TWI344963B - Clottable concentrate of platelet growth factors and preparation method thereof - Google Patents

Description

1344963 九、發明說明： 【發明所屬之技術領域】 本發明係關於血小板衍生物的領域，更特別是關於 從血小板得出之生長因子濃厚液的領域。本發明亦關於 製備這類生長因子濃厚液的方法，以及可凝結之血小板 生長因子濃厚液在治療性及/或美容性應用上的用途。 【先前技術】 指揮組織傷口療癒及組織再生的機制及路徑已有 非常詳細的研究，特別顯示出創傷後的細胞及分子活動 (event)結果大多會由體内不同組織共同承受。因此， 一般性的機制包括了早期及晚期發炎反應期、細胞之增 生及移動、血管新生、顆粒化組織形成及最後基質形成 及再塑（remodelling)。 有趣的是，這些反應過程（cascade)會在受傷後立 刻由血凝塊（cl〇t)的形成所啟動，其中前述血凝塊的 特徵是有相互交聯的纖維蛋白（fibrin)及多種蛋白，如 透明黏連蛋白（vitronectin )、纖維黏連蛋白（fibr〇nectin ) 及凝企酶敏感蛋白（thrombospondin)’而這個血凝塊會 預防再度流血，並作為入侵細胞的基質，同時提供一道 抵抗病原體入侵的障壁。此外，這個初始金凝塊可用來 儲存在療癒後期所需的細胞衍生物。 特定言之，當我們假定組織修復的所有時期都是由 許多因子、細胞激素及蛋白居中調控的，而它們是透過 和k跨細胞膜上的受器（reCept〇r)功能域（d〇main)真 接物理***互作用來調整細胞功能。之後，二次多 (secondary)之訊息轉換子（transducer)則控制細胞朽 5 1344963 的多方面的生物反應（subcellular biology )。雖然目前與 組織再生有關的機制和成分所扮演的角色只有一部份 被詮釋出來’但大多數的潛在優點則已被證實，特別是 在血小板衍生物的優點，尤其是血小板衍生之生長因子 (platelet-derived growth factor )方面的優點。 眾所皆知，血小板衍生之生長因子會展現出趨化性 及細胞***之性質，且顯現出其與軟組織及硬組織的療 癒與再生（如趨化性、細胞增生、血管新生、細胞外基 質沉澱及重塑）及增強細胞增生有直接的相關。進一步 來說，血小板衍生物和一些密切相關之生物功能也有間 接的關聯，例如由旁觀細胞（bystander cell)如纖維母 細胞、巨噬細胞、上皮細胞或淋巴細胞等刺激趨化因子 (chemokine)及細胞激素的產生。 因此，富含血小板之製劑、以及血小板凝膠、黏膠 及其釋出物在單獨使用或與移植生物材料合##用姑1344963 IX. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to the field of platelet derivatives, and more particularly to the field of growth factor thick fluids derived from platelets. The invention also relates to methods of preparing such growth factor concentrates, and to the use of clotting platelet growth factor concentrates in therapeutic and/or cosmetic applications. [Prior Art] The mechanism and path of directing tissue healing and tissue regeneration have been studied in great detail, and it has been shown that most of the cellular and molecular event results after trauma are shared by different tissues in the body. Thus, general mechanisms include early and late inflammatory response periods, cell growth and migration, angiogenesis, granulated tissue formation, and finally matrix formation and remodelling. Interestingly, these cascades are initiated immediately by the formation of a blood clot (cl〇t), which is characterized by fibrin and multiple proteins that crosslink each other. Such as vitronectin, fibrectin, and thrombospondin, and this blood clot prevents re-bleeding and acts as a substrate for invading cells while providing a resistance The barrier to pathogen invasion. In addition, this initial gold clot can be used to store the cellular derivatives needed in the later stages of healing. In particular, when we assume that all stages of tissue repair are centrally regulated by a number of factors, cytokines, and proteins, they are trans-receptors (reCept〇r) functional domains (d〇main) across the cell membrane. Real physical interactions to adjust cell function. Later, the secondary message transducer controls the multicellular biology of cell decay 5 1344963. Although only a portion of the roles and mechanisms involved in tissue regeneration are currently being interpreted', most of the potential benefits have been demonstrated, particularly in the advantages of platelet derivatives, especially platelet-derived growth factors ( Platelet-derived growth factor) advantages. It is well known that platelet-derived growth factors exhibit chemotaxis and cell division properties and exhibit healing and regeneration with soft tissues and hard tissues (eg chemotaxis, cell proliferation, angiogenesis, extracellular) There is a direct correlation between matrix deposition and remodeling) and enhanced cell proliferation. Further, platelet derivatives are also indirectly related to some closely related biological functions, such as chemokine by bystander cells such as fibroblasts, macrophages, epithelial cells or lymphocytes. Production of cytokines. Therefore, platelet-rich preparations, as well as platelet gels, viscose and their eliminators, are used alone or in combination with transplanted biomaterials.

再生及傷口療癒方面逐漸増力〇 注意和支持 ❸避UxWvo)擴增及分化 内皮細胞及纖維母細胞增生 3寺別的關注，因此它在軟骨 拿力σ的治療優點也逐漸受到 6 1344963 局部血·小板源製劑（如凝膠、黏膠及釋出物）之製 - 備通常係使血小板濃厚液與能夠引發血小板顆粒内容 ‘ 物釋出的活化劑混合，以模擬血小板之生理活化情形。 活化步驟一般係藉由直接加入外源性凝血酶、或透過氯 化鈣（CaCls)的使用來進行，這會抵消在收集血液或血 - 小板期間加入抗凝血劑的效應’而引發血液凝集連鎖反 應（coagulation cascade)，並釋出内源性凝血酶。内源 . 性及外源性凝血酶也會誘發纖維蛋白原（fibrinogen)之 聚合作用，並形成以纖維蛋白為主的生物材料（血凝 鲁 塊），從而導致血小板内媪含的多種捧合物（multifaceted blend)如多種血小板生長因子的釋出。 近來已開發出新的研究方法，從習知表現系統中生 產重組血小板生長因子，如Regranex (人類pDGF_bb ) • ( Janssen Cilag Internat.)。然而，可用的重組生長因子 在數量上仍然相當地少，一部份是因為這些生長因子在 分離、鑑定、選殖及表現上的難度。進一步來說，由於 我巧可=觀察到多種生長因子合併的協同效果，因此，' 相較於單一重組因子的使用，血小板源製劑仍提供了一 φ 種補充性的優點。 包含血小板或血小板源之既有製劑的主要缺點之 * 一是這類製劑缺乏合適的標準化及定義，這會讓具有多 種=療效果的富含血小板之產物特徵產生變動。用血小 板濃度來預估血小板衍生物中生長因子之量的這項簡 單假設確實是不明確的，因為用以收集及/或活化血小板 之方法的技術會影響生長因子之含量及所得之臨床效 果。所彳于產物之異質性（heterogeneity )可能取決於下 列不同參數的差異：如血小板濃度、起始材料中的白血 球、起始材料之種類、存放時間、儲存條件、以及活化 的方式。因此，這些變因顯然會引起產物間的重大差 7 ^44963 異’之後並產生生物性質及臨床效度（如療癒力）上的 區隔。 舉例來說’將包含完整血小板之溶液/膠直接送到傷 口的時候’血小板的活化係與纖維蛋白血凝塊之快速形 成，同樣的，也和血小板的不完全溶解有關。是以， ^ 1之完整血小板仍然被包於纖維蛋白血凝塊内 部’所以很難去測定真正之生長因子釋出量。 t進二步來說’當血小板凝膠及/或生長因子製劑係藉 由凝血酶活化同時使血小板激發而得到的，故當血小板 知生，產生時，纖維蛋白原已完全耗盡，不再是可凝結 的狀態’因此，需在送到傷口位置之前與外源性之天然 或合成產物混合。Regeneration and wound healing gradually gradually pay attention to and support the avoidance of UxWvo) amplification and differentiation of endothelial cells and fibroblast proliferation 3 temples, so its therapeutic advantages in cartilage σ are gradually affected by 6 1344963 local blood • Preparation of small plate source preparations (such as gels, gels, and eliminators) - usually by mixing platelet concentrate with an activator that triggers the release of platelet granules to mimic the physiological activation of platelets. The activation step is generally carried out by the direct addition of exogenous thrombin or by the use of calcium chloride (CaCls), which counteracts the effect of adding anticoagulant during blood or blood-small plate collection, which triggers blood agglutination. A coagulation cascade and release of endogenous thrombin. Endogenous. Sexual and exogenous thrombin also induces the polymerization of fibrinogen and forms fibrin-based biomaterials (blood clots), resulting in multiple combinations of platelet inclusions. Multifaceted blends such as the release of various platelet growth factors. Recently, new research methods have been developed to produce recombinant platelet growth factors such as Regranex (human pDGF_bb) (Jassen Cilag Internat.) from a conventional expression system. However, the available recombinant growth factors are still relatively small in number, in part because of the difficulty in isolation, identification, colonization and performance of these growth factors. Further, since I was able to observe the synergistic effect of the combination of multiple growth factors, the platelet source preparation still provided a supplemental advantage of φ compared to the use of a single recombinant factor. The main drawbacks of existing formulations containing platelets or platelet sources are that one of these formulations lacks proper standardization and definition, which can result in variations in the characteristics of platelet-rich products with multiple therapeutic effects. The simple hypothesis of using platelet concentrations to estimate the amount of growth factors in platelet derivatives is indeed unclear, as the techniques used to collect and/or activate platelets affect the growth factor content and the resulting clinical effects. The heterogeneity of the product may depend on differences in the following parameters: platelet concentration, white blood cells in the starting material, type of starting material, storage time, storage conditions, and manner of activation. Therefore, these variables apparently cause significant differences between products 7 ^ 44963 after the occurrence of the biological properties and clinical validity (such as healing power). For example, when a solution/glue containing intact platelets is directly delivered to the wound, the activation of the platelets is rapidly formed with the fibrin clot, and, similarly, with the incomplete dissolution of the platelets. Therefore, the complete platelets of ^ 1 are still encapsulated inside the fibrin clots' so it is difficult to determine the true release of growth factors. In the second step, when the platelet gel and/or growth factor preparation is activated by thrombin activation and platelet stimulation, when the platelets are known, the fibrinogen is completely depleted, no longer The clotting state 'Thus, it needs to be mixed with exogenous natural or synthetic products before being sent to the wound site.

進「步來說，多數的治療性局部血小板衍生物係由 =體來源進行製備。也就是說，自體血小板濃厚液係由 w者在=術前幾小時或幾天所捐的血液進行製備，而用 =重點照護（p〇int_0f_care)。因此它們大多用於需要固 疋體積之血小板凝膠的計劃性手術，以及用於健康可捐 血的患者身上。此外，在手術部位製造自體製劑是有宜 缺點的，因為它們常常是在控财良的條件下進行，^ 缺乏應有的標準化來確保生長因子釋出及其臨床效度。 a ，吾人對允許提前製備血小板衍生物方法有發 上特1 是在標準化條件下提前製備血小板生书 ，子濃厚液的情形，如此可配製出—種量身定做的靠 劑，而用於各種特殊的生理病理狀況。 步來說，既有的血小板源產物的另—項重要海 Sir然/有完整血液細胞或重要血液細胞碎片 對捐贈者抗原之免疫反應。針4:異 8 1344963 應可能會引起嚴重的後果’如同輸金反應觀察到 象，並包括患者的自體免疫及溶血併發症。、 進一步來說，既有的血小板源產物的另一項重大缺 點是使用生物^物必然會有的相關感染風險。儘管在且 有南度發展之管理環境及血液收集服務的國家中，以 代技術來測試單一捐贈者之人類同種異體血小 液的安全性確實很高，儘管標準血庫方法（如血小又 離術（apheresis)或由全血得出的血小板製劑） 二 菌製備條件的可能，但是，使用Α小板衍生物時仍、^ 能發生病毒及細菌傳染。因此，血小板衍生物及血小板 生長因子的病毒無害性（inn〇cuity)是極為重要的， 部使用之同種異體血小板製劑有理想的病毒安 最後，老年人口及慢性疾病的增加成了一項重 =滋長㈣康問題’這在^的面 雖然目前已有多種治療性處理，包括例如手2面對：： Ϊ:生Ϊί補植，組織傷口 “及組 在密集研^，^濟之成分的需求仍在逐漸增長。 用後文所t ’發明人驚奇地發現，這些目的可以 成之。可凝結之血小板生長因子濃厚液及其製 衍生簡單、快速且有效的製備血小板 多數人i疋’可從個人血小板濃厚液或來自 、小板》辰厚液（pool)得到可凝紝之血丨缸4 長因子濃厚液。更特宗〜^㈣了小板生 較之下，本發日Λ方所屬領域之其他方法相 收，至少顯增加所有生長因子的回 別的θ生具=重要之血小板生長因子的回收’且更特 疋生㈣子PDGF、卿似EGF的回收。 9 進-步來說，本發明之方法可溶解脂質膜，所以會 將脂貝套膜病毒及其他病原體（如細8及寄生蟲，如原 蟲）去活化，並可移除在起始血小板濃厚液中存在的血 漿及血小板脂質。 是以，本發明之方法使之可能提供一種病毒去活化 的Ί~凝、、π之ΑλΙ、板生長目子濃厚液，其可被有效地標準 化，而用於治療性處理。 臨床上在靜脈内使用血小板來矯治數量性或功能 性血小板數罝低下症（thr〇mb〇cyt〇penia)時，由於血小 板的保存期限是5或7天（部分是因為細菌污染的風 險），所以每年通常都會廢棄大量保存時間大於5或7 天的血小板單位。在允許使用過期血小板儲存液來製備 血小板源生長因子濃厚液的情況下，本發明之方法最後 揭示了一種極具有經濟目標的願景。 【發明内容】 本發明之一目的是一種可凝結之血小板生長因子 濃厚液，其係用於治療性及/或美容性用途。 在一較佳實施態樣中，前述可凝結之企小板生長因 子濃厚液包含生長因子PDGF、TGF-β、IGF、EC5F、 CTGF、bFGF 及 VEGF。 在另一較佳實施態樣中，前述可凝結之血小板生長 因子濃厚液不會誘發jk液細胞相關之輸血反應。 在另一較佳實施態樣中，前述可凝結之血小板生長 因子濃厚液包含至少一種選自由纖維黏連蛋白、透明黏 連蛋白、凝血酶敏感蛋白、凡威勒伯氏因子（v〇nIn the "step", most of the therapeutic topical platelet derivatives are prepared from the source of the body. That is, the autologous platelet thick solution is prepared from the blood donated by the person in the hours or days before surgery. And use = focus care (p〇int_0f_care). Therefore, they are mostly used for planned surgery that requires a solid volume of platelet gel, as well as for patients who are healthy donors. In addition, the preparation of autologous preparations at the surgical site is There are disadvantages, because they are often carried out under good financial conditions, ^ lack of standardization to ensure growth factor release and its clinical validity. a, we have the method of allowing the preparation of platelet derivatives in advance Special 1 is the case of preparing platelet raw books and sub-concentrated liquids in advance under standardized conditions, so that a tailor-made agent can be prepared for various special physiological and pathological conditions. Step by step, existing platelets The other source of the source product is the important sea Sir Ran / there is an immune response of the intact blood cells or important blood cell debris to the donor antigen. Pin 4: X 8 1344963 may cause The serious consequences 'as observed in the gold-receiving reaction, and include the patient's autoimmune and hemolysis complications. Further, another major drawback of the existing platelet-derived products is the inevitable use of biological substances. Risk of related infections. Although in countries with a developmental management environment and blood collection services in the South, the safety of human allogeneic blood sap using a single donor to test a single donor is indeed high, despite standard blood bank methods (eg Apheresis or platelet preparation derived from whole blood. It is possible to prepare conditions for the second bacteria. However, when using a small plate derivative, virus and bacterial infection can occur. Therefore, platelet derivatives and The virus innocuousness of platelet growth factor is extremely important. The allogeneic platelet preparation used in the Ministry has the ideal virus. Finally, the increase in the elderly population and chronic diseases has become a serious problem. Although there are a variety of therapeutic treatments on the surface of the ^, including, for example, the hand 2 face:: Ϊ: Ϊ Ϊ 补 replantation, tissue wounds and groups intensive research The demand for ^, ^ Jizhi ingredients is still growing. The inventors have surprisingly discovered that these objectives can be achieved. Condensable platelet growth factor thick solution and its preparation are simple, rapid and effective for the preparation of platelets. Most people can obtain clotting blood from individual platelet thick liquid or from small plate. Cylinder 4 long factor thick liquid. More special 宗~^(4) Compared with Xiaobansheng, other methods in the field of this Λ日Λ方, at least significantly increase the growth of all growth factors θ 生==Recovery of important platelet growth factor' More special health (four) sub-PDGF, Qing-like EGF recovery. In the case of 9-step, the method of the present invention dissolves the lipid membrane, so the lipocapsid virus and other pathogens (such as fine 8 and parasites such as protozoa) are deactivated and the platelets in the starting platelet can be removed. Plasma and platelet lipids present in concentrated liquids. Therefore, the method of the present invention makes it possible to provide a virus deactivated Ί 凝, π Α Ι Ι, plate growth target concentrate, which can be effectively standardized for therapeutic treatment. Clinically, when platelet is used intravenously to treat quantitative or functional platelet count hypothyroidism (thr〇mb〇cyt〇penia), since the shelf life of platelets is 5 or 7 days (partly because of the risk of bacterial contamination), Therefore, a large number of platelet units with a storage time greater than 5 or 7 days are usually discarded each year. In the case where an expired platelet stock solution is allowed to prepare a platelet-derived growth factor concentrate, the method of the present invention finally reveals a vision of a very economical goal. SUMMARY OF THE INVENTION One object of the present invention is a clotting platelet growth factor concentrate for therapeutic and/or cosmetic use. In a preferred embodiment, the condensable platelet growth factor concentrate comprises growth factors PDGF, TGF-β, IGF, EC5F, CTGF, bFGF and VEGF. In another preferred embodiment, the aforementioned clotting platelet growth factor concentrate does not induce a transfusion reaction associated with jk fluid cells. In another preferred embodiment, the clotting platelet growth factor concentrate comprises at least one selected from the group consisting of fibronectin, transparent adhesion protein, thrombin sensitive protein, and van derberg factor (v〇n).

