A combination of proteins secreted by Meckel chondrocytes, a preparation method thereof, and applications thereof

Abstract

The present application provides a secreted protein composition, characterized in that the composition includes GRN protein and any other one or more proteins secreted by Meckel's cartilage; and its preparation method and applications are provided. Compared with the prior art, the advantages of the present application are as follows: the protein combination secreted by Meckel's cartilage cells mimics the microenvironment of nerve development, providing a more suitable bioactive material for nerve regeneration; the secreted protein combination can promote the directional extension of nerve fibers, and is expected to improve the accuracy of nerve regeneration and the effect of functional recovery; the secreted protein combination can more comprehensively meet the complex requirements of nerve regeneration; the research based on Meckel's cartilage cells provides a theoretical basis and experimental evidence for the development of new nerve regeneration materials.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and specifically relates to a combination of proteins secreted by Meckel chondrocytes, a preparation method thereof, and an application thereof. Background Art

[0002] Peripheral nerve injury is a common clinical problem that can lead to motor and sensory dysfunction, seriously affecting the quality of life of patients. Currently, the repair of peripheral nerve injury still faces great challenges, and one of the main reasons is the lack of effective biomaterials to promote nerve regeneration. An ideal nerve repair material should be able to mimic the microenvironment during nerve development and provide necessary biochemical and physical support for axon regeneration.

[0003] Currently, the following main deficiencies exist in the field of peripheral nerve injury repair:

[0004] (1) Lack of materials based on biological principles: Most of the existing nerve conduits and scaffold materials are synthetic materials, lacking biological activity and unable to fully mimic the microenvironment of nerve development.

[0005] (2) Poor directivity of nerve regeneration: Existing materials are difficult to effectively guide the directional growth of axons, resulting in poor accuracy of nerve regeneration and unsatisfactory functional recovery effects.

[0006] (3) Imperfect growth factor delivery system: Although neurotrophic factors have a promoting effect on nerve regeneration, how to achieve their continuous and stable release remains a challenge.

[0007] (4) Lack of multifunctional compositions: Single factors are difficult to meet the complex needs of nerve regeneration, and it is necessary to develop multifunctional compositions that can synergistically promote nerve regeneration.

[0008] Meckel cartilage is a transient product during embryonic development. It was initially thought that Meckel cartilage only played a scaffolding role during mandibular bone development and had no other functions. However, the applicant's research found that during the embryonic development of mammals such as humans and mice, the mandibular nerve distributes and extends around Meckel cartilage ( Figure 1 , Figure 2 A), and more importantly, the applicant found that mice lacking Meckel cartilage had abnormal nerve distribution and function ( Figure 2 ), suggesting that Meckel cartilage may be related to nerve development. Summary of the Invention

[0009] In view of the above technical limitations, this application proposes a combination of proteins secreted by Meckel chondrocytes, a preparation method thereof, and an application thereof; it overcomes the deficiencies and defects mentioned in the background art.

[0010] To achieve the above object, this application adopts the following technical solutions:

[0011] The inventive point of this application is to provide a secreted protein composition, which includes GRN protein and any one or more other proteins secreted by Meckel's cartilage.

[0012] Optionally, in the above-mentioned secreted protein composition, the composition includes GRN protein and any one or more of the following proteins: CLU, IGFBP5, IGFBP3, SPARC, APP, SEMA3A, IGFBP4, IGFBP2, and SEMA3F.

