狄、發明說明： 狄、發明說明：Di, invention description: Di, invention description:
發月所屬气技術領域】 申π付合美國法典第35卷119條有關美國國 内臨時申請之插6 ^ 、疋’編號爲No· 60/246,258，申請提出曰期 爲2003年11月n pj 0a ’申請内容通過對照檢索以並入本次公 開披露之詳細資料。 發明領域 Θ /V及的内I爲從Ϊ魚的軟骨組織和軟骨細胞中 σ蚕抗血管新生藥物的手段。 【先前技術】又 發明背景 &新生疋形成新血管的基本過程。它是生殖、發育 和創傷癒合科μ理現象的重要步驟。在正常情況下， 2生。然而，㈣缺乏規律⑽管新生也 „多疾病’包括癌症、固體腫瘤、風濕性關節炎、 月牛皮_、糖尿病視賴病變、老年黃斑病變、 吕=狹窄、卡波西氏肉瘤、白血病、腫瘤轉移、良性腫 瘤、遺傳性出血性毛細血管擴張症、心肌血管新生、斑塊 4血&化毛細血官擴張、血友病關節病症、血管纖維瘤、 匐知肉芽、青光眼、角膜移植之排斥、動脈硬化、硬皮病、 瘢痕瘤、炎症及胎兒受孕等。 例如，腫瘤的生長和轉移完全依賴于血管新生。大多 數原發性固體腫瘤都要經過一段相當長的無血管時期，在 此期間腫瘤的生長明顯處於休眠狀態’最大也只能達到直 徑1-2毫米左右。腫瘤生長到這樣大小之後，腫瘤細胞就可 以通過簡單的被動擴散吸收必要的氧和其他營養物質。這 些只能在顯微鏡下觀察到的腫瘤塊最後會“啓動”血管生 成，它們利用四周成熟的宿主血管，催生出新的血管和毛 細血管，而這些血管會朝著腫瘤塊的方向生長，並最終渗 透進去。這使得腫瘤塊和血液轉移不斷擴散，將病痛的危 險無情地散播到身體其他部位。在風濕性關節炎中，新生 成的毛細血管侵入關節，破壞軟骨組織。而在糖尿病視網 膜病變中，視網膜中形成新的毛細血管，侵入玻璃體和血 液，並且最終導致失明（弗克曼1995年《自然醫學》丄【丄】 期27-31頁；弗克曼與克萊格斯布魯恩忉们年《科學雜誌》 235期442-447頁；D. A.沃爾什測年《風濕病學》邓【2】 期i〇3-m頁；希爾利等1998年《人類生殖新知》4【5】期 736-740頁）。 ’ 生抑制劑和腫瘤;ϋ 腫瘤生長可以通過阻止腫瘤血管新生加以限制或預 防’特別是直接對準_生成而且沒有完全成熟的血管内 皮細胞。該方法可以預防腫瘤塊出現新的擴散，並使豆持 續萎縮至直徑毫米大小，這也是腫錢在無血管供血 的條件下存活的狀態。這樣的治療手段可以稱爲“誘導休 眠，目的是以缓慢的方式控制疾病。 已經發表的相關文獻都曾詳細描述過f魚軟骨在醫學 領域裏所具有的衆多功效，其中就包括其在血管生成方面 的特性。裝魚軟骨提取物-直作爲營養補充齊 售。但《魚軟骨可以有效治療晚期癌症的觀點==肖 批評意見（請參閱米勒物98年《臨床腫瘤 γ，、子在 3649-3655頁）。 ’ ’》16期 來自佛羅里達州薩拉索塔市莫特海 T具纖中心 爾· A·甘爾發現，有三種蛋白質可以從:篆魚軟骨中恢A 相信能夠起到阻礙腫瘤擴散甚至使其逐漸萎縮的作用人设， 而避免放射治療對人體的有害副作用（《洛杉機時^ ’攸 年8月27日B3版）。 T 1990 杜邦（美國專利號6,028，118)和巴拉薩（ 丨连、果國專利赛 4,822,607)都詳細描述了各自發現的一種1 " 有抗血管新峰 和消炎特性的！、魚軟骨提取物以及提取方法和治療中則 用。但這兩項專利都未曾專門介紹其抗血管新生成分的使 成和分子特性。杜邦和巴㈣都直接從、軟骨這 而:可再生的資源中直接進行提取。他們所獲得的固態或: 液悲提取物都只能算是活性物質的“大雜 、、稚環，而非已經分 離出來的特效化合物。 而根據梁女士的研究報告，她所分離出的業魚軟骨提 取物與魯爾、格魯伯、杜邦及巴拉薩等人提取的蛋白質不 同，相信應該是-種蛋白多糖(請參閱詹媒.梁胸年^〈從 装魚軟骨中提取血管新生抑制劑的分離、分鶴及評定》【= 生論文】加州大學弗雷斯諾分校）。該提取物已通過内皮: 胞化驗進行壯管新线財面的錢。 中直接進行提取，具體過程是使用鋼制沖孔工具取==、 200423951 軟骨椎體的中心部分，然後將沖取的圓形部分冷凍乾燥， 用攪拌機研磨成粉，最好用水進行提取(請參閱梁1997年同 上文第9頁和34頁）。 然而，鯊魚是一種有限資源，很難大量獲取。因此非 5常有必要對鯊魚軟骨細胞進行人工培養，使其能夠重復培 育並大量獲取。 格羅根和朗德曾經在這方面略有突破，他們從鯊魚和 魟魚身上成功地培養出免疫細胞(格羅根和朗德1990年《維 持並保證鯊魚和魟魚免疫細胞分裂繁殖的培育系統》，發表 10 於《魚類生物學期刊》第36期633-642頁）。此外，哈特曼等 也曾試圖培養多種鯊魚組織，但不包括軟骨(J·哈特曼等 1992年《軟骨魚細胞初步培養的常規設置》，發表於《體外 細胞發育生物學》28A期第77-79頁）。這些嘗試都沒有能夠 成功，從生物體組織中獲取的細胞雖然開始顯示出一定的 15 成長，但卻無法進入成熟期，而最終也都無法存活（請參閱 C·奥哈拉1995年《從人工培養的鯊魚軟骨細胞中獲取的血 管新生抑制劑》第12頁【學生論文】加州大學弗雷斯諾分 校）。 奥哈拉和王曾經詳細報告過他們在人工培養的成年鯊 2〇 魚軟骨細胞和培養基中分離出一種血管新生抑制劑的過程 (請參閱C·奥哈拉1995年同上文及奥哈拉和王1996年《人 工培養的鯊魚軟骨細胞分泌出的一種血管新生抑制劑》，加 州州立大學弗雷斯諾分校）。但研究顯示’人工培養的細胞 生長速度非常緩慢，因爲軟骨細胞無法自行附在培養組織 8 200423951 的内壁上（請參閱體外研究小組1996年《體外生物學會期 刊》32期第37頁《有關奥哈拉和王相關論著的報告》和奥 哈拉1995年同上文）。爲提高軟骨細胞的附著能力，他們使 用了 一種人造母體。這種方法雖然促進了細胞生長，但卻 5 無法達到可供實際使用的密度（請參閱奥哈拉1995年同上 文、奥哈拉和王1996年同上文、以及體外研究小組1996年 同上文）。根據這些研究，“鯊魚軟骨細胞培養可以産生某 些具有抑制作用的因數，但迄今爲止，完成鯊魚軟骨細胞 體外培養的成功案例尚未出現”（請參閱奥哈拉和王1996 10 年以及體外研究小組同上文）。 此外，成年鯊魚的軟骨過於脆硬，容易碎裂。因此必 須在培養之前先將其分解，以便釋放出軟骨細胞。在離解 過程中，需要將軟骨浸泡于含有膠原酶的溶液裏直至出現 分解，然後通過離心過濾將軟骨細胞從溶液中分離出來(請 15 參閱奥哈拉1995年及奥哈拉和王1996年同上文）。這種方法 的風險在於可能破壞軟骨細胞的完整性並增加外界污染的 幾率。 奥哈拉在其1995年論文中提出使用鯊魚胚胎作爲軟骨 細胞來源的建議，因爲這樣做細胞分化變異的程度會有所 20 降低。但奥哈拉本人並未提供任何實踐操作的方法，也沒 有涉及到更改初始材料可能導致的任何結果。 【發明内容】 發明概要 本發明的内容包括一種具有抗血管新生及消炎功效的 9 Γΐί取物及其提取方法。該提取物是從人工培養的軟骨 2 °軟骨細胞培養基中分離出來的，而前者來自當 專心胞生長液。該提取物包含由蛋衫糖和糖蛋 物约上的生物雜劑分子量爲1mGKD，其巾碳水化合 化合物=%’而蛋白質約占4.5%’活性物質則包含在碳水 在提取過財，首先從軟骨魚綱動物或其胚胎上取出 10 tr，缝骨進行研磨使其釋放出軟骨細胞，人工培 ^月、、’田胞’取後將抗血管新生提取物從人卫培養的軟骨 =和/或培養基中分離出來。有—點不能忽略，用來培養 权月細胞的專用生長液含有t,#、的血清。 本發明還包括-種抑制内皮細胞生長的方法，即向細 胞發出-定量的提取物，達到生長抑制的要求。本發明還 15 =-種抑制組織内部血管増生的方法，即向組織發出一 疋=的提取物達5仏_增生的要求。治療癌症和關節 炎等多種疾病的方法在此也_併提及。 本發明介紹的軟骨細胞培養方法不需要使用能夠促進 胚胎軟骨細胞的附著和生長的人造母體。軟骨細胞具有一 加定的附著能力，並且能夠在—周内出現匯合並形成塊體。 這是一個令人意外的結果，因爲來自業魚等成年軟骨魚綱 動物的軟骨細胞如果不借助於人造母體就無法附著，而且 使用人造母體後生長時間會加倍延長至6〇天，此後生長速 度將有所減慢並需要新_贿（請參w奥哈拉簡年同 10 200423951 上文第49頁）。 此外，我們還開發出一種含有軟骨魚血清的專用細胞 生長液。該血清從血液中濾出，含有軟骨細胞達到最佳生 長效果所需的天然營養成分和荷爾蒙，其中包括一種副甲 5 狀腺荷爾蒙相關蛋白質（PTHrp)，在軟骨細胞的分裂繁殖和 分化變異過程中具有重要作用（請參閱來自加拿大圭爾夫 市圭爾夫大學的J·格爾斯雷施特和J· A·穆西克在1998 年7月17日美國軟骨魚學會大會上的摘要和陳述以及J·格 爾斯雷施特和J· A·穆西克1999年發表於《實驗動物學期 10 刊》第284期549-556頁的文章）。 使用胚胎軟骨魚的軟骨組織代替成年軟骨魚作爲軟骨 細胞的來源具有更多的優點。首先，成年軟骨魚的軟骨組 織需要膠原酶幫助分解（請參閱奥哈拉1995年同上文第21 頁），而胚胎軟骨細胞則不需要借助於任何酶。這將明顯降 15 低軟骨細胞的完整性遭到破壞或培養過程受到污染的風 險。 圖式簡單說明 第1圖顯示從胚胎鯊魚軟骨中準備抗血管新生提取物 的方法。 20 第2圖顯示準備鯊魚血清的方法。 第3A及3B圖顯示從軟骨細胞及軟骨細胞培養基中分 離出血管新生抑制劑的方法。 I：實施方式3 較佳實施例之詳細說明 11 心佈的内谷中’引用的各種文獻、專利及已發 =料說明都以引文的方式表示，讀者可以-目了然。 $的内各中弓丨用這些文獻、專利及已發表的專利 况明目的是致7 $ 5準。 ’、、、更加完整地表述本項發明涉及的最高水 、月在κ踐刼作中（除非另有特別說明）使用分子 子㈦括細胞重組技術)、微生物學、細胞生物學、生物 水;'免ί孥的傳統技術，均未超出目前科技發展的最高 。4技術將在文獻資料中予以詳細而完整的解釋。 10相關定義 在本文中已對相關術語定義如下。 在專利說明及專利要求内容中，除非文中另行說明， 有單數形式均包含複數含義。例如，1個細胞，，雖 。爲單數开7式，但同樣包含該細胞的複數含義以及單數和 15複數混合使用的含義。 17 在本文中，“含有，，意指成分和方法既包括詳細列舉的 各個要素，也並沒有將其他元素排除在外。當“基本上 由……組成，，用於成分和方法的定義時，它的含義中不應將 其他與该化合物具有重要關係的元素排除在外。因此，— 20種基本上由本文詳述的元素組成的成分不應將分離和提純 方法中的微量污染物以及磷酸緩衝液、防腐劑及其他從藥 物學理論上可以接受的載體排除在外。“由……組成，，的含 義中排除在外的内容不僅限於其他成分的微量污染物和本 項發明中進行化合過程中的實際操作步驟。這些術語變化 12 200423951 所代表的具體内容都在本項發明的範疇之内。 包括酸驗度、溫度、時間、濃度和分子量在内的所有 歸及域值均爲近似值，誤差爲±G1。儘管不是每次都明 確提不’但所有數值前均帶有“大約，，的字$，而且本文提 到的試劑僅具有典型特徵，其㈣物均爲該研究 熟知。 ^ “分離，，是指從組成物、細胞及其他與化合物天然結合 的成分中單獨獲取。 σ 口 “培養基，，可时單數或複數形式，它是指由各種不同 10物質組成的用於培養細胞生長的化合物。 “個體”或“宿主，，爲脊椎祕’最好是哺乳動物或人 類。哺乳動物包括（但不僅限於）鼠類、猿猴類、人類、畜類、 競技類動物及寵物。 ‘‘癌症，，和“腫瘤，，可用作單數或複數形式，它是指發生 15惡性病變並對宿主機體造成病患的細胞。原代癌細胞（即惡 性病變部位附近獲得的細胞）可以通過組織檢查等先進的 ^測手段與非癌細胞區別開來。本文中涉及的癌細胞定義 不僅包括原代癌細胞，而且指源自癌細胞的任何細胞，包 括轉和癌細胞、體外培養、以及源自癌細胞的細胞株。談 20到某種類型的癌症，通常是表示固體腫瘤，而“臨床可以發 現”的腫瘤是指那些在腫瘤塊基礎上可以通過CAT掃描、磁 共振成像(MRI)、X光、超聲波或觸診等診療手段發現的腫 瘤。而單純依靠生化或免疫學手段發現的結果尚不足以符 合本定義的要求。 13 200423951 在本文中，“抑制”是指推遲、延緩或阻止内皮細胞的 生長、繁殖或細胞分裂或血管在組織内的形成。監測抑制 功效的方法包括（但不僅限於）内皮細胞繁殖化驗、通過測定 血液含量及測量血管床體積、以及定量測定血管結構的密 5 度。當培養物質爲細胞混合體時，監測新血管化的過程需 要通過定量測量能夠體現内皮細胞特定指標的細胞加以完 成，這些指標包括血管新生因數、蛋白水解酶、以及内皮 細胞的特定細胞枯附分子等。 “合成”是指活性劑與另外一種惰性（如可發現的製劑 10 或示蹤劑）或活性(如佐劑）化合物或成分的結合。 “藥物成分”包括活性劑與惰性或活性載體的結合物， 並使該成分適用於體外或體内的診斷或治療。 在本文中，“藥物學可接受載體”的含義包含磷酸緩衝 溶液、水、乳劑（如油/水乳劑或水/油乳劑）及各種濕潤劑等 15 任何標準的藥物學載體。該成分還應包括穩定劑和防腐 劑。有關載體、穩定劑和防腐劑的實例請參閱馬丁·雷明 頓1975年《藥物學標準》第15版(麥克米倫出版公司，伊斯 頓市）。 “有效量”是指能夠對有利或預期效果産生影響的充足 20 數量。例如，治療數量指能夠獲得預期療效的數量。該數 量可能與具有預防功效的數量相同或相異，後者是指能夠 防止疾病發作或疾病症狀的必要數量。有效量可以通過一 次或多次施用或服用得以完成。 本文涉及的“軟骨魚”或“魚”是指任何軟骨魚綱動物， 14 200423951 常見的有鯊魚、魟魚和縫魚等。 本文用於鯊魚或其他軟骨魚的“成年”字句是指已自然 分娩或爲卵生物種時已産出幼體（即非妊娠階段）的鯊魚或 其他軟骨魚。 5 英文“chondrocyte”指軟骨細胞。 “軟骨”指軟骨魚的整個内骨骼。 “抗血管新生”包括消炎特性。 本發明的内容涉及一種抗血管新生及消炎提取物以及 獲得這種提取物的方法。該提取物可以從軟骨魚的軟骨組 10 織經人工培養取得的軟骨細胞及軟骨細胞培養基中獲得。 本發明還涉及一種專用細胞生長液，用於培養那些能夠産 出新提取物的細胞。該提取物包含由蛋白多糖和糖蛋白組 成的生物活性劑，分子量爲1到10KD，其中碳水化合物約 占95.5%，而蛋白質約占4.5%，活性物質則包含在碳水化合 15 物當中。生物活性糖蛋白分子量也可爲10KD左右。 本發明還涉及一種獲得提取物的方法。該提取物可以 從鯊魚胚胎的軟骨組織釋放出的軟骨細胞或在培養基中人 工培養的軟骨細胞中獲取。生物活性成分從軟骨細胞分泌 到培養基中，然後可以從培養基或人工培養的軟骨細胞裏 20 分離出來。有一點不能忽略，用來培養軟骨細胞的專用生 長液含有從軟骨魚血液中提煉出來的血清。 僅通過實例可知，分離出所需提取物的過程包括對培 養基進行離心過濾以保留上層清液，然後使用篩檢程式對 上層清液進行無菌化處理，最後將上層清液分餾，從而分 15 200423951 離出分子量1到10KD左右的提取物。 在提取過程中，首先從軟骨魚綱動物或其胚胎上取出 軟骨組織，對軟骨進行研磨使其釋放出軟骨細胞，人工培 養軟骨細胞，最後將抗血管新生提取物從人工培養的軟骨 5細胞和/或培養基中分離出來。有一點不能忽略，用來培養 軟骨細胞的專用生長液含有鯊魚的血清。 另外一種方案是從軟骨魚胚胎的軟骨組織中分離出所 需提取物，首先從軟骨魚胚胎上取出軟骨組織，對軟骨進 行研磨使其釋放出軟骨細胞，將釋放出的軟骨細胞加水製 10成糊狀，把粘稠的軟骨細胞糊混入培養基中，並將混合物 放置于組織培養板上。 需再次強調的是，該方案需要把粘稠的軟骨細胞糊混 入培養基中並將軟骨細胞/培養基混合物放置于組織培養 板上進行培養。 15 培養過程中需將軟骨細胞/培養基混合物放置于富含 二氧化碳的環境中，以便增強軟骨細胞的附著力，而且在 必要時需將軟骨細胞/培養基混合物從培育環境中取出，更 換新鮮的培養基，以便利於軟骨細胞的生長。 還應當注意，培養過程中需將軟骨細胞/培養基混合物 20 放置於二氧化碳含量達到5%左右而且溫度爲37°C的環境 中，並需將混合物在此環境下放置24到48小時左右。 在實際操作中’培養基中含有從軟骨魚血液中提煉出 來的血清。 在另一方面’提取過程中還包括從培養板上釋放出軟 16 200423951 骨細胞/培養基混合物並重復培養軟骨細胞。 還有一方面，提取過程中還可以從培養板上釋放出軟 骨細胞/培養基混合物並對混合物進行離心過濾以便將培 養基從軟骨細胞中分離出來。 5 一方面，提取過程中還需對軟骨細胞進行速凍保存。 速凍保存包括以下主要步驟：將軟骨細胞與冷凍培養基混 合並在-80°C至-90°C的低溫下冷凍1天左右，然後將軟骨細 胞與冷凍培養基的混合物放置在液氮中長期保存。與軟骨 細胞混合的冷凍培養基可以含有從軟骨魚血液中提煉出來 10 的血清。 本項發明還涉及到一種軟骨魚的軟骨細胞培養基，這 種培養基含有從軟骨魚血液中提煉出來的血清，從軟骨魚 的胚胎制取而成。應當注意，血清與軟骨細胞應當來自同 一種軟骨魚，例如本次實驗中的血清與軟骨細胞就同樣來 15 自鯊魚胚胎。 血清的準備過程包括首先過濾軟骨魚血液以便分離出 淋巴液並濾走微粒，然後對淋巴液進行離心過濾並保留上 層清液，將上層清液加熱以便降低酶的活性。培養基中可 以加入有效數量的三甲胺N-氧化物和谷氨酸鹽。 20 本項發明的内容還包括一種通過向細胞施用具有生長 抑制功效的一定數量的提取物來抑制内皮細胞生長的方 法。發明還包括一種通過向組織施用具有抗血管增生功效 的一定數量的提取物來抑制組織内血管增生的方法。活性 成分可以通過口服、靜脈注射、腹腔注射、或透皮傳送施 17 用於個體。提取物可以經過加工以液體或粉末形式施用。 應當說明的是，個體爲寵物、家畜或人類病人組群中 挑選出來的動物。 本項發明還包括一種針對個體體内因血管增生引起的 5機能紊亂的治療方法，該方法是向個體施用具有某種療效 的-定數量的提取物。這類身體機能紊亂包括(但不僅限於) 癌症、關節炎、因血管增生引起的皮膚病、糖尿病視網膜 病、又、卡波西氏肉瘤、老年黃斑病變、血管再狹窄、毛細 血官擴張、青光眼、瘢痕瘤、角膜移植之排斥、創傷肉芽、 笞纖、准瘤、这傳性出血性毛細血管擴張症、心肌血管新 生、以及硬皮病中的任意一種。但與此同時，抑制血管新 生會阻礙或抑制哺乳動物處於蛀娠期的胎兒發育。 風濕性關節炎的發展與軟骨組織内的血管增生有直接 關係，正是後者直接導致了病情惡化。沃爾什在1998年發 I5表於《風濕病學》38(2)期第1〇3_112頁的文章中指出，病理 性血管新生是導致風濕性關節炎發展的主要原因。我們可 以这樣設想，向患病部位定量施用有效數量的血管增生抑 制劑能夠緩解關節炎症狀，並且可能改善風濕性關節炎的 病情。 20 有的意見認爲，糖尿病視網膜病變和老年黃斑病變都 可以通過血管增生抑制劑獲得有效的治療。衆所周知，糖 尿病視網膜病變是由視__的血管惡性增生引起的，會 導致永久性失明。