Platelet subset as well as preparation method and application thereof

Abstract

The invention discloses a platelet subgroup as well as a preparation method and application thereof. A surface labeled molecule CD59 or CD59a of the platelet subgroup is positive. The platelet subgroups enriched by the identified surface marker molecules can remarkably improve the skin tissue repair efficiency, so that a more effective repair promoting strategy is provided for patients to relieve health pressure and economic burden caused by wounds.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and in particular, to a platelet subpopulation and a preparation method and application thereof. Background Art

[0002] Platelets (PLTs) are produced from megakaryocytes and are important terminal cell components in the blood system. They are characterized by large number, small size, and simple structure. Maintaining the body's coagulation balance is the most classic function of platelets. In recent years, with the deepening of the exploration of blood ecology, the function of platelets has been continuously expanded. However, promoting tissue repair is another important function of platelets, and no corresponding functional subpopulation has been found to be able to more concentratedly exert its repair effect. In recent years, the application of platelet-promoting tissue repair ability has been based on the characteristics of a large amount of growth factors stored in it, and dressings or preparations are prepared in the form of platelet-rich serum, platelet-derived exosomes, and platelet lysis products. However, from previous reports, we found that the existing platelet products still have the limitations of long cycles and limited repair effects for wound repair, especially the repair of difficult-to-heal skin wounds under diabetic conditions. Therefore, extracting platelet subpopulations that play a role in promoting tissue repair and more concentratedly amplifying their repair-promoting ability will provide a more efficient healing strategy. However, to date, there has been no research reporting on platelet subpopulations that can exert more effective abilities to promote tissue repair. Summary of the invention

[0003] Technical issues solved: One aspect of the present disclosure is to address the shortcomings of the prior art in that there is a lack of better platelet products for promoting skin tissue repair, and to provide a platelet subpopulation and a preparation method and application thereof.

[0004] Specifically, the inventors creatively discovered a group of special platelet subpopulations. After sorting and enriching them, they evaluated their ability to promote tissue repair in mice and found that they had strong ability and efficacy in promoting skin tissue repair, while also having the advantages of low usage, thereby solving the above-mentioned technical problems.

[0005] Technical solutions provided: The invention discloses a use of a platelet subpopulation in preparing a drug for promoting skin tissue repair, wherein the surface marker molecule CD59 or CD59a of the platelet subpopulation is positive.

[0006] Another aspect of the present disclosure is to provide use of a platelet subpopulation in preparing a skin care product for promoting skin tissue repair, wherein the surface marker molecule CD59 or CD59a of the platelet subpopulation is positive.

[0007] Another aspect of the present disclosure is to provide the use of a platelet subpopulation in the preparation of a medicament for treating skin tissue damage caused by abrasions, burns, scalds, contusions, punctures, lacerations, sunburn, cuts or contact with chemical substances or caused by diseases, wherein the surface marker molecules CD59 or CD59a of the platelet subpopulation are positive.

[0008] Further, in some embodiments, the above-mentioned chemical contact may include but is not limited to the contact of the following chemical substances with skin tissue: strong acid (e.g., sulfuric acid, hydrochloric acid, nitric acid, etc.), strong base (e.g., sodium hydroxide, etc.), yellow phosphorus, ammonia, hydrofluoric acid, dust, etc. In some embodiments, the above-mentioned disease may be one or more selected from the following diseases: diabetes, contact dermatitis, eczema, psoriasis, lupus erythematosus, dermatomyositis, scleroderma, bullous dermatosis, erythema protuberans persistent, facial granuloma, squamous cell carcinoma, basal cell carcinoma, T-cell lymphoma.

[0009] Furthermore, in some embodiments, the amount of the platelet subset is 3×10 6 ~5×10 6 For example, in some embodiments, the amount of the platelet subsets can be about 3×10 6 pcs, about 3.2×10 6 pcs, about 3.4×10 6 pcs, about 3.6×10 6 pcs, about 3.8×10 6 pcs, about 4×10 6 pcs, about 4.2×10 6 pcs, about 4.4×10 6 pcs, about 4.6×10 6 pcs, about 4.8×10 6 pcs, about 5×10 6 The inventors have found that in the repair of skin tissue damage using the platelet subsets disclosed in the present invention, a maximum of 5×10 6 Meanwhile, in some other embodiments, those skilled in the art can use more of the above-mentioned platelet subsets as needed without paying extra creative labor, for example, using about 5.5×10 6 pcs, about 6×10 6 pcs, about 6.5×10 6 pcs, about 7×10 6 pcs, about 7.5×10 6 pcs, about 8×10 6 pcs, about 8.5×10 6 pcs, about 9×10 6 pcs, about 9.5×10 6 pcs, about 1×107 The above dosage is also considered to be included in the protection scope of the present disclosure. In some embodiments, the use of the above platelet subsets can be used once or repeatedly. In the embodiments of the present disclosure, the above dosage is a single dosage of the platelet subset.

