阅读说明：本技术 凝血酶的制备方法 (Process for preparing thrombin ) 是由 刘强 白卓娅 林锋 马海波 于 2021-10-09 设计创作，主要内容包括：本发明公开了属于生物医用工程技术领域的一种凝血酶的制备方法。该方法是使用PRP管抽取全血制成抗凝全血,在抗凝全血中加入钙盐使混合均匀,在混合物中加入埃洛石纳米管(HNTs)静置,静置让充分接触后混合物中形成凝块,通过离心使得凝块和埃洛石纳米管(HNTs)位于管底部,上部形成上清液,吸取上清液从而制得凝血酶溶液。本发明的制备工艺简单,制备的凝血酶活性高,用制备的凝血酶激活富血小板血浆(PRP)形成凝胶的用时短、凝胶体积大。(The invention discloses a preparation method of thrombin, belonging to the technical field of biomedical engineering. The method comprises the steps of extracting whole blood by using a PRP (platelet-rich plasma) tube to prepare anticoagulated whole blood, adding calcium salt into the anticoagulated whole blood to uniformly mix, adding Halloysite Nanotubes (HNTs) into a mixture to stand, standing to enable the mixture to be in full contact to form a clot, enabling the clot and the Halloysite Nanotubes (HNTs) to be located at the bottom of the tube through centrifugation, forming supernatant on the upper portion of the tube, and absorbing the supernatant to prepare thrombin solution. The preparation process is simple, the prepared thrombin has high activity, and the time for activating Platelet Rich Plasma (PRP) to form gel by the prepared thrombin is short and the gel volume is large.)

1. A method for preparing thrombin, comprising the steps of:

step a: selecting a vacuum PRP tube, wherein the PRP tube contains separation gel and anticoagulant;

step b: whole blood was drawn using PRP tubing;

step c: mixing the whole blood in the PRP tube with an anticoagulant to produce anticoagulated whole blood;

step d: adding calcium salt into the anticoagulated whole blood and uniformly mixing;

step e: d, adding halloysite nanotubes into the mixture obtained in the step d, mixing, standing, and forming a clot in the mixture after the mixture is sufficiently contacted with the halloysite nanotubes;

step f: and e, centrifuging the mixture obtained in the step e to enable the clot and the halloysite nanotubes to be positioned at the bottom of the tube, forming a supernatant on the upper part of the tube, and sucking the supernatant to prepare the thrombin solution.

2. The method of claim 1, comprising the steps of:

step c 1: and c, centrifuging the anticoagulated whole blood prepared in the step c, wherein the centrifuged PRP tube sequentially comprises the following components from bottom to top: red blood cells, separation gel, a leucocyte layer and blood plasma;

step c 2: collecting the upper plasma obtained in step c1 to obtain platelet poor plasma;

step c 3: after step c2, inverting the PRP tube to suspend the buffy coat in the plasma to produce platelet rich plasma, and collecting the platelet rich plasma;

step d: c, adding calcium salt into the platelet poor plasma prepared in the step c2 and/or the platelet rich plasma collected in the step c3, and uniformly mixing;

step e: d, adding halloysite nanotubes into the mixture obtained in the step d, mixing, standing, and forming a clot in the mixture after the mixture is sufficiently contacted with the halloysite nanotubes;

step f: and e, centrifuging the mixture obtained in the step e to enable the clot and the halloysite nanotubes to be positioned at the bottom of the tube, forming a supernatant on the upper part of the tube, and sucking the supernatant to prepare the thrombin solution.

3. The method of claim 2, comprising the steps of:

step d: c2, adding calcium salt into the platelet poor plasma prepared in the step c, and uniformly mixing;

step e: d, adding halloysite nanotubes into the mixture obtained in the step d, mixing, standing, and forming a clot in the mixture after the mixture is sufficiently contacted with the halloysite nanotubes;

step f: and e, centrifuging the mixture prepared in the step e to enable the clot and the halloysite nanotubes to be positioned at the bottom of the tube, forming a supernatant on the upper part of the tube, and sucking the supernatant to prepare the thrombin solution.

