注射B16F0/TGF-β1-RNAi腫瘤細胞的老鼠,在注射後第21天觀察到轉移至肺部的腫瘤細胞明顯減少。 結論: 我們所設計的轉型生長因子β1寡核酸能抑制黑色素瘤細胞轉型生長因子β1的表現,利用核糖核酸干擾抑制技術來降低黑色素瘤轉型生長因子β1的產生,可以抑制此腫瘤細胞在小黑鼠皮下的生長與肺臟的轉移,這個發現有助於未來腫瘤基因治療的應用。 |
英文摘要 | Background: Transforming growth factor-beta (TGF-β) is the key molecule implicated in impaired immune function in human patients with malignant melanoma. We examined the effects of TGF-β1 protein knockdown by RNA interference on the growth and metastasis of melanoma in C57BL/6 mice induced by the B16F0 cell line. Aim and Objectives: The purpose of this work is to provide preclinical assessment of the therapeutic potential of TGF-β1 protein knockdown by RNA interference in melanoma. Materials and Methods: The TGF-β1 hairpin oligonucleotide was cloned into retroviral vector pSM2. The resulting plasmid (TGF-β1 -RNAi/pSM2) was stably introduced into murine melanoma cell line, B16F0, and designated as B16F0/TGF-β1-RNAi cells. The vector plasmid was transfected into B16F0 cells and designed as B16F0/vector-control cells served as a control. Five million B16F0 cells, B16F0/vector-control cells and B16F0/TGF-β1-RNAi cells were injected subcutaneously into the right flanks of adult female syngeneic mice C57BL/6 respectively. The growth rate of the parental cell and genetically modified murine melanoma cells were compared. C57BL/6 mice were also evaluated for pulmonary metastasis following tail vein injection of two million B16F0 cells, B16F0/vector-control cells and B16F0/TGF-β1-RNAi cells. Results: TGF-β1 expression was reduced in B16F0/TGF-β1-RNAi cells compared with B16F0 cells and B16F0/vector-control cells. The proliferation rate of B16F0/TGF-β1-RNAi cells was similar to that of the B16F0 cells and B16F0/vector-control cells in vitro. The tumor sizes were 495.32±77.25 mm^3, 516.65±73.71 mm^3 and 326.72±97.34 mm^3 at the fourteenth day in the mice receiving B16F0 cells, B16F0/vector-control cells and B16F0/TGF β1-RNAi cells respectively. The p value was less than 0.05 by one-way ANOVA. TGF-β1 knockdown in B16F0 cells enhanced the infiltration of CD4(superscript +) and CD8(superscript +) T cells in the tumor regions. The blood vessel density of the tumors markedly reduced in B16F0/TGF β1-RNAi tumors. The pulmonary metastasis also reduced significantly on 21day in mice injected with B16F0/TGF β1-RNAi tumors compared with mice injected with B16F0 cells and B16F0/vector-control cells. Conclusions: We found that TGF-β1 protein expression was significantly reduced from B16F0 cells after TGF-β1 hairpin oligonucleotide transduction. Silencing of TGF-β1 expression in B16F0 cells by RNA interference technology can inhibit the growth and metastasis of this tumor after being transplanted to C57BL/6 mice. Overall, our results have important implications for the clinical use of RNA interference targeting TGF-β1 as cancer gene therapy. |
本系統中英文摘要資訊取自各篇刊載內容。