1Z^4623 五、發明說明（） 背景 、2為世界上第二常見的癌症，並在如日本、韓國及印 =寺遠東國家高度流行。台灣與癌症相關的死因中，胃癌 系居一⑸名之癌症。僅有約百分之四十之患者可接受可 能治愈胃癌的手術，且胃淚电 a癌患者的五年存活率是很低的。 概要 本發月係基於發現文試者胃腸分泌蛋白（⑽p)表現量與 胃癌風險相關。因此，GISp表現可用於預測是否受試者 處於胃癌風險中或罹患胃癌。 因此，本發明特徵在於藉測定受試者生物樣本中Gisp表 見里並將▲ I與GISP表現之閥值量比較以判斷受試者是 否處於胃癌風險中之方法。受試者之GISp表現量大於閥 值量顯示罹患胃癌的風險。 菖實施本發明之方法，Gisp表現量可藉生物樣本中gisp 蛋白質I量來測定，及閥值量可為於未罹病受試者，如一 個健康受試者，所測得GISP蛋白質之量。一個未罹病受 試者為一個尚未罹患胃癌，而且在足夠期間（如，至少6, 9, 12，18, 24, 30, 36或更多個月）後沒有發展出胃癌之受試 者。GISP蛋白質之量可使用任何適合之分析法測定，如 使用抗-GISP專一抗體，如，’酶連接免疫吸收分析 (ELISA)，輻射免疫分析（RIA)，西方（Westem)墨點法或免 疫組織化學方法。或者，GISP表現量可藉生物樣本中GISp -4 本紙張尺度適用中國國家標準（CNS)A4規格（21〇 x 297公釐） (請先閱讀背面之注音？事項再填寫本頁) _--------訂---------線參 1224623 mRNA之量測定，而閥值量可為於未罹病受試者，如一個 未罹患胃癌並在足夠期間（如，至少6, 9，12，18, 24, 3〇, ％ 或更^個月）後/又有發展出胃癌之受試者，所測得Gap蛋 白質之量。由於GISP cDNA之核苷酸序列係為已知的（參 閱，如美國專利案第號），GISp mRNA量可使用 任何分析而輕易測定，如北方（N〇rthern)墨點法，rt_ PCR，或生物晶片。適用於本發明方法之生物樣本包括胃 腫瘤之固體組織樣本、排泄物，及如消化液及血液之液 體。 GISP表現閥值量可被測定，例如藉比較罹患胃癌之受試 者與彼等受試者之GISP表現，其中大於6〇%(如至少約6〇, 70, 75, 80, 85, 90,或95。/〇)之受試是在足夠期間（如，至少6, 9, 12, 18, 24, 30, 36或更多個月後）未發展出癌症。排除或 包括於風險群組之表現閥值量可設定為達到未發展成胃癌 之受試者之預先測定量。較佳地，處於胃癌風險之受試者 具有至少約10%，20%，40%大於閥值量之GISp表現量。 本發明之方法提供預測受試者發展成胃癌的風險及鑑別 罹患胃癌之受試者之手段。因此，本發明對受試者及他或 她的醫療保健提供者提供預後資訊以確保較佳資訊的醫療 決定。 在另一觀點，本發明特徵在於測定化合物治療胃癌有效 性的方法。該方法包含將一種化合物與胃癌細胞接觸，並 -5- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) — — — — — — — ^--------線秦 經濟部智慧財產局員工消費合作社印製 經濟部智慧財產局員工消費合作社印製 1224623 A7 B7_ 五、發明說明（3 ) 比較胃細胞與此化合物接觸前與接觸後之GISp表現量。 與化合物接觸後，GISP表現量之降低顯示該化合物對治 療胃癌是有效的。化合物對治療胃癌之有效性可在活體外 或活體内評估。 當在活體外實行本發明之方法，胃癌細胞可衍生自胃癌 細胞系。適合的胃癌細胞系之實例包括AGS細胞系， KATO III 細胞系，SNU-16 細胞系，RF_48 細胞系，NCI_N87 細胞系，NUGC細胞系，及az-521細胞系。或者，胃癌細 胞可在罹患胃癌的動物體内。例如，該化合物可投藥至罹 患胃癌之動物，及投藥後動物體内之GISp表現量可與投 藥岫動物體内之GISP表現量比較。動物體内之GISp表現 量可在活體内或活體外測定。例如，GISp量可在體外藉 知自動物之生物樣本而測定，如，投藥化合物前及後之腫 瘤固體組織樣本、排泄物、血液或消化液。測定動物中 GISP表現量之其它方法係描述於此中。可使用各種胃癌 動物模型如原位組織移植人類胃癌組織之SC]；D小鼠。1Z ^ 4623 V. Description of the invention () Background, 2 is the second most common cancer in the world, and it is highly prevalent in countries such as Japan, South Korea, and India. Among cancer-related causes of death in Taiwan, gastric cancer is an unknown cancer. Only about forty percent of patients can undergo surgery that may cure gastric cancer, and the five-year survival rate of patients with gastric lacrimal carcinoma a is very low. Summary This month is based on finding that the expression of gastrointestinal secreted protein () p) in test subjects is related to the risk of gastric cancer. Therefore, GISp performance can be used to predict whether a subject is at risk of or suffering from gastric cancer. Therefore, the present invention is characterized by a method for determining whether a subject is at risk of gastric cancer by measuring Gisp expression in a subject's biological sample and comparing the threshold amount of ▲ I with the expression of GISP. A subject's GISp manifestation greater than a threshold amount indicates a risk of developing gastric cancer.菖 Implementing the method of the present invention, the expression of Gisp can be determined by the amount of gisp protein I in the biological sample, and the threshold amount can be the amount of GISP protein measured in a non-diseased subject, such as a healthy subject. An unaffected subject is a subject who has not developed gastric cancer and has not developed gastric cancer after a sufficient period of time (eg, at least 6, 9, 12, 18, 24, 30, 36 or more months). The amount of GISP protein can be determined using any suitable analytical method, such as using an anti-GISP specific antibody, such as' enzyme-linked immunosorbent analysis (ELISA), radiation immunoassay (RIA), Westem blotting method, or immune tissue chemical method. Alternatively, the expression of GISP can be based on the GISp -4 in the biological sample. