Screening method of recombinant protein for inhibiting phagocytic function of macrophages on myelin sheath protein and hydrogel preparation

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a screening method of a recombinant protein for inhibiting the phagocytic function of macrophages on myelin sheath protein and a hydrogel preparation. The invention firstly provides a screening method of the recombinant protein, which comprises the following steps: co-culturing a recombinant protein solution and macrophages in an environment rich in myelin protein, and detecting various indexes to screen out the recombinant protein capable of remarkably inhibiting the phagocytic function of the macrophages on the myelin protein. The invention discloses regulation and control of the macrophage phenotypic change and the relationship between the macrophage phenotypic change and the MD phagocytosis function of the specific recombinant protein for the first time, and provides a new thought for screening the recombinant keratin with a specific function.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a method for screening a recombinant protein that inhibits the phagocytic function of macrophages on myelin proteins and a hydrogel preparation. Background Art

[0002] Spinal cord injury (SCI) is a central nervous system disease with a high disability rate, which often leads to persistent local accumulation of myelin debris (MD). Macrophages that dominate its clearance often form foam cells due to phagocytosis of excessive MD, resulting in a slowdown in the phagocytosis of local apoptotic neutrophils, thereby inhibiting neuronal regeneration and prolonging the duration of the inflammatory response.

[0003] Previous studies have found that recombinant human hair keratin plays a role in regulating neuroinflammation and promoting neural regeneration after spinal cord injury. However, existing treatments for SCI have significant limitations, primarily due to the inability of commonly used local intraperitoneal injections of modulators to precisely regulate local spinal cord injury. Furthermore, there are currently no reports on the effect of recombinant human hair keratin on macrophage phagocytosis of MD, making it difficult to regulate the phagocytic function of macrophages in the injured area.

[0004] In summary, it is necessary to propose new strategies and improvements to address the shortcomings of existing technologies. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for screening recombinant keratin that inhibits the phagocytic function of macrophages and a hydrogel preparation containing the recombinant keratin, so as to partially solve or alleviate the above-mentioned deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0006] The first aspect of the present invention is to provide a method for screening recombinant keratin that can inhibit the phagocytosis of MD by macrophages.

[0007] A method for screening a recombinant protein that inhibits the phagocytic function of macrophages on myelin proteins comprises the following steps: S01: preparing a variety of recombinant human hair keratins, formulating the recombinant human hair keratins into a protein solution and co-culturing the solution with macrophages in an environment rich in myelin proteins; S02: performing a first indicator test, wherein the first indicator is the phagocytosis rate of myelin sheath protein by macrophages; screening out recombinant human hair keratin types whose phagocytosis rate of myelin sheath protein by macrophages is less than 20%; S03: Performing a second indicator test on the recombinant human hair keratin types screened in S02, wherein the second indicator is the transcription level of foam cell-specific marker protein; screening out recombinant human hair keratin types that can significantly reduce foam cell-specific marker protein; the foam cell-specific marker protein includes one or more of ABCA1, ABCG1, CD9, GPNMB, MMP9, OLR1, SPP1 and / or TREM2.

[0008] Preferably, the foam cell-specific marker proteins include ABCA1, ABCG1, CD9, GPNMB, MMP9, OLR1, SPP1 and TREM2.

[0009] Furthermore, the screening method may further include a third indicator, which is intra-macrophage lipid staining; performing intra-macrophage lipid staining on the recombinant human hair keratin screened by S02; and screening out the recombinant human hair keratin type that reduces the intra-macrophage lipid staining area by approximately 2 / 3.

[0010] As a most preferred embodiment, the method for screening a recombinant protein that inhibits the phagocytic function of macrophages on myelin proteins comprises the following steps: S01: preparing a variety of recombinant human hair keratins, formulating the recombinant human hair keratins into a protein solution and co-culturing the solution with macrophages in an environment rich in myelin proteins; S02: performing a first indicator and a second indicator test, wherein the first indicator is the phagocytosis rate of myelin sheath proteins by macrophages, and the second indicator is lipid staining in macrophages; screening for recombinant human hair keratin types that simultaneously meet the requirements of a macrophage phagocytosis rate of myelin sheath proteins less than 20% and a reduction in the lipid staining area in macrophages by approximately 2 / 3; S03: Perform a third indicator test (verification) on the recombinant human hair keratin types screened in S02, wherein the third indicator is the transcription level of foam cell-specific marker proteins; screen out recombinant human hair keratin types that can significantly reduce foam cell-specific marker proteins; the foam cell-specific marker proteins include ABCA1, ABCG1, CD9, GPNMB, MMP9, OLR1, SPP1 and TREM2. Furthermore, the plurality of recombinant human hair keratins described in S01 include RK86, RK85, RK84, RK83, RK82, RK81, RK40, RK39, RK38, RK37, RK36, RK35, RK34, RK33B, RK33A, RK32, and RK31.