4. The method of preparing thrombin according to any one of claims 1 to 3, comprising the steps of:

step g: and f, adding platelet-rich plasma into the thrombin solution prepared in the step f, and mixing the platelet-rich plasma and the thrombin solution according to a volume ratio of 1-3: 10 are mixed to form a gel.

5. The method of preparing thrombin according to claim 2 or 3, comprising the steps of: step g: adding the platelet-rich plasma obtained in the step c3 into the thrombin solution obtained in the step f, and mixing the platelet-rich plasma and the thrombin solution according to a volume ratio of 1-3: 10 are mixed to form a gel.

6. The method for producing thrombin according to claim 1,

the anticoagulant is a sodium citrate solution, and the concentration of the sodium citrate is 0.1-0.12 mol/L.

7. The method for producing thrombin according to any one of claims 1 to 3,

the calcium salt is one or a mixture of more than two of calcium chloride, calcium carbonate, calcium sulfate and calcium gluconate.

8. The method for producing thrombin according to claim 7,

the calcium salt is a mixture of calcium chloride with the concentration of 10% and/or calcium gluconate with the concentration of 10%.

9. The method for producing thrombin according to any one of claims 1 to 3,

the volume ratio of the calcium salt to the anticoagulated whole blood, the platelet poor plasma and/or the platelet rich plasma is: 1: 3 to 7.

10. The method for producing thrombin according to any one of claims 1 to 3,

in each milliliter of the anticoagulated whole blood, the platelet poor plasma and/or the platelet rich plasma, the halloysite nanotube has the following mass: 0.25mg to 2 mg.

11. The method for producing thrombin according to any one of claims 1 to 3,

and purifying the halloysite nanotube.

12. The method for producing thrombin according to claim 11,

the purification treatment of the halloysite nanotube comprises the following steps:

the method comprises the following steps: preparing a halloysite nanotube into an aqueous solution, wherein the concentration of the halloysite nanotube is 5% -20%;

step two: adding sodium hexametaphosphate into the aqueous solution of the halloysite nanotube prepared in the step I, wherein the concentration of the sodium hexametaphosphate is as follows: 0.02% -0.10%, stirring for more than 2 hours, standing for 20-60 minutes, and layering the aqueous solution;

step three: after the aqueous solution prepared in the step two is stood for layering, filtering and removing impurities and sediments at the bottom, and collecting a suspension solution at the upper part;

step IV: and (4) carrying out centrifugal filtration on the upper suspension solution collected in the step (iii), and carrying out freeze drying on the halloysite nanotube obtained after centrifugal filtration.

13. The method for producing thrombin according to claim 12,

freeze-drying the halloysite nanotube in the step (IV), wherein the freeze-drying temperature is as follows: -80 ℃ to-20 ℃ for: 12 to 24 hours.

14. The method for producing thrombin according to claim 12,

the concentration of the halloysite nanotube is 8% -15%, and the concentration of the sodium hexametaphosphate is 0.04% -0.08%.

15. The method for producing thrombin according to any one of claims 1 to 3,

the whole blood is autologous blood.

Technical Field

The invention belongs to the technical field of biomedical engineering, and particularly relates to a preparation method of thrombin.

Background

Thrombin is a multifunctional serine protease that is capable of activating a variety of coagulation factors and can activate platelets. Thrombin can be generated by cleavage of two sites on prothrombin by activated factor X (Xa). The activity of factor Xa can be enhanced by binding to activated factor v (va), which is called the prothrombin complex. Once thrombin is formed, it proteolytically digests fibrinogen into fibrin monomers, initiating the coagulation cascade, which initiates the formation of a clot, the first step in the wound repair process. Thrombin also functions as a chemo-attractant for cells involved in wound healing (chemo-attractant), having a chemo-attractant effect on cells involved in the wound repair process, and the resulting fibrin network has a number of functions, including: fibroblasts produced from collagen act as scaffolds, increase phagocytosis, promote angiogenesis, and bind growth factors. Platelets, when activated, convert from a non-binding state to a binding state to the fibrin network. Thus, thrombin plays a very important role in the hemostasis process as a procoagulant.