而老年黃斑病變也與惡性血管新生^ 關，並同樣引起永久性視力喪失。根據預測，使用血管增 18 200423951 生抑制劑能夠防止血管新生的發作和持續，是治療這兩種 病症的有效手段。 在感染牛皮癖的病人身上，牛皮癖的症狀與不斷擴大 的血管新生也存在必然聯繫。我們可以這樣設想，增加血 5 管新生抑制劑的有效數量將抵消這種血管反常增生的不良 影響，從而防止牛皮癬症狀的出現。 本項發明還涉及一種篩選抑制血管增生的治療製劑的 方法，包括用適當的細胞或組織樣本接觸該製劑（樣本A)， 然後用另外的細胞或組織樣本接觸具有療效的一定數量的 10 提取物（樣本B)，將樣本A的增生情況與樣本B進行比較，樣 本A中抑制增生程度與樣本B相同或相似的任何製劑都是 抑制血管增生或内皮細胞生長的治療製劑。接觸方式可以 爲體外或體内兩種。 本項發明還涉及一整套針對宿主體内因病理性血管增 15 生引起的機能紊亂的治療方法，該方法是按照使用說明向 宿主施用具有某種療效的一定數量的提取物。 材料與方法 獲取方法 第1圖爲顯示從鯊魚胚胎軟骨組織中獲取抗血管新生 20 提取物的方法的示意圖。相關步驟包括：1)從鯊魚胚胎上 取出軟骨組織，2)對軟骨進行研磨使其釋放出軟骨細胞， 3)人工培養軟骨細胞，最好使用含有鯊魚血清的專用細胞 生長液，4)將抗血管新生提取物從人工培養的軟骨細胞和/ 或培養基中分離出來。 19 200423951 鯊魚屬於軟骨魚科動物，或稱爲軟骨魚，因爲其内骨 骼完全由軟骨組成。儘管本次實驗以鯊魚作爲軟骨細胞的 主要來源，但我們相信包括鯊魚、魟魚和縫魚在内的任何 軟骨魚都可以使用。屬於軟骨魚科的白斑角鯊、陰影絨毛 5 鯊和淺海長尾鯊都特別適宜獲取胚胎軟骨細胞，因爲它們 數量衆多，便於捕捉。 一旦它們胚胎的内骨骼發育完成，就可以作爲軟骨細 胞的重要來源。使用這些胚胎軟骨細胞的最佳時期需根據 物種不同而確定。白斑角鯊懷孕4至12個月期間爲使用其胚 10 胎的最佳時間，因爲從第四個月起胚胎的内骨骼就已經發 育完成。這種鯊魚的胚胎數量各不相同，每頭鯊魚可懷孕1 到10個胚胎。作爲卵生魚種，陰影絨毛鯊胚胎的發育期根 據水溫不同大約爲7.5到10個月左右。 對於白斑角鯊這樣的胎生鯊魚來說，胚胎將從懷孕的 15 鯊魚體内直接取出，最好是在消毒條件下使用保護罩進行 操作。不幸的是，目前這種獲取胚胎的方式常常導致母體 的死亡，這主要是出於同情憐憫的目的。如果是陰影絨毛 餐这樣的印生物種，打開受精印就可以獲得胚胎，不需要 犧牲母體的生命。 20 一旦獲得鯊魚胚胎，應將其内骨骼完整取出，並將骨 骼周圍的肉、皮膚和其他器官清理乾淨。内骨骼經過簡單 清理之後應放入消毒罩，以便進一步清潔和整理。 方案1 清理過的胚胎内骨骼應保存在含有羥乙基呱嗪-N-2乙 20 200423951 烧石K fee/氫氧化鈉、葡萄糖、氣化鈉、氣化鉀、鱗酸氫二鈉、 尿素和三甲胺N-氧化物的溶液中。更適宜儲存的溶液（以 下簡稱“溶液B”)應大致由11.92克pH值爲7.6的羥乙基,瓜嗓 县2乙燒磺酸/氫氧化鈉、7·1克葡萄糖、15.84克氯化鈉、〇 22 5克氣化鉀、0·81克磷酸氫二鈉、20克尿素和6克三甲胺队氧 化物組成。這樣的溶液可用來防止脫水，因爲鯊魚長期生 活在高鹽度環境下，因此其血液中含鹽濃度非常高。相信 二曱胺Ν-氧化物可以使鯊魚的酶在如此高鹽度環境下繼續 發揮作用（格羅根和朗德1990年同上文）。 胚胎内骨絡從保存溶液中取出後應在無菌的溶液Β中 漂洗乾淨。然後用刀將浸濕的内骨骼切成細微的小方塊， 並將其研磨成顆粒細微的糊狀，可以使用消過毒的研绰和 杵槌。這樣可以釋放出胚胎内骨骼的軟骨細胞。 使軟骨細胞懸浮在5毫升改良過的最佳最低必須培養 15 基(〇Pti-Mem)20E中，培養基中最好能夠含有來自鯊魚血清 (下文詳述）的專用細胞生長液。使用吸量管可將軟骨細胞混 入培養基。改良過的Opti-Mem 20E培養基成分更加適宜， 每500¾升培養基大約應當含有：20毫升10X 〇pti-Mem培養 基(可向美國GibcoBRL公司購買）；1.99克尿素；14.02克氯 20 化鈉；2毫升青黴素和鏈黴素，均爲每毫升500單位；2毫升 濃度爲7.5%的磷酸氫二鈉（這樣pH值可達到7.2);含有1.4 克三甲胺N-氧化物（氧化三甲胺）、2毫升200 mM谷氨酸鹽 和5到20毫升經加熱降低活性處理的鯊魚血清組成的專用 細胞生長液；以及將溶液稀釋至500毫升的二次蒸餾水。除 21 200423951 鯊魚血清之外，其他所有成分均可以向Gibc〇 BRI4uSigma 這樣的公司購買，並最好進行無菌過濾，然後存放在達到 組織培養等級的無菌玻璃器皿中。 專用細胞生長液中經加熱降低活性處理的鯊魚血清可 5以爲軟骨細胞提供2到3天的營養。氧化三甲胺可用來類比 咼鹽度水下生長環境中的正常生長條件（請參閱格羅根和 朗德1990年同上文），而且谷氨酸鹽也是細胞生長液中通常 使用的一種必不可少的氨基酸，它能夠促進胚胎軟骨細胞 的生長發育。鯊魚血清由鯊魚血液濾出的淋巴液（濾液）組 10成。圖2爲顯示獲取鯊魚血清方法的示意圖。用來過濾蓄# 血液的微孔篩檢程式孔徑爲100毫微米左右。過濾的目的是 清除所有細菌和真菌微粒以及衣原體，減少血色素和内毒 素數量，因爲這些物質都將對細胞生長液造成影響。也可 以使用孔徑達到50毫微米的更小的篩檢程式，以便進一步 15濾出血液中的所有病毒微粒。濾液在過濾之後需要經過離 心過濾才能獲得由血清組成的上層清液。然後將上層清液 加熱以便降低酶的活性，從而獲得加熱降低活性處理的鯊 魚血清。所需熱量根據不同類型的鯊魚而隨之變化。依照 最佳程式，使用淺海長尾鯊進行實驗時應將上層清液在68 2〇 C的溫度下加熱30分鐘左右。 相信使用鯊魚血清與奥哈拉使用的傳統胎牛血清（請 參閱奥哈拉1995年同上文第101頁）相比更能夠促進細胞生 長’因爲前者含有最適宜鯊魚軟骨細胞生長的天然營養成 刀和激素。鯊魚血清還含有一種甲狀旁腺激素相關蛋白 22 200423951 (PTHrp)，這種激素在軟骨細胞的繁殖和分化變異過程中具 有主要作用（請參閱J·格爾斯雷施特和j. Α·穆西克在 1998年7月17曰美國軟骨魚學會大會上的摘要和陳述以及 J·格爾斯雷施特和J· Α·穆西克1999年同上文）。使用胚胎 5鯊魚血清的效果優於成年鯊魚血清，因爲前者與胚胎軟骨 細胞的天然生長環境更爲相似，而且能夠提供更加適合軟 骨細胞生長的營養成分。 改良後的培養基和粘稠狀軟骨細胞糊應放置在康寧組 織培養24-培養盤上的各培養槽内，在二氧化碳濃度爲5%和 10 37°C溫度下培育24至48小時，直到軟骨細胞産生附著力。 如有必要，應使用真空抽取的方式將用過的培養基換成新 培養基(根據生長狀況通常每2到3天更換一次）。添加培養基 的數里要根據培養器皿的大小而定（組織培養盤上每個培 養槽大約0.5毫升，而組織培養瓶内則添加3至5毫升）。細胞 15生長過程中應定期查看其匯合程度（即培養盤表面完全被 細胞覆蓋時可通過掃描顯微鏡進行觀察）。培養過程中的第 一步是將軟骨細胞與培養基混合，在此之後大約1周時間内 即達到匯合程度。出現匯合現象（一個培養盤中每毫升增加 到108個細胞）時，加入胰島素解除細胞與盤底的附著。這此 20細胞便可以重復培養、分裂、儲存或重新使用、分析或進 一步提取並分離出血管新生抑制劑。 經過培養和胰蛋白酶化之後，軟骨細胞可以通過離心 分離（最好在4°C溫度下以每分鐘1000轉的速度旋轉5分鐘) 加以濃縮，分離出的固體物質可以重新懸浮在水中。重新 23 200423951 懸浮在水裏的這部分物質之後可以;東幹，以便除去更多的 水分，從而將軟骨細胞冷凍乾燥，以後可以根據需要重新 與水化合。以下將討論另外一種使用、分析或進一步提取 並分離出抗血管新生成分的程式。 5 爲了在胰蛋白酶化之後進行重復培養，細胞最好能夠 在二氧化碳濃度爲5%和37°C溫度下培育5分鐘，然後分裂 到不同的組織培養盤上。這時需要再次增加新的培養基以 便促進細胞生長。如有必要，該程式可重復進行。 從理論上說，一份胚胎鯊魚軟骨的樣本可以培養出無 10 限量的軟骨細胞，但這取決於該樣本上分離並生長出的細 胞是否具有強壯的生命力。有些細胞株的生命力要強于其 他同伴。 如果能夠發育出比較成功的細胞株，這份樣本應在-80 °C的低溫下速凍保存，以便將來再次使用，而其餘的細胞 15 可以繼續生長和使用。將細胞速凍也能夠保證在任何突發 情況下照常提供胚胎軟骨細胞。 爲了對某些細胞進行速凍保存，應當在細胞達到匯合 階段時通過胰蛋白酶化將其從培養基中取出，並且需要進 行離心過濾’然後與冷;東培養基混合。混合物經冷束後可 20以在_80至_90°C的低溫下長期保存。該冷凍培養基約含10% 的二甲基亞砜(DMSO)和90%的鯊魚血清。相信鯊魚血清在 重新培養冷凍細胞方面相當有益。整個冷凍過程需在冰點 溫度以下（〜〇。〇進行以保持細胞生命力。 爲防止污染，避免因操作不當導致人工培養的軟骨細 24 200423951 肊活動出現任何退化，最好儘快將半成品放置在代的溫度 下進灯保存。此外，整個過程都應儘量實現無菌化摔作以 避免污染。 w 木作以 耍的分離 5分離出的軟骨細胞和培養基㈣存在血管新生抑制 劑’因爲敕骨細胞也會分泌血管新生抑制劑到培養基裏。 經過=濾=分離’抑制劑只會以㈣kd的分餾物形式存 在，刀子ΐ約爲10 kd左右。抑制劑的成分包括含巧外碳 水化合物和4.5%蛋白質的蛋白多糖或糖蛋白、含有局部碳 10水化合物的俩角f蛋白、以及縮氨酸單^此外碳= 化合物與縮氨酸之間很可能形成队糖苦結合。相信抑制劑 的功效與其濃度有很大關係，而且對過高或過低的阳值非 常敏感。 第3A圖顯示了通過胰蛋白酶化和離心過濾（參見第1圖） 15將軟骨細胞從培養基中分離出之後從中提煉抑制劑的方 法。首先，分離出的軟骨細胞應進行細胞溶解以便釋放出 有效成分。完成這一步驟需要將細胞懸浮在濃度爲〇·5%_ι% 的Triton X-loo活化劑和磷酸緩衝液(pBS)(Sigm_司出品） 中，然後將溶液放在冰上5到15分鐘，再次進行離心過濾（在 20臺式離心分離機上以14,〇〇〇轉的速率轉動2分鐘）以便清除 液態提取物中的細胞殘骸。然後應對液態提取物進行分 餾，可以使用微孔過濾膜分離出1到l〇kd的分顧物。 第3B圖顯示了通過胰蛋白酶化和離心過濾（參見第丄圖） 將人工培養的軟骨細胞從培養基中分離出之後再從剩餘的 25 200423951 培養基中提煉抑制劑的方法。首先，需要再次對培養基進 行離心過濾（在4°c溫度下以1〇,〇〇〇轉的速率轉動30分鐘左 右）以便甩出凝結劑。然後使用〇·2微米的篩檢程式對立立養美 進行過濾消毒，並使用微孔過濾膜對其分餾，分離出工到 5 10kd的分餾物° 通過凝膠過濾層析技術可以進一步從1到10]^的分餾 物中分離出分子量爲10 kd的成分。首選的方法是將一個直 徑爲1·5釐米的玻璃管柱裝滿高度爲30釐米的Sephacryl S-300 HR丙烯葡聚糖凝膠(Sigma公司出品），並使用pBS或 1〇二次蒸餾水等缓衝液對其進行平衡。1毫克1至l〇kd的樣本 可以在2毫升同樣的緩衝液中溶解，將此樣本加入玻璃管 柱。每小時使用15毫升同樣的緩衝液即可實現洗提並獲得2 毫升的分餾成分。使用凝膠過濾分子量參照標準（Sigma& 司出品）對管柱進行校準。也可以使用鹽酸胍(GuHC1)(〜6m) 15作爲緩衝液。在這種情況下，獲得的分鶴物可以在二次蒸 德水中進行透析（1，〇〇〇 MW，Spectra 7)以便清除鹽分。 相信分子之間相互作用以形成多分子簇的聚合作用將 會阻止通過上述分餾和凝膠過濾所進行的分離。用來分離 出1至10kd分餾物的分餾操作將會導致該範圍内某些分子 20的丟失’因爲這些分子會産生聚合從而超出此範圍。分離 10kd成分也會導致聚合分子發生類似的丟失現象。在凝膠 過濾中使用鹽酸胍將有助於溶解這些聚合物。也可以在最 初1至10kd分子的分餾過程中使用鹽酸胍減少聚合現象。不 過相彳°鹽酸胍的作用過於強烈，會降低化合物的質量。 26 200423951 因此，蒸餾水和PBS應該是首選緩衝液。 培養胚胎鯊魚軟骨細胞與培養成年鯊魚軟骨細胞兩種方法 之間的對比 目前已發表的文獻中基本上介紹的都是從成年鯊魚軟 5 骨組織中培養軟骨細胞的方法。這種方法需要建立人工的 生長環境以便在軟骨細胞開始生長之前培養其附著力和滲 透力（請參閱奥哈拉1995年同上文、奥哈拉和王1996年同上 文）。根據奥哈拉的研究，使用瓊脂糖培養細胞的附著力需 要2到3周的時間，這也是一種最佳的人工生長環境。這些 10 細胞的倍增時間約爲60天，在此之後其生長速度會有所下 降(請參閱奥哈拉1995年同上文第49頁）。一旦生長速度出現 下降，含有這些細胞的瓊脂糖就必須倒入另外一個裝有新 鮮瓊脂糖的培養盤中。此外，使用瓊脂糖會增加細胞釋放 的難度(請參閱奥哈拉1995年同上文第53頁）。 15 另一方面，本項發明中培育的胚胎軟骨細胞不需要依 靠人工的生長環境使其能夠附著在組織培養器皿的内壁 上，而且其生長速度與成年鯊魚的軟骨細胞相比也顯著提 高（大約1周左右）。相信是因爲胚胎軟骨組織仍然處於生長 和發育階段。所以它還含有許多健康生長和發育所必需的 20 酶、生長激素和其他信號因數。而成年的軟骨組織已經發 育完成，處於相對平衡甚至逐漸退化的階段，所以它所含 有的這類因數從數量和種類上都無法同前者相比。此外， 使用含有鯊魚血清的專用細胞生長液相信也可以進一步提 高軟骨細胞的生長速度。 27 200423951 由於成年策魚的軟骨組織脆硬易碎，在處理過程中很 可能破壞軟骨細胞的完整性。成年震魚的軟骨組織需要用 刀切開之後使用㈣機進行研磨，而不應使料白和抑 棱。研磨後的軟骨組織須經溶解使其軟化至可以培養出個 5體細胞的程度。要達到這一目標，需要對軟骨組織施用含 有膠原酶的溶液(請參閱奥哈拉咖年同上文第耶）。根據 軟骨的大小可將其在溶液中浸泡3〇分鐘到12小時不等使 其可以充分溶解。然後對溶液進行離心過渡，分離出細胞 之後將溶液完全倒掉，將其餘的細胞微粒重新懸浮在改良 10 後的〇pti-Mem 20E培養基。 使用膠原酶等溶解製劑會産生幾個問題。首先，它有 可能破壞軟骨細胞的完整性並增加污染的風險。如果軟骨 組織在溶液中浸泡時間過長也會導致過度溶解。此外，增 加額外的設備、化學品和操作步驟也會使成本上升。不僅 15如此，成年軟骨組織較硬，所以最初開始清洗的時候更加 不便。 與此相反，胚胎軟骨組織不需要進行溶解，因爲它本 身就彳艮柔軟，具有出衆的延展性。只要通過簡單的切割並 使用研臼和杵槌進行研磨就足以釋放出軟骨細胞。 20 的A制劑治聲身患腫瘤^老鼠 根據梁1997年同上文中介紹的方法從成年長角鯊的軟 月組織提取物中分離出的1到10分餾物曾經在老鼠身上做 過實驗，這只老鼠在頸部長了一個顯眼的腫瘤，最初的直 &約爲3釐米。研究人員對它進行了爲期3個月的觀察，在 28 200423951 此期間這只老鼠喝的水裏每毫升含有1毫克提取物。 讀每毫升含有1毫克提取物的溶液最初在三個星期 内服用4毫升之後，腫瘤開始萎縮。經過4個星期，老鼠的 打爲和活動出現了明顯好轉，腫瘤變黑並繼續萎縮。8個星 5期之後，腫瘤與最初的大小相比已經萎縮了超過一半（直徑 約爲1.5釐米）。而經過1〇周的時間，腫瘤已經萎縮到〇.5羞 米，而且老氣的行爲和活動出現了顯著改善。在研究結束 的第13周，腫瘤依然在繼續萎縮，而老鼠則表現得非常活 躍。 10 15 本此研究的結果總結如下 --—-----一― I 在老鼠身上進行的其他實驗也顯示，服用成年裳魚軟 骨細胞提取物可以使老鼠的壽命最少延長12%。 胚胎装魚提取的製劑可以用於之前那些製劑使用的範 圍。不過新製劑在生產方面效率更高，而且更加實用，相 信新製劑所具有的抑制特性絕不低於之前使用的其他製劑。 抑制劑可以通過不同形式使用，包括靜脈注射i溶液 形式以及粉。可喊壯㈣料行治療的 病症包括癌症、實體腫瘤、風濕性關節炎、骨_ I、牛 皮癬、糖尿減網膜㈣、老年黃斑錢、血管再狹窄、 卡波西氏肉瘤、白血病、腫瘤轉移、良性腫瘤、遺傳性出 錄毛細血管擴張症、心肌血管新生、輯^管化、毛 29 20 200423951 細血管擴張、血友病關節病症、血管纖維瘤、創傷肉芽、 青光眼、角膜移植排斥、動脈硬化、硬皮病和瘢痕瘤等。 此外，抗血管新生製劑還可用於消炎、抗溶膠原、體内抗 腫瘤增生擴散、以及直接體外抗腫瘤增生擴散(請參閱杜邦 5 美國專利號6,028，118)。由於血管新生是胎兒發育的重要因 素，因此抗血管新生製劑也可能會用於節育（請參閱N·克 勞伯等人1997年《抗血管新生抑制劑可抑制女性生殖過程 中的一些重要因素》AGM-1470《國家醫學雜誌》第3(4)期 443-446頁）。 10 儘管上述發明已經通過說明和實例進行了詳細的描 述，但在這一領域内其他專業人士的眼裏，還需進一步的 優化和改進。因此，本文所做的描述和例證不應被視爲對 本項發明涉及範圍的限制，這一點我們也在附加的申請專 利中進行了必要的闡述。 15 【圖式簡單說明】 第1圖顯示從胚胎鯊魚軟骨中準備抗血管新生提取物 的方法。 第2圖顯示準備鯊魚血清的方法。 第3A及3B圖顯示從軟骨細胞及軟骨細胞培養基中分 20 離出血管新生抑制劑的方法。 