CN114081899A

CN114081899A - Preparation method of leukocyte-rich and platelet-rich plasma

Info

Publication number: CN114081899A
Application number: CN202111287905.8A
Authority: CN
Inventors: 袁馨
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2021-11-02
Filing date: 2021-11-02
Publication date: 2022-02-25

Abstract

The invention relates to the technical field of blood products, and discloses a preparation method of leukocyte-rich and platelet-rich plasma (L-PRP), which comprises the steps of collecting blood and preparing the L-PRP; also discloses the application of the L-PRP in wound treatment. The platelet content of the L-PRP prepared by the invention is about 4 times of that of whole blood, and the L-PRP is rich in white blood cells and interleukin 6(IL-6), and the L-PRP is found to promote keratinocyte migration to accelerate wound re-epithelialization by up-regulating IL-6 transcription level and activating signal transduction and transcriptional activation protein 3(STAT 3); the L-PRP prepared by the invention and the research on the L-PRP refine the classification and application scenes of platelet-rich plasma (PRP), discover a new mechanism for promoting wound healing by the L-PRP, provide a new solution for clinically solving the difficult wound, and have important clinical practice significance.

Description

Preparation method of leukocyte-rich and platelet-rich plasma

Technical Field

The invention relates to the technical field of blood products, in particular to a preparation method of leukocyte-rich and platelet-rich plasma.

Background

Refractory wounds refer to the phenomena of delayed or interrupted physiological healing due to insufficient angiogenesis, nerve loss, cell migration disorder, etc., and are generally defined as wounds with healing time longer than 3 months. Many diseases can affect wound healing, and with the more serious problems of aging, smoking, obesity, diabetes and the like all over the world, the incidence of the wound which is difficult to heal is gradually increased. This not only causes pain, infection, and even disability, amputation, and psychological problems for the patient, but also places a huge economic burden on society. Therefore, how to promote the wound healing becomes an important proposition to be solved urgently by clinicians.

Restoration of wound epithelialization to an intact skin barrier is an essential feature of wound healing completion, also known as re-epithelialization. The directed migration of keratinocytes is critical for wound epithelialization, and the deficiency in this function is closely associated with chronic refractory wounds. The re-epithelialization of the wound is achieved as a result of keratinocyte migration, proliferation and differentiation, which begins with the release of cell-cell and cell-matrix contact; next, the basal layer keratinocytes start to polarize and initiate migration on the wound matrix, while the keratinocytes start to proliferate by division next to the wound margins; finally, the newly formed epidermis is differentiated into a multilayered structure to restore the function of the epidermis. The most limiting step in the re-epithelialization process is the migration of keratinocytes. Parakeratosis and hyperkeratosis are characteristics of keratinocytes of a chronic wound, and the functional defect of migration of the keratinocytes is most related to chronic hard-to-heal wounds, so that the regulation of the migration of the keratinocytes in the process of wound healing and re-epithelialization is a key factor for promoting the healing of the chronic wound.

Research shows that the IL-6/STAT3 pathway is very critical in promoting wound re-epithelialization and skin barrier recovery, the IL-6-deficient mouse wound re-epithelialization is remarkably delayed, and IL-6 gene is introduced into keratinocytes to promote the proliferation of the keratinocytes. IL-6 is a typical upstream ligand of STAT3 signal transduction pathway, and STAT3 phosphorylation and nuclear translocation occur within 30 minutes after stimulation after keratinocytes are treated with recombinant IL-6 in vitro, and the intracellular phosphorylation signaling and activator of transcription 3(pSTAT3) content increases with time (30min to 6 h). IL-6 activates STAT3 in skin fibroblasts, causing it to secrete soluble factors, which in turn mediate keratinocyte migration. IL-6 mediated pSTAT3 signaling drives transcription of the keratinized epithelium Skint (selection and up-keep of intraepithelial T-cells) gene of acute wounds of young mice, activates Dendritic Epidermal T Cells (DETCs) to promote wound healing, and weakens the signal path in the wounds of delayed healing of old mice. Skint expression is directly affected by STAT3 signaling, knocking out the epidermal STAT3 gene affects the number and activation of periwound DETCs, which in turn affects delayed wound healing, STAT3 is its key role in keratinocyte migration signaling, rather than its proliferation, and STAT3 is essential for skin remodeling, including hair cycle and wound healing. In addition, IL-6 can obviously promote the migration of corneal epithelial cells in vitro, and after 6 hours of treatment, the content of pSTAT3 can be detected to be remarkably increased. Therefore, the IL-6/STAT3 pathway is very important for keratinocyte migration, and activation of the pathway can effectively promote re-epithelialization and accelerate wound healing.

