摘要：實驗目的: 利用抑制性扣減雜交/微陣列(SSH/microarray)及一般cDNA 微陣列(cDNAmicroarray)來找出早期胃癌與晚期胃癌病患之間的差異性表現基因以利後續分析並找出與胃癌發展有關之基因並且希望能夠在胃癌的臨床治療有所貢獻。另利用小鼠超音波系統建立一個非侵入性的腫瘤診斷與追蹤治療的方法。實驗材料與方法: 利用抑制性扣減雜交/微陣列(SSH/microarray)及一般cDNA 微陣列(cDNA microarray)來找出早期及晚期胃癌病患差異性表現基因，並利用半定量或及時定量聚合酶連鎖反應來進一步確認基因表現量。與血管新生(angiogenesis)、細胞遷移(migration)、侵襲(invasion)和癌症發展(cancer progression)的基因將被篩選並進行後續細胞與動物實驗來驗證這些基因在胃癌發展中的角色。目前我們已建立了抑制性扣減雜交/微陣列(SSH/microarray)及一般cDNA 微陣列(cDNA microarray)差異性表現基因分析系統並找出了具有差異性表現之基因。本計畫將在細胞與動物實驗上更進一步的研究基因功能並另在早、晚期胃癌病患中證實這些差異性基因表現與癌症發展之相關性。另外我們將利用Vevo770 Ultrasound system 來觀察小鼠腫瘤的生長（growth）與胃癌的發展（gastric cancer progression）。我們將分三年進行此計畫:第一年:我們將篩選與血管新生(angiogenesis)、細胞遷移(migration)、侵襲(invasion)和癌症發展(cancer progression)的基因並利用半定量(semi-quantitative RT-PCR)或及時定量反轉錄聚合酶連鎖反應(real-time RT-PCR)以進一步確認基因的差異性表現量並同時在另一組早、晚期胃癌病患中證實這些基因於癌症發展中的相關性。第二年:我們第二年的目標是在細胞內建立in vitro 系統來觀察差異性表現基因的功能以及這些基因在胃癌發展中所扮演的角色。第三年:我們將利用in vivo 動物系統來研究差異性表現基因的致病機制以建立未來可能可以治療的標地與預後的目標並利用小鼠超音波儀器建立非侵入性診斷系統。
Abstract: Gastric cancer is one of the most frequent cancers in the world, and it is the fifth mostcommon cancer in Taiwan. Early stage disease tends to be diagnosed in country in which endoscopic screening is more common; however, there still exist patients whose tumor is advanced at the time of diagnosis. Patients with stage I disease have a good prognosis, and those with stage III & IV disease show a very poor prognosis. However, the cellular and molecular heterogeneity of gastric cancers underlie diversity of biological behavior and make therapeutic result unpredictable. The large number of genes potentially involved in the multi-step process of gastric cancer pathogenesis and tumor progression, and the importance of studying multiple genetic alterations in concert between early and advanced gastric cancer cannot be overemphasized to profile gene expression related with clinical outcome. To evaluate the growth of tumor using a non-invasive method, an ultrasound system may be the solution to clearly visualize in live SCID mice tumor progression. Therefore, this non-invasive system may provide insight into tumor growth, vascularity while minimizing mouse-to-mouse difference.Purpose: The aim of this study is to identify differentially expressed genes in early and advanced gastric cancer patients using suppression subtractive hybridization/microarray as well as regular cDNA microarray. After identification of gastric cancer progression related differentially expressed genes, further studies on the clinical implications can be carried out to assess the potential prognostic markers. The second aim of this three year study is to setup a non-invasive ultrasound system to visualize tumor in SCID mice while tracking tumor growth and observe the effectiveness of potential therapeutic drugs.Materials and methods: Early and advanced gastric cancer patient tissue specimens have been collected during curative intent gastrectomy and RNA purified. The SSH/microarray system have been setup to identify differential expressed genes between early and advanced gastric cancer patients. We will confirm the differential expression using semi-quantitative RT-PCR or real-time RT-PCR to further confirm gene expression as well as comparing to another set of patients as test model to determine the differential gene expression in early and advanced patients. Genes of interest include those that are related to cancer progression, angiogenesis, migration, and invasion. In vitro studies will be carried out to select stable expression clones and their function tested using cell cultures and in vivo using mice models. Vevo770 ultrasound system will be used to observe gastric tumor growth in SCID mice using B-mode as well as the power Doppler mode.We will achieve the above goals in three years:First year:We will use semi-quantitative RT-PCR or real-time RT-PCR to confirm differential gene expression in early and advanced gastric cancer patients as well as using another set of gastric cancer patients as test models to determine the role of our differentially expressed genes in cancer progression.Second year:We will setup an in vitro cell culture model to investigate the role of these differential expressed genes by selecting stable expression clones and determine their role in invasion,migration, angiogenesis, cell proliferation and apoptosis.Third year:We will setup an in vivo animal model to study the role of differentially expressed genes in cancer progression, angiogenesis, invasion, migration and as well as determine the possible prognostic value of these genes. Apart from the invasive animal model, we will also setup the Vevo770 ultrasound system for the non-invasive observation of tumor progression in SCID mice.
Suppression subtractive hybridization/microarray