血管新生微列陣的構建及在人類胃癌的研究(1/2)

腫瘤的生長是須要血管新生才能繼續成長和造成腫瘤細胞的轉移，人類癌症 的血管新生的評估最常用的方法是微細血管密度，最近我們用彩色杜卜勒超音波 來偵測腸胃道癌症的血管新生，而這個方法─彩色杜卜勒血管密度，可用於活體 病人使用，可重覆偵測，且不具侵犯性，而且超音波是容易獲得的檢查儀器，這 是微細血管密度無法做到的。我們利用這個方法評估大腸癌病人發現彩色杜卜勒 血管密度可預測大腸癌病人的遠處轉移和存活。 在胃癌病人方面，彩色杜卜勒血管密度是一個獨立的預後因子，更有趣旳是 當彩色杜卜勒血管密度大於11%，可預測第三期胃癌病人有很差的預後，且術後 皆於兩年內死亡，但是小於11%的病人，其三年存活率卻高於60%。所以彩色 杜卜勒血管密度可挑選出第三期胃癌病人中，真正的高危險病人，這對臨床上胃 癌病人的治療是非常重要，可以挑險選出應該接受進一步治療的病人 但是彩色 杜卜勒超音波對胃癌的可偵測率不高，僅約39%，且無法偵測早期胃癌，因此無 法適用所有胃癌病人。 目前為止有關血管新生的研究中發現腫瘤的血管新生，其實是由很多不同種 類的細胞互相作用造成的，其中包括腫瘤細胞本身，間質細胞、免疫細胞、血管 內皮細胞等，而這些細胞的互動則須靠很多調節因子的相互作用，與血管新生相 關的至少包括(1)血管促進因子(proangiogenic factors)，如血管內皮生長因子 (VEGF)、胎盤生長因子(PlGF)等。(2)血管抑制因子(antiangiogenic factors)，如 angiostatin，endostatin 和thrombospondin 等。(3)腫瘤基因(oncogenes)，如ras， myc，src，eIF4 等。(4)腫瘤抑制基因(tumor suppressor genes)，如P53(5)發炎相關 基因(inflammation-related genes)，如環氧化(COX)和氧化氮生成(NOS)等。(6) 蛋白質分解(protease)基因，如MMP。(7)黏附基因(adhesive molecules)，如 selectin 家族，integrin 家族等。(8)Interleukin 家族，如IL-1，IL-6 和IL-8 等。(9)CD 家族，如CD31，34 等。(10) 腫瘤內皮細胞特有基因。由於單一分子(molecule) 僅是這個複雜網路的一員，很少單一分子可被利用當作選擇高危險病人的指標， 尤其是針對單一病人少有選擇性。 我們在臨床上胃癌的COX-1，COX-2，iNOS，VEGF，PLGF 和eIF4E 等相 關血管促進因子的研究中發現COX-2，iNOS，PLGF 和微細血管密度有顯著相 關，但僅iNOS 是一預後因子，但它卻無法像彩色杜卜勒血管密度(CDVI)，對單 一病人有很好的選擇性，即iNOS 可預測某一人群人的整體存活，但CDVI 卻有 更強的能力去預測單一病人的存活，為何CDVI 有此能力，我們並不清礎，我們 發現CDVI 與上述6 種血管新生相關因子並無顯著相關，這可能意謂著CDVI 是 由眾多的血管新生相關基因一起作用而產生的結果，並不是少數的基因就可決 定，因此，若分析大量血管新生相關基因的表現特徵或尋找真正重點基因(hot spot genes) 或許可提供這個問題的答案。因此我們期望可以建構一血管新生相關基 因微陣列，以提供一準確且可廣泛利用的方法去偵測胃癌的基因表現。 在本年度的計畫中我們已完成血管新生微列陣(angiochip)的構建。我們收集 了前述的十大類與血管新生相關的基因，和一部份未知的內皮細胞的特殊基因， 再加上之前我們利用9600 個基因的微列陣方法，比較CDVI 高低不同的胃癌的 基因表現而篩選出的重要基因，總共約350 個與血管新生有重要關係的基因。在 本院的第三共同研究室（包括微列陣專門人員，相關技術和儀器設備）進行這個 以血管新生相關基因所構建的血管新生微列陣(angiochip)。同時並以兩組胃癌病 人的組織抽取RNA 製備cDNA probe 進行此血管新生微列陣的測試，並找出此 血管新生微列陣最適之偵測條件，確保下一年度計劃能順利進行。Angiogenesis is a very complex phenomenon and essential for the growth of solid tumors measuring more than a few millimeter1s .It permits rapid tumor growth and potential presence of tumor metastasis. At present the most widely used method to assess angiogenesis in human malignancies is the quantification of microvessel density (MVD) of primary tumors using specific markers for enadlo tcheelllis including factor VIII-related antigen, CD31 and CD34. Recently we have developed a new method, color Doppler vascularity index (CDVI), to evaluate tumor angiogenesis. This is a non -invasive, in vivo, easily accessible method that can be performed repeatedly in the patients preoperatively. Using CDVI, we can predict distant metastasis and survival in colon cancer patients. In gastric cancer patients, CDVI were independent prognostic factors. There was an intriguing finding that the stage III gastric cancer patients with the CDVI > 11% could’tn survive beyond 2 years after curative resection, however, 3-yaer survival rate of those with CDVI > 11% was greater 60%. Therefore, CDVI can select high risky patients with gastric cancer, and this is very important for gastric