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (21 × 297 mm) (please read the note on the back? Matters before filling out this page) _-- ------ Order --------- The measurement of the amount of mRNA of line ginseng 1224623, and the threshold amount can be in a non-diseased subject, such as a person not suffering from gastric cancer and for a sufficient period of time (eg, at least 6, 9, 12, 18, 24, 30,% or ^ months) / in subjects who have developed gastric cancer, the amount of Gap protein measured. Since the nucleotide sequence of the GISP cDNA is known (see, for example, US Patent No.), the amount of GISp mRNA can be easily determined using any analysis, such as the Northern blot method, rt_PCR, or Biochip. Biological samples suitable for the method of the present invention include solid tissue samples of gastric tumors, feces, and fluids such as digestive juices and blood. The GISP performance threshold can be determined, for example, by comparing the GISP performance of subjects with gastric cancer and their subjects, where greater than 60% (such as at least about 60, 70, 75, 80, 85, 90, Or 95% /%) of the subjects did not develop cancer within a sufficient period of time (eg, at least 6, 9, 12, 18, 24, 30, 36 or more months). The performance threshold amount that is excluded or included in the risk group can be set to reach a pre-measured amount in subjects who have not developed gastric cancer. Preferably, subjects at risk for gastric cancer have at least about 10%, 20%, and 40% of GISp manifestations greater than a threshold amount. The method of the present invention provides a means for predicting the risk of a subject developing gastric cancer and identifying a subject with gastric cancer. Accordingly, the present invention provides prognostic information to a subject and his or her healthcare provider to ensure better informed medical decisions. In another aspect, the invention features a method for determining the effectiveness of a compound for treating gastric cancer. This method involves contacting a compound with gastric cancer cells, and this paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page) — — — — — — — ^ -------- Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224623 A7 B7_ V. Description of the invention (3) Comparison of gastric cells with this GISp manifestations before and after compound exposure. A reduction in the expression of GISP after contact with the compound indicates that the compound is effective in treating gastric cancer. The effectiveness of the compounds in the treatment of gastric cancer can be assessed in vitro or in vivo. When the method of the present invention is performed in vitro, gastric cancer cells can be derived from gastric cancer cell lines. Examples of suitable gastric cancer cell lines include AGS cell line, KATO III cell line, SNU-16 cell line, RF_48 cell line, NCI_N87 cell line, NUGC cell line, and az-521 cell line. Alternatively, the gastric cancer cells may be in an animal suffering from gastric cancer. For example, the compound can be administered to animals suffering from gastric cancer, and the expression of GISp in the animal after administration can be compared with the expression of GISP in the animal. GISp expression in animals can be measured in vivo or in vitro. For example, the amount of GISp can be measured in vitro from biological samples of animals, such as tumor solid tissue samples, excreta, blood or digestive fluids before and after administration of the compound. Other methods for measuring GISP expression in animals are described herein. Various gastric cancer animal models, such as orthotopic tissue transplantation of SC in human gastric cancer tissues can be used; D mice.