[0013] CLU: Clusterin, also known as apolipoprotein J, is a novel adipokine that participates in regulating various biological processes, such as lipid transport, cell adhesion, and complement regulation;

[0014] GRN: Granulin, which participates in inflammation, wound healing, and cell proliferation, regulates the transport of proteins to lysosomes, as well as the activity of lysosomal enzymes, and also promotes the acidification of lysosomes;

[0015] IGFBP5: Regulates IGF-mediated cellular processes, including proliferation, differentiation, and apoptosis, increases the proliferation of osteoblasts, intestinal smooth muscle cells, and neuroblastoma cells, and enhances the adhesion and survival of epithelial cells;

[0016] IGFBP3: Inhibits the positive effect of human insulin sensitivity and promotes the apoptosis of testicular germ cells;

[0017] SPARC: Interacts with the extracellular matrix and cytokines to regulate cell growth, binds calcium and copper, various types of collagen, albumin, thrombospondin, PDGF, and cell membranes;

[0018] APP: As a cell surface receptor, it performs physiological functions related to neurite growth, neuronal adhesion, and axonogenesis on the surface of neurons. The interaction between APP molecules on adjacent cells promotes synapse formation;

[0019] SEMA3A: Induces the disintegration and paralysis of neuronal growth cones and guides the direction of growth cones;

[0020] IGFBP4: Can both inhibit and stimulate the growth-promoting effect of IGF on cells and regulate the interaction between IGF and its cell surface receptor;

[0021] IGFBP2: When mitochondrial damage occurs, it promotes the apoptosis of podocyte ITGA5, and then activates the phosphorylation of FAK-mediated mitochondrial damage;

[0022] SEMA3F: Regulates cell motility and cell adhesion.

[0023] Optionally, for a combination of Merkel chondrocyte-secreted proteins as described above, in the selection of the ratio of each protein in the secreted protein combination, the ratio of CLU:GRN:IGFBP5:IGFBP3:SPARC:APP:SEMA3A:IGFBP4:IGFBP2:SEMA3F is (0 - 1.5):(0.3 - 0.5):(0 - 0.5):(0 - 0.5):(0 - 0.5):(0 - 0.5):(0 - 0.3):(0 - 0.2):(0 - 0.1):(0 - 0.1), preferably 1:0.439:0.397:0.384:0.384:0.384:0.2:0.132:0.062:0.038.

[0024] The second inventive point of the present application is to provide a method for preparing the above-mentioned combination of Merkel chondrocyte-secreted proteins, comprising the following steps:

[0025] Culture of primary Merkel chondrocytes:

[0026] Rinse the Merkel cartilage isolated from the mandible, filter it after digestion, collect the cell suspension, discard the supernatant after centrifuging the cell suspension and resuspend the cell pellet, and inoculate for culture.

[0027] Collection of secreted proteins from primary Merkel chondrocytes:

[0028] Collect the culture medium of primary Merkel chondrocytes obtained by culturing in step S1.

[0029] Determination of secreted proteins from primary Merkel chondrocytes:

[0030] Digest the proteins in the culture medium containing secreted proteins from primary Merkel chondrocytes collected in step S2 into peptide segments with a protease, then preliminarily separate the peptide segments by chromatography, and detect the separated peptide segments with a mass spectrometer to obtain peptide segment information.

[0031] Optionally, in the above preparation method, in step S1, the rinsing is carried out using a PBS buffer solution containing antibiotics (gibco, 15140122, penicillin-streptomycin mixture 100×), and the impurities and blood stains are repeatedly rinsed off, and the cartilage is cut into small pieces; the digestion is carried out using type II collagenase (Thermo Fisher, P5667270), the digestion temperature is 35°C - 40°C, the digestion time is 50 min - 70 min, and continuous shaking is required during the digestion process. The digestion is terminated by using a cartilage culture medium containing 8% - 12% serum (Procell, human cartilage complete medium CM-H107); the filtration is carried out using a 35μm - 45μm filter screen, and the purpose of filtration is to remove undigested tissue fragments and collect the cell suspension; the centrifugation speed is 1000 rpm - 1500 rpm, the centrifugation time is 4 min - 6 min, and the supernatant is discarded after centrifugation; the cell precipitate is resuspended, and the cells are inoculated into a culture flask or culture plate. The culture conditions after cell inoculation are 35°C - 40°C, 4% - 6% CO 2 , and then the cell growth state is observed regularly.