Willebrand factor)與第 Π、V、VII、Vin、IX、X 及 XI 1344963 凝血因子所組成之組群的蛋白。 本發明之另一目的是一種製備本 血小板生長因子濃厚液的方法，其可/旋結之 小板濃厚液與溶劑及/或清潔劑接觸 二=T ·使血 圍内之pH值及在從2°C至50ec銘R…主乃乂υ I巳 培養至少5分鐘至6小時的時間共同 45°c範圍内之严声推耔.u从丄役佳為在從25 c至 式來移除前述溶或清_肖油萃取及/或層析方 在一較佳實施態樣中，用於本發 恤類、具有不同St: 類所組成之組群；錢佳為三正丁基碟酸醋Willebrand factor) is a group of proteins consisting of 凝血, V, VII, Vin, IX, X and XI 1344963 coagulation factors. Another object of the present invention is a method for preparing the present platelet growth factor thick solution, which can be used to contact the solvent and/or detergent in a thick plated liquid. 2 ° C to 50 ec Ming R... Main 乂υ I 巳 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少 至少In the preferred embodiment, the above-mentioned solution or clear oil extraction and/or chromatography is used in the present invention, and has a group consisting of different St: classes; Qian Jia is a tri-n-butyl acid vinegar

传撰佳實施祕中，用於本發明之方法的清潔劑 類1pa:i丨曰肪酸之、聚氧乙烯衍生物、山梨糖醇酐之偏S 其甜菜^ e/te=)、_子性清潔劑、去氧膽酸鋼及續 $類（sulfGbetaines)所組叙組群；且又更佳為 址群 45 ' ™tGn X_1GG及T_ 80所組成之 劑及/在^射1於轉3狀紐的前述溶 積％潔劑的個別最終濃度範圍為從〇·2至5體 濃厚液t麟為從02至2體積0/〇，其係以前述起始血小板 ί^ί之ff為基礎計算。在本發明之方法的較佳實施 ΐ 起始血小板濃厚液倾單用之2%Tnm>接 血小ΤΐΐΒΡ及1% THt〇n Χ·45接觸，其係以前述 血j板;辰厚液之體積為基礎計算。 荦蓉在本發明之較佳實施態樣中，油萃取係以醫 /、 、 /來進行，且前述油之用量為從2至20重量％、 11 1344963 或從5至15重量％、或從5至1〇重量％，其係以前述 t ΐ板濃厚液與前述溶劑及/或清潔劑之混合物的重量 為基礎計算。 f另一較佳實施態樣中’層析方式係使用如包含 C18二氧化矽襞填材料、或SDR (溶劑_清潔劑移除，In the method of the present invention, the detergents used in the method of the present invention 1pa: i fatty acid, polyoxyethylene derivatives, sorbitan partial S, beet ^ e / te =), _ sub Sex cleansers, deoxycholic acid steel and sulphide group (sulfGbetaines) group; and better group of 45 'TMtGn X_1GG and T_ 80 and / in ^ ^ 1 turn 3 The individual final concentration range of the aforementioned dissolution % detergent is from 〇·2 to 5 body thick liquid t Lin is from 02 to 2 volume 0/〇, which is based on the aforementioned starting platelet ί^ί ff Calculation. In the preferred embodiment of the method of the present invention, the starting platelet thick solution is used for 2% Tnm> blood transfusion and 1% THt〇n Χ·45 contact, which is the same as the blood plate; Volume based calculation. In the preferred embodiment of the present invention, the oil extraction is carried out in the form of medicinal /, /, and the foregoing oil is used in an amount of from 2 to 20% by weight, 11 1344963 or from 5 to 15% by weight, or from 5 to 1% by weight, based on the weight of the mixture of the aforementioned t-plate thick liquid and the aforementioned solvent and/or detergent. In another preferred embodiment, the chromatographic method is used, for example, comprising a C18 ceria filler material, or SDR (solvent_cleaner removal,

Solvent-Detergent removal) hyperD 來移除前述溶劑及/ 或清潔劑。 在—較佳實施態樣中，本發明之方法進一步包含一 步驟，使用10至75_nm孔徑之濾膜或類似之病毒移除 膜對所得可凝結之血小板生長因子濃厚液進行奈米過 濾。 ’、 在一較佳實施態樣中，本發明之方法包含一製備起 ，血小板濃厚液之初步步驟，前述起始血小板濃厚液係 藉由血小板分離術或血沉棕黃層（buffy-coat)分離法從 全血加以製備，且係為新鮮或過期且以液態儲存、或過 期且冷凍儲存的狀態。 〆 本發明之另一目的是一種形成A凝塊的方法，其係 將本發明或藉由本發明之方法所得的血小板生長^子 >辰厚液與凝企酶（thrombin)混合。較佳為，在前述形 成血凝塊的方法中所使用之凝血酶係得自人類。在一特 別實施態樣中’係將0.1至1體積之活性範圍為從2〇 IU/ml至1〇〇〇 iu/ml的凝血酶與1體積之本發明或藉由 本發明之方法所得的可凝結之如小板生長因子濃厚液 混合。 本發明之另一目的是一種醫藥產物或人工鷹架，其 包含本發明或藉由本發明之方法所得的可凝結之'如小 板生長因子濃厚液。 u 12 本發明之另一目的是本發明或藉由本發明之方法 所知的可破結之血小板生長因子濃厚液的用途，其係用 於形成血凝塊、或用於骨骼再生或傷口療癒、或^於活 體外研究（h v办Ό)或活體研究（ex Wv0)之細胞培養\ 【實施方式】 本發明之一目的是一種可凝結之血小板生長因子 濃厚液’其係用於治療性及/或美容性用途。 在本發明中所使用之「可凝結」一詞係指本發明或 藉由本發明之方法所得的血小板生長因子濃厚液包含 纖維蛋白原（fibrinogen)及第XIII凝血因子，故當有 冶療性應用需求時，其可與合適的活化劑（如凝血酶 混合而生成血凝塊。 在一特別實施態财，本發明之可凝結之血小板生 長因子濃厚液中的纖維蛋白原濃度較佳為高於丨 “更佳為高於L5g/L濃厚液’又更佳為高於2·5二 =旱液，且其第ΧΠΙ因子濃度較佳為高於G.5 lU/ml、、農 ==高於π51·濃厚液，又更佳為高㈣ μ在所使狀「美雜用途」的說法係指-種 種體表部分接觸的處理方式，特別是與皮 黏膜接觸&曱、嘴唇、外生殖11官、或與牙齒及口腔 =觸，而使之清潔芳香、並有保護及改變的作用， 或使之維持在良好的狀態。 在本發明中所使用之「治療性用途 種治療性或預防性處理方式來處理人類及 匕 症’其係透過醫藥、免疫或代謝效用來回復'修正= 13 1344963 變其生理功能。 . 本發明或藉由本發明之方法所得的可凝結之血小 板生長因子濃厚液特別適用於處理人類及/或動物的病 症、及/或用於與其身體之體表部分接觸，這是因為它已 被清除溶劑及清潔劑兩者，立具有病毒安全性。 y ' 「清除〉谷劑及/或清潔劑J的說法係指可凝結之金小 板生長因子濃厚液中的溶劑及/或清潔劑量極低，且較佳 為其量無法偵測。確實，所屬領域具有通常知識者已知 φ 溶劑及/或清潔劑濃度升高與長期毒性是有直接連結 的，且特別是與神經性病症的發生有直接連結（如揭示 於：J.P.R. Pelletier，S. Transue，E.L. Snyder. Pathogen inactivation techniques. Best Practice & Research ClinicalSolvent-Detergent removal) hyperD to remove the aforementioned solvents and/or cleaners. In a preferred embodiment, the method of the present invention further comprises the step of subjecting the resulting clotable platelet growth factor concentrate to nanofiltration using a 10 to 75 mm aperture filter or similar virus removal membrane. In a preferred embodiment, the method of the present invention comprises a preliminary step of preparing a platelet thick solution which is separated by platelet separation or buffy-coat. The method is prepared from whole blood and is in a state of being fresh or expired and stored in a liquid state, or expired and stored frozen. Another object of the present invention is a method for forming an A clot which is obtained by mixing the platelet growth and the thickening solution obtained by the method of the present invention with a thrombin. Preferably, the thrombin used in the aforementioned method of forming a blood clot is obtained from a human. In a particular embodiment, the amount of 0.1 to 1 volume of activity is from 2 〇 IU/ml to 1 〇〇〇 iu/ml of thrombin and 1 volume of the invention or obtained by the method of the invention. Condensed as a small plate growth factor thick liquid mixed. Another object of the invention is a pharmaceutical product or artificial scaffold comprising a condensable <RTIgt; platelet growth factor thick liquid of the invention or obtained by the method of the invention. U 12 Another object of the invention is the use of a cleavable platelet growth factor thick solution of the invention or by the method of the invention for forming a blood clot, or for bone regeneration or wound healing Or in vitro study (hv) or in vivo study (ex Wv0) cell culture \ [Embodiment] One of the objects of the present invention is a clotting platelet growth factor thick liquid, which is used for therapeutic and / or cosmetic use. The term "coagulation" as used in the present invention means that the platelet growth factor thick liquid obtained by the present invention or by the method of the present invention contains fibrinogen and XIII coagulation factor, so that there is a therapeutic application. When required, it can be mixed with a suitable activator (such as thrombin to form a blood clot. In a particular embodiment, the fibrinogen concentration in the clotting platelet growth factor concentrate of the present invention is preferably higher than丨 “Better than L5g/L thick liquid” is better than 2·5 2 = dry liquid, and its ΧΠΙ factor concentration is preferably higher than G.5 lU/ml, agriculture == high In the case of π51·dense liquid, it is more preferable that it is high (four) μ in the form of “beauty use” refers to the treatment of contact with various body parts, especially contact with the skin mucosa & 曱 嘴唇, lips, external reproduction 11 official, or with the teeth and mouth = touch, so that it is clean and aromatic, and has the effect of protection and change, or to maintain it in a good state. "Therapeutic use of therapeutic use or prevention in the present invention" Sexual treatment to deal with humans and snoring Recovering 'correction= 13 1344963 by medicinal, immunological or metabolic effects to change its physiological function. The coagulated platelet growth factor concentrate obtained by the present invention or by the method of the present invention is particularly suitable for treating human and/or animal diseases, And/or for contact with the body part of the body, because it has been cleaned of both solvent and detergent, and is virally safe. y ' "Clearing" granules and / or detergent J means The solvent and/or cleaning dose in the condensable gold plate growth factor concentrate is extremely low, and preferably the amount cannot be detected. Indeed, it is known to those skilled in the art that the concentration of φ solvent and/or detergent is increased. High and long-term toxicity are directly linked, and in particular directly linked to the occurrence of neurological disorders (as disclosed in: JPR Pelletier, S. Transue, EL Snyder. Pathogen inactivation techniques. Best Practice & Research Clinical

Haematology Vol. 19, No. 1，pp. 205-242, 2006)。 • 故本發明中「清除溶劑」的說法係指該溶劑之量小 於100 ppm ’較佳為小於5〇 ppm，更佳為小於2〇 ppm， 又更佳為小於10 ppm、小於5 ppm，且再更佳為小於i ppm ° • 進一步來說，本發明中「清除清潔劑」的說法係指 清潔劑之量小於500 ppm，較佳為小於25〇 ppm，更佳 為小於lOOppm，又更佳為小於5〇ppm，且再更佳為小 於 10 ppm。 「病毒安全性」的說法係指<凝結之血小板生長 子濃厚液實質上不帶有感染性病毒，特別是不帶有含脂 質之病毒’舉例來說，如人類免疫不全病毒（HIV)、二 型肝炎病秦⑽v)、c型肝炎病毒（HCV)、西 毒（WNV)、TT病毒、登革熱病毒、巨細胞病毒（CMV)、 Epstein Barr 赫（EBV)、人類祕病毒_8 (hhv 8)、 u 1344963 猿猴泡沫病毒 '嚴重急性呼吸道症候群病毒（SARs冠 • 狀病毒）以及其他脂質套膜肝炎病毒、巨細胞病毒、乳 酸脫氫酶病毒、疱疹群病毒（Herpes group virus)、棒狀 病毒（rhabdovirus)、白血病病毒（leuk〇virus)、黏液病 毒、α病毒、蟲媒病毒（arbovirus )、副黏液病毒、沙狀 • 病毒（arenavirus)及冠狀病毒。「實質上不帶有」係指 可凝結之血·小板生長因子濃厚液之病毒去活化程度至 少大於4 log10’這是強力病毒減毒步驟所定出的標準（由 鲁 歐洲醫藥產物評量機構（European Agency for the Evaluation of Medicinal Products，EMEA)的專利醫藥產 物委員會（Committee for proprietary medicinal products，CPMP)在病毒批核研究基準（Note f〇r guidance on virus validation studies)定出，參考文獻 • CPMP/BWP/268/95)，且更佳為大於5 log1()或更進一步 大於6 log1() ’因此，它不大可能會在輸血給患者時造成 脂質套膜病毒的血源性感染。 本發明或藉由本發明之方法所得的可凝結之血小 • 板生長因子濃厚液進一步包含下列功能性生長因子：血 小板源生長因子（PDGF)，其為A及B鏈之異二聚體及 . /或 A-A 及/或 B-B 鏈之同二聚體（PDGF-AA、PDGF-AB、 PDGF-BB);轉形生長因子（TGF-β)超級家族，其特別 ' 包含TGF-βΙ及/或TGF-P2及/或骨形態發生蛋白（bone moi*phogenetic protein，BMP )，·胰島素樣生長因子 (IGF );表皮生長因子（EGF );結締組織生長因子 (CTGF);基層纖維母細胞生長因子（bFGF)及血管内 皮生長因子（VEGF )。 在一特別實施態樣中，本發明之可凝結之血小板生 15 1344963 長因子濃厚液令的PDGF濃度係高於9〇ng/ml濃厚液， 杈佳為南於100 ng/ml濃厚液，更佳為高於12〇 ng/ml 濃厚液，又更佳為高於150 ng/ml濃厚液，再更佳為高 於180 ng/ml濃厚液，且又再更佳為高於25〇吨編濃厚 液0 在一特別實施態樣中，本發明之可凝結之血小板生 長因子濃厚液中的TGF-βΙ濃度係高於1〇〇 ng/ml濃厚 液’較佳為高於140叩/1111濃厚液，更佳為高於16〇11§/1111Haematology Vol. 19, No. 1, pp. 205-242, 2006). The term "clearing solvent" in the present invention means that the amount of the solvent is less than 100 ppm', preferably less than 5 〇 ppm, more preferably less than 2 〇 ppm, still more preferably less than 10 ppm, less than 5 ppm, and More preferably, it is less than i ppm ° • Further, the term "clearing the cleaning agent" in the present invention means that the amount of the cleaning agent is less than 500 ppm, preferably less than 25 〇 ppm, more preferably less than 100 ppm, and even more preferably It is less than 5 〇 ppm, and even more preferably less than 10 ppm. "Virus safety" means that the "condensed platelet growth agent thick liquid is substantially free of infectious virus, especially without a lipid-containing virus", such as human immunodeficiency virus (HIV), for example. Hepatitis B virus (10) v), hepatitis C virus (HCV), western poison (WNV), TT virus, dengue virus, cytomegalovirus (CMV), Epstein Barr (EBV), human secret virus _8 (hhv 8 ), u 1344963 Colobus foam virus 'Severe Acute Respiratory Syndrome Virus (SARs Coronavirus) and other lipid envelope hepatitis viruses, cytomegalovirus, lactate dehydrogenase virus, Herpes group virus, baculovirus (rhabdovirus), leuk virus (leuk〇virus), mucus virus, alphavirus, arbovirus, paramucus virus, arenavirus, and coronavirus. "Substantially without" means that the degree of virus deactivation of the condensable blood and small plate growth factor thick solution is at least greater than 4 log10'. This is the standard set by the attenuating step of the strong virus (by the European Pharmaceutical Products Assessment Agency) (Committee for proprietary medicinal products (CPMP) (European Agency for the Evaluation of Medicinal Products, EMEA) is defined in the Note f〇r guidance on virus validation studies, references • CPMP /BWP/268/95), and more preferably greater than 5 log1 () or further greater than 6 log1 () ' Therefore, it is unlikely to cause a blood-borne infection of the lipid envelope virus when transfused to a patient. The condensable blood platelet growth factor concentrate obtained by the method of the present invention or the method of the present invention further comprises the following functional growth factors: platelet-derived growth factor (PDGF), which is a heterodimer of A and B chains and. / or homodimer of AA and / or BB chain (PDGF-AA, PDGF-AB, PDGF-BB); Transgenic growth factor (TGF-β) superfamily, which specifically contains TGF-βΙ and/or TGF -P2 and/or bone morphogenetic protein (BMP), insulin-like growth factor (IGF); epidermal growth factor (EGF); connective tissue growth factor (CTGF); primary fibroblast growth factor ( bFGF) and vascular endothelial growth factor (VEGF). In a special embodiment, the condensable platelet of the present invention has a PDGF concentration of 15 1344963 long-factor thick solution higher than 9 ng/ml thick solution, and is preferably about 100 ng/ml thick liquid, more Preferably, it is higher than 12〇ng/ml thick liquid, and more preferably higher than 150 ng/ml thick liquid, more preferably higher than 180 ng/ml thick liquid, and even more preferably higher than 25 tons Thick liquid 0 In a special embodiment, the concentration of TGF-βΙ in the condensable platelet growth factor thick solution of the present invention is higher than 1 ng/ml thick liquid 'preferably higher than 140 叩 / 1111 thick Liquid, more preferably higher than 16〇11§/1111

濃厚液，又更佳為高於180 ng/ml濃厚液，再更佳為高 於250 ng/ml濃厚液。 在一特別實施態樣中，本發明之可凝結之血小板生 長因子濃厚液中的IGF濃度係至少與起始血小板濃厚液 中者類似’且較佳為高於65 ng/ml濃厚液，更佳為高於 75 ng/ml濃厚液，又更佳為高於8〇 ng/ml濃厚液，再更 佳為高於100 ng/ml濃厚液。The thick liquid is more preferably a concentrated liquid higher than 180 ng/ml, and even more preferably a thick liquid higher than 250 ng/ml. In a particular embodiment, the concentration of IGF in the clotting platelet growth factor concentrate of the present invention is at least similar to that of the starting platelet concentrate and is preferably greater than 65 ng/ml thick, more preferably It is more than 75 ng/ml thick liquid, more preferably higher than 8 ng/ml thick liquid, and even more preferably higher than 100 ng/ml thick liquid.