化合物治療胃癌的能力可在活體内藉投藥化合物至罹患 同癌之動物並測定此化合物對腫瘤大小，生長或轉移潛力 之影響而確認D 本發明之其他特徵或優點將由下列詳述，及申請專利範 圍而明顯化。 圖式簡要說明 -6- 本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） li!i--------tT---------i (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1224623 A7 B7_ 五、發明說明（4) 圖1描述人類GISP之胺基酸序列及核酸序列。 圖2提供在各種胃癌細胞系之GISP基因表現之北方墨 點。 圖3說明人類GISP在5對不同之人類胃癌組織中（T1至T5 ) 及與它們對應之相鄰正常黏膜組織（N1 to N5)之RT-PCR表 現。 發明詳細說明 本發明係關於利用GISP表現作為發展成胃癌之風險之決 定因素之預期藥物及利用GISP作為胃癌之決定因素之診 斷藥物。本發明預期之範圍是將GISP表現之分析作為評 估病人狀態及預後之藥物遣傳學方法（如使用生物晶片）的 一部分。 與細胞中GISP基因相對應之mRNA量可在定位或活體外 的形式測定。 可用於雜交或擴增分析之GISP mRNA探針包含，但不限 於，南方（Southern)或北方（Northern)分析，聚合酶鏈式反 應分析及探針陣列。用於測定mRNA量的診斷方法包含將 mRNA與一個可與欲測定基因所編碼之mRNA雜交之核酸 分子（探針）接觸。核酸探針可為，例如，一個全長的 GISP核酸或其部分，如長度至少為7，10，15, 30, 50，100, 250或500個核苷酸，且在嚴格條件下足以與GISP mRNA、 cDNA或其部分特異性雜交之寡聚核苷酸。在此中所使用 -7- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) 訂---------線在 1224623 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（） 者，用語’’在嚴格條件下雜交’’ 一詞說明了雜交及洗滌條 件。嚴格的條件為熟諳此藝者所熟知者，並可見於Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989)，6.3.1至6.3.6。探針可以可測定之試劑標記以利於 探針之鑑定。有用的試劑包括，但不限於，放射性，螢光 染齊〗或催化可檢測產物之酵素。 在一種形式中，mRNA (或cDNA )被固定於平面上並與探 針接觸，例如將分離的mRNA置於瓊脂糖凝膠上，並將此 mRNA由凝膠轉移到一個膜上，如硝基纖維素。在另一替 代形式中，探針被固定於平面上及mRNA (或cDNA )與探針 接觸，例如，在一個二維基因晶片陣列中。一個熟習此技 藝者可改變已知的mRAN檢測方法而用於檢測由GISP基因 編碼之mRNA量。 由GISP核酸所編碼之樣本中之mRNA量可由核酸擴增方 式檢測，如以RT-PCR(美國專利案第4,683,2〇2號），連接 酶鏈反應（Barany，Proc. Natl. Acad. Sci. USA 88:189-193, 1991)，self sustained序列複製（Guatelli等人，Proc. Natl. AcadThe ability of a compound to treat gastric cancer can be confirmed in vivo by administering the compound to an animal suffering from the same cancer and measuring the effect of the compound on tumor size, growth or metastatic potential. D Other features or advantages of the present invention will be described in detail below and applied for The scope is obvious. Brief description of the drawing-6- This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) li! I -------- tT --------- i (Please Read the notes on the back before filling this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224623 A7 B7_ V. Description of the invention (4) Figure 1 depicts the amino acid sequence and nucleic acid sequence of human GISP. Figure 2 provides the northern dots of the GISP gene expression in various gastric cancer cell lines. Figure 3 illustrates the RT-PCR performance of human GISP in five different pairs of human gastric cancer tissues (T1 to T5) and their corresponding adjacent normal mucosal tissues (N1 to N5). DETAILED DESCRIPTION OF THE INVENTION The present invention relates to prospective drugs that use GISP performance as a determining factor for the risk of developing gastric cancer, and diagnostic drugs that use GISP as a determining factor for gastric cancer. The scope of the present invention is expected to include analysis of GISP performance as part of a drug delivery method (such as the use of biochips) to assess patient status and prognosis. The amount of mRNA corresponding to the GISP gene in a