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研究生:劉書瑜
研究生(外文):Liu, Shu-Yu
論文名稱:缺氧誘導因子的失衡參與腎細胞癌對於紓癌特的抗藥性
論文名稱(外文):Imbalance of Hypoxia-Inducible Factors Involved in the Drug Resistance of Sunitinib treatment in Renal Cell Carcinoma
指導教授:查岱龍查岱龍引用關係史中
指導教授(外文):Cha, Tai-LungShih, Chung
口試委員:史中孫光煥查岱龍
口試委員(外文):Shih, ChungSun, Guang-HuanCha, Tai-Lung
口試日期:2012-05-24
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物及解剖學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:98
中文關鍵詞:缺氧誘導因子腎細胞癌紓癌特
外文關鍵詞:Hypoxia-Inducible FactorsRenal Cell CarcinomaSunitinib
相關次數:
  • 被引用被引用:0
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  • 下載下載:5
  • 收藏至我的研究室書目清單書目收藏:0
腎臟癌是一種屬於血管新生的疾病,並且對於化學及免疫療法有高度的抗性,因此抗血管新生療法已經成為轉移性腎臟癌的關鍵療法。酪胺酸激脢是細胞訊號傳遞中的重要蛋白,具有多種生物活性,可調控細胞增殖、轉移、及血管新生。參與血管新生中的酪胺酸激脢受體包含血管內皮生長因子受體、血小板衍生生長因子受體…等,抑制抗血管新生的酪胺酸激脢受體已經發展成一種癌症的系統性治療策略。紓癌特為抗血管新生之酪胺酸激脢受體的抑制劑,已針對轉移型腎清細胞癌使用於臨床第一線用藥,並證實可增進轉移型腎臟癌病患無惡化存活率及在臨床上具有顯著的毒性效應。

紓癌特用於腎清細胞癌治療具有生物上的理據,約有75 %的腎清細胞癌中含有不活化的逢希伯-林道症候群腫瘤抑制基因。當缺氧情況產生或逢希伯-林道症候群基因不活化時,缺氧誘導因子累積並增加缺氧誘導因子所調控之基因的表達量。這些血管新生因子相關基因會藉由旁分泌迴圈的方式與相對之酪胺酸激脢受體產生接合,促使細胞增殖與血管新生。

然而病患在使用一段時間之後往往開始產生抗性,因此臨床與科學上關於抗血管新生之酪胺酸激脢受體抑制劑抗性的迫切問題便衍生而出。我們的研究目的為缺氧誘導因子是否參與酪胺酸激脢受體抑制劑紓癌特在腎臟癌中的抗性。而我們的結果顯示紓癌特處理後可降低由缺氧導致的缺氧誘導因子-1mRNA及蛋白表達量的增加,但卻增加了缺氧誘導因子-2、血管內皮生長因子的mRNA表達量,及缺氧誘導因子-2蛋白表達量的持平。

此外,我們更進一步研究在模擬腫瘤微環境的狀態下,紓癌特處理所造成的影響。在使用共同培養系統將腎臟癌細胞與人類臍帶內皮細胞共同培養時,我們發現腎臟癌細胞會對於人類臍帶內皮細胞提供保護作用協助對抗紓癌特的處理。而當我們在紓癌特的處理下降低ACHN中缺氧誘導因子-2的表達量時,保護作用就消失了。這樣的現象提供了我們紓癌特在腎臟癌中產生抗性的可能機制。評估由缺氧誘導因子所調控的紓癌特在腎臟癌中產生抗性的機制可以提供未來解決在臨床上所面臨的酪胺酸激脢受體抑制劑療法的障礙。

Renal cell carcinoma (RCC) is an angiogenesis-dependent disease and highly resistant to chemo- and radiation therapies. Therefore, anti-angiogenesis therapy has emerged as a pivotal treatment of metastatic RCC. Tyrosine kinases are important cellular signaling proteins that have a variety of biological activities including cell proliferation, migration and angiogenesis. Multiple kinases are involved in angiogenesis, including receptor tyrosine kinases such as the vascular endothelial growth factor receptor (VEGFR), platelet derived growth factor (PDGFR), etc. Inhibition of angiogenic tyrosine kinases has been developed as a systemic treatment strategy for cancer. As an anti-angiogenic tyrosine kinase inhibitor (TKIs), Sunitinib is used in first-line therapies for metastatic clear cell RCC (ccRCC) and it has been shown to improve progression-free survival in patients with mRCC and caused clinically significant toxic effects.

A biological rationale exists for treating ccRCC with Sunitinib. Since the von Hippel–Lindau tumor-suppressor gene (VHL) is inactivated in at least 75% of RCC. Under hypoxic condition or VHL inactivation, hypoxia inducible factor-α (HIF-α) accumulates and increases the expression of HIF-regulated genes, including genes encoding angiogenic factors which act in a paracrine loop on their cognate receptor tyrosine kinases, stimulating cell proliferation and angiogenesis.

However, some patients are inherently resistant to these approaches and most, if not all, patients acquire resistance over time. Thus, a pressing clinical and scientific question arises about the mechanism of resistance to anti-angiogenic TKI therapies. In this study, we try to figure out whether HIF participates in TKI resistance of Sunitinib in RCC. Our data shows that Sunitinib treatment reduced hypoxia-induced rises HIF-1 on both mRNA and protein levels. In contrast, Sunitinib treatment can increase HIF-2, VEGF mRNA level and maintain HIF-2 protein level.