Disclosure of Invention

In view of the above problems, the present invention provides a method for preparing leukocyte-rich platelet-rich plasma (L-PRP) having a platelet content of about 4 times that of whole blood and being rich in leukocytes and IL-6.

In order to solve the technical problems, the invention provides a preparation method of L-PRP, which comprises the following specific steps: collecting 10mL of whole blood by using a 10mL EDTA vacuum blood collection purple head tube, then placing the purple head tube filled with the whole blood at 4 ℃ for 200g for centrifugation for 10min, clearly dividing the whole blood into three parts, sequentially forming a clear light yellow plasma layer, a white flocculent white membranous layer and a red blood cell layer with the maximum density from top to bottom, sucking the three parts to a position 2mm below the white membranous layer by using a 1mL sterile suction pipe, moving the three parts to a 15mL clean centrifugal tube, discarding the rest components, leaving 1mL of plasma in the centrifugal tube, carefully suspending cell masses at the bottom of the centrifugal tube by using the sterile suction pipe, gently blowing and uniformly suspending the suspension to obtain 1mL light blood color L-PRP, wherein the part in the centrifugal tube needs to contain all upper plasma layers and all white membranous layers, and then performing secondary centrifugation, and the secondary centrifugation condition is 200g for 10min at 4 ℃.

In order to solve the technical problems, the invention also provides the L-PRP.

In order to solve the technical problems, the invention also provides application of the L-PRP in a medicament for treating the wound surface which is difficult to heal.

Compared with the prior art, the invention has the beneficial effects that: the platelet content of the L-PRP prepared by the invention is about 4 times of that of whole blood, the L-PRP is rich in leukocytes and IL-6, and the L-PRP is found to promote keratinocyte migration to accelerate wound surface re-epithelialization by up-regulating IL-6 transcription level and activating STAT 3; the L-PRP prepared by the invention and the research on the L-PRP refine the classification and application scenes of the PRP, discover a new mechanism for promoting the healing of the wound surface by the L-PRP, provide a new solution for clinically solving the difficult wound surface, and have important clinical practice significance.

Drawings

FIG. 1 is a schematic diagram of the preparation process and content measurement of PRP in the embodiment of the present invention;

FIG. 2 is a graph showing the results of an experiment in which L-PRP promotes re-epithelialization of rat wound according to an embodiment of the present invention;

FIG. 3 is a graph showing the results of transcription levels of proinflammatory factors in periwound tissue according to embodiments of the invention;

FIG. 4 is a graph showing the effect of L-PRP on the transcription level of IL-6 from HaCaT cells according to an embodiment of the present invention;

FIG. 5 is a graph showing the results of a cell scratch test according to an embodiment of the present invention;

FIG. 6 is a graph showing the results of a cell migration experiment according to an embodiment of the present invention;

FIG. 7 is a graph showing the effect of L-PRP on the level of STAT3 activation by HaCaT cells in accordance with an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example (b):

in order to solve the problem of difficult healing of clinical wounds, the present example is a method for preparing leukocyte-rich platelet-rich plasma (L-PRP), wherein the content of platelets in the L-PRP prepared in the present example is about 4 times of that of whole blood, and the L-PRP is rich in leukocytes and IL-6, and experiments show that the L-PRP can activate STAT3 to promote migration of keratinocytes and accelerate re-epithelization of wounds by increasing the transcription level of IL-6, and the specific method is described below.

