Protein polymer and production process therefor

Abstract

A protein polymer and a production process therefor. The protein polymer is obtained by means of stimulating MSCs, lysing same, and separating and purifying same. The protein polymer has a strong capability to repair oxidative damage, and thus can reduce the range of cerebral infarction, improve neural functions, inhibit the level of neuroinflammation, and increase the number of surviving neurons.

Description

Protein polymer and production process thereof

[0001] The present disclosure claims priority to the Chinese patent application No. 202410944325.9, filed on July 15, 2024, entitled "Protein polymer and production process thereof", the whole content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of biotechnology, and particularly relates to a protein polymer and a production process thereof. BACKGROUND

[0003] Mesenchymal stem cells (MSCs) have self-replication and multi-directional differentiation potential, and widely exist in bone marrow, fat, synovial membrane, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amnion, peripheral blood, muscle, urine and other tissues, have characteristics of wide source, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, convenient collection, etc., can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), interferon (IFN), etc., participate in the regulation of cell growth, cell apoptosis, cell differentiation, antiviral, immune maturation and other processes, and can be used for immune regulation, tissue repair and treatment of diseases such as acute lung injury, severe pneumonia, acute respiratory distress syndrome, etc.

[0004] Mesenchymal stem cells can be cultured under different stimulation conditions to produce different stress proteins, and these stress protein polymers have complex physiological activities. How to use MSCs to produce protein polymers with specific biological activities is a very challenging work. SUMMARY

[0005] The present application aims to overcome at least one deficiency of the prior art, and provides a protein polymer and a production process thereof.

[0006] The technical solution adopted by the present application is as follows:

[0007] In a first aspect, the present application provides:

[0008] A protein polymer and a production process thereof, comprising:

[0009] S1) culturing mesenchymal stem cells and using ultraviolet irradiation to create a stress environment;

[0010] S2) lysing the mesenchymal stem cells and isolating and purifying to obtain a protein polymer;

[0011] In some examples of the protein polymer, it comprises at least the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens.

[0012] Preferably, the above two proteins account for more than 40% of the total mass of the protein polymer.

[0013] Preferably, the content of the Serum albumin protein is at least 38% of the total content of the protein polymer, and the content of the Serotransferrin protein is at least 2% of the total content of the protein polymer.

[0014] Preferably, further comprising at least one of the following proteins: sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens; sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens; sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens; sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens; sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens; sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0015] In some examples of the protein polymer, the time of the ultraviolet irradiation to stimulate the mesenchymal stem cells is 1 h to 30 h, preferably, the irradiation time is 10 h to 30 h, more preferably 6 h to 18 h.

[0016] Preferably, the intensity of the ultraviolet irradiation to stimulate is 10 μW / cm 2 ~ 100 μW / cm 2The wavelength of the ultraviolet light is preferably 290nm to 340nm.

[0017] In a preferred example, the wavelength of the ultraviolet light is 290nm to 325nm.

[0018] In a specific and preferred example, the wavelength of the ultraviolet light is 300nm to 320nm.

[0019] In a specific and preferred example, the wavelength of the ultraviolet light is 300nm to 316nm.

[0020] Preferably, the medium used in the stimulation of the irradiation of the ultraviolet light is a serum-free MSCs culture medium.

[0021] In some examples of the protein polymer, the mesenchymal stem cells are selected from umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells.

[0022] In some examples of the protein polymer, the mesenchymal stem cells are selected from human umbilical cord mesenchymal stem cells, human amniotic membrane mesenchymal stem cells.

[0023] In some examples of the protein polymer, the protein polymer is derived from cells.

[0024] In some examples of the protein polymer, the protein polymer is derived from the supernatant of the culture medium after the culture of the stem cells.

[0025] In some examples of the protein polymer, the protein polymer is derived from the supernatant of the culture medium after the culture of the stem cells and from the cells.

[0026] In some examples of the protein polymer, the lysis is performed using pure water.

[0027] The above features can be combined arbitrarily without conflict.

[0028] In a second aspect of the present application, there is provided:

[0029] A production process of a protein polymer, comprising the expansion of MSCs, the irradiation of the MSCs in the culture medium using ultraviolet light, the stress treatment, the collection of the MSCs after the stress treatment, the lysis treatment, the separation and purification of the proteins to obtain the protein polymer.

[0030] In some examples of the production process, the time for the irradiation of the mesenchymal stem cells using ultraviolet light is 1h to 30h, preferably, the irradiation time is 10h to 30h, more preferably, 6h to 18h.

