A preservative solution for prolonging the in vitro storage period of platelets and a method for preparing the same

Abstract

The present application relates to a kind of preservative solution for prolonging the in vitro storage period of platelet and its preparation method, containing baicalin 4.50~6.00g, baicalein 3.00~3.40g, sodium citrate 1.50~2.00g, citric acid 0.20~0.50g, glucose 7.90~8.50g per liter of preservative solution, and the remaining component is distilled water.The preservative solution of the present application adds a certain proportion of baicalin and baicalein, and the composition has a synergistic effect on inhibiting platelet activation, protection and inhibition of platelet aggregation, can prolong the in vitro platelet normal temperature storage time, and the normal temperature storage time can reach 16d.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a preservation solution for prolonging the in vitro preservation period of platelets and its preparation method. Background Technology

[0002] Platelets are a widely used and irreplaceable component of blood for hemostasis and coagulation in clinical practice. In recent years, they have also been widely used in tissue repair (PRP) and basic research (regulating immune function). Currently, the People's Republic of China Health Industry Standard (WS399-2012) stipulates that the in vitro preservation method for platelets is as follows: the preservation solution uses anticoagulants such as ACD (citric acid-sodium citrate-glucose), CPD (citric acid-sodium citrate-sodium dihydrogen phosphate-glucose), or CPDA-1, CPDA-2 (citric acid-sodium citrate-sodium dihydrogen phosphate-glucose-adenine) to autologous plasma, and is stored at (22±2)℃ with continuous shaking for only 5 days.

[0003] The short shelf life of platelets limits their collection and use, leading to waste or shortages of this valuable resource, while large-scale storage can easily result in expiration and spoilage. Clinically, the inability to obtain platelet products promptly often delays emergency treatment, seriously threatening patients' lives. Therefore, researching platelet preservation solutions to extend their in vitro shelf life has been a topic of great interest both domestically and internationally.

[0004] In recent years, some studies have reported on extending the in vitro preservation period of platelets using 5% DMSO, long-term storage at -80℃, refrigerated storage at 4℃ for 10-14 days, and modified PAS-IIIM preservation solution, M-SO1 preservation solution, sodium citrate / sodium acetate / sodium phosphate / glucose preservation solution, and preservation solutions containing multiple components such as trehalose. However, most of these studies require storage at low or even ultra-low temperatures, which can lead to the drawback of platelet hypothermia.

[0005] Chinese patent CN 201410410653.7 discloses a platelet preservation solution. The solution is prepared by mixing dimethyl sulfoxide (DMSO) with fresh distilled water to a volume concentration of 100-50%, then stirring thoroughly and adding pharmaceutical-grade charcoal. The resulting solution is then filtered through a titanium rod to remove carbon, followed by filtration through a 0.45 μm microporous membrane and then through a 0.22 μm microporous membrane to obtain the platelet preservation solution. This solution can maintain the physiological function of platelets at low temperatures, but the cryopreservation survival rate is low, and DMSO has certain toxic side effects on the recipient.

[0006] Chinese patent CN 201410155266.3 discloses a platelet cryopreservation solution containing trehalose and its application. The solution contains Na3-citric acid, NaH2PO4, Na2HPO4, KCl, MgCl2, NaCl, and trehalose. This platelet cryopreservation solution allows for the preservation of platelets at relatively low temperatures for extended periods; however, cryopreservation can lead to platelet damage due to low temperatures. Summary of the Invention

[0007] The purpose of this invention is to provide a preservation solution for extending the in vitro preservation period of platelets, which has no toxic side effects on the recipient and can extend the in vitro platelet preservation time at room temperature to 16 days.

[0008] The technical solution adopted by this invention to solve the above problems is: a preservation solution for prolonging the in vitro preservation period of platelets, wherein each liter of the preservation solution contains

[0009] Baicalin 4.50~6.00g

[0010] Baicalein 3.00~3.40g

[0011] Sodium citrate 1.50~2.00g

[0012] Citric acid 0.20~0.50g

[0013] Glucose 7.90~8.50g

[0014] The remaining component is distilled water.