cell can be determined in a localized or ex vivo manner. GISP mRNA probes that can be used for hybridization or amplification analysis include, but are not limited to, Southern or Northern analysis, polymerase chain reaction analysis, and probe arrays. The diagnostic method for determining the amount of mRNA involves contacting the mRNA with a nucleic acid molecule (probe) that can hybridize with the mRNA encoded by the gene to be determined. The nucleic acid probe may be, for example, a full-length GISP nucleic acid or a portion thereof, such as at least 7, 10, 15, 30, 50, 100, 250, or 500 nucleotides in length, and under stringent conditions is sufficient to interact with the GISP mRNA Oligos that specifically hybridize to cDNA or parts thereof. Used in this paper-7- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page) Order --------- Printed on line 1224623 in the Intellectual Property Bureau of the Ministry of Economic Affairs, the Consumer Cooperatives printed A7 B7 V. Inventor (), the term "hybridization under strict conditions" used to describe hybridization and washing conditions. Stringent conditions are familiar to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 to 6.3.6. The probe can be labeled with a measurable reagent to facilitate the identification of the probe. Useful reagents include, but are not limited to, radioactive, fluorescently stained, or enzymes that catalyze detectable products. In one form, the mRNA (or cDNA) is immobilized on a flat surface and in contact with the probe. For example, the isolated mRNA is placed on an agarose gel and the mRNA is transferred from the gel to a membrane, such as a nitro group. Cellulose. In another alternative, the probe is immobilized on a flat surface and the mRNA (or cDNA) is in contact with the probe, for example, in a two-dimensional array of gene wafers. A person skilled in the art can change the known mRAN detection method to detect the amount of mRNA encoded by the GISP gene. The amount of mRNA in the sample encoded by the GISP nucleic acid can be detected by nucleic acid amplification methods, such as RT-PCR (U.S. Patent No. 4,683,202) and ligase chain reaction (Barany, Proc. Natl. Acad. Sci USA 88: 189-193, 1991), self sustained sequence replication (Guatelli et al., Proc. Natl. Acad
Sci. USA 87:1874-1878，1990)，轉錄擴增系統（Kwoh 等人， Proc. Natl. Acad. Sci· USA 86:1173-1177, 1989)，Q_Beta 複製驗 (Lizardi 等人，Bio/Technology6:1197，1988)，旋轉循環複製 (美國專利案第5,854,〇33號），或任何其他之核酸擴増方 法，接著使用此技藝中所熟知的技術檢測擴增之分予。 -8- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -----------——訂---------I ~ (請先閱讀背面之注意事項再填寫本頁) 61224623 A7 五、發明說明（ 此中所使用者，擴增引子被定義為可鍊合至基因$，或區 段之核酸分子對(分別為正或負股，或反之亦然），並在之 間包吕個短區段。一般而言，擴增引子之長度約為⑺ 至30個核黎酸，兩侧並與約5〇至2〇〇個核答酸長度之區域 相接。在適當的條件並與適當試劑作用下，此等引子可使 核酸分子擴增，包括引物兩侧之核苷酸序列。 在足位方法中，細胞或組織樣本可經製備/處理並固定 於支持物上，典型為玻璃載片，並接著以可與欲分析之 GISP基因編碼之mRNA雜交之探針接觸。 在另具μ施例中，本方法進一步包含將控制樣本與 一種可们則GISP mRNA，或cDNA之化合物或試劑接觸， 及比較控制樣本與測試樣本中GISp恤财或a·之存 在。 許多種方法可用於測定由GISp基因編碼之蛋白質量。一 般而T，此等方法包含將樣本和選擇性與蛋白質結合之試 劑，如抗體，接觸以評估樣本中之蛋白質量。在一個具體 實施例中，抗體具有可檢測標記。抗體可為多株或單株。 員 費 印 訂 可使用％整抗體，或其片段（如Fab4F(ab，)2)。用語，，已標 記”，係關於探針或抗體，係欲包含藉偶合（如，物理連 接）可檢測物質至探針或抗體而直接標記探針或抗體，及 藉可檢測物質反應而間接標記探針或抗體。 該測定法可用於測定在活體外及活體内之生物樣本之 -9 本紙張尺度刺巾關家鮮（CNS)A伐格挪公釐)_ 1224623 A7 _B7 五、發明說明（7) (請先閱讀背面之注意事項再填寫本頁) GISP蛋白質。活體外測定GISP蛋白質的技術包括酵素連 結免疫吸收測定法（ELISAs)，免疫沈澱法，免疫螢光法， 酵素免疫測定法（EIA)，輻射免疫測定法（RIA)，及西方 (Western)墨點分析。活體内測定GISP蛋白質技術包括將標 記之抗GISP抗體導入受試者體内。例如，此抗體可以放 射線活性記號（其存在並位於受試者）標記並藉標準成像技 術檢測。 在另一具體實施例中，該方法進一步包括將控制组樣本 與可測定GISP蛋白質之化合物或試劑接觸，及比較控制 組樣本及測試樣本中GISP蛋白質的存在。 