【圖式之主要元件代表符號表】 (無） 30The field of gas technology belongs to the United States Code of Article 35 of the United States Code of Article 119 concerning the provisional application of the US domestic interim application 6 ^, 疋 'number is No. 60 / 246,258, the application is filed in November 2003 n pj 0a 'The content of the application was incorporated into this public disclosure through cross-reference search. Field of the Invention Θ / V and EndoI are means for σ silkworm antiangiogenesis drugs from cartilage tissue and chondrocytes of sturgeon. [Prior Art] and Background of the Invention & The basic process of new blood vessels forming new blood vessels. It is an important step in the microphysical phenomenon of the reproductive, developmental and wound healing departments. Under normal circumstances, 2 lives. However, there is a lack of regularity. Newborns also have multiple diseases including cancer, solid tumors, rheumatoid arthritis, lupus, diabetic dependent disease, macular degeneration in the elderly, Lu = stenosis, Kaposi's sarcoma, leukemia, tumor Metastases, benign tumors, hereditary hemorrhagic telangiectasias, myocardial angiogenesis, plaque 4 blood & telangiectasia, hemophilia joint disorders, hemangiofibroma, unknown granulation, glaucoma, corneal transplant rejection , Arteriosclerosis, scleroderma, keloid, inflammation, and fetal pregnancy, etc. For example, tumor growth and metastasis are completely dependent on angiogenesis. Most primary solid tumors go through a considerable period of angioplasty, here The growth of the tumor during this period is obviously in a dormant state. The maximum can only reach about 1-2 mm in diameter. After the tumor grows to this size, the tumor cells can absorb the necessary oxygen and other nutrients through simple passive diffusion. These can only be The tumor mass observed under the microscope will eventually “initiate” angiogenesis, and they use the mature host blood around them. Tubes give birth to new blood vessels and capillaries, and these blood vessels will grow in the direction of the tumor mass and eventually penetrate into it. This allows the tumor mass and blood metastasis to spread and spread the danger of disease to other parts of the body relentlessly. In rheumatoid arthritis, newly formed capillaries invade joints and destroy cartilage tissue. In diabetic retinopathy, new capillaries are formed in the retina, invade the vitreous body and blood, and eventually cause blindness (Falkman 1995, " Natural Medicine, 丄 [丄], pp. 27-31; Volkman and Craigsbrunn, Science Magazine, 235, pp. 442-447; D. A. Walsh Dating, Rheumatology, Deng [2], p. 03-m; Hillley et al., 1998, New Knowledge of Human Reproduction, 4 [5], pp. 736-740). ‘Bioinhibitors and tumors; ϋ Tumor growth can be limited or prevented by preventing tumor angiogenesis’, especially by direct targeting and generation of vascular endothelial cells that are not fully mature. This method can prevent the new spread of tumor mass, and make the beans continue to shrink to the diameter of millimeters in diameter, which is also the state that the swollen money survives without blood supply. Such a treatment can be called "induced dormancy, the purpose of which is to control the disease in a slow manner. The published literature has described in detail the many functions of fish cartilage in the medical field, including its role in angiogenesis. Aspects. Fish cartilage extract-sold directly as a nutritional supplement. But "The view that fish cartilage can effectively treat advanced cancer = = Xiao criticism (see Miller's 1998" Clinical Tumor γ, Zizi 3649 -3655 pages). '' Issue No. 16 from T. Fiber Center, Mortsea, Sarasota, Florida, found that there are three kinds of proteins that can be recovered from carp cartilage, which is believed to be a hindrance. The spread of tumors even causes them to gradually shrink, while avoiding the harmful side effects of radiation therapy on the human body ("Los Angeles Time ^ 'August 27, B3 Edition". T 1990 DuPont (US Patent No. 6,028,118) And Balassa (丨 Lian, Guoguo Patent Race 4,822,607) both described in detail one of the types found 1 " anti-vascular new peak and anti-inflammatory properties !, fish cartilage extract and extraction method It is used in treatment. However, neither of these two patents specifically describes the formation and molecular properties of its anti-angiogenic component. DuPont and Bab are directly extracted from cartilage: renewable resources. What they have obtained The solid or liquid extracts can only be regarded as "large heterozygous, immature rings of active substances, not specific compounds that have been isolated. According to Ms. Liang's research report, the extracted cartilage extract of her fish is different from the protein extracted by Ruhr, Gruber, DuPont, and Balassa, etc. It is believed to be a proteoglycan (see Zhan Media. Liang Niannian ^ "Isolation, classification and evaluation of angiogenesis inhibitors extracted from fish cartilage" [= Thesis] University of California Fresno). The extract has undergone endothelial: cytochemical assays to carry out a strong management of money. Extraction is carried out directly. The specific process is to use a steel punching tool to take the central part of the cartilage vertebral body, and then freeze-dry the punched circular part, and grind it into a powder with a mixer. It is best to use water for extraction (please (See Liang 1997, supra, pp. 9 and 34). However, sharks are a limited resource and difficult to obtain in large quantities. Therefore, it is often necessary to artificially culture shark chondrocytes so that they can be repeatedly cultured and obtained in large quantities. Grogan and Lande have made a slight breakthrough in this regard, and they successfully cultivated immune cells from sharks and catfish (Grogan and Lande 1990 "Cultivation System for Maintaining and Ensuring Shark and Catfish Immune Cell Division and Multiplication" , Published 10 in "Journal of Fish Biology" 36th pages 633-642). In addition, Hartman et al. Have also tried to cultivate a variety of shark tissues, but not cartilage (J. Hartman et al., 1992, "General Settings for Initial Culture of Cartilage Fish Cells", published in "In vitro Cell Development Biology" 28A No. 77-79). None of these attempts were successful. Although cells obtained from the organism's tissues began to show a certain growth, they failed to enter the mature stage and eventually failed to survive (see C. O'Hara, 1995 Angiogenesis Inhibitors Obtained from Cultured Shark Chondrocytes, "page 12 [Student Thesis] University of California Fresno). O'Hara and Wang have reported in detail the process of their isolation of an angiogenesis inhibitor in artificially cultured adult shark 20 fish chondrocytes and culture medium (see C. O'Hara 1995 in Wang 1996 "An angiogenesis inhibitor secreted by artificially cultured shark chondrocytes" (California State University Fresno). However, research has shown that 'artificially grown cells grow very slowly because chondrocytes cannot attach themselves to the inner wall of culture tissue 8 200423951 (see in vitro research group 1996 Journal of the In vitro Biology Society, Issue 32, p. 37, "About Oha Reports of Related Works by La and Wang, and O'Hara (1995, supra). To improve the ability of chondrocytes to attach, they used an artificial mother. Although this method promotes cell growth, it does not reach a practically usable density (see O'Hara 1995, supra, O'Hara and Wang 1996, supra, and In vitro Research Group 1996, supra) . According to these studies, "shark chondrocyte culture can produce certain inhibitory factors, but to date, successful cases of in vitro culture of shark chondrocytes have not yet appeared" (see O'Hara and Wang 1996 10 years and in vitro research groups Same as above). In addition, the cartilage of adult sharks is too brittle and brittle. It must therefore be broken down before culturing in order to release chondrocytes. During the dissociation process, cartilage needs to be immersed in a solution containing collagenase until decomposition occurs, and then the chondrocytes are separated from the solution by centrifugal filtration (please refer to Ohara 1995 and Ohara and Wang 1996 Ibid.) Text). The risk of this method is that it may damage the integrity of chondrocytes and increase the chance of external contamination. O'Hara proposed in his 1995 paper the use of shark embryos as a source of chondrocytes because doing so would reduce the degree of cell differentiation and variation by 20%. However, O'Hara himself did not provide any practical methods, nor did it involve any consequences that may result from changing the initial materials. [Summary of the Invention] Summary of the Invention The content of the present invention includes a 9 Γΐί extract with anti-angiogenesis and anti-inflammatory effects and an extraction method thereof. The extract was isolated from artificially cultured cartilage 2 ° chondrocyte culture medium, while the former came from Dangxinxin cell growth fluid. The extract contains approximately 2,000 mg of biological miscellaneous agent with a molecular weight of 1mGKD, a carbohydrate compound =% ′ and a protein of approximately 4. 