cancer diagnosis and treatment. The major technical limitation of CDVI is low detectability rate. Only 39% of gastric cancer can be visualized in abdominal ultrasound examination. We ’ct anapply this method to evaluate all gastric cancer patients. Tumor angiogenesis is controlled by different cell types, including cancer cell itself, stromal cells, immune cells, and endothelial cells in the tumor tissue. The interaction of these cells is mediated by many regulatory factors such as (1n) gpiorgoeanic factors: vascular endothelial growth facto (rVEGF), placenta growth factor(PlGF), and et al. (2) antiangiogenic factors: angiostatin, endostatin, thrombospondin, and et al. (3) oncogenes: ras, myc, src, eIF4E, and et al. (4) tumor suppressor genes: p53. (5) inflammation related genes: cyclooxygenase-1(COX-1, -2(COX-2), inducible nitric oxide synthase(iNOS), and et al. (6) protease genes: matrix metalloprotease family and et al. (7) adhesive molecules: selectin family, integrin family, and e8t) al. ( interleukin: IL-1, IL-6, IL-8, and et al. (9) CD family: CD31, CD34, CD44, et al. (10) tumor specific endothelial antigens: known and unknown genes. In our previous preliminary study on clinicopathologic correlation of COX-1, COX-2, iNOS, VEGF, PlGF, and eIF4E with angiogenesis and clinical outcome in human gastric cancer, iNOS expression was significantly correlated with microvessel density and patients’ survival. INOS is an important gene in angiogenesis and its expression is significantly correlated with gastric cancer patients’ survival, but it is not as specific as CDVI to single patient. So we can predict a flock of patie’sn tsurvival by INOS. However it is better to predict a patie’ns t survival by CDVI than INOS. We do’tn know why CDVI has such ability. But we found CDVI wasn’t significantly correlated with above six angiogenesis factors. This means that CDVI is determined by a lot of angiogenesis-related genes not just a few genes. Therefore, from analysis the feature of the expression of large angiogenesis-related genes and finding out hot spot genes for angiogenesis may provide the answer of this question.w Seo would like to develop an angiogenesis related microarray to provide an unbiased and broad approach to detecting gene expression profile of gastric cancer. In st1 year study, we have constructed an angiogenesis microarray (Angiochip) including 350 genes of above 10 categories, and genes that were obtained from 9600-gene microarray differential expression of high and low CDVI gastric cancer to evaluate angiogenesis of gastric cancer. We optimized conditions of RNA extraction from human gastric cancer tissues anpdr epared cDNA probes from two pairs of gastric cancer tissue and corresponding adjacent noncancerous gastric tissue RNA to test of this newly developed angiochip and optimized angiomicroarray testing conditions. This will make sure experimental progress in the next year study