Protein polymer and use thereof in treatment of alzheimer's disease

Abstract

A protein polymer and a use thereof in the treatment of Alzheimer's disease. The protein polymer is obtained by stimulating MSCs, and then lysing the MSCs and performing separation and purification. The protein polymer has a strong oxidative damage repair ability, can improve Alzheimer's disease-related biomarker abnormalities, and has a significant treatment effect on Alzheimer's disease.

Description

Protein polymer and its application in treating Alzheimer's disease

[0001] The present disclosure claims priority to the Chinese patent application No. 202410944325.9, filed on July 15, 2024, entitled "A protein polymer and its production process", the whole content of the above application 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 its application in treating Alzheimer's disease. 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, amniotic membrane, 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, easy 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 produce different stress proteins under different stimulation conditions, 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 its production process.

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

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

[0008] A protein polymer, and its production process comprises:

[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 the 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 1h-30h, preferably, the irradiation time is 10h-30h, more preferably 6h-18h;

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

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

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

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

[0020] Preferably, the medium used in the stimulation of the irradiation of the ultraviolet light is 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.

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

[0026] In some examples of the protein polymer, the lysis is performed with 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 MSCs expansion, irradiation of the culture medium with ultraviolet light to stimulate the MSCs, stress treatment, collection of the MSCs after the stress treatment, lysis treatment, separation and purification of the protein to obtain the protein polymer.

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

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

[0032] Preferably, the medium used for 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, the present application provides:

[0035] The application of the protein polymer of the first aspect of the present application includes the preparation of a medicament for treating neurodegenerative diseases and stroke. Further, the neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and different types of spinocerebellar ataxia (SCA).

[0036] The present application has the following beneficial effects:

[0037] The protein polymer of some examples of the present application has good cell damage repair effect and is expected to be used for treating neurodegenerative diseases and stroke, in particular, the neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and different types of spinocerebellar ataxia (SCA).

[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 high freezing and recovery activity 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 culture 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] Fig. 4 is a cell state photo before ultraviolet irradiation of MSCs in culture test 1-2.

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

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

[0048] Figure 7 is the SDS-PAGE result of the intracellular protein and medium supernatant protein harvested 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 in Experiment Four on the secretion of inflammatory factor IL-6 by RAW cells.

[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] Figure 12 is an immunofluorescence graph of p-Tau protein of damaged motor neurons.

[0054] Figure 13 is a statistical graph of the average fluorescence intensity of Figure 12. DETAILED DESCRIPTION

[0055] The technical solutions of the present application will be further illustrated below in combination with experimental examples.

[0056] Experiment One: Effect of Different Treatments on Protein Expression

[0057] 1-1 Culture Test One

[0058] Human umbilical cord mesenchymal stem cells (HUC-MSC) were cultured in 2L of HK-G050 (PRF) 3D culture 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.

[0059] The cells were irradiated with LED ultraviolet light under the following conditions: 60μW / cm 2 Samples were taken at 8h, 12h, 16h, 18h, 24h, and 30h, and the cell morphology was observed and the intracellular protein was collected for protein concentration measurement.

[0060] The state of the cells after UV irradiation for 8h is shown in Figure 2. As can be seen from Figures 1 and 2, the microcarriers are substantially covered with cells at 0h. After UV irradiation for a period of time, the cell morphology is affected, and the cells are subjected to a survival stress, and the cells can produce stress proteins under this stress environment.

[0061] After the irradiation, the cells were harvested at different time points to obtain the protein aggregates. Specifically, for the 0h, 8h, 12h, 16h, 18h, 24h, and 30h groups, 8mL of the culture was sampled and the following operations were performed: the microcarriers were intercepted by a 300-mesh filter bag, the supernatant was centrifuged at 1200rpm for 6min, and the cell pellet was stored at 4°C. The cell pellet was washed with 150mL of normal saline for 3 times, lysed with 14mL of pure water, and filtered through a 0.22μm filter membrane. The harvested protein was stored at -80°C (if used for a short period of time, it can be stored at 4°C). The harvested protein was subjected to gel electrophoresis analysis, and the SDS-PAGE results are shown in Figure 3.