在一特別實施態樣中，本發明之可凝結之血小板 長因子濃厚液中的ECT濃錢高於G.5 ng/ml濃厚液， 較佳為高於lng/ml濃厚液，更佳為高於15吨/如濃 液，又更佳為高於2ng/ml濃厚液，且再更佳為高於25 ng/ml濃厚液。 在一特別實施態樣中，本發明或藉由本發明之方法 所得的可凝結之血小板生長因子濃厚液適用於 脂質含量的病症。 、而受低 本發明所使用之「需要低脂質含量的病症」的說法 係指本發明或藉由本發明之方法所得的可凝梦之灰 板生長因子浪厚液係已除去脂質（depleted迅'；； 亦即，在本發明或藉由本發明之方法所得的可凝結之血 16 3 : T濃厚液中’膽固醇、三酸甘油酯、hdl及 於其始血小板濃厚液中之量， 質量極低以=小板源生長因子濃厚液中的脂 用於特Ϊ的治療性用途，舉例來說，如 病症的^ ^症風險㈣患者、或用於高心臟風險之 量的合=因咸為、’：已接受治療的患者提供極低 ^ "大大減少由動脈及靜脈系統中脂肪斑 (Plaq=)形成所引起之血管阻塞的風險。 社之Πί本發明或藉由本發明之方法所得的可凝 子濃厚液巾，膽轉的量係低於⑽ g可凝、，Ό之血小板生長因子濃厚液，較佳為低於5〇 —凝結之===:液且更佳為低於 結之生本!可凝 丨OOmg/dl可凝結之血小板生長因液= : = 5 〇 mg/dl可凝結之血小板生長之車乂佳為低於 於3 —凝結…、板=以、夜且更佳為低 結之得的可凝 mg/cU可凝結之μ板生㈣子濃厚液，低於30 mg/dl可凝結之血小板生長因子濃厚液，且〗·=於15 10mg/d〗可凝結之血小板生長因子濃厚液。更盒為低於 更佳者，在本發明或藉由本發明 結之血小板生㈣子濃厚咖 17 1344963 mg/dl可凝結之血小板生長因子濃厚液，較佳為低於％ mg/dl可凝結之血小板生長因子濃厚液，更佳為低於2〇 mg/dl可凝結之血小板生長因子濃厚液，且又更佳為低 於5 mg/dl可凝結之血小板生長因子濃厚液。 進一步來說，本發明或藉由本發明之方法所得的可 凝結之血小板生長因子濃厚液中的脂質量非常低及/或 無法偵測，故有較佳之穩定性，當大規模純化血小板生 長因子時’會使該方法的進行較為容易。 在特別實加態樣中，本發明或藉由本發明之方法 所得的可凝結之血小板生長因子濃厚液不會引起血液 細胞相關之輸血反應。「血液細胞相關之輸血反應」係 指這種可凝結之生長因子濃厚液不帶有完整的活細胞 (例如紅血球、血小板及白血球），故可避免在患者身 上引起一义範圍之已知輸反應，包括免疫（如異體免 疫(Alloimmunizaticm))及溶血性併發症（如參見Str〇ncek et Rebulla，“Platelet transfusions”，The Lancet，August 4, 2007; vol‘ 370 : 427-438)。確實，免疫能力正常的受贈 者通¥會對捐贈者之血液細胞抗原表現出免疫反應，而 引起多種取決於相關血液細胞及特異抗原的臨床結 果。最普遍的相關抗原係選自下列種類··（”第一型 HLA，係血小板及白血球共有者；⑺第二型HLA，在某 些白血球有表現者；（3)顆粒性白a球特異抗原；（4)血小 板特異抗原（舉例來說，如人類血小板抗原ΉρΑ);以 及（5)紅血球特異抗原。 更特定言之’在本發明之製備方法中，活的血液細 ，（紅血球、白血球及血小板）會被溶劑_清潔劑摧毁/ 溶胞，從而減少（且更佳為預防）患者暴露於外來抗原 1344963 到的u、板3 mu法可從由患者得 小板:製^凝結之血二生長因子以種異… 所得的可中’本發明或藉由本發明之方法 少一種選自由連Λ長I子濃厚液進一步包含至 感蛋白及/或第π (、疑血虫論^透明黏連蛋白、凝血酶敏 VIII ^ IX ^ (P-thrombin) ) . V . VII . 組群的蛋自。凝血因子及凡威勒伯氏因子所組成之 施態樣中，本發明之可凝結之血小板生 ㈣連蛋㈣濃度料為高於〇2 g/L /辰厗液，且更佳為約〇.3 g/L濃厚液。 · 生長板源 rc佳為高於。.一濃厚液:且更佳= 別實施態樣中，本發明之可凝結之血小板生 長因子：厚液中第vm凝血因子及/或凡威勒伯氏因子 〇.9IU/mU農厚液，且又更佳為約丨m/ml濃厚液為回於 在另一實施態樣巾，藉由本發明之方法所得的可凝 ===厚液進-步包含免疫球蛋白，如 在-特別實施態樣中，本發明之可凝結之血小板生 長因子濃厚液中免疫球蛋白IgG的濃度較佳為高於5 g/L濃厚液，且更佳為約1〇 g/L濃厚液。 在另-實施態樣中，本發明或藉由本發明之方法所 19 1344963 得的可凝結之血小板生長因子濃厚液進一步包含至少 • 一種選自由下列各項所組成之組群的生長因子：cCN家 族成員、結締組織活化蛋白-3 (CTAP-3)、PF4、血小板 源血管新生因子（PDAF)、内皮細胞生長抑制劑、早期 懷孕因子（early pregnancy fact〇r，EPF )、表皮生長抑制 . 劑（EG]〇、角質細胞生長因子（KGF)、血管生成素樣 蛋白-6 (ANGPTL6)、IGFBP-3、***受器相關蛋白、 纖維母細胞源内皮細胞生長因子（f_ECGF)、肝細胞生 • 長因子（HGF)、組織肢釋出因子、人類膠原蛋白酶抑制 劑、血小板抗菌蛋白-1 (PMP-1)、經凝血酶誘發之血小 板抗菌蛋白-1 ( t-PMP )、凝血肽· 1 ( throinbocidin-1，TC1) 及凝血肽-2 (thrombocidin-2，TC2)。 在另一實施態樣中，本發明或藉由本發明之方法所 • 得的可凝結之血小板生長因子濃厚液進一步包含至少 一種選自由下列各項所組成之組群的蛋白：血清素、組 織蛋白酶（cathepsin)、白蛋白、血小板鹼性蛋白_pBp (CXCL7)、嗜中性球活化蛋白_2及_4 (NAP-2、-4)、 _ 體抑素（somat〇statin ’ SST)、RANTES、CTAP-3、胎盤 蛋白 14(PP14)、SCUBE卜膜聯蛋白 U (annexinlia)i - 熱休克蛋白27 ( HSP27)、及熱休克蛋白60 (HSP60)。 • 在一特別實施態樣中’本發明之可凝結之血小板生 長因子濃厚液中的白蛋白濃度較佳為高於3〇 g/L濃厚 液。 在另一貫施態樣中，本發明或藉由本發明之方法所 付的可滅結之血小板生長因子濃厚液進一步包含至少 一種選自由下列各項所組成之組群的酶：膠原蛋白酶、 超氧化物歧化酶（SUperoxide dismutase ’ SOD )、肝素酶、 20 金屬蛋白酶MMP-卜-2、-9、-13、自泌性ERK ( ext.Ceil reg. kinase)、自泌性及旁泌性蛋白C (PC)、以及微量 的酶如醛縮酶（aldolase)、羧基胜肽酶、酸性磷酸酶、 芳基硫酸酯酶（arylsulphatase)、β-半乳糖苷酶、β-葡萄 糖酸·酸酶、β-甘油磷_酸酶（P_glycer〇lph〇sphatase)、oc/β-匍萄糖苦酶、α/β-岩藻糖普酶（a/p_fUc〇sidase )、α-甘露 糖苷酶（α-mannosidase ) 及α-***糖苷酶 (α-arabinosidase) ° 在另一實施態樣中’本發明或藉由本發明之方法所 得的可凝結之血小板生長因子濃厚液進一步包含組織 胺、ADAMTS-13、α1-α2抗胰蛋白酶、α2-抗胞漿素 (a2-antiplasmin )、α2_ 巨球蛋白、ci-INH、誘發性 ΒΜΡ-2、-6、-7 ( TGF-β超級家族）、ECM 再塑因子（ECM remodelling factor ’ 已誘發之 ΜΜΡ、TNF-α、彈性蛋白 酶......）、自泌性及旁泌性溶血磷脂酸（lysophosphatidic acid ’ LPA )、HMGB1 (兩性調節蛋白（amphiregulin))、 ATP、ADP、GPT、GDP、Ca2+、Mg2+及/或 Zn2+。 本發明之另一目的是一種醫藥產物，其包含本發明 或藉由本發明之方法所得的可凝結之血小板生長因子 濃厚液。 「醫樂產物」係指金小板凝膠（plateiet gei)、血小 板黏膠（platelet glue)、富含生長因子之纖維蛋白黏膠 及/或密封劑（sealant)、人工鷹架。 本發明之另一目的是本發明或藉由本發明之方法 所得的可凝結之血小板生長因子在培養基中的用途，其 中如述培養基係適用於纖維母細胞、軟骨細胞、成骨細 胞、角質細胞、幹細胞及/或移植細胞之活體外或活體研 21 究的培養。本發明之另—目 胞、軟骨細胞、成骨細胞、角種適用於纖維母細 細胞之活體外或活體研究=紀、幹細胞及/或移植 發明或藉由本發明之；;錢，且其包含本 因子。 女所件的可凝結之血小板生長 小板濃厚液麟紐财/ 值及在從2。。至5^^ ^ ^ 分鐘至6小時的時間，更佳為在從25 柄ϋ C _ ^之溫度進行；以及藉由醇取及/或層 析方式來移除前述溶劑及/或清潔劑。 在較佳貫把態樣中，進行培養的時間範圍為從2 至4小時，其係於生理1)11值下進行，如在從pH7〇至 ΡΗ 7·5範圍内之1>11值（當起始血小板為新鮮的血小板 時）、或在從ΡΗ 6.8至8.2範圍内之ρΗ值（當起始血小 f為過期及/或冷/東企小板時）進行。有利的是，培養溫 度為約31°C。 在本發明之方法中所使用的合適溶劑為二_或三烷 ，碟醆酯類，如三-(η-丁基)磷酸酯、三-(t-丁基)磷酸酯、 〜已基)填酸酯、三_(2_乙基己基)磷酸酯、三_(n_癸基) 曰、二_(n_丁基)填酸醋、二_(t-丁基)碟暖醋、二_(n_ 己基)磷酸酯、二-(2-乙基己基)磷酸酯、二-(I癸基)磷酸 略及具有不同烷基鏈之二烷基磷酸酯類。可使用具有不 同燒基鏈之二或三烷基磷酸酯類，例如二-(II-丁基)磷酸 乙酷。特佳為三烷基磷酸酯為三_(n- 丁基）磷酸酯 22 (ΤηΒΡ)〇 在本發明之士·、4· 酸之聚氧乙稀衍^中=用的合適清潔劑包括脂肪 屯Γτ 0 物'山梨糖醇酐之偏酯類（例如品名 馬 iween 80 丨 / , βΛχ χ Γ 、也稱為「聚山梨醇醋80 (polysorbate 14 ii Aweel20 j ^ ^#) (^ $「τ .口口販"的氧乙基化之烷基酚：「Triton Χ-100」 或 Iriton X-4S 、、仏 ^ _ 」）。進一步考慮的清潔劑是去氧膽酸鈉 菜驗類，如N-十二基-n，n-二曱基-2鲁1-乙烧 % ^ ( N-d〇decyl-N,N-dimethyl-2-ammonio-l-ethane SUlph〇nate )。特佳清潔劑為「Triton X-45」、「Triton X-100」及「Tween 80」。 、在本發明之特別實施態樣中，起始血小板濃厚液係 與洛劑及清潔劑共同培養，較佳為與TnBp及THt〇n X-45共同培養。 &有利的是，前述溶劑或前述清潔劑之最終濃度、或 前述溶劑及前述清潔劑的個別最終濃度係包含於從〇. 2 至^體積内，較佳為包含於從G 2至2體積％ =範圍内=其係以前述血小板濃厚液之體積為基礎計 算。在-1父佳實施態樣中’前述起始血小板濃厚液係與 2% ΤηΒΡ共同培養。在另—較佳實施態樣中，前述起始 血小板濃厚液係與1% ΤηΒΡ及l% Triton χ_45共同培 養。 ^前述溶劑及/或清潔劑可藉由以醫藥等級油進行油 萃取及域其财法（如f柱層析）*從生物性流體中萃 出，如此則，厚液中的溶劑及/或清潔劑已被除去。 前述醫藥等級油可以是天然油（例如從植物或動物 萃取出來的油）或結構類似的合成化合物。合適的天然 23 1344963 油包括麻油（castor oil，或稱rjcinus 〇ii)、大豆、由、 葵花油、棉籽油。較佳之合成化合物為合成性三酸甘由 酯。合適的合成性三酸甘油酯範例包括三捽 (triolein )、三硬脂精（tristearin )、三棕櫚= (tripgf !?ltln)、二肉苴蔻精（trimyristin)、及其合併物。 醫藥等級油的量為可萃取至少8 〇 %脂溶性製程化學 物，（process chemical)的量，所用油量為從2至&In a special embodiment, the ECT rich in the clotting platelet long-factor thick solution of the present invention is higher than the G.5 ng/ml thick liquid, preferably higher than lng/ml thick liquid, more preferably high. It is preferably more than 2 ng/ml thick liquid, and more preferably more than 25 ng/ml thick liquid, at 15 ton / as a dope. In a particular embodiment, the condensable platelet growth factor concentrate of the present invention or obtained by the method of the present invention is suitable for use in a condition of lipid content. And the phrase "a condition requiring a low lipid content" used in the present invention means that the condensable liquid growth factor of the present invention or the method of the present invention has been removed from the lipid (depleted Xun) The amount of 'cholesterol, triglyceride, hdl and its initial platelet thick concentrate in the condensable blood 16 3 : T thick solution obtained by the present invention or by the method of the present invention is extremely low in quality. The lipid in the thick platelet growth factor concentrate is used for the therapeutic use of the sputum, for example, the risk of the disease (4), or the amount of the risk for high heart risk = salty, ': Patients who have received treatment offer extremely low levels " greatly reduce the risk of vascular occlusion caused by the formation of fatty plaques (Plaq=) in the arterial and venous systems. The invention may be obtained by the method of the invention or by the method of the invention. Condensed thick liquid towel, the amount of bile rotation is less than (10) g coagulable, sputum platelet growth factor thick liquid, preferably less than 5 〇 - condensed ===: liquid and more preferably lower than the knot Birth! Can condense OOmg/dl clotting platelet growth factor = : = 5 〇mg/dl condensable platelet growth 乂 preferably less than 3 - condensate..., plate = 、, night and better for the low condensable mg/cU condensable μ plate Raw (four) sub-thick liquid, less than 30 mg / dl clotting platelet growth factor thick liquid, and 〗 〖= 15 10mg / d condensable platelet growth factor thick liquid. More box is lower than better, in The present invention or the platelet-derived (four) sub-rich coffee 17 1344963 mg / dl clotting platelet growth factor thick solution, preferably less than % mg / dl clotting platelet growth factor thick liquid, more preferably low a platelet growth factor thick solution which is condensable at 2 mg/dl, and more preferably a platelet growth factor thick solution which is less than 5 mg/dl. Further, the present invention or the method obtained by the method of the present invention The quality of the lipid in the condensed platelet growth factor thick solution is very low and/or undetectable, so it has better stability. When the platelet growth factor is purified on a large scale, the method is easier to carry out. In the present invention, or by the method of the present invention The clotting platelet growth factor thick solution obtained by the method does not cause blood cell-related transfusion reaction. "Blood cell-related transfusion reaction" means that the clotting growth factor thick liquid does not have intact living cells (such as red blood cells). , platelets and white blood cells), so it can avoid causing a known range of known transfusion reactions in patients, including immunization (such as alloimmunizaticm) and hemolytic complications (see, for example, Str〇ncek et Rebulla, "Platelet transfusions" , The Lancet, August 4, 2007; vol' 370: 427-438). Indeed, recipients with normal immune abilities will develop an immune response to the donor's blood cell antigens, resulting in a variety of clinical outcomes depending on the relevant blood cells and specific antigens. The most common related antigens are selected from the following types: ("type 1 HLA, platelet and white blood cell co-owners; (7) type 2 HLA, in some white blood cells; (3) granular white a ball specific antigen (4) platelet-specific antigens (for example, human platelet antigens ;ρΑ); and (5) red blood cell-specific antigens. More specifically, 'in the preparation method of the present invention, live blood is fine, (red blood cells, white blood cells and Platelets) will be destroyed/dissolved by solvent_cleaner, thereby reducing (and better preventing) patients exposed to foreign antigen 1344963 to u, plate 3 mu method can be obtained from patients: small plate: condensed blood two The growth factor is different from the present invention. The invention may be further selected from the method of the present invention to be further contained from the sorghum I long thick liquid to the sensitizing protein and/or the π (the suspected worm) transparent adhesion Protein, thrombin sensitivity VIII ^ IX ^ (P-thrombin) ) V. VII. The group of eggs from the coagulation factor and the composition of the Willer's factor, the clotting platelets of the present invention The raw material of the raw (four) even egg (four) is higher than 〇 2 g / L / Chen sputum, and more preferably about 3 g / L thick liquid. · Growth plate source rc is better than.. a thick liquid: and better = other embodiments, the condensable of the present invention Platelet growth factor: a vm clotting factor and/or a van derrick factor 〇.9 IU/mU auxac thick liquid in a thick liquid, and more preferably about 丨m/ml thick liquid is in another embodiment The sample towel, the curable === thick liquid step obtained by the method of the invention comprises immunoglobulin, as in the special embodiment, the immunoglobulin IgG in the clotting platelet growth factor thick solution of the invention The concentration is preferably greater than 5 g/L of concentrated liquid, and more preferably about 1 〇g/L of concentrated liquid. In another embodiment, the present invention or condensable by the method of the present invention 19 1344963 The platelet growth factor concentrate further comprises at least one growth factor selected from the group consisting of cCN family members, connective tissue activating protein-3 (CTAP-3), PF4, platelet-derived angiogenic factor (PDAF) ), endothelial cell growth inhibitor, early pregnancy factor (EPF), Skin growth inhibition. Agent (EG) 〇, keratinocyte growth factor (KGF), angiopoietin-like protein-6 (ANGPTL6), IGFBP-3, estrogen receptor-associated protein, fibroblast-derived endothelial cell growth factor (f_ECGF) ), hepatocyte growth factor (HGF), tissue limb release factor, human collagenase inhibitor, platelet antibacterial protein-1 (PMP-1), thrombin-induced platelet antibacterial protein-1 (t-PMP) , thrombin peptide 1 (throinbocidin-1, TC1) and thrombin-2 (TC2). In another embodiment, the condensable platelet growth factor concentrate obtained by the present invention or by the method of the present invention further comprises at least one protein selected from the group consisting of serotonin, cathepsin (cathepsin), albumin, platelet basic protein _pBp (CXCL7), neutrophil activating protein 2 and _4 (NAP-2, -4), _ somatostatin (somat〇statin 'SST), RANTES , CTAP-3, placental protein 14 (PP14), SCUBE annexin U (heatinensis) i - heat shock protein 27 (HSP27), and heat shock protein 60 (HSP60). • In a particular embodiment, the albumin concentration in the condensable platelet growth factor concentrate of the present invention is preferably higher than 3 〇 g/L thick. In another embodiment, the indestructible platelet growth factor concentrate of the present invention or by the method of the present invention further comprises at least one enzyme selected from the group consisting of collagenase, superoxide SUperoxide dismutase ' SOD , heparinase , 20 metalloproteinase MMP-b-2, -9, -13, autocrine ERK (ext.Ceil reg. kinase), autocrine and paracrine protein C (PC), and trace enzymes such as aldolase, carboxyl peptide, acid phosphatase, arylsulphatase, β-galactosidase, β-gluconate acidase, --glycerophospho-acidase (P_glycer〇lph〇sphatase), oc/β-glucosidase, α/β-fucosylase (a/p_fUc〇sidase), α-mannosidase (α- Mannosidase and α-arabinosidase ° In another embodiment, the condensable platelet growth factor concentrate obtained by the present invention or by the method of the present invention further comprises histamine, ADAMTS-13, α1 -α2 antitrypsin, α2-antiplasmin, α2_ giant ball Protein, ci-INH, induced sputum-2, -6, -7 (TGF-β superfamily), ECM remodeling factor (ECM remodelling factor 'induced sputum, TNF-α, elastase..... .), autosomal and paralytic lysophosphatidic acid (LPA), HMGB1 (amphibiegulin), ATP, ADP, GPT, GDP, Ca2+, Mg2+ and/or Zn2+. Another object of the invention is a pharmaceutical product comprising the condensable platelet growth factor concentrate of the invention or obtained by the method of the invention. "Medical products" means plateiet gei, platelet glue, growth factor-rich fibrin glue and/or sealant, artificial eagle. Another object of the present invention is the use of the coagulated platelet growth factor of the present invention or obtained by the method of the present invention in a medium, wherein the medium is suitable for use in fibroblasts, chondrocytes, osteoblasts, keratinocytes, In vitro or in vivo culture of stem cells and/or transplanted cells. Another object of the present invention, a cell, a chondrocyte, an osteoblast, a horn, is suitable for in vitro or in vivo study of fibroblasts, a stem cell, and/or a transplant invention or by the present invention; This factor. The condensable platelet growth of the female part of the small plate thick liquid Lin Nui Cai / value and in from 2. . Up to 5^^^^ minutes to 6 hours, more preferably at a temperature of from 25 ϋC _ ^; and removal of the aforementioned solvent and/or cleaning agent by alcohol extraction and/or stratification. In a preferred embodiment, the culture is carried out for a time ranging from 2 to 4 hours, which is carried out at a physiological 1) 11 value, such as 1 > 11 in the range from pH 7 ΡΗ to ΡΗ 7·5 ( When the starting platelets are fresh platelets, or at a value ranging from 6.8 6.8 to 8.2 (when the initial blood small f is expired and/or cold/dong small plate). Advantageously, the culture temperature is about 31 °C. Suitable solvents for use in the process of the invention are di- or tri-alkanes, dish esters such as tris-(η-butyl)phosphate, tris-(t-butyl)phosphate, ~hexyl) Filler, tris(2-ethylhexyl)phosphate, tris(n-mercapto)phosphonium, di-(n-butyl) acid vinegar, di-(t-butyl) dish warm vinegar, Di-(n-hexyl)phosphate, di-(2-ethylhexyl)phosphate, di-(Iinyl)phosphoric acid and dialkyl phosphates having different alkyl chains. Di- or trialkyl phosphates having different alkyl groups may be used, such as bis-(II-butyl)phosphoric acid. Particularly preferred is a trialkyl phosphate which is tris-(n-butyl)phosphate 22 (ΤηΒΡ). In the present invention, a suitable cleaning agent for the use of a polyoxyethylene derivative of the acid = fat屯Γτ 0 The partial ester of sorbitol (for example, the name iween 80 丨 / , βΛχ Γ 、 , also known as "polysorbate 14 ii Aweel20 j ^ ^ #) (^ $ "τ .Oral ethoxylated alkyl phenol: "Triton Χ-100" or Iriton X-4S, 仏^ _"). Further consideration of the cleaning agent is sodium deoxycholate. For example, N-dodecyl-n,n-dimercapto-2-l-hexan-N,N-dimethyl-2-ammonio-l-ethane SUlph〇nate. "Triton X-45", "Triton X-100" and "Tween 80". In a special embodiment of the present invention, the starting platelet thick liquid is co-cultured with an agent and a detergent, preferably TnBp and THt〇n X-45 are co-cultured. & Advantageously, the final concentration of the solvent or the aforementioned cleaning agent, or the solvent and the individual final concentration of the aforementioned cleaning agent are contained in a volume of from 0.2 to 2. More It is included in the range from G 2 to 2% by volume = which is based on the volume of the aforementioned platelet thick liquid. In the -1 parent implementation, 'the aforementioned initial platelet thick liquid system is co-cultured with 2% ΤηΒΡ In another preferred embodiment, the starting platelet thick liquor is co-cultured with 1% ΤηΒΡ and 1% Triton χ_45. ^ The aforementioned solvent and/or detergent can be extracted by oil with a pharmaceutical grade oil and The financial method (such as f-column chromatography)* is extracted from the biological fluid, so that the solvent and/or detergent in the thick liquid has been removed. The aforementioned pharmaceutical grade oil may be a natural oil (for example, from plants or animals). An extracted oil) or a structurally similar synthetic compound. Suitable natural 23 1344963 oils include castor oil (or rjcinus 〇 ii), soybean, sunflower, sunflower oil, cottonseed oil. Preferred synthetic compounds are synthetic triacids. Examples of suitable synthetic triglycerides include triolein, tristearin, tripartre = (tripgf!?ltln), trimyristin, and combinations thereof. Medicine, medicine, etc. The amount of oil to be extracted at least 8 square lipid soluble process chemicals%, (process chemical) in the amount of, the fuel consumption of from 2 to &

重置％，其係以血小板濃厚液之溶胞產物的重量 ^算；較佳為從5至15重量％，且更佳為從5至 量％。油萃取可以進行一次，較佳為兩次，且 次’其係特別取決於油的濃度。 土‘、《' — 前述溶劑及/或清潔劑也可藉由管柱層析而從血 板，厚液之溶胞產物中移除。管柱層析也可在油萃取後 進行，或直接在溶劑及/或清潔劑處理後進行。The % is reset, which is calculated as the weight of the lysate of the platelet thick solution; preferably from 5 to 15% by weight, and more preferably from 5 to 5% by weight. The oil extraction can be carried out once, preferably twice, and the second is particularly dependent on the concentration of the oil. Soil ‘, ''—The aforementioned solvents and/or cleaners can also be removed from the blood plate, thick liquid lysate by column chromatography. Column chromatography can also be carried out after oil extraction or directly after solvent and/or detergent treatment.

