TWI772734B - Method of preparing platelet lysate and use thereof for treating vocal cord disease - Google Patents

Abstract

A method of preparing a platelet lysate and a use thereof are provided. The method includes the following steps. A collected whole blood is placed in a centrifuge tube containing a separation gel. A first centrifugation process is performed on the centrifuge tube containing the whole blood and the separation gel to separate the whole blood into a first upper portion, a middle portion, and a lower portion. The first upper portion and the middle portion are mixed to obtain a platelet-rich plasma fraction. The platelet-rich plasma fraction is clotted to obtain a platelet-rich fibrin gel. The platelet-rich fibrin gel is separated to obtain a platelet lysate.

Description

Translated fromChinese

製備血小板裂解液的方法及其用於治療聲帶疾病的用途Method for preparing platelet lysate and its use for treating vocal cord disease

本發明是有關於一種製備富含血小板血漿(platelet-rich plasma，PRP)的衍生物的方法及其用途，且特別是有關於一種製備血小板裂解液的方法及其用於治療聲帶疾病的用途。The present invention relates to a method for preparing a derivative of platelet-rich plasma (PRP) and its use, and in particular to a method for preparing a platelet lysate and its use for treating vocal cord diseases.

富含血小板血漿(platelet-rich plasma，PRP)為藉由對人類或動物的全血離心而得到的具有高濃度血小板的血漿，而血小板裂解液為富含血小板血漿經進一步處理的衍生物，血小板裂解液含有多種與再生有關的生長因子。因此富含血小板血漿及血小板裂解液已知可被應用在再生醫學。Platelet-rich plasma (PRP) is plasma with a high concentration of platelets obtained by centrifugation of human or animal whole blood, and platelet lysate is a further processed derivative of platelet-rich plasma. The lysate contains a variety of growth factors associated with regeneration. Therefore, platelet-rich plasma and platelet lysate are known to be used in regenerative medicine.

目前已知製備血小板裂解液的傳統方法為：將PRP進一步加入活化因子後，使用重複解凍冷凍或超音波方式取得血小板裂解液。而上述的傳統方法可能具有以下缺點：收取全血量過多、因分離不完全導致的紅血球汙染、因抗凝劑作用而需重複活化、不易進行濃縮等問題。上述問題會導致收取後的血小板裂解液的生長因子量不足。此外，在製備的血小板裂解液的傳統方式中，需要將PRP自離心管內取出並進行活化，因此在無菌性的效果堪慮，且以冷凍解凍或超音波震盪方式取得血小板裂解液所花的時間過長。因此，如何快速地製備一種具有高濃度生長因子的血小板裂解液，是目前研究人員急欲解決的問題。At present, the traditional method for preparing platelet lysate is as follows: after further adding activating factor to PRP, the platelet lysate is obtained by repeated thawing and freezing or ultrasound. The above-mentioned traditional methods may have the following disadvantages: excessive collection of whole blood, red blood cell contamination due to incomplete separation, repeated activation due to the effect of anticoagulants,It is not easy to carry out problems such as concentration. The above problems can lead to insufficient growth factors in the harvested platelet lysate. In addition, in the traditional way of preparing the platelet lysate, PRP needs to be taken out from the centrifuge tube and activated, so the effect of sterility is not considered, and it takes a long time to obtain the platelet lysate by freezing and thawing or ultrasonic shock. too long. Therefore, how to quickly prepare a platelet lysate with a high concentration of growth factors is a problem that researchers are eager to solve.

聲帶(門)閉合(鎖)不全(Vocal fold insufficiency；Glottic insufficiency)為一種當發音時，聲帶不完全閉合的症狀，會導致當發音時有不適當的空氣通過聲門，進一步造成聲音嘶啞、發音不長等現象，甚至導致嗆傷的危險，吞嚥困難、痙攣性發聲障礙及因嗆傷而造成之反覆性胸腔感染等都可能因此而產生。然而，造成聲帶閉合不全的成因相當多元，包含：單側或雙側之聲帶麻痺、聲帶萎縮、聲帶溝、聲帶瘢痕或畸形等，其中聲帶瘢痕或畸形可能是由先天性或原發性之疾病、次發性的醫源性損傷如：過長時間的插管或咽喉腫瘤的切除等損傷。Vocal fold insufficiency (Glottic insufficiency) is a symptom of incomplete closure of the vocal cords during pronunciation, resulting in inappropriate air passing through the glottis during pronunciation, further causing hoarseness and insufficiency of pronunciation. It may even lead to the danger of choking injury, dysphagia, spasmodic dysphonia, and repeated chest infections caused by choking injury. However, the causes of vocal cord insufficiency are quite diverse, including: unilateral or bilateral vocal cord paralysis, vocal cord atrophy, vocal cord groove, vocal cord scarring or deformity, etc., where vocal cord scarring or deformity may be caused by congenital or primary diseases , Secondary iatrogenic injury such as: prolonged intubation or throat tumor resection and other injuries.

目前聲帶的治療包括音聲治療、玻尿酸或自體脂肪的注射和甲狀軟骨成形術(Thyroplasty)。然而，治療結果並不令人完全滿意，因為聲帶振動最重要的固有層細胞外的基質成分沒有改變，且填充物易被人體吸收，無法有效的維持。Current vocal cord treatments include sonic therapy, injection of hyaluronic acid or autologous fat, and Thyroplasty. However, the treatment results were not completely satisfactory because the extracellular matrix composition of the lamina propria, the most important part of vocal cord vibration, was not changed, and the filler was easily absorbed by the body and could not be maintained effectively.

本發明提供一種製備血小板裂解液的方法，可在無菌的條件下快速地製備具有高濃度生長因子的血小板裂解液。The present invention provides a method for preparing platelet lysate, which can be used in sterileRapid preparation of platelet lysates with high concentrations of growth factors under conditions.

本發明提供一種包括富含血小板血漿及/或富含血小板血漿的衍生物的醫藥組成物，可有效地修補聲帶。The present invention provides a pharmaceutical composition comprising platelet-rich plasma and/or derivatives of platelet-rich plasma, which can effectively repair vocal cords.

本發明的實施例提供一種製備血小板裂解液的方法，所述方法包括以下步驟：步驟a)，將採集的全血放置於裝有分離膠體的離心管中，其中分離膠體的密度為1.030g/cm³至1.093g/cm³；步驟b)，對裝有全血及分離膠體的離心管進行第一離心製程，以使全血分離成第一上層部分、中間部分以及下層部分，其中分離膠體位於中間部分與下層部分之間；步驟c)，將第一上層部分與中間部分進行混合，以得到富含血小板血漿(platelet-rich plasma，PRP)部分；步驟d)，使富含血小板血漿部分凝結，以得到富含血小板纖維蛋白(platelet-rich fibrin，PRF)膠體；以及步驟e)，分離富含血小板纖維蛋白膠體，以得到血小板裂解液(platelet lysate，PL)，其中在步驟a)至步驟e)中未添加抗凝劑。An embodiment of the present invention provides a method for preparing a platelet lysate, the method comprising the following steps: step a), placing the collected whole blood in a centrifuge tube containing a separation colloid, wherein the separation colloid has a density of 1.030 g/g cm³ to 1.093 g/cm³ ; step b), performing a first centrifugation process on the centrifuge tube containing the whole blood and the separation colloid, so that the whole blood is separated into a first upper part, a middle part and a lower part, wherein the colloid is separated between the middle part and the lower part; step c), mixing the first upper part with the middle part to obtain a platelet-rich plasma (PRP) part; step d), making the platelet-rich plasma part Coagulation to obtain platelet-rich fibrin (PRF) colloid; and step e), separating the platelet-rich fibrin colloid to obtain platelet lysate (platelet lysate, PL), wherein in step a) to No anticoagulant is added in step e).

在本發明的一實施例中，在進行步驟c)之前，可更包括移除部分的第一上層部分，以得到第二上層部分，其中第一上層部分與第二上層部分的體積比率為1：0.01~0.99。In an embodiment of the present invention, before performing step c), it may further comprise removing a part of the first upper layer part to obtain a second upper layer part, wherein the volume ratio of the first upper layer part to the second upper layer part is 1 : 0.01~0.99.

在本發明的一實施例中，上述的抗凝劑可包括抗凝血複方檸檬酸鈉溶液A(Anticoagulant Citrate Dextrose Solution,Solution A，ACD-A)、抗凝血複方檸檬酸鈉溶液B(Anticoagulant Citrate Dextrose Solution,Solution B，ACD-B)、抗凝血複方檸檬酸鈉溶液C(Anticoagulant Citrate Dextrose Solution,Solution C，ACD-C)、檸檬酸-磷酸-葡萄溶液(Citrate-phosphate-dextrose Solution，CPD)、乙二胺四乙酸(Ethylenediaminetetraacetic acid，EDTA)、肝素(Heparin)、檸檬酸鈉(Sodium Citrate)或其組合。In an embodiment of the present invention, the above-mentioned anticoagulant may include anticoagulant compound sodium citrate solution A (Anticoagulant Citrate Dextrose Solution, Solution A, ACD-A), anticoagulant compound sodium citrate solution B (Anticoagulant Citrate Dextrose Solution, Solution A, ACD-A) Citrate Dextrose Solution, Solution B, ACD-B), anticoagulant compound sodium citrate solution C (Anticoagulant Citrate Dextrose Solution, Solution C,ACD-C), Citrate-phosphate-dextrose Solution (CPD), Ethylenediaminetetraacetic acid (EDTA), Heparin, Sodium Citrate or a combination thereof .