[0011] Furthermore, the concentration of the protein solution prepared in S01 is 0.2 mg / mL.

[0012] Furthermore, the concentration of myelin protein in S01 was 10 mg / mL.

[0013] The recombinant human hair keratin obtained by the above screening method, which inhibits the phagocytosis of myelin proteins by macrophages, is RK31, and the amino acid sequence is shown in SEQ ID NO.1.

[0014] Another aspect of the present invention is to provide a recombinant human hair keratin hydrogel preparation capable of accurately repairing local SCI damage.

[0015] A recombinant keratin hydrogel preparation for alleviating inflammatory response after spinal cord injury, the recombinant keratin hydrogel preparation comprising recombinant human hair keratin RK31 and a light-curable hydrogel; the amino acid sequence of the recombinant human hair keratin RK31 is shown in SEQ ID NO.1; and the light-curable hydrogel is methacrylated gelatin.

[0016] Furthermore, the recombinant keratin hydrogel preparation has the effect of reducing the inflammatory response after spinal cord injury by inhibiting macrophages at the spinal cord injury site from phagocytizing myelin proteins to form foam cells.

[0017] Furthermore, the recombinant keratin hydrogel preparation has a structure that couples with the irregular wound at the spinal cord injury site.

[0018] Preferably, the volume ratio of the recombinant human keratin RK31 to the light-curable hydrogel is 2:1.

[0019] Furthermore, a photoinitiator is added to the photocurable hydrogel, and the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

[0020] Beneficial technical effects: (1) The present invention first provides a method for screening functional recombinant human hair keratin based on the investigation of the phagocytic function of macrophages on MD. The method provided by the present invention involves the detection of multiple indicators to screen out recombinant human hair keratin that can significantly inhibit the phagocytic function of macrophages on myelin proteins; the detection indicators include the phagocytic rate of macrophages on MD and / or the accumulation of intracellular lipids of macrophages in an MD-rich environment, and the transcription level of foam cell-specific marker proteins. The first two indicators can be used to see from a relatively intuitive level whether a specific keratin has an inhibitory effect on the phagocytosis of MD by macrophages; the last indicator can accurately reflect whether a specific keratin inhibits the transcription level of foam cell-specific marker proteins, thereby screening out recombinant human hair keratin that can significantly inhibit the phagocytic function of macrophages on myelin proteins for practical application. It is worth noting that the present invention discloses for the first time the relationship between the regulation of macrophage phenotypic changes by specific recombinant keratin subtypes and the relationship between macrophage phenotypic changes and their phagocytic function of MD, providing a new idea for screening recombinant human hair keratin with specific functions.

[0021] (2) Secondly, based on the screened recombinant human hair keratin RK31 that can significantly inhibit the phagocytic function of macrophages on myelin proteins, the present invention also provides a hydrogel preparation loaded with high concentrations of recombinant human hair keratin RK31. The hydrogel preparation can be coupled with complex spinal cord injury wounds to achieve sustained release at the injury site, and achieve efficient repair of spinal cord injury by controlling the inflammatory response. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work.

