中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/50299
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    题名: 發展以攜帶骨結合素啟動子驅動PML腫瘤抑制基因之腺病毒載體作為抗腎細胞癌之新型治療策略
    A novel adenoviral vector armed with human osteonectin promoter driven PML inhibits human renal cell carcinoma growth
    作者: 李曉涵;Hsiao-Han Li
    贡献者: 癌症生物學研究所碩士班
    关键词: 腎臟癌;逢希伯-林道症候群;早幼粒細胞白血病蛋白;骨結合素;RCC;VHL;PML;Osteonectin
    日期: 2013-06-18
    上传时间: 2013-10-02 11:19:20 (UTC+8)
    出版者: 中國醫藥大學
    摘要: 研究背景：
    腎臟細胞癌（RCC）為具有極強烈的抗常規治療方法的癌症之一。晚期腎臟癌患者十分難以治療，因而導致病患的存活率不到1年，所以目前迫切需要開發新的療法來克服這種疾病。早幼粒細胞白血病基因PML是一種多效性抑制腫瘤基因，在缺氧環境下可對抗mTOR-HIF1α-VEGF血管內皮生長信號傳遞。我們推測PML可以對腎臟細胞癌做標靶對抗mTOR的基因治療，這是一種很有前途的治療劑對於腎臟細胞腫瘤進展的關鍵。骨粘連蛋白是一個分泌細胞蛋白質與人類惡性腫瘤血管生成、侵襲和轉移有關，目前hON啟動子相關研究已用於在許多癌症中，但它並沒有在腎臟細胞癌中研究。因此，我們測試hON 啟動子標靶對抗腎臟細胞癌的可行性，利用hON啟動子攜帶野生型或抗降解型的突變（S518A）PML製作腺病毒，來研究抑制腎臟細胞腫瘤的生成作用。
    材料方法：
    利用西方墨點法驗證hON與PML在正常腎臟細胞(HRE)和惡性腎臟細胞（RCC42，RCC52）的含量，並在臨床標本的組織染色切片做進一步驗證。實驗室先前已建構的522-bp hON啟動子(522E)，利用螢光素酶基因分析在腫瘤細胞株中的轉錄活性。不同的PML基因結構是由陳瑞華博士提供（Cancer cell 20, 214-228, August 16, 2011）(中央研究院生物化學研究所)。因而hON啟動子攜帶抑制腫瘤基因PML轉殖到腺病毒載體製作出病毒，利用在體外細胞毒殺實驗比較不同Ad-ON-PML在腎癌細胞株的細胞毒性。
    結果：
    利用西方墨點法得到結果發現RCC42與RCC52比正常HRE細胞的PML表現量較低。 組織染色切片結果發現hON在惡性腎臟組織比正常組織的表達量更高包含癌細胞與周圍基質細胞。hON啟動子在RCC42 RCC52細胞的表現量平均為14 與44倍，比對照與pGL3載體表現出強烈的轉錄活性。我們建構腺病毒利用hON啟動子分別攜帶PML-1和PML-4，無論是野生型或突變型(S518A)，並確認其在腎癌細胞在有氧和缺氧條件下的表現量。我們發現利用10-100 MOI的劑量之下Ad-hON-PML無論野生型或突變型皆可有效殺死腎臟癌細胞，特別的是在有氧無氧的環境下毒殺能力無明顯差異性。
    結論：
    我們證明在細胞實驗中腫瘤抑制基因PML的表達利用522 bp-hON啟動子的控制下可抑制腎癌細胞的生長。我們未來預想利用Ad-hON-PML用於治療腎癌在原位人類腎細胞癌小鼠模型之可行性。
    Introduction: Advanced renal cell carcinoma (RCC) is highly resistant to conventional therapies. Patients with advanced RCC have an extremely poor outcome with an estimated median survival of less than 1 year. It is medically urgent to develop new therapies to overcome this disease. The promyelocytic leukaemia gene PML is a pleiotropic tumor suppressor, which opposes mTOR-HIF1??-VEGF signaling in hypoxia. We hypothesize that PML may be a promising therapeutic agent for RCC targeted gene therapy as mTOR is critical for RCC tumor progression. Osteonectin is a matricellular protein and is implicated in the angiogenesis, invasion, and metastasis of human malignancies. Human osteonectin (hON) promoter has been used for gene delivery to certain types of cancer, but it has not been investigated in RCC. Herein, we tested the feasibility of hON promoter for RCC transcriptional targeting, and examined the anti-tumor effect of adenoviral vectors carrying either wild type or degradation-resistant mutant (S518A) PML driven by hON promoter toward RCC.
    Materials and Methods: The expression of hON and PML in normal (HRE) and malignant renal cell lines (RCC42, RCC52) was determined using western blot, and further validated in clinical specimen by IHC. A previously constructed 522-bp hON promoter was tested in cancer cell lines for transcription activity using luciferase-reporter assay. The different PML gene constructs (Cancer cell 20, 214-228, August 16, 2011) were kindly provided by Dr. Ruey-Hwa Chen at Institute of Biological Chemistry, Academia Sinica, and then subcloned into adenoviral vector with hON promoter. In vitro cell killing assay was performed to compare the cytotoxicity of cancer cell lines by PML-based adenoviral vectors.
    Results: Western blotting demonstrated a significant downregulation of PML in RCC42 and RCC52 compared to normal HRE cells. Immunohistochemcial staning of hON in normal kidney and malignant kidney tissues revealed a higher level of hON expression in both RCC cells and cancer adjacent stroma. In consistent with the endogenous hON expression, the 522 bp-hON promoter exhibited a strong transcriptional activity in RCC42 and RCC52 cells with an average of 14- and 44-fold higher luciferase reporter expression than control pGL3 vector, respectively.
    Adenovirus vectors carrying hON promoter-driven PML-1 and PML-4, either wild type or S518A mutant were constructed and confirmed their expression in RCC cells under both normoxia and hypoxia conditions. We found that Ad-hON-PML, regardless of wild type or mutant efficiently kill RCC42 and RCC52 cells at a moi rang of 30-100 in a dose-dependent manner. No significant different in cytotoxicity of RCC cells by Ad-hON-PML between normoxia and hypoxia culture condition.
    Conclusion: We have shown that expression of tumor suppressor PML gene under the control of 522 bp- hON promoter led to the inhition of RCC cell growth in vitro. We are currently investigating the feasibility of using Ad-hON-PML for the treatment of RCC in an orthotopic mouse model of human RCC.
    显示于类别:[癌症生物學研究所] 博碩士論文

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