可^合適層析管柱包含反相（疏水***互反應） 土貝、或蛋白吸附基質如離子交換（陰離子 基質及親和力（如免疫·親和力或固定化肝素子& 尺，排除（size-exclusion )基質。較佳之反相基質為C U 二氧化矽裝填材料、SDR (溶劑_清潔劑移除）hyper D (Pall corporation )、聚苯乙烯吸收劑（)及Suitable chromatographic columns include reversed phase (hydrophobic interaction), shellfish, or protein-adsorbing matrices such as ion exchange (anionic matrix and affinity (eg, immunological affinity or immobilized heparin & ruler, size-exclusion) Base. The preferred reverse phase substrate is CU cerium oxide filling material, SDR (solvent_cleaner removal) hyper D (Pall corporation), polystyrene absorbent () and

Ambedyte ( Rohm )。即便c! 8係一般用於工業規模層析 的較佳選擇，亦將tC18二氧化矽裝填材料列入考慮。這 些吸附物質係在前述溶劑及清潔劑於前緣部分 (breakthrough fraction )流洗出來的時候用來與前述生 長因子作結合。陰離子交換基質係取決於所要純化之生 長因子，較佳為陰離子交換凝膠，如DEAE_Sephadex A-50 ^ DEAE- Sepharose FF ^ Q-Sepharose > DEAE- 24Ambedyte ( Rohm ). Even though the c! 8 series is generally preferred for industrial scale chromatography, the tC18 ceria loading material is also considered. These adsorbing substances are used in combination with the aforementioned growth factor when the aforementioned solvent and detergent are washed out in the leading fraction. The anion exchange matrix depends on the growth factor to be purified, preferably an anion exchange gel such as DEAE_Sephadex A-50 ^ DEAE-Sepharose FF ^ Q-Sepharose > DEAE-24

Toyopearl 650M、DEAE-Hyper D。這些吸附物質係在前 述溶劑及清潔劑於前緣部分中洗提出來的時候用來與 生長因子作結合。 在一較佳實施態樣中，油萃取及/或層析後的溶劑量 為小於100 ppm，較佳為小於50 ppm，更佳為小於20 ppm ’又更佳為小於1 〇 ppm，小於5 ppm，且再更佳為 小於1 ppm ° 在一較佳實施惑樣中，油萃取及/或層析後的清潔劑 量為小於500 ppm，較佳為小於250 ppm，更佳為小於 100 ppm，又更佳為小於50 ppm，且再更佳為小於1〇 ppm ° 在藉由油萃取及/或層析來移除溶劑及/或清潔劑 後，可進一步加入一步驟，來使非套膜病毒（如小病毒 B19、或者也可能是a型肝炎病毒(HAV))去活化或將 之移除，例如奈米過濾，且更特別是使用75_nm、35_nm、 2〇-nm、l5_nm或l〇-nm孔徑之濾膜來進行奈米過濾。 在另一較佳實施態樣中，係在油萃取及/或層析後進 行離心步驟，以移除細胞碎片。有利的是，前述離心步 驟係於800至20000 X g進行至3〇分鐘，且較佳為 於10000 X g進行15分鐘。 ,在另一較佳實施態樣中，係在油萃取及/或層析後進 行過濾清除步驟’以移除細胞碎片。有利的是，前述過 遽步驟係以從1 μιη i 〇 2 μιη漸次變化的過渡器來進 行，這也會移除細菌。 在一特別實施態樣中，在本發明之方法中用作起始 材料的血小板》辰厚液係單一或混合之標準金 小板濃厚液，例如製備作輸血用途的血小板濃厚液。血 25 1344963 小板濃厚液亦可得自全血之血沉棕黃層分離法。一單位 得自血沉棕黃層之血小板濃厚液一般係30至50 ml。得 自血沉棕黃層之血小板可用作起始材料，其係單一單位 或多個單一年位之混合，如在形成一治療性單位時，其 係4至6個早一早位的混合（如Guide to the preparation， use and quality assurance of blood components - 13th edition (2007)，edited by the Council of Europe 之揭示）〇Toyopearl 650M, DEAE-Hyper D. These adsorbing substances are used for binding to growth factors when the aforementioned solvent and detergent are eluted in the leading edge portion. In a preferred embodiment, the amount of solvent after oil extraction and/or chromatography is less than 100 ppm, preferably less than 50 ppm, more preferably less than 20 ppm, and even more preferably less than 1 〇 ppm, less than 5 Ppm, and more preferably less than 1 ppm ° In a preferred embodiment, the cleaning dose after oil extraction and/or chromatography is less than 500 ppm, preferably less than 250 ppm, more preferably less than 100 ppm, More preferably less than 50 ppm, and even more preferably less than 1 〇 ppm °. After removing the solvent and/or detergent by oil extraction and/or chromatography, a further step can be added to make the non-sleeve The virus (such as the small virus B19, or possibly the hepatitis A virus (HAV)) is deactivated or removed, such as nanofiltration, and more particularly 75_nm, 35_nm, 2〇-nm, l5_nm or l〇 Nanofiltration was carried out using a -nm pore size filter. In another preferred embodiment, a centrifugation step is performed after oil extraction and/or chromatography to remove cellular debris. Advantageously, the centrifugation step is carried out at 800 to 20,000 x g for 3 minutes, and preferably at 10,000 x for 15 minutes. In another preferred embodiment, a filtration removal step is performed after oil extraction and/or chromatography to remove cell debris. Advantageously, the aforementioned passage step is carried out with a transitioner that gradually changes from 1 μηη i 〇 2 μηη, which also removes bacteria. In a particular embodiment, the platelet thickening liquid used as a starting material in the method of the present invention is a single or mixed standard gold plate thick liquid, for example, a platelet thick liquid for blood transfusion use. Blood 25 1344963 Small plate thick liquid can also be obtained from the whole blood buffy brown layer separation method. One unit of platelet concentrate derived from the buffy coat is typically 30 to 50 ml. Platelets derived from buffy coats can be used as starting materials, which are a single unit or a mixture of multiple single years, such as when forming a therapeutic unit, which is a mixture of 4 to 6 early and early positions (eg, Guide to the preparation, use and quality assurance of blood components - 13th edition (2007), edited by the Council of Europe

血小板濃厚液可藉由血液分離術（指透過全血捐輸 的血小板製備法）、細胞分離術（cytapheresis )或血小 板分離術（plateletpheresis)標準程序（如參見Guideto the preparation, use and quality assurance of blood components, 13th edition (2007), edited by the Council of Europe)得出，且可藉由使用 MCS+ (Haemonetics)、Platelet concentrate can be obtained by blood separation (referred to as platelet preparation by whole blood donation), cytapheresis or plateletpheresis (see, for example, Guideto the preparation, use and quality assurance of blood components). , 13th edition (2007), edited by the Council of Europe), and by using MCS+ (Haemonetics),

Trima Accell 或 COBE Spectra ( Gambro)或 Amicus (Baxter)而得出。與透過血沉棕黃層分離法程序所得 者相較之下，血小板分離術一般會從每個捐贈者身上得 到較大的體積（相當於3〇〇 mi血小板濃厚液）。From Trima Accell or COBE Spectra (Gambro) or Amicus (Baxter). In contrast to those obtained through the buffy coat separation procedure, platelet separation typically results in a larger volume (equivalent to 3 〇〇 mi platelet concentrate) from each donor.

在一权佳貫施態樣中，本發明之方法包含一製備起 始板濃厚液之初步步驟。有利的是，製備起始血小 板濃厚液之方法包括但不限於：標準血庫程序（如血小 =分離術或透過全血捐輸的血小板製備法）及重點照護 私序（如那些使用血球保存劑/分離器或桌上裝置者 f本發明之方法中用作起始材料之血小板濃厚液 可以是新鮮（即收集後少於5或7天）、過期（即枚集 後多於5或7天）、或過期且冷凍或以下數週 時間。 在特別實施態樣中，在本發明之方法中用作起始 26 材料之血小板濃厚液可進一步包含白血球及/或紅血 球。故该起始血小板濃厚液可能包含數種已分化且未活 化之白血球，如淋巴細胞、嗜中性之顆粒性白血球及單 ，球二更特定言之，嗜中性球及單核球富含内有骨髓過 氧化酶（myeloperoxidase)之顆粒，這種酶會催化氯化 物的氧化作用，而生成次氯酸（hypochlorous acid)及 $他反應性氧衍生物，其中前述反應性氧衍生物係作為 殺菌氧化劑’對微生物及黴菌具有毒性。In a preferred embodiment, the method of the present invention comprises a preliminary step of preparing a starting plate thick liquor. Advantageously, methods for preparing a starting platelet concentrate include, but are not limited to, standard blood bank procedures (eg, small blood = separation or platelet preparation via whole blood donation) and focus on private procedures (eg, using blood cell preservatives / Separator or table device f The platelet concentrate used as a starting material in the method of the invention may be fresh (ie less than 5 or 7 days after collection), expired (ie more than 5 or 7 days after collection) Or expired and frozen or for a few weeks. In a particular embodiment, the platelet concentrate used as the starting material 26 in the method of the present invention may further comprise white blood cells and/or red blood cells. Therefore, the starting platelet thick liquid It may contain several differentiated and unactivated white blood cells, such as lymphocytes, neutrophilic granulocytes, and spheroids. In particular, neutrophils and mononuclear spheres are rich in bone marrow peroxidase ( a particle of myeloperoxidase) which catalyzes the oxidation of chloride to form hypochlorous acid and a derivative of its reactive oxygen species, wherein the aforementioned reactive oxygen derivative is Bacteria oxidant 'toxic to microorganisms and fungi.

因此，當起始血小板濃厚液包含數種已分化且未活 化之白血球時，藉由本發明之方法所得的可凝結之血小 板生長因子濃厚液進一步包含得自白血球之抗微生物 成分。 在一較佳實施態樣中，用作起始材料之血小板濃厚 液中的白血球會被消滅，更佳為藉由白血球去除作用 (leukoreduction)來進行，以避免白血球中之蛋白酶及 酸水解酶發生促發炎反應。白血球去除作用也可降低普 利子蛋白（prion)污染的風險。 _曰Thus, when the starting platelet concentrate comprises several differentiated and non-activated white blood cells, the condensable platelet growth factor concentrate obtained by the method of the present invention further comprises an antimicrobial component derived from white blood cells. In a preferred embodiment, the white blood cells in the platelet concentrate used as the starting material are eliminated, more preferably by leukoreduction, to avoid the occurrence of proteases and acid hydrolases in the white blood cells. Promotes an inflammatory response. Leukocyte removal also reduces the risk of prion contamination. _曰

健康人的正常血小板計數一般係每mm3血液中包 含150000至400000個血小板，亦即15〇至4〇〇 X ι〇9 個血小板/L。這個「正常」血小板計數在約％%健康人 身上是如此，而剩下的5%可能會有統計上不正常^血 小板§十數（非常低或非常高）。 ,當以血小板分離術收集血小板時，在一袋25〇 ml 當中的血小板計數—般係高於每ml冑1.2 X 1〇9個血小 板’其企小板計數相當於每袋（單位）有多於約3 x 1〇n 個小板。 在-較佳實施態射，在起始到、板濃厚液中的血 27 小板數目要比血液中通常可見的數目高出3至10倍。 本發明之另一目的是一種形成血凝塊的方法，其係 由下列步驟所組成：將本發明或藉由本發明之方法所得 的可凝結之血小板生長因子濃厚液與凝血酶混合，或與 另一種血液凝集連鎖反應之活化劑混合，如已活化之 FVH、或者牛或人類的凝血活酶（thromboplastin) 〇 在以本發明或藉由本發明之方法所得的可凝結之 企小板生長因子濃厚液形成血凝塊之前，可在本發明或 藉由本發明之方法所得的可凝結之血小板生長因子濃 厚液中加入抗纖維蛋白溶解劑，來抑制或減緩纖維蛋白 ;谷解糸統中的天然蛋白水解酶（如胞漿素(plasmin))分 解纖維蛋白企凝塊。 在一特別實施態樣中，前述抗纖維蛋白溶解劑為抑 胰肽（aprotinin )，但濃度 > 1 〇 mg/ml 之傳明酸（tranexamic acid)或8••胺基己酸（epsilon aminocaproic acid)也可以 用作抑胰肽的替代化合物。在液體形式中，所提供之抑 胰肽濃度通常為3000 KIU或以下。在可凝結之血小板 生長因子濃厚液及凝血酶成分混合後，抑胰肽之最終漢 度一般是起始溶液的一半。 本發明或藉由本發明之方法所得的可凝結之血小 板生長因子濃厚液可進一步與人工鷹架（例如以膠原蛋 =、幾丁聚醣（chit〇san)、陶瓷所製造者）混合，或與 知·自血聚之纖維蛋白黏膠或纖維蛋白密封劑混合。 在與凝血酶混合之前，亦可在本發明之可凝結之血 L板生長因子濃厚液中加入額外的化合物。這類額外成 分特別包含但不限於：化療劑、抗生素及/或激素。 將本發明或藉由本發明之方法所得的可凝結之血 28 板生長因子〉農厚液與;疑血酶混合在__起會重現血液 凝集連鎖反應的最後步驟，且㈣維蛋白原逐漸聚合， 而形成血凝塊或不溶性纖維蛋白。 在本發明中所使用的「凝血酶」一詞係關於可得自 任來源之凝血酶，且較佳係指牛凝血酶；若用於人類 的冶療性應用，更佳為人類凝血酶。經CaC12活化之人 ，血漿或蝮蛇類凝血酶（batr〇x〇bin，另一種纖維蛋白原 j血蛋白轉，係從蛇办加心印$ 所⑽尽⑼出得出的蛇 毋）、已活化之FVII、或者人類或牛的凝血活酶也可用 作凝血酶之替代化合物。 在一較佳實施態樣中，將至丨體積之凝血酶加 到1體積之本發明或藉由本發明之方法所得的可凝結之 血小板生長因子濃厚液中。 在一較佳實施態樣中，凝血酶濃度範圍係從2〇 IU/ml至1〇〇〇 iu/m卜更佳者，凝血酶之最終濃度為約 500 IU/ml，以確保連續且快速的纖維蛋白原聚合作用會 先得出可溶的纖維蛋白，之後再得出穩定的（不溶性） 纖維蛋白，或者當在特殊手術情形中想要減缓聚合作用 時’其最終濃度會較低，大約為4至25 IU/m卜 兩種成分（亦即本發明或藉由本發明之方法所得的 可凝結之血小板生長因子濃厚液和凝血酶）都可藉由雙 針筒系統而先後施用或同時施用在要修復的位置上，其 可使用或不使用内視鏡遞送裝置來協助，且使用喷灑方 式或易吸收的海棉來進行（參見Radosevich et al.， Fibrin sealant · Scientific Rationale, Production Methods,A healthy person's normal platelet count typically contains between 150,000 and 400,000 platelets per mm3 of blood, i.e., 15 to 4 X X 〇 9 platelets/L. This "normal" platelet count is about 5% of healthy people, and the remaining 5% may be statistically abnormal. The blood plate is § ten (very low or very high). When platelets are collected by platelet separation, the platelet count in a bag of 25 〇ml is generally higher than 1.2 X 1 〇 9 platelets per ml ' 'the plate count is equivalent to more per bag (unit) About 3 x 1〇n small plates. In the preferred embodiment, the number of blood platelets in the initial, plate thick liquid is 3 to 10 times higher than the number normally seen in blood. Another object of the present invention is a method of forming a blood clot, which comprises the steps of: mixing the condensable platelet growth factor thick solution obtained by the present invention or by the method of the present invention with thrombin, or A mixture of activators of a blood agglutination chain reaction, such as activated FVH, or bovine or human thromboplastin, a condensable platelet growth factor concentrate obtained by the present invention or by the method of the present invention Prior to the formation of a blood clot, an antifibrinolytic agent may be added to the condensable platelet growth factor concentrate obtained by the method of the present invention or the method of the present invention to inhibit or slow down fibrin; natural proteolysis in glutathione Enzymes such as plasmin break down fibrin clots. In a particular embodiment, the antifibrinolytic agent is aprotinin, but the concentration is > 1 〇mg/ml of tranexamic acid or 8 •• aminocaproic acid (epsilon aminocaproic) Acid) can also be used as an alternative compound to trypsin. In the liquid form, the concentration of the inhibitor peptide is usually 3000 KIU or less. After mixing the clotting platelet growth factor concentrate and thrombin components, the final degree of the pancreatic peptide is typically half that of the starting solution. The condensable platelet growth factor thick solution obtained by the present invention or by the method of the present invention may be further mixed with an artificial scaffold (for example, a manufacturer of collagen egg=, chitosan, ceramics), or Know the self-blooded fibrin glue or fibrin sealant. Additional compounds may also be added to the condensable blood L-plate growth factor concentrate of the present invention prior to mixing with thrombin. Such additional components include, but are not limited to, chemotherapeutic agents, antibiotics, and/or hormones. The condensable blood 28 plate growth factor> agricultural thick liquid obtained by the present invention or by the method of the present invention and the suspected blood enzyme mixed in the __ will reproduce the final step of the blood agglutination chain reaction, and (4) the protein progenitor gradually Polymerization forms a blood clot or insoluble fibrin. The term "thrombin" as used in the present invention relates to thrombin which can be obtained from a source, and preferably refers to bovine thrombin; and if it is used for therapeutic use in humans, it is more preferably human thrombin. A person activated by CaC12, plasma or python thrombin (batr〇x〇bin, another fibrinogen j blood protein transfer, from the snake to add the heart of the $10 (10) to the snakes) Activated FVII, or human or bovine thromboplastin can also be used as a replacement for thrombin. In a preferred embodiment, thrombin to volume is added to one volume of the condensable platelet growth factor concentrate of the present invention or obtained by the method of the present invention. In a preferred embodiment, the thrombin concentration ranges from 2 〇 IU/ml to 1 〇〇〇 iu/m 卜, and the final concentration of thrombin is about 500 IU/ml to ensure continuous and rapid. The fibrinogen polymerization will result in soluble fibrin, followed by stable (insoluble) fibrin, or when the specific surgery is desired to slow the polymerization, the final concentration will be lower. The two components of about 4 to 25 IU/m (i.e., the clotting platelet growth factor concentrate and thrombin obtained by the method of the present invention or by the method of the present invention) can be administered sequentially or simultaneously by a double syringe system. Apply at the location to be repaired, with or without the use of an endoscopic delivery device, and using spray or absorbable sponges (see Radosevich et al., Fibrin sealant · Scientific Rationale, Production Methods ,