5% 'active substance is contained in carbohydrates for extraction. First, remove 10 tr from cartilage fishes or their embryos, grind the suture bone to release the chondrocytes, artificially cultivate the cells The anti-angiogenesis extract is then separated from the cartilage culture and / or culture medium of the human health. Yes-point can not be ignored, the special growth solution used to culture right moon cells contains t, #, serum. The invention also includes a method for inhibiting the growth of endothelial cells, that is, sending out a quantitative extract to the cells to meet the requirements for growth inhibition. The present invention also provides a method for inhibiting the proliferation of blood vessels in the tissue, that is, sending an extract of 疋 = to the tissue to meet the requirement of 5 仏 _proliferation. Methods for treating various diseases such as cancer and arthritis are also mentioned here. The chondrocyte culture method described in the present invention does not require the use of an artificial mother body capable of promoting the attachment and growth of embryonic chondrocytes. Chondrocytes have a fixed ability to attach and can converge to form masses within a week. This is an unexpected result, because chondrocytes from adult cartilage fishes such as industry fish cannot be attached without the aid of artificial mothers, and the growth time will be doubled to 60 days after using artificial mothers, after which the growth rate will be doubled. Will slow down and require new bribes (please see O'Hara Janian and 10 200423951, page 49 above). In addition, we have developed a special cell growth fluid containing cartilage fish serum. The serum is filtered from the blood and contains the natural nutrients and hormones needed for optimal growth of chondrocytes, including a parathyroid hormone-associated protein (PTHrp), which is involved in the division, reproduction and differentiation of chondrocytes. Play an important role (see the abstracts and presentations of J. Gelsrest and J. A. Musik from the University of Guelph, Guelph, Canada, at the American Cartilage Society Congress on July 17, 1998 and (Statement and article by J. Gersrest and J. A. Musik, 1999, 10th issue of the Experimental Animal Semester, pages 284, 549-556). The use of cartilage tissue from embryonic cartilage fish instead of adult cartilage fish as a source of chondrocytes has more advantages. First, the cartilage tissue of adult chondrocytes requires collagenase to help break down (see O'Hara 1995 and p. 21 above), while embryonic chondrocytes do not require any enzymes. This will significantly reduce the risk of chondrocyte integrity being compromised or contamination of the culture process. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a method for preparing an antiangiogenic extract from embryonic shark cartilage. 20 Figure 2 shows how to prepare shark serum. Figures 3A and 3B show a method for separating an angiogenesis inhibitor from chondrocytes and chondrocyte culture medium. I: Detailed description of the preferred embodiment of the third embodiment 11 The various documents, patents, and issued materials cited in the inner valley of the heart cloth are indicated by citations, and the reader can see it at a glance. The internal bows of $ 1 use these documents, patents, and published patents for the purpose of achieving $ 7 accurate. ',,, a more complete description of the highest water involved in this invention, the use of molecular progenitors (including cell recombination technology) in microbes (unless otherwise specified), microbiology, cell biology, and biological water; None of the traditional technologies of free-lever have exceeded the current highest level of technological development. 4 The technology will be explained in detail and in the literature. 10 Related definitions The related terms have been defined in this article as follows. In the patent description and patent requirements, unless otherwise stated in the text, the singular forms include plural meanings. For example, 1 cell, though. Open 7 for the singular, but also include the plural meaning of the cell and the meaning of the mixed use of the singular and 15 plurals. 17 In this article, "contains," meaning that the ingredients and methods include both the elements listed in detail, and do not exclude other elements. When "consisting essentially of, used in the definition of ingredients and methods, Its meaning should not exclude other elements that have an important relationship with the compound. Therefore, the 20 constituents consisting essentially of the elements detailed herein should not exclude trace contaminants, phosphate buffers, preservatives, and other pharmacologically acceptable carriers from separation and purification methods. "Consisting of", the content excluded is not limited to the trace pollutants of other ingredients and the actual operating steps in the compounding process of this invention. The specific content represented by these term changes 12 200423951 is in this item Within the scope of the invention. All attribute values including acidity, temperature, time, concentration, and molecular weight are approximate values with an error of ± G1. Although not explicitly mentioned each time, all values are preceded by There are "about," words, and the reagents mentioned in this article have only typical characteristics, and the compounds are well known in this research. ^ "Isolated" means separately obtained from the composition, cells, and other components that naturally bind to the compound. Σ "" medium, can be singular or plural, it refers to a variety of 10 substances used for culture Cell growth compounds. "Individuals" or "hosts" are spinal secrets, preferably mammals or humans. Mammals include (but are not limited to) rodents, simians, humans, livestock, competitive animals, and pets. '' Cancer, and "Tumor, which can be used in the singular or plural, refers to cells that develop 15 malignant lesions and cause disease in the host body. Primary cancer cells (ie, cells obtained near the site of a malignant lesion) can be distinguished from non-cancer cells by advanced detection methods such as tissue examination. The definition of cancer cell referred to herein includes not only primary cancer cells, but also any cell derived from cancer cells, including transgenic and cancer cells, in vitro culture, and cell lines derived from cancer cells. Talking about 20 to some types of cancer usually means solid tumors, and "clinically detectable" tumors are those that can be detected by CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation based on the tumor mass. Tumors found in other medical treatments. However, the findings based on biochemical or immunological methods alone are not enough to meet the requirements of this definition. 