[0023] Figure 1 This is an SDS-PAGE image of 17 recombinant human hair keratins synthesized in one embodiment of the present invention; Figure 2 This is a verification of the ability of 17 recombinant human hair keratins synthesized in one embodiment of the present invention to phagocytose MD by macrophages; Figure 3 This is a verification of the effects of 17 recombinant human hair keratins synthesized in one embodiment of the present invention on the intracellular lipid accumulation of macrophages in an MD-rich environment; Figure 4This is one of the examples of the present invention, which verifies the transcription level of maeker protein in foam cells of macrophages in a MD-rich environment by RK31; Figure 5 In one of the embodiments of the present invention, the effect of RK31 on the transcriptional level of macrophage MD phagocytosis targets was verified; Figure 6 In one embodiment of the present invention, the effect of macrophages on MD phagocytosis in the presence of an antagonist is verified; Figure 7 This is the effect of RK31 with a gradient decreasing concentration on various indicators of macrophages in an MD-rich environment in one embodiment of the present invention; Figure 8 The photocuring process of the RK31-loaded hydrogel in one embodiment of the present invention and the pore image under a scanning electron microscope (scale bar: 300 μm); Figure 9 This is a Fourier transform infrared spectrum of the RK31-loaded hydrogel in one embodiment of the present invention; Figure 10 This is a verification of the mechanical properties of the RK31-loaded hydrogel in one of the embodiments of the present invention; Figure 11 The footprint test (4 weeks and 8 weeks), BBB score (1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 weeks) and ramp test (8 weeks) at different time points after implantation of RK31-loaded hydrogel in a rat SCI hemisection model according to one embodiment of the present invention; Figure 12 The accumulation of foam cells at different time points (4 weeks and 8 weeks) after implantation of RK31-loaded hydrogel in a rat SCI hemisection model in one embodiment of the present invention; Figure 13 This is the local nerve regeneration 8 weeks after the RK31-loaded hydrogel in one embodiment of the present invention was implanted into a rat SCI hemisection model; Figure 14 The results of differential expression of local inflammatory factors 8 weeks after implantation of RK31-loaded hydrogel in a rat SCI hemisection model in one of the embodiments of the present invention. DETAILED DESCRIPTION

[0024] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0026] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0027] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0028] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0029] Example 1 In this example, 17 keratin subtypes were recombinantly expressed using a prokaryotic recombinant expression system. After successful expression was verified, they were added to a macrophage culture environment rich in MD to further explore the effects of different keratin subtypes on the macrophage phagocytosis of MD and whether they affect foam cell formation.

[0030] First, this example synthesized 17 recombinant human hair keratin subtypes based on the prokaryotic recombinant expression system, namely RK86, RK85, RK84, RK83, RK82, RK81, RK40, RK39, RK38, RK37, RK36, RK35, RK34, RK33B, RK33A, RK32 and RK31, and their SDS-PAGE images are shown in FIG. Figure 1 .

[0031] Furthermore, we investigated the effects of the 17 recombinant human hair keratins on the phagocytic function of macrophages in an MD-rich environment. The method was as follows: 17 recombinant human hair keratins were co-cultured with macrophages in an MD-rich environment at 0.20 mg / mL, where the MD content in the MD-rich environment was 10 mg / mL. The cells were cultured for 24 hours, and the phagocytic rate of macrophages on MD was detected by flow cytometry. Figure 2 The results showed that the RK31 group had the lowest proportion of MD.

[0032] Further, we investigated the effects of the above 17 recombinant human hair keratins on the intracellular lipid accumulation of macrophages in a MD-rich environment. Figure 3 . Figure 3 The left picture shows Nile red staining, which can mark lipid droplets and cell membranes and help determine the content of intracellular lipid droplets. Figure 3 The right image shows Oil Red O staining, which marks intracellular lipid droplets. Both images show that the RK31 group had the smallest lipid droplet area, demonstrating that recombinant protein RK31 can effectively reduce lipid accumulation in macrophages.

[0033] Based on the above experimental results, we further explored the effect of RK31 on the transcriptional level of maeker protein in foam cells of macrophages in a MD-rich environment. Figure 4 The results showed that most of the protein markers in the RK31+MD group were reduced, proving that RK31 can effectively inhibit the phagocytosis of MD by macrophages.

[0034] Example 2 In this example, by adding antagonist components and gradient concentration of RK31, the mechanism of action of RK31 in reducing excessive phagocytosis of MD and reducing foam cell formation by inhibiting LRP1 expression in macrophages was explored.

[0035] In this example, the antagonist used was LRPAP1 protein at a concentration of 2 μg / mL. The incubation time with macrophages, MD, and RK31 was 2 hours. The starting concentration of RK31 protein was set at 0.20 mg / mL, and the concentration gradient was set at 0.20 mg / mL, 0.13 mg / mL, 0.07 mg / mL, and 0. Flow cytometry was then used to examine the inhibitory effect of LRPAP1 protein on MD phagocytosis by macrophages.

