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題名 | Inhibition of Melanoma Cell Growth and Pulmonary Metastasis in an Ex-vivo Animal Model Using Adenovirus Armed Short Hairpin RNA Targeting Transforming Growth Factor-Beta1 Encoding mRNA=以腺病毒為載體針對轉型生長因子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; 洪茂欽; 孟凡傑; 孫暐傑; 王健興; | 書刊名 | 慈濟技術學院學報 |
卷期 | 16 2011.03[民100.03] |
頁次 | 頁375-396 |
分類號 | 415.78 |
關鍵詞 | 腺病毒載體; 核糖核酸干擾抑制; 轉型生長因子β1; 黑色素瘤; Adenoviral vector; RNA interference; TGF-β1 melanoma; |
語文 | 英文(English) |
中文摘要 | 目的:核糖核酸干擾抑制技術被應用在抑制特定基因的表現上,已是一個相當重要的技術,本研究之目的是要以腺病毒載體攜帶轉型生長因子β1 寡核苷酸,藉此達成抑制小黑鼠黑色素瘤轉型生長因子β1 的表現,並觀察利用這樣的方法在抑制轉型生長因 子β1 的表現後,黑色素瘤在小黑鼠皮下的生長與轉移情形。 材料與方法:我們將設計好的轉型生長因子β1 寡核苷酸構築到一個腺病毒載體,將此重組腺病毒稱為Ad/TGF-β1-RNAi,之後感染小黑鼠黑色素瘤細胞—B16F0 細胞株,感染後的細胞命名為B16F0/TGF-β1-RNAi 細胞,另外也將不帶任何轉殖基因的空白腺病毒載體感染小黑鼠黑色素瘤細胞並命名為B16F0/vector-control 細胞,作為實驗中的載體控制組細胞,分別將上述3 × 106 個野生型腫瘤細胞(B16F0)、載體控制組細胞(B16F0/vector-control)或是抑制TGF-β1 表現的腫瘤細胞(B16F0/TGF-β1-RNAi)植入六至八週大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。上述細胞也以尾靜脈注射方式注射到C57BL/6 老鼠體內,來評估這些腫瘤細胞轉移到肺臟的能力。 結果:與野生型腫瘤細胞(B16F0)或是載體控制組細胞(B16F0/vector-control)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756.09 ± 65.35 mm3 , 植入載體控制組細胞( B16F0/vector-control)的小黑鼠腫瘤細胞其大小為798.48 ± 78.77 mm3,植入B16F0/TGF-β1-RNAi 細胞的小黑鼠腫瘤細胞其大小則只有203.55 ± 24.56 mm3,明顯降低許多,以one-way ANOVA 統計分析 p<0.01,在B16F0/TGF-β1-RNAi 腫瘤內發現有明顯的CD4+及CD8+T 細胞浸潤。尾靜脈注射B16F0/TGF-β1-RNAi 腫瘤細胞的老鼠,在注射後第14 及第21 天都觀察到轉移至肺部的腫瘤細胞明顯減少。 結論:我們所設計帶著轉型生長因子β1 寡核苷酸的重組腺病毒在感染黑色素瘤細胞(B16F0 細胞株)後,明顯抑制B16F0 細胞株轉型生長因子β1 的表現,並抑制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臨床上腫瘤治療的應用。 |
英文摘要 | Objectives: In this study, we used an adenovirus-based shRNA expression system and successfully constructed Ad/TGF-β1-RNAi which mediated the RNAi for TGF-β1 gene silencing. 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. Materials and Methods: The TGF-β1 hairpin oligonucleotide was cloned into adenoviral vector. The resulting recombinant adenoviruses infected murine melanoma cell line, B16F0, and designated as B16F0/TGF-β1-RNAi cells. The blank adenoviral vector also infected B16F0 cells and designed as B16F0/vector-control cells served as a control. Three million wild type 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. C57BL/6 mice were 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 tumor sizes were 756.09 ± 65.35 mm3, 798.48 ± 78.77 mm3 and 203.55 ± 24.56 mm3 at the fourteenth day in the mice receiving B16F0 cells, B16F0/vector-control cells and B16F0/TGF-β1-RNAi cells respectively. TGF-β1 knockdown in B16F0 cells enhanced the infiltration of CD4+ and CD8+ T cells in the tumor regions. The pulmonary metastasis also reduced significantly on 14 day and 21day in mice injected with B16F0/TGFβ1-RNAi tumors. Conclusions: Our results showed that Ad/TGF-β1-RNAi could induce silencing of the TGF-β1 gene effectively. Silencing of TGF-β1 expression in B16F0 cells by RNA interference can inhibit the growth and metastasis of melanoma cells in syngeneic C57BL/6 mice. RNA interference targeting TGF-β1 by adenoviral vector might be a promising vector for cancer therapy. |
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