在本發明的一實施例中，上述的分離膠體的材料可包括含矽聚合物、丙烯酸聚合物或聚酯類聚合物。In an embodiment of the present invention, the material for the separation colloid may include a silicon-containing polymer, an acrylic polymer or a polyester polymer.

在本發明的一實施例中，上述的第一離心製程的離心轉速例如是1000g至4000g，離心時間例如是3分鐘至10分鐘。In an embodiment of the present invention, the centrifugation speed of the above-mentioned first centrifugation process is, for example, 1000 g to 4000 g, and the centrifugation time is, for example, 3 minutes to 10 minutes.

在本發明的一實施例中，上述的第一上層部分為包含血小板的血漿層，中間部分為包含血小板及白血球的白膜層(buffy coat)，以及下層部分為包含紅血球的紅血球層。In an embodiment of the present invention, the first upper part is a plasma layer containing platelets, the middle part is a buffy coat containing platelets and white blood cells, and the lower part is an erythrocyte layer containing red blood cells.

在本發明的一實施例中，使富含血小板血漿部分凝結包括對富含血小板血漿部分進行第二離心製程。In one embodiment of the invention, coagulating the platelet-rich plasma fraction includes subjecting the platelet-rich plasma fraction to a second centrifugation process.

在本發明的一實施例中，上述的第二離心製程的離心轉速例如是1000g至4000g，離心時間例如是3分鐘至10分鐘。In an embodiment of the present invention, the centrifugation speed of the second centrifugation process is, for example, 1000 g to 4000 g, and the centrifugation time is, for example, 3 minutes to 10 minutes.

在本發明的一實施例中，分離富含血小板纖維蛋白膠體包括對富含血小板纖維蛋白膠體進行第三離心製程。In an embodiment of the present invention, separating the platelet-rich fibrin colloid includes performing a third centrifugation process on the platelet-rich fibrin colloid.

在本發明的一實施例中，上述的所述第三離心製程包括：對富含血小板纖維蛋白膠體進行單次離心，其中離心轉速為1000g至4000g，離心時間為3分鐘至45分鐘，或者對富含血小板纖維蛋白膠體進行多次離心，其中離心轉速為1000g至4000g，每次的離心時間為3分鐘至15分鐘。In an embodiment of the present invention, the above-mentioned third centrifugation process includes: performing a single centrifugation on the platelet-rich fibrin colloid, wherein the centrifugation speed is 1000g to 4000g, and the centrifugation time is 3 minutes to 45 minutes, or The platelet-rich fibrin colloid is centrifuged for several times, wherein the centrifugation speed is 1000g to 4000g, and the centrifugation time is 3 minutes to 15 minutes each time.

在本發明的一實施例中，製備血小板裂解液的整個製程時間為10分鐘至60分鐘。In an embodiment of the present invention, the entire process of preparing platelet lysateThe time is 10 minutes to 60 minutes.

在本發明的一實施例中，在進行步驟e)之前，更包括對富含血小板纖維蛋白膠體按壓、擠壓、過濾、超音波震盪或進行冷凍解凍循環，以使得血小板裂解液自富含血小板纖維蛋白膠體釋放。In an embodiment of the present invention, before step e) is performed, it further includes pressing, squeezing, filtering, ultrasonically shaking or performing a freeze-thaw cycle on the platelet-rich fibrin colloid, so that the platelet lysate is freed from the platelet-rich fibrin colloid. Fibrin colloid release.

在本發明的一實施例中，上述的血小板裂解液可包括由TGF-β1、PDGF-BB、PDGF-AB、FGF2、EGF、VEGF及VEGFA中選出的至少一種生長因子，其中TGF-β1的濃度為1ng/ml至10000ng/ml，PDGF-BB的濃度10pg/ml至500000pg/ml，PDGF-AB的濃度為10pg/ml至500000pg/ml，FGF2的濃度為1pg/ml至10000pg/ml，EGF的濃度為1pg/ml至10000pg/ml，VEGF的濃度為1pg/ml至10000pg/ml，VEGFA的濃度為0.1pg/ml至5000pg/ml。In an embodiment of the present invention, the above-mentioned platelet lysate may include at least one growth factor selected from TGF-β1, PDGF-BB, PDGF-AB, FGF2, EGF, VEGF and VEGFA, wherein the concentration of TGF-β1 1 ng/ml to 10,000 ng/ml, PDGF-BB 10 pg/ml to 500,000 pg/ml, PDGF-AB 10 pg/ml to 500,000 pg/ml, FGF2 1 pg/ml to 10,000 pg/ml, EGF The concentration is 1 pg/ml to 10000 pg/ml, the concentration of VEGF is 1 pg/ml to 10000 pg/ml, and the concentration of VEGFA is 0.1 pg/ml to 5000 pg/ml.

本發明提供一種醫藥組成物用於治療聲帶疾病之藥物的用途，其中所述醫藥組成物包括富含血小板血漿及/或富含血小板血漿的衍生物。The present invention provides the use of a pharmaceutical composition for the treatment of vocal cord diseases, wherein the pharmaceutical composition comprises platelet-rich plasma and/or derivatives of platelet-rich plasma.

在本發明的一實施例中，上述的富含血小板血漿的衍生物包括血小板裂解液。In an embodiment of the present invention, the above-mentioned derivative of platelet-rich plasma includes platelet lysate.

在本發明的一實施例中，上述的聲帶疾病包括聲帶閉合不全。In an embodiment of the present invention, the above-mentioned vocal cord disease includes vocal cord insufficiency.

在本發明的一實施例中，上述的富含血小板血漿及所述富含血小板血漿的衍生物包括由TGF-β1、PDGF-BB、PDGF-AB、FGF2、EGF、VEGF及VEGFA中選出的至少一種生長因子。In an embodiment of the present invention, the above-mentioned platelet-rich plasma and the derivatives of the platelet-rich plasma include TGF-β1, PDGF-BB, PDGF-AB,At least one growth factor selected from FGF2, EGF, VEGF and VEGFA.

在本發明的一實施例中，上述的TGF-β1在醫藥組成物中的濃度為0.7ng/ml至7000ng/ml，PDGF-BB在醫藥組成物中的濃度7pg/ml至350000pg/ml，PDGF-AB在醫藥組成物中的濃度為7pg/ml至350000pg/ml，FGF2在醫藥組成物中的濃度為0.7pg/ml至7000pg/ml，EGF在醫藥組成物中的濃度為0.7pg/ml至7000pg/ml，VEGF在醫藥組成物中的濃度為0.7pg/ml至7000pg/ml，VEGFA在醫藥組成物中的濃度為0.06pg/ml至3500pg/ml。In an embodiment of the present invention, the concentration of the above-mentioned TGF-β1 in the pharmaceutical composition is 0.7ng/ml to 7000ng/ml, the concentration of PDGF-BB in the pharmaceutical composition is 7pg/ml to 350000pg/ml, the PDGF -The concentration of AB in the pharmaceutical composition is 7 pg/ml to 350,000 pg/ml, the concentration of FGF2 in the pharmaceutical composition is 0.7 pg/ml to 7000 pg/ml, and the concentration of EGF in the pharmaceutical composition is 0.7 pg/ml to 0.7 pg/ml 7000 pg/ml, the concentration of VEGF in the pharmaceutical composition is 0.7 pg/ml to 7000 pg/ml, and the concentration of VEGFA in the pharmaceutical composition is 0.06 pg/ml to 3500 pg/ml.

在本發明的一實施例中，上述的醫藥組成物更包括選自玻尿酸、生理食鹽水、自體脂肪及自體幹細胞中的至少一者。In an embodiment of the present invention, the above-mentioned pharmaceutical composition further comprises at least one selected from the group consisting of hyaluronic acid, physiological saline, autologous fat and autologous stem cells.

在本發明的一實施例中，上述的醫藥組成物是以注射方式給藥至聲帶。In an embodiment of the present invention, the above-mentioned pharmaceutical composition is administered to the vocal cords by injection.

基於上述，由於本發明的製備血小板裂解液的方法在整個製程皆未添加抗凝劑，因此並沒有將血小板裂解液自離心管取出並重覆活化的額外步驟，藉此可縮短製程時間。此外，本發明的製備血小板裂解液的整個製程皆在同一個離心管中進行操作，因此具有避免污染且具有無菌性的優點。Based on the above, since the method for preparing platelet lysate of the present invention does not add anticoagulant in the whole process, there is no additional step of taking out the platelet lysate from the centrifuge tube and repeating activation, thereby shortening the process time. In addition, the whole process of preparing the platelet lysate of the present invention is performed in the same centrifuge tube, so it has the advantages of avoiding contamination and having sterility.

