TWI786362B - Method of preparing platelet lysate and use thereof for improving female pregnancy success rate - 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

Method for preparing platelet lysate and its use for improving female pregnancy rate

The present invention relates to a method for preparing derivatives of platelet-rich plasma (PRP) and its use, and in particular to a method for preparing platelet lysate and its use for increasing the pregnancy rate of females .

Platelet-rich plasma (platelet-rich plasma, PRP) is obtained by centrifuging human or animal whole blood with high concentration of platelets, and platelet lysate is a further processed derivative of platelet-rich plasma, platelet The lysate contains a variety of growth factors involved in regeneration. Platelet-rich plasma and platelet lysates are therefore known to be useful in regenerative medicine.

Currently known traditional methods for preparing platelet lysates are: after adding PRP to further activating factors, the platelet lysates are obtained by repeated thawing and freezing or ultrasonic waves. And above-mentioned traditional method may have following shortcoming: collect the whole blood volume too much, Contamination of red blood cells due to incomplete separation, repeated activation due to the action of anticoagulants, difficulty in concentrating, etc. The above problems will lead to insufficient amount of growth factors in the collected platelet lysate. In addition, in the traditional way of preparing platelet lysate, PRP needs to be taken out from the centrifuge tube and activated, so the effect on sterility is negligible, and the platelet lysate obtained by freezing and thawing or ultrasonic vibration takes a long time. 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.

In addition, it is known that many factors may cause female infertility, and the 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 infertile patients.

The invention provides a method for preparing platelet lysate, which can quickly prepare platelet lyse with high concentration of growth factors under aseptic conditions.

The present invention provides a pharmaceutical composition comprising platelet-rich plasma and/or derivatives of platelet-rich plasma, which can be used to improve the success rate of female pregnancy.

Embodiments of the present invention provide a method for preparing platelet lysate, said method comprising the following steps: step a), placing the collected whole blood in a centrifuge tube filled with separating colloid, wherein the density of the separating colloid is 1.030g/ cm ³ to 1.093g/cm ³ ; 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 layer part, a middle part and a lower layer part, wherein the separating 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 (platelet-rich plasma, PRP) part; step d), making the platelet-rich plasma part Coagulation to obtain platelet-rich fibrin (platelet-rich fibrin, PRF) colloid; and step e), separating platelet-rich fibrin colloid to obtain platelet lysate (platelet lysate, PL), wherein in steps a) to No anticoagulant is added in step e).

In an embodiment of the present invention, before performing step c), it may further include removing the first upper layer portion of the 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.

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 B, ACD-B), anticoagulant compound sodium citrate solution C (Anticoagulant Citrate Dextrose Solution, Solution C, ACD-C), citric acid-phosphate-grape solution (Citrate-phosphate-dextrose Solution, CPD), ethylenediaminetetraacetic acid (EDTA), heparin, sodium citrate or a combination thereof.

In an embodiment of the present invention, the above-mentioned separating colloid material may include silicon-containing polymers, acrylic polymers or polyester polymers.

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.

In one embodiment of the present invention, the above-mentioned first upper layer is a plasma layer containing platelets, and the middle part is a buffy coat layer containing platelets and white blood cells. coat), and the lower part is the red blood cell layer containing red blood cells.

In one embodiment of the invention, coagulating the platelet rich plasma fraction comprises performing a second centrifugation process on the platelet rich plasma fraction.

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.

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.

In an embodiment of the present invention, the above-mentioned third centrifugation process includes: centrifuging the platelet-rich fibrin colloid for a single time, 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 multiple times, wherein the centrifugation speed is 1000g to 4000g, and the centrifugation time is 3 minutes to 15 minutes each time.

In an embodiment of the present invention, the entire process time for preparing the platelet lysate is 10 minutes to 60 minutes.

In one embodiment of the present invention, before step e), the platelet-rich fibrin colloid is pressed, squeezed, filtered, ultrasonically oscillated, or subjected to freeze-thaw cycles, so that the platelet lysate is obtained from the platelet-rich fibrin colloid. Fibrin colloid release.