本發明進一步提供測定治療胃癌之化合物之有效性之方 法（在此也稱為’’篩選測定法（screening assays) ”），如候選或 測試化合物。可用於測試之候選化合物實例包括蛋白質， 肽，肽模擬物（peptidomimetics)，類肽物（peptoids)，核酸， 對GISP表現具抑制效果之小分子或其他藥物。 經濟部智慧財產局員工消費合作社印製 GISP表現之抑制劑可用已知方法鑑定。例如，可將一種 候選化合物與胃癌細胞接觸，並評估GISP mRNA或蛋白質 之表現量與缺乏候選化合物時GISP mRNA或蛋白質表現量 之相對值。當候選化合物存在時之GISP mRNA或蛋白質之 表現低於（統計上顯著地低於）它不存在時，此候選化合物 便鑑別為對於治療胃癌可能有效之GISP mRNA或蛋白質表 現抑制劑。GIP mRNA或蛋白質表現可依此處所述測定 -10- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 經濟部智慧財產局員工消費合作社印製 1224623 A7 B7_ 五、發明說明（8) GISP mRNA或蛋白質之方法測定。 GISP抑制劑可以上述任何分析物鑑定，而此GISP抑制劑 治療癌症之能力可於活體中確認，如在一種為胃癌動物模 型的動物體内。例如，可評估此化合物對胃腫瘤大小，成 長和/或轉移潛力之效果。胃癌之動物模型可為，如，植 入人類胃癌組織之SCID鼠。 本發明亦特徵在於治療胃癌的方法。本方法包括對罹患 胃癌之受試者投藥足夠治療胃癌量之GISP抑制劑。此 GISP抑制劑可依此所述之篩選方法鑑定。經篩選可對 GISP表現產生抑制效果之候選化合物實例包括蛋白質， 肽、肽模擬物（peptidomimetics)，類肽物（peptoids)，核酸、 抗體及小分子。此等化合物可併入醫藥組合物中。此等組 合物典型上包括核酸、蛋白質、抗體或小分子及一種醫藥 上可接受之載劑。與藥物投藥路徑相容之醫藥上可接受載 劑包含溶劑，分散介質、包覆劑、抗細菌及抗真菌劑、等 張及延遲吸收劑，及其類似物。 實例 實例1 . GISP基因序列之鑑定 BLAST伺服器程式（2.0.4版）由美國國家衛生研究院 (National Institute of Health)之國家生物資訊中心（National Center for Biological Information(NCBI))取得，並建立於 SGI (Origin-200)伺服器。預設之BLAST參數被用於分析 -11 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 丨！——秦--------^---------線拿 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1224623 A7 B7 ___ 五、發明說明（9) (e=l〇，v=50，b=50及w=0)。取得人類及鼠類之dbEST資料 並用於基因鑑定。人類lithostathine及PAP蛋白質序列用於 進行對照人類dbEST資料庫之tblastn搜索查詢，而顯著之 EST序列以MacVector程式組合。序列校準及種系分析以 PowerMac G3系統之CLUSTAL X及PHYLIP電腦程式執行。 實例II :藉RT_PCR對人類GISP基因之分子選殖 藉資料庫比對鑑定GISP基因後，自組合之核酸序列設計 PCR引子對。如林（Lin)等人，J· Biomed· Sci. 5:101-110，1998 及林（Lin)等人，Clin. Cancer res· 5:1745-1751，1999 所述， cDNA係製備自人類胃癌細胞系。簡單言之，逆轉錄以總 共 20 微克（Mg)之 mRNA，寡聚（dT)15 及得自 Promega Madison， WI之MMLV逆轉錄酶進行。RT產品之品質藉瓊脂糖凝膠 電泳及藉GAPDH專一性引子之PCR反應檢測。PCR引子係 用於擴增缺口區段。PCR反應在94°C下5分鐘，94X：下1 5 秒，58°C下30秒鐘及70°C下2分鐘之35次循環及在72°C下 10分鐘之最終延伸相，以Perkin Elmer之2400PCR反應機 (thermocycler)及 Takara Taq 聚合酶（Shiga，Japan)進行。最終 的PCR產物以1%之瓊脂糖凝膠電泳分析。擴增的片段自 凝膠中沖提出，並次選殖至得自Invitrogen (Carlsbad，CA)之 pCR2.1 T/A選殖載體。在選殖程序後，隨機篩選，純化數 個選殖物，並藉ABI 377自動定序儀測定它們的序列。 鑑定人類GISP基因後，搜尋老鼠EST序列之向同源基 -12- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 1224623 A7 B7 10 五、發明說明（） 因。鑑定數個分離之菌落。此等菌落係得自IMAGE聯盟， 並藉ABI 377自動定序儀測定它們的序列。 (請先閱讀背面之注意事項再填寫本頁) 實例III，RNA萃取 胃癌組織及其正常胃黏膜組織係得自接受過胃切除術之 病患。於所有病患取得告知同意書。切除後立即將樣本以 液態氮冷康。所有的RNA以直接脈異硫氰酸鹽析（direct guanidine isothiocyanate lysis)及如林等人，Clin. Cancer res. 5:1745-1751，1999所述之氯化铯分離方法而萃取自胃癌及 胃黏膜組織。如上所述進行RT-PCR實驗。 實例IV .組織分佈分析 在組織表現及分佈分析中，多重之組織mRNA墨點（MTN I-III)及 RNA 人類主墨點係得自 Clontech (Palo Alto, CA)。得 自已確認序列之GISP純系之32Ρ·標記片段用於依照供應商 所建議之雜交程序進行之雜交反應。 經濟部智慧財產局員工消費合作社印製 GISP可在人類的胰臟，小腸，胃，及結腸組織中高度表 現。此外，GISP可在人類胃癌細胞系AGS中過度表現。參 照圖2。以RT-PCR方法檢測數種人類胃癌組織後，在人類 胃癌組織中可發現較正常胃皮膜高之GISP表現量。參照 圖3。 在未進一步闡述下，熟諳此藝者可依上述揭示内容及其 下所述之實例為基礎，將本發明利用至其最大程度。下述 實例僅可供說明熟諳此藝者如何操作本發明之方法，並非 -13- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1224623 A7 B7 11 五、發明說明（） 以任何方式限制其餘揭示内容。任何本揭示所引用之出版 物係併入於此中作為參考。 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 -14- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）Sci. USA 87: 1874-1878, 1990), Transcription Amplification System (Kwoh et al., Proc. Natl. Acad. Sci · USA 86: 1173-1177, 1989), Q_Beta Replication Test (Lizardi et al., Bio / Technology6 : 1197, 1988), rotary cyclic replication (U.S. Pat. No. 5,854,033), or any other method of nucleic acid amplification, followed by detection of amplified fractions using techniques well known in the art. -8- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) --------------- Order --------- I ~ (please first Read the notes on the