Properties，and Current Clinical use，，，Vox Sanguinis，1997, 72:133-143 以及 Marx G，“Evolution of fibrin glue 29 1344963 applicators”，Transfus Med Rev，2003; 17(4):287-98 所述 . 之應用方法，以上係合併於本文作為參考文獻）。 - 因此，本發明之另一目的是本發明或藉由本發明之 方法所付的可;旋結之血小板生長因子濃厚液在治療性 應用上的用途、以及形成血凝塊的用途、或在活體外或 . 活體研究的細胞培養用途。當其用於活體外或活體研究 - 的細胞培養時，本發明或藉由本發明之方法所得的可凝 結之血小板生長因子濃厚液會以從1%至3〇%範圍的量 • 出現在培養基中’更佳為從2%至20%，且又更佳為從 3%至10%，其係以培養基之體積為基礎計算。 前述濃厚液/凝血酶混合物之主要治療性應用包含 • 但不限於：牙科手術、植入、骨科及口腔手術、整型手 • 術、軟組織及硬組織的療癒及重建、心血管、胸腔、或 胃腸道手術、神經手術、一般手術/創傷外科、眼科、耳 鼻喉科、泌尿科、以及在抗凝血患者或凝血不良之患者 身上進行的手術。 、 本發明之上述及其他目的、特徵及優點將因以下敘 鲁述、參考文獻及後附圖式而變得更加顯明。 實施例 Ϊ·材料及方法 1.1-分離術血小板（apheresis platelet)的收集 起始血小板濃厚液（PC)係徵得志願捐贈者同意後 使用 MCS+多成分系統（Haemonetics，Braintree，USA) 收集而來。全血係透過靜脈導管取得，使用間歇性流動 ^ intermittent flow)及抗凝血劑（lml抗凝血檸檬酸右 旋葡萄糖容液配方_每i〇mi血液）。富含灰小板的血漿 30 1344963 (PRP )會自動藉由離心而與其他血液成分分離，並收 集在一個無菌、單次使用的拋棄式袋子裡，而紅血球及 企漿會再回到捐贈者體内。重複此一循環，直到得到預 定體積之PRP (約300 ml)為止。起始血小板濃厚液係 如下所述，在收集後24小時内進行處理。 1.2-血液細胞計數 血小板、白血球（WBC)及紅血球計數係使用細胞Properties, and Current Clinical use,,, Vox Sanguinis, 1997, 72: 133-143 and Marx G, "Evolution of fibrin glue 29 1344963 applicators", Transfus Med Rev, 2003; 17(4): 287-98. The method of application, the above is incorporated herein by reference. - Therefore, another object of the present invention is the use of the present invention or by the method of the present invention; the use of a spinnated platelet growth factor thick solution for therapeutic applications, and the use of a blood clot to form, or in vivo External or . Cell culture use for in vivo studies. When it is used for cell culture in vitro or in vivo studies, the clotting platelet growth factor concentrate obtained by the present invention or by the method of the present invention may be present in the medium in an amount ranging from 1% to 3%. 'More preferably from 2% to 20%, and even more preferably from 3% to 10%, based on the volume of the medium. The primary therapeutic applications of the aforementioned thick liquid/thrombin mixture include, but are not limited to, dental surgery, implantation, orthopedics and oral surgery, hand surgery, soft tissue and hard tissue healing and reconstruction, cardiovascular, thoracic, Or gastrointestinal surgery, neurosurgery, general surgery/trauma surgery, ophthalmology, otolaryngology, urology, and surgery in patients with anticoagulation or coagulopathy. The above and other objects, features, and advantages of the present invention will become more apparent from the description and appended claims. EXAMPLES Materials and Methods 1.1 - Collection of apheresis platelet The initial platelet thick solution (PC) was collected by a volunteer donor using the MCS+ multi-component system (Haemonetics, Braintree, USA). The whole blood is obtained through an intravenous catheter, using intermittent flow and anticoagulant (lml anticoagulant dextroglucose solution formula _ per 〇mi blood). The gray plate-rich plasma 30 1344963 (PRP) is automatically separated from other blood components by centrifugation and collected in a sterile, single-use disposable bag, and the red blood cells and the plasma will return to the donor. in vivo. This cycle is repeated until a predetermined volume of PRP (about 300 ml) is obtained. The starting platelet thick liquor was processed within 24 hours of collection as described below. 1.2- Blood cell counts Platelets, white blood cells (WBC) and red blood cell counts use cells

計數器（ABC Vet Automatic Blood Counter，ABXCounter (ABC Vet Automatic Blood Counter, ABX

Diagnostics，France)來進行測定。 1.3-起始血小板濃厚液的處理 α-研究設計Diagnostics, France) for the determination. 1.3- Treatment of starting platelet thick solution α-research design

起始血小板濃厚液係根據第一圖之研究設計來處 理。簡言之，將相同捐贈者起始血小板濃厚液（3〇〇ml) 輕輕混合，並分成兩個等體積的子集池（15〇ml)。子集 池1係直接接受S/D處理，沒有先活化。子集池2係^ 23 mMCaCl2及玻璃球粒（beads)存在時，於會使血小 板凝膠形成的條件下活化，如下所述；用吸量 的方式小心、地回收所得之釋出物（平均體積相當於120The starting platelet thick liquor was processed according to the study design of the first panel. Briefly, the same donor starting platelet concentrate (3 〇〇 ml) was gently mixed and divided into two equal volumes of pool (15 〇 ml). Subset Pool 1 is directly subjected to S/D processing and is not activated first. When the subset pool 2 is in the presence of 23 mM CaCl 2 and glass beads, it is activated under conditions that cause platelet gel formation, as described below; the resulting release is carefully and carefully recovered by pipetting (average Volume is equivalent to 120

ml’因為5 3〇ml損失在血小板凝膠中），之後接受S/D ’ Ϊί 萃取。從起始血小板濃厚液所得之樣 本中，有經S/D處理後的未活化之血小板子隼幻，= 化後的子集池2，以及經處理後的經活化子 31 b-血小板活化： 在玻璃球粒存在的情況下，在血小板濃厚液（子集 池 2)中加入 1 MCaCl2 (Sigma，批號 〇56k0688)來進 行/舌化’其表終遭度為23 mM。將混合物輕輕地旋轉混 合’直到金凝塊形成為止，這一般會在5至8分鐘内形 成。使混合物再活化6〇分鐘的時間，在這些實驗條件 下’這個時間會使最理想的血小板源生長因子釋出。將 上清液倒出’與玻璃球粒/已形成之纖維蛋白血凝塊分 離。所回收之上清液平均體積為起始血小板濃厚液之約 80%。該上清液會進一步做s/D處理。 c-S/D處理： 為了方便起見，未活化之子集池1及已活化之子集 池2的S/D處理係如EP 1 685 852所述，在袋中進行。 簡言之，將 TnBP ( Merck KGaA，Darmstadt，Germany ) 及 TritonX-45 ( Sigma, Missouri，USA.)之 50%/50%混合 物加入各個子集池中15分鐘，並持續混合，以使其最 終濃度（v/v)為1%ΤηΒΡ及1% TritonX-45。在完全加 入後，將S/D-血小板子集池之混合物猛烈震盪1分鐘。 之後將處理袋完全地浸入水浴，將S/D血小板混合物加 溫至25 ±0.5 °C，之後在持續溫和的攪動下處理1小時。 S/D處理完成時，在S/D-血小板子集池中加入大豆油 (Sigma，Missouri，USA )’ 使之最終濃度為 10% ( v/v )。 將上述袋子猛烈震盪1分鐘，之後置入旋轉震盪器處理 15分鐘。以倒出的方式將血小板子集池（下層）從油（上 層）移除（20分鐘），並藉由重力移入第二袋，再重複 進行三次油萃取。此一油萃取程序可使ΤηΒΡ及Triton 32 1344963 X-45分別降到小於10及100 ppm。 1.4-離心與溶胞劑種類的影響 為了研究血小板含量及S/D試劑種類對血小板源生 長因子釋出的影響，進行了 一項實驗：將血小板濃厚液 _( 300 ml)次分為兩個150 ml的子集池。 .其中一個子集池進行高速（ 10000 X g)離心’使 血小板成為一團塊（pellet )，並使上清液接受1% φ TnBP-l%TritonX-45的處理。另一個未離心之子集池再 分為兩個75 ml的子集池，其中一個接受1% ΤιιΒΡ-1% TritonX-45處理’另一個接受2%TnBP處理。S/D處理 (培養及油萃取）係如前述來進行。 - 1.5-牛凝血酶活化實驗 為了排除CaCl2活化不會使血小板完全活化的假 設，如前述將兩種血小板濃厚液（14 ml)在0.23 M CaCb 及玻璃球粒存在的情況下進行活化。 φ 於室溫溫和旋轉混合60分鐘後，回收l〇ml之如小 板濃厚液釋出物，並加入0.5 ml之1000國際單位（IU) _ /ml 的牛凝血酶（Thrombin_JMI，52604-7102-1，J〇nesMl' was lost in the platelet gel due to 5 3 〇 ml) and then subjected to S/D ' Ϊί extraction. Among the samples obtained from the starting platelet thick solution, there were unactivated platelet sputum after S/D treatment, = subset pool 2 after treatment, and treated activator 31 b-platelet activation: In the presence of glass pellets, 1 MCaCl2 (Sigma, lot number k56k0688) was added to the platelet concentrate (subset pool 2) for/tongueization with a final degree of 23 mM. The mixture is gently spun and mixed until the gold clot forms, which typically takes place in 5 to 8 minutes. The mixture was allowed to reactivate for a further 6 minutes, under which conditions the optimal platelet-derived growth factor was released. The supernatant was poured out' separated from the glass pellet/formed fibrin clot. The average supernatant recovered was about 80% of the starting platelet concentrate. The supernatant will be further processed by s/D. c-S/D treatment: For the sake of convenience, the S/D treatment of the unactivated subset pool 1 and the activated subset pool 2 is carried out in a bag as described in EP 1 685 852. Briefly, a 50%/50% mixture of TnBP (Merck KGaA, Darmstadt, Germany) and Triton X-45 (Sigma, Missouri, USA.) was added to each subset pool for 15 minutes and continued to mix to finalize The concentration (v/v) was 1% ΤηΒΡ and 1% Triton X-45. After complete addition, the mixture of S/D-platelet subset pools was shaken vigorously for 1 minute. The treatment bag was then completely immersed in a water bath and the S/D platelet mixture was warmed to 25 ± 0.5 °C and then treated for 1 hour with gentle agitation. Upon completion of the S/D treatment, soybean oil (Sigma, Missouri, USA) was added to the S/D-platelet subset pool to give a final concentration of 10% (v/v). The bag was shaken vigorously for 1 minute and then placed in a rotary shaker for 15 minutes. The platelet subset pool (lower layer) was removed from the oil (upper layer) in a pour-out manner (20 minutes), and transferred to the second bag by gravity, and the oil extraction was repeated three times. This oil extraction procedure reduces ΤηΒΡ and Triton 32 1344963 X-45 to less than 10 and 100 ppm, respectively. 1.4-Effects of centrifugation and lysing agent types In order to study the effect of platelet content and S/D reagent types on the release of platelet-derived growth factors, an experiment was conducted: dividing platelet concentrate _ (300 ml) into two A pool of 150 ml subsets. One of the subset pools was subjected to high speed (10000 X g) centrifugation to make the platelets a pellet and the supernatant was treated with 1% φ TnBP-1% Triton X-45. Another uncentrifuged subset pool was subdivided into two 75 ml subset pools, one of which received 1% ΤιιΒΡ-1% TritonX-45 treatment and the other received 2% TnBP treatment. S/D treatment (culture and oil extraction) was carried out as described above. - 1.5-Bovine Thrombin Activation Experiment In order to rule out the fact that CaCl2 activation does not completely activate platelets, two platelet concentrates (14 ml) were activated as described above in the presence of 0.23 M CaCb and glass spherules. After φ is gently mixed and vortexed at room temperature for 60 minutes, l〇ml of a small plate thick liquid release is recovered, and 0.5 ml of 1000 international units (IU) _ /ml of bovine thrombin (Thrombin_JMI, 52604-7102- 1, J〇nes

Pharma，Saint-Louis, MO )，使其最終濃度為約 48 IU/ml。將混合物於室溫溫和地旋轉混合60分鐘。之後 進行l°/〇TnBP-l%TritonX-45處理及油萃取。在平行實 驗中，亦將兩種血小板濃厚液樣本（10 ml)以0.5瓜1 之相同牛凝血酶直接活化，在數秒内凝膠形成，接著輕 輕旋轉混合60分鐘。在實驗方法之多個步驟中採取樣 本’於10000 X g離心並於_8〇°C冷凍，直到進行血小 33 1344963 板源生長因子分析為止。 1.6-生長因子測定 在程序之各個步驟中取出樣本。將之於1〇〇卯 X g 離心 15 分鐘（Microfuge® 22R，Beckman c〇ulterPharma, Saint-Louis, MO) to a final concentration of about 48 IU/ml. The mixture was gently mixed and spun at room temperature for 60 minutes. Thereafter, l°/〇TnBP-l% Triton X-45 treatment and oil extraction were carried out. In a parallel experiment, two platelet concentrate samples (10 ml) were also directly activated with the same bovine thrombin of 0.5 meg, and gelled in a few seconds, followed by gentle spin mixing for 60 minutes. Samples were taken at various steps in the experimental procedure's centrifugation at 10,000 xg and frozen at _8 〇 °C until blood platelet 33 1344963 plate source growth factor analysis. 1.6-Growth Factor Determination Samples were taken at each step of the procedure. Centrifuge at 1 〇〇卯 X g for 15 minutes (Microfuge® 22R, Beckman c〇ulter

Fullerton，CA)，使血小板及/或細胞碎片成為一團塊，並 付出無細胞之上清液進行血小板源生長因子測量。另' 外，也以800 xg之離心力進行15分鐘的平行實驗。之 後上清液立即於-8CTC冷束。 樣本係於37°C解凍，並在1小時内以敏感且具專 一性的市售免疫測定法進行分析。標準品及樣本係進行 二重複測定，並計算平均值。結果乘以樣本所用的稀釋 係數。Fullerton, CA), makes platelets and/or cell debris a mass, and delivers cell-free supernatant for platelet-derived growth factor measurements. In addition, a parallel experiment of 15 minutes was also performed with a centrifugal force of 800 xg. Immediately thereafter, the supernatant was cold-bound at -8 CTC. Samples were thawed at 37 ° C and analyzed in a sensitive and specific commercial immunoassay within 1 hour. Standards and sample lines were subjected to two replicate measurements and the average was calculated. The result is multiplied by the dilution factor used for the sample.

a-PDGF-AB 使用 Quantikine ELISA kit ( #.DHD00B，R&D SYSTEMs，Minneapolis, MN)來測定 PDGF-AB。樣本以 Calibrator稀釋劑（RD6-11 )稀釋100倍。將孔盤培養2 小時、清洗、並和與酶共輛結合之PDGF-AB抗體於室 溫再共同培養3小時。使用清洗緩衝液（Wash Buffer ；) 來清洗盤孔’之後於室溫加入受質溶液（Substrate Solution) 20-30分鐘。盤孔係避光保護。在各盤孔中加 入停止溶液（Stop Solution) ’並使用微滴定盤讀取儀來 測定450 nm之吸光值。最小可偵測的劑量為1.7 pg/m卜 b-TGF-βΙa-PDGF-AB PDGF-AB was assayed using a Quantikine ELISA kit (#.DHD00B, R&D SYSTEMs, Minneapolis, MN). The sample was diluted 100 times with Calibrator diluent (RD6-11). The wells were incubated for 2 hours, washed, and co-cultured with the enzyme-bound PDGF-AB antibody for 3 hours at room temperature. The cleaning buffer (Wash Buffer;) was used to clean the wells' and then the Substrate Solution was added at room temperature for 20-30 minutes. The hole is protected from light. A Stop Solution was added to each well and the absorbance at 450 nm was measured using a microtiter plate reader. The minimum detectable dose is 1.7 pg/m b b-TGF-βΙ

使用 Quantikine ELISA kit ( DB100B, R&D 34 1344963 SYSTEMS)來測定TGF-βΙ。樣本以Calibrator稀釋劑 (RD5-26)稀釋1〇〇倍。在塗覆有TGF令受器π之96 孔微滴定盤中製備體積為1〇〇-μ1的TGF-βΙ標準品 ( 890207)稀釋序列。在TGF-βΙ分析之前，進行酸活 化及中和反應’以將潛性TGF-β 1活化到免疫活性形成 狀態。為違此目的’將0.5 ml樣本與〇.1 m丨之in HC1 混合’於室溫培養10分鐘，加入0.1 ml之i 2NNaOH/ 0.5M HEPES (N-[2-羥基乙基]派畊-Ν0·[2-乙烷磺酸]) (Sigma ’ Η-7523 )力口以中和，再行離心。之後測定上 清液部分之總TGF-βΙ含量。將各等分（5〇μ1)以二重 複的方式加到微滴定盤中，之後蓋上蓋子，於室溫培養 2小時。之後清洗盤孔’加入與酶共軛結合之TGF-βΙ 多株抗體，並於室溫持續培養1.5小時。如前述完成測 量。TGF-βΙ之偵測限值為4.61 pg/m卜TGF-βΙ was determined using a Quantikine ELISA kit (DB100B, R&D 34 1344963 SYSTEMS). The sample was diluted 1 fold with Calibrator diluent (RD5-26). A dilution sequence of TGF-βΙ standard (890207) having a volume of 1〇〇-μ1 was prepared in a 96-well microtiter plate coated with TGF. Prior to TGF-βΙ analysis, acid activation and neutralization reactions were performed to activate latent TGF-β 1 to an immunologically active state. For this purpose 'mix 0.5 ml sample with 〇.1 m丨 in HC1' for 10 minutes at room temperature, add 0.1 ml of i 2NNaOH / 0.5M HEPES (N-[2-hydroxyethyl] Ν0·[2-ethanesulfonic acid]) (Sigma 'Η-7523) was neutralized and centrifuged. The total TGF-βΙ content of the supernatant fraction was then determined. Aliquots (5 μl) were added to the microtiter plate in duplicate, then capped and incubated for 2 hours at room temperature. Thereafter, the wells were conjugated with TGF-βΙ conjugated to the enzyme, and cultured at room temperature for 1.5 hours. The measurement is completed as described above. The detection limit of TGF-βΙ is 4.61 pg/m

c-EGF 使用 Quantikine ELISA kit ( #.DEG00，R&D SYSTEMS, Minneapolis，MN)來測定 EGF。樣本以 Calibrator稀釋劑（RJD6N)稀釋20倍。將200 μΐ之標 準品、對照品或樣本加入盤孔中。將孔盤於室溫培養2 小時。各盤孔經過抽吸’並填入清洗緩衝液來加以清 洗。在各盤孔中加入EGF共軛結合物，並於室溫培養2 小時。使用清洗緩衝液清洗盤孔，並在各盤孔中加入受 質溶液（200 μΐ)。混合物於室溫避光培養2〇分鐘。在 各盤孔中加入停止溶液（50 μΐ)。各盤孔之光學密度係 使用微孔盤讀取儀（VersaMax ™ microplate reader，c-EGF EGF was determined using a Quantikine ELISA kit (#.DEG00, R&D SYSTEMS, Minneapolis, MN). The sample was diluted 20-fold with Calibrator diluent (RJD6N). Add 200 μΐ of the standard, control or sample to the well. The wells were incubated for 2 hours at room temperature. Each well was pumped' and filled with wash buffer for cleaning. EGF conjugates were added to each well and incubated for 2 hours at room temperature. The wells were washed with a washing buffer, and a solution (200 μM) was added to each well. The mixture was incubated at room temperature for 2 minutes in the dark. A stop solution (50 μM) was added to each well. The optical density of each well is a VersaMaxTM microplate reader (VersaMaxTM microplate reader,

