TW200930726A - Clottable concentrate of platelet growth factors and preparation method thereof - Google Patents
Clottable concentrate of platelet growth factors and preparation method thereof Download PDFInfo
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200930726 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】 指揮組織傷口療療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, —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(clot)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(fibronectin ) 及凝血酶敏感蛋白(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,這個初始血凝塊可用來 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,而它們是透遞 和橫跨細胞膜上的受器(receptor)功能域(domain) # 接物理性交互作用來調整細胞功能。之後,二次多 (secondary )之訊息轉換子(transducer )則控制細胞内 5 200930726 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―部份 被證釋出來’但大多數的潛在優點則已被證實,特別^ 在血小板衍生物的優點’尤其是灰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 ' 及細胞分裂之性質,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J Ό 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併使用的 情形都在增加,它們在多種臨床性或治療性應用上作為 血小板衍生物或血小板生長因子的來源。 ⑩ 現已發現,這類包含血小板或血小板源之製劑特別 有利於處理大型骨骼損傷、複雜頭顱成形術(c〇mplex cranioplasties)及慢性傷口(例如口腔及顎面手術、骨 科手術、牙周病科手術及整形手術):且在處理慢性潰 瘍、牙科之骨骼及軟組織再生及口腔植體時亦特別有 利;以及處理肌肉骨骼病症時亦是 而且在間葉幹細胞之活體(以WVC>)擴增及分化 方面、以及加速軟骨細胞、内皮細胞及纖維母細胞增生 方面’血小板釋出物也受到__注,因此它在軟骨 再t及?口療癒方面逐漸增加的治療優點也逐漸受到 200930726 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(CaCl2)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 巧及外源性凝灰酶也會誘發纖維蛋白原(fibrin〇gen)之 聚合作用,並形成以纖維蛋白為主的生物材料(血凝 塊)’從而導致灰小板内蘊含的多種掺合物(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) f們可以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, (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200930726 IX. INSTRUCTIONS 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 concentrates 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 wound 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, the 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 after the injury by the formation of blood clots, which are characterized by fibrin and various proteins that cross each other. The blood clots (vitronectin), fibronectin, and thrombospondin's blood clots prevent re-bleeding and serve as a substrate for invading cells while providing a barrier against pathogen invasion. In addition, this initial blood 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 receptors that transduce and cross the cell membrane. Sexual interactions to adjust cell function. Later, the secondary message transducer controls the multicellular biology of intracellular 5 200930726. Although the roles and mechanisms involved in tissue regeneration are only partially “released”, most of the potential advantages have been confirmed, especially in the advantages of platelet derivatives, especially from gray platelets. Advantages of the platelet-derived growth factor. 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, cells). There is a direct correlation between extracellular matrix precipitation and remodeling) and enhanced cell proliferation. In addition, platelet derivatives are also indirectly related to some closely related biological functions, such as chemokine stimulated by bystander cells such as fibroblasts, macrophages, epithelial cells or lymphocytes. ) and the production of cytokines. Thus, platelet-rich formulations, as well as platelet gels, viscose and their eliminators, are used alone or in combination with transplanted biomaterials, and they serve as platelet derivatives in a variety of clinical or therapeutic applications. Or the source of platelet growth factor. 10 It has been found that such preparations containing platelet or platelet sources are particularly advantageous for the treatment of large bone injuries, complex cranioplasties and chronic wounds (eg oral and facial surgery, orthopedic surgery, periodontal disease). Surgery and plastic surgery): It is also particularly beneficial in the treatment of chronic ulcers, dental bone and soft tissue regeneration and oral implants; and in the treatment of musculoskeletal conditions and in the expansion of mesenchymal stem cells (by WVC) In terms of differentiation, and accelerating the proliferation of chondrocytes, endothelial cells and fibroblasts, platelet release is also subject to __ note, so its therapeutic advantages in cartilage re-treatment and gradual healing are also gradually affected by 200930726 local platelets. Source preparations (e.g., gels, gels, and elixirs) are typically prepared by mixing a platelet concentrate with an activator capable of eliciting release of the contents of the platelet particles to mimic the physiological activation of the platelets. The activation step is generally carried out by direct addition of exogenous thrombin or by the use of calcium chloride (CaCl2), which counteracts the effect of adding an anticoagulant during the collection of blood or platelets, triggering a blood agglutination chain reaction ( Coagulation cascade) 'and release endogenous thrombin. Endogenous and exogenous coagulase also induce fibrinogen (fibrin〇gen) polymerization and form fibrin-based biomaterials (blood clots), resulting in a variety of gray plate 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) from conventional expression systems, and the synergistic effects of various growth factors can be observed. Therefore, compared with single recombinant factors, (Janssen Cilag Internat.). 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, because
μ Ht- ra , ......吨廿你什、以及活化 的方式。因此,這些變因顯然會引起產㈣的重大差 起始材料之種類如=度儲;=料 7 200930726 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 成有關,同樣的,也和血小板的不完全溶解有關。是以, 相當量之完整血小板仍然被包於纖維蛋白血凝掄Λ 部,所以很難去測定真正之生長因子釋出量。μ Ht- ra , ... tons of you, and the way of activation. Therefore, these variables obviously cause a significant difference in production (IV). The type of starting material, such as = degree storage; = material 7 200930726, and then the division of biological properties and clinical validity (such as healing power). For example, when a solution/gel containing intact platelets is delivered directly to the wound, the activation of platelets is associated with the rapid formation of fibrin clots, as well as the incomplete dissolution of platelets. Therefore, a considerable amount of intact platelets are still encased in fibrin hemagglutination, so it is difficult to determine the true release of growth factors.
進一步來說,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企小板激發而得到的,故當灰小'板 衍生物產生時,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 鈇 或合成產物混合。 =「芡|詋,夕数的治療性局部血小板衍生物係由 自體來源進行製備。也就是說,自體血小板濃车' 幾小Λ或幾1所捐的血液進行製備,而用 定體積之到、板凝膠的計劃性手術, 固。 缺點的,因為它們常常是在控制不良的 有其 缺乏f有的鮮化來確保生長因子㈣及其臨J度故 烈的ίί,物方法有強 因子濃厚液的情形,如此可製板生長 劑,而用於各種特殊的生理病理狀況。種里身定做的製 點來自於㈣會有完# 1重要缺 的存在’從而在血小板源產物血液細胞碎片 對捐贈者抗原之免疫反應。針源㈣發針 、同種異體抗原之免疫反 200930726 f可H丨if重的後果’如同輸血反應觀察到的現 象’並包括患者的自體免疫及溶也併發症。J的現 點fί况既有的血小板源產物的另一項重大缺 ❹ ❿ 由全血得出的血小板製劑)保2: 能’但是,使用血小板衍生物時仍有; ίίίϊ:ϊ=ί染。因此,血小板衍生物及灰小板 U部使用之同種異體血小板製劑有“ 最後,老年人口及慢性疾病的增加成了一會 逐漸滋長的健康問題,這在不久的將來必須要 ^。 雖然目前已有多種治療性處理,包括例如手術充生 Ϊ:生=補植,但對於組織傷口療癒及: 在密集研究後,發明人驚奇地發現,這些目斬=以 用後文所述之可凝結之血小板生長因子 備方法來達成。 什狀久,、裂 „方法可以簡單、快速且有效的製備金小板 何生物,且更特別的是,可從個人血]、板濃厚液或 多數人的企小板濃厚液(ροο1)得到可凝結之血小 長因子濃厚液。更特定言之,與關領域之1他方 較之下,本發明之方法會明顯增加所有生 4 收’至少是比較重要之血小板生長因子的回收,且3 別的是生長因子PDGF、TGF-β及EGF的回收。 付 9 200930726 c膜病毒及其他病原體蟲所:: 脂r多除在起始血小板濃厚液中存在的: 化’而用於治療性處理。Further, when the platelet gel and/or the growth factor preparation is obtained by the activation of thrombin and the excitation of the platelet, the fibrinogen is completely depleted when the gray small plate derivative is produced, The condensable state is therefore required to be mixed with exogenous hydrazine or synthetic product prior to delivery to the wound site. = "芡|詋, the therapeutic local platelet derivatives of the cerebral number are prepared from an autologous source. That is to say, the autologous platelet car is prepared by a few small or a few donated blood, and a fixed volume is used. The planned operation of the plate gel, solid. Disadvantages, because they are often in poor control, there is a lack of freshness to ensure that the growth factor (4) and its pre-J degree are ίί, In the case of strong factor thick liquid, it can be used to make plate growth agent, and it can be used for various special physiological and pathological conditions. The system is made up of (4) there will be the existence of #1 important deficiency, and thus in the blood of platelet source products. The immune response of cell debris to donor antigens. Acupuncture (4) Hair injection, allogeneic antigen immunity 200930726 f can be H丨if heavy consequences 'as observed in the transfusion reaction' and include the patient's autoimmune and dissolution Complications. J's current point is another major deficiency of existing platelet-derived products. 血小板 Platelet preparations derived from whole blood.) 2: Yes, but still when using platelet derivatives; ίίίϊ:ϊ = 染染. This allogeneic platelet formulations of platelet derivatives and gray small plate portion U "Finally, the increase in the aging population and chronic diseases will gradually grow became a health problem, which must be in the near future ^. Although there are a variety of therapeutic treatments available, including, for example, surgical sputum: birth = replantation, but for tissue wound healing and: after intensive research, the inventors were surprised to find that these targets = as described later The clotting platelet growth factor preparation method is achieved. The method can be simple, fast and effective in the preparation of gold platelets and organisms, and more particularly, can be obtained from individual blood], plate thick liquid or most people's small plate thick liquid (ροο1). Condensable blood small long-term factor thick liquid. More specifically, in comparison with the field of the related art, the method of the present invention will significantly increase the recovery of all the raw materials, at least the more important platelet growth factor, and 3 Others are the recovery of growth factors PDGF, TGF-β and EGF. Pay 9 200930726 c membrane virus and other pathogens:: The fat r is present in the initial platelet thick solution: .
臨床上在靜脈内使用血小板來矯治數量性或功 *小板數量低下症(thromb〇cytopenia )時,由於血,丨 =保二期限是5 · 7天(部分是因為細菌污染的風 險),所以母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,前述可凝結之血小板生長因 子濃厚液包含生長因子PDGF、TGF-P、IGF、EGF、 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血液細胞相關之輸血反應。Clinically, when platelet is used intravenously to treat Quantitative or Thromb〇cytopenia, due to blood, 丨=保二期 is 5 · 7 days (partly because of the risk of bacterial contamination), so A large number of platelet units with a storage time greater than 5 or 7 days are usually discarded in the mother 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 thick liquid' for use in therapeutic and/or cosmetic applications. In a preferred embodiment, the condensable platelet growth factor concentrate comprises growth factors PDGF, TGF-P, IGF, EGF, CTGF, bFGF and VEGF. In another preferred embodiment, the aforementioned clotting platelet growth factor concentrate does not induce blood cell related transfusion reactions.