[0015] The addition of citric acid and sodium citrate to the preservation solution has an anticoagulant effect and keeps the preservation solution in a weakly alkaline environment, preventing platelet activation and ensuring platelet activity.

[0016] Adding glucose to the preservation solution provides nutrients, which can provide sufficient energy for platelet metabolism and maintain platelet activity and function.

[0017] Preferably, the weight ratio of baicalin to baicalein is 4~3:2.

[0018] Another object of the present invention is to provide a method for preparing the above-mentioned preservation solution for prolonging the in vitro preservation period of platelets, comprising the following steps:

[0019] (1) Sodium citrate, citric acid and glucose are mixed, and distilled water is added and stirred to obtain the first mixture;

[0020] (2) Baicalin and baicalein are mixed, and distilled water is added and stirred to obtain a second mixture;

[0021] (3) Mix the first mixture and the second mixture, and then add distilled water to make up to 1000 mL to obtain the preservation solution.

[0022] Preferably, the weight ratio of distilled water in step (1) to distilled water in step (2) is 1~2:5~7.

[0023] Another object of the present invention is to provide the application of the above-mentioned preservation solution for prolonging the in vitro preservation period of platelets in platelets.

[0024] The above application is as follows: the above preservation solution is added to the collected platelets and stored by shaking at (22±2)℃ (vibration frequency of 60 times / min, amplitude of 5cm).

[0025] Compared with the prior art, the advantages of the present invention are as follows:

[0026] The preservation solution of the present invention contains a certain proportion of baicalin and baicalein. The composition has a synergistic effect on inhibiting platelet activation, protecting and inhibiting platelet aggregation, and can prolong the in vitro platelet storage time at room temperature, up to 16 days. Implementation

[0027] The present invention will be further described in detail below with reference to the embodiments. Example 1

[0028] A preservation solution for prolonging the in vitro preservation period of platelets, containing [missing information] per liter of preservation solution.

[0029] Baicalin 4.50g

[0030] Baicalein 3.00g

[0031] Sodium citrate 1.50g

[0032] Citric acid 0.30g

[0033] 8.00g of glucose

[0034] The remaining component is distilled water.

[0035] A method for preparing the above-mentioned preservation solution for prolonging the in vitro preservation period of platelets includes the following steps:

[0036] (1) Sodium citrate, citric acid and glucose are mixed and 130g of distilled water is added and stirred evenly to obtain the first mixture;

[0037] (2) Mix baicalin and baicalein, add 820g of distilled water and stir until homogeneous to obtain a second mixture;

[0038] (3) After mixing the first mixture and the second mixture, add distilled water to make up to 1000 mL to obtain the preservation solution. Example 2

[0039] A preservation solution for prolonging the in vitro preservation period of platelets, containing [missing information] per liter of preservation solution.

[0040] Baicalin 6.00g

[0041] Baicalein 3.00g

[0042] Sodium citrate 2.00g

[0043] Citric acid 0.50g

[0044] 8.50g of glucose

[0045] The remaining component is distilled water.

[0046] A method for preparing the above-mentioned preservation solution for prolonging the in vitro preservation period of platelets includes the following steps:

[0047] (1) Sodium citrate, citric acid and glucose are mixed and 110g of distilled water is added and stirred evenly to obtain the first mixture;

[0048] (2) Mix baicalin and baicalein, add 750g of distilled water and stir until homogeneous to obtain a second mixture;

[0049] (3) After mixing the first mixture and the second mixture, add distilled water to make up to 1000 mL to obtain the preservation solution. Example 3

[0050] A preservation solution for prolonging the in vitro preservation period of platelets, containing [missing information] per liter of preservation solution.

[0051] Baicalin 4.80g

[0052] Baicalein 3.20g

[0053] Sodium citrate 1.80g

[0054] Citric acid 0.40g

[0055] 8.30g of glucose

[0056] The remaining component is distilled water.

[0057] A method for preparing the above-mentioned preservation solution for prolonging the in vitro preservation period of platelets includes the following steps:

[0058] (1) Sodium citrate, citric acid and glucose are mixed and 125g of distilled water is added and stirred to obtain the first mixture;

[0059] (2) Mix baicalin and baicalein, add 800g of distilled water and stir to obtain a second mixture;

[0060] (3) Mix the first mixture and the second mixture, and then add distilled water to make up to 1000 mL to obtain the preservation solution.