13 200423951 In this context, "inhibition" refers to delaying, retarding or preventing the growth, reproduction or cell division of endothelial cells or the formation of blood vessels in tissues. Methods to monitor the efficacy of inhibition include (but are not limited to) endothelial cell proliferation tests, measurement of blood content and measurement of vascular bed volume, and quantitative determination of vascular structure density. When the culture material is a mixture of cells, monitoring the process of neovascularization needs to be accomplished by quantitatively measuring cells that can reflect specific indicators of endothelial cells. These indicators include angiogenesis factor, proteolytic enzymes, and specific cell degeneration molecules of endothelial cells Wait. "Synthetic" refers to the combination of an active agent with another inert (eg, discoverable formulation 10 or tracer) or active (eg, adjuvant) compound or ingredient. "Pharmaceutical ingredient" includes a combination of an active agent and an inert or active carrier, and makes the ingredient suitable for use in diagnosis or treatment in vitro or in vivo. In this context, the meaning of "pharmacologically acceptable carrier" includes phosphate buffered solution, water, emulsions (such as oil / water emulsions or water / oil emulsions), and various wetting agents. 15 Any standard pharmaceutical carrier. This ingredient should also include stabilizers and preservatives. For examples of carriers, stabilizers and preservatives, see Martin Remington, 1975, Pharmacological Standards, 15th Edition (Macmillan Publishing Company, Easton). "Effective amount" means an amount sufficient to have a beneficial or expected effect. For example, the number of treatments refers to the number that will achieve the desired effect. This number may be the same as or different from the amount that has a preventive effect, the latter being the amount necessary to prevent the onset or symptoms of the disease. An effective amount can be accomplished by one or more administrations or administrations. The term "cartilage fish" or "fish" referred to in this article refers to any cartilage fishes. 14 200423951 Sharks, catfishes, and sea fishes are common. The term “adult” used in this document for sharks or other chondrocytes refers to sharks or other chondrocytes that have given birth to larvae (ie, non-pregnant stages) when they have naturally given birth or are egg species. 5 English "chondrocyte" refers to chondrocytes. "Cartilage" refers to the entire endoskeleton of a cartilage fish. "Anti-angiogenesis" includes anti-inflammatory properties. The present invention relates to an antiangiogenic and anti-inflammatory extract and a method for obtaining such an extract. The extract can be obtained from chondrocytes and chondrocyte culture medium obtained by weaving the cartilage group of cartilage fish. The present invention also relates to a special cell growth fluid for culturing cells capable of producing a new extract. The extract contains a bioactive agent composed of proteoglycan and glycoprotein, and has a molecular weight of 1 to 10KD, of which the carbohydrate accounts for about 95. 5% and about 4. 5%, the active substance is contained in the carbohydrate 15. The molecular weight of the bioactive glycoprotein can also be about 10KD. The invention also relates to a method for obtaining an extract. The extract can be obtained from chondrocytes released from cartilage tissue of shark embryos or chondrocytes cultured in culture in culture medium. Bioactive ingredients are secreted from the chondrocytes into the culture medium and can then be isolated from the culture medium or artificially chondrocytes20. One thing that cannot be ignored is that the special growth fluid used to culture chondrocytes contains serum extracted from the cartilage fish blood. It can be known only through examples that the process of separating the required extracts includes centrifuging the culture medium to retain the supernatant, then using a screening program to sterilize the supernatant, and finally fractionating the supernatant to obtain 15 200423951 An extract having a molecular weight of about 1 to 10 KD is isolated. In the extraction process, the cartilage tissue is first removed from the cartilage fish or its embryo, the cartilage is ground to release the chondrocytes, the chondrocytes are cultured artificially, and the anti-angiogenesis extract is finally extracted from the artificially cultured cartilage 5 cells and And / or isolated from the culture medium. One thing that cannot be ignored is that the special growth fluid used to culture chondrocytes contains shark serum. Another solution is to isolate the desired extract from the cartilage tissue of the cartilage fish embryo. First, remove the cartilage tissue from the cartilage fish embryo, grind the cartilage to release the chondrocytes, and add 10% of the released chondrocytes to water. To paste, mix the thick chondrocyte paste into the culture medium and place the mixture on a tissue culture plate. It should be emphasized again that this protocol requires mixing a thick chondrocyte paste into the culture medium and placing the chondrocyte / medium mixture on a tissue culture plate for culture. 15 During the cultivation process, the chondrocyte / medium mixture needs to be placed in a carbon dioxide-rich environment in order to enhance the adhesion of the chondrocytes. When necessary, the chondrocyte / medium mixture needs to be removed from the cultivation environment and replaced with fresh medium. To facilitate the growth of chondrocytes. It should also be noted that the chondrocyte / medium mixture 20 should be placed in an environment with a carbon dioxide content of about 5% and a temperature of 37 ° C during the culture process, and the mixture should be left in this environment for about 24 to 48 hours. In the actual operation, the culture medium contains serum extracted from the cartilage fish blood. In another aspect, the extraction process also includes the release of soft 16 200423951 osteocyte / medium mixture from the culture plate and repeated culture of chondrocytes. In another aspect, the chondrocyte / medium mixture can be released from the culture plate during the extraction process and the mixture can be centrifuged to separate the culture medium from the chondrocytes. 5 On the one hand, chondrocytes need to be frozen and stored during extraction. Frozen storage includes the following main steps: mixing chondrocytes with frozen medium and freezing at a low temperature of -80 ° C to -90 ° C for about one day, and then storing the mixture of chondrocytes and frozen medium in liquid nitrogen for long-term storage. The frozen medium mixed with chondrocytes may contain serum extracted from the cartilage fish blood. The invention also relates to a chondrocyte culture medium of a chondrocyte, which contains serum extracted from the blood of the chondrocyte and is prepared from the embryo of the chondrocyte. It should be noted that the serum and chondrocytes should be from the same cartilage fish. For example, the serum and chondrocytes in this experiment were also derived from shark embryos. The serum preparation process involves first filtering the cartilage fish blood to separate the lymph fluid and filtering off the particles, then centrifuging the lymph fluid to retain the supernatant, and heating the supernatant to reduce the enzyme activity. An effective amount of trimethylamine N-oxide and glutamate can be added to the medium. 20 This invention also includes a method for inhibiting the growth of endothelial cells by administering a certain amount of extracts with growth-inhibiting effects to the cells. The invention also includes a method of inhibiting angiogenesis in a tissue by administering a certain amount of an extract having anti-angiogenic effect to the tissue. The active ingredient can be administered to an individual by oral, intravenous, intraperitoneal, or transdermal delivery 17. The extract can be processed and applied in liquid or powder form. It should be noted that the individual is an animal selected from a group of pets, livestock, or human patients. The invention also includes a method for treating a 5 dysfunction caused by angiogenesis in an individual, which method comprises administering to the individual a certain amount of extract. This type of physical dysfunction includes (but is not limited to) cancer, arthritis, skin diseases caused by angiogenesis, diabetic retinopathy, Kaposi's sarcoma, age-related macular degeneration, vascular restenosis, telangiectasia, glaucoma , Keloids, rejection of corneal transplants, traumatic granulation, patellar fibrils, quasi tumors, this type of hemorrhagic capillary telangiectasia, myocardial angiogenesis, and scleroderma. At the same time, however, the inhibition of angiogenesis can impede or inhibit fetal development in mammals during gestation. The development of rheumatoid arthritis is directly related to the proliferation of blood vessels in the cartilage tissue, which is directly responsible for the deterioration of the condition. In a 1998 article I5 published in Rheumatology, 38 (2), pages 103-112, Walsh pointed out that pathological angiogenesis is the main cause of the development of rheumatoid arthritis. We can imagine that quantitatively administering an effective amount of an angiogenesis inhibitor to the affected area can alleviate the symptoms of arthritis and may improve the condition of rheumatoid arthritis. 