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(von Willebrand factor)與第 π、V、VII、VIII、IX、X 及 XI 200930726 凝血因子所組成之組群的蛋白。 Ο 本發明之另一目的是一種製備本發明之可凝結之 長因子濃厚液的方法,其包含下列步驟:使血 ’、板?辰厚液與溶劑及/或清潔劑接觸;使前述血小板濃厚 ^與前述溶劑及/或清潔劑於維持在從約6 〇至約9 〇範 之值及在從2。(:至5〇沱範圍内之溫度下共同 至y 5分鐘至6小時的時間,較佳為在從25。€至 45 C範圍内之溫度進行;以及藉由油萃取及/或層析方 式來移除前述溶劑及/或清潔劑。 較^實施態樣中’用於本發明之方法的溶劑係 或三烷基磷酸酯類、具有不同烷基鏈之二或三 類所組成之組群;且較佳為三正丁基磷酸酯 較佳實施態樣巾,用於本發明之方法的清潔劑 類、/ /旨肪酸之聚氧乙烯衍生物、山梨糖醇酐之偏醋 S)、祕子性清_、去氧膽酸納及續 類(―s)所組成之組群;且又更佳ί 45、Trit〇n χ-ι〇〇 及―0 所組成之 心在;1 較佳實鋪财,綠本發明之方法的前述溶 S H清潔躺侧最終濃度範圍為從g .2至5體 ίί’ΛΙ 2至2體積%,其係以前述起始血小板 離基礎計算。在本發明之方法的較佳實施 1%TritonX_45接觸,其係以前述 板/農厚液之體積為基礎計算。 藥等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2〇重量 200930726 或從5 ^ 15重量%、或從5至1〇重量%,其係以前述 血小板濃,液與前述溶劑及/或清潔劑之混合物的^量 為基礎計算。 在,一較佳實施態樣中,層析方式係使用如包含 ^18 一乳化矽裝填材料、或SDR (溶劑_清潔劑移除, o:t-Detergent removal) hyperD 來移除前 或清潔劑。 各鸮1幻 在一較佳實施態樣中,本發明之方法進一步 一 ❹ ❹ g所二至75-nm,之滤膜或類似之病毒i除 ,對所侍可旋結之血小板生長因子濃厚液進行奈米過 在一較佳實施態樣中,本發明之方法包含一 始血小板濃厚液之初步步驟,前述起始血小板 藉由血小板分離術或血沉棕黃層(buffy-coat)分離法從 ,血加以製備,且係為新鮮或過期且以液態儲存、 期且冷减儲存的狀態。 / 士發明之另-目的是一種形成血凝塊的方法,豆係 或藉由本發明之方法所得的血小板生長C /辰厚液與凝血酶(thrombin)混合。較佳為,户‘ 成血凝塊的方法中所使用之凝血^係得自又類。二二= 別實施態樣中,係將(U至i體積之活性 , =明之方麟得的可凝結之血 本發明之另一目的是一種醫藥產物或人工 包含本發明或藉由本發明之方法所得的可凝結之'血f 板生長因子濃厚液。 、 12 200930726 本發明之另一目的是本發明或藉由本發 所得的可凝結之血小板生長因子濃厚液的用途,方法 於形成血凝塊、或用於骨骼再生或傷口療癒、ς係用 體外研究(k w加)或活體研究(exvivo)之細胞拉=活 【實施方式】 本發明之一目的是一種可凝結之血小板生 濃厚液’其係用於治療性及/或美容性用途。 因子 Ο Ο 在本發明中所使用之「可凝結」一詞係指 精由本發明之方法所得的血小板生長因子濃,月或 纖維蛋白原(flbrin〇gen)及第凝血因子,文包含 治療性應用需求時,其可與合適的活化劑(如盔故當有 混合而生成血凝塊。 成轉)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 von Willebrand factor. A protein of the group consisting of π, V, VII, VIII, IX, X, and XI 200930726 coagulation factors.另一 Another object of the present invention is a method for preparing a clotting long-factor thick liquid of the present invention, comprising the steps of: contacting blood, a thick liquid with a solvent and/or a detergent; and making the aforementioned platelets thick ^ with the aforementioned solvent and / or detergent maintained at a value from about 6 〇 to about 9 及 and at from 2. (: to a temperature within the range of 5 共同 to y 5 minutes to 6 hours, preferably at a temperature ranging from 25 to 45 C; and by oil extraction and/or chromatography To remove the aforementioned solvent and/or detergent. In the embodiment, the solvent or trialkyl phosphate used in the method of the present invention, the group consisting of two or three different alkyl chains And preferably a preferred embodiment of tri-n-butyl phosphate, a detergent for use in the method of the present invention, a polyoxyethylene derivative of fatty acid, and a partial vinegar of sorbitan S) , the group consisting of secretive _, deoxycholic acid and continuation (-s); and better ί 45, Trit〇n χ-ι〇〇 and ―0 are composed of hearts; Preferably, the final concentration of the above-mentioned dissolved SH cleansing side of the method of the present invention ranges from g. 2 to 5 ίί' ΛΙ 2 to 2% by volume, which is calculated based on the aforementioned initial platelet. A preferred embodiment of the method of the invention is 1% Triton X_45 contact, which is calculated based on the volume of the aforementioned plate/agricultural thick liquor. In a preferred embodiment of the invention, the oil extraction is carried out by a doctor, and the aforementioned oil is used in an amount of from 2 to 2 2009, 200930726 or from 5 to 15% by weight, or from 5 to 1% by weight, It is calculated based on the amount of the above-mentioned platelet concentrate, the mixture of the liquid and the solvent and/or detergent. In a preferred embodiment, the chromatographic method uses, for example, an 18-emulsified ruthenium filling material. Or SDR (solvent_detergent removal, o: t-Detergent removal) hyperD to remove the pre-cleaning agent. Each 鸮1 illusion is a preferred embodiment, the method of the present invention further ❹ 所 g Up to 75-nm, a filter or similar virus i, in addition to the spinel platelet growth factor thick solution, in a preferred embodiment, the method of the present invention comprises an initial platelet thick solution In a preliminary step, the aforementioned starting platelets are prepared from blood by platelet separation or buffy-coat separation, and are in a state of being fresh or expired and stored in a liquid state, and stored in a reduced state. / Invented another - the purpose is to form a blood coagulation The method of blocking, the bean line or the platelet growth C / chen thick liquid obtained by the method of the present invention is mixed with thrombin. Preferably, the clotting method used in the method of forming a blood clot is obtained from Further, in the second embodiment, the activity of the U to i volume, = the condensable blood of the square of the invention, is another object of the invention being a pharmaceutical product or artificially comprising the invention or by the invention The condensable 'blood f-plate growth factor thick liquid obtained by the method. 12 200930726 Another object of the present invention is to use the present invention or the use of the coagulated platelet growth factor thick liquid obtained by the present invention to form blood coagulation Block, or for cell regeneration or wound healing, in vitro study (kw plus) or in vivo study (exvivo) of cell pull = live [embodiment] One of the objects of the present invention is a clotting platelet thick concentrate 'It is used for therapeutic and/or cosmetic purposes. Factor Ο Ο The term "coagulation" as used in the present invention refers to a platelet growth factor concentrate, moon or fiber egg obtained by the method of the present invention. Original (flbrin〇gen) and a second coagulation factor, when the message contains a therapeutic applications, it may be with a suitable activating agent (e.g., when the helmet so that there is generated by mixing blood clot. To turn)
在一特別實施態樣中’本發明之可凝結之 長因子濃厚液中的纖維蛋白原濃度較佳為高於丨小板生 厚液’更佳為高於1.5 g/L濃厚液’又更佳為高於g/L濃 濃厚液;且其第XIII因子濃度較佳為高於〇 5 ' .5g/L 厚液,更佳為高於〇.75IU/ml濃厚液,又更佳如濃 IU/ml濃厚液。 ⑽鬲於1.0 在本文中所使用之「美容性用途」的說法 會與人體之多種體表部分接觸的處理方式,特別Γΐ 膚、頭髮、指甲、嘴唇、外生殖器官、或與^與皮 ,膜接觸,而使之清潔芳香、並有保護及 =口脸 或使之維持在良好的狀態。 雙的作用, 、在本發明中所使用之r治療性用途」的說 種/σ療性或預防性處理方式來處理人類及/或動从、 症,其係、透過醫藥、免疫或代謝效絲回復、 13 200930726 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,且具有病毒安全性。 「清除溶劑及/或清潔劑」的說法係指可凝結之血小 - 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 ❹ 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 ClinicalIn a particular embodiment, the concentration of fibrinogen in the condensable long-factor thick liquid of the present invention is preferably higher than that of the small plate thick liquid, and more preferably higher than 1.5 g/L thick liquid. Preferably, it is higher than g/L thick thick liquid; and its concentration of factor XIII is preferably higher than 〇5 '.5g/L thick liquid, more preferably higher than 〇.75 IU/ml thick liquid, and more preferably thick IU/ml thick solution. (10) 1.0 1.0 The term “cosmetic use” as used in this article refers to the treatment of various body parts of the human body, especially skin, hair, nails, lips, external reproductive organs, or with skin and skin. The membrane is in contact with it to make it clean and fragrant, and to protect and keep the face in good condition. The role of the dual, the therapeutic use of the therapeutic use of the invention, or the therapeutic or prophylactic treatment of the human, and/or the motility, the disease, the medicinal, the immune or the metabolic effect Silk rejuvenation, 13 200930726 changed its physiological function. The condensable 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 conditions, and/or for contact with body parts of the body because it has been removed Both solvent and detergent, and are viral safe. The term "clearing solvent and/or detergent" means that the amount of solvent and/or cleaning agent in the condensable blood small-plate growth factor thick solution is extremely low, and is preferable. Indeed, it is known to those of ordinary skill in the art that elevated concentrations of solvents and/or detergents are directly linked to long-term toxicity and, in particular, directly linked to the development 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50 ppm,更佳為小於20 ppm, 又更佳為小於10 ppm、小於5 ppm,且再更佳為小於夏 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250 ppm,更佳 為小於100 ppm ’又更佳為小於50 ppm,且再更佳為小 • 於 10 ppm。 「病毒安全性」的說法係指可凝結之血小板生長因 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、B 型肝炎病毒(HBV)、C型肝炎病毒(HCV)、西尼羅病 毒(WNV)、TT病毒、登革熱病毒、巨細胞病毒(CMV)、Haematology Vol. 19, No. 1, pp. 205-242, 2006). Therefore, the term "clearing solvent" in the present invention means that the amount of the solvent is less than 100 ppm', preferably less than 50 ppm, more preferably less than 20 ppm, still more preferably less than 10 ppm, less than 5 ppm, and even better. Further, in the present invention, the term "cleaning agent" means that the amount of the cleaning agent is less than 500 ppm, preferably less than 250 ppm, more preferably less than 100 ppm, and even more preferably less than 50. Ppm, and even more preferably small at 10 ppm. "Virus safety" means that the clotting platelet growth factor thick liquid is substantially free of infectious virus, especially without lipid-containing viruses, such as human immunodeficiency virus, hepatitis B virus. (HBV), hepatitis C virus (HCV), West Nile virus (WNV), TT virus, dengue virus, cytomegalovirus (CMV),
Epstein Barr 病毒(EBV)、人類疱疹病毒-8 (HHV-8)、 200930726 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 西夂脫風酶病毋、疮療群病毒(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(arb〇virus )、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 logl◦,這是強力病毒減毒步驟所定出的標準(由 ❹Epstein Barr virus (EBV), human herpesvirus-8 (HHV-8), 200930726 simian foam virus, severe acute respiratory syndrome virus (SARS coronavirus) and other lipid envelope hepatitis viruses, cytomegalovirus, milky scorpion Enzymatic disease, Herpes group virus, rhabdovirus, leuk〇virus, mucus virus, alphavirus, arbvirus, paramyxovirus, squamous virus (arenavirus) and coronavirus. “Substantially without” means that the degree of virus deactivation of the clotting platelet growth factor thick solution is at least greater than 4 logl ◦, which is the standard set by the powerful virus attenuating step (by ❹
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(N〇te f〇r guidance on vims validation studies)定出,參考文獻 CPMP/BWP/268/95 ) ’且更佳為大於5 l〇g1G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:金 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^ve、 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^b〇ne morphogenetic 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)及金管内 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 200930726 長因子,厚液中的PDGF濃度係高於9〇ng/mUf厚液, 較佳為高於1〇〇 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/mi濃厚液,再更佳為高 於180 ng/mi濃厚液,且又再更佳為高於25〇吨如丨濃厚 液。 ❹ ❿ 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 ng/ml濃厚液,更佳為高於16〇 ng/ml 濃厚液,又更佳為高於18〇 ng/mi濃厚液,再 於250 ng/ml濃厚液。 句冋 在一特別實施態樣中,本發明之可凝結之血小 長因子濃厚液中的IGF濃度係至少與起始血小板 中者,二較佳為高於65 ng/ml濃厚液,更 75^-濃厚液’又更佳為高於心祕濃厚液更 佳為南於100 ng/ml濃厚液。 在:特別實施態樣中,本發明之可凝結之 長因子濃厚液中# EGF濃度係高於〇 5 η_濃^生 ,為焉於1,1濃厚液,更佳為高於1.5 ng/心戽 液,又更佳為尚於2ng/ml濃厚液,且再 」 ng/ml濃厚液。 為问於2.5 在-特別實施紐t,本發i崎由 == 血小板生長因子漠厚液適用於需= 本發明所使用之「需要低脂質含 係指本發明或藉由本發明之方法所 板生長因子濃厚液係已除去脂f (deplet ^之血小 亦即,在本㈣料發R^ 16 200930726 =生$因子漠厚液t,膽固醇、三酸甘油醋、皿及 之篁日顯低於其於起始血小板濃厚液 =Γΐ日=、_化領域中已知之生長因子濃厚液中之 凝結之血小板源生長因子濃厚液中的脂 二極,(特別是膽固醇、三酸甘油醋及LDL量極低〕 2該Γί液適用於較的治療性用途,舉例來說,如 用於心^併發症風險高的患者、或用於高心臟風險之 ❹ ❷ 者I廷疋因為’對已接受治療的患者提供極低 ,、日^日大大減少由動脈及靜脈系統中脂肪斑 (plaque)形成所引起之血管阻塞的風險。 結之=生==本=方法所得的可凝 me/dl^r;&姓Λ厚中,膽固醇的量係低於100 可凝:之血:板t’較佳為低於5〇 心_可凝結之且更佳為低於 結之ί=生本發::方法所得的可凝 100 mg/dl可凝結之q、板生中長因^酸曲甘油酯的量係低於 5〇續可凝結之▲小板生長因子濃 更^、板綱切歧 結之發^枝麟的可凝 mg/cn可凝/Λ Ϊ厚液中,HDL的量係低於30 mg= 了凝結之血小板生長因子濃 液一為低於 本發明或#由本發明之方法所得的可凝 結之金小板生長因子濃厚液中,咖的量係低的於= 17 200930726 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。 φ 在特別實施態樣中,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不會引起血液 細胞相關之輸血反應。