[0036] The experimental results are shown in Figure 5-Figure 7 . Figure 5 The RT-qPCR results show changes in gene transcription levels, among which RK31 significantly inhibited the transcription of the macrophage LRP1 gene, proving that RK31 affects the transcription level of macrophage MD phagocytic targets. Figure 6 It was shown that LRP1 antagonist significantly inhibited the phagocytosis of MD by macrophages. Figure 7 It was shown that the gradient-decreasing concentration of RK31 gradually restored the LRP1 expression, phagocytic activity of MD, and intracellular lipid accumulation of macrophages in an MD-rich environment, and gradually lost its inhibitory effect on foam cell formation.

[0037] Example 3 This embodiment provides a hydrogel preparation containing recombinant keratin RK31 and functional verification of the hydrogel preparation.

[0038] First, a method for preparing a hydrogel formulation containing recombinant keratin RK31 is provided: 20 μL of a 10 mg / mL RK31 aqueous solution, 250 μL of a 20% w / v GelMA solution, and 630 μL of ddH2O are mixed, 100 μL of LAP (1.5% w / v) is added, the mixture is mixed thoroughly, and the mixture is UV-cured to produce a hydrogel containing a low concentration of recombinant keratin RK31, labeled Gel-RK31(L). 500 μL of a 10 mg / mL RK31 aqueous solution, 250 μL of a 20% w / v GelMA solution, and 150 μL of ddH2O are mixed, 100 μL of LAP (1.5% w / v) is added, the mixture is mixed thoroughly, and the mixture is UV-cured to produce a hydrogel containing a low concentration of recombinant keratin RK31, labeled Gel-RK31(H). The amino acid sequence of the recombinant keratin RK31 used in this example is set forth in SEQ ID NO. 1.

[0039] Table 1 The hydrogel loaded with recombinant keratin RK31 prepared above was verified, and the results are shown in Figures 8-10 The results showed that the pore size of the hydrogel loaded with recombinant keratin RK31 was 300 μm. Larger pores can promote the exchange of hydrogel substances and cell colonization. Fourier transform infrared spectroscopy showed the appearance of characteristic peaks: amide A (2800 - 4000 cm -1 ), Amide I band (1600 - 1700 cm -1 ), amide II band (1480 - 1580 cm -1 ), amide III band (1220 - 1330 cm -1 ), amide B band (3069 cm -1 Mechanical properties of the recombinant keratin RK31-loaded hydrogel were tested, showing a 24-hour swelling rate of 1500%, a 4-week degradation rate of 50%, and a compression modulus of 6 kPa. This excellent swelling rate allows the hydrogel to adhere to irregular wounds.

[0040] The above results show that the hydrogel loaded with recombinant keratin RK31 has excellent swelling properties and can couple with irregular wounds of spinal cord injury in a short period of time to achieve sustained release and efficient and precise repair; in addition, the prepared hybrid hydrogel maximizes the retention of the excellent performance of GelMA hydrogel and the chemical structure characteristics of RK31, without significantly damaging the mechanical properties of GelMA.

[0041] Example 4 This example further verifies the effect of the recombinant keratin RK31-loaded hydrogel prepared in Example 3 in an injury animal model.

[0042] (1) RK31-GelMA hydrogel was implanted into a rat spinal cord injury hemisection model and the recovery of motor function in the rats was verified.

[0043] Experimental Methods: The BBB method was used to evaluate the recovery of motor function of the affected hind limb in rats under open conditions. Two independent researchers systematically evaluated and recorded scores weekly for 8 weeks after surgery.

[0044] Footprint experiment: At 4 and 8 weeks after SCI, rats were placed in a specially designed tunnel with a darkroom at the end to train them to move within the tunnel. White paper was laid on the tunnel floor to track the rats' footprints. To facilitate tracking, the instep of each rat's hind limb was evenly painted with red wash paint, while the sole was painted with blue wash paint. Prior to the experiment, the rats were trained to move from the beginning of the tunnel to the darkroom, so their footprints could be collected and photographed for analysis.

[0045] Slope test: 8 weeks after SCI, rats were placed on an inclined wooden board covered with a rubber mat. The longitudinal axis of each rat's body was parallel to the inclined board, with the head facing the elevated end. The maximum angle the rat could maintain on the inclined board for 5 seconds was recorded, starting from 0° and gradually increasing in increments of 2°. Five measurements were performed on each rat, and the average value was calculated and recorded. Results are shown in Figure 11 .

[0046] Table 2 BBB scoring results (2) At 4 and 8 weeks after spinal cord injury, the effect of hydrogel implantation on foam cell production at the injury site was investigated.