Thrombin can be prepared from a number of sources including: allogenic, and autologous. Among them, the use of thrombin derived from calf plasma (total bovine serum) for human therapy poses the risk of infection with pathogenic protein particles (prion), and even causes severe immune reactions and fatal bleeding. If thrombin made from allogeneic (volunteer) plasma is used, there may be immunological rejection reactions, viral infections and other side effects.

Halloysite nanotubes (chemical formula: Al)₂Si₂O₅(OH)₄·nH₂O, abbreviated as "HNTs") is a widely-existing natural clay nanotube, which has a hollow tubular structure with an inner diameter of about (10-40) nm, an outer diameter of about (40-70) nm, and a length of about (500-1500) nm. The HNTs has super-hydrophilicity and a unique tubular nano structure, so that water in blood components can enter a cavity and an interlayer of the HNTs after the HNTs is contacted with the blood components, and the HNTs adsorbs blood coagulation proteins in plasma on the surface of the HNTs through electrostatic acting force, thereby being beneficial to adsorption between platelets and the HNTs. At present, research of HNTs in the biomedical field draws attention of scientists, the HNTs have good biocompatibility, have the characteristic of hemostasis, and can adsorb lysozyme with positive charges through physical adsorption and electrostatic interaction due to negative charges carried on the surfaces of the HNTs. Based on good biocompatibility and biological safety, the hemostaticIn the field, how to develop thrombin with stronger hemostatic function by using HNTs becomes a research hotspot.

Disclosure of Invention

Aiming at the problems of complex process, high impurity, poor hemostatic effect and the like of thrombin preparation in the prior art, the invention aims to provide a preparation method which has simple process, high activity of prepared thrombin and quick gel.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the invention relates to a preparation method of thrombin, which comprises the following steps:

step a: selecting a vacuum PRP tube, wherein the PRP tube contains separation gel and anticoagulant;

step b: whole blood was drawn using PRP tubing;

step c: mixing the whole blood in the PRP tube with an anticoagulant to prepare anticoagulated whole blood;

step d: adding calcium salt into anticoagulated whole blood, and mixing;

step e: d, adding halloysite nanotubes into the mixture obtained in the step d, mixing, standing, and forming a clot in the mixture after the mixture is sufficiently contacted with the halloysite nanotubes;

step f: and e, centrifuging the mixture obtained in the step e to enable the clot and the halloysite nanotubes to be positioned at the bottom of the tube, forming a supernatant on the upper part of the tube, and sucking the supernatant to prepare the thrombin solution.

Preferably, step c 1: and c, centrifuging the anticoagulated whole blood prepared in the step c, wherein the components in the PRP tube after centrifugation are as follows in sequence from bottom to top: red blood cells, separation gel, a leucocyte layer and blood plasma;

step c 2: collecting Platelet Poor Plasma (PPP) from the upper plasma produced in step c 1;

step c 3: after step c2, inverting the PRP tube to suspend the buffy coat in plasma to produce Platelet Rich Plasma (PRP), and collecting platelet rich plasma;

step d: c, adding calcium salt into the platelet poor plasma prepared in the step c2 and/or the platelet rich plasma collected in the step c3, and uniformly mixing;

step e: d, adding halloysite nanotubes into the mixture obtained in the step d, mixing, standing, and forming a clot in the mixture after the mixture is sufficiently contacted with the halloysite nanotubes;

step f: and e, centrifuging the mixture obtained in the step e to enable the clot and the halloysite nanotubes to be positioned at the bottom of the tube, forming a supernatant on the upper part of the tube, and sucking the supernatant to prepare the thrombin solution.

Preferably, step d: c2, adding calcium salt into the platelet poor plasma prepared in the step c, and uniformly mixing;

step e: d, adding halloysite nanotubes into the mixture obtained in the step d, mixing, standing, and forming a clot in the mixture after the mixture is sufficiently contacted with the halloysite nanotubes;

step f: and e, centrifuging the mixture prepared in the step e to enable the clot and the halloysite nanotubes to be positioned at the bottom of the tube, forming a supernatant on the upper part of the tube, and sucking the supernatant to prepare the thrombin solution.