[0061] Comparative Example 1

[0062] The only difference from Example 1 is that 7.50g of baicalein was added, but baicalin was not added.

[0063] Comparative Example 2

[0064] The only difference from Example 1 is that 7.50g of baicalin was added, but baicalein was not added.

[0065] Comparative Example 3

[0066] The only difference from Example 1 is that 6.25g of baicalin and 1.25g of baicalein were added, that is, the weight ratio of baicalin to baicalein was 5:1.

[0067] The preservation solutions of Examples 1-3 and Comparative Examples 1-3 were used for in vitro platelet preservation, and the efficacy of the corresponding preservation solutions was recorded.

[0068] Sample preparation:

[0069] The apheresis platelets were divided into 7 bags using a sterile dispensing machine and labeled as experimental group 1, experimental group 2, experimental group 3, control group 1, control group 2, control group 3, and control group.

[0070] Under aseptic conditions in a clean bench, the corresponding preservation solutions obtained in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were injected into Experimental Group 1, Experimental Group 2, Experimental Group 3, and Comparative Example 3, respectively, using disposable syringes. The solutions were then shaken and preserved at 22±2℃.

[0071] The control group was stored with shaking at 22±2℃.

[0072] Sample collection:

[0073] Platelet bags from experimental group 1 were shaken at 1d, 3d, 5d, 8d, 11d, 14d, and 16d. The bags were first disinfected with 3% iodine tincture, and after the iodine tincture dried, the bags were disinfected twice with 75% alcohol. The operation was performed aseptically in a clean bench, and 20mL of platelets were drawn from the bags each time using a disposable syringe and injected into a test tube for later use in testing. After sampling, the bags and tubes were immediately heat-sealed and stored at 22±2℃ with shaking until the next sampling. Platelet samples from experimental group 2, experimental group 3, control group 1, control group 2, control group 3, and control group were collected using the same method.

[0074] In vitro experiments:

[0075] (1) TEG (thromboela-stogram) detection

[0076] TEG detection was performed using a thromboelastography (TEG) instrument (CFMS LEPU-8800) and its matching reagent kit (Beijing Lepu Diagnostics Technology Co., Ltd.).

[0077] (2) CD62p detection

[0078] Collect 5 μL of platelet sample per unit and dilute with sheath fluid (Beckman Coulter, USA) to adjust the platelet count to 0.5~1×10⁻⁶. 6 / L, with the matching negative reagent (BD Biosciences, USA) as a control, take 100μL of diluted sample into a special test tube, add 10μL of CD62p fluorescently labeled antibody, react in the dark for 20min, adjust the voltage to 50V, and detect CD62p expression by flow cytometry (DxFLEX).

[0079] (3) JC-1 detection

[0080] 1) Preparation of 1× buffer: Distilled water and 10× buffer of the mitochondrial membrane potential detection kit (JC-1) were prepared at a ratio of 9:1;

[0081] 2) Set up a positive control: Mix the CCCP reagent (Immuno Chemistry Technologies, USA) provided with the kit with distilled water at a ratio of 1:5000, dilute to 10 μmol / L, add 5 μL of platelets, incubate at 37℃ for 20 min, and then perform instrumental detection (normal cells show orange fluorescence after JC-1 staining, while after the above treatment, JC-1 is completely lost, and the cells show green fluorescence after staining).

[0082] 3) Sample testing: Collect approximately 10g of platelets. 6For each sample, add 5 μL of JC-1 reagent and 1 mL of 1× buffer, invert and mix 3 times, incubate at 37°C for 20 min, centrifuge at 600 g for 3-4 min, discard the supernatant, wash twice with 2 mL of 1× buffer, resuspend in 200 μL of 1× buffer, and detect JC-1 using a flow cytometer (DxFLEX).