reverse side and fill out this page) 61224623 A7 V. Description of the invention (The users in this article are defined as a pair of nucleic acid molecules (positive or negative strands) that can be chained to gene $, or segments. , Or vice versa), and include a short segment between them. In general, the length of amplification primers is about ⑺ to 30 nucleotides, and flanked by about 50 to 200 nuclei. The region of the acid length is contiguous. Under appropriate conditions and with the appropriate reagents, these primers can amplify nucleic acid molecules, including the nucleotide sequences on both sides of the primer. In the foot position method, a cell or tissue sample can be Prepared / treated and fixed to a support, typically a glass slide, and then contacted with a probe that can hybridize to the mRNA encoded by the GISP gene to be analyzed. In another μ embodiment, the method further includes controlling the The sample is contacted with a compound or reagent of GISP mRNA, or cDNA, and the GISp in the control sample is compared with the test sample. There are many methods that can be used to determine the quality of the protein encoded by the GISp gene. Generally, these methods involve contacting the sample with a reagent that selectively binds to the protein, such as an antibody, to evaluate the protein in the sample. In a specific embodiment, the antibody has a detectable label. The antibody can be multiple strains or single strains. Staffing can use% whole antibodies, or fragments thereof (such as Fab4F (ab,) 2). Terms ,, “Labeled” refers to probes or antibodies, and is intended to include direct labeling of probes or antibodies by coupling (eg, physically connecting) detectable substances to probes or antibodies, and indirect labeling of probes or antibodies by reaction of detectable substances. Antibodies. This assay can be used for the determination of biological samples in vitro and in vivo -9 paper-sized stab towels Guan Jiaxian (CNS) A vagner millimeter) _ 1224623 A7 _B7 V. Description of the invention (7) ( (Please read the notes on the back before filling this page) GISP protein. Techniques for measuring GISP protein in vitro include enzyme-linked immunosorbent assays (ELISAs), immunoprecipitation, immunofluorescence, and enzyme immunization Assay (EIA), Radiation Immunoassay (RIA), and Western blot analysis. In vivo measurement of GISP protein technology involves introducing a labeled anti-GISP antibody into a subject. For example, this antibody can be radioactive A marker (which is present and located in the subject) is labeled and detected by standard imaging techniques. In another specific embodiment, the method further comprises contacting the control group sample with a compound or reagent capable of measuring the GISP protein, and comparing the control group sample And the presence of GISP protein in test samples. The present invention further provides methods (also referred to herein as "screening assays") for determining the effectiveness of compounds for treating gastric cancer, such as candidate or test compounds. Examples of candidate compounds that can be used for testing include proteins, peptides, peptidomimetics, peptoids, nucleic acids, small molecules or other drugs with inhibitory effects on GISP performance. Inhibitors of GISP performance printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs can be identified by known methods. For example, a candidate compound can be contacted