TW202126315A - Method of preparing platelet lysate and use thereof for improving female pregnancy success rate - Google Patents
Method of preparing platelet lysate and use thereof for improving female pregnancy success rate Download PDFInfo
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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提升雌性懷孕率的用途。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 improving female pregnancy rate .
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 centrifuging human or animal whole blood, while platelet lysate is a further processed derivative of platelet-rich plasma. The lysate contains a variety of growth factors related to 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 PRP is further added with activating factor, 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, and difficulty in concentration. The above-mentioned problems will lead to insufficient growth factors in the collected platelet lysate. In addition, in the traditional method of preparing platelet lysate, PRP needs to be taken out of the centrifuge tube and activated, so the effect of sterility is worrying, and the cost of obtaining platelet lysate by freezing and thawing or ultrasonic vibration Time is 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.
此外,已知有眾多因素可能造成雌性不孕症,而目前的治療不孕症的方法有人工受精或體外受精。然而,現有的治療方式對於提升不孕症患者的懷孕效果仍不佳。In addition, many factors are known to cause female infertility, and current methods of treating infertility include artificial insemination or in vitro fertilization. However, the existing treatment methods are still not effective in improving the pregnancy of patients with infertility.
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 invention provides a method for preparing a platelet lysate, which can quickly prepare a platelet lysate with a high concentration of growth factors under aseptic 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,可用於提升雌性懷孕成功率。The present invention provides a pharmaceutical composition comprising platelet-rich plasma and/or platelet-rich plasma derivatives, which can be used to improve the pregnancy success rate of females.
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1.030 g/cm3 至1.093 g/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。The 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 separating colloid, wherein the density of the separating colloid is 1.030 g/ cm 3 to 1.093 g/cm 3 ; step b), perform the first centrifugation process on the centrifuge tube containing the whole blood and the separation colloid, so that the whole blood is separated into the first upper part, the middle part and the lower part, wherein the separation colloid Located 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) to separate platelet-rich fibrin colloid to obtain platelet lysate (PL), wherein in steps 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 include removing part of the first upper layer portion to obtain a second upper layer portion, wherein the volume ratio of the first upper layer portion to the second upper layer portion 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 aforementioned anticoagulant may include anticoagulant citrate compound sodium citrate solution A (Anticoagulant Citrate Dextrose Solution, Solution A, ACD-A), and anticoagulant compound sodium citrate solution B (Anticoagulant Citrate Dextrose Solution, Solution A, ACD-A). Citrate Dextrose Solution, Solution B, ACD-B), 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 aforementioned separation colloid material 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1000 g至4000 g,離心時間例如是3分鐘至10分鐘。In an embodiment of the present invention, the centrifugal rotation 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 a red blood cell 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 an embodiment of the present invention, coagulating the platelet-rich plasma fraction includes performing a second centrifugation process on the platelet-rich plasma fraction.
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1000 g至4000 g,離心時間例如是3分鐘至10分鐘。In an embodiment of the present invention, the centrifugal rotation speed of the above-mentioned 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1000 g至4000 g,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1000 g至4000 g,每次的離心時間為3分鐘至15分鐘。In an embodiment of the present invention, the aforementioned third centrifugation process includes: performing a single centrifugation on the platelet-rich fibrin colloid, wherein the centrifugation speed is 1000 g to 4000 g, and the centrifugation time is 3 minutes to 45 minutes, Or, the platelet-rich fibrin colloid is centrifuged multiple times, wherein the centrifugal speed is 1000 g to 4000 g, 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 time for preparing the platelet lysate 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 performing step e), it further comprises pressing, squeezing, filtering, ultrasonic vibration or freezing and thawing cycle of the platelet-rich fibrin gel, so that the platelet lysate is self-enriched with platelets. Fibrin colloid is released.
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 1 ng/ml至 10000 ng/ml,PDGF-BB的濃度10 pg/ml至500000 pg/ml,PDGF-AB的濃度為10 pg/ml至500000 pg/ml,FGF2的濃度為1 pg/ml至10000 pg/ml,EGF的濃度為1 pg/ml至10000 pg/ml,VEGF的濃度為1 pg/ml至10000 pg/ml,VEGFA的濃度為0.1 pg/ml至5000 pg/ml。In an embodiment of the present invention, the aforementioned 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 From 1 ng/ml to 10000 ng/ml, the concentration of PDGF-BB is 10 pg/ml to 500000 pg/ml, the concentration of PDGF-AB is 10 pg/ml to 500000 pg/ml, and the concentration of FGF2 is 1 pg/ml To 10000 pg/ml, the concentration of EGF 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製備提升雌性懷孕成功率之藥物的用途,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 a use of a medicinal composition for preparing a medicine for improving the pregnancy success rate of females, wherein the medicinal composition includes platelet-rich plasma and/or a derivative 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可包括血小板裂解液。In an embodiment of the present invention, the aforementioned derivative of platelet-rich plasma may include platelet lysate.