Preferably, step g: and f, adding platelet-rich plasma into the thrombin solution prepared in the step f, and mixing the platelet-rich plasma and the thrombin solution according to the volume ratio of 1-3: 10 are mixed to form a gel.

Preferably, step g: adding the platelet-rich plasma obtained in the step c3 into the thrombin solution obtained in the step f, and mixing the platelet-rich plasma and the thrombin solution according to the volume ratio of 1-3: 10 are mixed to form a gel.

Preferably, the anticoagulant is a sodium citrate solution, and the concentration of the sodium citrate is 0.1-0.12 mol/L.

Preferably, the calcium salt is one or a mixture of two or more of calcium chloride, calcium carbonate, calcium sulfate and calcium gluconate.

Preferably, the calcium salt is a mixture of calcium chloride at a concentration of 10% and/or calcium gluconate at a concentration of 10%.

Preferably, the volume ratio of the calcium salt to the anticoagulated whole blood, platelet poor plasma and/or platelet rich plasma is: 1: 3 to 7.

Preferably, the halloysite nanotubes have a mass per ml of anticoagulated whole blood, platelet poor plasma and/or platelet rich plasma of: 0.25mg to 2 mg.

Preferably, the halloysite nanotubes are purified.

Preferably, the purification treatment of the halloysite nanotubes comprises the following steps:

the method comprises the following steps: preparing the halloysite nanotube into an aqueous solution, wherein the concentration of the halloysite nanotube is 5-20%;

step two: adding sodium hexametaphosphate into the aqueous solution of the halloysite nanotube prepared in the step I, wherein the concentration of the sodium hexametaphosphate is as follows: 0.02% -0.10%, stirring for more than 2 hours, standing for 20-60 minutes, and layering the aqueous solution;

step three: after the aqueous solution prepared in the step two is stood for layering, filtering and removing impurities and sediments at the bottom, and collecting a suspension solution at the upper part;

step IV: and (4) carrying out centrifugal filtration on the upper suspension solution collected in the step (iii), and freeze-drying the halloysite nanotube obtained after centrifugal filtration.

Preferably, the halloysite nanotubes are freeze-dried in the step (iv), wherein the freeze-drying temperature is as follows: -80 ℃ to-20 ℃ for: 12 to 24 hours.

Preferably, the concentration of the halloysite nanotube is 8% -15%, and the concentration of the sodium hexametaphosphate is 0.04% -0.08%.

Preferably, the whole blood is autologous blood.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the preparation process is simple, the prepared thrombin has high activity, and the time for activating PRP to form gel by using the prepared thrombin is short and the gel volume is large.

Drawings

FIG. 1 is a flow diagram of the preparation of thrombin from anticoagulated whole blood;

FIG. 2 is a flow chart of the process for preparing thrombin from platelet poor plasma and platelet rich plasma after centrifugation of anticoagulated whole blood;

figure 3 is a schematic representation of a gel.

Detailed Description

The present invention will be further described in detail with reference to specific examples, but the embodiments of the present invention are not limited thereto.

The process for preparing thrombin according to the present invention is specifically described in examples 1 and 2.

Example 1

The thrombin of the present invention is prepared as shown in FIG. 1. FIG. 1 is a flow chart of the preparation of thrombin from anticoagulated whole blood, which comprises the following steps:

step a: vacuum PRP tubes were selected. The PRP tube comprises a tube body and a rubber plug for sealing the opening of the tube body, wherein the tube body is internally provided with separation rubber and anticoagulant, and the PRP tube is preset to be vacuum.

Wherein the anticoagulant is citrate, and sodium citrate solution can be selected, and the concentration of the sodium citrate is as follows: (0.1-0.12) mol/L.

Step b: the amount of whole blood drawn using the PRP tube was (8-10) ml.

Step c: the hand-held PRP tube is gently inverted repeatedly to mix the whole blood with the anticoagulant. When the sodium citrate solution can be used as the anticoagulant, the prepared anticoagulated whole blood is citric acid anticoagulated whole blood.

Step d: calcium salt is added to anticoagulated whole blood.