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 1ng/ml to 10000ng/ml, PDGF-BB concentration 10pg/ml to 500000pg/ml, PDGF-AB The concentration of 10pg/ml to 500000pg/ml, the concentration of FGF2 is 1pg/ml to 10000pg/ml, the concentration of EGF is 1pg/ml to 10000pg/ml, the concentration of VEGF is 1pg/ml to 10000pg/ml, the concentration of VEGFA From 0.1pg/ml to 5000pg/ml.

The present invention provides an application of a pharmaceutical composition for preparing a drug for improving the pregnancy success rate of females, wherein the pharmaceutical composition includes platelet-rich plasma and/or derivatives of platelet-rich plasma.

In an embodiment of the present invention, the above-mentioned derivatives of platelet-rich plasma may include platelet lysate.

In an embodiment of the present invention, the above-mentioned pharmaceutical composition can be administered into the uterine cavity by infusion.

In one embodiment of the present invention, the dosage of the above-mentioned pharmaceutical composition is, for example, 0.1 mL to 2 mL.

In one embodiment of the present invention, the above-mentioned pharmaceutical composition is administered at the follicular phase, ovulation phase or luteal phase of the natural cycle (menstrual cycle) or hormone replacement therapy (Hormone Replacement Therapy, HRT) The 8th to 16th day.

In one embodiment of the present invention, the administration cycle of the above-mentioned pharmaceutical composition is once every 2-3 days, at least 1-3 times.

In one embodiment of the present invention, the above-mentioned female is, for example, a female with infertility caused by uterine factors or ovarian factors.

Based on the above, due to the method for preparing platelet lysate of the present invention in the whole No anticoagulant is added to the process, so there is no additional step of removing the platelet lysate from the centrifuge tube and repeating the 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 pollution and having sterility.

In addition, the pharmaceutical composition comprising platelet-rich plasma and/or platelet-rich plasma derivatives of the present invention can significantly increase the success rate of female pregnancy.

10: whole blood

12: First upper part

13: Second upper part

14: middle part

15: Platelet-rich plasma fraction

16: Lower part

17: Platelet-rich fibrin colloid

18: platelet lysate

19: colloid part

20: Separation colloid

30: centrifuge tube

FIG. 1 is a flowchart of a method for preparing a platelet lysate according to an embodiment of the present invention.

The invention provides a method for preparing platelet lysate, which can obtain platelet lysate with high concentration of growth factors under specific operating conditions, and the method has simple operation process, short process time, and the whole process does not need to add any anticoagulant. In addition, the pharmaceutical composition containing platelet-rich plasma and/or derivatives of platelet-rich plasma provided by the present invention can be used to improve the success rate of female pregnancy.

In order to thoroughly understand the present invention, the process steps for preparing the platelet lysate will be described in detail below. However, well-known compositions or process steps have not been described in detail in order to avoid limiting the invention. Preferred embodiments of the present invention will be described in detail below, but the present invention is not limited thereto, and the present invention can also be widely implemented in other In the embodiment, and the scope of the present invention is not limited, the following patent scope shall prevail.

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 platelet lysate of the present invention comprises the following steps.

Firstly, step a) is performed, and the collected whole blood 10 is placed in the centrifuge tube 30 filled with the separating colloid 20 . In one embodiment, the whole blood 10 is, for example, fresh mammalian whole blood. In this embodiment, the whole blood 10 is, for example, fresh human whole blood.

In this embodiment, the density of the separation colloid 20 is 1.030 g/cm ³ to 1.093 g/cm ³ . In this embodiment, the material of the separation colloid 20 is, for example, silicon-containing polymer, acrylic polymer or polyester polymer.

Next, step b) is performed to perform the first centrifugation process on the centrifuge tube 30 containing the whole blood 10 and the separating colloid 20, so that the whole blood 10 is separated into a first upper layer part 12, a middle part 14 and a lower layer part 16, wherein the separated The gel 20 is located between the middle portion 14 and the lower 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, 1500g to 3000g. 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.