[0010] Another aspect of the present disclosure is to provide a platelet preparation, which includes a platelet subpopulation that is positive for surface marker molecules CD59 or CD59a. In some embodiments, the platelet subpopulation that is positive for surface marker molecules CD59 or CD59a in the above-mentioned platelet preparation can also be used in combination with other substances for promoting skin tissue repair, for example, medical film dressings, recombinant collagen dressings, recombinant human epidermal growth factor gel, platelet-rich plasma, etc. In some embodiments, the above-mentioned platelet preparation can also include conventional pharmaceutical excipients, for example, including but not limited to preservatives, anticoagulants, diluents, antibacterial agents, antioxidants, thickeners, enzyme preparations, nutritional enhancers, etc. In some embodiments, the above-mentioned platelet preparation can also include a pharmaceutically acceptable carrier, for example, a culture fluid, etc.

[0011] Further, in some embodiments, the platelet preparation can be composed of a platelet subpopulation positive for surface marker molecules CD59 or CD59a, pharmaceutical excipients, and pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical excipients include, but are not limited to, preservatives, anticoagulants, diluents, antibacterial agents, antioxidants, thickeners, enzyme preparations, nutritional enhancers, etc. In some embodiments, the pharmaceutically acceptable carrier, for example, culture fluid, etc.

[0012] In some embodiments, the platelet preparation can be prepared in any suitable form. For example, the platelet preparation can be in the form of a smear, a subcutaneous injection, a gel, or a patch. Preferably, in one embodiment of the present disclosure, the platelet preparation is in the form of a smear.

[0013] Furthermore, in some embodiments, the content of the platelet subpopulation positive for surface marker molecules CD59 or CD59a in the platelet preparation may be 3×10 6 ~5×10 6 For example, in some embodiments, the content of the platelet subpopulation can be about 3×10 6 pcs, about 3.2×10 6 pcs, about 3.4×10 6 pcs, about 3.6×10 6 pcs, about 3.8×10 6 pcs, about 4×10 6pcs, about 4.2×10 6 pcs, about 4.4×10 6 pcs, about 4.6×10 6 pcs, about 4.8×10 6 pcs, about 5×10 6 The inventors have found that in the repair of skin tissue damage using the platelet subsets disclosed in the present invention, a maximum of 5×10 6 Meanwhile, in some other embodiments, those skilled in the art can adjust the content of the platelet subpopulation in the platelet preparation as needed without paying extra creative labor. For example, it can be adjusted to about 5.5×10 6 pcs, about 6×10 6 pcs, about 6.5×10 6 pcs, about 7×10 6 pcs, about 7.5×10 6 pcs, about 8×10 6 pcs, about 8.5×10 6 pcs, about 9×10 6 pcs, about 9.5×10 6 pcs, about 1×10 7 The above content is also considered to be included in the protection scope of the present disclosure. In the embodiment of the present disclosure, the above dosage is a single dosage of the platelet subset.

[0014] Another aspect of the present disclosure is to provide use of the above-mentioned platelet preparation in the preparation of a drug for promoting skin tissue repair.