「血液細胞相關之輪血反應」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 上引起一疋範圍之已知輸血反應,包括免疫(如異體免 疫(Alloimmunization))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 ❹ 者通常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:(1)第一型 • HLA,係血小板及白血球共有者;(2)第二型HLA,在某 些白血球有表現者;(3)顆粒性白血球特異抗原;(4)血小 板特異抗原(舉例來說,如人類血小板抗原HpA);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 胞(紅血球、白血球及血小板)會被溶劑清潔劑摧毀/ 溶胞’從而減少(且更佳為預防)患者暴露於外來抗原 18The European Agency for the Evaluation of Medicinal Products (EMEA), the Committee for the Evaluation of Medicinal Products (CPMP), is based on the guidelines for the approval of viruses (N〇te f〇r guidance on vims validation studies). ), the reference CPMP/BWP/268/95) 'and more preferably greater than 5 l〇g1G or further greater than 6 log1(), so 'it is unlikely to cause a lipid envelope virus when transfused to a patient Blood-borne infection. The clotting platelet growth factor concentrate obtained by the present invention or by the method of the present invention further comprises the following functional growth factor: gold platelet growth factor (PDGF), which is a heterodimer of A and B chains and/or a homodimer of AA and/or BB chain (PDGF-AA, PDGF^ve, PDGF-BB); a superfamily of transforming growth factor (TGF-β), which specifically comprises TGF-βΙ and/or TGF-P2 and / or bone morphogenetic protein ^b〇ne morphogenetic protein, BMP); insulin-like growth factor (IGF); epidermal growth factor (EGF); connective tissue growth factor (CTGF); primary fibroblast growth factor (bFGF) and gold tube Endothelial growth factor (VEGF). In a special embodiment, the clotting platelet of the present invention 15 200930726 long factor, the concentration of PDGF in the thick liquid is higher than 9 ng/mUf thick liquid, preferably higher than 1 ng/ml thick. Preferably, the liquid is more than 12 ng/ml thick liquid, more preferably higher than 150 ng/mi thick liquid, more preferably higher than 180 ng/mi thick liquid, and even more preferably higher than 25 〇 tons such as 丨 thick liquid. ❹ ❿ 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 ng/ml thick liquid More preferably, it is higher than 16 ng/ml thick liquid, and more preferably higher than 18 ng/mi thick liquid, and then 250 ng/ml thick liquid. In a special embodiment, the concentration of IGF in the condensable blood small factor thick solution of the present invention is at least the same as that of the starting platelets, and preferably more than 65 ng/ml thick solution, and more 75 ^-Thick liquid 'is better than the thick liquid of the heart secret is better than 100 ng / ml thick liquid. In a special embodiment, the # EGF concentration in the condensable long-factor concentrated liquid of the present invention is higher than 〇5 η_concentration, and is 11,1 thick liquid, more preferably higher than 1.5 ng/ The heart palpital solution is more preferably a 2 ng/ml thick solution and a ng/ml thick solution. In order to ask 2.5 in the special implementation of neot, the present invention is a platelet growth factor == platelet growth factor indifference liquid is suitable for use = "requires low lipid content" as used in the present invention or refers to the method of the present invention Growth factor thick liquid system has been removed fat f (deplet ^ blood is small, that is, in this (four) material R ^ 16 200930726 = raw $ factor indifference liquid t, cholesterol, triglyceride, vinegar, dish and the day after the day is low a lipid bipolar in a concentrated platelet-derived growth factor thick solution in a concentrated growth factor thick solution known as the starting platelet thick solution=Γΐ日=, _chemical field (especially cholesterol, triglyceride and LDL) The amount is extremely low. 2 The Γ 液 liquid is suitable for more therapeutic use, for example, for patients with high risk of cardiac complications, or for high risk of heart disease. Patients treated are extremely low, and the risk of vascular occlusion caused by the formation of plaques in the arteries and venous systems is greatly reduced. Conclusion ================================================================ r; & surname Λ thick, the amount of cholesterol is less than 100 can be condensed: blood: plate t' is preferably less than 5 〇 heart _ condensable and more preferably lower than the knot ί = raw hair:: method of condensable 100 mg / dl condensable q, plate medium length due to acid The amount of glycerol ester is less than 5 可 condensable ▲ small plate growth factor concentration ^, plate cleavage ^ 枝 麟 的 的 的 mg mg mg mg / / / mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg mg H H H H H H The amount of coffee is less than 30 mg = the amount of coagulated platelet growth factor concentrate is lower than the present invention or # condensable gold plate growth factor thick solution obtained by the method of the present invention, the amount of coffee is lower than = 17 200930726 mg/dl clotting platelet growth factor thick solution, preferably less than mg/dl clotting platelet growth factor thick solution, more preferably less than 2 〇mg/dl clotting platelet growth factor thick solution, and Still more preferably less than 5 mg/dl of clotting platelet growth factor concentrate. Further, the quality of the lipid in the clotting platelet growth factor concentrate obtained by the method of the invention or by the method of the invention is very low and/or • Undetectable, so there is better stability, when large-scale purification of platelet growth factor The method is relatively easy to perform. φ In a special embodiment, the condensable platelet growth factor thick solution obtained by the present invention or by the method of the present invention does not cause blood cell-related transfusion reaction. "This clotting growth factor concentrate does not contain intact living cells (such as red blood cells, platelets, and white blood cells), so it can avoid causing a range of known transfusion reactions in patients, including immunity (such as allogeneic immunity (" Alloimmunization) 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 typically exhibit 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 categories: (1) type 1 • HLA, which is a combination of platelets and white blood cells; (2) type 2 HLA, which is expressed in some white blood cells; (3) granulocyte-specific An antigen; (4) a platelet-specific antigen (for example, a human platelet antigen HpA); and (5) a red blood cell-specific antigen. More specifically, in the preparation method of the present invention, living blood cells (red blood cells, white blood cells, and platelets) are destroyed/lysed by solvent detergents to reduce (and more preferably prevent) patients from exposure to foreign antigens.
200930726 ΐΪίίί細胞的風險。故本發明之方法可從由患者得 小板來制I (用來讀患者本身)、或從同種異體之血 J板末:備可凝結之血小板生長因子濃厚液。 二特別實施祕巾,本發明或―本發明之方法 少二種選ΓΓί血小板生長因子濃厚液進—步包含至 ί 蛋二二 1=白X:XI凝血因子及凡威勒伯氏因子所組成之 且ΠΕίΐ:特別實施態樣中,本發明之可凝結之血小板生 長因子^農厚液中纖維黏連蛋白的濃度較佳為高於〇2 g/L濃厚液,且更佳為約〇 3 g/L濃厚液。 在-特施祕中,本發明之可凝結之到、板源 生長因子濃厚液中第11、IX、X、VII及/或XI凝血因子、 ==佳為高於0.51腕1濃厚液’且更佳為❸ 在-特別實施紐中,本發明之可凝結之血小板生 長因子濃厚液中第vm凝血因子及/或凡威勒伯氏因子 的個別濃度較佳為高於0.5 iu/mi濃厚液,更佳為古 〇. 9·濃厚液,且又更佳為…二:為-於 在另-實施態樣巾’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免疫球蛋白,如 IgG、IgM 及/或 IgA。 在一特別實施態樣中,本發明之可凝結之血小板生 長因子濃厚液中免疫球蛋自IgG❺濃度較佳為高於5 g/L ;辰厚液’且更佳為約1 〇 g/L濃厚液。 在另一實施態樣中,本發明或藉由本發明之方法所 19 200930726 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 factor ’ EPF )、表皮生長抑制 劑(EGI)、角質細胞生長因子(KGF)、金管生成素樣 蛋白_6 (ANGPTL6)、IGFBP-3、雌激素受器相關蛋白、 - 纖維母細胞源内皮細胞生長因子(f-ECGF)、肝細胞生 ❹ 長因子(HGF)、組織胺釋出因子、人類膠原蛋白酶抑制 劑、血小板抗_蛋白-1 (PMP-1)、經凝血酶誘發之血小 板抗菌蛋白-1(1>1>1^0>)、凝血肽_1(如〇11115〇(^11-1,1^1) 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 仔的可疑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S# ( cathepsin )、白蛋白、血小板臉性蛋白_pjgp (CXCL7)、嗜中性球活化蛋白_2及_4 (NAp_2、_4)、 ❹ 體抑素(somat〇statin,SST)、RANTES、CTAP-3、胎盤 蛋白 14(PP14)、SCUBE卜膜聯蛋白 n(annexinll口)、 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30 g/L濃厚 液。 〜在另一實施態樣中,本發明或藉由本發明之方法所 :的可凝結之血小板生長因子濃厚液進一步包含至少 一,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 200930726 金屬蛋白酶 MMP-l、-2、-9、-13、自泌性 ERK ( ext.Cell reg. kinase)、自泌性及旁泌性蛋白C (PC)、以及微量 的酶如搭縮酶(aldolase)、叛基胜肽酶、酸性構酸酶、 芳基硫酸酯酶(arylsulphatase)、β-半乳糖苷酶、β_葡萄 糖醒·酸酶、β-甘油填酸S# ( β-glycerolphosphatase)、α/β· 葡萄糖音酶、α/β-岩藻糖普酶(α/β-fucosidase )、oc-甘露 ' 糖苷酶(α-mannosidase ) 及α_阿拉伯糖苷酶 . (α-arabinosidase )。 0 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、al-oc2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;必性溶血填脂酸(lysophosphatidic acid ’ LPA)、HMGB1 (兩性調節蛋白(amphiregulin))、 ATP、ADP、GPT、GDP、Ca2+、Mg2+及/或 Zn2+。 ❿ 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。 - 醫藥產物」係指金小板凝膠(platelet gel )、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)、人工鹰架。 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 中前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 200930726 究的培養。本發明夕另一曰沾 胞、軟骨細胞、成二、角的質纖維# 發明或藉由本發明之方法所本 因子。 r嘁結之血小板生長 本發明之另—目的是一種製 金小板生長叫農厚㈣方法ϋ本人 ==凝1之 〇 始血小板濃厚液與_及/或清 3义起2 小板濃厚液與前述溶劑及/戋清接觸,使則达起始血 約9·。範圍内之ΡΗ= 及在以約6.。至 了共同培養至少5分鐘至6切度 C至45°c範圍內之、、田声推并·、 TU文佳為在從25 析方式來移除前述溶或清藉由油萃取及/或層 至 4:;較= =、=ΓΛ8至8.2範圍内之pH值(當起始血小 或梅小板時)進行。有利的是,培養溫 在本發明之方法中所使用的合 ς-(η_己基)填酸醋、三-(2-乙基己基)碟酸酯、1 ^ 己$酵二如丁基)她1、二♦丁基)磷‘、3200930726 ΐΪίίί Cell risk. Therefore, the method of the present invention can be made from a small plate prepared by a patient (for reading the patient itself), or from the blood of the allogeneic J plate: a clotting platelet growth factor thick solution. 2. Specially implemented secret towel, the present invention or the method of the present invention has two kinds of selective ΓΓ platelet growth factor thick liquid into the step consisting of ί egg 22 = white X: XI coagulation factor and Van der Boer factor Further, in a special embodiment, the concentration of fibronectin in the clotting factor of the platelet growth factor of the present invention is preferably higher than that of the 〇2 g/L thick solution, and more preferably about 〇3. g/L thick liquid. In the secret of the present invention, the clotting factor of the 11th, IX, X, VII and/or XI in the condensable, plate source growth factor thick solution of the present invention is preferably higher than 0.51 wrist 1 thick liquid' More preferably, in the special implementation, the individual concentration of the vm coagulation factor and/or the van der Waals factor in the condensable platelet growth factor thick solution of the present invention is preferably higher than 0.5 iu/mi thick liquid. More preferably, it is an ancient sputum. 9· thick liquid, and more preferably... two: for-in another embodiment, the condensable platelet growth factor thick liquid obtained by the method of the present invention further comprises an immunoglobulin Proteins such as IgG, IgM and/or IgA. In a specific embodiment, the concentration of the immunoglobulin from the IgG 可 in the condensable platelet growth factor thick solution of the present invention is preferably higher than 5 g/L; and the thick liquid solution is more preferably about 1 〇g/L. Thick liquid. In another embodiment, the condensable platelet growth factor concentrate obtained by the present invention or by the method of the present invention 19 200930726 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), epidermal growth inhibitor (EGI), keratin Cell 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), histamine release factor, human collagenase inhibitor, platelet anti-protein-1 (PMP-1), thrombin-induced platelet antibacterial protein-1 (1>1>1^0>), coagulation Peptide_1 (such as 〇11115〇(^11-1,1^1) and thrombincidin-2 (TC2). In another embodiment, the invention is exemplified by the method of the invention Suspected platelet growth factor thick solution Further comprising at least one protein selected from the group consisting of serotonin, tissue protein S# (cathepsin), albumin, platelet facial protein _pjgp (CXCL7), neutrophil activating protein-2 and _4 (NAp_2, _4), somatostatin (SST), RANTES, CTAP-3, placental protein 14 (PP14), SCUBE annexin n (annexinll), heat shock protein 27 (HSP27) And heat shock protein 60 (HSP60) - In a particular embodiment, the concentration of albumin in the clotting platelet growth factor concentrate of the present invention is preferably greater than 30 g/L of concentrated liquid. In another embodiment, the condensable platelet growth factor concentrate of the present invention or 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 200930726 metalloproteinases MMP-1, -2, -9, -13, autocrine ERK (ext.Cell reg. kinase), autocrine and paracrine Protein C (PC), and trace amounts of enzymes such as aldolase Defensin, acid structase, arylsulphatase, β-galactosidase, β-glucose acetoin, β-glycerolphosphatase, α/ β·glucoine enzyme, α/β-fucosylase (α/β-fucosidase), oc-mannose's glycosidase (α-mannosidase) and α_arabinosidase. (α-arabinosidase). 0 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, al-oc2 antitrypsin, α2_anti-cytosolic ( A2-antiplasmin), α2-macroglobulin, ci-INH, induced sputum-2, -6, -7 (TGF-β superfamily), ECM remodeling factor (ECM remodelling factor), induced sputum, TNF- α, elastase......), autocrine and para; lysophosphatidic acid (LPA), HMGB1 (amphiregulin), ATP, ADP, GPT, GDP, Ca2+ , Mg2+ and/or Zn2+. Another object of the present invention is a pharmaceutical product comprising the condensable platelet growth factor • concentrated solution of the present invention or obtained by the method of the present invention. - "Pharmaceutical product" means a platelet gel, a platelet glue, a growth factor-rich fibrin glue and/or a sealant, an artificial eagle. Another object of the present invention is the use of the coagulated platelet growth factor of the present invention or the method of the present invention in a medium, wherein the medium is suitable for use in fibroblasts, chondrocytes, osteoblasts, keratinocytes, stem cells And/or transplantation of cells in vitro or in vivo. In the present invention, another factor, a cell, a chondrocyte, a nucleus, or a horn of the nucleus fiber # invention or the method of the present invention. Platelet growth of r嘁 knot Another object of the present invention is to grow a small plate of gold called a thick layer (four) method ϋ I == condensed 1 start platelet thick liquid and _ and / or Qing 3 from 2 small plate thick liquid Contact with the solvent and / / 戋 clear, so that the initial blood is about 9 ·. Within the range = and at about 6. To co-culture at least 5 minutes to 6 degrees C to 45 ° C, Tian Sheng Push and · TU Wen Jia is to remove the aforementioned solution or clear by oil extraction and / or Layers to 4:; pH values in the range of ==, = ΓΛ8 to 8.2 (when the initial blood is small or plum plate). Advantageously, the culture enthalpy-(η-hexyl) acid vinegar, tris-(2-ethylhexyl) oleate, 1 ^ hexanol, such as butyl, used in the process of the invention. She 1, 2 ♦ butyl) phosphorus ', 3