[0031] Preferably, the intensity of the irradiation of the ultraviolet light is 10μW / cm 2 ~100μW / cm 2The wavelength of the ultraviolet light is preferably 290nm to 340nm.

[0032] Preferably, the culture medium used when stimulating the ultraviolet irradiation is a serum-free MSCs culture medium.

[0033] The above features can be combined arbitrarily without conflict.

[0034] In a third aspect of the present application, there is provided:

[0035] The application of the protein polymer in the first aspect of the present application includes the use for preparing a medicament for treating a neurodegenerative disease or a stroke.

[0036] The present application has the following advantages:

[0037] The protein polymer of some examples of the present application has a good cell damage repair effect and is expected to be used for treating a neurodegenerative disease or a stroke.

[0038] The production process of some examples of the present application can effectively overcome the differences between different batches of MSCs, obtain more stable MSCs with small batch differences, and greatly ensure the quality and yield of the protein polymer.

[0039] The production process of some examples of the present application can better ensure the activity of umbilical cord-derived MSCs and BMSCs, and is conducive to improving the original amount of MSCs obtained.

[0040] The production process of some examples of the present application has a high freezing and recovery activity rate of MSCs.

[0041] The production process of some examples of the present application can well separate and purify the protein polymer. BRIEF DESCRIPTION OF DRAWINGS

[0042] Fig. 1 is a growth state photo of MSCs after being cultured in Tang Yi 3D culture medium in culture test 1-1.

[0043] Fig. 2 is a cell state photo after 8h ultraviolet irradiation in culture test 1-1.

[0044] Fig. 3 is the SDS-PAGE result of the protein harvested in culture test 1-1.

[0045] Figure 4 is a photograph of the cell state before UV irradiation of MSCs in culture test 1-2.

[0046] Figure 5 is a photograph of the cell state after 6 hours of weak intensity UV irradiation of MSCs in culture test 1-2.

[0047] Figure 6 is the SDS-PAGE result of the harvested proteins in culture test 1-2.

[0048] Figure 7 is the SDS-PAGE result of the harvested intracellular proteins and medium supernatant proteins in culture test 1-3.

[0049] Figure 8 is a statistical column chart of the repair ability of damaged nerve cells by the protein polymers obtained in Experiment Two.

[0050] Figure 9 is a column chart of the proportion of motor neurons forming stress granules (SG) after sample treatment SA injury in different treatment groups in Experiment Three.

[0051] Figure 10 is the effect of protein polymers obtained in different treatment groups on the secretion of inflammatory factor IL-6 by RAW cells in Experiment Four.

[0052] Figure 11 is a statistical column chart of the repair ability of damaged nerve cells by the protein polymers obtained in different treatment groups in Experiment Five.

[0053] Figures 12 and 13 are the effects of different treatments on the infarction area of rats.

[0054] Figure 14 is the effect of different treatments on the neurological function of rats.

[0055] Figure 15 is the effect of intrathecal administration of protein polymers combined with intravenous administration on neurons. DETAILED DESCRIPTION

[0056] The technical solutions of the present application will be further illustrated below in combination with experimental examples.

[0057] Experiment One: Effect of Different Treatments on Protein Expression

[0058] 1-1 Culture Test One

[0059] Human umbilical cord mesenchymal stem cells (HUC-MSCs) were cultured in 2L of HK-G050 (PRF) 3D medium from Tang Yihui Biological Technology Co., Ltd. The total number of cells was about 5x10 8 The cells were stained and observed, and the microcarriers were basically covered with cells. The results are shown in Figure 1.

[0060] The cells were irradiated with LED ultraviolet light under the following conditions: 60μW / cm 2After 8h, 12h, 16h, 18h, 24h, 30h, sample, observe cell morphology and collect intracellular protein, and measure protein concentration.

[0061] Taking 8h ultraviolet irradiation as an example, the cell state after irradiation is shown in Figure 2. As can be seen from Figure 1 and Figure 2, the microcarriers are basically covered with cells at 0h. After ultraviolet irradiation for a period of time, the cell morphology is affected, which causes stress to the cells, and then the cells can produce stress proteins in this stress environment.