[0015] Beneficial effects: The surface marker molecules screened by the present invention can simply and effectively identify platelet subpopulations with stronger ability to promote tissue repair; and the platelet subpopulation suspension enriched by the marker molecules has many advantages over previous platelet repair products. First, from the analysis of the components of the platelet suspension, the platelet subpopulation suspension prepared by the present invention eliminates the stimulation of fibrinogen in plasma, avoiding the entry of fibrinogen or coagulation factors into the circulatory system through damaged blood vessels during the repair process, leading to coagulation disorders; secondly, in terms of efficacy, after applying the platelet subpopulation suspension prepared by the present invention, the repair speed and healing ratio of tissue damage such as the skin are significantly better than those of other platelet suspensions; in addition, in terms of dosage, the platelet products previously used for repair conventionally contain at least 1×10 7 PLTs, and the platelet suspension prepared in the present invention only needs 5×10 6PLTs can achieve significantly better results than PBS and the control group. In summary, the platelet subpopulations enriched by the surface marker molecules identified in the present invention can significantly improve the efficiency of tissue repair, thereby providing patients with a more effective repair-promoting strategy to reduce the health pressure and economic burden caused by trauma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The original scatter plot and proportion statistical graph of the expression ratio of CD59 or CD59a on the surface of mouse peripheral blood platelets detected by flow cytometry in the embodiments of the present disclosure, wherein the upper figure is the original scatter plot and proportion statistical graph of the CD59 expression ratio, and the lower figure is the original scatter plot and proportion statistical graph of the CD59a expression ratio; Figure 2 This is a result diagram of immunofluorescence staining to verify the expression of CD59a in platelets in the embodiments of the present disclosure; Figure 3 CD59a in the disclosed embodiment + PLTs and CD59a - Comparison of the expression levels of collagen component proteins COL1a1 and COLIII, the skin repair effector molecules, in PLTs; Figure 4 CD59a in the disclosed embodiment + PLTs and CD59a - Comparison of mean fluorescence intensity of mitochondrial probes in PLTs; Figure 5 Schematic diagram of the construction of the full-thickness skin wound model in the disclosed embodiment and the collection of CD59a by flow cytometry + PLTs, CD59a - Schematic diagram of the sorting gate strategy for PLTs and overall PLTs; Figure 6 For the disclosed embodiment, CD59a is applied + PLTs, CD59a - Representative diagram of the dynamic recording of PLTs, whole PLTs and the degree of skin wound healing in diabetic mice after PBS; Figure 7 For the disclosed embodiment, CD59a is applied + PLTs, CD59a - Comparison line graph of the healing ratio of skin wounds in diabetic mice after PLTs, whole PLTs and PBS and the statistical result graph corresponding to each time point; Figure 8 For the embodiment of the present disclosure, CD59a is applied + PLTs, CD59a -Representative diagram of the dynamic recording of the degree of skin wound healing in wild-type C57BL / 6J mice after PLTs, whole PLTs, and PBS; Fig. 9 For the embodiment of the present disclosure, CD59a is applied + PLTs, CD59a - Line graph comparing the healing ratios of skin wounds of wild-type C57BL / 6J mice after PLTs, whole PLTs and PBS, and the statistical results corresponding to each time point. DETAILED DESCRIPTION

[0017] The present invention discloses a platelet subpopulation and a preparation method and application thereof. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention, and relevant personnel can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0018] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" etc. will be understood to include the elements or components stated, without excluding other elements or other components. The term "a", "an" and "the" include plural indicators. The term "multiple" refers to two or more. The terms "such as", "for example", etc. are intended to refer to exemplary embodiments, and are not intended to limit the scope of the present disclosure.

[0019] In the present disclosure, when a range of values ​​is provided, it should be understood that the endpoints are included in the range and each intervening value between the upper and lower limits of the range and any other specified value or intervening value in the specified range and any value in the smaller range between the specified values ​​are included unless the context clearly dictates otherwise.

[0020] In this disclosure, the term "about" generally refers to variations within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0021] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The definition of common terms in molecular biology can be found in Lewin's GENES, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Publisher: Jones & Bartlett Learning. The definition of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: WH Freeman. The definition of common terms in cell biology can be found in Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Publisher: Garland Science. The definition of common terms in genetics can be found in Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Publisher: Jones & Bartlett Learning.

[0022] Unless otherwise specified, the experimental techniques herein employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found in standard books such as the following: Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook, etc.