20 Some have suggested that both diabetic retinopathy and age-related macular degeneration can be effectively treated with angiogenesis inhibitors. It is well known that diabetic retinopathy is caused by malignant hyperplasia of blood vessels, which can lead to permanent blindness. Age-related macular degeneration is also associated with malignant angiogenesis ^ and also causes permanent vision loss. According to predictions, the use of angiogenesis inhibitors can prevent the onset and persistence of angiogenesis, and is an effective treatment for these two conditions. In patients infected with psoriasis, the symptoms of psoriasis are also necessarily related to expanding angiogenesis. We can imagine that increasing the effective amount of angiogenesis inhibitors will offset the adverse effects of this abnormal proliferation of blood vessels, thereby preventing the appearance of psoriasis symptoms. The invention also relates to a method for screening a therapeutic agent for inhibiting angiogenesis, which comprises contacting the agent with an appropriate cell or tissue sample (sample A), and then contacting another cell or tissue sample with a certain amount of 10 extracts having a curative effect (Sample B): Compare the proliferation of sample A with sample B. Any agent in sample A that has the same or similar degree of inhibition of proliferation as sample B is a therapeutic agent that inhibits vascular proliferation or endothelial cell growth. Contact can be in vitro or in vivo. The invention also relates to a set of treatment methods for the functional disorders caused by pathological angiogenesis in the host, which method is to administer a certain amount of extracts with certain curative effect to the host according to the instructions for use. Materials and methods Obtaining method Figure 1 is a schematic diagram showing a method for obtaining anti-angiogenesis 20 extract from shark embryo cartilage tissue. The relevant steps include: 1) removing the cartilage tissue from the shark embryo, 2) grinding the cartilage to release the chondrocytes, 3) artificially cultivating the chondrocytes, preferably using a special cell growth solution containing shark serum, 4) Angiogenesis extracts are isolated from cultured chondrocytes and / or culture media. 19 200423951 Sharks belong to the cartilage fish family, or cartilage fish, because their internal bones are composed entirely of cartilage. Although this experiment used sharks as the main source of chondrocytes, we believe that any cartilage fish, including sharks, catfish and sea fish, can be used. White-spotted sharks, shadow villi 5 sharks, and shallow sea sharks belonging to the chondridae family are particularly suitable for obtaining embryonic chondrocytes because they are numerous and easy to capture. Once their embryonic endoskeleton development is complete, they can serve as an important source of cartilage cells. The optimal period for using these embryonic chondrocytes depends on the species. The white spot shark is the best time to use 10 embryos from 4 to 12 months of pregnancy, as the embryo's endoskeleton has been developed from the fourth month. The number of embryos in this shark varies, and each shark can conceive between 1 and 10 embryos. As an oviparous fish species, the developmental period of shadow villi shark embryos is about 7. About 5 to 10 months. For a viviparous shark like a white spotted shark, the embryos will be taken directly from the pregnant 15 shark, and it is best to use a protective cover under sterile conditions. Unfortunately, the current method of obtaining embryos often results in maternal deaths, primarily for the purpose of compassion and compassion. If it is a printed species such as shadow villous meal, the embryo can be obtained by opening the fertilization print without sacrificing the life of the mother. 20 Once a shark embryo is obtained, its endoskeleton should be completely removed and the meat, skin, and other organs surrounding the bone should be cleaned up. The endoskeleton should be placed in a disinfection hood after a simple cleanup for further cleaning and organization. Scheme 1 The cleaned embryonic endoskeleton should be stored in a solution containing hydroxyethylxazine-N-2 ethyl 20 200423951 burnt stone K fee / sodium hydroxide, glucose, sodium gasification, potassium gasification, disodium hydrogen phosphate, urea And trimethylamine N-oxide solution. A more suitable solution for storage (hereinafter referred to as "solution B") should be approximately 11. 92 grams pH 7. 6 hydroxyethyl, guar county 2 ethanesulfonic acid / sodium hydroxide, 7.1 g glucose, 15. It consisted of 84 g of sodium chloride, 0 22 g of potassium gasification, 0.81 g of disodium hydrogen phosphate, 20 g of urea and 6 g of trimethylamine team oxide. Such a solution can be used to prevent dehydration, as sharks live in high salinity environments for a long time, so their blood has a very high salt concentration. Diamine N-oxide is believed to allow shark enzymes to continue to function in such high salinity environments (Grogan and Lande, 1990, supra). After the embryonic bones are removed from the preservation solution, they should be rinsed in sterile solution B. Then use a knife to cut the soaked endoskeleton into small cubes and grind it into a fine granular paste. You can use a sterile paste and pestle. This releases chondrocytes from the embryo's endoskeleton. Chondrocytes are suspended in 5 ml of a modified optimal minimum culture medium of 15 (OPti-Mem) 20E. The medium should preferably contain a special cell growth solution derived from shark serum (described in detail below). Chondrocytes can be mixed into the culture medium using a pipette. The composition of the modified Opti-Mem 20E medium is more suitable, and about 500 ¾ liters of medium should contain: 20 ml of 10X 〇pti-Mem medium (can be purchased from GibcoBRL, USA); 1. 99 grams of urea; 14. 02 grams of sodium chloride 20; 2 ml of penicillin and streptomycin, 500 units per ml; 2 ml at a concentration of 7. 5% disodium hydrogen phosphate (so that the pH can reach 7. 