[0083] The results of the in vitro experiments are shown below. Table 1 shows the TEG index results of platelets preserved in the experimental group, control group, and control group. Table 2 shows the CD62p results of platelets preserved in the experimental group, control group, and control group. Table 3 shows the JC-1 results of platelets preserved in the experimental group, control group, and control group.

[0084] Table 1. TEG parameters of preserved platelets in the experimental group, control group and control group.

[0085]

[0086] Note: All data in the table were analyzed using SPSS 21.0 statistical software and are expressed as "mean ± standard deviation".

[0087] As shown in Table 1, in the experimental group, the R value, K value, and α angle were within the normal range from day 1 to day 14, while they were slightly abnormal on day 16. In the control group, the R value and α angle exceeded the normal range from day 11 to day 16. In the control group, the R value, K value, and α angle all exceeded the normal range from day 8 to day 16. The MA value in the experimental group from day 1 to day 16 was generally within the normal range of 50-70, while in the control group, the MA value was <50 from day 11 to day 16, and in the control group, the MA value was <50 from day 8 to day 16. This indicates that the preservation solution containing baicalin and baicalein can significantly prolong the in vitro preservation period of platelets. Furthermore, the results from experimental group 1, control group 1, and control group 2 show that baicalin and baicalein have a synergistic effect in protecting and inhibiting platelet aggregation.

[0088] Table 2. CD62p expression rate of preserved platelets in experimental group, control group and control group

[0089]

[0090] Note: All data in the table were analyzed using SPSS 21.0 statistical software and are expressed as "mean ± standard deviation".

[0091] As shown in Table 2, the CD62p expression rate in the experimental group was >60% on days 11-14 and >80% on day 16; in the control group, the CD62p expression rate was >60% on day 8 and >80% on days 11-16; and in the control group, the CD62p expression rate was >80% on days 5-16. This indicates that the preservation solution containing baicalin and baicalein can significantly prolong the in vitro preservation period of platelets. Furthermore, the results from experimental group 1, control group 1, and control group 2 show that baicalin and baicalein have a synergistic effect in protecting and inhibiting platelet activation.

[0092] Table 3. JC-1 results of preserved platelets in the experimental group, control group, and control group.

[0093]

[0094] Note: All data in the table were analyzed using SPSS 21.0 statistical software and are expressed as "mean ± standard deviation".

[0095] Table 3 shows that the apoptosis rate of cells in the experimental group was <50% from day 1 to day 16; the apoptosis rate of cells in the control group was <50% from day 1 to day 14 and >50% from day 16; and the apoptosis rate of cells in the control group was >50% from day 11 to day 16. This indicates that the preservation solution containing baicalin and baicalein can significantly prolong the in vitro preservation period of platelets. Furthermore, the results from experimental group 1, control group 1, and control group 2 show that baicalin and baicalein have a synergistic effect in protecting and inhibiting platelet apoptosis.

[0096] In addition to the above embodiments, the present invention also includes other embodiments. All technical solutions formed by equivalent transformation or equivalent substitution should fall within the protection scope of the claims of the present invention.

Claims

1. A preservation solution for prolonging the in vitro preservation period of platelets, characterized in that: Each liter of preservation solution contains Baicalin 4.50~6.00g Baicalein 3.00~3.40g Sodium citrate 1.50~2.00g Citric acid 0.20~0.50g Glucose 7.90~8.50g The remaining component is distilled water; The weight ratio of baicalin to baicalein is 4~3:

2.

2. A method for preparing a preservation solution for prolonging the in vitro preservation period of platelets according to claim 1, characterized in that: Includes the following steps: (1) Sodium citrate, citric acid, and glucose are mixed, and distilled water is added and stirred to obtain the first mixture; (2) Mix baicalin and baicalein, add distilled water and stir to obtain a second mixture; (3) Mix the first mixture and the second mixture together, and add distilled water to make up to 1000 mL to obtain the preservation solution.

3. The method for preparing the preservation solution for prolonging the in vitro preservation period of platelets according to claim 2, characterized in that: The weight ratio of distilled water in step (1) to distilled water in step (2) is 1~2:5~7.

4. The application of the preservation solution according to claim 1 for prolonging the in vitro preservation period of platelets in platelets.

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