St不同烧基鏈之二烧_類:== 土之一或一烷基磷酸酯類,例如二-(η-丁基)磷酸 曰特佳為二烷基磷酸酯為三-(η- 丁基)磷酸酯 22 200930726 (ΤηΒΡ)〇 在本發明之方法中所使用的合適清潔劑包括脂肪 酸之聚氧乙烯衍生物、山梨糖醇酐之偏酯類(例如品名 為 Tween 80」(也稱為「聚山梨醇g旨80 (polysorbate 80)」)、「Tween 20」之市售產品)及非離子性清潔劑(如 以下列品名販售的氧乙基化之烷基酚:「Trit〇n χ_ι〇〇」 ' 或「Triton X-45」)。進一步考慮的清潔劑是去氧膽酸鈉 • 及磺基甜菜鹼類,如N-十二基-n,N-二曱基-2-銨-1-乙烷 ❾ ㉖酸鹽(N-dodecyl-N,N-dimethyl-2.-ammonio-l-ethane sulphonate )。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,起始金小板濃厚液係 與溶劑及清潔劑共同培養,較佳為與ΤηΒΡ及Triton 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0.2 至5體積%之範圍内’較佳為包含於從0.2至2體積% ❹ 之範圍内’其係以前述血小板濃厚液之體積為基礎計 算。在一較佳實施態樣中,前述起始血小板濃厚液係與 2% ΤηΒΡ共同培養。在另一較佳實施態樣中,前述起始 • 金小板濃厚液係與1% ΤηΒΡ及1% 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/或其他方法(如管柱層析)而從生物性流體中萃 出,如此則濃厚液中的溶劑及/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 23St. Burning base chain of the second burning class _ class: = = soil one or alkyl phosphates, such as bis-(η-butyl) phosphonium phthalate is preferably dialkyl phosphate is tri-(η-丁Phosphate 22 200930726 (ΤηΒΡ) 合适 Suitable cleaning agents for use in the process of the invention include polyoxyethylene derivatives of fatty acids, partial esters of sorbitan (for example, the product name Tween 80) (also known as "Polysorbate 80" ("polysorbate 80"), "Tween 20", and non-ionic detergents (such as oxyethylated alkylphenols sold under the following product names: "Trit〇n Χ_ι〇〇” or “Triton X-45”). Further considerations for the detergents are sodium deoxycholate and sulfobetaines such as N-dodedo-n,N-didecyl-2-ammonium-1-ethanephthalate (N-dodecyl) -N,N-dimethyl-2.-ammonio-l-ethane sulphonate ). The best cleaning agents are "Triton X-45", "Triton X-100" and "Tween 80". In a particular embodiment of the invention, the initial gold plate thick liquid is co-cultured with a solvent and a detergent, preferably co-cultured with ΤηΒΡ and Triton X-45. Advantageously, the final concentration of the aforementioned solvent or the aforementioned cleaning agent, or the respective final concentration of the aforementioned solvent and the aforementioned cleaning agent is included in the range of from 0.2 to 5% by volume, preferably contained in from 0.2 to 2% by volume. Within the range of 'based on the volume of the aforementioned platelet thick liquid. In a preferred embodiment, the aforementioned initial platelet rich liquid system is co-cultured with 2% ΤηΒΡ. In another preferred embodiment, the aforementioned initial • gold platelet thick liquor is co-cultured with 1% ΤηΒΡ and 1% Triton Χ-45. The solvent and/or detergent may be extracted from the biological fluid by oil extraction with a pharmaceutical grade oil and/or other methods such as column chromatography, such that the solvent and/or detergent in the thick liquid Has been removed. The aforementioned pharmaceutical grade oil may be a natural oil (e.g., an oil extracted from plants or animals) or a structurally similar synthetic compound. Suitable natural 23
200930726 油包括蓖麻油(castor oU,或稱ridnus 〇u)、大豆油、 葵花油、棉籽油β較佳之合成化合物為合成性三酸甘油 酯。.合適的合成性三酸甘油酯範例包括三油精 triolein )、二硬脂精(tristeadn )、三棕櫚精 (tnpdmitin )、三肉蓋謹精㈤㈣咖)、及其合併物。 醫樂等級油的量為可萃取至少8〇%脂溶性製程化學 物,(process chemica!)的量,所用油量為從2至2〇 ff% ’其細到、板濃厚液之溶胞產物的重量為基礎 ^舁,較,為從5至15重量%,且更佳為從5至ι〇重 量%。油萃取可以進行—次,較佳為兩次,且更佳為三 次,其係特別取決於油的濃度。 前述溶劑及/或清潔劑也可藉由管柱層析而從企小 板,厚液之溶胞趋中移除。管㈣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。 可用的合適層析管柱包含反相(疏水性交互反應) ς質、或蛋白吸附基質如離子交換(陰離子及陽離子) 基質及親和力(如免疫-親和力或固定化肝素)基質或 尺=排除(size-exclusion )基質。較佳之反相基質為ci8 :乳化石夕裝填材料、SDR (溶劑__清潔劑移除)如柯D 0>aU c〇rporation )、聚苯乙烯吸收劑(ν&Γί^ )及200930726 Oil includes castor oil (castor oU, or ridnus 〇u), soybean oil, sunflower oil, cottonseed oil β. The preferred synthetic compound is synthetic triglyceride. Examples of suitable synthetic triglycerides include triolein, tristeadn, tnpdmitin, triterpenoid (f), and combinations thereof. The amount of the medical grade oil is extractable at least 8% by weight of the fat-soluble process chemical, (process chemica!), the amount of oil used is from 2 to 2 〇 ff% 'the fine lysate of the plate thick liquid The basis weight is from 5 to 15% by weight, and more preferably from 5 to ι by weight. The oil extraction can be carried out once, preferably twice, and more preferably three times, depending in particular on the concentration of the oil. The aforementioned solvent and/or cleaning agent can also be removed from the smear of the thick plate by means of column chromatography. Tube (iv) analysis can also be carried out after oil extraction or directly after solvent and/or detergent treatment. Suitable chromatographic columns available include reversed phase (hydrophobic interaction) tannins, or protein-adsorbing matrices such as ion exchange (anionic and cationic) matrices and affinity (eg, immuno-affinity or immobilized heparin) matrix or ruler = exclusion ( Size-exclusion) matrix. Preferably, the reverse phase substrate is ci8: emulsified Shishi loading material, SDR (solvent__cleaner removal) such as Ke D 0> aU c〇rporation), polystyrene absorbent (ν & Γί^ ) and
Amberlyte ( Rohm)。即便c丄8係一般用於工業規模層析 的較佳選擇’亦將謂二氧切裝填㈣列人考慮。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_S叩hadex A-50 . DEAE- Sepharose FF > Q-Sepharose ^ DEAE- 24 200930726Amberlyte (Rohm). Even if the c丄8 series is generally used for the better choice of industrial scale chromatography, it will be considered as a dioxo-filling (four). 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_S叩hadex A-50. DEAE-Sepharose FF > Q-Sepharose ^ DEAE- 24 200930726
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^ 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& ppm ’又更佳為小於1 〇 ppm ’小於5 ppm,且再更佳為 小於1 ppm。Toyopearl 650M, DEAE-Hyper D. These adsorbing substances are used for the binding of the growth factor 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 & ppm' and even more preferably less than 1 〇ppm' Less than 5 ppm, and even more preferably less than 1 ppm.
在一較佳實施態樣中,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^ 100 ppm’又更佳為小於50 ppm,且再更佳為小於 ppm 〇 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/病主 B19、或者也可能是A型肝炎病毒(HAV))去活化或: 之移除,例如奈米過濾’且更特別是使用75_nm、35_nm、 20-nm、15-nm或10-譲孔徑之濾膜來進行奈米過滹。 在另一較佳實施態樣中,係在油萃取及/或層析^進 行離心步驟,以移除細胞碎片。有利的是,前述離+ 驟係於800至20000 X g進行1〇至3〇分鐘,且較 於10000 X g進行15分鐘。 馬 在另一敉住貫施態樣中,係在油萃取及/或層析 行過濾清除步驟,以移除細胞碎片。有利的是,前 濾步驟係以從1 μπι至0.2 μιη漸次變化的過濾器 行,這也會移除細菌。 〜 在-特別實施態樣中’在本發明之方法中 材料的血小板濃厚液係單一或混合(p〇〇led)之 : 小板濃厚液’例如製備作輸▲用途的血小板濃厚^。企 25 200930726 小板濃厚液亦可得自全血之血沉棕黃層分離法。一單位 付自血沉掠育層之血小板濃厚液一般係3〇至5〇 mi。得 自jit >儿掠百層之血小板可用作起始材料,其係單一單位 或多個單一單位之混合,如在形成一治療性單位時,其 係4至6個單一單位的混合(如Guide t〇 the扒叩虹此仙', use and quality assurance of blood components _ 13th edition (2007),edited by the Council of Europe 之揭示)。 金小板濃厚液可藉由jk液分離術(指透過全血捐輸 φ 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 )、 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300 ml血小板濃厚液)。 ❹ 在一較佳實施態樣中,本發明之方法包含一製備起 始血小板》辰厚液之初步步驟。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 , 板分離術或透過全血捐輸的血小板製備法)及重點照護 程序(如那些使用血球保存劑/分離器或桌上裝置者)。 在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(即收集 後多於5或7天)、或過期且冷凍於_2〇。(:或以下數週的 時間。 在一特別實施態樣中,在本發明之方法中用作起始 26 200930726 材料之血小板濃厚液可進一步包含白血球及/或紅血 球。故該起始企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。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(hyp〇chlorous acid)及 其他反應性氧衍生物,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。 ❹ 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。 〇 健康人的正常血小板計數一般係每 mm3血液中包 含150000至400000個金小板,亦即150至400 X 1〇9 個血小板/L。這個「正常」血小板計數在約95%健康人 ' 身上疋如此,而剩下的5%可能會有統計上不正常的血 小板計數(非常低或非常高)。 當以血小板分離術收集血小板時,在一袋250 ml 當中的血小板計數一般係高於每ml有1.2 X 1〇9個血小 板’其血小板計數相當於每袋(單位)有多於約3 χ 1〇" 個血小板。 在較佳實施態樣中’在起始灰小板濃厚液中的血 27 200930726 小板數^要比灰液中通常可見的數目高出3至1〇倍。 本發明之另1的是—種形成血凝塊的方法,其係 由下列步驟顺成: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 VII或者牛或人類的凝金活酶(thromboplastin)。 在以本發明或藉由本發明之方法所得的可凝結之 =小板生長因子濃厚液形成血凝塊之前,可在本發明或 〇 藉由本發明之方法所得的可凝結之血小板生長因子濃 ^液,加入抗纖維蛋白溶解劑,來抑制或減緩纖維蛋白 /谷解糸、,’克中的天然蛋白水解酶(如胞漿素(plasmid))分 解纖維蛋白血凝塊。 在一特別實施態樣中,前述抗纖維蛋白溶解劑為抑 腺肽(aprotinin)’但濃度>1〇 mg/ml之傳明酸(加狀仰― acid)或ε-胺基己酸(epsil〇namin〇capr〇icaeid)也可以 用作抑姨肽的替代化合物。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 ❹ 生長因子濃厚液及凝血酶成分混合後,抑胰肽之最終濃 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 白、幾丁聚醣(chitosan)、陶瓷所製造者)混合,或與 得自血漿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 小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。 將本發明或错由本發明之方法所得的可凝結之如 28 200930726 小板生長因子濃厚液與凝血酶混合在一起會重現血液 凝集連鎖反應的最後步驟,且使纖維蛋白原逐漸聚合, 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 任一來源之凝血酶,且較佳係指牛凝血酶;若用於人類 的m療性應用,更佳為人類凝血^酶。經匚&(^12活化之人 類血漿或蝮蛇類凝血酶(batroxobin,另一種纖維蛋白原 凝血蛋白姆’係從蛇^^仏⑺^加尸似历⑻公⑼出得出的蛇 ❹ 毒)、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,將〇1至1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 ▲•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20 IU/ml至1000 IU/m卜更佳者,凝血酶之最終濃度為約 500 IU/m卜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) Ο 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/ml。 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Rad〇sevich et al., Fibrin sealant · Scientific Rationale, Production Methods, Properties, and Current Clinical use55, Vox Sanguinis, 1997, 72:133-143 以及 Marx G,“Evolution of fibrin glue 29 200930726 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 方法所得的可凝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30%範圍的量 f| 出現在培養基中,.更佳為從2%至20%,且又更伟兔舛 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 月腸道手術、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 ❹ 述、參考文獻及後附圖式而變得更加顯明。 資施例 I·材料及方法 1.1-分離術jk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十多成分系統(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(1 ml抗凝血檸檬酸右 旋葡萄糖溶液配方··每10ml血液)。富含血小板的血漿 30 200930726 (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 血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、白企球(WBC )及紅企球計數係使用細胞 q 計數器(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 a-研究設計 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(150 ml)。子集 池1係直接接受S/D處理’沒有先活化。子集池2係在 ❹ 23 mMCaCl2及玻璃球粒(beads)存在時,於會使金小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', and even more preferably less than 50 ppm, and further More preferably less than ppm. After removing the solvent and/or detergent by oil extraction and/or chromatography, a further step can be added to make the non-enveloped virus (such as small/ill B19, or possibly Is Hepatitis A Virus (HAV)) deactivated or: removed, such as nanofiltration' and more particularly using 75_nm, 35_nm, 20-nm, 15-nm or 10-inch pore size membranes for nanoparticles Too much. In another preferred embodiment, the centrifugation step is performed on oil extraction and/or chromatography to remove cell debris. Advantageously, the foregoing is carried out at 800 to 20000 X g for 1 to 3 minutes and for 15 minutes for 10,000 X. Horses In another mode of treatment, the oil extraction and/or chromatography is followed by a filtration removal step to remove cell debris. Advantageously, the pre-filtration step is performed with a filter that varies gradually from 1 μm to 0.2 μm, which also removes bacteria. ~ In a special embodiment, in the method of the present invention, the platelet thick liquid of the material is single or mixed: a small plate thick liquid, for example, a platelet thickening for use in the production of ▲. Enterprise 25 200930726 Small plate thick liquid can also be obtained from the whole blood buffy brown layer separation method. A unit of platelet thick liquid from the blood sedimentation layer is generally 3 to 5 〇 mi. Platelets obtained from jit > granules can be used as a starting material, which is a single unit or a mixture of a plurality of single units, such as a mixture of 4 to 6 single units when forming a therapeutic unit ( For example, Guide t〇the扒叩虹此,, and use assurance of blood components _ 13th edition (2007), edited by the Council of Europe. Gold plate thickening can be performed by jk liquid separation (referred to as platelet preparation by whole blood donation of φ), cell separation (cytapheresis) or plateletpheresis standard procedure (see, for example, Guideto the preparation, use and quality Assurance of blood components, 13th edition (2007), edited by the Council of Europe) is derived and can be derived by using MCS+ (Haemonetics), 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 300 ml of platelet concentrate) from each donor. In a preferred embodiment, the method of the present invention comprises a preliminary step of preparing a starting platelet cullet. Advantageously, methods for preparing a starting platelet concentrate include, but are not limited to, standard blood bank procedures (eg, small blood, plate separation, or platelet preparation via whole blood donation) and priority care procedures (eg, using blood cell preservatives/ Separator or table device). 