[0032] The preferred scheme of the above step S1 is: the rinsing is carried out using a PBS buffer solution containing antibiotics; the digestion is carried out using type II collagenase, the digestion temperature is 37°C, the digestion time is 60 min, and the digestion is terminated by using a cartilage culture medium containing 10% serum; the filtration is carried out using a 40μm filter screen; the centrifugation speed is 1200 rpm, the centrifugation time is 5 min; the culture conditions after cell inoculation are 37°C, 5% CO 2 .

[0033] In step S2, the culture medium is collected after culturing the primary Merkel cartilage cells for three days, ensuring that the serum and other factors are removed from the culture medium. Trypsin is used to digest the proteins into peptide segments for isotope labeling for quantitative comparison, etc.;

[0034] In step S3, chromatographic techniques are used to preliminarily separate the peptide segments; the separated peptide segments are introduced into a mass spectrometer for detection to obtain information such as the mass and charge of the peptide segments; the mass spectrometry data is processed and analyzed, including peptide segment identification, protein assembly, quantitative calculation, differential analysis, etc.

[0035] The third inventive point of the present application is to provide the use of the above-mentioned combination of proteins secreted by Merkel cartilage cells in the preparation of a drug for preventing / treating peripheral nerve injury.

[0036] The fourth inventive point of the present application is to provide the use of the above-mentioned combination of proteins secreted by Merkel cartilage cells in the preparation of a drug for promoting the directional growth and repair of peripheral nerves.

[0037] The fifth inventive point of the present application is to provide the use of the above-mentioned protein combination secreted by Merkel chondrocytes in the preparation of a biomaterial for promoting the directional growth of peripheral nerves, and the biomaterial is selected from one or more of nerve conduits and scaffold materials.

[0038] The sixth inventive point of the present application is to provide the use of the above-mentioned protein combination secreted by Merkel chondrocytes in the preparation of Merkel cartilage organoids.

[0039] The technology disclosed in the present application mainly includes the following contents:

[0040] 1) Composition of the protein combination secreted by Merkel chondrocytes: The specific components and ratios of the protein combination that can promote nerve development identified by secretory proteomics testing (as shown in Table 1 and Figure 3 ).

[0041] 2) Preparation method of the protein combination: including the culture conditions of Merkel chondrocytes, methods for collecting secreted proteins, etc.

[0042] 3) Mechanism of the protein combination promoting the directional growth of nerve fibers: Elucidate how this combination acts on nerve cells to promote their directional growth. That is, the secreted protein combination can promote the growth of neuronal processes, and through molecules such as CDC42 and RAC1, regulate the cytoskeleton remodeling of nerve fibers to make the neuronal processes tend to grow in the direction of Merkel chondrocytes ( Figure 4 , Figure 5 ).

[0043] Based on the above research, the present application proposes a nerve regeneration technology based on the protein combination secreted by Merkel chondrocytes, providing a new treatment strategy for the repair of peripheral nerve injuries.

[0044] A protein combination secreted by Merkel chondrocytes provided by the present application, its preparation method and application, compared with the prior art, have the following advantages:

[0045] 1) Provide a nerve regeneration material based on biological principles: The protein combination secreted by Merkel chondrocytes is a natural protein composition, rather than a synthetic material. It mimics the microenvironment of nerve development and provides a more suitable bioactive material for nerve regeneration;

[0046] 2) Improve the directivity of nerve regeneration: This secreted protein combination can promote the directional extension of nerve fibers, and is expected to improve the accuracy of nerve regeneration and the effect of functional recovery ( Figure 4 , Figure 5 );

[0047] 3) Provide a combination that promotes nerve regeneration through multi-functional synergy: The technology used in this application is a protein combination, rather than a single factor. This protein combination can more comprehensively meet the complex needs of nerve regeneration (Table 1, Figure 3);

[0048] 4) Provides new ideas for the development of nerve regeneration materials: The research based on Meckel chondrocytes provides a theoretical basis and experimental evidence for the development of new nerve regeneration materials. Brief Description of the Drawings

[0049] Figure 1 Schematic diagram of the nerve extending around Meckel cartilage during mandibular development.