In this embodiment, after step b), the whole blood 10 is separated into three layers, wherein the uppermost layer (i.e. the first upper layer part 12) is a plasma layer containing platelets, and the middle layer (i.e. the middle part 14) Buffy coat that contains platelets and white blood cells (buffy coat), the lowermost layer (ie, the lower layer part 16) is the red blood cell layer containing red blood cells.

In this embodiment, the separating colloid 20 of the present invention can effectively separate red blood cells and platelets, so that red blood cells are almost only present in the lower layer 16 , and platelets are present in the first upper layer 12 and the middle portion 14 . Specifically, the concentration of platelets in first upper portion 12 and middle portion 14 is higher than the concentration of platelets in whole blood and lower portion 16 .

In this embodiment, step c) may be followed after step b), but the present invention is not limited thereto. In another embodiment, after performing step b) and before performing step c), part of the first upper layer portion 12 may be optionally further removed to obtain the second upper layer portion 13 . In this embodiment, the volume ratio of the first upper layer portion 12 to the second upper 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 second upper 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 part 12 is, for example, piercing a needle into the first upper part 12 of the centrifuge tube 30 , and sucking part of the first upper part 12 to the outside of the centrifuge tube 30 .

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 second upper layer portion 13, the platelet-rich The platelet lysate separated from the fibrin colloid can be stored in a smaller volume of solution, thereby achieving the effect of concentration.

Then, carry out step c), the first upper layer part 12 (or the second upper layer part Part 13) is mixed with the middle part 14 to obtain a platelet-rich plasma (platelet-rich plasma, PRP) part 15 . In this embodiment, the method for mixing the first upper layer part 12 (or the second upper layer part 13) with the middle part 14 is, for example, to shake the centrifuge tube 30 evenly, so that the first upper layer part 12 (or the second upper layer part 13) ) mixed with the middle part 14. But the present invention is not limited thereto, and any method that can uniformly mix the first upper layer part 12 (or the second upper layer part 13 ) and the middle part 14 can be applied in this step.

Afterwards, step d) is performed to coagulate the platelet-rich plasma fraction 15 to obtain a platelet-rich fibrin (platelet-rich fibrin, PRF) colloid 17 . In this embodiment, the method for coagulating the platelet-rich plasma portion 15 is, for example, performing a second centrifugation process on the platelet-rich plasma portion 15 . In this embodiment, the centrifugation speed of the second centrifugation process in step d) is, for example, 1000 to 4000 g. In one embodiment, the centrifugation 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 portion 15 self-agglutinates into a platelet-rich fibrin (PRF) colloid 17 .

Then, step e) is performed to separate platelet-rich fibrin colloid 17 to obtain platelet lysate (platelet lysate, PL) 18 . In this embodiment, the method for isolating platelet-rich fibrin colloid 17 is, for example, The protein colloid 17 is subjected to the third centrifugation process. In one embodiment, the third centrifugation process is, for example, performing single centrifugation on 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 multiple times, wherein the centrifugation speed is 1000 g to 4000 g, 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 times to 15 times, but the present invention is not limited thereto, and the number of times of centrifugation can be adjusted according to requirements. During the third centrifugation process, the platelet lysate 18 is released from the platelet-rich fibrin colloid 17, and the colloid part 19 of the platelet-rich fibrin colloid 17 is compressed on the separation colloid 20 due to centrifugal force. This separates the platelet lysate 18 from the colloidal fraction 19 .

In this embodiment, after performing step d) and before performing step e), the platelet-rich fibrin colloid 17 can be further pressed, squeezed, filtered, ultrasonically oscillated, or subjected to freeze-thaw cycles, which helps to make the platelets The lysate 18 is released from the platelet-rich fibrin colloid 17 .