[0062] After irradiation, cells at different time points are harvested to obtain protein polymers. The specific operation is as follows: for 0h, 8h, 12h, 16h, 18h, 24h, 30h, 8mL culture is taken for sampling, and the following operations are performed: the microcarriers are intercepted by a 300-mesh filter bag. The supernatant is centrifuged at 1200rpm for 6min. Store at 4℃. The cell pellet is washed with 150mL normal saline for 3 times, lysed with 14mL pure water, and filtered through a 0.22μm filter membrane. The harvested protein is stored at -80℃ (if used for a short period of time, it can be stored at 4℃). The harvested protein is taken for gel electrophoresis analysis, and the SDS-PAGE result is shown in Figure 3.

[0063] 1-2 Culture Test Two

[0064] Recover a small crystal P8 generation HUC-MSC into 9 T25 culture bottles, add 2.5mL Huagang mesenchymal stem cell serum-free culture medium to each bottle, and inoculate the cells at a density of 1×10 5

[0065] Take 4 bottles of cells respectively, irradiate them with two different intensities of LED ultraviolet light mainly at 300nm-316nm, and irradiate them for 6h, 12h, 18h, 24h. The irradiation conditions are as follows. Carefully remove the supernatant from the cells, wash them twice with 1mL normal saline, add 660μL pure water to lyse the cells by repeated blowing for 10min, then filter them through a 0.22μm filter membrane and store them at 4℃.

[0066] The ultraviolet irradiation conditions are as follows:

[0067] The cell state before ultraviolet irradiation is shown in Figure 4, and the cell state after irradiation for 6h with weak intensity is shown in Figure 5. It can be seen that ultraviolet irradiation has an effect on the cell morphology, which causes stress to the cells, and then the cells can produce stress proteins in this stress environment. The method for measuring protein concentration is a conventional method known to those skilled in the art, for example, including Bradford method, BCA method, Lowry method, ultraviolet spectrophotometry and Kjeldahl nitrogen determination method. In the present application, the BCA method is used to measure the protein concentration at each time point. The protein concentration and volume of the harvested protein are shown in Table 1. ​

[0068] Table 1

[0069] The harvested protein SDS-PAGE results are shown in Figure 6. As can be seen from Figure 6, the expression of the target protein can be promoted under both strong and weak ultraviolet irradiation conditions. Under strong ultraviolet irradiation conditions, as the irradiation time increases, the target protein becomes purer, and when the irradiation time is about 18 h, the target protein has a high purity and a high concentration. Weak ultraviolet can effectively promote the expression of the target protein, but the irradiation time needs to be longer than that required by strong ultraviolet irradiation.

[0070] 1-3 Culture Test Three

[0071] Recover one small crystal P8 generation HUC-MSC into a T25 culture flask, add 2.5 mL of Huagang mesenchymal stem cell serum-free culture medium to each flask, and inoculate the cells at a density of 1 x 10 5

[0072] Take the cells and irradiate them under UVB ultraviolet conditions at a wavelength of 300 nm to 316 nm for 6 h, 12 h, 18 h, 24 h, and 30 h.

[0073] The ultraviolet irradiation conditions are as follows:

[0074] After the irradiation is completed, the cells are harvested at different time points to obtain protein polymers. The specific operation is as follows: take the culture medium supernatant irradiated for 6 h, 12 h, 18 h, 24 h, and 30 h, and perform the following operations: after the supernatant is filtered through a 0.22 μL filter membrane, it is stored at 4°C.

[0075] Then wash the remaining cells twice with 2 mL of normal saline, add 1 mL of pure water, and repeatedly blow the cells off the bottom of the bottle. Lyse the cells by repeatedly blowing for about 6 min, then filter them through a 0.22 μm filter membrane, and store them at 4°C for later use.

[0076] Take the harvested protein for gel electrophoresis analysis. The protein concentrations obtained under each culture condition are as follows in Table 2:

[0077] Table 2

[0078] The harvested intracellular protein and stem cell culture medium supernatant protein are analyzed using SDS-PAGE, and the results are shown in Figure 7. As shown in Figure 7, as the irradiation time increases, the content of the target protein contained in the intracellular protein gradually increases, and when the irradiation time is about 18 h, there are very few impurities, and most of the bands are target proteins. In the supernatant, there are also target protein bands.

[0079] ​The protein polymer is subjected to mass spectrometry analysis, and known proteins are matched according to the mass spectrometry data, and it is confirmed that the protein polymer contains the following two proteins: sp|P02768|ALBU_HUMAN Serum albumin OS = Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS = Homo sapiens;

[0080] Among them, the content of Serum albumin protein is at least 38% or more of the total content of the protein polymer, and the content of Serotransferrin protein is at least 2% or more of the total content of the protein polymer.