[0050] Figure 2 Abnormal nerve distribution and function in mice lacking Meckel cartilage; among them, Figure 2 A is the three-dimensional reconstruction diagram of the mandible, nerve, blood vessel, and Meckel cartilage of wild-type (WT) mice and mice lacking Meckel cartilage (Sox9 flox / flox ; Wnt1-Cre), Figure 2 B is the immunofluorescence staining diagram of three neurotransmitters (VAChT, Substance P, CGRP) in WT mice and mice lacking Meckel cartilage, Figure 2 C is the statistical chart of the fluorescence intensity of three neurotransmitters in WT mice and mice lacking Meckel cartilage.

[0051] Figure 3 Enrichment of secreted proteins from primary Meckel cartilage cells into pathways promoting nerve development; among them, Figure 3 A is the schematic diagram of the collection and mass spectrometry determination of secreted proteins from Meckel cartilage, Figure 3 B is the display of the pathway by which Meckel cartilage promotes nerve development.

[0052] Figure 4 Promotion of neurite outgrowth by secreted proteins from primary Meckel cartilage cells; among them, Figure 4 A is the schematic diagram of co-culturing Meckel cartilage medium with neurons, Figure 4 B is the MAP2 staining diagram of neurons in each group, Figure 4 C is the statistical chart of the neurite length of neurons in each group; Figure 4 Shows the effect of applying GRN alone, similar to adding MC medium (the average neurite length of the ordinate is 53, 80, 87 respectively), so it is considered that GRN mainly plays a key role.

[0053] Figure 5 Attraction of directional growth of nerve fibers by secreted proteins from Meckel cartilage cells through affecting the remodeling of the neuronal cytoskeleton; among them, Figure 5 A is the schematic diagram of co-culturing Meckel cartilage organoids with neurons, Figure 5 B is the identification of Meckel cartilage organoids, that is, expressing the marker of chondrocytes: Col2a1, Figure 5Figure C shows the immunofluorescence staining image after co - culturing Merkel cartilage organoids with neurons, indicating that the neuronal processes are attracted by Merkel cartilage. Figure 5 Figure D shows the immunofluorescence staining image of NF200 (nerve) and RAC1 (one of the indicators of cytoskeletal remodeling) in the mandible. Figure 5 Figure E is a graph with the center at MC (abscissa distance is 0), and the average RAC1 expression in the nerves of three circles with the same interval as the ordinate. Figure 5 Figure F shows the immunofluorescence staining image of NF200 (nerve) and CDC42 (one of the indicators of cytoskeletal remodeling) in the mandible. Figure 5 Figure G is a graph with the center at MC (abscissa distance is 0), and the average CDC42 expression in the nerves of three circles with the same interval as the ordinate. Detailed implementation mode

[0054] To make the purpose, technical solutions and advantages of this application clearer, the following further details this application. However, it should be understood that the description here is only for explaining this application and not for limiting the scope of this application.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art and will not be elaborated herein.

[0056] To further understand this application, the following further details this application in combination with the best embodiments. Embodiment

[0057] 1. Composition ratio of the secretory factor combination of Merkel cartilage cells:

[0058] The specific ratio is shown in Table 1.