Calcium salts are added to anticoagulated whole blood as crystalline salts in solid form or as salt solutions in solution, either as powders with HNTs pre-placed in PRP tubes or as a side solution before use. At room temperature, calcium salt is mixed with anticoagulated whole blood in a volume ratio of: 1: (3-7) in the above ratio. Optionally, 10% calcium chloride or 10% calcium gluconate is added to the anticoagulated whole blood, and after the PRP tube is slowly inclined, the mixture is gently shaken in the left-right direction or the up-down direction to be fully mixed, so that calcium ions are combined with blood components.

The calcium salt can be selected from calcium chloride, calcium carbonate, calcium sulfate, calcium gluconate and their mixture, and can be prepared by mixing one or more of calcium chloride 10% and calcium gluconate 10%. Because of the presence of an anticoagulant such as citrate in the anticoagulated whole blood, if more calcium is added than the amount of citrate-complexed calcium remaining in the anticoagulated whole blood, the remaining uncomplexed calcium ions can participate in the coagulation cascade and contribute to the generation of thrombin.

To coagulate blood components, Halloysite Nanotubes (HNTs) are added to the mixture of step d by step e, mixed well and left to stand. Adding halloysite nanotubes into per milliliter of anticoagulated whole blood, wherein the mass of the halloysite nanotubes is as follows: 0.25mg-2 mg. After allowing to rest for 30 minutes for sufficient contact, a clot formed in the mixture.

Mixing can be accomplished by inversion, shaking, stirring, or vortexing, and can be repeated once, multiple times, and periodically. After or during mixing, the anticoagulated whole blood is in intimate contact with calcium salts, Halloysite Nanotubes (HNTs), and after standing for a period of time, clots may form. For example, the time of contact standing can be 10 minutes to 20 minutes, and the temperature of contact standing can be 20 ℃ to 45 ℃, and can be room temperature or higher, and the temperature is more favorable for accelerating the formation of the clot.

Preparing a thrombin solution by step f. And e, centrifuging the mixture obtained in the step e to enable the clot and Halloysite Nanotubes (HNTs) to be positioned at the bottom of the test tube, forming a supernatant on the upper part of the test tube, and sucking the supernatant to prepare the thrombin solution.

To separate the clot to give a supernatant, the clot, Halloysite Nanotubes (HNTs), clot and any other residual cellular components can be pelleted to the bottom of the tube by centrifugation and the supernatant formed on the top, and the thrombin-containing supernatant can be poured off or aspirated by syringe. For example, the supernatant containing thrombin can be aspirated by a syringe or pipette after centrifugation at 1500g for 5 minutes. In order to maintain the activity for preparing thrombin, the thrombin solution may be stored at a low temperature, e.g., at (2-8) deg.C.

Example 2

The thrombin of the present invention is prepared as shown in FIG. 2. FIG. 2 shows that after centrifugation of anticoagulated whole blood obtained in step c of example 1 to obtain Platelet Poor Plasma (PPP) and Platelet Rich Plasma (PRP), thrombin is prepared from PPP and PRP, and thrombin can also be prepared from either PPP or PRP. The method specifically comprises the following steps:

the anticoagulated whole blood in the PRP tube in step c may further be centrifuged according to practical requirements. The method comprises the following steps:

step c 1: and c, centrifuging the anticoagulated whole blood prepared in the step c, wherein the components in the PRP tube after centrifugation from bottom to top are as follows: red blood cells, separation gel, a leucocyte layer and blood plasma;

step c 2: platelet Poor Plasma (PPP) prepared by collecting the upper plasma prepared in step c 1;

step c 3: after step c2 is completed, inverting the PRP tube to suspend the buffy coat in plasma to produce Platelet Rich Plasma (PRP), and collecting Platelet rich plasma;

step d: adding calcium salt into PRP and PPP.

The calcium salt may be mixed with PRP and/or PPP at a concentration of 10%, in volume ratios: 1: (3-7) mixing; optionally, 10% calcium chloride and/or 10% calcium gluconate can be added into PPP or PRP, and the calcium salt is mixed to account for the total volume: 1/8-1/4. After mixing at room temperature, the PRP tube was gently tilted, and then gently shaken in the left-right or up-down direction to mix them sufficiently, thereby binding calcium ions to blood components.