[0081] Further, in addition to the above two proteins, the protein polymer obtained by the present application further comprises at least one of the following proteins: sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens; sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens; sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens; sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens; sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens; sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0082] Experiment Two Pharmacodynamics Test of Protein Polymer for Repairing Nerve Cell Damage

[0083] Cell modeling and detection: SH-SY5Y cells were seeded for 24 h, and then divided into three groups: normal group, model group and drug administration group. The model group and drug administration group were damaged by 250 μM H2O2 for 30 min, and then the supernatant was discarded. The drug administration group was administered at a concentration of 100 ng / mL protein concentration, with 5 replicate wells for each sample. The model group and normal cell group were replaced with normal culture medium. Drug administration group 1 was the intracellular protein control group at 0 h in experiments 1-3; drug administration group 2 was the intracellular protein group at 6 h in experiments 1-3; drug administration group 3 was the intracellular protein group at 12 h in experiments 1-3; drug administration group 4 was the intracellular protein group at 18 h in experiments 1-3; drug administration group 5 was the intracellular protein group at 24 h in experiments 1-3; drug administration group 6 was the intracellular protein group at 30 h in experiments 1-3; drug administration group 7 was the supernatant protein control group at 0 h in experiments 1-3; drug administration group 8 was the supernatant protein group at 6 h in experiments 1-3; drug administration group 9 was the supernatant protein group at 12 h in experiments 1-3; drug administration group 10 was the supernatant protein group at 18 h in experiments 1-3; drug administration group 11 was the supernatant protein group at 24 h in experiments 1-3; and drug administration group 12 was the supernatant protein group at 30 h in experiments 1-3. The SH-SY5Y nerve cells were cultured for 72 h, and the cell viability was detected by CellTiter-glo luminescence method (Bi Yun Tian CellTiter-Luminescence Cell Viability Assay Kit, chemiluminescence method, detection wavelength 590 nm). TM II cell activity detection kit, chemiluminescence method, detection wavelength 590 nm).

[0084] The experimental results are shown in Figure 8. The intracellular proteins and supernatant proteins of stem cells without ultraviolet irradiation (0 h) have some nerve cell protection function, but the protection ability is very weak. The intracellular proteins and secreted proteins in the supernatant of stem cells cultured under ultraviolet irradiation for different times all have strong nerve cell repair and protection ability. In particular, the intracellular proteins of stem cells cultured under ultraviolet irradiation for more than 12 h and the secreted proteins secreted into the supernatant have the strongest nerve cell protection ability.

[0085] Experiment III: Effect of protein polymers on sodium arsenite (SA) damaged motor neurons to form stress granules (SG)

[0086] The same experimental method as in Experiment II was used to prepare the intracellular protein and supernatant protein of mesenchymal stem cells cultured under normal conditions for 18 h without ultraviolet irradiation. At the same time, the intracellular protein and supernatant protein of mesenchymal stem cells irradiated by ultraviolet for 18 h under the conditions described in Experiment II were prepared. At the same time, simple stem cell culture medium and albumin control samples with and without ultraviolet irradiation were prepared. The sample information is shown in Table 3:

[0087] Table 3: Experimental sample information

[0088] The pharmacodynamic experiment steps of the above-mentioned various samples are as follows:

[0089] The primary motor neurons (MN) were cultured to the 7th day (DIV7), and sodium arsenite (SA) injury was performed according to the following scheme:

[0090] (1) Model group (SA group): after SA 400 mM injury for 30 min, the complete culture medium was replaced and cultured for 1.5 h;

[0091] (2) Drug administration group: after SA 400 mM injury for 30 min, the culture medium containing sample 1, or sample 2, or sample 3, or sample 4, or sample 5, or sample 6, or sample 7, or sample 8 was added and cultured for 1.5 h;

[0092] (3) Normal control group (Ctr group): the whole process was operated in parallel by replacing the normal culture medium.

[0093] After the cells were fixed, stress granules were labeled by immunofluorescence staining. Fluorescence microscopy was used for photographing, and ImageJ was used to analyze the number of G3BP1 fluorescent granules (stress granules) of neurons positive for NeuN. The MN in the random field in different groups were counted for SG granules, and Graphpad was used for statistical analysis of the total score percentage, and the results were arranged into a column chart.