[0023] the term: The term "platelets" (PLTs) in this disclosure is produced by megakaryocytes and is an important terminal cell component in the blood system. It has the characteristics of large number, small size, and simple structure. The blood circulation of a healthy adult contains nearly 1×10 12 The total number of platelets in the peripheral blood of adult mice can reach 1~2×10 9. In the body, each megakaryocyte can release an average of 2,000 to 3,000 daughter platelets. Therefore, although megakaryocytes only account for 0.05% to 0.1% of the bone marrow, their daughter platelets can maintain a sufficient number in the peripheral blood circulation to maintain the stability of the blood ecology. Compared with other blood cells, platelets are the smallest members of the blood system, with an average diameter of only 2 to 4 μm. When they were first discovered, they were once described as "blood dust" released by megakaryocytes, and were later officially named platelets based on their disc-shaped morphology. In addition, as terminal cells that directly perform functions, platelets have a very simple structure. There is no nuclear component inside, and only ultrastructures such as mitochondria, lysosomes, α granules and dense granules that directly perform functions are retained. The lightened morphological structure allows a large number of platelets to more flexibly travel through blood vessels at all levels and respond quickly and promptly to body stimuli.

[0024] Maintaining the body's coagulation balance is the most classic function of platelets. In recent years, with the deepening of the exploration of blood ecology, the function of platelets has been continuously expanded. For example, in addition to maintaining the body's coagulation balance, in the body's immune defense process, platelets can participate in innate immunity and acquired immunity to build a defense line for the maintenance of the body's homeostasis. In the immune response process, platelets can release inflammatory mediators and active peptides stored in the granules to regulate leukocyte recruitment, adhesion, proliferation and differentiation. On the other hand, platelets can also participate in pathogen clearance through the interaction of three types of pattern recognition receptors (PRR) on the surface, TLR, CLR, and NLR, with various immune cells. In addition, in addition to participating in the repair of vascular ruptures to maintain the integrity of the vascular wall during the coagulation process, platelets also play an important role in the reconstruction of the body's vascular system. For example, in the tumor environment, the abundant α granules and dense granules in platelets can release a large number of cytokines that regulate tumor angiogenesis, such as PDGF, TEGF, etc., promote the regeneration of blood vessels by sprouting, intussusception, etc., increase the blood supply of solid tumor tissues, build structural components for tumor infiltration and migration, and provide nutritional support. In the repair of skin and other tissues, platelets can also promote the proliferation and migration of fibroblasts in tissue defects, promote the regeneration of collagen and elastin, and promote skin re-epithelialization with the help of their abundant internal growth factors such as EGF and TGF-β and surface glycoproteins such as GPIb, thereby playing its repair-promoting function.

[0025] As the understanding of platelet functions has gradually deepened, many hypotheses have been proposed in the field in recent years about how platelets perform various functions. More and more researchers believe that platelets do not perform different functions such as coagulation and immunity as a whole, but there are different functional subgroups that perform different functions in a targeted manner. For example, based on the immunoregulatory function of platelets, studies have successively found that there are different subgroups of platelets that perform different immune defense functions. The Looney team in the United States found that splenic megakaryocytes will produce a group of special platelet subgroups with high expression of CD40L to exert immune regulatory functions; Liu et al. also found that under inflammatory stress conditions, CD48 in peripheral blood + The proportion of platelet subpopulations will increase, and CD48-positive platelets are a group of platelets with immune regulatory tendencies; the above results indicate that platelets are not a homogeneous group, but can perform corresponding functions in specific functional subpopulations.

[0026] The terms "subpopulation" and "cell subset" in this disclosure can be used interchangeably. It refers to different cell populations formed during the development, differentiation or function of cells due to differences in gene expression patterns, protein expression or other biological characteristics. The classification methods of cell subsets include but are not limited to classification based on cell surface differentiation antigens, cell surface receptors, functions, and differences in antigen response. For example, in the immune system, T cells can be divided into CD4 T cells and CD5 T cells based on the differences in their surface differentiation antigens CD4 T cells. + and CD8 + Two major subgroups; NK cells can be divided into different subtypes according to the differences in their surface markers and functions. The platelet subgroups in the present disclosure are divided by the surface marker protein molecules they express. The main surface marker molecules of platelets, for example, CD41, CD42a, CD42b, CD61, etc. Methods for detecting cell surface marker molecules, for example, flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay, immunoblotting, etc. In one embodiment of the present disclosure, the method for detecting or enriching platelets that are positive for the surface marker molecule CD59a is flow cytometry.