此外，本發明的包括富含血小板血漿及/或富含血小板血漿的衍生物的醫藥組成物可有效地修補聲帶。In addition, the pharmaceutical compositions of the present invention comprising platelet-rich plasma and/or derivatives of platelet-rich plasma can effectively repair vocal cords.

10:全血10: Whole blood

12:第一上層部分12: First Upper Section

13:第二上層部分13: Second Upper Section

14:中間部分14: Middle Section

15:富含血小板血漿部分15: Platelet rich plasma fraction

16:下層部分16: Lower part

17:富含血小板纖維蛋白膠體17: Platelet-Rich Fibrin Colloid

18:血小板裂解液18: Platelet Lysate

19:膠體部分19: Colloid part

20:分離膠體20: Separation of colloids

30:離心管30: Centrifuge tube

圖1為依照本發明的一實施例的製備血小板裂解液的方法的流程圖。FIG. 1 is a flow chart of a method for preparing a platelet lysate according to an embodiment of the present invention.

本發明提供一種製備血小板裂解液的方法，可在特定的操作條件下得到具有高濃度生長因子的血小板裂解液，且此方法操作過程簡單、製程時間短，且整個過程無需添加任何抗凝劑。The present invention provides a method for preparing platelet lysate, which can obtain platelet lysate with high concentration of growth factors under specific operating conditions.

此外，本發明所提供的含有富含血小板血漿及/或富含血小板血漿的衍生物的醫藥組成物，可有效地修補聲帶。In addition, the pharmaceutical composition containing platelet-rich plasma and/or the derivatives of platelet-rich plasma provided by the present invention can effectively repair the vocal cords.

為了能徹底地了解本發明，將在以下詳盡描述所述製備血小板裂解液的製程步驟。然而，眾所皆知的組成或製程步驟並未描述於細節中，以避免限制本發明。本發明的較佳實施例會詳細描述如下，但本發明不限於此，本發明還可廣泛地施行在其他的實施例中，且本發明的範圍不受限定，以其後的專利範圍為準。For a thorough understanding of the present invention, the process steps for preparing the platelet lysate will be described in detail below. However, well-known components or process steps are not described in detail in order to avoid limiting the present invention. The preferred embodiments of the present invention will be described in detail as follows, but the present invention is not limited thereto, the present invention can also be widely implemented in other embodiments, and the scope of the present invention is not limited, and the following patent scope shall prevail.

圖1為依照本發明的一實施例的製備血小板裂解液的方法的流程圖。本發明的製備血小板裂解液的方法包括以下步驟。FIG. 1 is a flow chart of a method for preparing a platelet lysate according to an embodiment of the present invention. The method for preparing platelet lysate of the present invention includes the following steps.

首先，進行步驟a)，將採集的全血10放置於裝有分離膠體20的離心管30中。在一實施例中，全血10例如是新鮮的哺乳類動物的全血。在本實施例中，全血10例如是新鮮的人類全血。First, step a) is performed, and the collectedwhole blood 10 is placed in thecentrifuge tube 30 containing theseparation colloid 20 . In one embodiment,whole blood 10 is, for example, fresh mammalian whole blood. In this embodiment, thewhole blood 10 is, for example, fresh human whole blood.

在本實施例中，分離膠體20的密度為1.030g/cm³至1.093g/cm³。在本實施例中，分離膠體20的材料例如是含矽聚合物、丙烯酸聚合物或聚酯類聚合物。In this embodiment, the density of the separatingcolloid 20 is 1.030 g/cm³ to 1.093 g/cm³ . In this embodiment, the material of theseparation colloid 20 is, for example, a silicon-containing polymer, an acrylic polymer or a polyester polymer.

接著，進行步驟b)，對裝有全血10及分離膠體20的離心管30進行第一離心製程，以使全血10分離成第一上層部分12、中間部分14以及下層部分16，其中分離膠體20位於中間部分14與下層部分16之間。在本實施例中，步驟b)的第一離心製程的離心轉速例如是1000g至4000g。在一實施例中，步驟b)的第一離心製程的離心轉速例如是1500g至3000g。在本實施例中，步驟b)的第一離心製程的離心時間例如是3分鐘至10分鐘。在一實施例中，步驟b)的第一離心製程的離心時間例如是4分鐘至8分鐘。在一實施例中，步驟b)的第一離心製程的離心轉速例如是1500g，離心時間例如是5分鐘。Next, step b) is performed, and a first centrifugation process is performed on thecentrifuge tube 30 containing thewhole blood 10 and theseparation colloid 20, so that thewhole blood 10 is separated into the first upper part 12, themiddle part 14 and thelower part 16, wherein the separation Thegel 20 is located between themiddle portion 14 and thelower portion 16 . In this embodiment, the centrifugation speed of the first centrifugation process in step b) is, for example, 1000 g to 4000 g. In one embodiment, the centrifugation speed of the first centrifugation process in step b) is, for example, 1500 g to 3000 g. In this embodiment, the centrifugation time of the first centrifugation process in step b) is, for example, 3 minutes to 10 minutes. In one embodiment, the centrifugation time of the first centrifugation process in step b) is, for example, 4 minutes to 8 minutes. In one embodiment, the centrifugation speed of the first centrifugation process in step b) is, for example, 1500 g, and the centrifugation time is, for example, 5 minutes.

在本實施例中，在進行步驟b)後，使得全血10分離成三層的程度，其中最上層(即第一上層部分12)為包含血小板的血漿層，中間層(即中間部分14)為包含血小板及白血球的白膜層(buffy coat)，最下層(即下層部分16)為包含紅血球的紅血球層。In this embodiment, after step b) is performed, thewhole blood 10 is separated into three layers, wherein the uppermost layer (ie, the first upper layer portion 12 ) is the plasma layer containing platelets, and the middle layer (ie, the middle portion 14 ) It is a buffy coat including platelets and leukocytes, and the lowermost layer (ie, the lower layer portion 16 ) is an erythrocyte layer including erythrocytes.

在本實施例中，本發明的分離膠體20可有效地隔離紅血球與血小板，使得紅血球幾乎僅存在於下層部分16中，以及使得血小板存在於第一上層部分12及中間部分14中。具體來說，第一上層部分12及中間部分14中的血小板濃度高於全血及下層部分16中的血小板濃度。In this embodiment, the separatingcolloid 20 of the present invention can effectively isolate red blood cells and platelets, so that red blood cells exist almost only in thelower part 16 , and platelets exist in the first upper part 12 and themiddle part 14 . Specifically, the platelet concentration in the first upper portion 12 and themiddle portion 14 is higher than the platelet concentration in the whole blood and thelower portion 16 .

在本實施例中，在進行步驟b)後，可接著進行步驟c)，但本發明不限於此。在另一實施例中，在進行步驟b)之後且進行步驟c)之前，可選擇性地進一步移除部分的第一上層部分12，以得到第二上層部分13。在本實施例中，第一上層部分12與第二上層部分13的體積比率例如是1：0.01~0.99。在一實施例中，第一上層部分12與第二上層部分13的體積比率例如是1：0.1-0.8。在上述的體積比例範圍內，可得到較高濃度的血小板裂解液。在本實施例中，移除部分的第一上層部分12的方法例如是將針頭刺入離心管30的第一上層部分12處，並將部分的第一上層部分12吸取至離心管30外部。In this embodiment, after step b) is performed, step c) may be performed next, but the present invention is not limited thereto. In another embodiment, after performing step b) and performingBefore step c), a portion of the first upper layer portion 12 may optionally be further removed to obtain a secondupper layer portion 13 . In this embodiment, the volume ratio of the first upper layer portion 12 to the secondupper layer portion 13 is, for example, 1:0.01˜0.99. In one embodiment, the volume ratio of the first upper layer portion 12 to the secondupper layer portion 13 is, for example, 1:0.1-0.8. Within the above volume ratio range, a higher concentration of platelet lysate can be obtained. In this embodiment, the method of removing part of the first upper layer part 12 is, for example, piercing a needle into the first upper layer part 12 of thecentrifuge tube 30 , and sucking part of the first upper layer part 12 to the outside of thecentrifuge tube 30 .

在本實施例中，在進行步驟b)之後且進行步驟c)之前，由於移除了部分的第一上層部分12而得到較小體積的第二上層部分13，因此在後續製程自富含血小板纖維蛋白膠體所分離的血小板裂解液可存在較小體積的溶液中，藉此達成濃縮的效果。In this embodiment, after performing step b) and before performing step c), since part of the first upper layer portion 12 is removed to obtain a smaller volume of the secondupper layer portion 13 , the platelet-rich platelet-rich portion is obtained in the subsequent process. The platelet lysate separated by the fibrin colloid can be stored in a smaller volume of solution, thereby achieving a concentrated effect.