In one embodiment, the 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 1 ng/ml to 10000ng/ml, the concentration of PDGF-BB is 10pg/ml to 500000pg/ml, the concentration of PDGF-AB is 10pg/ml to 500000pg/ml, the concentration of FGF2 is 1pg/ml to 10000pg/ml, the concentration of EGF is 1pg/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.

In another embodiment, the 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 to 3000ng/ml, the concentration of PDGF-BB is 50pg/ml to 50000pg/ml, the concentration of PDGF-AB is 100pg/ml to 50000pg/ml, the concentration of FGF2 is 20pg/ml to 6000pg/ml, and the concentration of EGF is 1.5pg /ml to 2000pg/ml, the concentration of VEGF is 15pg/ml to 6000pg/ml, and the concentration of VEGFA is 0.2pg/ml to 1500pg/ml.

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, Solution C, ACD-C), citric acid-phosphate-dextrose solution (Citrate-phosphate-dextrose Solution, CPD), ethylenediaminetetraacetic acid (Ethylenediaminetetraacetic acid , EDTA), heparin (Heparin), sodium citrate (Sodium Citrate) or a combination thereof.

In this embodiment, since no anticoagulant is added in the whole process of preparing the platelet lysate of the present invention, there is no additional step of taking the platelet lysate out of 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 obviously 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 operated in the same centrifuge tube, so it has the advantage of avoiding contamination. It has the advantage of being sterile and sterile.

The present invention provides a pharmaceutical composition for improving the success rate of female pregnancy, which includes platelet-rich plasma and/or derivatives 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, the platelet lysate prepared by the method for preparing the platelet lysate described above.

In one embodiment, the pharmaceutical composition may further include pharmaceutically acceptable salts or carriers of the pharmaceutical composition.

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 1ng/ml to 10000ng /ml, the concentration of PDGF-BB is 10pg/ml to 500000pg/ml, the concentration of PDGF-AB is 10pg/ml to 500000pg/ml, the concentration of FGF2 is 1pg/ml to 10000pg/ml, the concentration of EGF is 1pg/ml to 10000pg/ml, the concentration of VEGF is 1pg/ml to 10000pg/ml, the concentration of VEGFA is 0.1pg/ml to 5000pg/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 5ng/ml to 3000ng /ml, the concentration of PDGF-BB is 50pg/ml to 50000pg/ml, the concentration of PDGF-AB is 100pg/ml to 50000pg/ml, the concentration of FGF2 is 20pg/ml to 6000pg/ml, the concentration of EGF is 1.5pg/ml to 2000 pg/ml, the concentration of VEGF was 15 pg/ml to 6000 pg/ml, and the concentration of VEGFA was 0.2 pg/ml to 1500 pg/ml.

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, human females who are infertile due to uterine factors or ovarian factors.

In this embodiment, the pharmaceutical composition can be made into a sterile injectable solution. In one embodiment, the pharmaceutical composition is administered, for example, into the uterine cavity by infusion. In one embodiment, the pharmaceutical composition can be administered into the uterine cavity by perfusion using an artificial insemination (Intrauterine insemination, IUI) tube or an embryo transfer (Embryo Transfer, ET) tube. In one embodiment, the dosage of the pharmaceutical composition is, for example, 0.1 mL to 2 mL.

In this embodiment, the administration time point of the pharmaceutical composition is the follicular phase, ovulation phase or luteal phase of the natural cycle (menstrual cycle), or the 8th to 16th phase of hormone replacement therapy (Hormone Replacement Therapy, HRT). day, but the present invention is not limited thereto. 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 used in the hormone supplement therapy is, for example, Estradiol, Progesterone, or a combination of both. In this embodiment, the administration cycle of the pharmaceutical composition is once every 2-3 days, at least 1-3 times.

The pharmaceutical composition of the present invention can be used to improve the success rate of female pregnancy, specifically, can be used to improve the success rate of female artificial insemination. In one embodiment, the pharmaceutical composition of the present invention can help endometrium thickening in females.