2); Contains 1. Dedicated cell growth solution consisting of 4 g of trimethylamine N-oxide (trimethylamine oxide), 2 ml of 200 mM glutamate, and 5 to 20 ml of heat-reduced shark serum; and a solution diluted to 500 ml of Distilled water. With the exception of 21 200423951 shark serum, all other ingredients can be purchased from companies such as Gibaco BRI4uSigma and are preferably sterile filtered and then stored in sterile glassware that has reached tissue culture grade. The shark serum treated with heat-reducing activity in special cell growth fluid can provide chondrocytes with nutrition for 2 to 3 days. Trimethylamine oxide can be used to simulate the normal growth conditions in the salinity underwater growth environment (see above, Grogan and Lande, 1990), and glutamate is also an essential component of cell growth fluids. Amino acids, which can promote the growth and development of embryonic chondrocytes. Shark serum is composed of 10% of lymph fluid (filtrate) filtered from shark blood. Fig. 2 is a schematic diagram showing a method for obtaining shark serum. The pore size of the micropore screening program used to filter the stored blood is about 100 nm. The purpose of filtration is to remove all bacterial and fungal particles and chlamydia, and reduce the amount of hemoglobin and endotoxin, as these substances will affect the cell growth fluid. A smaller screening procedure with a pore size of 50 nm can also be used to further filter out all virus particles in the blood. After filtration, the filtrate needs to be subjected to centrifugal filtration to obtain a supernatant composed of serum. The supernatant was then heated to reduce the activity of the enzyme, thereby obtaining a heat-reduced shark serum. The amount of heat required varies according to the type of shark. According to the best formula, when using shallow sea sharks for experiments, the supernatant should be heated at 68 2 ° C for about 30 minutes. It is believed that the use of shark serum can promote cell growth more than the traditional fetal bovine serum used by O'Hara (see O'Hara 1995 vs. page 101 above) because the former contains a natural nutrient-forming knife that is most suitable for the growth of shark chondrocytes And hormones. Shark serum also contains a parathyroid hormone-related protein 22 200423951 (PTHrp), a hormone that plays a major role in the proliferation and differentiation of chondrocytes (see J. Gersrester and j. A. Musik's summary and presentation at the American Cartilage Society Congress on July 17, 1998, and J. Gersrest and J. A. Musik, 1999, supra). The effect of using embryo 5 shark serum is better than adult shark serum, because the former is more similar to the natural growth environment of embryonic chondrocytes, and it can provide nutrients suitable for the growth of chondrocytes. The modified medium and viscous chondrocyte paste should be placed in each culture tank on Corning's tissue culture 24-culture dish, and incubated at a carbon dioxide concentration of 5% and a temperature of 10 37 ° C for 24 to 48 hours until the chondrocytes Generates adhesion. If necessary, the used medium should be replaced with a new one by vacuum extraction (usually every 2 to 3 days depending on the growth conditions). The number of medium additions depends on the size of the culture vessel (each culture tank on the tissue culture plate is about 0. 5 ml, and 3 to 5 ml in the tissue culture flask). During the growth of cells 15, the degree of confluence should be checked regularly (that is, when the surface of the culture plate is completely covered by cells, it can be observed through a scanning microscope). The first step in the culture process is to mix the chondrocytes with the culture medium, and confluence is reached in about 1 week after that. When confluence occurs (increasing to 108 cells per milliliter in a culture plate), add insulin to release the cells from the bottom of the plate. These 20 cells can then be repeatedly cultured, divided, stored or reused, analyzed or further extracted and isolated from the angiogenesis inhibitor. After culture and trypsinization, the chondrocytes can be concentrated by centrifugation (preferably at 5 ° C for 5 minutes at 4 ° C), and the separated solid can be resuspended in water. Re-2004200423951 This part of the substance suspended in water can be used later; dried in order to remove more water, so that the chondrocytes can be freeze-dried, and can be rehydrated with water later if needed. An alternative procedure for using, analyzing, or further extracting and isolating antiangiogenic components is discussed below. 5 For repeated cultures after trypsinization, cells should preferably be incubated for 5 minutes at 5% CO2 and 37 ° C, and then split into different tissue culture dishes. At this time, new medium needs to be added again to promote cell growth. The procedure can be repeated if necessary. In theory, a sample of embryonic shark cartilage can produce chondrocytes without a limit of 10, but this depends on whether the cells isolated and grown from the sample have strong vitality. Some cell lines are more viable than others. If a more successful cell line can be developed, this sample should be frozen and stored at -80 ° C for reuse in the future, while the remaining cells can continue to grow and be used. Freezing the cells will also ensure that embryonic chondrocytes are provided as usual in any emergency. In order to quickly freeze certain cells, they should be removed from the culture medium by trypsinization when they reach the confluent stage, and they need to be filtered by centrifugation 'and then mixed with cold medium. The mixture can be stored at a temperature of _80 to _90 ° C for a long time after being cold-stranded. This frozen medium contains approximately 10% dimethyl sulfoxide (DMSO) and 90% shark serum. Shark serum is believed to be quite beneficial in reculturing frozen cells. The entire freezing process needs to be performed below the freezing temperature (~ 0.〇) to maintain the vitality of the cells. To prevent contamination and avoid artificial cartilage fines caused by improper operation 24 200423951 任何 any degradation of activities, it is best to place the semi-finished products as soon as possible Store in a lamp at a temperature. In addition, the entire process should be aseptic as possible to avoid contamination. W Separation of wooden chondrocytes and culture media 5 Angiogenesis inhibitors exist because sacral cells will also Angiogenesis inhibitors are secreted into the culture medium. After = filtration = separation 'inhibitors only exist in the form of a fraction of ㈣kd, the knife ΐ is about 10 kd. The composition of the inhibitor includes extra carbohydrates and 4. Proteoglycans or glycoproteins of 5% protein, keratin f protein containing local carbohydrates, and peptides ^ In addition, carbon = compounds and peptides are likely to form a sugar-bitter binding. It is believed that the efficacy of an inhibitor is strongly related to its concentration and is very sensitive to excessively high or low positive values. Figure 3A shows a method for extracting inhibitors from trypsinization and centrifugation (see Figure 1) 15 after chondrocytes have been isolated from the culture medium. First, the isolated chondrocytes should be lysed to release the active ingredient. To complete this step, cells are suspended in Triton X-loo Activator and Phosphate Buffer (pBS) (produced by Sigm_) at a concentration of 0.5% _5%, and then the solution is placed on ice for 5 to 15 minutes Centrifuge again (rotate for 2 minutes at 14,000 rpm on a 20 benchtop centrifuge) to remove cell debris from the liquid extract. The liquid extract should then be fractionated, and 1 to 10 kd fractions can be separated using a microporous membrane. Figure 3B shows a method for extracting inhibitors from the remaining 25 200423951 culture medium by separating the cultured chondrocytes from the culture medium by trypsinization and centrifugation (see figure 丄). First, the culture medium needs to be subjected to centrifugal filtration again (rotating at 10,000 rpm for 30 minutes at 4 ° C) to shake off the coagulant. Then use a 0.2 micron screening program to filter and sterilize Li Li Yang Mei, and use a microporous filter membrane to fractionate it to separate 5 to 10 kd fractions. The gel filtration chromatography technology can be further used 10] ^ fraction was separated from the component with a molecular weight of 10 kd. The preferred method is to fill a 1.5 cm diameter glass tube column with a 30 cm height Sephacryl S-300 HR acryldextran gel (produced by Sigma), and use pBS or 10 secondary distilled water, etc. Buffer to equilibrate it. 1 mg of 1 to 10 kd sample can be dissolved in 2 ml of the same buffer. This sample is applied to a glass column. Elution is achieved with 15 ml of the same buffer per hour and 2 ml of fractionated fractions are obtained. The column was calibrated using a gel filtration molecular weight reference standard (produced by Sigma & Company). Guanidine hydrochloride (GuHC1) (~ 6m) 15 can also be used as a buffer. In this case, the obtained fractions can be dialyzed in secondary distilled water (1,000 MW, Spectra 7) to remove salts. It is believed that the polymerization of molecular interactions to form multi-molecular clusters will prevent the separation by the aforementioned fractionation and gel filtration. The fractionation operation used to separate the 1 to 10 kd fractions will result in the loss of certain molecules 20 in this range 'because these molecules will polymerize beyond this range. Separation of 10kd components will also cause similar loss of polymer molecules. The use of guanidine hydrochloride in gel filtration will help dissolve these polymers. It is also possible to use guanidine hydrochloride during the initial fractionation of 1 to 10 kd molecules to reduce polymerization. However, the effect of guanidine hydrochloride is too strong, which will reduce the quality of the compound. 