[0094] Experimental results: as shown in FIG. 9, compared with the normal control, SA stimulation treatment can increase the content of stress granules in nerve cells. The intracellular proteins and supernatant proteins of the stem cells subjected to ultraviolet irradiation can repair the increase of stress granules in neurons caused by SA. However, the samples in each control group have no function of protecting and repairing neurons. The experimental results suggest that the specific proteins produced by MSC subjected to ultraviolet irradiation can alleviate the damage of neurons caused by SG pathological aggregation, and have therapeutic potential for nerve cell damage or related neurodegenerative diseases.

[0095] Experiment four: the protein products obtained after the mesenchymal stem cells are cultured under ultraviolet irradiation have anti-inflammatory efficacy

[0096] According to the experimental conditions and experimental methods described in experiments 1-3, human umbilical cord mesenchymal stem cells or human amniotic membrane mesenchymal stem cells were cultured under UV B ultraviolet irradiation for 18 h, and then the culture medium supernatant was taken. The supernatant was filtered through a 0.22 μL filter membrane and stored at 4°C. Then the remaining cells were washed twice with 2 mL of normal saline, 1 mL of pure water was added and repeatedly blown, the cells were blown off from the bottom of the bottle, and the cells were repeatedly blown for about 6 min to lyse the cells, then filtered through a 0.22 μm filter membrane and stored at 4°C for standby.

[0097] RAW cells are a common inflammatory cell model. This experiment used RAW cells as the inflammatory model cells. RAW cells were seeded at a density of 20,000 cells / well in a 96-well plate. The inflammatory cell model was established by stimulating the RAW cells with LPS (500 ng / mL) for 24 hours. The LPS supernatant was then discarded, and samples (corresponding to samples with 500 ng / mL protein content) were added. Fresh culture medium was added to the model group. The cell supernatant was collected after 24 hours. The supernatant was diluted 15-20 times and the IL-6 content was measured according to the ELISA kit instructions.

[0098] IL-6 is the most common inflammatory factor. During an inflammatory response, IL-6 levels rise, and the ability to reduce IL-6 levels indicates an anti-inflammatory function. The experimental results are shown in Figure 10. The intracellular proteins and supernatant proteins of the umbilical cord mesenchymal stem cells or amniotic mesenchymal stem cells described in this invention, after being cultured under UV stress for a certain period, can inhibit the inflammatory response in model cells, demonstrating anti-inflammatory function.

[0099] Experiment 5: Intracellular protein products and supernatant protein products of amniotic mesenchymal stem cells cultured under UV irradiation exhibit nerve cell repair function.

[0100] Human amniotic mesenchymal stem cells were collected using standard methods. The simplified steps are as follows: Amniotic tissue was isolated from the human placental amniotic membrane, minced with surgical scissors, and primary amniotic mesenchymal stem cells were isolated and cultured using a tissue adherence method. After 5 days of tissue adherence culture, a large number of primary cells migrated out. Cell passage was performed using trypsin digestion. When the cell confluence reached 80%–90%, cells were passaged at a rate of 3000 cells / cm³. 2 Passaging at a specific density yields human amniotic mesenchymal stem cells. This is achieved by passage at a density of 5 × 10⁻⁶ cells / year. 6 Cells were cryopreserved per tube for future use.

[0101] Following the experimental conditions and methods described in Experiments 1-3, human amniotic mesenchymal stem cells were cultured under UV B irradiation for 18 hours or without UV irradiation for 18 hours (i.e., the 0h group in Experiments 1-3). The supernatant from each culture was then collected, filtered through a 0.22 μL filter membrane, and stored at 4℃. The remaining cells were then washed twice with 2 mL of physiological saline, followed by repeated pipetting with 1 mL of pure water to lyse the cells by blowing them down from the bottom of the flask for approximately 6 minutes. The lysed cells were then filtered through a 0.22 μm filter membrane and stored at 4℃ for later use.

[0102] Cell modeling and detection: After 24h of SH-SY5Y cell plating, three groups were set, normal group, model group and drug administration group. The model group and the drug administration group were damaged by 250μM H2O2 for 30min, and then the supernatant was discarded. The drug administration group was administered at a concentration of 100ng / mL protein concentration, with 5 replicate wells for each sample. The model group and the normal cell group were replaced with normal culture medium. Among them, the drug administration group 1 was the intracellular protein control group obtained by culturing mesenchymal stem cells for 18h without ultraviolet irradiation (i.e. 0h group); the drug administration group 2 was the intracellular protein group obtained by culturing mesenchymal stem cells for 18h under ultraviolet irradiation; the drug administration group 3 was the supernatant protein control group obtained by culturing mesenchymal stem cells for 18h without ultraviolet irradiation (i.e. 0h group); and the drug administration group 4 was the supernatant protein group obtained by culturing mesenchymal stem cells for 18h under ultraviolet irradiation. The SH-SY5Y nerve cells were cultured for 72h, and then the cell viability was detected by CellTiter-glo luminescence method (bielun CellTiter-LummiTMII cell activity detection kit, chemiluminescence method, 590nm wavelength detection of cell viability).

