臺灣博碩士論文加值系統

癌症的轉移是腫瘤發展過程中最難治療的致死原因。目前對於腫瘤細胞轉移與生長的新興療法是以腫瘤內血管新生的特性作為標的物，試圖透過抑制血管新生來達到抑制腫瘤生長及轉移的目的。然而，在這些進程中，腫瘤細胞與腫瘤微環境的發炎現象也扮演著一定的角色。我們利用慢病毒 (Lentivirus) 載體攜帶kallistatin基因 (LV-Kallistatin) 以進行小鼠肺癌 (LL/2) 轉移與生長的治療。在利用以LV-Kallistatin感染細胞後所取得的條件培養液與人類臍靜脈內皮細胞 (HUVEC) 或LL/2作用後發現，細胞的轉移、增生、黏附和侵噬的能力有被抑制的現象。此外，先以LV-Kallistatin感染穩定表現冷光的肺癌細胞株 (LL-Luc) 後篩選出可以穩定表現這兩種蛋白質的肺癌細胞株 (LL-Luc-Kallistatin) ，再以尾靜脈給予的方式來分析kallistatin在小鼠體內對腫瘤細胞的影響。實驗證實表現kallistatin的肺癌細胞株生長有趨緩的現象。在直接以慢病毒傳送kallistatin基因對模擬腫瘤轉移實驗模式 (experimental metastasis model) 進行治療後，從肺臟腫瘤組織的重量評估與存活期的觀察顯示了LV-Kallistatin在癌症基因治療中的扮演著重要的角色。利用酵素連結免疫檢測 (enzyme-linked immunoassay, ELISA) 及組織免疫染色 (immunohistochemistry, IHC) 的方式，我們也進一步發現與腫瘤生長、黏附至血管細胞及發炎相關的腫瘤壞死因子-α (tumor necrosis factor-α, TNF-α)、轉型生長因子-β (transforming growth factor-β, TGF-β) 的表現在給予LV-Kallistatin治療的組別中和對照組有所不同之現象，因此推測kallistatin可能會透過這類細胞激素來調控腫瘤微環境。並發現巨噬細胞 (macrophage) 在LV-Kallistatin治療組中有較少的浸潤，暗示了給予治療後腫瘤組織的發炎反應有趨緩的現象。綜合以上的實驗結果，我們認為kallistatin可能透過抑制血管新生 (anti-angiogenesis) 及調控發炎反應 (anti-inflammation) 進而對癌症產生有效的治療效果。

Induction of angiogenesis plays an important role in the development and progression of most human tumors, including lung cancer. Currently available therapies for malignant lung cancer produce low response rates in patients. Therefore, more effective treatment modalities are needed. Therapeutic targeting of angiogenesis has recently been explored to inhibit malignant tumor growth and metastasis. In this study, the expression of human gene kallistatin in Lewis Lung carcinoma cells (LL/2), resulting in inhibiting the metastatic tumor growth and prolonging the survival of tumor bearing mice. Meanwhile, we constructed lentivirus vectors carrying human gene kallistatin (LV-Kallistatin) for gene therapy. By using the conditioned medium from LV-Kallistatin infected cells, we demonstrated that migration and proliferation of endothelial cell were inhibited. The similar results were also observed in migration, invasion and adhesion of LL/2. In experimental metastatic study, the wet lung weights from animals that received LV-Kallistatin were decreased compared with control-treated mice. Besides, the survival rate was dramatically extended in LV-Kallistatin treated group. By using the Enzyme-linked immunoassay (ELISA), we analyzed the cytokine expression at the tumor site. Tumor necrosis factor-α (TNF-α) was lower in LV-Kallistatin treated group compared with control group. On the contrary, transforming growth factor-β (TGF-β) was slightly higher in LV Kallistatin treated group than the control group. According to our findings, lentivirus mediated kallistatin expression has provided a promising therapeutic potential in cancer metastasis therapy.

中文摘要 --------------------------------------------------------------------------- I
英文摘要------------------------------------------------------------------------------ III
誌謝 ---------------------------------------------------------------------------------- IV
目錄 ----------------------------------------------------------------------------------- V
圖目錄 ----------------------------------------------------------------------------- VIII
縮寫與符號 ------------------------------------------------------------------------- IX
緒論
A. 肺癌 (Lung cancer) ------------------------------------------------------- 1
B. 血管新生與轉移 (Angiogenesis and metastasis) ---------------------- 2
1. 血管新生的過程與調控 ---------------------------------------------- 3
C. 癌細胞與轉移 (Tumor and metastasis) ------------------------------ 4
D. 發炎反應與癌細胞的生長 (Inflammation and tumor promotion) -- 5
E. 基因治療 (Gene therapy) -------------------------------------------------- 7
1. 慢病毒載體 (Lentivirus vector) -------------------------------------- 8
F. 絲氨酸蛋白酶抑制物 (Serine proteinase inhibitor, serpin) --------- 10
G. 研究的目的與實驗的策略 ---------------------------------------------- 12
材料與方法
A. 材料
1. 質體 -------------------------------------------------------------------- 13
2. 細胞株 ----------------------------------------------------------------- 13
3. 實驗動物 -------------------------------------------------------------- 14
B. 方法
1. 慢病毒系統質體之構築 (pWPXL-kallistatin construction ) -- 14
2. 慢病毒的生產 -------------------------------------------------------- 15
3. 慢病毒效價偵測 (Virus titration ) --------------------------------- 16
4. 細胞條件培養液配製 (Preparation of conditioned medium)--- 17
5 慢病毒調節的kallistatin表現 --------------------------------------- 17
6. 細胞增生分析（Proliferation assay）----------------------------- 18
7. 細胞移動分析 (Migration assay) ---------------------------------- 18
8. 細胞侵噬分析 (Invasion assay) ------------------------------------ 19
9. 細胞黏附分析 (Adhesion assay) ----------------------------------- 20
10. Stable clone的選殖 -------------------------------------------------- 20
11. 動物實驗
11.1 癌細胞轉移模擬實驗之穩定表現基因株實驗 --------- 21
11.2 癌細胞轉移模擬實驗之病毒治療實驗 ------------------ 21
12. 免疫組織染色(Immunohistochemistry) -------------------------- 22
13. 統計學 (statistical analysis) --------------------------------------- 22
結果
A. 構築帶有kallistatin的慢病毒載體 -------------------------------------- 23
B. LV-Kallistatin可以穩定的在細胞及生物體內表現kallistatin ------- 23
C. 被LV-Kallistatin感染後的細胞條件培養液抑制人類臍靜脈內皮細胞的爬行和增生能力 ---------------------------------------------------------- 25
D. 被LV-Kallistatin感染後的細胞條件培養液可以減緩肺癌細胞的爬行、侵噬、黏附及增生 ---------------------------------------------------------- 26
E. Kallistatin蛋白影響生物體內肺癌的轉移與生長 --------------------- 28
F. LV-Kallistatin可以有效的抑制腫瘤的生長及延長存活率 ----------- 29
G. LV-Kallistatin抑制血管新生 ---------------------------------------------- 30
H. LV-Kallistatin的感染降低了細胞激素的表現及巨噬細胞的浸潤 - 31
討論 ---------------------------------------------------------------------------------- 33
參考文獻 ---------------------------------------------------------------------------- 42
圖 ------------------------------------------------------------------------------------- 48

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