26 200423951 Therefore, distilled water and PBS should be the preferred buffers. The comparison between two methods of culturing embryonic shark chondrocytes and adult shark chondrocytes. The published literature basically describes the method of culturing chondrocytes from the bone tissue of adult shark cartilage. This method requires the establishment of an artificial growth environment to cultivate the adhesion and permeability of chondrocytes before they begin to grow (see O'Hara 1995, supra, O'Hara and Wang 1996, supra). According to O'Hara's research, using agarose to cultivate cell adhesion requires 2 to 3 weeks, which is also an optimal artificial growth environment. The doubling time of these 10 cells is about 60 days, after which their growth rate will decrease (see O'Hara 1995, p. 49 above). Once the growth rate decreases, the agarose containing these cells must be poured into another culture dish containing fresh agarose. In addition, the use of agarose increases the difficulty of cell release (see O'Hara 1995, p. 53 above). 15 On the other hand, the embryonic chondrocytes cultivated in this invention do not need to rely on an artificial growth environment to enable them to attach to the inner wall of tissue culture vessels, and their growth rate is significantly higher than that of adult shark chondrocytes (approximately 1 week or so). This is believed to be because embryonic cartilage tissue is still in the growth and development stage. So it also contains many of the 20 enzymes, growth hormones and other signal factors necessary for healthy growth and development. Adult cartilage tissue has been developed and is in a relatively balanced or even gradual degrading stage. Therefore, the factors it contains cannot be compared with the former in terms of quantity and type. In addition, the use of a special cell growth solution containing shark serum is believed to further increase the growth rate of chondrocytes. 27 200423951 Because the cartilage tissue of adult fish is brittle, hard and brittle, it is likely to destroy the integrity of chondrocytes during processing. The cartilage tissue of adult quake fish needs to be cut with a knife after being cut with a knife, and should not be whitened and stiffened. After grinding, the cartilage tissue must be lysed to soften it to the extent that it can grow 5 somatic cells. To achieve this, a solution containing collagenase is required for cartilage tissue (see O'Halaka Nyan as above). Depending on the size of the cartilage, it can be soaked in the solution for 30 minutes to 12 hours so that it can be fully dissolved. Then the solution was subjected to centrifugal transition. After the cells were separated, the solution was completely discarded, and the remaining cell particles were resuspended in the modified 10pti-Mem 20E medium. The use of solubilizing preparations such as collagenase creates several problems. First, it has the potential to disrupt the integrity of chondrocytes and increase the risk of contamination. Excessive dissolution can also result if cartilage tissue is soaked in the solution for too long. In addition, the addition of additional equipment, chemicals, and operating procedures can increase costs. Not only that, but the adult cartilage tissue is harder, so it is more inconvenient when initially cleaning. In contrast, embryonic cartilage tissue does not need to be lysed because it is soft and has excellent ductility. Simply cutting and grinding with a mortar and pestle is sufficient to release chondrocytes. Preparation A of 20 cures tumors in mice ^ Rats 1 to 10 fractions isolated from soft moon tissue extracts of adult longhorn sharks according to the method described above by Liang in 1997 have been tested on mice. The mice developed a prominent tumor in the neck, initially straight & about 3 cm. Researchers observed it for a period of 3 months, during which time the mice drank 1 milligram of extract per milliliter of water. After a solution containing 1 mg of extract per ml was initially taken in 4 ml in three weeks, the tumor began to shrink. After 4 weeks, the rats' behavior and activity improved markedly, and the tumors became black and continued to shrink. After 8 stars, the tumor has shrunk by more than half (about 1. 5 cm). And after 10 weeks, the tumor has shrunk to 0. 5 years old, and there have been significant improvements in old-fashioned behavior and activities. By the 13th week of the end of the study, the tumors continued to shrink, and the mice were very active. 10 15 The results of this study are summarized as follows --------------- Other experiments performed on mice have also shown that taking adult chondrocyte chondrocyte extract can extend the life span of mice by at least 12%. Embryo fish extract preparations can be used in the range of those previously used. However, the new formulation is more efficient in production and more practical, and it is believed that the new formulation will never have lower inhibitory properties than other formulations used before. Inhibitors can be used in different forms, including intravenous solutions and powders. Disorders that can be treated include cancer, solid tumors, rheumatoid arthritis, bone I, psoriasis, diabetic diminished omentum, age-related macular degeneration, vascular restenosis, Kaposi's sarcoma, leukemia, tumor metastasis, Benign tumors, hereditary telangiectasias, myocardial angiogenesis, tuberculosis, hair 29 20 200423951 telangiectasias, hemophilia joint disorders, hemangiofibroma, wound granulation, glaucoma, corneal transplant rejection, arteriosclerosis , Scleroderma and keloids. In addition, anti-angiogenesis agents can be used for anti-inflammatory, anti-sologen, in vivo anti-tumor proliferation, and direct in vitro anti-tumor proliferation (see DuPont 5 US Patent No. 6,028,118). Since angiogenesis is an important factor in fetal development, anti-angiogenesis agents may also be used for birth control (see N. Klauber et al. 1997 "Anti-angiogenesis inhibitors can inhibit some important factors in female reproduction" AGM-1470, National Medical Journal, 3 (4), pp. 443-446). 10 Although the above invention has been described in detail through illustrations and examples, in the eyes of other professionals in this field, further optimization and improvement are needed. Therefore, the descriptions and illustrations made herein should not be considered as limiting the scope of the invention, and we have also made the necessary explanations in the attached patent application. 15 [Schematic description] Figure 1 shows a method for preparing an antiangiogenic extract from embryonic shark cartilage. Figure 2 shows how to prepare shark serum. Figures 3A and 3B show a method for separating angiogenesis inhibitors from chondrocytes and chondrocyte culture medium. [Representative symbol table for main elements of the diagram] (None) 30