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 at _2 Hey. (: or the following weeks. In a particular embodiment, the platelet concentrate used as the starting material 26 200930726 in the method of the present invention may further comprise white blood cells and/or red blood cells. Thick liquids may contain several differentiated and unactivated white blood cells, such as lymphocytes, neutrophilic granulocytes, and single and globules. More specifically, neutrophils and mononuclear spheres are rich in bone marrow peroxidation. a particle of myeloperoxidase, which catalyzes the oxidation of chlorides to produce hypochlorous acid and other reactive oxygen derivatives, wherein the reactive oxygen derivative is used as a bactericidal oxidant, Microorganisms and molds are toxic. ❹ Therefore, when the starting platelet concentrate contains several differentiated and unactivated white blood cells, the condensable platelet growth factor thick solution obtained by the method of the present invention further comprises an antimicrobial derived from white blood cells. In a preferred embodiment, the white blood cells in the platelet concentrate used as the starting material are destroyed, preferably by Leukoreduction is used to 'avoid the pro-inflammatory response of proteases and acid hydrolases in white blood cells. Leukocyte depletion also reduces the risk of prion contamination. 〇 Normal human platelet counts are generally The mm3 blood contains 150,000 to 400,000 gold platelets, which is 150 to 400 X 1 〇 9 platelets/L. This "normal" platelet count is about 95% healthy people's, and the remaining 5% may There will be statistically abnormal platelet counts (very low or very high). When platelets are collected by platelet separation, the platelet count in a bag of 250 ml is generally higher than 1.2 X 1 〇 9 platelets per ml. The platelet count is equivalent to more than about 3 χ 1 〇 " platelets per bag (unit). In the preferred embodiment, 'the blood in the initial gray plate thick liquid 27 200930726 small plate number ^ is better than The number generally seen in ash is 3 to 1 times higher. Another aspect of the invention is a method of forming a blood clot which is carried out by the following steps: the invention or by the method of the invention The resulting clotting platelet growth factor concentrate is mixed with thrombin or with another activator of blood agglutination, such as activated VII or bovine or human thromboplastin. Or the condensable platelet growth factor concentrate obtained by the method of the present invention or the anti-fiber may be added to the condensable platelet growth factor concentrate obtained by the method of the present invention or the blood clot formed by the method of the present invention. A protein solubilizing agent to inhibit or slow down fibrin/glutamate, a natural proteolytic enzyme (such as plasmid) that breaks down fibrin clots. In a particular embodiment, the anti-fibrinolytic agent is aprotinin 'but concentration> 1 〇 mg/ml of tranexamic acid (additional acid - acid) or ε-aminohexanoic acid ( Epsiil〇namin〇capr〇icaeid) can also be used as an alternative compound for ruthenium peptides. In the liquid form, the concentration of the inhibitor peptide is usually 3000 KIU or less. After mixing the clotable platelet ❹ growth factor concentrate and thrombin components, the final concentration of the pancreatic peptide is typically half that of the starting solution. The condensable blood small, plate growth factor thick solution obtained by the method of the present invention or the method of the present invention can be further mixed with an artificial scaffold (for example, a manufacturer of collagen, chitosan, ceramics), or A mixture of plasma fibrin glue or fibrin sealant. Additional compounds may also be added to the condensable platelet 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. Mixing the condensable, such as 28 200930726 small plate growth factor thick solution obtained by the method of the present invention with thrombin, reproduces the final step of the blood coagulation chain reaction, and gradually polymerizes fibrinogen to form Blood clots or insoluble fibrin. The term "thrombin" as used in the present invention relates to thrombin which can be obtained from any source, and preferably refers to bovine thrombin; if it is used for human therapeutic applications, it is more preferably human coagulase . ❹ amp & (^12 activated human plasma or python thrombin (batroxobin, another fibrinogen coagulation protein ' ' from the snake ^ ^ 仏 (7) ^ plus corpse (8) public (9) Toxic), activated FVII, or human or bovine thromboplastin can also be used as a replacement for thrombin. In a preferred embodiment, 1 to 1 volume of thrombin is added to 1 volume. The invention or the condensable ▲ small plate growth factor thick solution obtained by the method of the invention. In a preferred embodiment, the thrombin concentration range is from 20 IU/ml to 1000 IU/m. , the final concentration of thrombin is about 500 IU / m to ensure that continuous and rapid fibrinogen polymerization will first obtain soluble fibrin, and then obtain stable (insoluble) Ο fibrin; or when In the case of special surgery, in order to slow down the polymerization, the final concentration will be lower, about 4 to 25 IU/ml. Two components (ie, the present invention or the clotting platelet growth obtained by the method of the present invention) Both factor thick and thrombin) can be double The cartridge system is applied sequentially or simultaneously at the location to be repaired 'with or without the use of an endoscope delivery device, and is sprayed or easily absorbed (see Rad sevich et al. , Fibrin sealant · Scientific Rationale, Production Methods, Properties, and Current Clinical use55, Vox Sanguinis, 1997, 72:133-143 and Marx G, "Evolution of fibrin glue 29 200930726 applicators", Transfus Med Rev, 2003; 17 (4 The application method described in 287-98 is incorporated herein by reference. Therefore, another object of the present invention is the therapeutic use of the condensable platelet growth factor thick solution obtained by the present invention or by the method of the present invention. Uses, and uses for forming blood clots, or cell culture applications for in vitro or in vivo studies. When used for cell culture in vitro or in vivo studies, the present invention or the coagulation obtained by the method of the present invention • The platelet growth factor thick solution will appear in the medium in an amount from 1% to 30%, preferably from 2% to 20%, and Also more rabbits 舛 3 ° /. to 10%, based on the volume of the medium. The main therapeutic applications of the aforementioned thick liquid / thrombin mixture include but not limited to: dental surgery, implantation, orthopedics and oral surgery , surgery, soft tissue and hard tissue healing and reconstruction, cardiovascular, thoracic, or monthly bowel surgery, neurosurgery, general surgery / trauma surgery, ophthalmology, otolaryngology, urology, and in patients with anticoagulation Or surgery on a patient with poor coagulopathy. The above and other objects, features, and advantages of the present invention will become more apparent from the description and appended claims. Pharmacology I. Materials and Methods 1.1 - Separation of the akes platelet The initial platelet concentrate (PC) was obtained using the MCS ten-component system (Haemonetics, Braintree, USA) with the consent of the volunteer donor. Collected. Whole blood is obtained through an intravenous catheter using intermittent flow and anticoagulant (1 ml anticoagulant citrate dextrose solution formulation per 10 ml of blood). Platelet-rich plasma 30 200930726 (PRP) is automatically separated from other blood components by centrifugation and collected in a sterile, single-use disposable bag, and red blood cells and plasma are returned to the donor. 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 count Platelets, white balls (WBC) and red flag counts were measured using a cell q counter (ABC Vet Automatic Blood Counter, ABX Diagnostics, France). 1.3- Treatment of the starting platelet concentrate a-Study design The initial platelet thick solution was processed according to the study design of the first figure. Briefly' The same donor starting platelet concentrate (3 〇〇 ml) was gently mixed' and divided into two equal volumes of pool (150 ml). Subset Pool 1 is directly subjected to S/D processing' without prior activation. The subset pool 2 is in the presence of ❹ 23 mM CaCl 2 and glass beads (beads), which will make the gold small
板凝膠形成的條件下活化,如下所述;用吸量(pipetting) 的方式小心地回收所得之釋出物(平均體積相當於12〇 - m卜因為有30 ml損失在企小板凝膠中),之後接受S/D 處理,接著進行油萃取。從起始血小板濃厚液所得之樣 本中,有經S/D處理後的未活化之灰小板子集池丨,血 小板活化後的子集池2,以及經S/D處理後的經活化子 集池2。 200930726 b-jhi小板活化: 在玻璃球粒存在的情況下,在jii小板濃厚液(子集 池 2 )中加入 1 M CaCl2 ( Sigma,批號 056k0688 )來進 行活化,其最終濃度為23 mM。將混合物輕輕地旋轉混 合,直到血凝塊形成為止,這一般會在5至8分鐘内形 成。使混合物再活化60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 0 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 φ 終濃度(v/v)為1% TnBP及1% Triton X-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’之後在持續溫和的擾動下處理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TnBP及Triton 32 200930726 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i5〇ml的子集池。 其中—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ι% ❹ 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% TnBP-1% Triton X-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(H ml)在0.23 M CaCl2 及玻璃球粒存在的情況下進行活化。 G 於室溫溫和旋轉混合60分鐘後,回收10 ml之血小 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,Jones • 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1% TnBP-1% Triton X-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 ml 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-80°C冷凍,直到進行血小 33 200930726 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丨—⑹樣本。將之於1〇〇〇〇 X g 離心 15 分鐘(Microfuge® 22R,Beckman Coulter,The plate gel is activated under conditions of formation, as described below; the resulting release is carefully recovered by pipetting (average volume is equivalent to 12 〇-m b because there is a loss of 30 ml in the small plate gel Medium), then subjected to S/D treatment followed by oil extraction. Among the samples obtained from the starting platelet thick solution, there are S/D-treated unactivated gray plate subset pools, platelet-activated subset pool 2, and S/D-treated activated subsets. Pool 2. 200930726 b-jhi platelet activation: In the presence of glass pellets, 1 M CaCl2 (Sigma, lot 056k0688) was added to the jii platelet concentrate (subset pool 2) for activation to a final concentration of 23 mM. . The mixture is gently spun and mixed until a blood clot forms, which typically takes place in 5 to 8 minutes. The mixture was allowed to reactivate for a period of 60 minutes, at which time the optimal platelet-derived growth factor was released under these experimental conditions. • The supernatant is poured out and 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 maximize The final concentration of φ (v/v) was 1% TnBP 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 continued mild disturbance. Upon completion of the S/D treatment, soybean oil (Sigma, Missouri, USA) was added to the S/D-platelet subset pool to 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 TnBP and Triton 32 200930726 X_45 to less than 10 and 100 ppm, respectively. 1.4- Effects of centrifugation and lysing agent types In order to study the effects 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 i5 〇ml subset pool. One of the subsets was centrifuged at high speed (10000 X g) to make the platelets a pellet and the supernatant was treated with ι % ❹ TnBP-l% Triton X-45. Another uncentrifuged subset pool was subdivided into two 75 ml subset pools, one of which received 1% TnBP-1% Triton X-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 Assay Assuming that CaCl2 activation does not completely activate platelets, the two platelet concentrates (H ml) were activated as described above in the presence of 0.23 M CaCl2 and glass spherules. G After gently mixing for 60 minutes at room temperature, 10 ml of platelet thick liquor release was recovered, and 0.5 ml of 1000 IU/ml bovine thrombin (Thrombin-JMI, 52604-7102-1, was added. Jones • Pharma, Saint_Louis, MO) to a final concentration of approximately 48 IU/ml. The mixture was gently mixed and spun at room temperature for 60 minutes. After that, 1% TnBP-1% Triton X-45 treatment and oil extraction were performed. In a parallel experiment, two platelet concentrate samples (10 ml) were also directly activated with 0.5 ml of the same bovine thrombin, gelled in a few seconds, and then gently mixed for 60 minutes. Samples were taken in various steps of the experimental method, centrifuged at 10,000 xg and frozen at -80 °C until blood platelet growth analysis was performed. 1.6-Growth Factor Determination The 丨-(6) sample was taken at each step of the procedure. Centrifuge at 1 〇〇〇〇 X g for 15 minutes (Microfuge® 22R, Beckman Coulter,