然後，進行步驟c)，將第一上層部分12(或第二上層部分13)與中間部分14進行混合，以得到富含血小板血漿(platelet-rich plasma，PRP)部分15。在本實施例中，使第一上層部分12(或第二上層部分13)與中間部分14進行混合的方法例如是將離心管30均勻搖晃，使得第一上層部分12(或第二上層部分13)與中間部分14混合。但本發明不限於此，任何可使第一上層部分12(或第二上層部分13)與中間部分14均勻混合的方式皆可應用在此步驟中。Then, in step c), the first upper layer part 12 (or the second upper layer part 13 ) is mixed with themiddle part 14 to obtain a platelet-rich plasma (PRP)part 15 . In this embodiment, the method of mixing the first upper layer part 12 (or the second upper layer part 13 ) with themiddle part 14 is, for example, by shaking thecentrifuge tube 30 evenly, so that the first upper layer part 12 (or the second upper layer part 13 ) ) mixed with themiddle portion 14. However, the present invention is not limited to this, and any method that can uniformly mix the first upper layer part 12 (or the second upper layer part 13 ) and themiddle part 14 can be applied in this step.

之後，進行步驟d)，使富含血小板血漿部分15凝結，以得到富含血小板纖維蛋白(platelet-rich fibrin，PRF)膠體17。在本實施例中，使富含血小板血漿部分15凝結的方法例如是對富含血小板血漿部分15進行第二離心製程。在本實施例中，步驟d)的第二離心製程的離心轉速例如是1000至4000g。在一實施例中，步驟d)的第二離心製程的離心轉速例如是1500至3000g。在本實施例中，步驟d)的第二離心製程的離心時間例如是3分鐘至10分鐘。在一實施例中，步驟d)的第一離心製程的離心時間例如是4分鐘至8分鐘。在一實施例中，步驟d)的第一離心製程的離心轉速例如是1500g，離心時間例如是5分鐘。在進行第二離心製程的過程中，血小板血漿部分15會自我凝結成富含血小板纖維蛋白(platelet-rich fibrin，PRF)膠體17。After that, step d) is performed to coagulate the platelet-rich plasma fraction 15 to obtain a platelet-rich fibrin (PRF) colloid 17 . existIn this embodiment, the method for coagulating the platelet-rich plasma fraction 15 is, for example, performing a second centrifugation process on the platelet-rich plasma fraction 15 . In this embodiment, the centrifugal rotation speed of the second centrifugation process in step d) is, for example, 1000 to 4000 g. In one embodiment, the centrifugal rotation speed of the second centrifugation process in step d) is, for example, 1500 to 3000 g. In this embodiment, the centrifugation time of the second centrifugation process in step d) is, for example, 3 minutes to 10 minutes. In one embodiment, the centrifugation time of the first centrifugation process in step d) is, for example, 4 minutes to 8 minutes. In one embodiment, the centrifugation speed of the first centrifugation process in step d) is, for example, 1500 g, and the centrifugation time is, for example, 5 minutes. During the second centrifugation process, the platelet-plasma fraction 15 will self-aggregate into a platelet-rich fibrin (PRF) colloid 17 .

然後，進行步驟e)，分離富含血小板纖維蛋白膠體17，以得到血小板裂解液(platelet lysate，PL)18。在本實施例中，分離富含血小板纖維蛋白膠體17的方法例如是對富含血小板纖維蛋白膠體17進行第三離心製程。在一實施例中，第三離心製程例如是對富含血小板纖維蛋白膠體17進行單次離心，其中離心轉速為1000g至4000g，離心時間為3分鐘至45分鐘。在另一實施例中，第三離心製程例如是對富含血小板纖維蛋白膠體17進行多次離心，其中離心轉速為1000g至4000g，每次的離心時間為3分鐘至15分鐘。在本實施例中，多次離心的離心次數例如是5次至15次，但本發明不限於此，可視需求而調整離心的次數。在進行第三離心製程的過程中，血小板裂解液18自富含血小板纖維蛋白膠體17中釋放出來，同時富含血小板纖維蛋白膠體17的膠體部分19因離心力而被壓縮在分離膠體20上，藉此使得血小板裂解液18與膠體部分19分離。Then, step e) is performed, and the platelet-rich fibrin colloid 17 is separated to obtain a platelet lysate (platelet lysate, PL) 18 . In this embodiment, the method for separating the platelet-rich fibrin colloid 17 is, for example, performing a third centrifugation process on the platelet-rich fibrin colloid 17 . In one embodiment, the third centrifugation process is, for example, a single centrifugation of the platelet-rich fibrin colloid 17 , wherein the centrifugation speed is 1000 g to 4000 g, and the centrifugation time is 3 minutes to 45 minutes. In another embodiment, the third centrifugation process is, for example, centrifuging the platelet-rich fibrin colloid 17 for several times, wherein the centrifugation speed is 1000g to 4000g, and the centrifugation time is 3 minutes to 15 minutes each time. In this embodiment, the number of times of centrifugation is, for example, 5 to 15 times, but the present invention is not limited to this, and the number of times of centrifugation can be adjusted as required. During the third centrifugation process, theplatelet lysate 18 is released from the platelet-rich fibrin colloid 17 , and the colloid portion of the platelet-rich fibrin colloid 17 isThefraction 19 is compressed on theseparation colloid 20 by centrifugal force, thereby separating theplatelet lysate 18 from thecolloid fraction 19 .

在本實施例中，在進行步驟d)之後且進行步驟e)之前，可進一步對富含血小板纖維蛋白膠體17按壓、擠壓、過濾、超音波震盪或進行冷凍解凍循環，有助於使血小板裂解液18自富含血小板纖維蛋白膠體17釋放出來。In this embodiment, after step d) and before step e), the platelet-rich fibrin colloid 17 can be further pressed, squeezed, filtered, ultrasonically shaken, or subjected to a freeze-thaw cycle, which is helpful for the platelet-rich fibrin colloid 17Lysate 18 is released from platelet-rich fibrin colloid 17 .

在一實施例中，血小板裂解液18包括由TGF-β1、PDGF-BB、PDGF-AB、FGF2、EGF、VEGF及VEGFA中選出的至少一種生長因子，其中TGF-β1的濃度為1ng/ml至10000ng/ml，PDGF-BB的濃度10pg/ml至500000pg/ml，PDGF-AB的濃度為10pg/ml至500000pg/ml，FGF2的濃度為1pg/ml至10000pg/ml，EGF的濃度為1pg/ml至10000pg/ml，VEGF的濃度為1pg/ml至10000pg/ml，VEGFA的濃度為0.1pg/ml至5000pg/ml。In one embodiment, theplatelet lysate 18 includes at least one growth factor selected from TGF-β1, PDGF-BB, PDGF-AB, FGF2, EGF, VEGF, and VEGFA, wherein the concentration of TGF-β1 ranges from 1 ng/ml to 10000ng/ml, PDGF-BB at 10pg/ml to 500000pg/ml, PDGF-AB at 10pg/ml to 500000pg/ml, FGF2 at 1pg/ml to 10000pg/ml, EGF at 1pg/ml To 10000 pg/ml, the concentration of VEGF was 1 pg/ml to 10000 pg/ml, and the concentration of VEGFA was 0.1 pg/ml to 5000 pg/ml.

在另一實施例中，血小板裂解液18包括由TGF-β1、PDGF-BB、PDGF-AB、FGF2、EGF、VEGF及VEGFA中選出的至少一種生長因子，其中TGF-β1的濃度為5ng/ml至3000ng/ml，PDGF-BB的濃度50pg/ml至50000pg/ml，PDGF-AB的濃度為100pg/ml至50000pg/ml，FGF2的濃度為20pg/ml至6000pg/ml，EGF的濃度為1.5pg/ml至2000pg/ml，VEGF的濃度為15pg/ml至6000pg/ml，VEGFA的濃度為0.2pg/ml至1500pg/ml。In another embodiment,platelet lysate 18 includes at least one growth factor selected from TGF-β1, PDGF-BB, PDGF-AB, FGF2, EGF, VEGF, and VEGFA, wherein the concentration of TGF-β1 is 5 ng/ml Up to 3000ng/ml, PDGF-BB at 50pg/ml to 50000pg/ml, PDGF-AB at 100pg/ml to 50000pg/ml, FGF2 at 20pg/ml to 6000pg/ml, EGF at 1.5pg /ml to 2000 pg/ml, VEGF concentrations from 15 pg/ml to 6000 pg/ml, and VEGFA concentrations from 0.2 pg/ml to 1500 pg/ml.