[0027] The terms "CD59" and "CD59a" in the present disclosure are marker protein molecules on the surface of platelets, which are expressed on the surface of human platelets and mouse platelets, respectively, and are homologous genes. Among them, humans have 1 CD59 gene, while mice have 2 homologous genes, CD59a and CD59b. Compared with CD59b, mouse CD59a has a higher similarity with human CD59 molecules in terms of results, tissue distribution and functional characteristics. Therefore, when studying the human CD59-related mechanism using mice as a model, CD59a is used for research. The mouse CD59a molecule and the human CD59 molecule have the same characteristics at the amino acid level: ① Both are glycoproteins with a relative molecular mass of about 18-25kDa, both are composed of about 100 amino acid residues, and both contain a single N-terminal glycosylation site, and their C-terminals are anchored to the cell surface by glycosylphosphatidylinositol. ② The main structures of both are relatively compact in space and spherical, which is conducive to their corresponding functions on the cell surface, such as interacting with other molecules. ③ They are similar in structure in the functional region, and both have sites that bind to complement C7, C8 or C9. Through these sites, they bind to complement components, thereby inhibiting the formation of the attack complex and protecting the cells from complement damage. (Qin X, et al., Mamm Genome. 2001. doi: 10.1007 / s00335-001-2060-8; Qian YM, et al., J Immunol. 2000. doi: 10.4049 / jimmunol.165.5.2528) Therefore, based on the common knowledge in the above prior art, those skilled in the art can undoubtedly know that CD59 and CD59a have the same biological functions.

[0028] The term "skin care products" in the present disclosure refers to skin care products that have the effect of protecting the normal skin. Its basic functions include, for example, beautifying and nourishing the skin, replenishing moisture, moisturizing, delaying aging, etc. In some embodiments, in addition to the above basic functions, the skin care products or skin care product compositions also have the effect of promoting skin tissue repair. Example

[0029] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0030] Example 1: Collection of mouse peripheral blood In order to meet the needs of subsequent surface marker molecule detection, effector molecule detection and functional verification samples, this example collects mouse peripheral blood in the following manner to ensure that the platelets in the peripheral blood samples remain in a resting state and are not activated and degraded due to human manipulation. The specific experimental operation is as follows: After anesthetizing 8-10 week old female mice with 100 μl of 10% chloral hydrate by intraperitoneal injection, perform laparotomy along the linea alba, expose the inferior vena cava, and use a 1 ml syringe to draw 100 μl of ACD anticoagulant and repeatedly aspirate to soak the syringe needle and inner wall. Insert the needle parallel to the blood vessel, and use a rinsed syringe to draw blood from the inferior vena cava, first fast and then slow, to avoid fractures and vascular collapse. After the blood draw is completed, slowly withdraw the needle and transfer the collected peripheral blood to a centrifuge tube, and flick to mix.

[0031] Example 2: CD59a + PLTs / CD59 + Detection of PLTs ratio In order to detect the expression ratio of CD59a / CD59 in platelets, this example used flow cytometry to detect and count the expression of CD59a / CD59 on the surface of platelets in peripheral blood of human and mouse. The specific experimental operation is as follows: After collecting peripheral blood of mice in the manner described in Example 1, 1 μl of peripheral blood was added to 100 μl of normal temperature benchtop solution, and 1 μl of CD41, Ter119, CD59a antibodies and Hoechst nucleic acid dye were added. After incubation at room temperature in the dark for 30 minutes, 100 μl of normal temperature benchtop solution was added for dilution, and all samples were transferred to a flow cytometer to detect Hoechst. - Ter119 - CD41 + The expression level of CD59a in the PLTs population. Figure 1 The results showed that CD59a in the peripheral blood of mice + The proportion of PLTs was about 19.87%. The expression of CD59 in human platelets was detected by the same antibody incubation method, and the positive proportion was also about 20%.