在本發明的一實施例中,上述的醫藥組成物可以灌注方式給藥至子宮腔內。In an embodiment of the present invention, the above-mentioned medical composition can be administered into the uterine cavity by infusion.
在本發明的一實施例中,上述的醫藥組成物的給藥量例如是0.1 mL至2 mL。In an embodiment of the present invention, the dosage of the aforementioned pharmaceutical composition is, for example, 0.1 mL to 2 mL.
在本發明的一實施例中,上述的醫藥組成物的給藥時間點為自然週期(menstrual cycle)的濾泡期、排卵期或黃體期或賀爾蒙補充療法(Hormone Replacement Therapy,HRT)用藥的第8~16天。In an embodiment of the present invention, the time point of administration of the above-mentioned pharmaceutical composition is the follicular phase, ovulation phase or luteal phase of the menstrual cycle, or Hormone Replacement Therapy (HRT) medication The 8th to 16th days.
在本發明的一實施例中,上述的醫藥組成物的給藥的週期為2~3天一次,至少1~3次。In an embodiment of the present invention, the period of administration of the aforementioned pharmaceutical composition is once every 2 to 3 days, at least 1 to 3 times.
在本發明的一實施例中,上述的雌性例如是因子宮因素或卵巢因素所造成的不孕症的雌性。In an embodiment of the present invention, the above-mentioned female is, for example, a female with infertility caused by uterine factors or ovarian factors.
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 an anticoagulant in the entire process, there is no additional step of removing the platelet lysate from the centrifuge tube and repeating activation, thereby shortening the process time. In addition, the entire 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明顯提升雌性懷孕成功率。In addition, the pharmaceutical composition of the present invention including platelet-rich plasma and/or platelet-rich plasma derivatives can significantly improve the pregnancy success rate of females.
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。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,可用於提升雌性懷孕成功率。The invention provides a method for preparing a platelet lysate, which can obtain a platelet lysate with a high concentration of growth factors under specific operating conditions, and the method has a simple operation process, a short process time, and the entire process does not need to add any anticoagulant. In addition, the pharmaceutical composition containing platelet-rich plasma and/or platelet-rich plasma derivatives provided by the present invention can be used to improve the success rate of female pregnancy.
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。In order to thoroughly understand the present invention, the process steps for preparing the platelet lysate will be described in detail below. However, the well-known composition or process steps are not described in details 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 subsequent 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 flowchart of a method for preparing a platelet lysate according to an embodiment of the present invention. The method for preparing a 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, perform step a), and place the collected
在本實施例中,分離膠體20的密度為1.030 g/cm3
至1.093 g/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
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1000 g至4000 g。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3000 g。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1500 g,離心時間例如是5分鐘。Next, proceed to step b). The
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, the
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 separation colloid 20 of the present invention can effectively isolate red blood cells and platelets, so that red blood cells are almost only present in the
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) can be followed, but the present invention is not limited to this. In another embodiment, after performing step b) and before performing step c), a portion of the first
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
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, step c) is performed to mix the first upper part 12 (or the second upper part 13) with the
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4000 g。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3000 g。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1500 g,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-
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,其中其中離心轉速為1000 g至4000 g,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1000 g至4000 g,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, proceed to step e) to separate the platelet-rich fibrin colloid 17 to obtain a platelet lysate (PL) 18. In this embodiment, the method for separating the platelet-
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 performing step d) and before performing step e), the platelet-
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 1 ng/ml至 10000 ng/ml,PDGF-BB的濃度10 pg/ml至500000 pg/ml,PDGF-AB的濃度為10 pg/ml至500000 pg/ml,FGF2的濃度為1 pg/ml至10000 pg/ml,EGF的濃度為1 pg/ml至10000 pg/ml,VEGF的濃度為1 pg/ml至10000 pg/ml,VEGFA的濃度為0.1 pg/ml至5000 pg/ml。In one embodiment, the
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5 ng/ml至3000 ng/ml,PDGF-BB的濃度50 pg/ml至50000 pg/ml,PDGF-AB的濃度為100 pg/ml至50000 pg/ml,FGF2的濃度為20 pg/ml至6000 pg/ml,EGF的濃度為1.5 pg/ml至2000 pg/ml,VEGF的濃度為15 pg/ml至6000 pg/ml,VEGFA的濃度為0.2 pg/ml至1500 pg/ml。In another embodiment, the
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 entire process of preparing the platelet lysate. The anticoagulant is, for example, Anticoagulant Citrate Dextrose Solution, Solution A (ACD-A), and Anticoagulant Citrate Dextrose Solution, Solution B (ACD-B). , 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 the entire process of preparing the platelet lysate of the present invention does not add an anticoagulant, there is no additional step of removing the platelet lysate from the centrifuge tube and repeating the 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 entire 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提升雌性懷孕成功率的醫藥組成物,其包括富含血小板血漿及/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。在本實施例中,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所製備的血小板裂解液。The present invention provides a medical composition for improving the success rate of female pregnancy, which comprises platelet-rich plasma and/or a derivative of platelet-rich plasma. In one embodiment, the derivative of platelet-rich plasma is, for example, platelet lysate. In this embodiment, the derivative of platelet-rich plasma is, for example, a platelet lysate prepared by the above method for preparing a platelet lysate.