在本實施例中，在整個製備血小板裂解液的過程中未添加任何抗凝劑。抗凝劑例如是抗凝血複方檸檬酸鈉溶液A(Anticoagulant Citrate Dextrose Solution,Solution A，ACD-A)、抗凝血複方檸檬酸鈉溶液B(Anticoagulant Citrate Dextrose Solution,Solution B，ACD-B)、抗凝血複方檸檬酸鈉溶液C(Anticoagulant Citrate Dextrose Solution,Solution C，ACD-C)、檸檬酸-磷酸-葡萄溶液(Citrate-phosphate-dextrose Solution，CPD)、乙二胺四乙酸(Ethylenediaminetetraacetic acid，EDTA)、肝素(Heparin)、檸檬酸鈉(Sodium Citrate)或其組合。In this example, no anticoagulant was added during the whole process of preparing the platelet lysate. Anticoagulants such as anticoagulant compound sodium citrate solution A(Anticoagulant Citrate Dextrose Solution, Solution A, ACD-A), Anticoagulant Compound Sodium Citrate Solution B (Anticoagulant Citrate Dextrose Solution, Solution B, ACD-B), Anticoagulant Compound Sodium Citrate Solution C (Anticoagulant Citrate Dextrose Solution B) Solution,Solution C, ACD-C), citric acid-phosphate-dextrose solution (CPD), ethylenediaminetetraacetic acid (Ethylenediaminetetraacetic acid, EDTA), heparin (Heparin), sodium citrate (Sodium citrate) Citrate) or a combination thereof.

在本實施例中，由於本發明的製備血小板裂解液的整個過程皆未添加抗凝劑，因此並沒有將血小板裂解液自離心管取出並重覆活化的額外步驟，藉此可縮短製程時間。在本實施例中，製備血小板裂解液的整個製程時間為10分鐘至60分鐘，明顯短於傳統製備血小板裂解液的方法。此外，本發明的製備血小板裂解液的整個製程皆在同一個離心管中進行操作，因此具有避免污染且具有無菌性的優點。In this embodiment, since no anticoagulant is added in the whole process of preparing the platelet lysate according to the present invention, there is no additional step of removing the platelet lysate from the centrifuge tube and repeating activation, thereby shortening the process time. In this embodiment, the entire process time for preparing the platelet lysate is 10 minutes to 60 minutes, which is significantly shorter than the traditional method for preparing the platelet lysate. In addition, the whole process of preparing the platelet lysate of the present invention is performed in the same centrifuge tube, so it has the advantages of avoiding contamination and having sterility.

本發明提供一種用於治療聲帶疾病的醫藥組成物，其包括富含血小板血漿(platelet-rich plasma，PRP)及/或富含血小板血漿的衍生物。在一實施例中，富含血小板血漿的衍生物例如是血小板裂解液(platelet lysate，PL)。The present invention provides a pharmaceutical composition for treating vocal cord diseases, which comprises platelet-rich plasma (PRP) and/or derivatives of platelet-rich plasma. In one embodiment, the derivative of platelet-rich plasma is, for example, platelet lysate (PL).

在一實施例中，製備富含血小板血漿的方法例如是將自體的血液進行離心，以使全血分離成血漿層、白膜層以及紅血球層，並將所取得到血漿層及白膜層混合以得到富含血小板血漿。在一實施例中，製備富含血小板血漿的方法例如是如上述製備血小板裂解液的方法中的步驟a)至步驟c)的方式製備富含血小板血漿。In one embodiment, the method for preparing platelet-rich plasma is, for example, centrifuging autologous blood to separate the whole blood into plasma layer, buffy coat and red blood cell layer, and separating the obtained plasma layer and buffy coat. Mix to obtain platelet rich plasma. In one embodiment, the method for preparing platelet-rich plasma is, for example, preparing blood as described above.Platelet-rich plasma is prepared in the manner of step a) to step c) in the method for the platelet lysate.

在一實施例中，製備血小板裂解液的方法例如是將富含血小板血漿加入氯化鈣溶液並混合均勻，接著進行冷凍解凍循環，以得到血小板裂解液。在一實施例中，富含血小板血漿的衍生物例如是由上述製備血小板裂解液的方法(步驟a)至步驟e))所製備的血小板裂解液。In one embodiment, the method for preparing the platelet lysate is, for example, adding the platelet-rich plasma to the calcium chloride solution and mixing evenly, followed by a freeze-thaw cycle to obtain the platelet lysate. In one embodiment, the derivative of platelet-rich plasma is, for example, the platelet lysate prepared by the above-mentioned method for preparing a platelet lysate (step a) to step e)).

在一實施例中，富含血小板血漿及富含血小板血漿的衍生物可包括由TGF-β1、PDGF-BB、PDGF-AB、FGF2、EGF、VEGF及VEGFA中選出的至少一種生長因子。In one embodiment, platelet-rich plasma and derivatives of platelet-rich plasma may include at least one growth factor selected from TGF-β1, PDGF-BB, PDGF-AB, FGF2, EGF, VEGF, and VEGFA.

在本發明的一實施例中，上述的TGF-β1在醫藥組成物中的濃度為0.7ng/ml至7000ng/ml，PDGF-BB在醫藥組成物中的濃度7pg/ml至350000pg/ml，PDGF-AB在醫藥組成物中的濃度為7pg/ml至350000pg/ml，FGF2在醫藥組成物中的濃度為0.7pg/ml至7000pg/ml，EGF在醫藥組成物中的濃度為0.7pg/ml至7000pg/ml，VEGF在醫藥組成物中的濃度為0.7pg/ml至7000pg/ml，VEGFA在醫藥組成物中的濃度為0.06pg/ml至3500pg/ml。In an embodiment of the present invention, the concentration of the above-mentioned TGF-β1 in the pharmaceutical composition is 0.7ng/ml to 7000ng/ml, the concentration of PDGF-BB in the pharmaceutical composition is 7pg/ml to 350000pg/ml, the PDGF -The concentration of AB in the pharmaceutical composition is 7 pg/ml to 350,000 pg/ml, the concentration of FGF2 in the pharmaceutical composition is 0.7 pg/ml to 7000 pg/ml, and the concentration of EGF in the pharmaceutical composition is 0.7 pg/ml to 0.7 pg/ml 7000 pg/ml, the concentration of VEGF in the pharmaceutical composition is 0.7 pg/ml to 7000 pg/ml, and the concentration of VEGFA in the pharmaceutical composition is 0.06 pg/ml to 3500 pg/ml.

在一實施例中，TGF-β1在醫藥組成物中的濃度為3ng/ml至2000ng/ml，PDGF-BB在醫藥組成物中的濃度35pg/ml至35000pg/ml，PDGF-AB在醫藥組成物中的濃度為70pg/ml至35000pg/ml，FGF2在醫藥組成物中的濃度為15pg/ml至4000pg/ml，EGF在醫藥組成物中的濃度為1pg/ml至1400pg/ml，VEGF在醫藥組成物中的濃度為10pg/ml至4000pg/ml，VEGFA在醫藥組成物中的濃度為0.1pg/ml至1000pg/ml。In one embodiment, the concentration of TGF-β1 in the pharmaceutical composition is 3ng/ml to 2000ng/ml, the concentration of PDGF-BB in the pharmaceutical composition is 35pg/ml to 35000pg/ml, and the concentration of PDGF-AB in the pharmaceutical composition The concentration of FGF2 in the pharmaceutical composition is 70pg/ml to 35000pg/ml, the concentration of FGF2 in the pharmaceutical composition is 15pg/ml to 4000pg/ml, and the concentration of EGF in the pharmaceutical composition is 1pg/ml to 1400pg/ml.The concentration in the pharmaceutical composition is 10 pg/ml to 4000 pg/ml, and the concentration of VEGFA in the pharmaceutical composition is 0.1 pg/ml to 1000 pg/ml.

富含血小板血漿(或富含血小板血漿的衍生物)為一種富含生長因子、細胞黏附分子以及細胞因子的自體血液產物。這些生長因子可將未分化的細胞吸引到聲帶的新形成的基質中來促進癒合，從而引發細胞分裂並有利於血管生成及聲帶修補。Platelet-rich plasma (or derivatives of platelet-rich plasma) is an autologous blood product rich in growth factors, cell adhesion molecules, and cytokines. These growth factors promote healing by attracting undifferentiated cells to the newly formed stroma of the vocal cords, thereby triggering cell division and facilitating angiogenesis and vocal cord repair.

在一實施例中，醫藥組成物可更包括選自玻尿酸、生理食鹽水、自體脂肪及自體幹細胞中的至少一者。在本實施例中，上述玻尿酸、生理食鹽水、自體脂肪及自體幹細胞可作為填充物，用以填充聲帶損傷處的基質區域。In one embodiment, the pharmaceutical composition may further comprise at least one selected from the group consisting of hyaluronic acid, physiological saline, autologous fat and autologous stem cells. In this embodiment, the above-mentioned hyaluronic acid, physiological saline, autologous fat and autologous stem cells can be used as fillers to fill the matrix area at the damaged vocal cords.

在一實施例中，自體幹細胞例如是脂肪幹細胞或間質幹細胞，但本發明不限於此。在一實施例中，自體脂肪例如是自體腹部脂肪。In one embodiment, the autologous stem cells are, for example, adipose stem cells or mesenchymal stem cells, but the present invention is not limited thereto. In one embodiment, the autologous fat is, for example, autologous abdominal fat.