The present invention will be described in detail below with experimental examples, but the parameters and data results of the experimental examples are only used to illustrate the effect of the present invention, rather than to limit the present invention range.

[Preparation of platelet lysate]

Example 1

First, 10 ml of venous blood was taken from the subject, and fresh whole blood was injected into a centrifuge tube containing separating colloid with a density of 1.030 g/cm ³ to 1.093 g/cm ³ 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 layer and red blood cell layer, wherein the plasma layer is a small amount of platelet-poor plasma (Platelet-Poor Plasma, PPP).

Next, 2 mL of the plasma layer was removed using an 18G needle connected to a 10 mL syringe. The centrifuge tube was evenly shaken up and down, so that the remaining plasma layer was mixed with the buffy coat layer to obtain platelet-rich plasma (PRP).

Then, put the same centrifuge tube into a centrifuge and centrifuge at 1500 g for 5 minutes to obtain platelet-rich fibrin (PRF) colloid.

Afterwards, the same centrifuge tube was put into a centrifuge and centrifuged at 1500 g for 5 minutes. Repeat the centrifugation conditions above for 10 times to obtain the platelet lysate at the top of the centrifuge tube.

Comparative example 1

First, 10 ml of venous blood was collected from the subject, and fresh whole blood was injected into a centrifuge tube containing anticoagulant (ACD Solution-A, ACDA) 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 layer and red blood cell layer.

Next, take out the plasma layer and buffy coat layer, move 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 left at room temperature for one hour, then placed in a -80°C refrigerator for freezing, and then taken out after 4 hours for growth factor determination.

[Determination of growth factors]

In this example, the growth factor TGF- The contents of β1, PDGF-AB, PDGF-BB, VEGF and VEGFA were recorded in Table 1. The results in Table 1 are expressed as <mean ± standard deviation>, and there is a significant difference (P<0.05) between each growth factor embodiment and the comparative example.

As can be seen from the contents of Table 1, compared with the platelet lysate prepared by the traditional method (i.e. Comparative Example 1), the platelet lysate (i.e. Example 1) prepared by the method for preparing platelet lysate of the present invention has a higher concentration of growth factor. In addition, since the method for preparing the platelet lysate of the present invention does not add anticoagulant throughout the process, there is no additional step of taking the platelet lysate out of the centrifuge tube and repeating the 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 pollution and having sterility.

[Efficacy of platelet lysate for infertile patients]

Example 2

In this example, the subjects were 10 infertile female patients with an average age of over 40, and the platelet lysate prepared in Example 1 was used as the pharmaceutical composition of this example, and the platelet lysis was performed in the following manner The evaluation of the pregnancy success rate of the test subjects.

First, the initial thickness of the endometrium of the subject was measured on day 10-12 of the natural cycle. The syringe containing the platelet lysate (0.5-1 ml) of Example 1 was connected to an artificial insemination (Intrauterine insemination, IUI) tube, and administered into the uterine cavity of the subject by perfusion. Subjects were required to lie flat for 30 minutes after administration. 48 hours after administration, the second administration was performed by infusion. 48 hours after the second administration, the thickness of the endometrium of the subject was measured. Embryo implantation can be performed when the thickness of the endometrium of the subject reaches 7mm.

Then, embryo implantation was performed on the subject after administration, and after embryo implantation After admission, continue to cooperate with hormone supplement therapy, taking progesterone and estrogen for 2 weeks. Then, detect the beta-choriogonadotropin (β-HCG) value in the blood of the test subject to confirm the implantation status of the fertilized egg. The pregnancy results and endometrial thickness of the test subject are shown in Table 2 and Table 2 respectively. 3.

P<0.01 when compared to before administration.

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.

Generally speaking, the success rate of artificial insemination is only 30%. However, as the age gradually 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 perfusion, the pregnancy success rate of the overall subject increased from 7.7% to 50%.