In addition, we further investigate the effect of Suntinib treatment in RCC cells under mimicing tumor microenvironment. By co-culturing RCC cells with human umbilcal vessel endothelial cells (HUVEC), we found that RCC cells will provide HUVEC protection effect to against Sunitinib treatment. And the protection effect would be disappeared when we knockdown HIF-2 in ACHN upon Sunitinib treatment. This phenomenon provided us the possible mechanism of Sunitinib resistance in RCC through HIF-2regulation. Evaluating the mechanisms involved in HIF-mediated resistance to Sunitinib treatment in RCC could provide further resolution in dealing with the clinical obstacle upon TKI therapy.


正文目錄............................................................................................VII
『圖』目錄............................................................................................XI
『附錄』目錄..........................................................................................XII
中文摘要............................................................................................XIII
英文摘要............................................................................................XV
第一章、緒言............................................................................................1
第一節、腎細胞癌 (Renal cell carcinoma, RCC)之簡介........................................................1
壹、腎臟癌症病患之現況....................................................................................1
貳、腎細胞癌之類型 ......................................................................................2
第二節、逢希伯-林道症候群 (von Hippel-Lindau, VHL)簡介....................................................2
壹、VHL 疾病介紹........................................................................................2
貳、pVHL 生物活性.......................................................................................3
第三節、缺氧誘發因子 (Hypoxia-inducible factors, HIFs)簡介................................................3
第四節、腫瘤血管新生 (Tumor Angiogenesis)之簡介..........................................................5
第五節、紓癌特 (Sunitinib, Sutent®)的臨床應用............................................................7
第六節、研究背景與目的..................................................................................8
第二章、材料與方法 .....................................................................................11
第一節、藥品........................................................................................11
第二節、細胞株與細胞培養液............................................................................11
第三節、抗體........................................................................................12
第四節、模擬缺氧情況 (Hypoxia).......................................................................12
第五節、細胞生長曲線分析(Sulforhodamine B (SRB) colorimetric assay)...................................13
第六節、反轉錄訊息核醣即時核酸表現程度之分析(Real-time Quantitative Polymerase. Chain Reaction analysis)..13
壹、RNA之分離(RNA isolation)..........................................................................14
貳、反轉錄反應(Reverse transcription, RT) ...........................................................14
參、Taqmen ® 定量聚合酵素連鎖反應 (Taqmen ® Quantitative Polymerase chain reaction, QPCR)..............15
第七節、西方墨點法(Western blotting)................................................................15
壹、人類腎臟癌細胞株藥物處理............................................................................16
貳、蛋白質萃取(Protein extraction)................................................................16
參、蛋白質濃度測定 ............................................................................16
肆、SDS-PAGE(Sodiumdodecyl Sulfate Polyacrylaminde Gel ......................................17
伍、西方墨點法Western blotting................................................................17
第八節、共同培養系統 (Co-culture system)...........................................................18
第九節、HIF-2小干擾RNA (Small interference RNA(siRNA))轉染(Transfection)及HIF-2轉譯抑制劑處理之調整培養基........................................................................................19
壹、小干擾RNA (Small interference RNA(siRNA))轉染 ....................................................19
貳、HIF-2轉譯抑制劑處理 ............................................................................20
參、調整培養基對於HUVEC增殖之影響 ................................................................20
第十節、人類Cytokine與cytokine receptor interaction PrimerArray .................................20
第十一節、酵素連結免疫吸附分析法 (Enzyme-Linked Immunosorbent Assay ELISA) .........................21
第三章、結果 ....................................................23
第一節、Sunitinib在生理作用濃度上對於RCC和HUVEC並無明顯細胞毒性......................................23
第二節、建立共同培養系統模擬腫瘤微環境以觀察Sunitinib對於HUVEC增殖的影響....................................24
第三節、Sunitinib對於RCC的HIF和VEGF mRNA表現程度的影響....................................................26
第四節、Sunitinib對於RCC的HIF蛋白表現程度的影響....................................................27
第五節、Sunitinib處理下HIF-2對HIF-1的影響、VEGF mRNA表現程度及HUVEC的增殖情況...........................30
第六節、HIF-2轉譯抑制劑對Sunitinib處理下之HIF和VEGF mRNA表現程度及HUVEC的增殖情況.........................32
第七節、Sunitinib處理對人類腎細胞癌株的Cytokine-cytokine receptor interaction影響........................34
第八節、Sunitinib處理對人類腎細胞癌株VEGF分泌量的影響....................................................35
第四章、討論....................................................37
第一節、Sunitinib在生理作用濃度上對於RCC和HUVEC並無細胞毒性................................................37
第二節、觀察Sunitinib在共同培養系統模擬腫瘤微環境下對於HUVEC增殖的影響 ................................38
第三節、Sunitinib對於RCC的HIF和VEGF mRNA表現程度及HIF蛋白表現程度的影響..............................39
第四節、Sunitinib處理下HIF-2對HIF-1的影響、VEGF mRNA表現程度及HUVEC的增殖情況......................41
第五節、HIF-2轉譯抑制劑影響在Sunitinib處理下HIF和VEGF mRNA表現程度及HUVEC的增殖情況 .................42
第六節、Sunitinib影響人類腎清細胞癌株的Cytokine-cytokine receptor interaction.......................43
第五章、結論........................................................................................45
第六章、參考文獻.....................................................................................47
第七章、結果圖表.....................................................................................53
附錄..............................................................................................75


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