在一實施例中，醫藥組成物可更包括自體脂肪，其中自體脂肪與富含血小板血漿(或富含血小板血漿的衍生物)的體積比為0.5~10。在一實施例中，自體脂肪與富含血小板血漿(或富含血小板血漿的衍生物)的體積比為0.5~2。In one embodiment, the pharmaceutical composition may further include autologous fat, wherein the volume ratio of autologous fat to platelet-rich plasma (or a derivative of platelet-rich plasma) is 0.5-10. In one embodiment, the volume ratio of autologous fat to platelet-rich plasma (or a derivative of platelet-rich plasma) is 0.5-2.

自體脂肪包括高含量的脂肪幹細胞(ADSC)，脂肪幹細胞可分化成傷口癒合細胞(例如纖維細胞及角質形成細胞)且具有促進血管生成的特性。然而，脂肪移植物的維持率是高度變化的，原因是脂肪的移植與血管再生不同，自體脂肪容易於被人體吸收，因此失去其填充及注射的意義。而在本發明的醫藥組成物同時包括血小板血漿(及/或富含血小板血漿的衍生物)以及自體脂肪的情況下，血小板血漿(及/或富含血小板血漿的衍生物)可改善可以對脂肪的癒合潛力產生協同效應，並可增加脂肪的維持率，進而達到一個長效填充及協助修復功能。Autologous fat includes a high content of adipose stem cells (ADSCs), which can differentiate into wound healing cells such as fibroblasts and keratinocytes and have properties that promote angiogenesis. However, the maintenance rate of fat grafts is highly variable because, unlike revascularization, autologous fat is easily absorbed by the body and thus loses its significance for filling and injection. And in the pharmaceutical composition of the present inventionIn the case of including both platelet plasma (and/or platelet-rich plasma derivatives) and autologous fat, platelet plasma (and/or platelet-rich plasma derivatives) can improve the healing potential of fat can have a synergistic effect, It can increase the maintenance rate of fat, thereby achieving a long-term filling and assisting repair function.

在一實施例中，聲帶疾病包括聲帶閉合不全。在一實施例中，聲帶閉合不全例如是由聲帶萎縮、聲帶麻痺、聲帶外傷或聲帶老化所導致。In one embodiment, the vocal cord disorder comprises vocal cord insufficiency. In one embodiment, the vocal cord insufficiency is caused by, for example, vocal cord atrophy, vocal cord paralysis, vocal cord trauma, or vocal cord aging.

在本實施例中，醫藥組成物可製作成無菌注射溶液。在一實施例中，醫藥組成物例如是以注射方式給藥至聲帶。具體來說，醫藥組成物例如是以注射方式給藥至聲帶萎縮處。在一實施例中，可使用彎曲注射器(美敦力公司(Medtronic))將醫藥組成物注射至聲帶萎縮處。在一實施例中，醫藥組成物的給藥量例如是0.5mL至5mL。In this embodiment, the pharmaceutical composition can be made into a sterile injectable solution. In one embodiment, the pharmaceutical composition is administered to the vocal cords, for example, by injection. Specifically, the pharmaceutical composition is administered, for example, by injection to the atrophy of the vocal cords. In one embodiment, a curved syringe (Medtronic) can be used to inject the pharmaceutical composition into the atrophy of the vocal cords. In one embodiment, the dosage of the pharmaceutical composition is, for example, 0.5 mL to 5 mL.

以下將以實驗例具體說明本發明，但實驗例之參數與其數據結果僅是用來說明本發明的功效，而並非是用以限定本發明之範圍。The present invention will be specifically described below with experimental examples, but the parameters and data results of the experimental examples are only used to illustrate the efficacy of the present invention, but not to limit the scope of the present invention.

[血小板裂解液的製備][Preparation of platelet lysate]實驗例1Experimental example 1

首先，自受試者採取靜脈血液10毫升，並以18G的針頭將新鮮全血注入含有分離膠體的離心管中。將離心管放入離心機內，以1500g的條件離心5分鐘，以使全血分離成血漿層、白膜層以及紅血球層，其中血漿層為少量血小板血漿(Platelet-Poor Plasma，PPP)。First, 10 ml of venous blood was taken from the subject, and fresh whole blood was injected into a centrifuge tube containing separation colloid with an 18G needle. Put the centrifuge tube into the centrifuge and centrifuge at 1500g for 5 minutes to separate the whole blood into plasma layer, buffy coat and red blood cell layer, wherein the plasma layer is a small amount of platelet plasma (Platelet-Poor).Plasma, PPP).

接著，利用18G針頭連接10毫升針筒移除2毫升的血漿層。將離心管上下均勻搖晃，使得剩餘的血漿層與白膜層混合，以得到富含血小板血漿(platelet-rich plasma，PRP)。Next, 2 ml of the plasma layer was removed using an 18G needle attached to a 10 ml syringe. The centrifuge tube was shaken up and down evenly to mix the remaining plasma layer with the buffy coat layer to obtain platelet-rich plasma (PRP).

然後，將同一離心管放入離心機內，以1500g的條件離心5分鐘，以得到富含血小板纖維蛋白(platelet-rich fibrin，PRF)膠體。Then, the same centrifuge tube was put into a centrifuge and centrifuged at 1500 g for 5 minutes to obtain platelet-rich fibrin (PRF) colloid.

之後，將同一離心管放入離心機內，以1500g的條件離心5分鐘。重覆上述離心條件離心10次，藉此得到位於離心管頂部的血小板裂解液。Then, the same centrifuge tube was put into a centrifuge and centrifuged at 1500 g for 5 minutes. The above centrifugation conditions were repeated 10 times, thereby obtaining the platelet lysate at the top of the centrifuge tube.

比較例1Comparative Example 1

首先，自受試者採取靜脈血液10毫升，並以18G的針頭將新鮮全血注入含有抗凝劑(ACD Solution-A，ACDA)的離心管中。將離心管放入離心機內，以1500g的條件離心5分鐘，以使全血分離成血漿層、白膜層以及紅血球層。First, 10 ml of venous blood was taken from the subject, and fresh whole blood was injected into a centrifuge tube containing an anticoagulant (ACD Solution-A, ACDA) with an 18G needle. The centrifuge tube was put into a centrifuge and centrifuged at 1500 g for 5 minutes to separate the whole blood into a plasma layer, a buffy coat layer and an erythrocyte layer.

接著，取出血漿層及白膜層，移至另一無菌容器內，加入氯化鈣溶液並混合均勻，此步驟為活化血小板。接下來進行冷凍解凍循環，將此活化的溶液放置室溫一小時，接著放進-80度冰箱進行冷凍，於4小時後取出，即可進行生長因子測定。Next, take out the plasma layer and buffy coat layer, move it to another sterile container, add calcium chloride solution and mix well, this step is to activate platelets. Next, a freeze-thaw cycle was performed, and the activated solution was placed at room temperature for one hour, then placed in a -80 degree refrigerator for freezing, and taken out after 4 hours for growth factor determination.

[生長因子的測定][Determination of Growth Factors]

在本實施例中，使用酵素連結免疫吸附法(Enzyme-linked Immuno-Sorbent Assay，ELISA)測定實驗例1的富含血小板血漿、實驗例1的血小板裂解液與比較例1的血小板裂解液中生長因子TGF-β1、PDGF-AB、PDGF-BB、VEGF及VEGFA的含量，並將結果記載於表1。表1中的結果皆以<平均值±標準差>表示，每個生長因子實施例與比較例間有顯著差異(P<0.05)。In this example, the platelet-rich plasma of Experimental Example 1 was measured by enzyme-linked Immuno-Sorbent Assay (ELISA).Table 1 shows the contents of growth factors TGF-β1, PDGF-AB, PDGF-BB, VEGF and VEGFA in the platelet lysate of Experimental Example 1 and the platelet lysate of Comparative Example 1. The results in Table 1 are all expressed as <mean ± standard deviation>, and there is a significant difference between each growth factor example and the comparative example (P<0.05).

由表1的內容可知，相對於傳統方法所製備的血小板裂解液(即比較例1的PL)，由本發明的製備血小板裂解液的方法所製備的血小板裂解液(即實驗例1的PL)以及富含血小板血漿(即實驗例1的PRP)皆具有較高濃度的生長因子。此外，本發明的製備血小板裂解液的方法由於在整個製程皆未添加抗凝劑，因此並沒有將血小板裂解液自離心管取出並重覆活化的額外步驟，藉此可縮短製程時間。此外，本發明的製備血小板裂解液的整個製程皆在同一個離心管中進行操作，因此具有避免污染且具有無菌性的優點。As can be seen from the contents of Table 1, compared with the platelet lysate prepared by the traditional method (ie, the PL of Comparative Example 1), the platelet lysate prepared by the method for preparing a platelet lysate of the present invention (ie, the PL of Experimental Example 1) and Platelet-rich plasma (ie, PRP of Experimental Example 1) all had higher concentrations of growth factors. In addition, since the method for preparing platelet lysate of the present invention does not add anticoagulant in the whole process, there is no additional step of taking out the platelet lysate from the centrifuge tube and repeating activation, thereby shortening the process time. In addition, the whole process of preparing the platelet lysate of the present invention is operated in the same centrifuge tube, so it has the advantages of avoiding contamination and having the advantages ofHas the advantage of sterility.