Molecular Devices, USA )在 30 分鐘内於 450 nm 進行測 35 1344963 定。最小可偵測的劑量為0.7 pg/πύ。 . d-IGF-1 使用 Quantikine ELISA kit (DG100，得自 R&d SYSTEMS)來定量IGF-1。樣本以Calibrator稀釋劑 ‘ （RD5-22)稀釋1〇〇倍。如製造商所說，其最小可偵測 的劑量範園為從〇.⑻7至0.056 ng/m卜且平均MDD為 0.026 ng/ml。在各盤孔中加入150 μΐ之測定稀釋劑 φ (RD1-53)，接著是50 μΐ之標準品（890775)。以黏膠 條覆蓋孔盤，並於2-8°C培養2小時。將盤孔清洗3次， 之後和與酶共軛結合之IGF-1於2-8°C共同培養1小 時。如前述完成測量。 1.7 -統計分析 所有系列實驗之數據係以平均值、標準差、以及最 小值及最大值來呈現。並以雙尾配對學生檢定（two-tailed paired Student test) 來進行統計上 的比較 。 p 值小 • 於〇.〇5係用來估算血小板製備程序之不同步驟中平均 PGF 濃度之明顯差異（the Significance 〇f the - differences)。其值係表示為不明顯（NS，<〇.〇5)、<0.01 或<0.00卜當其值接近0.05時，會顯示確實的p值。 I.8-PC、S/D-PC及Act-PC比較之樣本的製備及分離 分別對應於起始金小板濃厚液（PC)、經溶劑/清潔 ，（1%1^及1%Trit〇nX_45)處理之起始血小板濃 厚液（S/D-PC)、以及經CaCl2活化之起始血小板濃厚液 (ACt_PC)的樣本係用SDS-PAGE加以分離，以比較其 36 1344963 蛋白含量。 • 將20pg的各樣本與5 plNuPAGELDS樣本緩衝液 . (4x ) ( Invitrogen )、2 μΐ NuPAGE 還原劑（ι〇χ ) (Invitrogen)及去離子水混合，所得最終體積為2〇 μΐ (已活化之血小板濃厚液的樣本為21 μΐ)。之後將所得 混合物於70°C加熱10分鐘，並使用4-12%聚丙稀醯胺 _ 梯度凝膠（NuPAGE Bis-Tris，Invitrogen)來進行 SDS_ PAGE。 φ 蛋白分離係在恆定電壓200V之下進行35分鐘，其 中預定電流為150 mA/凝膠。所得凝膠以考馬斯藍R_25〇 (coomassie blue R-250 )加以染色。 蛋白標記^1&也12之未染色標準品（11^杜〇弘11)係 用以測疋樣本中之蛋白的分子量。Mark 12標記係裝入 帶有 MES 之 NuPAGE novex 4-12% Bis-Tris 凝膠 (Invitrogen )上，並在分離後以考馬斯藍R_25〇加以^ 色。對應結果係揭示於第四圖。 經溶劑/清潔劑方法處理之起始血小板濃厚液的蛋 • 白圖譜（pr〇file)顯示它與未處理之起始血小板濃厚液 並沒有很大的差異，而經活化之血小板漠厚液的蛋白圖 - 譜則有明顯的差異，在40至70咖區域中的條帶（band) 消失了，這些條帶係對應到纖維蛋白原之α、P及次單 元，分別是63.5、56及47 kDa。 1.9-生長因子活性測定 為了測定血小板S/D處理（1% TnBp_1% Tdt〇n X-45)所得的生長因子是否能保持活性，使用人類成骨 細胞樣MG-63細胞株來進行活體外細胞培養研究。 37 1344963 MG-63細胞接受從S/D-PC或從Act-PC所得之生長因子 • 濃厚液處理，其細胞反應係以細胞形態學及存活率來加 以評估。 將105至106個細胞在35-mm Petri培養盤中，使用 90% Minimum essential medium Eagle (MEM)進行培 -養，其中前述培養基係帶有2mM-麩醯胺酸、Eagle’sBSS (Balanced Salt Solution )、調整至 1.5 g/L 之碳酸氫納、 0.1 mM之非必須胺基酸、1.0 mM之丙酮酸鈉、10%之 φ 加熱去活化的胎牛血清，並選擇性帶有5% S/D-PC或Molecular Devices, USA) measured 35 1344963 at 450 nm in 30 minutes. The minimum detectable dose is 0.7 pg/πύ. d-IGF-1 IGF-1 was quantified using a Quantikine ELISA kit (DG100 from R&d SYSTEMS). The sample was diluted 1〇〇 with Calibrator Diluent ‘ (RD5-22). As stated by the manufacturer, the minimum detectable dose range is from 〇.(8)7 to 0.056 ng/m and the mean MDD is 0.026 ng/ml. 150 μM of the measurement diluent φ (RD1-53) was added to each well, followed by a 50 μΐ standard (890775). The well plate was covered with a strip of glue and incubated at 2-8 ° C for 2 hours. The wells were washed 3 times, and then IGF-1 conjugated with the enzyme was co-cultured at 2-8 ° C for 1 hour. The measurement was completed as described above. 1.7 - Statistical Analysis The data for all series of experiments is presented as mean, standard deviation, and minimum and maximum values. A statistical comparison was made with a two-tailed paired Student test. The p value is small • The 〇.〇5 series is used to estimate the difference in the average PGF concentration in the different steps of the platelet preparation procedure (the Significance 〇f the - differences). The value is expressed as inconspicuous (NS, < 〇.〇5), <0.01 or <0.00, and when the value is close to 0.05, a true p value is displayed. The preparation and separation of samples comparing I.8-PC, S/D-PC and Act-PC correspond to the initial gold plate thick solution (PC), solvent/cleaning, (1%1^ and 1%Trit). 〇nX_45) The treated initial platelet concentrate (S/D-PC) and the CaCl2-activated starting platelet concentrate (ACt_PC) were separated by SDS-PAGE to compare the 36 1344963 protein content. • Mix 20 pg of each sample with 5 plNuPAGELDS sample buffer. (4x ) ( Invitrogen ), 2 μΐ NuPAGE reducing agent (ι) (Invitrogen) and deionized water to give a final volume of 2 μμΐ (activated) The sample of platelet concentrate was 21 μΐ). The resulting mixture was then heated at 70 ° C for 10 minutes and subjected to SDS_PAGE using 4-12% polyacrylamide gradient gel (NuPAGE Bis-Tris, Invitrogen). The φ protein separation was carried out for 35 minutes under a constant voltage of 200 V, wherein the predetermined current was 150 mA/gel. The resulting gel was stained with Coomassie blue R-250. The protein marker ^1 & also 12 unstained standard (11^Du Honghong 11) is used to measure the molecular weight of the protein in the sample. The Mark 12 marker was loaded onto a NuPAGE novex 4-12% Bis-Tris gel (Invitrogen) with MES and separated by Coomassie Blue R_25® after separation. The corresponding results are disclosed in the fourth figure. The egg white map (pr〇file) of the initial platelet concentrate treated with the solvent/detergent method showed no significant difference from the untreated initial platelet concentrate, whereas the activated platelet desert solution The protein map - the spectrum is significantly different, the bands in the 40 to 70 coffee area disappear, and these bands correspond to the alpha, P and subunits of fibrinogen, 63.5, 56 and 47 respectively. kDa. 1.9-Growth factor activity assay In order to determine whether the growth factor obtained by platelet S/D treatment (1% TnBp_1% Tdt〇n X-45) can maintain activity, human osteoblast-like MG-63 cell line was used for in vitro cells. Cultivate research. 37 1344963 MG-63 cells were treated with growth factors obtained from S/D-PC or from Act-PC • concentrated, and cell responses were assessed by cell morphology and survival. 105 to 106 cells were cultured in a 35-mm Petri dish using 90% Minimum essential medium Eagle (MEM) with 2 mM-glutamic acid, Eagle's BSS (Balanced Salt Solution) ), adjusted to 1.5 g / L sodium bicarbonate, 0.1 mM non-essential amino acid, 1.0 mM sodium pyruvate, 10% φ heated deactivated fetal bovine serum, and optionally with 5% S / D-PC or

Act-PC生長因子濃厚液。於37°C在包含5%C02及95% 空氣之潮溼大氣中培養後，以磷酸缓衝鹽溶液（PBS， Gibco，UK)清洗細胞，之後用胰蛋白酶-EDTA溶液 (0.25%胰蛋白酶）於37〇C作用5分鐘，使細胞脫落， _ 之後離心並懸浮，以進行進一步的細胞測試。 使用3-(4,5-二曱基噻唑-2-基)-2,5-二苯基四唑鑌溴 化物（MTT)測定法來偵測活/死細胞（存活率測定法）。 另亦進行電子顯微鏡觀察來研究細胞形態。 • 使用3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯 基)-2-(4-續苯基)-2H-四唾鑌（MTS )測定法來監測細胞 增生。簡言之，在各樣本中加入500 μΐ之MTS ( Celliter 96PromegaCorp.，USA)。於 37°C 培養 60 分鐘後，使用 微孔盤讀取儀來測量490 nm之吸光值。 1.10-病毒去活化測定 用於起始血小板濃厚液溶胞作用之溶劑及/或清潔 劑處理的病毒效力研究係以規模較低的實驗來進行，其 係根據國際基準如EMEA及WHO的建議來進行。分別 38 將50 ml之起始血小 類免疫不全病毒）及^^、^相關病毒㈤V，人 毒性腹篇病毒（BVDf】广的儲存懸⑦液·牛病 性狂犬病病毒(PRV));=型肝炎病毒之模型；假 時，有時用作=有其他方便活體外模型病毒 (VSV)。將這些病毒二，儲文及水泡性口炎病毒 板濃厚液，並加入儲存懸浮液引入起始血小 病毒感染力財_ f f ^ % tdtGn X_45之混合物。 的時間點進行估瞀，一/月泳"丨處理之前及處理期間不同 活體外細胞培養;毒的動力學。進行 值來表示。所得數據二 數據係以TCIDw/ml之 内使4種病主員不，S/D處理會在處理後5分鐘 ㈣制母凡全去活化（ 未毒感染力。丄’ 二為6.4 log10，而在vsv為>7 〇 1〇以。故溶 =$溶胞處理會確保可能存在於起始血小^ 液洛胞產物中之脂質套膜病毒當t強壯的病毒去活化旱 1.11-過期冷凍血小板的生長因子組成物 、將過期（收集後超過5天）血小板濃厚液移入· c冷凍庫儲存一個月。之後將它們在3rc水洛中解 並進行前述與新鮮血小板相同的實驗。 借％ 工的生長因子含量，對應結果係揭示於第 1.12-灰减塊形成測定 從經S/D處理之起始血小板濃厚液得出的生長因子 39 遭厚液係藉由油萃取來移除溶劑及清潔劑之後進行回 收。將5 ml所得生長因子濃厚液引入一 5-ml針筒中。 將5 ml之500 IU/ml的牛凝血酶引入另一 5-ml針筒中。 將兩個針筒置於一個以單一喷嘴連接的雙針筒供給 器。讓這兩種成分從中通過。血小板凝膠會在5秒内形 成。 II·結果 II. 1-細胞計數 對從十名不同的捐贈者所得之十份血小板分離術 濃厚液進行研究。得自血小板分離術之血小板濃厚液結 果平均為1064 ± m2 X 106個血小板/mi (範圍：782_ 1358 X 106個血小板/ml)，平均WBC計數為0.1125 土 0.025 X 10個/ml (枕圍· 0.0-1.5 ) ’且平均RBC含量為 0.0212 ± 0.025 X 1〇6 個/m卜 II.2-生長因子含量 PDGF-AB、TGF-P1、EGF及咖]在多種血小板 製備中的平均濃度±標準差（SD)、最小值及最大值、以 及p值係示於表卜從各系列實驗所得之個別數據點可 見於第二圖。Act-PC Growth Factor Thick Liquid. After culturing in a humidified atmosphere containing 5% CO 2 and 95% air at 37 ° C, the cells were washed with phosphate buffered saline (PBS, Gibco, UK), followed by trypsin-EDTA solution (0.25% trypsin). After 37 min of C 作用C, the cells were detached, and then centrifuged and suspended for further cell testing. The live/dead cells (survival assay) were detected using the 3-(4,5-dimercaptothiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Electron microscopy was also performed to study cell morphology. • Use of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-continuous phenyl)-2H-tetrasporin (MTS) Assays to monitor cell proliferation. Briefly, 500 μM of MTS (Celliter 96 Promega Corp., USA) was added to each sample. After incubation at 37 ° C for 60 minutes, the absorbance at 490 nm was measured using a microplate reader. 1.10-Virus Deactivation Assay The solvent potency study for solvent and/or detergent treatments used to initiate platelet thick lysis is performed on a lower scale experiment based on international benchmarks such as EMEA and WHO recommendations. get on. 38 (50 ml of the initial blood subtype of immunodeficiency virus) and ^^, ^ related virus (5) V, human toxicity of the abdominal virus (BVDf) widely stored 7 liquid · bovine rabies virus (PRV); Hepatitis virus model; when used, sometimes used = there are other convenient in vitro model viruses (VSV). These viruses were stored in a concentrated solution of vesicular stomatitis virus plate and added to the stock suspension to introduce a mixture of the initial blood virulence virus _f f ^ % tdtGn X_45. The time points were estimated, one/month of swimming " 丨 before treatment and during treatment during different in vitro cell culture; kinetics of toxicity. Value is used to indicate. The data obtained from the data are within TCIDw/ml so that the four disease controllers do not, and the S/D treatment will be activated 5 minutes after the treatment (four), and the mother will be completely deactivated (non-toxic infection force. 丄' two is 6.4 log10, and The vsv is >7 〇1〇. Therefore, the solution = $ lysate will ensure that the lipid envelope virus may be present in the initial blood sputum cell product when the t strong virus deactivates the drought 1.11 expires frozen Platelet growth factor composition, the platelet thick solution which was expired (more than 5 days after collection) was transferred to the c library for one month, and then they were decomposed in 3rc water and subjected to the same experiment as the fresh platelets described above. Factor content, the corresponding results are revealed in the 1.12 - gray reduction block formation assay from the S / D treatment of the initial platelet thick solution derived from the growth factor 39 thick liquid is extracted by oil to remove the solvent and detergent Recycling was carried out. 5 ml of the resulting growth factor concentrate was introduced into a 5-ml syringe. 5 ml of 500 IU/ml bovine thrombin was introduced into another 5-ml syringe. Double syringe feeder connected to the nozzle. Let these two The components pass through. The platelet gel will form within 5 seconds. II. Results II. 1-cell counts Ten platelet separation concentrates from ten different donors were studied. Platelets from platelet separation The results of the concentrated solution averaged 1064 ± m2 X 106 platelets/mi (range: 782_ 1358 X 106 platelets/ml), and the average WBC count was 0.1125 ± 0.025 X 10 cells/ml (pillow circumference 0.0-1.5 ) 'and average RBC content is 0.0212 ± 0.025 X 1 〇 6 / m 卜 II. 2 - growth factor content PDGF-AB, TGF-P1, EGF and coffee] average concentration in standard platelet preparation ± standard deviation (SD), minimum The maximum value and the p value are shown in Table 2. The individual data points obtained from each series of experiments can be found in the second figure.