with gastric cancer cells and the relative expression of the expression of GISP mRNA or protein in the absence of the candidate compound can be evaluated. When the expression of a GISP mRNA or protein in the presence of a candidate compound is lower (statistically significantly lower) than it does not exist, the candidate compound is identified as a GISP mRNA or protein expression inhibitor that may be effective in treating gastric cancer. The expression of GIP mRNA or protein can be determined as described here-10- This paper size applies Chinese National Standard (CNS) A4 specifications (210 X 297 mm) Printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs 1224623 A7 B7_ V. Invention Note (8) Determination of GISP mRNA or protein. GISP inhibitors can be identified with any of the analytes described above, and the ability of this GISP inhibitor to treat cancer can be confirmed in vivo, such as in an animal model of a gastric cancer animal. For example, the effect of this compound on gastric tumor size, growth, and / or metastatic potential can be evaluated. An animal model of gastric cancer may be, for example, a SCID mouse implanted into human gastric cancer tissue. The invention also features a method for treating gastric cancer. The method includes administering to a subject suffering from gastric cancer a sufficient amount of a GISP inhibitor to treat gastric cancer. This GISP inhibitor can be identified by the screening method described herein. Examples of candidate compounds that can be screened to inhibit GISP performance include proteins, peptides, peptide mimetics, peptoids, nucleic acids, antibodies, and small molecules. These compounds can be incorporated into pharmaceutical compositions. These compositions typically include nucleic acids, proteins, antibodies or small molecules and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers compatible with the route of administration of the drug include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and delayed absorption agents, and the like. Examples Example 1. Identification of GISP gene sequence The BLAST server program (version 2.0.4) was obtained and established by the National Center for Biological Information (NCBI) of the National Institute of Health On SGI (Origin-200) server. The preset BLAST parameters are used for analysis. -11-This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 丨! ——Qin -------- ^ --------- Line take (Please read the notes on the back before filling this page) Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1224623 A7 B7 ___ V. Description of the invention (9) (e = 10, v = 50, b = 50 and w = 0). Obtain human and rodent dbEST data and use it for genetic identification. Human lithostathine and PAP protein sequences were used to perform tblastn searches against the human dbEST database, and significant EST sequences were combined using the MacVector program. Sequence calibration and germline analysis were performed using the CLUSTAL X and PHYLIP computer programs of the PowerMac G3 system. Example II: Molecular selection of human GISP gene by RT-PCR After identifying the GISP gene by database comparison, design PCR primer pairs from the combined nucleic acid sequences. As described by Lin et al., J. Biomed. Sci. 5: 101-110, 1998 and Lin et al., Clin. Cancer res. 5: 1745-1751, 1999, the cDNA line was prepared from human gastric cancer Cell line. In brief, reverse transcription was performed with a total of 20 