[醫藥組成物對於聲帶萎縮患者的效用][Effect of pharmaceutical composition on patients with vocal cord atrophy]實驗例2Experimental example 2

在本實施例中，受試者為10位年齡介於25至80歲被診斷出嗓音障礙指數(Voice Handicap Index-10,VHI-10)評分

11的聲帶萎縮患者。此外，本實施例以與實驗例1相同的製備方法製備富含血小板血漿並作為醫藥組成物。In this example, the subjects were 10 people between the ages of 25 and 80 who were diagnosed with a Voice Handicap Index-10 (VHI-10) score

11 patients with vocal cord atrophy. In addition, in this example, platelet-rich plasma was prepared by the same preparation method as in Experimental Example 1 as a pharmaceutical composition.

首先，將受試者進行全身麻醉後，藉由纖維鏡監測器(fiberscopic monitoring)並使用彎曲注射器將1mL~2mL的所製備的富含血小板血漿注射至受試者的聲帶萎縮處，並在注射後記錄受試者的聲帶膨脹的情況。First, after subjecting the subject to general anesthesia, 1mL~2mL of the prepared platelet-rich plasma was injected into the subject's vocal cord atrophy by fiberscopic monitoring using a curved syringe, and the injection After recording the swelling of the vocal cords of the subjects.

在進行注射後3、6個月，測定受試者的嗓音障礙指數。At 3 and 6 months after the injection, the subjects' voice disturbance index was determined.

實驗例3Experimental example 3

在本實施例中，受試者為10位年齡介於25至80歲被診斷出嗓音障礙指數(Voice Handicap Index-10,VHI-10)評分

11的聲帶萎縮患者。以與實驗例1相同的製備方法製備血小板裂解液並作為醫藥組成物。In this example, the subjects were 10 people between the ages of 25 and 80 who were diagnosed with a Voice Handicap Index-10 (VHI-10) score

11 patients with vocal cord atrophy. A platelet lysate was prepared by the same preparation method as in Experimental Example 1 as a pharmaceutical composition.

首先，將受試者進行全身麻醉後，藉由纖維鏡監測器(fiberscopic monitoring)並使用彎曲注射器將1mL~2mL的所製備的血小板裂解液注射至受試者的聲帶萎縮處，並在注射後記錄受試者的聲帶膨脹的情況。First, after subjecting the subject to general anesthesia, 1mL~2mL of the prepared platelet lysate was injected into the subject's vocal cord atrophy by fiberscopic monitoring and using a curved syringe, and after the injection The dilation of the subject's vocal cords was recorded.

在進行注射後3、6個月，測定受試者的嗓音障礙指數。At 3 and 6 months after the injection, the subjects' voice disturbance index was determined.

實驗例2與實驗例3所測定的嗓音障礙指數如下表2所示。The voice disorder indices measured in Experimental Example 2 and Experimental Example 3 are shown in Table 2 below.

由表2的結果可以看出，將本發明的包括富含血小板血漿(即實驗例2)或血小板裂解液(即實驗例3)的醫藥組成物注射至患者的聲帶萎縮處後，患者的VHI明顯下降，藉此可證實本發明的包括富含血小板血漿或血小板裂解液的醫藥組成物可有效地修補聲帶。As can be seen from the results in Table 2, after the pharmaceutical composition comprising platelet-rich plasma (i.e. experimental example 2) or platelet lysate (i.e. experimental example 3) of the present invention was injected into the patient's vocal cord atrophy, the patient's VHI Significantly decreased, thereby confirming that the pharmaceutical composition comprising platelet-rich plasma or platelet lysate of the present invention can effectively repair vocal cords.

10:全血10: Whole blood

12:第一上層部分12: First Upper Section

13:第二上層部分13: Second Upper Section

14:中間部分14: Middle Section

15:富含血小板血漿部分15: Platelet rich plasma fraction

16:下層部分16: Lower part

17:富含血小板纖維蛋白膠體17: Platelet-Rich Fibrin Colloid

18:血小板裂解液18: Platelet Lysate

19:膠體部分19: Colloid part

20:分離膠體20: Separation of colloids

30:離心管30: Centrifuge tube

Claims (19)