To further coagulate the blood components, Halloysite Nanotubes (HNTs) were added to the calcium salt mixture of step d by step e, mixed and left to stand. Upon sufficient contact, a clot forms in the mixture. Adding HNTs into PPP and PRP per ml, wherein the mass of the HNTs is as follows: 0.25mg-2 mg. After allowing to rest for 30 minutes for sufficient contact, a clot formed in the mixture.

A thrombin solution can likewise be prepared by step f. And e, centrifuging the mixture obtained in the step e to enable the clot and HNTs to be positioned at the bottom of the test tube, forming a supernatant at the upper part, and sucking the supernatant containing the thrombin to prepare the thrombin solution.

As Halloysite Nanotubes (HNTs) are a widely-existing natural clay nanotube with a hollow tubular structure, the outermost chemical property and silicon dioxide (SiO)₂) Similarly, the chemical properties of the inner tubular surface are similar to those of aluminum oxide (Al)₂O₃) Similarly. HNTs activate secondary factors and platelets in plasma, as well as some other components, to produce thrombin, but these components in blood are certain,if too much HNTs are added, it is wasted. Conversely, if too little HNTs are added, even sufficient supernatant will cause insufficient activation of these components in the blood.

Although HNTs have good biocompatibility and low cytotoxicity, HNTs can be purified before use in order to further improve the activity and purity of the prepared thrombin.

The Halloysite Nanotube (HNTs) purification treatment method comprises the following steps:

the method comprises the following steps: preparing the halloysite nanotubes into an aqueous solution, wherein the concentration of the halloysite nanotubes is 5-20%, and the preferred concentration of the halloysite nanotubes is 8-15%;

step two: adding sodium hexametaphosphate and other phosphate into the aqueous solution of the halloysite nanotube prepared in the step I. Wherein, the concentration of the sodium hexametaphosphate is as follows: 0.02% -0.10%, preferably the concentration of sodium hexametaphosphate is 0.04% -0.08%. Stirring for more than 2 hours, standing for 20-60 minutes at room temperature, and layering the aqueous solution;

step three: after the aqueous solution prepared in the step two is stood for layering, removing impurities and precipitates at the bottom in a filtering mode, and collecting a suspension solution at the upper part;

step IV: and (4) centrifugally filtering the upper suspension solution collected in the step (iii), and freeze-drying halloysite nano particles obtained after centrifugal filtering to form particles so as to prevent the halloysite nano tubes from being coagulated into blocks.

Freeze-drying the halloysite nanotube in the step (IV), wherein the freeze-drying temperature is as follows: -80 ℃ to-20 ℃ for: 12 hours to 24 hours.

Since thrombin can activate platelets to release growth factors, cytokines, chemokines, etc., thrombin can convert fibrinogen in PRP to fibrin, thereby forming a gel.

In order to further prepare the thrombin prepared in the above examples 1 and 2 into gel, the method specifically comprises the following steps:

the thrombin solution prepared in step f can be added with PRP through step g, so that the volume ratio of the thrombin solution to the PRP is as follows: (1-3): 10 are mixed to form a gel. Here, the PRP may be the PRP collected in the step c3 after the anticoagulated whole blood in the PRP tube is centrifuged in example 2, or may be the PRP collected by centrifuging the anticoagulated whole blood in the PRP tube separately.

Thrombin binds to platelets to form a gel, the shape of which is shown in figure 3. Wherein PRP is added to thrombin prepared by using calcium salt and HNTs as reagents to form a gel, which is shown in (a) of FIG. 3; PRP was added to thrombin prepared using calcium salt as a reagent to form a gel, which is shown in FIG. 3 (b). The thrombin prepared by combining calcium salt and Halloysite Nanotubes (HNTs) has high activity. Under otherwise identical conditions, a gel was formed after PRP activation with thrombin, and the gel was significantly larger in volume and shorter in activation time in FIG. (a).

The following experimental procedures were used to prepare thrombin and further prepare gels, and the experimental conditions used to test the gels were used.