In summary, since the method for preparing the platelet lysate of the present invention does not add anticoagulant throughout the process, there is no additional step of taking the platelet lysate out of the centrifuge tube and reactivating it, thereby shortening the process time. In addition, the present invention The whole process of preparing platelet lysate is carried out in the same centrifuge tube, so it has the advantages of avoiding pollution and having sterility.

In addition, the pharmaceutical composition comprising platelet-rich plasma and/or platelet-rich plasma derivatives of the present invention can significantly increase the success rate of female pregnancy.

10: whole blood

12: 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: colloid part

20: Separation colloid

30: centrifuge tube

Claims (6)

The use of a pharmaceutical composition for preparing a drug for improving the success rate of female pregnancy, wherein the pharmaceutical composition includes a platelet lysate, wherein the platelet lysate is prepared by a method comprising the following steps: Step a), the collected The whole blood is placed in a centrifuge tube containing the separating colloid, wherein the density of the separating colloid is 1.030g/cm ³ to 1.093g/cm ³ ; step b), for the centrifuge tube containing the whole blood and the separating colloid The centrifuge tube is subjected to a first centrifugation process to separate the whole blood into a first upper part, a middle part and a lower part, wherein the separating colloid is located between the middle part and the lower part, wherein the The first upper part is a plasma layer containing platelets, the middle part is a buffy coat layer containing platelets and white blood cells, and the lower part is a red blood cell layer containing red blood cells, and the centrifugal speed of the first centrifugation process is 1000g to 4000g, centrifugation time is 3 minutes to 10 minutes; After carrying out described step b), be to further remove the described first upper layer part of part to obtain the second upper layer part, wherein the first upper layer part and the first upper layer part The volume ratio of the two upper layer parts is 1:0.1-0.8, and the method for removing the first upper layer part is to pierce a needle into the first upper layer part of the centrifuge tube, and part of the first upper layer part The upper part is sucked to the outside of the centrifuge tube; step c), mixing the second upper part with the middle part to obtain a platelet-rich plasma part; step d), under the condition that no platelet activating factor is added Next, the platelet-rich plasma fraction is subjected to a second centrifugation process to obtain a platelet-rich fibrin colloid, wherein during the second centrifugation process, the platelet-rich plasma fraction self-coagulates into the The platelet-rich fibrin colloid , wherein the centrifugation speed of the second centrifugation process is 1000g to 4000g, and the centrifugation time is 3 minutes to 10 minutes; and step e), separating the platelet-rich fibrin colloid to obtain the The platelet lysate, wherein the method for separating the platelet-rich fibrin colloid is to perform a third centrifugation process, wherein the third centrifugation process includes performing a single centrifugation on the platelet-rich fibrin colloid, and the first The centrifugation speed of the three-centrifugation process is 1000g to 4000g, and the centrifugation time is 3 minutes to 45 minutes, wherein no anticoagulant and the activation factor are added in the step a) to the step e). The use of the pharmaceutical composition as described in item 1 of the patent application for preparing a drug for improving the pregnancy success rate of females, wherein the pharmaceutical composition is administered into the uterine cavity by infusion. Use of the pharmaceutical composition as described in item 1 of the patent application for preparing a drug for improving female pregnancy success rate, wherein the dosage of the pharmaceutical composition is 0.1 mL to 2 mL. The use of the pharmaceutical composition as described in item 1 of the scope of the patent application for the preparation of a drug for improving the success rate of female pregnancy, wherein the administration time point of the pharmaceutical composition is the follicular phase of the natural cycle (menstrual cycle), ovulation The 8th to 16th day of the luteal phase or Hormone Replacement Therapy (HRT) medication. Use of the pharmaceutical composition described in item 1 of the patent application for the preparation of a drug for improving the success rate of female pregnancy, wherein the administration cycle of the pharmaceutical composition is once every 2-3 days, at least 1-3 times. Use of the pharmaceutical composition described in item 1 of the patent application for preparing a drug for improving the pregnancy success rate of females, who are females with infertility caused by uterine factors or ovarian factors.

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