Fullerton,CA) ’使金小板及/或細胞碎片成為一團塊,並 得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 X g之離心力進行15分鐘的平行實驗。之 後上清液立即於-80°C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) 'A small plate and/or cell debris is made into a mass, and a cell-free supernatant is obtained for platelet-derived growth factor measurement. In addition, a parallel experiment of 15 minutes was carried out with a centrifugal force of 800 x g. Immediately thereafter, the supernatant was cold-bundled at -80 °C. 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 ❹ Calibrator稀釋劑(RD6-11 )稀釋100倍。將孔盤培養2 小時、清洗、並和與酶共軛結合之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(Substratea-PDGF-AB PDGF-AB was assayed using a Quantikine ELISA kit (#.DHD00B, R&D SYSTEMS, Minneapolis, MN). The sample was diluted 100-fold with ❹ Calibrator diluent (RD6-11). The wells were incubated for 2 hours, washed, and conjugated with the enzyme conjugated PDGF-AB antibody for further 3 hours at room temperature. Use Wash Buffer - to clean the wells, then add the substrate at room temperature (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。最小可彳貞測的劑量為1.7 pg/ml。 b-TGF-filSolution) 20_30 minutes. The hole is protected from light. Stop solution was added to each well and the absorbance at 450 nm was measured using a microtiter plate reader. The minimum detectable dose was 1.7 pg/ml. b-TGF-fil
使用 Quantikine ELISA kit ( DB100B,R&D 34 200930726 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_p受器π之% 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_pi分析之前’進行酸活 化及中和反應,以將潛性TGF_pi活化到免疫活性形成 狀態。為達此目的’將0.5 ml樣本與〇.1 ml之in HC1 ' 混合’於室溫培養10分鐘’加入0.1 ml之1.2N NaOH/ 0.5M HEPES (N-[2-羥基乙基]〇辰啡_n〇_[2-乙烧磺酸]) ❹ (Slgma ’ H-7523 )加以中和,再行離心。之後測定上 清液部分之總TGF-β 1含量。將各等分(5〇 μΐ)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,加入與酶共輕結合之TGF—pi 多株抗體,並於室溫持續培養i 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 200930726 SYSTEMS). The sample was diluted 1 〇〇 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 microtiter plate coated with a TGF_p receptor π. Acid activation and neutralization reactions were performed prior to TGF_pi analysis to activate latent TGF_pi to an immunologically active state. For this purpose 'mix 0.5 ml of sample with 1.1 ml of in HC1 'mixed at room temperature for 10 minutes' Add 0.1 ml of 1.2N NaOH / 0.5M HEPES (N-[2-hydroxyethyl] 〇辰Morphine _n〇_[2-ethanesulfonic acid]) ❹ (Slgma 'H-7523) was neutralized and centrifuged. The total TGF-β 1 content of the supernatant fraction was then determined. Aliquots (5 μ μΐ) were added to the microtiter plate in duplicate, then capped and incubated for 2 hours at room temperature. After that, the wells were washed, and TGF-pi polyclonal antibody, which was lightly bound to the enzyme, was added, and cultured for 5 hours at room temperature. As described above, the detection limit of TGF-βΙ is 4 61 pg/m
c-EGF 使用 Quantikine ELISA kit ( #.DEG00,R&D ❹ SYSTEMs,Minneapolis, MN )來測定 EGF。樣本以 Calibrator稀釋劑(rD6n)稀釋2〇倍。將20〇 μ1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 ❹ SYSTEMs, Minneapolis, MN). The sample was diluted 2 times with Calibrator diluent (rD6n). Add 20 μl of the standard * reference, reference or sample to the well. The well plates were incubated for 2 - hour at room temperature. Each well was suctioned and filled with a 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 200930726 定。最小可偵測的劑量為0.7 pg/ml。 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100倍。如製造商所說,其最小可债測 的劑量範圍為從0.007至0.056 ng/ml,且平均MDD為 0.026 ng/ml。在各盤孔中加入15〇 μ1之測定稀釋劑 (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, 之後和與酶共輛結合之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(tw〇_ tailed paired Student test)來進行統計上的比較。p值小 於0.05係用來估算企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心Molecular Devices, USA ) Tested at 450 nm in 30 minutes 35 200930726. The minimum detectable dose is 0.7 pg/ml. d-IGF-1 IGF-1 was quantified using a Quantikine ELISA kit (DG100 from r&d SYSTEMS). The sample was diluted 100-fold with Calibrator diluent (RD5-22). As stated by the manufacturer, the minimum bond dose ranged from 0.007 to 0.056 ng/ml with an average MDD of 0.026 ng/ml. 15 μ μl of the assay diluent (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 combined 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 tw〇_tailed paired Student test. A p-value less than 0.05 is used to estimate the significant difference in mean PGF concentrations in the different steps of the plate preparation process (the significance 〇f heart)
differences)。其值係表示為不明顯(Ns,<〇 〇5)、<〇 〇1 或<0.001。當其值接近〇·〇5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 分別對應於起始血小板濃厚液(pc)、經溶劑/清潔 劑(1% ΤηΒΡ及1% Trit〇n χ_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)的樣本係用皿_削£加以分離以比較其 36 200930726 蛋白含量。 將20 pg的各樣本與5 plNuPAGELDS樣本緩衝液 (4x ) ( Invitrogen )、2 μΐ NuPAGE 還原劑(10x ) (Invitrogen)及去離子水混合,所得最終體積為2〇 μ1 (已活化之jk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%聚丙烯醯胺 梯度凝膠(NuPAGE Bis-Tris, Invitrogen)來進行 SDS- • PAGE。 f) 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Mark 12之未染色標準品(Invhr〇gen)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 25〇加以毕 色。對應結果係揭示於第四圖。 μ 經溶劑/清潔劑方法處理之起始血小板濃厚液的蛋 © 自圖譜(Ρπ)ί!1〇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^】、板遠厚液的蛋白圖 譜則有明顯的差異,在40至70kDa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% ΤηΒΐΜ% τ_η χ_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 200930726 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-mmPetri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2 mM-麵酿胺酸、Eagle’s BSS * ( 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 0 加熱去活化的胎牛血清,並選擇性帶有5% S/D-PC或Differences). The value is expressed as insignificant (Ns, < 〇 〇 5), < 〇 〇 1 or < 0.001. When the value is close to 〇·〇5, the exact p value is displayed. The preparation and separation of samples comparing I.8_PC, S/D-PC and Act_PC correspond to the initial platelet thick solution (pc), treated with solvent/cleaner (1% ΤηΒΡ and 1% Trit〇n χ_45). Samples of the initial platelet thick solution (S/D-PC) and the CaCl2-activated starting platelet concentrate (Act-PC) were separated by a dish to compare their 36 200930726 protein content. 20 pg of each sample was mixed with 5 pl NuPAGELDS sample buffer (4x) (Invitrogen), 2 μΐ NuPAGE reducing agent (10x) (Invitrogen) and deionized water to give a final volume of 2 μμ1 (activated jk platelet) The sample for the thick solution is 21 μΐ). The resulting mixture was then heated at 70 ° C for 10 minutes and subjected to SDS- • PAGE using a 4-12% polyacrylamide gradient gel (NuPAGE Bis-Tris, Invitrogen). f) Protein separation was performed at a constant voltage of 200 V for 35 minutes with a predetermined current of 150 mA/gel. The resulting gel was stained with Coomassie blue R-250. The protein-labeled Mark 12 unstained standard (Invhr〇gen) is used to measure the molecular weight of the protein in the sample. The Mark 12 label was loaded onto a NuPAGE novex 4-12% Bis-Tris gel (Invitrogen) with MES and separated by Coomassie Blue R 25 毕. The corresponding results are disclosed in the fourth figure. μ Eggs from the initial platelet thick solution treated by the solvent/detergent method. Self-map (Ρπ) ί!1〇 shows that it is not significantly different from the untreated initial platelet concentrate, but activated. The protein profiles of the plate and the thick liquid are obviously different. The bands in the 40 to 70 kDa region disappear. These bands correspond to the α, p and subunits of fibrinogen, which are respectively 63.5. , 56 and 47 kDa. 1.9 - Growth factor activity measurement In order to determine whether the growth factor obtained by platelet S/D treatment (1% ΤηΒΐΜ% τ_η χ _45) can maintain activity, a human osteoblast-like MG-63 cell line was used for in vitro cell culture studies. 37 200930726 MG-63 cells were treated with growth factor concentrates obtained from S/D-PC or from Act-PC, 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-face acid, Eagle's BSS* ( Balanced Salt Solution), Adjust to 1.5 g/L sodium bicarbonate, 0. 1 mM non-essential amino acid, 1.0 mM sodium pyruvate, 10% 0 heat 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,以磷酸緩衝鹽溶液(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°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)-2,5-二苯基四唑鏽溴 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。 Ο 使用3-(4,5-二曱基噻唑-2-基)-5-(3-羧基甲氧基苯 基)-2-(4-磺苯基)-2H-四唑鏽(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 - 96 PromegaCorp” USA)。於 370C 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 200930726 ΐ免疫1小板濃厚液攙人相關病毒(ΗΙν,人 毒性腹浮液:牛病 性狂犬病Γ 财―毒之模型;假 時,有時二T^V)’在沒有其他方便活體外模型病毒 叶有%用作HBV之模型;以 病毒的高效價儲存懸 Ϊ 人1% ΤηΒΡ及1% _ 之混合物。 Ο Ο 的時間點進行估ί 及處理期間不同 活體外細胞培養來測活化的動力學。進行 ^ _ 來/則疋感木力,數據係以TCID5〇/ml之 =殘餘病毒感染力。所得=:7:: :=跡為>5.6〜相一 主logl〇 ’ *在vsv為>7 〇‘〇。故溶劑及/或 處理會確保可能存在於起始血小板濃厚 胞產物巾之脂質套膜病毒當+餘的病毒去活化。 L11_過期冷凍血小板的生長因子組成物 =過期(收集後超過5天)血小板濃厚液移入-20。 二Ϊ ί儲存一個月。之後將它們在3 5。C水浴中解束, ^行如述與新鮮血小板相同的實驗。測量多個製備物 的生長因子含量,對應結果係揭示於第五圖及第六 圖0 1.12-血凝塊形成測定 從經S/D處理之起始血小板濃厚液得出的生長因子 39 200930726 濃厚液係#由油萃取來移除_及清_之後進行回 收。將5 ml所得生長因子遭厚液引入一 5 n^筒中。 將5 mi之500 IU/ml的牛凝血酶引入另一 5 mi針筒中。 將兩個針筒置於一個以單—噴嘴連接的雙針筒供仏 器。讓這兩種成分從中通過。到、板凝膠會在5秒^ II·結果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) at 37 The cells were allowed to detach for 5 minutes at °C, 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-diphenyltetrazole rust bromide (MTT) assay. Electron microscopy was also performed to study cell morphology. Ο 3-(4,5-Dimercaptothiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole rust (MTS) Assay to monitor cell-proliferation. Briefly, 500 μM of MTS (Celliter - 96 PromegaCorp USA) was added to each sample. After 60 minutes of incubation at 370 C, the absorbance at 490 nm was measured using a microplate reader. 1.10-Virus Deactivation assay The study of the efficacy of the solvent and/or detergent treatment for the initiation of platelet thick liquor lysis is carried out on a lower scale experiment based on international benchmarks such as EMEA and WHO recommendations. 38 200930726 ΐ Immune 1 small plate thick liquid 搀 human-associated virus (ΗΙν, human toxic abdomen floating liquid: bovine rabies Γ ― - poisonous model; fake, sometimes two T ^ V) ' in the absence of other convenient in vitro model virus leaves % was used as a model for HBV; a mixture of 1% ΤηΒΡ and 1% _ was stored at a high titer of the virus. The kinetics of activation were measured by different in vitro cell cultures during the time of Ο 及 and during treatment. Perform ^ _ to / then 疋 sense wood force, the data is TCID5 〇 / ml = residual virus infectivity. The resulting =: 7:: : = trace is > 5.6 ~ phase one main logl 〇 ' * in vsv for > ;7 〇'〇. So solvent and / or treatment will ensure It may be present in the lipid envelope virus of the initial platelet thick cell product towel when the + remaining virus is deactivated. L11_ expired frozen platelet growth factor composition = expired (more than 5 days after collection) platelet thick liquid is transferred to -20. Ϊ ί store for one month. Then they are unbundled in a 35 ° C water bath. The same experiment as fresh platelets is performed. The growth factor content of multiple preparations is measured. The corresponding results are shown in the fifth and the first Figure 6 1.12 - Blood clot formation assay Growth factor 39 derived from S/D treated initial platelet concentrate 2009 30726 Thick liquid system # is extracted by oil to remove _ and clear _ after recovery. 5 ml The resulting growth factor was introduced into a 5 n^ cartridge by thick liquid. 5 μM of 500 IU/ml bovine thrombin was introduced into another 5 mi syringe. The two syringes were placed in a double syringe connected by a single nozzle. Supply the device. Let the two components pass through. The plate gel will be in 5 seconds ^ II. Results
❹ ΙΙ·1-細胞計數 =十名不同的捐贈者所得之十份血小板分離術 行研究。得自血小板分離術之血小板濃厚液結 =均為'064 ± 235.2 X⑽個血小板/ml (範圍:782_ 1358 X 1〇個血小板/ml),平均WBC計數為〇 ιΐ25 + 0.025 X Μ個/ml (範圍:〇 〇七), — ⑽ν,±0·025 χ 1〇6個/ml。 置為 II.2-生長因子含量 储lD=IGF_P1、咖及咖1在多種到、板 二備::平均濃度土標準差(SD)、最小值及最大值、以 目二笛:、Γ於表1。從各系列實驗所得之個別數據點可 見於弟二圖。 200930726❹ ΙΙ·1-cell count = ten platelet separation studies obtained from ten different donors. The platelet thick solution obtained from platelet separation = '064 ± 235.2 X (10) platelets / ml (range: 782_ 1358 X 1 platelets / ml), the average WBC count is 〇ιΐ25 + 0.025 X Μ / ml ( Range: 〇〇7), — (10) ν, ±0·025 χ 1〇6/ml. Set II.2-Growth factor content storage lD=IGF_P1, coffee and coffee 1 in a variety of to, plate two preparation:: average concentration soil standard deviation (SD), minimum and maximum, to the two flute: Table 1. Individual data points from each series of experiments can be found in the second figure. 200930726