在一實施例中,醫藥組成物可進一步包括醫藥組成物在藥學上可接受的鹽類或載劑。In one embodiment, the pharmaceutical composition may further include a pharmaceutically acceptable salt or carrier of the pharmaceutical composition.
在一實施例中,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TGF-β1的濃度為 1 ng/ml至 10000 ng/ml,PDGF-BB的濃度10 pg/ml至500000 pg/ml,PDGF-AB的濃度為10 pg/ml至500000 pg/ml,FGF2的濃度為1 pg/ml至10000 pg/ml,EGF的濃度為1 pg/ml至10000 pg/ml,VEGF的濃度為1 pg/ml至10000 pg/ml,VEGFA的濃度為0.1 pg/ml至5000 pg/ml。In one embodiment, the platelet lysate 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 1 ng/ml to 10000 ng/ml, the concentration of PDGF-BB is 10 pg/ml to 500000 pg/ml, the concentration of PDGF-AB is 10 pg/ml to 500000 pg/ml, the concentration of FGF2 is 1 pg/ml to 10000 pg/ml, The concentration of EGF 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.
在另一實施例中,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TGF-β1的濃度5 ng/ml至3000 ng/ml,PDGF-BB的濃度50 pg/ml至50000 pg/ml,PDGF-AB的濃度為100 pg/ml至50000 pg/ml,FGF2的濃度為20 pg/ml至6000 pg/ml,EGF的濃度為1.5 pg/ml至2000 pg/ml,VEGF的濃度為15 pg/ml至6000 pg/ml,VEGFA的濃度為0.2 pg/ml至1500 pg/ml。In another embodiment, the platelet lysate 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 to 3000 ng/ml, the concentration of PDGF-BB is 50 pg/ml to 50000 pg/ml, the concentration of PDGF-AB is 100 pg/ml to 50000 pg/ml, the concentration of FGF2 is 20 pg/ml to 6000 pg/ml, The concentration of EGF is 1.5 pg/ml to 2000 pg/ml, the concentration of VEGF is 15 pg/ml to 6000 pg/ml, and the concentration of VEGFA is 0.2 pg/ml to 1500 pg/ml.
在本實施例中,雌性例如是因子宮因素或卵巢因素所造成的不孕症的雌性。更具體來說,雌性例如是因子宮因素或卵巢因素所造成的不孕症的雌性人類。In this embodiment, the female is, for example, a female with infertility caused by uterine factors or ovarian factors. More specifically, females are, for example, female humans who are infertile due to uterine factors or ovarian factors.
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灌注方式給藥至子宮腔內。在一實施例中,可使用人工授精(Intrauterine insemination,IUI)管或胚胎移植(Embryo Transfer,ET)管將醫藥組成物以灌注方式給藥至子宮腔內。在一實施例中,醫藥組成物的給藥量例如是0.1 mL至2 mL。In this embodiment, the medical composition can be made into a sterile injection solution. In one embodiment, the pharmaceutical composition is administered into the uterine cavity by infusion, for example. In one embodiment, an artificial insemination (Intrauterine insemination, IUI) tube or an embryo transfer (Embryo Transfer, ET) tube may be used to administer the pharmaceutical composition into the uterine cavity by perfusion. In one embodiment, the dosage of the pharmaceutical composition is, for example, 0.1 mL to 2 mL.