PDGF-AB TGF-βΙ EGF IGF (N=10) (N=10) (N 二 8) (N = 6) m 13.8 16.6 <0.0007 83.4 A SD 14.3 14.3 - 32.8 Min. 2.2 1.1 60.1 Max. 49.5 36.0 - 162.7 m 184.4 192.2 2.2 88.4 B SD 80.2 37.4 1.6 33.5 Min. 107.7 140.4 0.7 68.8 Max. 392.6 272.6 5.0 170.1 m 84.6 63.8 0.9 117.2 C SD 35.5 14.1 0.6 34.9 Min. 52.8 48.6 0.2 82.3 Max. 209.5 88.5 1.6 195.3 m 88.3 68.6 1.40 112.4 D SD 45.9 27.2 1.0 39.7 Min. 52.0 38.3 0.5 73.0 Max. 209.5 132.1 3.0 202.1 B vs A <0.001 <0.001 <0.05 0.025 C vs A <0.001 <0.001 <0.05 <0.001 P值 B vs C <0.001 <0.001 0.044 <0.001 B vs D <0.001 <0.001 0.087 <0.01 CvsD NS NS NS NS 表1 :在起始血小板中（A)、在l%TnBP-l%TritonX-45 1344963 處理後(B)、以及在CaCl2活化後(C)接著對釋出物進行 1% TnBP-1% Triton X-45處理(D)的生長因子平均濃度 (ng/ml)。NS :無明顯差異。 在起始血小板濃厚液中的平均PDGF-AB含量為 13.8 士 14.3 ng/ml (N=10)。在以 TnBP-TritonX-45 進行 直接S/D處理後，其含量會明顯增加（p<0.001)至184,4 ± 80.2 ng/ml，相當於比起始血小板濃厚液增加大約13 倍。當起始血小板濃厚液首先被CaCl2活化時，其含量 41 也會明顯增加（84.6 ± 35.5，p<〇.〇〇l) ’但增加量較少 (約增加6倍），而且它在後續S/D處理期間保持實質 上未改變的狀態（88.3±45.9，NS)。在未活化的經^ 處理之血小板中，PGDF-AB含量明顯高於其在已活化、 以及已活化/經S/D處理之血小板中的含量 在TGF-βΙ方面也有類似的數據。在起始血小板濃 厚液中的平均TGF-βΙ含量為16,6 ± 143 ng/= (N=10)。直接S/D處理後，TGF_pi含量會比起始pc 增加近 12 倍’為 192.2 ± 37.4 ng/ml (p<〇.〇〇1 )。當起始 血小板濃厚液首先被CaCl2活化時，含量會增加^ 4 ^ (63.8± 14.1 ng/ml) (p<〇.001)並在後續 S/D 處理期 ^ 保持在沒有明顯不同的狀態（68.6±27.2ng/ml)。又: 在經S/D處理之血小板中，TGF_pi平均含量明顯高於 其在已活化、以及已活化再經S/D處理之血小板中=值 (p<0.001)。 在起始血小板濃厚液中，無法偵測到 pg/ml)，但在S/D處理後會變成可偵測到，且平均 含量（2.2±1.6ng/ml ’ .6)明顯高於（p<〇 〇5) CaCl2 活化後所得之值（0.9 ±0.6 ng/ml) (p<〇 〇5)，或者明顯 高於在CaCb活化後接著進行s/D處理所得之值（丨4 + 1.0 ng/ml)〇 在起始血小板濃厚液中，IGF-1平均含量為83 4 + 32.8ng/ml(N=8)。相對於其他血小板源生長因子來說了 它在S/D處理後似乎沒有明顯的增加（88 4 ±幻5， p=0.025)。在CaCh活化後之血小板製備中，它的平均 含量甚至還稍高（P^.OOl) (ll7.2± 34 9 ng/ml)，且在 後續S/D處理之後保持穩定（H2.4 ± 39.7 ng/ml)。 42 1344963 在150 ml血小板濃厚液中，直接S/D處理後（153 - ml)所回從的 PDGF-AB、TGF-β卜 EGF 及 IGF-1 總量 分別為 28213、29406、336 及 13525 ng ;而在 CaCl2 活 . 化後則分別為1〇152、7150、1〇8及14064 ng;血小板 凝膠形成（120 ml)所引起的20%平均體積損失亦計算 在内。這確5忍了 S/D處理在釋出pDGF-AB、TGF-βΙ及 EGF方面的效度會比CaCl2活化來得高。 表2比較了血小板源生長因子在起始新鮮血小板濃 厚液中（A)、在離心後（離心成一團塊並移除血小板）接 響著對上清液進行TnBP-Tdton X-45處理時⑼、及以1% ΤηΒΡ-1 % Triton X-45對起始血小板濃厚液進行S/D處理 後(C)或以2%TnBP對之進行S/D處理後(D)的含量。 PDGF-AB、TGF-β卜EGF及IGF-1在未處理之血小板 - 濃厚液(Α)中的含量與先前數據一致（表1)。當無血小 板之上清液（Β)接受TnBP_Triton Χ-45處理時（Β)， PDGF-AB、TGF-βΙ或EGF之含量保持低量或無法偵測 (3.9 ; 1.1 ; p<〇.〇〇l) ’其中IGIM之含量高，但與其在 φ 起始血小板濃厚液中的量類似（75·4對72.；2 ng/ml)。故， PDGF_AB、TGF-βΙ及EGF在S/D處理期間的釋出係取 決於金小板的存在。在經1% TnBP-1% Triton X-45或2% ΤηΒΡ處理之血小板溶胞產物中’血小板源生長因子的 含量增加是類似的。進一步來說，血小板濃厚液接受1% tritoiiX-45或l%tritonX-100處理後，後續清潔劑處理 會在沒有油萃取情況下藉由tC18吸附作用（SPlT45tC18 及SPlT100tC18)移除；這顯示，單以2% ΤηΒΡ或以 1% ΤηΒΡ及1% Triton Χ-45處理後所觀察到的生長因子 PDGF-AB、TGF-βΙ、IGF及EGF釋出情形係在一範圍 43 1344963 内，故而指出血小板溶胞的程度實質上係與使用此一溶 劑及清潔劑合併物或單用溶劑或清潔劑的情況是相同 的。 最後，數據（未顯示）指出，樣本於x g或 800 X g下離心不會影響在此測定中所測量到的血小板 源生長因子之量。 PDGF-AB TGF-P1 egf IGF-1 A 2.9 1.1 <0.0007 72.2 B 3.9 1.1 <0.0007 75.4 C 128.7 207.9 0.8 79.9 D 133.7 185.6 1.2 89.8 表2·多種生長因子在起始血小板濃厚液中(a)、經離心 之無血小板上清液在TnBP-Triton X-45處理後(B)、在 TnBP-Triton X-45處理後(C)或在2% ΤηΒΡ處理後(D)之 起始企小板濃厚液+的濃度（ng/ml)。 進一步來說，纖維蛋白原、纖維黏連蛋白、白蛋白、 免疫球蛋白 IgG 及第 II、VII、VIII、IX、X、XI、XIII 凝血因子以及凡威勒伯氏因子的濃度係在經S/D處理之 起始血小板濃厚液及經CaCl2活化之血小板濃厚液所得 之生長因子濃厚液當t進行測量。這些結果係揭示於表 44 蛋白 S/D-PC Act-PC 纖維蛋白原（g/L) 2.6 0.2 纖維黏連蛋白（g/L) 0.33 0.12 第 VIII 因子(IU/ml) 0.92 <0.1 第II因子(IU/ml) 1.2 <0.1 第IX因子(IU/ml) 1.02 <0.1 第X因子（IU/ml) L15 <0.1 第 VII 因子（IU/ml) 1.08 <0.1 第 XIII 因子(IU/ml) 0.87 <0.1 第XI因子(IU/ml) 1.35 <0.1 凡威勒伯氏因子（IU/ml) 0.95 <0.1 白蛋白（g/L) 33.8 32.5 IgG (g/L) 11.5 11.2 1344963 表3 :在得自以本發明之l%TnBP-l%TritonX-45處理的 起始血小板濃厚液（S/D-PC)、或得自以CaCl2活化之起 始血小板濃厚液（Act-PC)的生長因子濃厚液中的多種 蛋白漢度比較。 故顯示經活化之血小板濃厚液會幾乎完全除去纖 維蛋白原及凝血因子。 Π.3-牛凝血酶活化實驗PDGF-AB TGF-βΙ EGF IGF (N=10) (N=10) (N 2 8) (N = 6) m 13.8 16.6 <0.0007 83.4 A SD 14.3 14.3 - 32.8 Min. 2.2 1.1 60.1 Max. 49.5 36.0 - 162.7 m 184.4 192.2 2.2 88.4 B SD 80.2 37.4 1.6 33.5 Min. 107.7 140.4 0.7 68.8 Max. 392.6 272.6 5.0 170.1 m 84.6 63.8 0.9 117.2 C SD 35.5 14.1 0.6 34.9 Min. 52.8 48.6 0.2 82.3 Max. 209.5 88.5 1.6 195.3 m 88.3 68.6 1.40 112.4 D SD 45.9 27.2 1.0 39.7 Min. 52.0 38.3 0.5 73.0 Max. 209.5 132.1 3.0 202.1 B vs A < 0.001 < 0.001 < 0.05 0.025 C vs A < 0.001 < 0.001 < 0.05 < 0.001 P Value B vs C < 0.001 < 0.001 0.044 < 0.001 B vs D < 0.001 < 0.001 0.087 < 0.01 CvsD NS NS NS NS Table 1: In the starting platelets (A) at 1% TnBP-l %TritonX-45 1344963 After treatment (B), and after CaCl2 activation (C), the release compound was subjected to 1% TnBP-1% Triton X-45 treatment (D) for the average growth factor concentration (ng/ml). NS: No significant difference. The average PDGF-AB content in the starting platelet concentrate was 13.8 ± 14.3 ng/ml (N = 10). After direct S/D treatment with TnBP-TritonX-45, its content increased significantly (p<0.001) to 184,4 ± 80.2 ng/ml, which is equivalent to approximately 13-fold increase over the initial platelet concentrate. When the initial platelet concentrate is first activated by CaCl2, its content 41 will also increase significantly (84.6 ± 35.5, p < 〇.〇〇l) 'but the increase is less (about 6 times increase), and it is in the subsequent S The /D process remains substantially unchanged (88.3 ± 45.9, NS). In the unactivated treated platelets, the PGDF-AB content was significantly higher than that in the activated, and activated/S/D treated platelets. Similar data were also found for TGF-βΙ. The average TGF-βΙ content in the starting platelet concentrate was 16,6 ± 143 ng/= (N=10). After direct S/D treatment, the TGF_pi content will increase by nearly 12 times that of the starting pc' to 192.2 ± 37.4 ng/ml (p<〇.〇〇1). When the initial platelet concentrate is first activated by CaCl2, the content is increased by ^ 4 ^ (63.8 ± 14.1 ng/ml) (p < 〇.001) and remains in a distinctly different state during the subsequent S/D treatment period ( 68.6 ± 27.2 ng / ml). Also: In the S/D-treated platelets, the average TGF_pi content was significantly higher than that in the activated, and activated, S/D-treated platelets (p<0.001). In the initial platelet thick solution, pg/ml could not be detected, but it became detectable after S/D treatment, and the average content (2.2±1.6 ng/ml ' .6) was significantly higher than (p&lt ;〇〇5) The value obtained after CaCl2 activation (0.9 ±0.6 ng/ml) (p<〇〇5), or significantly higher than the value obtained after SiO2 activation followed by s/D treatment (丨4 + 1.0 ng) /ml) The average level of IGF-1 in the starting platelet concentrate was 83 4 + 32.8 ng/ml (N=8). Compared to other platelet-derived growth factors, it did not appear to have a significant increase after S/D treatment (88 4 ± phantom 5, p = 0.025). In the preparation of platelets after CaCh activation, its average content was even slightly higher (P^.OOl) (ll7.2±34 9 ng/ml) and remained stable after subsequent S/D treatment (H2.4 ± 39.7 ng/ml). 42 1344963 The total amount of PDGF-AB, TGF-β, EGF and IGF-1 returned by direct S/D treatment (153 - ml) in 150 ml platelet concentrate was 28213, 29406, 336 and 13525 ng, respectively. After CaCl2 was activated, it was 1〇152, 7150, 1〇8 and 14064 ng, respectively; the 20% average volume loss caused by platelet gel formation (120 ml) was also counted. This does not allow the S/D treatment to be more effective in releasing pDGF-AB, TGF-βΙ and EGF than CaCl2 activation. Table 2 compares platelet-derived growth factor in the initial fresh platelet concentrate (A), after centrifugation (centrifugation into a mass and removal of platelets), when the supernatant is subjected to TnBP-Tdton X-45 treatment (9) And the content of the initial platelet thick solution after S/D treatment (C) or S/D treatment (D) with 2% TnBP at 1% ΤηΒΡ-1% Triton X-45. The levels of PDGF-AB, TGF-β, EGF and IGF-1 in untreated platelet-thick liquor (Α) were consistent with previous data (Table 1). When the platelet-free supernatant (Β) was treated with TnBP_Triton®-45 (Β), the content of PDGF-AB, TGF-βΙ or EGF remained low or could not be detected (3.9; 1.1; p<〇.〇〇) l) 'The content of IGIM is high, but similar to the amount in φ starting platelet concentrate (75·4 vs. 72.; 2 ng/ml). Therefore, the release of PDGF_AB, TGF-βΙ and EGF during S/D treatment depends on the presence of gold platelets. The increase in 'platelet-derived growth factor' content in platelet lysates treated with 1% TnBP-1% Triton X-45 or 2% ΤηΒΡ was similar. Further, after the platelet concentrate is treated with 1% tritoiiX-45 or 1% tritonX-100, the subsequent detergent treatment is removed by tC18 adsorption (SPlT45tC18 and SPlT100tC18) without oil extraction; The growth factors PDGF-AB, TGF-βΙ, IGF and EGF observed after treatment with 2% ΤηΒΡ or 1% tonηΒΡ and 1% Triton Χ-45 were in a range of 43 1344963, indicating that platelet dissolution The extent of the cells is substantially the same as in the case of using such a solvent and detergent combination or a solvent or detergent alone. Finally, the data (not shown) indicate that centrifugation of the sample at xg or 800 Xg does not affect the amount of platelet-derived growth factor measured in this assay. PDGF-AB TGF-P1 egf IGF-1 A 2.9 1.1 <0.0007 72.2 B 3.9 1.1 <0.0007 75.4 C 128.7 207.9 0.8 79.9 D 133.7 185.6 1.2 89.8 Table 2. Various growth factors in the starting platelet concentrate (a) The centrifuged platelet-free supernatant is treated with TnBP-Triton X-45 (B), after TnBP-Triton X-45 treatment (C) or after 2% ΤηΒΡ treatment (D) Concentration of concentrated liquid + (ng/ml). Further, the concentrations of fibrinogen, fibronectin, albumin, immunoglobulin IgG, and II, VII, VIII, IX, X, XI, XIII coagulation factors and Van derrick's factor are in the S The growth factor thick solution obtained from the /D-treated initial platelet concentrate and the CaCl2-activated platelet concentrate was measured as t. These results are disclosed in Table 44 Protein S/D-PC Act-PC Fibrinogen (g/L) 2.6 0.2 Fibronectin (g/L) 0.33 0.12 Factor VIII (IU/ml) 0.92 <0.1 Factor II (IU/ml) 1.2 <0.1 Factor IX (IU/ml) 1.02 <0.1 Factor X (IU/ml) L15 <0.1 Factor VII (IU/ml) 1.08 <0.1 Factor XIII (IU/ml) 0.87 <0.1 Factor XI (IU/ml) 1.35 <0.1 Van Wyber's Factor (IU/ml) 0.95 <0.1 Albumin (g/L) 33.8 32.5 IgG (g/L 11.5 11.2 1344963 Table 3: Starting platelet concentrate (S/D-PC) obtained from 1% TnBP-1% Triton X-45 of the present invention, or starting platelet concentrate obtained by activation with CaCl2 (Act-PC) Comparison of various protein hanrosities in growth factor concentrates. Therefore, it is shown that the activated platelet concentrate almost completely removes fibrinogen and clotting factors. Π.3-bovine thrombin activation experiment

CaCl2/玻璃球粒處理只會部分活化血小板，這可能 可以解釋其釋出物中的PDGF-AB'TGF-βΙ及EGF含量 要比S/D溶胞產物中來得低；為了破除上述假設，故測 定這些血小板源生長因子在血小板濃厚液之釋出物中 45 1344963 的含量（第三圖），#中前述血小板濃厚液之釋出物首 先被CaCh活化，之後接受牛凝血酶（Act_T)及凝血酶 =進一步處理，並接受進一步S/D處理（Act_T_s/D)。 第二圖顯示：PDGFAB (A)、TGF-βΙ (B)及 EGF (C)在起 始亡小板濃厚液中（起始）、在經CaC12活化後（Act) 接著進行牛凝血酶活化（Act-T )及S/D處理（Act_T_SD )CaCl2/glass pellet treatment only partially activates platelets, which may explain the PDGF-AB'TGF-βΙ and EGF content in the release is lower than that in the S/D lysate; in order to break the above assumptions, The content of these platelet-derived growth factors in the release of platelet thick liquor 45 1344963 (third image), the release of the aforementioned platelet thick solution was first activated by CaCh, followed by bovine thrombin (Act_T) and coagulation Enzyme = further processing and further S/D treatment (Act_T_s/D). The second panel shows that PDGFAB (A), TGF-βΙ (B), and EGF (C) are activated in the initial platelet thickening (starting), after CaC12 activation (Act) followed by bovine thrombin activation ( Act-T) and S/D processing (Act_T_SD)

時的含量。可以看到，與CaCU活化相較之下，再加上 牛凝血酶活化不會增加pdgf ab (a)、TGf EGF(C)之釋出。 P 廷是首度顯示，對血小板進行S/D處理時，血小板 生長因子的釋出（特別是PDGF-AB'TGF-βΙ及EGF的 釋出）會比用鈣及/或凝血酶來活化血小板時要高得多。 S/D處理理應會誘發富含脂質之血小板膜溶解，而使血 小板源生長因子從細胞内α顆粒中釋出。事實是，當先 進行凝血酶活化時，那些血小板源生長@子的釋出可能 會比較低，這可能是因為，凝集之血小板及部分釋出的 ，小板源生長因子會被抓在纖維蛋白網絡（稱為血小板 凝膠）中，該網絡係由起始企小板濃厚液中之纖維蛋白 ，被凝血酶誘發產生聚合作用而形成的。確實，CaCi2 只，致部分活化、以及内源性凝企酶引起的血凝塊形成 不凡全的可旎性，可以被在已活化之血小板釋出物中進 步加入約48 NIH單位之牛凝血酶/w後，仍不會導致 血小板源生長因子釋出量繼續增加的事實來加以排 除。血小板的完全活化亦可用纖維蛋白原及凝血因子濃 度極低（或無法偵測）的情形來加以確認，如上表3所 示。 同樣地（未顯示），我們發現，當使用牛凝血酶活 46 量是厚液時’在釋出液中的血小板生長因子的含 持為實Γ1改ί們=現，1GF_1含量在s/D處理後保 加。這項凝血酶活化後只有輕微地增 出絕大多數的IGF係以自由循環 的事實存在於錢中，只有極小比例存在於4小板中 我們的數翻*，與其他方法如凝血酶活化、冷陳 • I東週期及/或冷凍乾燥相較之下，以S/D處理來進行 2、板溶胞作用是使血小板生長因子從血小板細胞内 •立釋出到渗出液中最有效的手段。猶早的研究業已發 ，，^類全血血清中的平均PDGF量為17.5 ng/ml，即 :/個血小板含〇 〇6 ng之量。這個量相當於我們的研 究當中的分離術血小板濃厚液6〇 ng/m卜而我們發現， 經S/D處理之企小板濃厚液中的pDGF量平均約三倍以 上。我們的數據進一步顯示，要使pDGF_AB、TGF-pj 及EGF從血小板釋出量增加，可藉由使用1%TnBp_1〇/〇The amount of time. It can be seen that, in contrast to CaCU activation, bovine thrombin activation does not increase the release of pdgf ab (a) and TGf EGF (C). P Ting is the first to show that when S/D is treated with platelets, the release of platelet growth factor (especially the release of PDGF-AB'TGF-βΙ and EGF) will activate platelets with calcium and/or thrombin. It is much higher. S/D treatment should induce the dissolution of lipid-rich platelet membranes, and release platelet-derived growth factors from intracellular alpha particles. The fact is that when thrombin activation is first performed, the release of those platelet source growth may be lower, probably because agglutinated platelets and partially released, small plate source growth factors are caught in the fibrin network. In a platelet gel (called a platelet gel), the network is formed by the initiation of polymerization by fibrin in the thick liquid of the initial plate. Indeed, CaCi2 only, partial activation, and endogenous coagulase-induced blood clot formation of extraordinary collaterality, can be promoted in activated platelet release to add about 48 NIH units of bovine thrombin After /w, the fact that platelet-derived growth factor release continues to increase is still not ruled out. Complete activation of platelets can also be confirmed by the extremely low (or undetectable) concentration of fibrinogen and clotting factors, as shown in Table 3 above. Similarly (not shown), we found that when the amount of bovine thrombin was used, the amount of platelet growth factor in the effluent was Γ1 ̄ ̄ ̄ = now, 1GF_1 content in s/D After processing, it will be added. This thrombin activation only slightly increases the fact that most of the IGFs are free to circulate in the money, only a very small percentage of the 4 small plates are in our number*, and other methods such as thrombin activation, Cold Chen • I Eastern cycle and / or freeze-dried compared to S / D treatment 2, plate lysis is the most effective release of platelet growth factor from platelet cells to exudate means. In the early research, the average amount of PDGF in the whole blood serum was 17.5 ng/ml, that is, the amount of platelets containing 〇 〇 6 ng. This amount is equivalent to 6 ng/m of the separation platelet concentrate in our study and we found that the amount of pDGF in the S/D-treated slab thick solution was on average about three times higher. Our data further show that to increase the release of pDGF_AB, TGF-pj and EGF from platelets, 1% TnBp_1〇/〇 can be used.

Triton X-45 合併物、2% ΤηΒΡ、或 i〇/0 triton X-45 或 Triton X-100的S/D處理來進行，且以油萃取處理來移除S/D， 不會降低血小板溶胞產物中4種血小板生長因子的含 量。 II.4-生長因子濃厚液中之脂質含量的比較 起始血小板濃厚液經S/D處理或Caci2活化後，血 小板生長因子濃厚液中的脂質含量結果係經測量，並與 起始血小板濃厚液中的含量進行比較，如表4所示。 47 1344963 膽固醇 三酸甘油酉旨 HDL LDL (mg/dl) (mg/dl) (mg/dl) (mg/dl) 起始PC 164 170 33 99 Trit-PC 160 162 101 62 S/D-PC 1 32 20 11 0 S/D-PC 3 33 43 2 0 Act-PC 150 156 31 97 表4:從相同起始血小板濃厚液製備之生長因子濃厚液^ 的脂質含量比較，其係使用0.5% Triton ⑽ (Trit-PC);或在本發明之S/D處理後，接著進行〜劣 油萃取（S/D-PC 1);或在本發明之S/D處理後，接 行三次油萃取（S/D-PC3);或者在血小板之caCl 違 2 /〇 後進行。 測 個血 脂質含量係藉由Hitachi臨床技術儀器來進行 試。各受試樣本係以pH值為7.2且包含886 X lQ3 τ χ 1〇6 小板/μΐ、0.1 X 103 個白血球（WBC) /μΐ 及 〇 〇8 個紅血球（RBC) /pl的相同起始血小板濃厚液來製1 如表4所示，與經Triton處理之血小板或與備。 之血小板相較之下，在藉由S/D處理再接著進行，化 所製備之S/D-PC生長因子濃厚液中’ ldl (低a萃取 蛋白）、HDL (尚密度脂蛋白）、三酸甘油酯及膦脂 明顯較低。進一步來說，經S/D處理溶胞之血^醇量 萃取來萃取一次或三次不會觀察到明顯的差異。板以油 本發明之生長因子濃厚液中膽固醇、三酸 LDL被除去，將引起臨床及治療目的方面报大的^贿及 48 J344963 因為目前已知三酸甘油酯、以及LDL-膽固醇合併物在 動脈粥狀硬化及透過動脈及靜脈系統中脂肪斑形成所 弓丨起的心血管疾病的發展中所扮演的角色，這些問題會 • 引起心臟病發作、中風及周邊血管疾病。 II.5-生長因子活性 顯微鏡觀察顯示細胞在形狀、大小及數目上的變 -化：當細胞與經S/D處理或CaCh活化之起始血小板濃 岸液所得的血小板源生長因子濃厚液共同培養時，會特 籲 別觀察到許多纺錘狀細胞。進一步來說，細胞計數顯 乔，與Act-PC或未與生長因子濃厚液共同培養之細胞 相較之下，當細胞與SD-PC生長因子濃厚液共同培養 時’ MG-63成骨細胞的數目會以時間及劑量依存性的方 式有明顯的增加。MTT分析結果也顯示，與經Act-PC 處理之細胞相較之下，SD-PC生長因子濃厚液的存在會 增加培養細胞的細胞活性。S/D-PC或Act-PC生長因子 濃厚液都不會對MG-63成骨細胞表現出細胞毒性。 • 故本研究的結果顯示’從經S/D處理之血小板或經Triton X-45 combination, 2% ΤηΒΡ, or i〇/0 triton X-45 or Triton X-100 S/D treatment, and oil extraction to remove S/D, does not reduce platelet dissolution The content of four platelet growth factors in the cell product. II. Comparison of lipid content in the growth factor thick solution After the initial platelet thick solution was activated by S/D or Caci2, the lipid content in the platelet growth factor thick solution was measured and compared with the starting platelet thick solution. The contents in the comparison are as shown in Table 4. 47 1344963 Cholesterol Triglyceride HHDL LDL (mg/dl) (mg/dl) (mg/dl) (mg/dl) Starting PC 164 170 33 99 Trit-PC 160 162 101 62 S/D-PC 1 32 20 11 0 S/D-PC 3 33 43 2 0 Act-PC 150 156 31 97 Table 4: Comparison of lipid content of growth factor concentrates prepared from the same starting platelet concentrate using 0.5% Triton (10) (Trit-PC); or after the S/D treatment of the present invention, followed by ~ poor oil extraction (S/D-PC 1); or after the S/D treatment of the present invention, three oil extractions (S /D-PC3); or after the platelet's caCl violates 2 /〇. Measuring blood lipid levels were tested by Hitachi clinical instrumentation. Each sample was based on pH 7.2 and contained 886 X lQ3 τ χ 1〇6 platelets/μΐ, 0.1 X 103 white blood cells (WBC) / μΐ and 〇〇 8 red blood cells (RBC) / pl. The initial platelet thick solution was prepared as shown in Table 4, and treated with Triton-treated platelets or prepared. In contrast to the platelets, in the S/D-PC growth factor concentrate prepared by S/D treatment, 'ldl (low a extract protein), HDL (still density lipoprotein), three Acid glycerides and phospholipids are significantly lower. Further, no significant difference was observed when one or three extractions were performed by S/D treatment of lysed blood. The plate is oil-extracted in the growth factor thick liquid of the present invention, and the cholesterol and the tri-acid LDL are removed, which will cause the clinical and therapeutic purposes to be reported as bribes and 48 J344963 because triglyceride and LDL-cholesterol combination are currently known. Atherosclerosis and the role of cardiovascular disease through the formation of fatty plaques in the arteries and venous systems that can cause heart attacks, strokes, and peripheral vascular disease. II.5-Growth factor activity Microscopic observation shows the change in shape, size and number of cells: when the cells are combined with platelet-derived growth factor concentrate obtained by S/D treatment or CaCh-activated initial platelet concentration solution When cultured, many spindle cells are specifically observed. Further, the cell count showed that, compared with Act-PC or cells not co-cultured with growth factor concentrate, when the cells were co-cultured with SD-PC growth factor concentrate, 'MG-63 osteoblasts The number will increase significantly in a time and dose dependent manner. MTT analysis also showed that the presence of SD-PC growth factor concentrate increased the cell viability of cultured cells compared to Act-PC treated cells. S/D-PC or Act-PC growth factor concentrates did not exhibit cytotoxicity to MG-63 osteoblasts. • The results of this study show that 'from S/D-treated platelets or