[0047] Experimental Methods: Rats were anesthetized with an intraperitoneal injection of pentobarbital (30 mg / kg) and secured in the supine position on an operating table. The xiphoid process was palpated upwards as a landmark. The thorax was gradually opened along the lower edge of the ribs using a sharp knife to expose the heart, lungs, and liver. Care was taken to avoid damaging the heart and lung lobes during the procedure, and to avoid contact with major thoracic vessels to prevent rat death. The infusion needle was slowly inserted into the heart from the left ventricle. A vascular clamp was then used to secure the needle to prevent it from slipping out. A careful incision was made in the right atrial appendage. Pre-chilled sterile saline was slowly perfused into the heart, initially at a slow rate and then increasing in speed, until the fluid flowing out of the right atrial appendage became clear. Following saline perfusion, pre-chilled 4% paraformaldehyde was then perfused. A minimum of 200 mL of 4% paraformaldehyde was administered to each rat. Successful perfusion was confirmed by limb twitching. The modeling site was exposed layer by layer using a sharp knife. A 2-3 cm section of the spinal column was resected, centered at the injury site. Spinal cord tissue was then carefully removed on ice using forceps and rongeurs and photographed according to group. The harvested spinal cord tissue was fixed in 4% paraformaldehyde for 6 h, then washed and dehydrated by immersion in 10%, 20%, and 30% sucrose solutions. After complete sedimentation, the tissue surface was blotted dry with filter paper, embedded in OCT embedding medium, and frozen at -80°C. The cryostat was set to -25°C. The embedded tissue was mounted on the cryostat stage and allowed to warm briefly before sectioning for 30 minutes. The cryostat section thickness was set to 25 μm (Oil Red O staining), and serial longitudinal sections were made parallel to the long axis of the spinal cord. Tissue sections were mounted on high-adhesion slides and examined for tissue integrity under a light microscope. Intact sections were labeled and sealed after the OCT embedding medium thawed and stored at -20°C. The processed sections were immersed in 60% isopropyl alcohol for 10 minutes. The isopropanol was then aspirated and the sample was immersed in the prepared Oil Red O staining working solution and incubated in the dark at room temperature for 15 min.

[0048] After incubation, the staining solution was discarded and the sample was washed three times with PBS for 5 minutes each time. 60% isopropanol was added and incubated for another 30 seconds. After removing the isopropanol, the sample was washed again. The slides were mounted with glycerol gelatin and observed and photographed under an optical microscope. Figure 12 .

[0049] (3) At 8 weeks after spinal cord injury, investigate the effect of hydrogel implantation on nerve regeneration and repair at the injured site.

[0050] Experimental method: The frozen spinal cord sections were taken out of the -20℃ refrigerator, dried thoroughly at room temperature, then placed in a 65℃ oven for 1 hour, taken out, and slowly cooled at room temperature. They were then immersed in PBS buffer and rinsed for 5 minutes, repeated 3 times (the processed sections can be used for Oil Red O staining and LFB staining), then immersed in Tris-EDTA antigen retrieval solution, and then set the microwave oven to medium heat: 8 minutes, low heat: 12 minutes, and then slowly cooled at room temperature. The processed sections can be subjected to tissue immunofluorescence staining. For immunohistochemical staining, 3% H2O2 solution needs to be added to the sections, and they need to be allowed to stand in a wet box at room temperature for 10 minutes, and then rinsed with PBS for 5 minutes, repeated 3 times. The processed sections were circled with an immunohistochemistry pen to mark the area to be stained, and then a permeabilization solution (containing 0.3% TritonX-100) was added to permeabilize the tissue for 10 minutes. After washing, the sections were blocked with a tissue blocking solution containing 5% BSA in a humidified chamber at room temperature for 1 hour. The NEUN / GFAP antibody diluted in proportion was added to the area to be stained after the tissue blocking solution was removed, slightly submerged, and placed in a humidified chamber for overnight incubation at 4°C. The humidified chamber was removed and placed at room temperature for 30 minutes to rewarm. The primary antibody was slowly removed and the sections were rinsed three times with PBST buffer for 15 minutes each. The tissue was covered with the diluted fluorescent-labeled secondary antibody and incubated in a humidified chamber at room temperature for 1 hour. The sections were then rinsed three times with PBST buffer in the dark for 15 minutes each. An appropriate amount of anti-fluorescence anti-fading DAPI mounting solution was added to the washed sections, and the tissue was sealed after slowly expelling bubbles with a coverslip. The sealed sections were scanned under a fluorescence microscope to obtain images for subsequent analysis. The results are shown in Figure 13 The damaged tissue was divided into three distinct zones: the injury zone (IA: composed of newly formed tissue), the scar zone (SA: composed of localized glial scars after injury), and the degenerative zone (DA: a zone retaining a large number of necrotic cells and MD). The figure shows that Gel-RK31(H) has a significant repair effect on the injury zone, with its repair effect significantly higher than that of Gel-RK31(L).