[0032] Example 3: Immunofluorescence verification of CD59a expression in platelets In order to more intuitively verify the expression and distribution of CD59a in platelets, this example observed CD59a positive platelets under a microscope using immunofluorescence staining. The specific operation is as follows: 500μl of blood was collected from the medial angular venous plexus, and then an equal volume of physiological saline preheated to 37°C was added and gently inverted to mix. After centrifugation at 1100rpm for 7 minutes, the supernatant was transferred to another 1.5ml EP tube and centrifuged again at 3500rpm for 5min. The supernatant was discarded, and 1ml of CGS solution preheated to 37°C was added (500ml CGS solution contains 3.6g Nacl, 3.3g D-glucose (H20), 2.165g sodium citrate (2H2O), 400ml ddH2O, and the volume was adjusted to 500ml after adjusting the pH to 6.5 with dilute hydrochloric acid) to resuspend the platelets. When resuspending, it is necessary to blow from the opposite side of the sediment at the bottom of the tube, and the platelets can be blown up in the form of quicksand. Then, prostaglandin E1 (PGE1) was added at a ratio of 1:1000, and the tube was gently inverted to mix, and then centrifuged at 2500rpm for 5min for washing. After repeating the CGS washing process, discard the supernatant, resuspend the precipitate with 100ul benchtop solution to prepare platelet suspension, add 1μl of CD41, CD59a, Ter119 antibodies and Hoechst nucleic acid dye, and incubate at room temperature in the dark for 30 minutes. Then add 100μl benchtop solution to dilute and centrifuge at 3500rpm for 5min to wash, finally resuspend the precipitate with 50ul benchtop solution, and use a pipette to draw 20μl of platelet suspension, drop it on the bottom of the confocal culture dish, and observe Hoechst under the microscope. - Ter119 - CD41 + CD59a expression in PLTs population. Figure 2 As shown, some platelets were CD59a positive under the microscope.

[0033] Example 4: CD59a + PLTs and CD59a - Detection of expression of repair-promoting effector molecules in PLT In order to detect whether the group of platelets marked by CD59a contains higher levels of tissue repair effector molecules, this example compares CD59a + PLTs and CD59a - Expression of COL1a1 and COLIII proteins in PLT. The specific experimental procedures are as follows: After collecting mouse peripheral blood in the manner described in Example 1, the peripheral blood was fixed with a final concentration of 1% PFA at room temperature for 15 minutes, and then an equal amount of room temperature benchtop solution was added and centrifuged at 300g for 5 minutes for washing. After discarding the supernatant, the precipitate was resuspended with 50ul of permeabilization solution and 1μl of COL1a1 / COLIII intracellular protein antibody was added and incubated at room temperature in the dark for 1.5h. 500μl of benchtop solution was added and centrifuged at 3500rpm for 5min for washing, and then resuspended with 50μl of benchtop solution. 1μl anti-rabbit secondary antibody, 1μl CD41, 1μl Ter119, and 0.5μl Hoechst fluorescent dye were added and incubated at room temperature in the dark for 45min. Finally, 500μl of benchtop solution was added for dilution and washing, and then transferred to the flow tube for detection. The results are as follows Figure 3 As shown, the results showed that the two collagen components COL1a1 and COLIII were expressed in CD59a + The expression ratio in PLTs was close to 100%, while in CD59a - In PLTs, only 70% and 60% were found, and in CD59a + In PLTs, the mean fluorescence intensity of COL1a1 and COLIII was also significantly higher than that of CD59a. - As we all know, collagen regeneration is one of the important links in tissue repair. High expression of collagen component proteins COL1a1 and COLIII indicates CD59a + PLTs have a stronger potential to promote tissue repair.

[0034] Example 5: CD59a + Detection of mitochondrial potential and mitochondrial volume in PLTs Previous studies have shown that active mitochondrial function is one of the mechanisms by which platelets promote tissue repair. + Whether PLTs are a group of platelets with more active mitochondrial function, this example conducts the following experiments: After collecting the platelet suspension in the same manner as in Example 3, mitochondrial deep red fluorescent probe and mitochondrial green fluorescent probe were added at a volume ratio of 1:5000 and 1:2500, respectively. 7 The cells need 1 μl of antibody and CD41 and Ter119 antibodies in a ratio of 1:100. Hoechst nucleic acid dye is added in a volume ratio of 1:100. After flicking to mix, incubate at room temperature in the dark for 30 minutes. Finally, add an equal volume of room temperature benchtop solution for dilution and washing, and detect Hoechst on the machine. - Ter119 - CD41 + The average fluorescence intensity of mitochondrial deep red fluorescent probe and mitochondrial green fluorescent probe in platelet population was compared with CD59 + PLTs and CD59- Mitochondrial potential and volume in PLT. Figure 4 As shown, the results showed that CD59a + The average fluorescence intensity of mitochondrial deep red fluorescent probe and mitochondrial green fluorescent probe in PLTs was higher than that of CD59a - PLTs, suggesting that the mitochondrial function inside them is more active.