Referring to fig. 1, fig. 1A and 1B are schematic diagrams of the preparation process of PRP of this embodiment, and the specific preparation process is as follows:

1.1. blood collection: drawing blood from SD rat: animals are fasted, the operation area is shaved, and the operation area is observed to be free from red swelling and bleeding and feasible operation; anesthesia: 0.3mL of chloral hydrate with the concentration of 10 percent per 100g is used for intraperitoneal injection anesthesia; and (3) disinfection: after the anesthesia works, the rat is fixed in a supine position, the abdominal skin preparation part is disinfected by alcohol, the abdominal exploration is performed, the inferior vena cava is found, the scalp needle is punctured into the inferior vena cava, after the blood returns, the tail end of the scalp needle is inserted into a 10mL EDTA vacuum blood sampling purple head tube, and about 10mL blood can be collected; collecting human venous blood by using a 10mL EDTA vacuum blood collection purple head tube;

1.2. preparation of L-PRP: carrying out first centrifugation on the collected whole blood, wherein the centrifugation condition is 200g centrifugation for 10min at 4 ℃, the whole blood is clearly divided into three parts, namely a clear light yellow plasma layer, a white flocculent white membrane layer and a red blood cell layer with the maximum density from top to bottom after centrifugation, then sucking the whole blood to a position 2mm below the white membrane layer by using a 1mL sterile suction tube, moving the whole blood to a 15mL clean centrifuge tube, abandoning the rest components, wherein the part in the centrifuge tube needs to contain all upper plasma layers and all white membrane layers, then carrying out second centrifugation, the second centrifugation condition is 200g centrifugation for 10min at 4 ℃, abandoning the upper layers, leaving 1mL plasma, carefully suspending cell masses at the bottom of the centrifuge tube by using the sterile suction tube, gently blowing and beating uniformly, and obtaining 1mL light blood color L-PRP after suspension.

1.3. Preparation of P-PRP (leukocyte-deficient platelet-rich plasma): centrifuging the collected whole blood for the first time, centrifuging the whole blood for 10min at 200g and 4 ℃ according to the first centrifugation parameters, carefully sucking the whole blood to the surface of a white membrane layer by using a 1mL suction tube, transferring the whole blood to a 15mL clean centrifugal tube, and discarding the rest components, wherein the part should contain all upper plasma; centrifuging for 10min at 200g and 4 ℃ for the second time according to the centrifugation parameters, discarding the upper layer, leaving 1mL of plasma, carefully suspending cell masses at the bottom of the centrifuge tube by using a pipette, gently blowing and beating uniformly, and suspending to obtain about 1mL of faint yellow P-PRP.

Referring to FIG. 1, the content of platelets, leukocytes and IL-6 in L-PRP and P-PRP was determined in this example, and referring to FIG. 1C, the platelet content in rat whole blood was 629. + -. 170X 10³Mu.l, platelet content in P-PRP 2381. + -. 141X 10³Mu.l, platelet content in L-PRP 2367. + -. 918X 10³Mu.l. The platelet content of the L-PRP and the P-PRP has no obvious difference, but the platelet content is obviously higher than that of the whole blood (about 4 times), and the conditions of the platelet-rich plasma are met. As shown in FIG. 1D, the white blood cell content in the rat whole blood is 5.3 + -2.2 × 10³Mu L, the white blood cell content in the P-PRP is lower than the detection threshold of a complete blood cell counter, and the white blood cell content in the L-PRP is 10.6 +/-2.3 multiplied by 10³Mu.l; the content of white blood cells in the L-PRP is obviously higher than that of the whole blood and the P-PRP, and the content of white blood cells in the whole blood is obviously higher than that of the P-PRP. Referring to FIG. 1E, ELISA was used to detect IL-6 content in rat L-PRP and P-PRP, IL-6 content in L-PRP was 53.95 + -16.65 pg/ml, IL-6 content in P-PRP was 21.34 + -11.69 pg/ml; the IL-6 content in L-PRP is obviously higher than that in P-PRP.

In this embodiment, after a rat creates a back wound model, the rat in this embodiment photographs 0 to 7d after surgery on the same scale daily, and the picture is imported into ImageJ to measure the wound area with the macroscopic wound edge for 3 times, and the average value is taken for statistical comparison to calculate the cumulative healing rate: the cumulative wound healing rate on Day X is (Day0 wound area-Day X wound area)/Day 0 wound area × 100%; calculating the daily healing rate: the daily healing rate of the wound on Day X is (Day (X-1) wound area-Day X100%) per Day (X-1) wound area).