在本實施例中,醫藥組成物的給藥時間點為自然週期(menstrual cycle)的濾泡期、排卵期或黃體期或賀爾蒙補充療法(Hormone Replacement Therapy,HRT)用藥的第8~16天,但本發明不限於此。在一實施例中,亦可依照冷凍胚胎移植或新鮮胚胎移植來決定給藥的時間點。在一實施例中,賀爾蒙補充療法中的賀爾蒙例如是雌二醇(Estradiol)、黃體激素(Progesterone)或兩者合併使用。在本實施例中,醫藥組成物的給藥的週期為2~3天一次,至少1~3次。In this embodiment, the time point of administration of the pharmaceutical composition is the follicular phase, ovulation phase, or luteal phase of the menstrual cycle, or the 8th to 16th of Hormone Replacement Therapy (HRT) medication. Days, but the present invention is not limited to this. In one embodiment, the time point of administration can also be determined according to frozen embryo transfer or fresh embryo transfer. In one embodiment, the hormone in hormone replacement therapy is, for example, estradiol (Estradiol), progesterone (Progesterone), or a combination of both. In this embodiment, the period of administration of the pharmaceutical composition is once every 2 to 3 days, at least 1 to 3 times.
本發明的醫藥組成物可用於提升雌性懷孕成功率,具體來說,可用於提升雌性人工受孕的成功率。在一實施例中,本發明的醫藥組成物可有助於雌性的子宮內膜增厚。The pharmaceutical composition of the present invention can be used to increase the success rate of female pregnancy, specifically, it can be used to increase the success rate of female artificial insemination. In one embodiment, the pharmaceutical composition of the present invention can help thicken the endometrium of females.
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。Hereinafter, an experimental example will be used to specifically illustrate the present invention, but the parameters and data results of the experimental example are only used to illustrate the efficacy of the present invention, 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Experimental example 11
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1500 g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 collected from the subject, and fresh whole blood was injected into a centrifuge tube containing separation colloid with an 18G needle. Put the centrifuge tube in a centrifuge and centrifuge at 1500 g for 5 minutes to separate the whole blood into a plasma layer, a white membrane layer and a red blood cell layer. 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, connect a 10 ml syringe with an 18G needle to remove the 2 ml plasma layer. Shake the centrifuge tube up and down evenly to mix the remaining plasma layer with the albuginea layer to obtain platelet-rich plasma (PRP).
然後,將同一離心管放入離心機內,以1500 g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, put the same centrifuge tube into a centrifuge and centrifuge at 1500 g for 5 minutes to obtain a platelet-rich fibrin (PRF) colloid.
之後,將同一離心管放入離心機內,以1500 g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。After that, put the same centrifuge tube into the centrifuge and centrifuge at 1500 g for 5 minutes. Repeat the above centrifugation conditions for 10 centrifugation, thereby obtaining the platelet lysate at the top of the centrifuge tube.
比較例Comparative example 11
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1500 g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, take 10 ml of venous blood from the subject, and inject fresh whole blood into a centrifuge tube containing anticoagulant (ACD Solution-A, ACDA) with an 18G needle. Put the centrifuge tube into the centrifuge and centrifuge at 1500 g for 5 minutes to separate the whole blood into a plasma layer, a white membrane layer, and a red blood cell 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。Then, take out the plasma layer and white film layer, move to another sterile container, add calcium chloride solution and mix well, this step is to activate platelets. Next, a freeze-thaw cycle is performed. The activated solution is placed at room temperature for one hour, and then placed in a -80 degree refrigerator for freezing. After 4 hours, it is taken out and the growth factor can be determined.
[[ 生長因子的測定Growth factor determination ]]
在本實施例中,使用酵素連結免疫吸附法(Enzyme-linked Immuno-Sorbent Assay,ELISA)測定實施例1的富含血小板血漿、實施例1與比較例1的血小板裂解液中的生長因子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 enzyme-linked Immuno-Sorbent Assay (ELISA) was used to determine the growth factor TGF- in the platelet-rich plasma of Example 1, and the platelet lysate of Example 1 and Comparative Example 1. The contents of β1, PDGF-AB, PDGF-BB, VEGF and VEGFA, and the results are shown in Table 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).
[表1]
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),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(即實施例1)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。It can be seen from Table 1 that compared with the platelet lysate prepared by the traditional method (ie Comparative Example 1), the platelet lysate prepared by the method for preparing platelet lysate of the present invention (ie Example 1) has a higher concentration of Growth factor. In addition, since the method for preparing platelet lysate of the present invention does not add anticoagulant in the entire process, there is no additional step of taking the platelet lysate from the centrifuge tube and repeating the activation, thereby shortening the process time. In addition, the entire 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.