[0035] Example 6: CD59a + PLTs Collection After peripheral blood was collected in the manner described in Example 1, the peripheral blood collected in the sodium citrate anticoagulant tube was diluted with an equal volume of physiological saline and centrifuged at 1100 rpm for 7 minutes. The supernatant was slowly transferred to another 1.5 ml EP tube to remove most of the nucleated cells in the peripheral blood. 7 The cells need 1 μl of antibody and add CD41, Ter119, and CD59a antibodies at a volume ratio of 1:100. Add Hoechst nucleic acid dye at room temperature and incubate for 30 minutes in the dark. Then add an equal volume of room temperature benchtop solution for dilution and washing, and finally transfer the platelet suspension to the flow sorting tube for sorting.

[0036] 6 ml of room temperature benchtop solution was added to a 15 ml centrifuge tube as a collection tube. After the main platelet population was determined by FSC and SSC parameters, the platelets were - Ter119 - CD41 + In the platelet population, CD59a positive and CD59a negative populations are distinguished based on isotype controls and sorted into collection tubes. - Ter119 - CD41 + The platelet population (whole PLTs) was used as a control. Figure 5 After sorting, the liquid in each collection tube was divided into two, ensuring that the liquid volume in each collection tube was less than 8 ml, and then centrifuged at 4000 rpm (the speed was set to 0, and the speed was set to 1) for 10 minutes, and then the supernatant was discarded. The platelet precipitate was resuspended with the remaining liquid at the bottom of the tube, and the multi-tube platelet suspension of the same sample was transferred to the same 1.5 ml EP tube, and then centrifuged at 3500 rpm (the speed was set to 0, and the speed was set to 1) for 10 minutes, and the supernatant was discarded. The precipitate was resuspended with room temperature PBS to prepare a platelet suspension.

[0037] Example 7: CD59a + Verification of the ability of PLTs to promote skin damage repair in diabetic mice Due to impaired local microcirculation and other reasons, it is often difficult for diabetic patients to heal their skin after injury. The health pressure and economic burden caused by chronic skin damage are major challenges in the management of chronic diseases in diabetic patients. The diabetic mouse model is a common model for studying the repair of chronic skin damage in the diabetic environment. To verify the CD59a + The repair-promoting ability of PLTs, in this example, a full-thickness skin injury model was constructed using db / db BKS diabetic mice, and the platelet-promoting repair ability was compared by dynamically monitoring the degree of wound healing after applying each group of platelet suspension. The specific experimental operation is as follows: First, after preparing platelet suspension as described in Example 5, CD59a + PLTs suspension, CD59a - 10 μl of each of the PLTs suspension and the whole PLTs suspension was added to 240 animal cell diluent for dilution, and then the platelet concentration was detected by a blood cell analyzer (Mindray, BC-5000Vet). PBS was added to dilute to 5×10 6 / 20μl concentration for later use.

[0038] After 12-week-old BKS db / db male mice were anesthetized with 10% chloral hydrate by intraperitoneal injection, the back skin was shaved and disinfected with 75% alcohol. Two symmetrical 6 mm full-thickness skin wounds were made at the prepared skin using a hole puncher. A ruler was placed next to the wound of each mouse and photographed in turn. The initial wound size (e.g., 1.0 mm / s) on the day of the wound (Day 0) was recorded. Figure 6 After the recording was completed, the model mice were divided into four groups. The first group was smeared with 20 μl of the above CD59a + PLTs suspension, the second group applied 20 μl of the above CD59a - PLTs suspension, the third group was smeared with whole PLTs suspension, and the fourth group was smeared with 20 μl sterile PBS solution as a control.

[0039] On the second day after the injury (Day 2), the above anesthesia operation was repeated and photos were taken again to record the size of each wound and compare its healing degree (such as Figure 6 and CD59a was collected again as described above. + PLTs, CD59a - Platelet suspension and PBS solution were prepared using PLTs and whole PLTs and applied on the wound surface of mice in the corresponding groups to enhance the comparison of effects among the groups.