活化之血小板兩者得到的生長因子，用所選之萃取方法 - 後仍保持活性。然而，我們的實驗顯示，當細胞與從S/D 處理得到的生長因子濃厚液共同培養時，細胞反應會明 顯增加。本發明之方法所得的濃厚液在MG-63細胞上的 效果有所改善，這可能是因為生長因子含量增加的關 係，特別是PDGF、TGF-βΙ及EGF (這些因子在傷口療 癒及組織再生方面的重要性係為已知）’及/或可能是因 為其他原本在經活化之jk小板濃厚液中不存在或表現 量低之生物活性物質的量增加的關係。 49 得的果ί確認了本發明或藉由本發明之方法所 性/旳if子濃厚液在治療的用途方面以及製備改良 、、-田月L培養基方面的重要潛力。 II.6-病毒去活化 此外，有許多證據顯示此處所使用的處理會 =i將血源性套膜病毒去活化，特別是及 HCV 〇 ,如上材料及方法所示，1% TnBP-l% Triton X-45合 ，物於3PC處理’會確保HIV、BVDV及PRV減縮係 數在處理後5分鐘内為>5.6、>6.6、及>6 4 一。，此外 也會快速地將>7.0 loglQ之vsv及辛德畢斯模型病毒 (Sindbis model virus)去活化。非套膜病毒（如ΉΑν 及小病毒Β19)無法藉由§/d處理而去活化，但它們只 對少數免疫功能改變的患者具有致病性。不做混合 (pooling)會大大降低單一捐贈者之同種異體血小板源 生長因子製劑污染在統計上的風險，但進一步奈米過渡 也可用來降低非套膜病毒污染的風險。顯然，此處所描 述之金小板源生長因子製劑將必須在優良製造標準 (GMP)的條件下生產，如血液機構之標準（參見 Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products. www.WHO.int. Geneva，2003:1-72)。這類血小 板製劑的S/D處理可在局部應用上提供一定程度的實用 優點。第一’同種異體捐輸的病毒安全性會有所改善， 而使這類產物擁有較廣的臨床潛力。第二，藉由S/D處 理來釋出效價較高的血小板源生長因子可能可以改善 50 1344963 使用血小板釋出物在程序上的成本效益問題。第三，在 不允許使用重組血小板源生長因子的情況下，病毒去活 化方法可以讓同種異體血小板源生長因子在應用上更 加方便；或者，在允許使用重組血小板源生長因子的情 況下（如用以處理某些下歧糖尿病潰癌時），當它盘天 然血小板源生長因子合併使用時，可得出有利的協同效 用。最後，經病毒去活化之血小板源生長因子在充分定 性後亦可併入以纖維蛋白為主的人工鷹架來作為局部 應用，以加速療癒，作為胎牛血清之替代物，或在細胞 工程研究、或間葉幹細胞 >舌體擴增（expansi〇n )及其分 化為骨細胞或軟骨細胞的過程中用作重組血小板&生 長因子。 ~ 【圖式簡單說明】 第一圖：研究設計：將捐輸的分離術血小 分成兩個子集池。子集池1係直接以1% TnBP-1% Triton X-45處理1小時’沒有先用CaCl：2活化。子集池2則先 用23 mM CaCl2及玻璃球粒加以活化，形成金小板凝 膠。經過1小時培養後，移除血凝塊，並將釋出物以1% TnBP-1% Triton X-45處理1小時。經S/D兩者（溶劑_ 清潔劑）處理之溶液經過油萃取（3次）’將溶劑及清潔 劑移除。測定起始血小板、活化後、S/D處理-油萃取後 的生長因子含量。 第二圖：（A) PDGF_AB、（B) TGF-βΙ、（〇 EGF 及 (D) IGF在起始血小板濃厚液中（起始）、在直接s/d處 理（1% TnBP_l% Triton X-45)後（S/D)、在起始血小 板濃厚液經CaCl2活化後（活化）、以及在經CaCl2活化 51 1344963 之血小板釋出物經S/D處理（1% TnBP- 1% Triton X-45) 後（活化+S/D)的含量（ng/ml)。s/D處理係如材料及 方法所述來進行，並包括3次油萃取來移除s/D用劑。 第三圖：（A) TOGF-AB、（B) TGF-βΙ 及(C) EGF 在 起始血小板濃厚液中（起始）、在起始血小板濃厚液經 CaCb活化後（Act)接著進行牛凝血酶活化（Act-T)及 S/D 處理（l%TnBP-l%TritonX-45)(ActT-S/D)時的 含量（ng/ml)。S/D處理係如材料及方法所述來進行， 並包括3次油萃取來移除S/D用劑。 第四圖：起始金小板濃厚液（第1行）、經本發明 之溶劑-清潔劑方法處理之相同血小板濃厚液（第2行） 及CaCl2活化後之相同血小板濃厚液（第3行）之間的 蛋白組成物比較。樣本係在4%-12%凝膠上以SDS-PAGE分離，之後蛋白以考馬斯藍染色。第M行相當於 蛋白標記（大小以千道爾頓(kiloDalton)計，標示在凝膠 左侧）。 第五圖：過期冷凍血小板分別接受S/D處理（1〇/〇The growth factors obtained from both activated platelets remain active after the selected extraction method. However, our experiments show that when cells are co-cultured with a growth factor concentrate obtained from S/D treatment, the cellular response is significantly increased. The effect of the concentrated solution obtained by the method of the present invention on MG-63 cells is improved, which may be due to an increase in the growth factor content, particularly PDGF, TGF-βΙ and EGF (these factors are in wound healing and tissue regeneration). The importance of the aspect is known to be 'and/or may be due to the increased amount of other biologically active substances that were not present in the activated jk platelet thick liquor or that exhibited a low amount of bioactive material. 49. The obtained fruit ί confirmed the important potential of the present invention or the method of the present invention for the use of the therapeutic substance and the preparation of the improved, Tianyue L medium. II.6-Virus Deactivation In addition, there is ample evidence that the treatment used here will =i deactivate the blood-derived envelope virus, especially HCV oxime, as indicated by the materials and methods above, 1% TnBP-l% Triton X-45 combined with 3PC treatment will ensure that the HIV, BVDV and PRV reduction factors are >5.6, >6.6, and > 6 4 within 5 minutes of treatment. In addition, the >7.0 loglQ vsv and Sindbis model virus will be quickly deactivated. Non-enveloped viruses (such as ΉΑν and small virus Β19) cannot be deactivated by §/d treatment, but they are only pathogenic to a small number of patients with altered immune function. No pooling can significantly reduce the statistical risk of contamination of a single donor's allogeneic platelet-derived growth factor formulation, but further nanotransition can also be used to reduce the risk of non-enveloped virus contamination. Obviously, the gold platelet growth factor formulation described herein will have to be produced under Good Manufacturing Practice (GMP) conditions, such as guidelines for viral inactivation and removal procedures intended to assure the viral safety of human blood. Plasma products. www.WHO.int. Geneva, 2003: 1-72). S/D treatment of such platelet formulations can provide a degree of practical advantage in topical applications. The safety of the first 'allogene-supplied virus will improve, giving these products a broader clinical potential. Second, the release of higher potency platelet-derived growth factors by S/D treatment may improve the procedural cost-effectiveness of using 50 1344963 platelet release. Third, in the case where recombinant platelet-derived growth factor is not allowed, the virus deactivation method can make the allogeneic platelet-derived growth factor more convenient to use; or, in the case of allowing the use of recombinant platelet-derived growth factor (such as In order to treat some of the hypothalamic cancers, when it is combined with natural platelet-derived growth factors, a favorable synergistic effect can be obtained. Finally, the virus-deactivated platelet-derived growth factor can be incorporated into a fibrin-based artificial eagle as a topical application to accelerate healing, as a substitute for fetal bovine serum, or in cell engineering. Research, or mesenchymal stem cells> lingual expansion (expansi〇n) and its differentiation into osteoblasts or chondrocytes are used as recombinant platelets & growth factors. ~ [Simple description of the diagram] The first picture: Study design: divide the blood of the donation into two subset pools. Subset pool 1 was treated directly with 1% TnBP-1% Triton X-45 for 1 hour without prior activation with CaCl:2. Sub-pool 2 was first activated with 23 mM CaCl2 and glass pellets to form a gold plate gel. After 1 hour of incubation, the blood clots were removed and the liberated material was treated with 1% TnBP-1% Triton X-45 for 1 hour. The solution treated with both S/D (solvent_detergent) was subjected to oil extraction (3 times) to remove the solvent and detergent. The growth factor content of the starting platelets, after activation, and after S/D treatment-oil extraction was measured. Figure 2: (A) PDGF_AB, (B) TGF-βΙ, (〇EGF and (D) IGF in the initial platelet concentrate (start), in direct s/d treatment (1% TnBP_l% Triton X- 45) After (S/D), after initiation of platelet thick solution by CaCl2 activation (activation), and in CaCl2 activation 51 1344963 platelet release by S/D treatment (1% TnBP-1% Triton X- 45) Post-(activation + S/D) content (ng/ml). The s/D treatment is carried out as described in Materials and Methods and includes 3 oil extractions to remove the s/D agent. :(A) TOGF-AB, (B) TGF-βΙ and (C) EGF in the initial platelet concentrate (start), after activation of CaCb in the initial platelet concentrate (Act) followed by bovine thrombin activation (Act-T) and S/D treatment (1% TnBP-l% Triton X-45) (ActT-S/D) content (ng/ml). S/D treatment is carried out as described in Materials and Methods And includes 3 oil extractions to remove the S/D agent. Figure 4: Start gold plate thick liquid (line 1), the same platelet thick solution treated by the solvent-cleaner method of the present invention (2nd) Line) and the same platelet thick solution after CaCl2 activation (3rd) Comparison of protein compositions between rows. Samples were separated by SDS-PAGE on 4%-12% gels, after which the proteins were stained with Coomassie blue. Line M corresponds to protein labeling (size in thousands of Daltons) (kiloDalton), indicated on the left side of the gel.) Figure 5: Overdue frozen platelets are treated with S/D (1〇/〇)

TnBP-1% Triton X-45) (S/D-PC)、或接受 CaCl2 活化 (Act-PC)、或選擇性在CaCl2活化接著進行S/D處理 (Act-PC+S/D)所得之濃厚液中，PDGF_AB(第五圖〇 及EGF (第五圖b )的組成物比較。 第六圈：過期冷凍血小板分別接受S/D處理（1%TnBP-1% Triton X-45) (S/D-PC), or accepting CaCl2 activation (Act-PC), or selective activation in CaCl2 followed by S/D treatment (Act-PC+S/D) In the thick solution, the composition of PDGF_AB (figure 5 and EGF (figure b) is compared. The sixth circle: expired frozen platelets are treated with S/D (1%)

TnBP-1% Triton X_45) (S/D-PC)或接受 CaCi2 活化 (Act-PC)所得之濃厚液中，igf_i(第六圖a)及 (第六圖b )的組成物比較。 【主要元件符號說明】 無 52The compositions of igf_i (sixth figure a) and (sixth figure b) were compared in a thick liquid obtained by TnBP-1% Triton X_45) (S/D-PC) or by CaCi2 activation (Act-PC). [Main component symbol description] None 52

Claims (1)

民國100年4月6日 十、申請專職® : 1. 一種病毒去活化的可凝結之血小板生長因子濃厚 液，其係用於治療性及/或美容性用途。 2·如申請專職圍第i項所述之病毒去活化的可凝結之 血小板生長因子濃厚液，其包含生長因子pDGF、 TGM、IGF、EGF、CTGF、bFGF 3. 如申請專利範圍第1項所述病毒去活化的之可凝結之 血小板生長因子濃厚液，其特徵在於沒有血液細胞相 關之輸血反應的風險。 4. 如申請專利範圍第1項所述之病毒去活化的可凝結之 血小板生長因子濃厚液，其進一步包含至少一種選自 由纖維黏連蛋白（fibroneetin )、透明黏連蛋白 (vitronectin)、凝血酶敏感蛋白（thromb〇sp〇ndin)、 凡威勒伯氏因子（v〇n Willebrand factor )與第II、V、 VII ' VIII、ιχ、χ及χι凝血因子所組成之組群的蛋 白。 5. 一種製備病毒去活化的可凝結之血小板生長因子濃 厚液的方法，其包含下列步驟： a) 使起始血小板濃厚液與溶劑及/或清潔劑接觸； b) 使前述起始血小板濃厚液與前述溶劑及/或清潔 劑於維持在從約6.0至約9.0範圍内之pH值及在 從2°C至50°C範圍内之溫度下共同培養至少5 分鐘至6小時的時間；以及 c) 藉由油萃取及/或層析方式來移除前述溶劑及/或 清潔劑。 民國100年4月6曰 6. 如申請專利範圍第5項所述之方法，其中前述溶劑係 選自由二-或三烷基磷酸酯類、具有不同烷基鏈之二 或三烷基磷酸酯類所組成之組群。 7. 如申請專利範圍第6項所述之方法，其中前述溶劑為 三正丁基磷酸酯（ΤηΒΡ)。 8. 如申請專利範圍第5項所述之方法，其中前述清潔劑 係選自由脂肪酸之聚氧乙烯衍生物、山梨糖醇酐之偏 酯類（partial esters )、非離子性清潔劑、去氧膽酸鈉 及磺基甜菜驗類（sulfobetaines)所組成之組群。 9. 如申請專利範圍第8項所述之方法，其中前述清潔劑 為 Triton X-45、Triton X-100 或 Tween 80。 10. 如申請專利範圍第5項所述之方法，其中前述溶劑及 /或前述清潔劑的個別最終濃度範圍為從〇. 2至5體積 %，其係以前述起始血小板濃厚液之體積為基礎計 算。 11. 如申請專利範圍第5項所述之方法，其中前述血小板 濃厚液係與單用之2% ΤηΒΡ接觸、或與1% ΤηΒΡ及 1 % Triton Χ-45接觸，其係以前述起始血小板濃厚液 之體積為基礎計异。 12. 如申請專利範圍第5項所述之方法，其中油萃取係以 醫藥等級油來進行，前述油之用量為從2至20重量 %、或從5至15重量％、或從5至10重量％，其係 以前述血小板濃厚液與前述溶劑及/或清潔劑之混合 物的重量為基礎計算。 13. 如申請專利範圍第5項所述之方法，其中前述層析方 式包含C18二氧化矽裝填材料、或SDR (溶劑-清潔 劑移除，Solvent-Detergent removal) hyper D。 】 民國丨00年4月6日 _ 4:二用利-=5項所述之方法，其進-步包含步 如申=範: :…包含-製備起始血=:::步前步述 16·如申請專利範圍S 15項所述之方 液係藉由血小板分離h、:：= 分離法而從全血力“乂 2濃厚液係為新鮮、過期'或過期且血 項所述之方法，其=製Τ 生長因子濃 1液用途的病甘去活化的可凝結之血小板 19.:種形成血凝塊的方法’其係 ^至17項中任-項所述之方法得出== 圍生長因子濃厚液、或如申請專利範 性用途的;广Λ 所述之用於治療性及/或美容 厚液與凝^混合舌化的可凝結之血小板生長因子濃 I::;:?圍第〗9項所述之方法，其係將。·ι至I 一積之活性乾圍為從2〇 117/111丨至1〇〇〇 因%=:述病毒去活化的可凝結之血二 第2°項所述之方法’其帽述凝血 2.種醫藥產物，其包含藉由如申請專利範圍第5至17 ^44963 民國100年4月6曰 項所述之方法得出的病毒去 = 反生長因子濃厚液、或如申請專利範圍第= ^中任-項所述之用於治療性及/或美容性 23. 可凝結之血小板生長因子濃厚液。 種错由如申請專利範圍第5至17項中任 24. 申請專利範圍第5至17項中任-項所述 方法侍出的血小板源生長因子 ^申請專利翻第！至4射任—項所述之 二或美容性用途的病毒去活化的可凝結== 25. 織液之用途，其係用於骨絡再生、或軟組 纖及硬組織的療癒。 -種藉由如申請專利範圍第5至17項中任一 m 套得出的病毒去活化的可凝結之血小板生長因 千/辰厚液、或如申請專利範圍第1至4項中任一項所 2用於治療性及/或美容性料的絲去活化的可 ^ '，、°之血小板生長因子遭厚液之用途，其係用於活體 26 — （明η⑽）或活體研究WV〇)之細胞培養。 種精由β如申請專利範圍第5至17項中任一項所述 出的病毒去活化的可凝結之血小板生長因 56April 6, 100, 100. Application for full-time®: 1. A virus-deactivated clotting platelet growth factor concentrate for therapeutic and/or cosmetic use. 2. For example, apply for a virus-deactivated clotting platelet growth factor thick solution containing the growth factor pDGF, TGM, IGF, EGF, CTGF, bFGF as described in item i of the full-time sub-paragraph i. A clotting platelet growth factor concentrate that is deactivated by the virus, characterized by the absence of a blood cell-associated transfusion reaction. 4. The virus-deactivated clotting platelet growth factor concentrate according to claim 1, further comprising at least one selected from the group consisting of fibroneetin, vitronectin, thrombin A protein consisting of a group of thromb〇sp〇ndin, v〇n Willebrand factor, and II, V, VII 'VIII, ιχ, χ, and χι coagulation factors. 5. A method of preparing a virus-deactivated clotable platelet growth factor concentrate comprising the steps of: a) contacting a starting platelet concentrate with a solvent and/or a cleaning agent; b) causing said starting platelet concentrate And co-cultivating the solvent and/or cleaning agent at a pH ranging from about 6.0 to about 9.0 and at a temperature ranging from 2 ° C to 50 ° C for at least 5 minutes to 6 hours; and The solvent and/or cleaning agent is removed by oil extraction and/or chromatography. The method of claim 5, wherein the solvent is selected from the group consisting of di- or trialkyl phosphates, di- or trialkyl phosphates having different alkyl chains. A group consisting of classes. 7. The method of claim 6, wherein the solvent is tri-n-butyl phosphate (ΤηΒΡ). 8. The method of claim 5, wherein the cleaning agent is selected from the group consisting of polyoxyethylene derivatives of fatty acids, partial esters of sorbitan, nonionic detergents, deoxygenated A group consisting of sodium cholate and sulfobetaines. 9. The method of claim 8, wherein the cleaning agent is Triton X-45, Triton X-100 or Tween 80. 10. The method of claim 5, wherein the respective final concentration of the solvent and/or the cleaning agent ranges from 0.2 to 5% by volume, which is the volume of the aforementioned starting platelet thick solution. Basic calculation. 11. The method according to claim 5, wherein the platelet thick solution is contacted with 2% ΤηΒΡ alone or with 1% ΤηΒΡ and 1% Triton Χ-45, which is the aforementioned initial platelet. The volume of the thick liquid is based on the basis. 12. The method of claim 5, wherein the oil extraction is carried out with a pharmaceutical grade oil, the amount of the oil being from 2 to 20% by weight, or from 5 to 15% by weight, or from 5 to 10 % by weight, based on the weight of the mixture of the aforementioned platelet concentrate and the aforementioned solvent and/or detergent. 13. The method of claim 5, wherein the chromatographic method comprises a C18 ceria loading material, or an SDR (Solvent-Detergent removal) hyper D. 】 民 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = The method described in claim 15 is to use the platelet separation h, ::: separation method from the whole blood force "乂 2 thick liquid system is fresh, expired" or expired and the blood is described The method of the method of Τ Τ Τ Τ Τ 液 液 用途 用途 去 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 == Peripheral growth factor thick liquid, or as claimed in the patent application; 可 之 可 用于 用于 用于 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗 治疗: The method described in Item 9 of the ninth item, which is from 〇 to I, the active dry circumference is from 2〇117/111丨 to 1〇〇〇%=: the clotting of the virus is activated. The method described in the 2nd item of the blood of the second section, the capping of the blood coagulation 2. The medical product, which is obtained by the method as described in the Patent Application No. 5 to 17 ^44963, Republic of China, April 6, 100 Disease Toxic = anti-growth factor thick solution, or as described in the scope of the patent application No. ^ ^, for therapeutic and / or cosmetic 23. Condensable platelet growth factor thick solution. Any of the range 5th to 17th. 24. The method of claim 5, wherein the method of applying the method of any one of the items 5 to 17 applies for a platelet-derived growth factor. For the use of viruses for deactivation of sexual use == 25. The use of vesicles for the regeneration of bone collaterals, or the healing of soft fibrils and hard tissues. - Examples of applications for patents 5 to 17 Any of the m sets of virus deactivated clotting platelet growth due to a thousand/thick thick liquid, or as used in any one of claims 1 to 4 for therapeutic and/or cosmetic materials The silk deactivated can be used for the thick liquid of the platelet growth factor, which is used for the cell culture of the living body 26 (bright η (10)) or the living body study WV 〇). Virus deactivated clotting platelet growth as described in any one of items 5 to 17 56