[0059] Table 1

[0060] Secretory factor (gene name) Ratio range Preferred ratio CLU 0-1.5 1 GRN 0.3-0.5 0.439 IGFBP5 0-0.5 0.397 IGFBP3 0-0.5 0.384 SPARC 0-0.5 0.384 APP 0-0.5 0.384 SEMA3A 0-0.3 0.2 IGFBP4 0-0.2 0.132 IGFBP2 0-0.1 0.062 SEMA3F 0-0.1 0.038

[0061] 2. Culture of primary Merkel cartilage cells and organoids:

[0062] (1) Culture of primary Merkel cartilage cells and collection of secreted proteins ( Figure 3 A):

[0063] 1) Culture of primary Merkel cartilage cells:

[0064] First, under sterile conditions, the Meckel's cartilage was separated from the mandible. The cartilage tissue was repeatedly rinsed with PBS buffer containing antibiotics to remove impurities and bloodstains. The cartilage was cut into small pieces and placed in type II collagenase, and digested at 35 - 40 °C (preferably 37 °C) for 50 - 70 min (preferably 60 min) with continuous shaking during this period. Subsequently, cartilage culture medium containing 8 - 12% (preferably 10%) serum was added to terminate the digestion. The mixture was filtered through a 35 - 45 μm (preferably 40 μm) filter to remove undigested tissue debris, and the cell suspension was collected. The cell suspension was centrifuged at 1000 - 1500 rpm (preferably 1200 rpm) for 4 - 6 minutes (preferably 5 minutes), and the supernatant was discarded. The cell pellet was resuspended, and the cells were seeded into culture flasks or plates and cultured at 35 - 40 °C (preferably 37 °C) and 4 - 6% (preferably 5%) CO 2 cultured under conditions, and the cell growth status was observed regularly.

[0065] 2) Collection of secreted proteins from primary Meckel's cartilage cells:

[0066] The culture medium was collected after culturing the primary Meckel's cartilage cells for three days;

[0067] 3) Determination of secreted proteins from primary Meckel's cartilage cells:

[0068] Trypsin was used to digest the proteins in the collected culture medium into peptide fragments; isotope labeling was performed for quantitative comparison, etc.; chromatographic techniques were used for preliminary separation of the peptide fragments; the separated peptide fragments were introduced into a mass spectrometer for detection to obtain information such as the mass and charge of the peptide fragments; the mass spectrometry data were processed and analyzed, including peptide fragment identification, protein assembly, quantitative calculation, differential analysis, etc.

[0069] (2) Culture of Meckel's cartilage organoids ( Figure 5 A):

[0070] First, Meckel's cartilage was separated, cut into pieces, digested with type II collagenase, and centrifuged at 3 - 5 °C and 1000 - 1500 rpm for 4 - 6 minutes to collect cells according to the method for culturing primary Meckel's cartilage cells. Next, the cell suspension was mixed with Matrigel (BD Matrigel, 354234) at a ratio of (0.5 - 1):(0.5 - 1) (preferably 1:1), seeded into 24-well plates, with 40 - 60 μl of the suspension in each well, and left to stand in an incubator for 25 - 35 minutes. After it solidified, Meckel's cartilage culture medium (Procell, Complete Medium for Human Chondrocytes CM-H107) was added to the 24-well plates. Example

[0071] 1. Culture of primary Meckel's cartilage cells:

[0072] (1)First, under sterile conditions, the Meckel's cartilage was separated from the mandible. The cartilage tissue was repeatedly rinsed with PBS buffer containing antibiotics to remove impurities and bloodstains.

[0073] (2)The cartilage was cut into small pieces and placed in type II collagenase for digestion at 37 °C for 1 hour, with continuous shaking during this period.

[0074] (3)Subsequently, cartilage medium containing 10% serum was added to terminate the digestion.

[0075] (4)It was filtered through a 40 μm filter to remove undigested tissue fragments, and the cell suspension was collected.

[0076] (5)The cell suspension was centrifuged at 1200 rmp for 5 minutes, and the supernatant was discarded.

[0077] (6)The cell pellet was resuspended, and the cells were seeded into culture flasks or culture plates and cultured under the conditions of 37 °C and 5% CO 2 The cell growth status was observed regularly; the concentration of GRN in the culture medium was determined by ELISA to be 10.23 nM / L (the rest can be calculated by proportion).