Experiment 1

Experiment temperature: concentration of platelets in whole blood of volunteers at 24 ℃: 233X 10⁹L, centrifuging to obtain PPP and PRP with platelet concentration of 712 × 10⁹/L。

Scheme 1: PPP and 10% calcium gluconate solution are mixed according to the volume ratio of 3: 1, mixing uniformly.

Scheme 2: adding 2ml of PPP and 3mg of purified HNTs into a test tube, uniformly mixing, and then adding a mixture of (PPP + HNTs) and 10% calcium gluconate solution in a volume ratio of 3: 1, mixing uniformly.

After mixing uniformly, the mixture was left to stand at 37 ℃ and thoroughly contacted for 15 minutes, and then centrifuged at 1500 g.times.5 min. After centrifugation, the supernatant was drawn off by syringe and the volume ratio of PRP to Autologous Thrombin (ATS) was 10: 1 and 10: 3 Lima activates PRP, records the time for PRP to gel, leaves the supernatant for a period of time, repeats the activation experiment, and records the activation time. The experimental results are as follows:

1. PRP was activated immediately after thrombin preparation and the time to gel PRP was as follows. Unit (second)

2. PRP was activated 30min after thrombin preparation, and was gelled as follows. Unit (second)

3. 60min after thrombin preparation, PRP was activated and the time for PRP to gel was as follows. Unit (second)

4 PRP was activated 120min after thrombin preparation and the time for PRP to gel was as follows. Unit (second)

5. PRP was activated 180min after thrombin preparation, and the time for PRP to gel was as follows. Unit (second)

6. At 240min after thrombin preparation, PRP was activated and allowed to gel for the following time. Unit (second)

From the above experimental results, it is known that the thrombin activity decreases with the increase of the standing time, resulting in a longer time for activating PRP to gel. Scheme 1 and scheme 2 activate PRP 180min after thrombin preparation, making the time difference between PRP gelation large, and scheme 2 forms a large volume of gel after PRP activation.

Experiment 2

Experiment temperature: platelet concentration in whole blood of volunteers at 24 ℃: 180 x 10⁹L, centrifuging to obtain PPP and PRP with platelet concentration of 305 × 10⁹/L。

Scheme 1: PPP and 10% calcium chloride solution were mixed at volume 5: 1, mixing uniformly.

Scheme 2: after 2ml of PPP and 1mg of purified HNTs were added to the tube and mixed well, the mixture was mixed by volume ratio of (PPP + HNTs) to 10% calcium chloride of 5: 1, mixing uniformly.

After mixing uniformly, the mixture was left to stand at 37 ℃. After sufficient contact for 10 minutes, it was then centrifuged at 1500 g.times.5 min. Extracting the supernatant by using a syringe, and mixing the supernatant and the thrombin according to the volume ratio of PRP to thrombin of 10: 1 and 10: 3 Lima activates PRP, records the time for PRP to gel, leaves the supernatant for a period of time, repeats the activation experiment, and records the activation time. The experimental results are as follows:

1. PRP was activated immediately after thrombin preparation and the time to gel PRP was as follows. Unit (second)

2. PRP was activated 30min after thrombin preparation and the time to gel PRP was as follows. Unit (second)

3. PRP was activated 60min after thrombin preparation and the time to gel PRP was as follows. Unit (second)

4. PRP was activated 90min after thrombin preparation and the time to gel PRP was as follows. Unit (second)

5. PRP was activated 120min after thrombin preparation and the time to gel PRP was as follows. Unit (second)

Experiment 2

Experiment temperature: platelet concentration in whole blood of volunteers at 25 ℃: 220 x 10⁹L, centrifuging to obtain PPP and PRP, wherein the platelet concentration in PRP is 370 × 10⁹/L。

Scheme 1: anticoagulated whole blood and a 10% calcium gluconate solution were mixed in a volume of 4: 1, mixing uniformly.

Scheme 2: after 2ml of PPP and 2mg of purified HNTs were added to the tube and mixed well, the volume ratio of (anticoagulated whole blood + HNTs) to 10% calcium gluconate was 4: 1, mixing uniformly.