[0051] Furthermore, the differential expression levels of local inflammatory factors at 8 weeks after the RK31-loaded hydrogel was implanted into the rat SCI hemisection model, including the changes in the pro-inflammatory polarization phenotype (CD86 and TNF-α) and anti-inflammatory polarization phenotype (CD206 and Arg-1) of macrophages, are shown in Figure 2. Figure 14 In the figure, I represents control, II represents Gel, III represents Gel-RK31(L), and IV represents Gel-RK31(H). The results showed that Gel-RK31(H) can significantly inhibit the expression level of the pro-inflammatory polarization phenotype of macrophages after spinal cord injury and promote the expression level of the anti-inflammatory polarization phenotype, thereby alleviating the level of inflammation after spinal cord injury.

[0052] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0053] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A method for screening a recombinant protein that inhibits the phagocytic function of macrophages on myelin proteins, characterized in that: The following steps are involved: S01: preparing a variety of recombinant human hair keratins, formulating the recombinant human hair keratins into a protein solution and co-culturing the solution with macrophages in an environment rich in myelin proteins; S02: performing a first indicator test, wherein the first indicator is the phagocytosis rate of myelin sheath protein by macrophages; screening out recombinant human hair keratin types whose phagocytosis rate of myelin sheath protein by macrophages is less than 20%; S03: Performing a second indicator test on the recombinant human hair keratin types screened in S02, wherein the second indicator is the transcription level of foam cell-specific marker protein; screening out recombinant human hair keratin types that can significantly reduce foam cell-specific marker protein; the foam cell-specific marker protein includes one or more of ABCA1, ABCG1, CD9, GPNMB, MMP9, OLR1, SPP1 and / or TREM2.

2. The screening method according to claim 1, wherein The screening method further includes a third indicator, which is intra-macrophage lipid staining; the recombinant human hair keratin types screened by S02 are subjected to intra-macrophage lipid staining; and the recombinant human hair keratin types that reduce the intra-macrophage lipid staining area by approximately 2 / 3 are screened.

3. The screening method according to claim 1, wherein The various recombinant human hair keratins described in S01 include RK86, RK85, RK84, RK83, RK82, RK81, RK40, RK39, RK38, RK37, RK36, RK35, RK34, RK33B, RK33A, RK32, and RK31.

4. The screening method according to claim 1, wherein The concentration of the protein solution prepared in S01 is 0.2 mg / mL.

5. The screening method according to claim 1, wherein The concentration of myelin protein in S01 was 10 mg / mL.

6. A recombinant keratin hydrogel preparation for alleviating inflammatory response after spinal cord injury, characterized in that: The recombinant keratin hydrogel preparation comprises recombinant human hair keratin RK31 and a light-curable hydrogel; the amino acid sequence of the recombinant human hair keratin RK31 is shown in SEQ ID NO.1; and the light-curable hydrogel is methacrylated gelatin.

7. The recombinant keratin hydrogel preparation for alleviating inflammatory response after spinal cord injury according to claim 6, characterized in that: The recombinant keratin hydrogel preparation has the effect of reducing the inflammatory response after spinal cord injury by inhibiting macrophages at the spinal cord injury site from phagocytizing myelin proteins to form foam cells.

8. The recombinant keratin hydrogel preparation for alleviating inflammatory response after spinal cord injury according to claim 6, characterized in that: The recombinant keratin hydrogel preparation has a structure that couples with the irregular wound at the spinal cord injury.

9. The recombinant keratin hydrogel preparation for alleviating inflammatory response after spinal cord injury according to claim 6, characterized in that: The volume ratio of the recombinant human hair keratin RK31 to the light-curable hydrogel is 2:

1.

10. The recombinant keratin hydrogel preparation for alleviating inflammatory response after spinal cord injury according to claim 6, characterized in that: A photoinitiator is added to the photocurable hydrogel, and the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

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