[[ 血小板裂解液對於不孕症患者的效用The effectiveness of platelet lysate for infertility patients ]]
實施例Example 33
在本實施例中,受試者為10位平均40歲以上具有不孕症的雌性患者,使用實施例1所製備的血小板裂解液作為本實施例的醫藥組成物,並如以下方式進行血小板裂解液對受試者懷孕成功率的評估。In this example, the subjects are 10 female patients with infertility over 40 years old on average. The platelet lysate prepared in Example 1 is used as the medical composition of this example, and platelet lysis is performed in the following manner The assessment of the success rate of pregnancy by the liquid.
首先,在自然週期第10-12天時量測受試者子宮內膜的初始厚度。將裝有實施例1的血小板裂解液(0.5-1毫升)的針筒連接人工受精(Intrauterine insemination,IUI)管,以灌注方式給藥至受試者的子宮腔內。給藥後受試者需保持平躺30分鐘。給藥48小時後,以灌注方式進行第二次的給藥。在第二次給藥48小時後,量測受試者的子宮內膜的厚度。當受試者的子宮內膜的厚度達7公分時,即可進行胚胎植入。First, measure the initial thickness of the subject's endometrium on days 10-12 of the natural cycle. Connect a syringe containing the platelet lysate (0.5-1 ml) of Example 1 to an artificial insemination (Intrauterine insemination, IUI) tube, and administer it into the uterine cavity of the subject by perfusion. Subjects need to lie down for 30 minutes after administration. 48 hours after the administration, the second administration was given by infusion. 48 hours after the second administration, the thickness of the subject's endometrium was measured. When the thickness of the subject's endometrium reaches 7 cm, the embryo can be implanted.
接著,對給藥後的受試者進行胚胎植入,並且於胚胎植入後持續搭配賀爾蒙補充療法,服用黃體素(Progesterone)及雌激素(estrogen),服用時間為2週。然後,檢測受試者的血液中的乙型絨毛膜促性腺激素(β-HCG)數值,以確認受精卵的著床狀態,受試者的懷孕結果及子宮內膜厚度分別如表2及表3所示。Next, embryo implantation was performed on the subjects after the administration, and hormone replacement therapy was continued after embryo implantation, taking progesterone and estrogen for 2 weeks. Then, test the beta-HCG value in the subject’s blood to confirm the implantation status of the fertilized egg. The subject’s pregnancy results and endometrial thickness are shown in Table 2 and Table respectively. 3 shown.
[表2]
[表3]
由表2可以看出,將血小板裂解液以灌注方式給藥至受試者的子宮腔內後,可增加受試者的子宮內膜厚度。It can be seen from Table 2 that after the platelet lysate is administered into the uterine cavity of the subject by perfusion, the thickness of the endometrium of the subject can be increased.
一般來說,人工受孕的成功機率僅為30%,然而隨著年齡漸增,40歲以上婦女人工受孕成功機率只有7.7%。而由表3可以看出,將血小板裂解液以灌注方式給藥至受試者的子宮腔內後,整體受試者的懷孕成功率由7.7%提升至50%。Generally speaking, the success rate of artificial insemination is only 30%. However, as the age increases, the success rate of artificial insemination for women over 40 years old is only 7.7%. It can be seen from Table 3 that after the platelet lysate was administered into the uterine cavity of the subject by infusion, the overall pregnancy success rate of the subject was increased from 7.7% to 50%.
綜上所述,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。In summary, since the method for preparing platelet lysate of the present invention does not add an anticoagulant in the entire process, there is no additional step of removing the platelet lysate from the centrifuge tube and repeating activation, thereby shortening the process time. In addition, the entire 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明顯提升雌性懷孕成功率。In addition, the pharmaceutical composition of the present invention including platelet-rich plasma and/or platelet-rich plasma derivatives can significantly improve the pregnancy success rate of females.
10:全血 12:第一上層部分 13:第二上層部分 14:中間部分 15:富含血小板血漿部分 16:下層部分 17:富含血小板纖維蛋白膠體 18:血小板裂解液 19:膠體部分 20:分離膠體 30:離心管10: Whole blood 12: The first upper part 13: The second upper part 14: middle part 15: Platelet-rich plasma fraction 16: Lower part 17: Platelet-rich fibrin colloid 18: Platelet lysate 19: colloidal part 20: Separation of colloid 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 flowchart of a method for preparing a platelet lysate according to an embodiment of the present invention.
10:全血10: Whole blood
12:第一上層部分12: The first upper part
13:第二上層部分13: The second upper part
14:中間部分14: middle part
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: colloidal part
20:分離膠體20: Separation of colloid
30:離心管30: Centrifuge tube
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