Translated fromChinese

一種製備血小板裂解液的方法，所述方法包括以下步驟：步驟a)，將採集的全血放置於裝有分離膠體的離心管中，其中所述分離膠體的密度為1.030g/cm³至1.093g/cm³；步驟b)，對裝有所述全血及所述分離膠體的所述離心管進行第一離心製程，以使所述全血分離成第一上層部分、中間部分以及下層部分，其中所述分離膠體位於所述中間部分與所述下層部分之間；步驟c)，將所述第一上層部分與所述中間部分進行混合，以得到富含血小板血漿(platelet-rich plasma，PRP)部分；步驟d)，在未添加血小板的活化因子的條件下，對所述富含血小板血漿部分進行第二離心製程，以得到富含血小板纖維蛋白(platelet-rich fibrin，PRF)膠體，其中在進行所述第二離心製程的過程中，所述富含血小板血漿部分會自我凝結成所述富含血小板纖維蛋白膠體；以及步驟e)，分離所述富含血小板纖維蛋白膠體，以得到血小板裂解液(platelet lysate，PL)，其中在所述步驟a)至所述步驟e)中未添加抗凝劑及所述活化因子。A method for preparing a platelet lysate, the method comprising the following steps: step a), placing the collected whole blood in a centrifuge tube containing a separation colloid, wherein the separation colloid has a density of 1.030g/^cm3 to 1.093 g/cm³ ; step b), performing a first centrifugation process on the centrifuge tube containing the whole blood and the separating colloid, so that the whole blood is separated into a first upper part, a middle part and a lower part , wherein the separation colloid is located between the middle portion and the lower portion; step c), mixing the first upper portion with the middle portion to obtain platelet-rich plasma (platelet-rich plasma, PRP) part; step d), under the condition of not adding platelet activating factor, a second centrifugation process is performed on the platelet-rich plasma part to obtain platelet-rich fibrin (platelet-rich fibrin, PRF) colloid, Wherein, during the second centrifugation process, the platelet-rich plasma fraction will self-aggregate into the platelet-rich fibrin colloid; and in step e), the platelet-rich fibrin colloid is separated to obtain Platelet lysate (PL), wherein no anticoagulant and the activating factor are added in the step a) to the step e).如申請專利範圍第1項所述的製備血小板裂解液的方法，其中在進行所述步驟c)之前，更包括移除部分的所述第一上層部分，以得到第二上層部分，其中所述第一上層部分與所述第二上層部分的體積比率為1：0.01~0.99。The method for preparing a platelet lysate solution according to item 1 of the claimed scope, wherein before performing the step c), it further comprises removing a part of the first upper layerpart to obtain a second upper layer part, wherein the volume ratio of the first upper layer part to the second upper layer part is 1:0.01~0.99.如申請專利範圍第1項所述的製備血小板裂解液的方法，其中所述抗凝劑包括抗凝血複方檸檬酸鈉溶液A(Anticoagulant Citrate Dextrose Solution,Solution A，ACD-A)、抗凝血複方檸檬酸鈉溶液B(Anticoagulant Citrate Dextrose Solution,Solution B，ACD-B)、抗凝血複方檸檬酸鈉溶液C(Anticoagulant Citrate Dextrose Solution,Solution C，ACD-C)、檸檬酸-磷酸-葡萄溶液(Citrate-phosphate-dextrose Solution，CPD)、乙二胺四乙酸(Ethylenediaminetetraacetic acid，EDTA)、肝素(Heparin)、檸檬酸鈉(Sodium Citrate)或其組合。The method for preparing a platelet lysate according to item 1 of the application scope, wherein the anticoagulant comprises anticoagulant compound sodium citrate solution A (Anticoagulant Citrate Dextrose Solution, Solution A, ACD-A), anticoagulant compound sodium citrate solution A (Anticoagulant Citrate Dextrose Solution, Solution A, ACD-A), Compound Sodium Citrate Solution B (Anticoagulant Citrate Dextrose Solution, Solution B, ACD-B), Anticoagulant Compound Sodium Citrate Solution C (Anticoagulant Citrate Dextrose Solution, Solution C, ACD-C), Citric Acid-Phosphoric Acid-Grape Solution (Citrate-phosphate-dextrose Solution, CPD), ethylenediaminetetraacetic acid (Ethylenediaminetetraacetic acid, EDTA), heparin (Heparin), sodium citrate (Sodium Citrate) or a combination thereof.如申請專利範圍第1項所述的製備血小板裂解液的方法，其中所述分離膠體的材料包括含矽聚合物、丙烯酸聚合物或聚酯類聚合物。The method for preparing a platelet lysate according to item 1 of the patent application scope, wherein the material of the separation colloid comprises a silicon-containing polymer, an acrylic polymer or a polyester polymer.如申請專利範圍第1項所述的製備血小板裂解液的方法，其中所述第一離心製程的離心轉速為1000g至4000g，離心時間為3分鐘至10分鐘。The method for preparing a platelet lysate according to item 1 of the patent application scope, wherein the centrifugation speed of the first centrifugation process is 1000g to 4000g, and the centrifugation time is 3 minutes to 10 minutes.如申請專利範圍第1項所述的製備血小板裂解液的方法，其中所述第一上層部分為包含血小板的血漿層，所述中間部分為包含血小板及白血球的白膜層(buffy coat)，以及所述下層部分為包含紅血球的紅血球層。The method for preparing a platelet lysate according to claim 1, wherein the first upper layer part is a plasma layer containing platelets, the middle part is a buffy coat containing platelets and leukocytes, and The lower portion is an erythrocyte layer containing erythrocytes.如申請專利範圍第1項所述的製備血小板裂解液的方法，其中所述第二離心製程的離心轉速為1000g至4000g，離心時間為3分鐘至10分鐘。The method for preparing a platelet lysate according to item 1 of the patent application scope, wherein the centrifugation speed of the second centrifugation process is 1000g to 4000g, and the centrifugation time is 3 minutes to 10 minutes.如申請專利範圍第1項所述的製備血小板裂解液的方法，其中分離所述富含血小板纖維蛋白膠體包括對所述富含血小板纖維蛋白膠體進行第三離心製程。The method for preparing a platelet lysate according to claim 1, wherein separating the platelet-rich fibrin colloid comprises performing a third centrifugation process on the platelet-rich fibrin colloid.如申請專利範圍第8項所述的製備血小板裂解液的方法，其中所述第三離心製程包括：對所述富含血小板纖維蛋白膠體進行單次離心，其中離心轉速為1000g至4000g，離心時間為3分鐘至45分鐘，或者對所述富含血小板纖維蛋白膠體進行多次離心，其中離心轉速為1000g至4000g，每次的離心時間為3分鐘至15分鐘。The method for preparing a platelet lysate according to item 8 of the patent application scope, wherein the third centrifugation process comprises: performing a single centrifugation on the platelet-rich fibrin colloid, wherein the centrifugation speed is 1000g to 4000g, and the centrifugation time is 3 minutes to 45 minutes, or the platelet-rich fibrin colloid is centrifuged for several times, wherein the centrifugation speed is 1000g to 4000g, and the centrifugation time is 3 minutes to 15 minutes each time.如申請專利範圍第1項所述的製備血小板裂解液的方法，其中製備所述血小板裂解液的整個製程時間為10分鐘至60分鐘。The method for preparing a platelet lysate according to item 1 of the claimed scope, wherein the entire process time for preparing the platelet lysate is 10 minutes to 60 minutes.如申請專利範圍第1項所述的製備血小板裂解液的方法，其中在進行所述步驟e)之前，更包括對所述富含血小板纖維蛋白膠體按壓、擠壓、過濾、超音波震盪或進行冷凍解凍循環，以使得所述血小板裂解液自所述富含血小板纖維蛋白膠體釋放。The method for preparing a platelet lysate according to item 1 of the claimed scope, wherein before performing the step e), the method further comprises pressing, squeezing, filtering, ultrasonically oscillating or performing the platelet-rich fibrin colloid Freeze-thaw cycles to release the platelet lysate from the platelet-rich fibrin colloid.如申請專利範圍第1項所述的製備血小板裂解液的方法，其中所述血小板裂解液包括由TGF-β1、PDGF-BB、PDGF-AB、FGF2、EGF、VEGF及VEGFA中選出的至少一種生長因子，其中所述TGF-β1的濃度為1ng/ml至10000ng/ml，所述PDGF-BB的濃度10pg/ml至500000pg/ml，所述PDGF-AB的濃度為10pg/ml至500000pg/ml，所述FGF2的濃度為1pg/ml至10000pg/ml，所述EGF的濃度為1pg/ml至10000pg/ml，所述VEGF的濃度為1pg/ml至10000pg/ml，VEGFA的濃度為0.1pg/ml至5000pg/ml。The method for preparing a platelet lysate according to claim 1, wherein the platelet lysate comprises at least one growth selected from TGF-β1, PDGF-BB, PDGF-AB, FGF2, EGF, VEGF and VEGFA factor, whereThe concentration of the TGF-β1 is 1ng/ml to 10000ng/ml, the concentration of the PDGF-BB is 10pg/ml to 500000pg/ml, the concentration of the PDGF-AB is 10pg/ml to 500000pg/ml, the concentration of the FGF2 The concentration is 1 pg/ml to 10000 pg/ml, the concentration of the EGF is 1 pg/ml to 10000 pg/ml, the concentration of the VEGF is 1 pg/ml to 10000 pg/ml, and the concentration of VEGFA is 0.1 pg/ml to 5000 pg/ml .一種醫藥組成物用於製備治療聲帶疾病之藥物的用途，其中所述醫藥組成物包括由如申請專利範圍第1項所述的製備血小板裂解液的方法所製備的血小板裂解液。A use of a pharmaceutical composition for preparing a drug for treating vocal cord diseases, wherein the pharmaceutical composition includes a platelet lysate prepared by the method for preparing a platelet lysate as described in item 1 of the patent application scope.如申請專利範圍第13項所述的醫藥組成物用於製備治療聲帶疾病之藥物的用途，其中所述聲帶疾病包括聲帶閉合不全。Use of the pharmaceutical composition according to item 13 of the application scope for preparing a medicament for the treatment of vocal cord disease, wherein the vocal cord disease includes vocal cord insufficiency.如申請專利範圍第13項所述的醫藥組成物用於製備治療聲帶疾病之藥物的用途，其中所述富含血小板血漿及所述富含血小板血漿的衍生物包括由TGF-β1、PDGF-BB、PDGF-AB、FGF2、EGF、VEGF及VEGFA中選出的至少一種生長因子。The use of the pharmaceutical composition according to item 13 of the application scope for preparing a drug for treating vocal cord diseases, wherein the platelet-rich plasma and the derivatives of the platelet-rich plasma include TGF-β1, PDGF-BB , at least one growth factor selected from PDGF-AB, FGF2, EGF, VEGF and VEGFA.如申請專利範圍第15項所述的醫藥組成物用於製備治療聲帶疾病之藥物的用途，其中所述TGF-β1在所述醫藥組成物中的濃度為0.7ng/ml至7000ng/ml，所述PDGF-BB在所述醫藥組成物中的濃度7pg/ml至350000pg/ml，所述PDGF-AB在所述醫藥組成物中的濃度為7pg/ml至350000pg/ml，所述FGF2在所述醫藥組成物中的濃度為0.7pg/ml至7000pg/ml，所述EGF在所述醫藥組成物中的濃度為0.7pg/ml至7000pg/ml，所述VEGF在所述醫藥組成物中的濃度為0.7pg/ml至7000pg/ml，所述VEGFA在所述醫藥組成物中的濃度為0.06pg/ml至3500pg/ml。The use of the pharmaceutical composition described in item 15 of the application scope for preparing a drug for treating vocal cord diseases, wherein the concentration of the TGF-β1 in the pharmaceutical composition is 0.7ng/ml to 7000ng/ml, and the The concentration of the PDGF-BB in the pharmaceutical composition is 7pg/ml to 350000pg/ml, the concentration of the PDGF-AB in the pharmaceutical composition is 7pg/ml to 350000pg/ml, and the FGF2 is in the pharmaceutical composition. The concentration in the pharmaceutical composition is 0.7pg/ml to 7000pg/ml, the concentration of the EGF in the pharmaceutical composition is 0.7pg/ml to 7000pg/ml, the VEGF in the pharmaceutical compositionThe concentration of VEGFA in the pharmaceutical composition is 0.7 pg/ml to 7000 pg/ml, and the concentration of the VEGFA in the pharmaceutical composition is 0.06 pg/ml to 3500 pg/ml.如申請專利範圍第13項所述的醫藥組成物用於製備治療聲帶疾病之藥物的用途，其中所述醫藥組成物更包括選自玻尿酸、生理食鹽水、自體脂肪及自體幹細胞中的至少一者。Use of the pharmaceutical composition described in item 13 of the scope of the application for preparing a drug for treating vocal cord diseases, wherein the pharmaceutical composition further comprises at least one selected from the group consisting of hyaluronic acid, physiological saline, autologous fat and autologous stem cells one.如申請專利範圍第17項所述的醫藥組成物用於製備治療聲帶疾病之藥物的用途，其中自體幹細胞包括脂肪幹細胞或間質幹細胞。The use of the pharmaceutical composition described in item 17 of the application scope for preparing a drug for treating vocal cord diseases, wherein the autologous stem cells include adipose stem cells or mesenchymal stem cells.如申請專利範圍第18項所述的醫藥組成物用於製備治療聲帶疾病之藥物的用途，其中所述醫藥組成物是以注射方式給藥至聲帶。The use of the pharmaceutical composition described in item 18 of the application scope for preparing a medicine for treating vocal cord diseases, wherein the pharmaceutical composition is administered to the vocal cords by injection.

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