[0078] 2. Three-dimensional reconstruction of HE-stained sections ( Figure 1 , Figure 2 A):

[0079] Considering that HE-stained sections can only show the positional relationship between Meckel's cartilage and structures such as nerves in this plane, the applicant further clarified the overall spatial position through three-dimensional reconstruction. The specific method is as follows:

[0080] The obtained human mandible was paraffin-embedded, then serially sectioned and HE-stained. The scanned HE-stained pictures were imported into mimics Research20, the section directions were adjusted, and the areas of interest (mandible, Meckel's cartilage, nerves, etc.) were circled. Subsequently, all consecutive sections were selected for three-dimensional reconstruction, and finally, the color and smoothness of the reconstructed images were adjusted.

[0081] 3. Culture of Meckel's cartilage organoids ( Figure 5 A):

[0082] (1)First, Meckel's cartilage was separated according to the method of primary cell culture of Meckel's cartilage, cut into pieces, digested with type II collagenase, and centrifuged at 4 °C and 1200 rpm for 5 minutes to collect cells.

[0083] (2)Next, the cell suspension was mixed with Matrigel at a ratio of 1:1, seeded in 24-well plates, 50 μl of the suspension was added to each well, and it was placed in an incubator and allowed to stand for 30 minutes. After it solidified, Meckel's cartilage medium was added to the 24-well plates.

[0084] Figure 5 In this study, Merkel chondroid organs were used for co-culture with neurons to verify whether Merkel cartilage attracts the processes of neurons (directive effect).

[0085] 4. Primary neuron cell culture:

[0086] (1) Take the cerebral cortex of mice at 0 - 3 days after birth, isolate the cortical tissue under a stereomicroscope, and remove the pia mater, blood vessels, and choroid plexus.

[0087] (2) Chop the brain tissue and digest it with 0.25% trypsin at 37°C with shaking for 10 minutes.

[0088] (3) Terminate the digestion with neuron medium containing 10% serum, and pipette the cell suspension until no tissue clumps are visible to the naked eye.

[0089] (4) Centrifuge at 100g, and filter the supernatant through a 40μm cell strainer.

[0090] (5) Centrifuge the filtrate at 400g for 12 minutes, discard the supernatant, resuspend the pellet and seed the cells. Use the serum-free neuron cell culture method for mixed neuron cell culture. According to the growth curve characteristics of astrocytes, add cytosine arabinoside at a final concentration of 5 μmol / L to the culture medium between 18 - 24 hours after cell seeding to inhibit the proliferation of astrocytes. Then change the culture medium by half every 3 days, and conduct the experiment on the 7th day.

[0091] 5. Co-culture of Merkel chondroid organs and primary neuron cells ( Figure 5 C):

[0092] Passage the Merkel chondroid organs (spherical organoids with a three-dimensional structure formed by culturing the cultured Merkel chondrocytes in Matrigel), and after passage, inoculate them into the primary neuron cells that have grown for 4 days, and then continue to culture for about 3 days. Fix and immunofluorescently stain the co-cultured Merkel chondroid organs and neurons, and take pictures with a confocal microscope to clarify the regulation of the orientation of neuron processes by Merkel chondroid organs.

[0093] 6. Co-culture of Merkel chondrocyte medium and primary neuron cells ( Figure 4 ):

[0094] To verify whether Merkel cartilage can promote the growth of neuron processes, a co-culture method of adding the medium of Merkel cartilage to neurons was adopted. The specific method is as follows:

[0095] The culture medium of primary cultured Merkel chondrocytes was added to the primary neurons that had been growing for 4 days, and the neurons were cultured for another three days. In the GRN group, 10 nM / L of GRN protein (Progranulin / PGRN protein, Human, purchased from MCE, HY-P74618) was added to the cultured neurons. The reason for choosing a concentration of 10 nM / L was that, as determined by an ELISA kit (purchased from Lianbo Biotechnology Co., Ltd., LBK-H04284), the concentration of GRN in the Merkel cartilage culture medium was approximately 10 nM / L. After three days, the cells in each group were fixed and immunofluorescently stained with MAP2 to statistically analyze the length of neuronal processes.