Referring to FIGS. 2A-B, the changes in wound area were recorded and it was seen that the wound area decreased with time for each group, and the callus was shed generally at 5-6 days post-surgery, with the L-PRP group wound healing most rapidly (FIG. 2A). Comparing the cumulative healing rates of all groups, the cumulative healing rates of 1-7d after operation and L-PRP group are obviously higher than those of P-PRP group and NS group; there was no significant difference in cumulative healing rates between the P-PRP and NS groups. Comparing the daily healing rates of the groups, the daily healing rate of the post-operative 1d, L-PRP group was significantly higher than that of the P-PRP group and the NS group (P < 0.001), and the daily healing rate of the post-operative 3d, 5d, L-PRP group was significantly higher than that of the P-PRP group (P <0.05), but the daily healing rate was not significantly different from that of the NS group (FIG. 2B).

In this example, wound tissue was obtained 3d and 7d after molding, and h.e. staining was performed to observe and measure the periwound epithelial tongue length. Taking the wound periphery as a boundary, taking the farthest vertical distance of the epithelial ligulate process from the boundary as the migration length of the epithelial ligulate process, measuring for 3 times, and taking the average value to perform statistical comparison.

As shown in FIGS. 2C-D, obvious epithelial ligulate processes in each group at periwound were observed at 3D after surgery, wherein the epithelial ligulate processes in the L-PRP group had migrated to the surface of the new granulation tissue, while the epithelial ligulate processes in the P-PRP group and NS group only covered the surface of the contracted periwound tissue; at 7d after operation, the epithelial tongue process was significantly elongated and thickened compared to 3d after operation, with the L-PRP group having the most significant elongation, and the P-PRP and NS groups covering only a small amount of granulation tissue (fig. 2C). The length of the epithelial tongue process in the 3d, L-PRP group is obviously longer than that in the P-PRP group (P is less than 0.001) and NS group (P is less than 0.001) after operation, and the length of the epithelial tongue process in the P-PRP group is obviously longer than that in the NS group (P is less than 0.01); the length of the epithelial tongue process in the L-PRP group was significantly longer than that in the P-PRP group (P <0.05) and NS group (P <0.01) after the operation (7D), and the length of the epithelial tongue process in the P-PRP group was not significantly different from that in the NS group (FIG. 2D).

This example also measured the transcription levels of proinflammatory factors in peri-invasive tissue, as shown in FIG. 3, wherein IL-6: 8h, the L-PRP group is obviously more than the P-PRP group (P <0.01) and the NS group (P < 0.05); 24h, L-PRP was significantly more than in the remaining two groups (p < 0.05); the rest have no obvious difference.

This example next investigated a new mechanism of L-PRP for promoting wound healing, and the cell experiments were grouped: iguratmod (Idesin/Iguratimod) was used as IL-6 inhibitor, and the following groups were performed:

as shown in figure 4, IL-6RT-PCR was performed to detect IL-6 transcript levels after incubation of HaCaT cells for 8h in different media. The expression level of the L group is obviously higher than that of the Li group (P is less than 0.05), the P group (P is less than 0.001) and the C group (P is less than 0.001); the P group and the C group have no obvious difference; the Li group, although expressing significantly lower levels than the L group, also showed significantly higher levels than the C group (p < 0.001).

In the embodiment, the influence of L-PRP on the migration capacity of HaCaT cells is researched through a cell scratching experiment and a cell migration experiment (Transwell experiment), the cell scratching experiment is shown in an attached figure 5, the HaCaT cells are paved on a 24-hole plate, a vertical scratch is made in the center of the hole plate after 24h, different culture mediums are replaced, the HaCaT migration distance under different PRP effects is observed at different time points, and the migration of the HaCaT cells can be obviously promoted in the L group at each time point compared with the P group and the C group; after the IL-6 inhibitor Iguratmod with the final concentration of 30 mug/mL is added, the effect of L-PRP on promoting the migration of HaCaT cells is obviously weakened. The relative migration distance was calculated as (0h initial distance-24 h distance)/0 h initial distance × 100%. At each time point, the relative migration distance of the L group is significantly greater than that of the P group; starting from 24h, the relative migration distances of the L groups are all significantly greater than those of the C group; group P performed better than group C only at 72 h; the inhibitor group added with Iguratmo has no difference from the group C at each time point, and shows remarkable inhibition effect on the migration capacity of HaCaT cells from 24h compared with the group L.