PDGF-AB (N=10) TGF-βΙ (N=10) EGF (N = 8) IGF (N = 6) m 13.8 16.6 <0.0007 83.4 A SD 14.3 14.3 - 32.8 A Min. 2.2 1.1 60.1 Max. 49.5 36.0 - 162.7 m 184.4 192.2 2.2 88.4 r> SD 80.2 37.4 1.6 33.5 J3 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 SD 35.5 14.1 0.6 34.9 U 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 ΤΛ SD 45.9 27.2 1.0 39.7 xJ 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 BvsC <0.001 <0.001 0.044 <0.001 B vs D <0.001 <0.001 0.087 <0.01 C vsD NS NS NS NS 〇 表1 :在起始血小板中(A)、在l%TnBP-l%TritonX-45 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 ± 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<〇.〇〇1 )至184.4 ± 80.2 ng/nu’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 200930726 也會明顯增加(84·6 ± 35·5,p<0.001),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S/D 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(p<〇.〇〇1)。 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 ± 14.3 ng/mlPDGF-AB (N=10) TGF-βΙ (N=10) EGF (N = 8) IGF (N = 6) m 13.8 16.6 <0.0007 83.4 A SD 14.3 14.3 - 32.8 A Min. 2.2 1.1 60.1 Max. 49.5 36.0 - 162.7 m 184.4 192.2 2.2 88.4 r> SD 80.2 37.4 1.6 33.5 J3 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 SD 35.5 14.1 0.6 34.9 U 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 ΤΛ SD 45.9 27.2 1.0 39.7 xJ 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 BvsC < 0.001 < 0.001 0.044 < 0.001 B vs D < 0.001 < 0.001 0.087 < 0.01 C vsD NS NS NS NS 〇 Table 1: In the starting platelets (A), at l %TnBP-l%TritonX-45 After treatment (B), and after CaCl2 activation (C), the release agent was subjected to 1% TnBP-1% Triton X-45 treatment (D) for the average concentration of growth factors, (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-Triton X-45, its content increased significantly (p<〇.〇〇1) to 184.4 ± 80.2 ng/nu', which is approximately 13 times greater than the initial platelet concentrate. When the initial platelet concentrate is first activated by CaCl2, its content 200930726 will also increase significantly (84·6 ± 35·5, p < 0.001), but the increase is less (about 6 times), and it is in the subsequent s The /D process remains substantially unchanged (88.3 ± 45.9, NS). In the unactivated S/D treated platelets, the PGDF-AB content was significantly higher than that in the activated, and activated/S/D treated platelets (p<〇.〇〇1). There are similar data on TGF-βΙ. The average TGF-βΙ content in the starting platelet thick • thick solution was 16.6 ± 14.3 ng/ml
. (N=10)。直接s/D處理後,TGF-βΙ含量會比起始PC ❹ 增加近 12 倍’為 192.2 ± 37.4 ng/ml (p<0.001 )。當起始 血小板濃厚液首先被CaCl2活化時,含量會增加約4倍 (63.8± 14.1 ng/ml) (p<〇.〇〇l)並在後續 S/D 處理期間 保持在沒有明顯不同的狀態(68.6 ± 27.2 ng/ml)。又, 在經S/D處理之血小板中,TGF-βΙ平均含量明顯高於 其在已活化、以及已活化再經S/D處理之血小板中的值 (p<0.001)。 在起始血小板濃厚液中,無法偵測到EGF (<〇7 pg/ml),但在S/D處理後會變成可偵測到,且平均EGF ❹ 含量(2.2± N=6)明顯高於(p<0.05) CaCl2 活化後所得之值(〇·9 ± 0.6 ng/ml) ( p<〇.〇5 ),或者明顯 尚於在CaCl2活化後接著進行s/D處理所得之值(1.4 土 〜 1.0 ng/ml)。 在起始血小板濃厚液中,IGF-1平均含量為83.4 土 32.8 ng/ml ( N-8 )。相對於其他血小板源生長因子來說, 它在S/D處理後似乎沒有明顯的增加(88 4 ± % 5, p=0.025)。在CaCU活化後之血小板製備中,它的平均 含量甚至還稍高(P<〇.001) ( 117.2 ±34.9 ng/ml),且在 後續S/D處理之後保持穩定(112.4 ± 39.7 ng/ml)。 42 200930726 在150 ml血小板濃厚液中,直接S/D處理後(153 ml)所回收的 PDGF-AB、TGF-βΙ、EGF 及 IGF-1 總量 分別為 28213、29406、336 及 13525 ng ;而在 CaCld^ 化後則分別為10152、7156、108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。這確認了 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企小板濃 φ 厚液中(A)、在離心後(離心成一團塊並移除血小板)接 著對上清液進行TnBP-Triton X-45處理時(B)、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-βΙ、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 ; ρ<〇.〇〇1 ) ’其中之含量高,但與其在 ❹ 起始血小板濃厚液中的量類似(75.4對72.2 ng/ml)。故, PDGF-AB、TGF-(31及EGF在S/D處理期間的釋出係取 - 決於血小板的存在。在經l%TnBP-l%TritonX-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1〇/〇 tritonX-45或l%trit〇nX-100處理後,後續清潔劑處理 會在沒有油卒取情況下藉由tC 18吸附作用(sp 1 T45tC 18 及SPlT100tC18)移除;這顯示’單以2% ΤηΒΡ或以 1%ΤιιΒΡ及l%TritonX-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 200930726 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10000 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-pl 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 起始血小板濃厚液中的濃度(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 凝因子以及凡威勒伯氏因子的濃度係在經S/D處理之 起成小板濃厚液及經CaCl2活化之血小板濃厚液所得 生長因子濃厚液當中進行測量。這些結果係揭示於表 3 〇 44 200930726 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) 1.15 <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 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 蛋白濃度比較。 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。 II.3-牛凝血酶活化實驗(N=10). After direct s/D treatment, the TGF-βΙ content increased by nearly 12-fold than the initial PC ’ to 192.2 ± 37.4 ng/ml (p<0.001). When the starting platelet concentrate is first activated by CaCl2, the content is increased by about 4 times (63.8 ± 14.1 ng/ml) (p<〇.〇〇l) and remains in a distinctly different state during subsequent S/D treatment. (68.6 ± 27.2 ng/ml). Further, in the S/D-treated platelets, the average content of TGF-βΙ was significantly higher than that in the activated and activated S/D-treated platelets (p<0.001). EGF (<〇7 pg/ml) could not be detected in the initial platelet thick solution, but became detectable after S/D treatment, and the average EGF ❹ content (2.2 ± N = 6) was significant. Higher than (p<0.05) the value obtained after CaCl2 activation (〇·9 ± 0.6 ng/ml) (p<〇.〇5), or apparently after the activation of CaCl2 followed by s/D treatment ( 1.4 soil ~ 1.0 ng / ml). In the starting platelet concentrate, the average IGF-1 content was 83.4 soil 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 ± % 5, p = 0.025). In platelet preparation after CaCU activation, its average content was even slightly higher (P<〇.001) (117.2 ± 34.9 ng/ml) and remained stable after subsequent S/D treatment (112.4 ± 39.7 ng/ml) ). 42 200930726 In 150 ml of platelet concentrate, the total amount of PDGF-AB, TGF-βΙ, EGF and IGF-1 recovered after direct S/D treatment (153 ml) were 28213, 29406, 336 and 13525 ng, respectively; After CaCld^, they were 10152, 7156, 108, and 14064 ng, respectively; the 20% average volume loss caused by platelet gel formation (120 ml) was also counted. This confirms that the S/D treatment is more effective in releasing PDGF-AB, TGF-βΙ and ^ EGF than CaCl2 activation. Table 2 compares the platelet-derived growth factor in the initial fresh plate of concentrated φ thick solution (A), after centrifugation (centrifugation into a mass and removal of platelets) followed by TnBP-Triton X-45 treatment of the supernatant Time (B), and after S/D treatment of the initial platelet thick solution with 1% ΤηΒΡ-1 % Triton X-45 (C) or after S/D treatment with 2% TnBP (D) . The levels of PDGF-AB, TGF-βΙ, EGF and IGF-1 in untreated platelet concentrate (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; ρ<〇. 〇〇1) 'The content is high, but similar to the amount in the sputum starting platelet concentrate (75.4 vs. 72.2 ng/ml). Therefore, PDGF-AB, TGF- (31 and EGF release during S/D treatment - depends on the presence of platelets. Platelet solution treated with 1% TnBP-l% TritonX-45 or 2%, ΤηΒΡ In the cell product, the increase in the content of platelet-derived growth factor is similar. Further, after the platelet concentrate is treated with 1〇/〇tritonX-45 or 1%trit〇nX-100, the subsequent detergent treatment will be in the absence of oil. In the case of removal by tC 18 adsorption (sp 1 T45tC 18 and SPlT100tC18); this shows that the growth factor PDGF-AB observed after treatment with 2% ΤηΒΡ or 1% ΤιιΒΡ and 1% TritonX-45 The release of TGF-βΙ, IGF and EGF is within a range of 43 200930726. Therefore, the extent of platelet lysis is essentially the same as the use of this solvent and detergent combination or solvent or detergent alone. The final 'data (not shown) indicates that 'centrifugation of the sample at 10000 X g or 800 X g does not affect the amount of platelet-derived growth factor measured in this assay. PDGF-AB TGF-pl 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: Multiple growth factors in the starting platelet concentrate (a), centrifuged platelet-free supernatant after TnBP-Triton X-45 treatment (B), at TnBP- Concentration (ng/ml) in the initial platelet concentrate after Triton X-45 treatment (C) or after 2% ΤηΒΡ treatment (D). Further, fibrinogen, fibronectin, albumin, The concentration of immunoglobulin IgG and II, VII, VIII, IX, X, XI, XIII coagulation factors and Van derrick's factor are in S/D-treated platelet concentrate and CaCl2-activated platelets. The thick liquid was measured in the growth factor thick solution. These results are shown in Table 3. 〇44 200930726 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) 1.15 <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 Table 3: Starting platelet concentrate obtained from 1% TnBP-1% Triton X-45 treated with the present invention (S) /D-PC), or a comparison of various protein concentrations in a growth factor concentrate obtained from CaCl2-activated starting platelet concentrate (Act-PC). Therefore, it is shown that the activated platelet concentrate almost completely removes fibrinogen and clotting factors. II.3-Bovine thrombin activation experiment
CaC^/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-pl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 200930726 的含量(第三圖),其中前述j6l小板濃厚液之釋出物首 先被CaCl2活化,之後接受牛凝血酶(Act-T)及凝血酶 之進一步處理,並接受進一步S/D處理(Act-T-S/D)。 第三圖顯示:PDGFAB (A)、TGF-βΙ (B)及 EGF (C)在起 始血小板濃厚液中(起始)、在經CaCl2活化後(Act) 接著進行牛凝血酶活化(Act-T)及S/D處理(Act-T-SD) • 時的含量。可以看到,與CaCl2活化相較之下,再加上 -牛凝血酶活化不會增加PDGF-AB (A)、TGF-pi (B)及 ⑩ EGF (C)之釋出。 這是首度顯示’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-pl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,那些血小板源生長因子的釋出可能 會比較低,這可能是因為,凝集之血小板及部分釋出的 血小板源生長因子會被抓在纖維蛋白網絡(稱為血小板 ❹ 滅勝)中,該網絡係由起始血小板濃厚液中之纖維蛋白 原被凝血酶誘發產生聚合作用而形成的。確實,CaQ2 • 只導致部分活化、以及内源性凝血酶引起的血凝塊形成 * 不元全的可月匕性,可以被在已活化之血小板釋出物中進 一步加入約48 NIH早位之牛凝血酶/mi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 200930726 化血小板濃厚液時,在釋出液中的血小板生長因子的含 置是類似的。我們也發現,IGIM含量在s/D處理後保 夺為、,質未改變,而在部/凝血酶活化後只有輕微地增 力,這項、、’σ果可也反映出絕大多數的係以自由循環 形式早已存在於灰漿中’只有極小比例存在於血小板中 的事實。 、我們的數據顯示,與其他方法如凝血酶活化、冷凍 ' -解凍週期及/或冷凍乾燥相較之下,以S/D處理來進行 © 血小板溶胞作用是使血小板生長因子從血小板細胞内 顆粒釋出到滲出液中最有效的手段。稍早的研究業已發 ^類全血血清中的平均PDGF量為17 5 ng/ml,即 每106個血小板含0.06ng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處理之金小板濃厚液中的pdgf量平均約三倍以 上。我們的數據進一步顯示,要使pDGF_AB、TGF-βΙ 及EGF從血小板釋出量增加,可藉由使用1%TnBP_1%CaC^/glass pellet treatment only partially activates platelets, which may explain the lower levels of PDGF-AB, TGF-pl and EGF in the release than in the S/D lysate; in order to break the above assumptions, Therefore, the content of these platelet-derived growth factors in the release of platelet thick liquor 45 200930726 (third image) was determined, wherein the release of the aforementioned j6l platelet thick solution was first activated by CaCl2, and then received bovine thrombin (Act- Further treatment of T) and thrombin and further S/D treatment (Act-TS/D). The third panel shows: PDGFAB (A), TGF-βΙ (B) and EGF (C) in the initial platelet concentrate (start), after CaCl2 activation (Act) followed by bovine thrombin activation (Act- T) and S/D treatment (Act-T-SD) • The content of the time. It can be seen that, in contrast to CaCl2 activation, the addition of bovine thrombin does not increase the release of PDGF-AB (A), TGF-pi (B) and 10 EGF (C). This is the first time that 'release of platelet growth factor (especially the release of PDGF-AB, TGF-pl and EGF) when platelets are S/D treated is more effective than calcium and/or thrombin to activate platelets. 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 platelet-derived growth factors may be lower, probably because agglutinated platelets and partially released platelet-derived growth factors are caught in the fibrin network (called In platelet sputum, the network is formed by the polymerization of fibrinogen in the starting platelet concentrate by thrombin. Indeed, CaQ2 • only causes partial activation, and the formation of blood clots caused by endogenous thrombin. * Unexpected, menstrual, can be further added to the activated platelet release to about 48 NIH. After bovine thrombin/mi, it still does not cause the fact that the release of platelet-derived growth factor continues to increase. 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 the platelet growth factor content in the effluent was similar when using bovine thrombin activity 46 200930726 platelet concentrate. We also found that the IGIM content was retained after s/D treatment, and the quality was unchanged, but only slightly increased after the activation of the part/thrombin. This, 'σ fruit can also reflect the vast majority. It is already in the form of free circulation in the mortar - the fact that only a very small proportion is present in the platelets. Our data show that, compared to other methods such as thrombin activation, cryo-thaw cycles and/or freeze-drying, S/D treatment is used to effect platelet lysis by platelet growth factor from platelet cells. The most effective means of releasing the particles into the exudate. Earlier studies have reported an average amount of PDGF of 17 5 ng/ml in whole blood serum, which is 0.06 ng per 106 platelets. This amount is equivalent to 6 ng/m b of the separation platelet concentrate in our study and we found that the amount of pdgf in the S/D treated gold plate thick solution was on average about three times higher. Our data further show that to increase the release of pDGF_AB, TGF-βΙ and EGF from platelets, 1% TnBP_1% can be used.