[0103] The experimental results are shown in Figure 11. The intracellular protein and supernatant protein of stem cells without ultraviolet irradiation (cultured for 18h without ultraviolet irradiation) have certain nerve cell protection function, but the protection ability is weak. The intracellular protein and supernatant protein of stem cells cultured under ultraviolet irradiation have strong nerve cell repair and protection ability.

[0104] Experiment six: application of protein polymer in the treatment of stroke

[0105] The protein polymer obtained in experiment one of the present application is used below to further study its biological activity.

[0106] After 7 days of drug administration to MCAO rats, the brain infarction was observed by TTC staining method. The brain infarction range of rats in the intrathecal and intravenous administration group of 36μg / kg protein polymer (protein polymer obtained in culture experiment two) was significantly reduced (p<0.05), while the administration of butylphthalide and edaravone dextrophan did not significantly reduce the brain infarction range (Figures 12 and 13).

[0107] The neurological function of the model rats was evaluated by blind method before and after drug administration, and the results showed that:

[0108] After 5-7 days of drug administration, the neurological function of rats in the intrathecal and intravenous administration group of protein polymer was better than that of the control group, and the neurological function of the butylphthalide and edaravone dextrophan administration group had no significant difference from that of the control group (Figure 14).

[0109] The results of detecting nerve inflammation and neuron marker in the rat brain by immunofluorescence method show that the intrathecal combination of the protein polymer and intravenous administration can inhibit the level of nerve inflammation and increase the survival number of neurons (Figure 15).

[0110] The above is a further detailed description of the present application, which cannot be considered as a specific implementation of the present application. For those skilled in the art to which the present application belongs, simple deduction or replacement without departing from the concept of the present application is within the protection scope of the present application.

Claims

1. A protein polymer, characterized by, The production process comprises: S1) culturing mesenchymal stem cells and using ultraviolet irradiation to create a stress environment; S2) lysing the mesenchymal stem cells and isolating and purifying the protein polymer.

2. The protein polymer of claim 1, wherein, The protein polymer comprises at least the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens; Preferably, the total amount of the above two proteins is more than 40% of the total mass of the protein polymer. Preferably, the protein polymer further comprises at least one of the following proteins: sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens; sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens; sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens; sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens; sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens; sp|P60709|ACTB_HUMAN Actin,cytoplasmic 1 OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

3. The protein polymer of claim 1, wherein The time for stimulating the mesenchymal stem cells by ultraviolet irradiation is 1h-30h, preferably, the irradiation time is 10h-30h, more preferably 6h-18h; Preferably, the intensity of the ultraviolet irradiation stimulus is 10 μW / cm 2 ~ 100 μW / cm 2 and the wavelength of the ultraviolet light is preferably 290 nm to 340 nm. Preferably, the culture medium used in the stimulation by ultraviolet irradiation is serum-free MSCs culture medium.

4. The protein polymer according to any one of claims 1 to 3, characterized in that The mesenchymal stem cells are selected from umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells and human placenta-derived mesenchymal stem cells.

5. A production process of protein polymer, comprising MSCs expansion, stimulating MSCs in culture medium by ultraviolet irradiation, stress treatment, collecting the MSCs after stress treatment for lysis treatment, separation and purification of protein to obtain protein polymer.

6. The production process according to claim 5, characterized in that, The time for stimulating the mesenchymal stem cells by ultraviolet irradiation is 1h-30h, preferably, the irradiation time is 10h-30h, more preferably 6h-18h; Preferably, the intensity of the ultraviolet irradiation stimulus is 10 μW / cm 2 ~ 100 μW / cm 2 The wavelength of the ultraviolet light is preferably 290 nm to 340 nm.

7. The production process according to claim 5 or 6, characterized in that, The culture medium used in the stimulation by ultraviolet irradiation is serum-free MSCs culture medium.

8. The use of the protein polymer according to any one of claims 1-4, wherein the use comprises preparing a medicament for treating stroke.

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