[0096] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present application shall be included within the protection scope of the present application.

Claims

1. Use of a secretory protein combination derived from Merkel chondrocytes in the preparation of a drug for preventing / treating peripheral nerve damage, characterized in that: The combination includes GRN protein and the following proteins: CLU, IGFBP5, IGFBP3, SPARC, APP, SEMA3A, IGFBP4, IGFBP2 and SEMA3F; the concentration ratio of each protein in the secretory protein combination is selected as CLU: GRN: IGFBP5: IGFBP3: SPARC: APP: SEMA3A: IGFBP4: IGFBP2: SEMA3F, which is 1: 0.439: 0.397: 0.384: 0.384: 0.384: 0.2: 0.132: 0.062: 0.

038.

2. Use of a secretory protein combination derived from Merkel chondrocytes in the preparation of a drug for promoting directional growth and repair of peripheral nerves, characterized in that: The combination includes GRN protein and the following proteins: CLU, IGFBP5, IGFBP3, SPARC, APP, SEMA3A, IGFBP4, IGFBP2 and SEMA3F; the concentration ratio of each protein in the secretory protein combination is selected as CLU: GRN: IGFBP5: IGFBP3: SPARC: APP: SEMA3A: IGFBP4: IGFBP2: SEMA3F, which is 1: 0.439: 0.397: 0.384: 0.384: 0.384: 0.2: 0.132: 0.062: 0.

038.

3. Use of a secretory protein combination derived from Merkel chondrocytes in the preparation of a biomaterial for promoting directional growth of peripheral nerves, characterized in that: The combination includes GRN protein and the following proteins: CLU, IGFBP5, IGFBP3, SPARC, APP, SEMA3A, IGFBP4, IGFBP2 and SEMA3F; the concentration ratio of each protein in the secretory protein combination is selected as CLU: GRN: IGFBP5: IGFBP3: SPARC: APP: SEMA3A: IGFBP4: IGFBP2: SEMA3F, which is 1: 0.439: 0.397: 0.384: 0.384: 0.384: 0.2: 0.132: 0.062: 0.038; the biomaterial is selected as a nerve conduit material or a scaffold material.

4. The use according to any one of claims 1 to 3, characterized in that: The method for preparing the secretory protein combination comprises the following steps: S1. Culture of primary Merkel cartilage cells: The Meckel's cartilage isolated from the mandible was rinsed, filtered after digestion, and the cell suspension was collected. After the cell suspension was centrifuged, the supernatant was discarded and the cell pellet was resuspended and inoculated for culture; S2. Collection of secretory proteins from Merkel's primary cartilage cells: Collect the culture medium of Merkel's primary cartilage cells cultured in step S1; S3. Determination of secretory proteins of primary Merkel cartilage cells: The culture medium containing secretory proteins of primary Merkel cartilage cells collected in step S2 is digested into peptides by protease, and then the peptides are preliminarily separated by chromatography. The separated peptides are detected by mass spectrometer to obtain peptide information.

5. The use according to claim 4, characterized in that: In step S1, the washing is performed using a PBS buffer containing antibiotics; Digestion was performed using 0.2% type II collagenase at a temperature of 35°C-40°C for 50 min-70 min, and digestion was terminated using cartilage culture medium containing 10% serum; filtration was performed using a 35μm-45μm filter; the centrifugal speed was 1000rpm-1500rpm, and the centrifugal time was 4min-6min; the culture conditions after cell inoculation were 35°C-40°C, 4%-6% CO2.

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