After being mixed evenly, the mixture is kept stand at normal temperature. After sufficient contact for 10 minutes, it was then centrifuged at 1500 g.times.5 min. The supernatant was extracted with a syringe and the volume ratio of PRP to Autologous Thrombin (ATS) was 10: 1 and 10: 3 Lima activates PRP, records the time for PRP to gel, leaves the supernatant for a period of time, repeats the activation experiment, and records the activation time. The experimental results are as follows:

1. PRP was activated immediately after thrombin preparation and the time to gel PRP was as follows. Unit (second)

Immediately after thrombin preparation, a 10: 3 (PRP: ATS), the gel formed in scheme 1 is voluminous and only a small portion of the supernatant; the gel formed in scheme 2 was larger in volume with little supernatant.

2. PRP was activated 30min after thrombin preparation and the time to gel PRP was as follows. Unit (second)

After 30min of thrombin preparation, the volume ratio was 10: 3 (PRP: ATS), scheme 1 forms a small gel volume and a large amount of supernatant; the gel formed in scheme 2 was large in volume and had a small supernatant.

3. PRP was activated 90min after thrombin preparation and the time to gel PRP was as follows. Unit (second)

4. PRP was activated 150min after thrombin preparation and the time to gel PRP was as follows. Unit (second)

To sum up: compared with the method that only calcium salt is used as an Autologous Thrombin (ATS) reagent, the method uses the calcium salt and Halloysite Nanotubes (HNTs) as thrombin preparation reagents, the activity of the prepared thrombin is high, the time for activating PRP under the same experimental conditions is short, and the formed clot is large in volume.

The invention uses calcium salt and Halloysite Nanotubes (HNTs) as reagents, and the coagulation cascade reaction can be started without being complexed by citrate and redundant calcium to generate thrombin. The tubular structure gives HNTs a negatively charged Si-O-Si outer surface and a positively charged Al-OH inner surface. The unique tubular structure and the extremely large length-diameter ratio of HNTs enable the HNTs to have large specific surface area and pore volume, so that the HNTs have more negative charges on the outer surface and can better activate the second factor and other substances in plasma to be converted into thrombin. In addition, the HNTs is in an anhydrous state when in use, and has super-hydrophilic performance and a unique tubular nano structure, so that water in blood components can enter a cavity and an interlayer of the HNTs after the HNTs is contacted with the blood components, and the HNTs has a large pore volume and a large length-diameter ratio to generate capillary action, thereby having a concentration effect on autologous blood components. The HNTs adsorbs the blood coagulation protein in the plasma on the surface thereof through electrostatic acting force, thereby being beneficial to the adsorption between platelets and the HNTs, and the unique tubular nano structure of the HNTs enables the HNTs to be more easily contacted with the platelets, thereby better activating the platelets. HNTs can generate thrombin by activating second factors and platelets and other components in plasma, and can concentrate blood components to prepare high-activity enzyme. Fibrin gel formed by thrombin solution and blood products can be used in surgical operations, such as incision, transplantation and repair parts in orthopedic operations, and the gel can close wounds, thereby helping repair of injured parts of tissues and helping the body to heal quickly.

The whole blood used in the present invention may be obtained from volunteers or from patients themselves. For example, whole blood may be collected from a patient and centrifuged, and then PPP or PRP after centrifugation may be used to prepare patient autologous thrombin. In order to improve the use effect, thrombin can be mixed with PRP, so that the thrombin further activates platelets in the PRP to release growth factors, cytokines, chemotactic factors and the like, and meanwhile, the autologous thrombin can convert fibrinogen in the PRP into fibrin, thereby forming fibrin gel. The prepared gel can be applied to wounds to serve as a tissue sealant and promote the repair of the wounds. The thrombin and PRP can be mixed prior to use, or can be mixed at the wound site. The prepared self-body thrombin article has no rejection problem of foreign body thrombin and no possibility of being infected by virus.

Compared with the preparation of thrombin by using calcium salt as a reagent, the thrombin prepared by combining the calcium salt with Halloysite Nanotubes (HNTs) has high activity. In addition, under the same experimental conditions, the gel formation by the activation of PRP with thrombin according to the invention is bulky and the activation time is short.

The foregoing is only a partial embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.