The cell migration experiment is shown in figure 6, the same amount of different culture media are placed in a Transwell lower chamber, the same amount of single cell suspension is inoculated in the Transwell chamber, the chamber is taken out after 24 hours of incubation under the conventional condition, formaldehyde is fixed, HaCaT cells migrating under the action of different PRPs can be displayed by crystal violet staining, and the fact that the migration number of the HaCaT cells of an L group, a Li group, a P group and a C group is reduced in sequence can be obviously found. Observed under a high-power lens field (400 x), the mobility is equal to the area occupied by the migrated cells/total area x 100%, and the mobility of the L group is obviously higher than that of the Li group (P is less than 0.05), the P group (P is less than 0.001) and the C group (P is less than 0.001); the mobility of the P group is obviously higher than that of the C group (P is less than 0.01); the Li group, although having a lower mobility than the L group, is still superior to the C group (p < 0.01).

In this example, the effect of L-PRP on the activation level of STAT3 in HaCaT cells was also studied, and the activation level of STAT3 was measured by Western blot after incubation of HaCaT cells for 8h in various media, wherein the protein content is expressed as protein gray value/GAPDH gray value, and the activation ratio of STAT3 is expressed as pSTAT3/STAT 3. As shown in figure 7, the STAT3 content between the L group, the P group and the C group is not obviously different, but the STAT3 content of the Li group is obviously reduced compared with the L group (P <0.05) and the C group (P < 0.001). The contents of pSTAT3 in the group C, the group P, the group Li and the group L are increased in sequence, and the difference is obvious: the L group (P <0.01), Li group (P < 0.001), and P group (P <0.01) were all significantly higher than the C group, and P group (P < 0.001) and Li group (P <0.05) were all significantly lower than the L group. STAT3 was activated at the highest rate in the Li group, with L (P < 0.001), Li (P < 0.001), and P (P < 0.001) all significantly higher than in the C group, and L was lower than in the Li group (P < 0.001) but significantly higher than in the P group (P < 0.001).

In conclusion, the L-PRP prepared in the embodiment has the platelet content about 4 times that of whole blood, is rich in white blood cells and IL-6, and is found to accelerate wound re-epithelialization by up-regulating IL-6 transcription level and activating STAT 3. The L-PRP prepared by the embodiment and the research on the L-PRP refine the classification and application scenes of the PRP, discover a new mechanism for promoting the healing of the wound by the L-PRP, provide a new solution for clinically solving the difficult wound, are expected to be applied to the medicine for treating the difficult wound, and have important clinical practice significance.

The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.

Claims

1. A preparation method of leukocyte-rich and platelet-rich plasma is characterized by comprising the following specific steps: collecting 10mL of whole blood by using a 10mL EDTA vacuum blood collection purple head tube, then placing the purple head tube filled with the whole blood at 4 ℃ for 200g for centrifugation for 10min, clearly dividing the whole blood into three parts, sequentially forming a clear light yellow plasma layer, a white flocculent white membranous layer and a red blood cell layer with the maximum density from top to bottom, sucking the three parts to a position 2mm below the white membranous layer by using a 1mL sterile suction pipe, moving the three parts to a 15mL clean centrifugal tube, discarding the rest components, leaving 1mL of plasma in the centrifugal tube, carefully suspending cell masses at the bottom of the centrifugal tube by using the sterile suction pipe, gently blowing and uniformly suspending the suspension to obtain 1mL light blood color L-PRP, wherein the part in the centrifugal tube needs to contain all upper plasma layers and all white membranous layers, and then performing secondary centrifugation, and the secondary centrifugation condition is 200g for 10min at 4 ℃.

2. An L-PRP produced by the production process according to claim 1.

3. Use of an L-PRP according to claim 2 in a medicament for the treatment of refractory wounds.