micrograms (Mg) of mRNA, oligo (dT) 15 and MMLV reverse transcriptase from Promega Madison, WI. The quality of RT products was tested by agarose gel electrophoresis and PCR reaction by GAPDH specific primers. PCR primers are used to amplify gaps. PCR reaction was performed at 94 ° C for 5 minutes, 94X: 15 seconds at 15 ° C, 30 seconds at 58 ° C, 35 cycles at 70 ° C and 2 minutes at 70 ° C, and the final extension phase at 72 ° C for 10 minutes. Elmer's 2400 PCR thermocycler and Takara Taq polymerase (Shiga, Japan). The final PCR products were analyzed by 1% agarose gel electrophoresis. The amplified fragment was eluted from the gel and subcultured to the pCR2.1 T / A colony vector from Invitrogen (Carlsbad, CA). After the selection procedure, several selections were randomly selected, purified, and sequenced using the ABI 377 autosequencer. After identifying the human GISP gene, search for the ortholog of the mouse EST sequence -12- This paper applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------------- ------ Order --------- line (Please read the precautions on the back before filling this page) 1224623 A7 B7 10 V. Description of the invention () Reason. Several isolated colonies were identified. These colonies were obtained from the IMAGE Alliance and their sequence was determined by an ABI 377 autosequencer. (Please read the precautions on the back before filling out this page) Example III, RNA extraction Gastric cancer tissue and its normal gastric mucosa tissue were obtained from patients who had undergone gastrectomy. Obtain informed consent from all patients. Immediately after excision, the samples were cooled with liquid nitrogen. All RNA was extracted from gastric cancer and stomach by direct guanidine isothiocyanate lysis and the cesium chloride separation method described in Lin et al., Clin. Cancer res. 5: 1745-1751, 1999. Mucosal tissue. RT-PCR experiments were performed as described above. Example IV. Tissue Distribution Analysis In tissue performance and distribution analysis, multiple tissue mRNA dots (MTN I-III) and RNA human primary dots were obtained from Clontech (Palo Alto, CA). The 32P · labeled fragment of the pure GISP line obtained from the confirmed sequence was used for the hybridization reaction according to the hybridization procedure recommended by the supplier. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, GISP is highly expressed in human pancreas, small intestine, stomach, and colon tissue. In addition, GISP can be overexpressed in the human gastric cancer cell line AGS. Refer to Figure 2. After detecting several types of human gastric cancer tissues by RT-PCR method, the expression of GISP in human gastric cancer tissues was found to be higher than that of normal gastric epithelium. Refer to Figure 3. Without further elaboration, those skilled in the art can use the present invention to its fullest extent based on the above disclosure and the examples described below. The following examples are only provided to illustrate how a skilled artist can operate the method of the present invention, not -13- This paper size is applicable to Chinese National Standard (CNS) A4 (210 X 297 mm) 1224623 A7 B7 11 V. Description of the invention ( ) Limit the rest of the disclosure in any way. Any publications cited in this disclosure are incorporated herein by reference. (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs -14- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)