Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52872
標題: 利用小鼠基因晶片剖析VEGF誘導基因轉殖鼠肺腫瘤生成之機制
Mechanism Study of Lung Tumorigenesis Induced by Human Vegf Over-Expression in Transgenic Mice Using Mouse Cdna and Cpg Islands Microarray
作者: 陳全木
關鍵字: 基礎研究
Human VEGF
生物技術, 基礎醫學類
人類血管內皮生長因子
基因轉殖動物
肺腫瘤生成
cDNA基因晶片
Transgenic animal
Lung tumorigenesis
cDNA microarray
摘要: Vascular endothelial growth factor (VEGF) family members consist of VEGF-A, VEGF-B,VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF). The VEGF-A isoform plays acentral role in vascular development. VEGF-A and its receptor are involved in carcinogenesis,invasion and distant metastasis as well as tumor angiogenesis. Clara cell secretory protein (CCSP)is a protein that is secreted by nonciliated, nonmucous clara cells in the pulmonary airway. CCSPplays a protective role against pulmonary inflammatory response. CCSP is a potent naturalimmunosuppressor and anti-inflammatory agent. In this study, a transgene of CCSP-Vegf-A165-SV40poly(A) was constructed for the production of lung-specific overexpressing VEGF-A165 transgenicmice. The previous results showed that human VEGF-A overexpression in the lung tissue willinitiate a tumor growth in the transgenic mice. The object of this research proposal is to investigatethe mechanism of pulmonary tumorigenesis induced by human VEGF-A overexpression in the lungtissue of transgenic mice using cDNA microarray and CpG islands genechip. The whole project wasorganized to be finished within 3 years. In the first year, the study will focus on the establishment ofdifferent lines of transgenic mice with high- and low-level expression of VEGF-A protein in lungClara cells. The time-course of pulmonary adenocarcinoma development will be dissected bypathological analysis. The different transgenic mice lines will be used to determine the humanVEGF-A mRNA and protein expression level. The molecular properties of vascular angiogenesis inthe tumor mass will be studied. In the second year, a home-made animal in vivo image system willbe performed to observe the synergetic effect of lung tumorigenesis after nicotine-derived NNKtreatment in the VEGF-A transgenic mice. In addition, the genome-wide gene expression profiles oftransgenic lung tumor will be analyzed by the 43K mouse cDNA microarray hybridization. Thetumorigenic pathways including cell survival, oncogenesis, anti-apoptosis, cell proliferation andmigration will be examined based on microarray information. In the third year, the whole genomeof epigenetic changes during VEGF-A induced lung adenocarcinoma in the transgenic mice will befurther investigated using a mouse CpG islands genechip. Hypermethylated tumor suppressor geneswill be isolated to evaluate their function contributing in the inhibition of lung tumorigenesis.Whenever this project has been successfully done, it could provide the groundwork for theunderstanding molecular mechanism of human VEGF-A in the promotion of pulmonarytumorigenesis.
應用基因轉殖科技產製癌症模式動物,已成為腫瘤生物學一項重要的研究工具。血管內皮成長因子(vascular endothelial growth factor, VEGF)之基因家族包括PIGF、VEGF-A、-B、-C、-D、-E共六個成員,其中VEGF-A分子具有促進血管增生(angiogenesis),增加血管壁通透性,促進細胞存活以及促進細胞遷移之功能,並於許多試驗中得知於腫瘤生長過程中VEGF-A表現量會聚集增加,因而促進腫瘤生長與腫瘤轉移(metastasis)之能力。在肺癌的研究中發現最主要的肺腺癌多數衍生自細支氣管非纖維性的Clara細胞,而該細胞會釋泌一種特殊的分泌蛋白(Clara cell secretory protein; CCSP),係一種保護性蛋白,具有抑制免疫反應,降低發炎反應之效能。基於上述特徵,本研究計畫採用小鼠CCSP 之啟動子銜接人類VEGF-A165 基因建構一個轉殖基因[CCSP-Vegf-A165-SV40 poly(A)],經鼠胚基因顯微注射建立一套於鼠肺部細支氣管上皮專一性表現VEGF-A之基因轉殖小鼠動物模式,初步試驗結果顯示VEGF-A過量表現可直接誘發基因轉殖小鼠於12月齡時產生肺部腫瘤,此一新穎性研究發現提供一個良好肺癌動物模式,作為探討VEGF-A誘導肺腫瘤生成之機轉。本計畫擬分三年逐步完成:第一年之重點於不同VEGF-A表現量之基因轉殖小鼠品系的建立,觀察肺部腫瘤生成的速率與VEGF-A含量的關係、利用組織病理切片分析肺部腫瘤組織之癌症特性、並度量血管新生之效應。第二年之重點則分別投予尼古丁衍生物NNK致突變劑,給予雙重誘癌因子探討VEGF-A基因轉殖小鼠之肺癌加成的衍生速度,並且利用動物活體螢光影像分析系統進行即時之腫瘤影像分析;此外,將以小鼠43K cDNA基因晶片進行基因轉殖肺癌鼠之全基因體表現分析,以瞭解VEGF-A誘導肺腫瘤生成過程中所牽涉之訊息傳遞途徑。第三年之重點則著重於利用CpG islands甲基化基因晶片探討VEGF-A誘導肺癌產生過程中所更動之基因體甲基異常修飾的腫瘤抑制基因群,並測試此群抑癌基因對於肺部之保護功能,本研究亦將投予基因轉殖肺癌鼠VEGF-A抗體與抑制劑,觀察小鼠肺部腫瘤之抑制程度。透過本計畫之順利執行,預期將可釐清人類VEGF-A在誘發肺部腫瘤生成過程中所扮演的角色,對於未來肺腺癌的預防與醫療將有所助益。
URI: http://hdl.handle.net/11455/52872
其他識別: NSC97-2313-B005-004-MY3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1985867&plan_no=NSC97-2313-B005-004-MY3&plan_year=99&projkey=PD9902-0073&target=plan&highStr=*&check=0&pnchDesc=%E5%88%A9%E7%94%A8%E5%B0%8F%E9%BC%A0%E5%9F%BA%E5%9B%A0%E6%99%B6%E7%89%87%E5%89%96%E6%9E%90VEGF%E8%AA%98%E5%B0%8E%E5%9F%BA%E5%9B%A0%E8%BD%89%E6%AE%96%E9%BC%A0%E8%82%BA%E8%85%AB%E7%98%A4%E7%94%9F%E6%88%90%E4%B9%8B%E6%A9%9F%E5%88%B6
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