Triton X-45 合併物、2% ΤηΒΡ、或 1% triton x_45 或 Triton 〇 X·*100的S/D處理來進行,且以油萃取處理來移除S/D, 不會降低金小板溶胞產物中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CaCl2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 200930726 三酸甘油酯 (mg/dl) HDL (mg/dl) LDL (mg/dl) — 170 33 99 162 ----- 101 62 20 ----— 11 0 43 ---—______ 2 0 156 ----------- 31 97 膽固醇 起始PC Trit-PC S/D-PC 1 S/D-PC 3 164 —--- 160 150 Ο 的脂質含量液製備之生長因子濃厚液 油萃取⑽H 處祕’接著進行一次 行三次油萃取(s=本^ 3 J理後’接著進 後進行。 C3),或者在血小板之CaCb活化 ★式。各受試由HltadU臨床技術儀器來進行測 4各又,本係以pH值為7 2且包含88“ 1〇3個血 ❹ 小板 μ、Ο·1 X 1〇3 個白血球(WBC) /μΐ 及 〇·〇8 χ 1〇6 個紅血球(RBC) /Ml的相同起始也小板濃厚液來製備。 ‘ 如表4所示,與經Triton處理之血小板或與已活化 之血小板相較之下,在藉由S/D處理再接著進行 所製備之S/D-PC生長因子濃厚液中,LDL (低 脂 蛋白)、HDL (高密度脂蛋白)、三酸甘油酯及麟固醇量 明顯較低。進一步來說,經S/D處理溶胞之血^板以油 萃取來萃取一次或三次不會觀察到明顯的差異。 / 本發明之生長因子濃厚液中膽固醇、三酸甘油醋及 LDL被除去,將引起臨床及治療目的方面报大的關主, 48 200930726 因為目前已知三酸甘油酯、以及LDL_膽固醇合併物 動脈粥狀硬化及透過動脈及靜脈系統中脂肪斑形成所 引起的心血管疾病的發展中所扮演的角色,這些問題合 引起心臟病發作、中風及周邊血管疾病。 日 Π.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 - 化:當細胞與經S/D處理或CaCl2活化之起始血小板濃 ❹ 賴所得的血小㈣生長®子濃隸共同培養時,會特 別觀察到許多纺錘狀細胞。進一步來說,細胞計數顯 不’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 時,MG-63成骨細胞的數目 式有明顯的增加。MTT分析結果也顯示,與經Act p(: 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。 ❹ 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 1% triton x_45 or Triton 〇X·*100 S/D treatment, and oil extraction to remove S/D, does not reduce gold plate dissolution Comparison of the content of four kinds of platelet growth factors in the cell product. II. The lipid content in the growth factor thick solution. The lipid in the platelet growth factor thick solution after the initial platelet thick solution was activated by S/D treatment or CaCl2 activation. The content results were measured and compared to the levels in the starting platelet concentrate, as shown in Table 4. 47 200930726 Triglyceride (mg/dl) HDL (mg/dl) LDL (mg/dl) — 170 33 99 162 ----- 101 62 20 ----— 11 0 43 ---—______ 2 0 156 ----------- 31 97 Cholesterol Startup PC Trit-PC S/D-PC 1 S/D-PC 3 164 —--- 160 150 Ο Lipid content liquid preparation growth factor Thick liquid oil extraction (10) H Department secret' followed by three times of oil extraction (s = this ^ 3 J after the 'after proceeding. C3), or CaCb activation in platelets. Each subject was tested by HltadU clinical technical instrument. The system has a pH of 7 2 and contains 88" 1 〇 3 blood ❹ small plates μ, Ο · 1 X 1 〇 3 white blood cells (WBC) / Μΐ and 〇·〇8 χ 1〇6 red blood cells (RBC) / Ml of the same starting also a small plate of thick liquid to prepare. ' As shown in Table 4, compared with Triton-treated platelets or activated platelets LDL (low-lipoprotein), HDL (high-density lipoprotein), triglyceride, and linalool in the S/D-PC growth factor concentrate prepared by S/D treatment. The amount is significantly lower. Further, no significant difference is observed in the extraction of the lysed blood plate by S/D with oil extraction for one or three times. / Cholesterol, triglyceride in the growth factor thick solution of the present invention The removal of vinegar and LDL will lead to a major cause of clinical and therapeutic goals, 48 200930726 because triglycerides and LDL-cholesterol combinations are known to be atherosclerotic and form fatty plaques in the arteries and venous systems. The role played by the development of cardiovascular disease, these problems Causes heart attack, stroke and peripheral vascular disease. Nippon.5-Growth factor activity microscopic observation shows the shape, size and number of cells: when the cells are concentrated with S/D or CaCl2 activated platelets A large number of spindle-shaped cells are particularly observed when co-cultured with the blood-small (4) growth® sub-concentration. Further, the cell count is not 'co-cultured with Act-PC or with growth factor concentrate. Compared with cells, when the cells were co-cultured with SD_PC growth factor thick solution, the number of MG-63 osteoblasts increased significantly. The results of MTT analysis also showed that compared with Act p (: treated cells) The presence of SD_PC growth factor concentrate increases the cell viability of cultured cells. S/D_pc or Act_pc growth factor concentrate does not exhibit cytotoxicity to MG-63 osteoblasts. 故 Therefore, the results of this study show that 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 , 處理制的生長因子濃厚液糾培養時,細胞反應會明 顯增加。本發明之方法所㈣濃職在觀⑹細胞上的 效果有所改善,現可能是因為生長因子含量增加的關 係’特別是PDGF、TGF_P1及咖(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血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 200930726 ,些結果也確認了本發明或藉由本發明之方法所 付的生長因子濃厚液在治療的用途方面以及製備改良 性細胞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S/D處理會有 效地將血源性套膜病毒去活化,特別是Hiv、hbV及 - HCV。 ❹ 如上材料及方法所示,1% TnBIM% Triton X-45合The growth factors obtained from both activated platelets remain active after the selected extraction method. However, our experiments show that when cells are incubated with S/D, a growth factor-rich thick solution, the cellular response increases significantly. The method of the present invention (4) has an improved effect on the cells of the (6) cells, and may now be due to an increase in the growth factor content, particularly PDGF, TGF_P1 and coffee (these factors are important in wound healing and tissue regeneration). The sexual system is known, and/or may be due to an increase in the amount of other biologically active substances that are not present or have a low amount of expression in the activated platelet concentrate. 49 200930726, these results also confirm the important potential of the present invention or the growth factor concentrates afforded by the method of the invention in terms of therapeutic use and in the preparation of modified cell culture media. II.6 - Virus Deactivation In addition, there is ample evidence that the S/D treatment used here effectively deactivates blood-borne envelope viruses, particularly Hiv, hbV and - HCV. 1 1% TnBIM% Triton X-45 combination as shown in the materials and methods above
併物於31°C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 log10,此外 也會快速地將27.0 l〇gl()之VSV及辛德畢斯模型病毒 (Sindbis model virus)去活化。非套膜病毒(如HAV 及小病毒B19)無法藉由S/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,但進一步奈米過濾 ❹ 也可用來降低非套膜病毒污染的風險。顯然,此處所描 述之血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jk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 200930726 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(如用以處理某些下肢糖尿病潰瘍時),當它與天 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,作為胎牛企清之替代物,或在細胞 工程研究、或間葉幹細胞活體擴增(exjpansi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源生 長因子。 【圖式簡單說明】 第一圈:研究設計:將捐輸的分離術血小板(N=8 ) 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CaCl2活化。子集池2則先 用23 niM CaCL及玻璃球粒加以活化’形成血小板凝 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。 第二圈:(A) PDGF-AB、(B) TGF-βΙ、(C) 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-1% 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 200930726 之血小板釋出物經S/D處理(1% ΤηΒΡ- 1% TritonX-45) 後(活化+S/D)的含量(ng/mi)。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。 第三圈:(A) PDGF-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 CaCl2活化後(Act)接著進行牛凝血酶活化(Act-T)及 ' S/D 處理(1 % ΤηΒΡ-1 % Triton X- 45 ) ( Act T-S/D )時的 , 含量(ng/ml)。S/D處理係如材料及方法所述來進行’ U 並包括3次油萃取來移除S/D用劑。 第四圈:起始血小板濃厚液(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CaCls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μ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:過期冷床血小板分別接受S/D處理(1% ❹ TnBP_l% 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 k (Act-PC+S/D)所得之濃厚液中,PDGF-AB (第五圖a) 及EGF (第五圖b)的組成物比較。 第六圖:過期冷凍血小板分別接受S/D處理(1%Treatment at 31 °C will ensure that the HIV, BVDV and PRV reduction factors are >5.6, >6.6, and >6.4 log10 within 5 minutes after treatment, and will also quickly 27.0 l〇gl() The VSV and Sindbis model virus are deactivated. Non-enveloped viruses (such as HAV and small virus B19) cannot be deactivated by S/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 nanofiltration can also be used to reduce the risk of non-enveloped virus contamination. Obviously, the platelet-derived growth factor preparations described herein will have to be produced under Good Manufacturing Practices (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). The S/D treatment of such jk platelets provides a degree of practical advantage in topical applications. First, the safety of viruses transmitted by allogeneic organisms 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 2009. 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, or, in the case of allowing the use of recombinant platelet-derived growth factor (such as To treat certain lower extremity diabetic ulcers, when used in combination with natural platelet-derived growth factors, a beneficial 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 cattle, or in cell engineering studies. Or a mesenchymal stem cell in vivo expansion (exjpansi〇n) and its differentiation into osteoblasts or chondrocytes as a recombinant platelet-derived growth factor. [Simple description of the diagram] The first circle: study design: the isolated platelets (N=8) were divided into two subset pools. Subset pool 1 was treated directly with 1% TnBP-1% Triton X-45 for 1 hour without first being activated with CaCl2. Sub-pool 2 was first activated with 23 niM CaCL and glass pellets to form a platelet 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. Second circumstance: (A) PDGF-AB, (B) TGF-βΙ, (C) EGF and (D) IGF in the initial platelet concentrate (start), in direct S/D treatment (1% TnBP- After 1% Triton X-45) (S/D), after initial platelet concentrate was activated by CaCl2 (activation), and after CaCl2 activation 51 200930726, platelet release was treated with S/D (1% ΤηΒΡ- 1% TritonX-45) Post (activation + S/D) content (ng/mi). The s/D treatment was carried out as described in Materials and Methods and included 3 oil extractions to remove the S/D agent. The third circle: (A) PDGF-AB, (B) TGF-βΙ and (C) EGF in the initial platelet thick solution (start), after the initial platelet thick solution is activated by CaCl2 (Act) followed by cattle Content (ng/ml) when thrombin activation (Act-T) and 'S/D treatment (1 % ΤηΒΡ-1 % Triton X- 45 ) (ACT TS/D ). The S/D treatment is performed as described in Materials and Methods and includes 3 oil extractions to remove the S/D agent. Fourth circle: between the starting platelet thick solution (line 1), the same platelet thick solution treated by the solvent-cleaner method of the present invention (line 2), and the same platelet thick solution (line 3) after activation of CaCls (line 3) Comparison of protein compositions. Samples were separated by SDS-PAGE on 4%-12% gels, after which the proteins were stained with Coomassie blue. The μ line corresponds to the protein label (size in kilodaltons, indicated on the left side of the gel). Figure 5: Excessive cold bed platelets are treated with S/D (1% ❹ TnBP_l% Triton X_45) (S/D-PC), or CaCl2 activation (Act-PC), or selective activation in CaCl2 followed by s The composition of PDGF-AB (figure a) and EGF (figure b) was compared in the thick solution obtained by /D treatment of k (Act-PC+S/D). Figure 6: Overdue frozen platelets are treated with S/D (1%)
TnBP-1% Triton X-45) (S/D-PC)或接受 CaCl2 活化 (Act-PC)所得之濃厚液中,IGF-1(第六圖a)及TGF-βΙ (第六圖b)的組成物比較。 【主要元件符號說明】 益 52TnBP-1% Triton X-45) (S/D-PC) or a concentrated solution obtained by CaCl2 activation (Act-PC), IGF-1 (sixth panel a) and TGF-βΙ (sixth panel b) Comparison of the composition. [Main component symbol description] Benefit 52
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