[0040] Then, the wounds of each group of mice were photographed and recorded on the fourth day (Day4), sixth day (Day6), and eighth day (Day8) after trauma, and the wound area was recorded to compare the degree of repair. The size of the wound area was statistically analyzed using ImageJ. The degree of repair was quantitatively calculated according to the healing ratio (%) = (W0-Wt) / W0×100%, where W0 is the initial wound area; Wt is the wound area dynamically monitored every two days from the second day to the eighth day after trauma. The results are shown in Figure 6 and Figure 7 As shown, first of all, the images recorded dynamically from the wound size clearly show that the smear CD59a + The wound healing degree after PLTs was significantly better than that of the control group starting from Day 2 after injury. Further quantitative and statistical analysis of the wound healing ratio showed that the application of CD59a + After PLTs, the wound healing rate can reach about 80% on Day 4, and the healing rate is close to 100% on the eighth day after the injury. - Although the degree of wound repair in the PLTs and whole PLTs groups was better than that in the PBS control group, the healing rate did not reach 80% until Day 8. This result is consistent with previous research reports. Products such as platelet-rich serum and platelet-derived extracellular particles can increase the wound healing rate on Day 8 in the diabetic mouse skin wound model from about 30% in the control group to about 60%, but they are not as good as the CD59a in the present invention. + PLTs suspension promotes tissue repair quickly and effectively, indicating that CD59a + PLTs are a subpopulation of platelets that have a stronger ability to promote skin damage repair.

[0041] Example 8: CD59a + Verification of the ability of PLTs to promote skin damage repair in wild-type mice In addition to chronic skin damage in diabetic settings, this example also verified the expression of CD59a in wild-type C57BL / 6J mice. + The specific experimental operation was similar to that described in Example 7, except that the 12-week-old db / db BKS male mice used as model recipients in Example 7 were replaced with 8-9-week-old wild-type C57BL / 6J female mice. The rest of the platelet suspension preparation, grouping, and wound dynamic detection methods were the same as those in Example 7. Figure 8 , Fig. 9 As shown, compared with CD59a - PLTs, whole PLTs and PBS control group, CD59a + PLTs have better pro-repair ability. +After PLTs, the repair efficiency of mouse wound surface exceeded 60% on Day 2, which was significantly higher than that of other groups, and was always higher than that of other control groups in subsequent dynamic monitoring, indicating that CD59a + PLTs have stronger ability to promote repair.

[0042] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of platelet subpopulations in the preparation of a drug for promoting skin tissue repair, characterized in that: The surface marker molecules CD59 or CD59a of the platelet subpopulation are positive.

2. Use of platelet subpopulations in the preparation of skin care products for promoting skin tissue repair, characterized in that: The surface marker molecules CD59 or CD59a of the platelet subpopulation are positive.

3. Use of platelet subpopulations in the preparation of a medicament for treating skin tissue damage caused by abrasions, burns, scalds, contusions, punctures, lacerations, sunburns, cuts or chemical contact or caused by diseases, characterized in that: The surface marker molecules CD59 or CD59a of the platelet subpopulation are positive.

4. The use according to claim 3, characterized in that The disease is one or more selected from the following diseases: diabetes, contact dermatitis, eczema, psoriasis, lupus erythematosus, dermatomyositis, scleroderma, bullous dermatosis, persistent raised erythema, facial granuloma, squamous cell carcinoma, basal cell carcinoma, T-cell lymphoma.

5. The use according to any one of claims 1 to 4, characterized in that: The amount of the platelet subset is 3×10 6 ~5×10 6 indivual.

6. A platelet preparation, characterized in that: The platelet preparation includes a platelet subpopulation whose surface marker molecules are positive for CD59 or CD59a.

7. The platelet preparation according to claim 6, characterized in that The platelet preparation consists of a platelet subpopulation with positive surface marker molecules CD59 or CD59a, pharmaceutical excipients and a pharmaceutically acceptable carrier.

8. The platelet preparation according to claim 6 or 7, characterized in that: The platelet preparation is in the form of a smear, a subcutaneous injection, a gel or a dressing.

9. The platelet preparation according to claim 6 or 7, characterized in that: The content of the platelet subpopulation positive for surface marker molecules CD59 or CD59a is 3×10 6 ~5×10 6 indivual.

10. Use of the platelet preparation according to any one of claims 6 to 9 in the preparation of a medicament for promoting skin tissue repair.

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