[0062] 1-2 Culture Test Two

[0063] One vial of P8 generation HUC-MSCs was recovered and added to 9 T25 culture bottles, 2.5mL of HuGuan mesenchymal stem cell serum-free culture medium was added to each bottle, and the cell density in each culture bottle was 1×10 5

[0064] Four bottles of cells were irradiated with two different intensities of LED ultraviolet light with a wavelength of about 300nm to 316nm, mainly UV B, for 6h, 12h, 18h, and 24h, respectively. The supernatant was carefully removed, washed twice with 1mL of normal saline, lysed by repeatedly blowing 660μL of pure water for 10min, and then filtered through a 0.22μm filter membrane and stored at 4°C.

[0065] The UV irradiation conditions are as follows:

[0066] The state of the cells before UV irradiation is shown in Figure 4, and the state of the cells after UV irradiation for 6h at a low intensity is shown in Figure 5. As can be seen, the UV irradiation affects the cell morphology and causes a survival stress to the cells, and the cells can produce stress proteins under this stress environment. The determination method of the protein concentration is a conventional method known to those skilled in the art, for example, including the Bradford method, the BCA method, the Lowry method, the ultraviolet spectrophotometry, and the Kjeldahl nitrogen determination method. In the present application, the BCA method is used to determine the protein concentration at each time point. The protein concentration and volume of the harvested protein are shown in Table 1.

[0067] Table 1

[0068] ​The harvested protein SDS-PAGE results are shown in Figure 6. As shown in Figure 6, the strong and weak ultraviolet irradiation conditions can promote the expression of the target protein. Under the strong ultraviolet irradiation condition, with the increase of the irradiation time, the target protein becomes purer, and when the irradiation time is about 18h, the target protein has high purity and high concentration. Weak ultraviolet can effectively promote the expression of the target protein, but the irradiation time needs to be longer than that of using strong ultraviolet irradiation.

[0069] 1-3 Culture Test Three

[0070] Recover a small crystal P8 generation HUC-MSC into a T25 culture flask, add 2.5mL of Huagankang mesenchymal stem cell serum-free culture medium to each flask, and inoculate the cells at a density of 1x10 5

[0071] Take the cells under the condition of UVB ultraviolet irradiation at a wavelength of 300nm-316nm for 6h, 12h, 18h, 24h and 30h.

[0072] The ultraviolet irradiation conditions are as follows:

[0073] After irradiation, the cells were harvested at different time points to obtain protein polymers. The specific operation is as follows: take the culture medium supernatant irradiated for 6h, 12h, 18h, 24h and 30h, and do the following operation: after the supernatant is filtered through a 0.22μL filter membrane, it is stored at 4°C.

[0074] Then wash the remaining cells with 2mL of normal saline for 2 times, add 1mL of pure water and repeatedly blow, blow the cells from the bottom of the bottle, repeatedly blow for about 6min to lyse the cells, then filter through a 0.22μm filter membrane and store at 4°C for standby.

[0075] Take the harvested protein for gel electrophoresis analysis. The protein concentration obtained under each culture condition is as follows:

[0076] Table 2

[0077] The above harvested intracellular protein and stem cell culture medium supernatant protein were analyzed by SDS-PAGE, and the results are shown in Figure 7. As shown in Figure 7, with the extension of the irradiation time, the content of the target protein contained in the intracellular protein gradually increases, and when the irradiation time is about 18h, there are few impurities and most of the bands are target proteins. In the supernatant, there are also target protein bands.

[0078] ​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;

[0079] 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.

[0080] Further, in addition to the above two proteins, the protein polymer obtained by the present application also includes 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.

[0081] Experiment two: pharmacodynamic test of protein polymer for repairing nerve cell damage

[0082] 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).

[0083] The experimental results are shown in Figure 8. The intracellular proteins and supernatant proteins of stem cells without ultraviolet irradiation (0 h) had some nerve cell protection function, but the protection ability was weak. The intracellular proteins and secreted proteins in the supernatant of stem cells cultured under ultraviolet irradiation for different times all had 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 had the strongest nerve cell protection ability.

[0084] Experiment III: Effect of protein polymers on the formation of stress granules (SG) in sodium arsenite (SA) damaged motor neurons

[0085] 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.

[0086] Table 3: Experimental sample information

[0087] The pharmacodynamic experiment steps of the above-mentioned various samples are as follows:

[0088] The primary motor neurons (MN) were cultured to the 7th day (DIV7), and sodium arsenite (SA) injury was performed according to the following scheme:

[0089] (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;

[0090] (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;

[0091] (3) Normal control group (Ctr group): the whole process was operated in parallel by replacing the normal culture medium.

[0092] 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 was counted for SG granules, and Graphpad was used to statistically analyze the total score percentage, and the results were arranged into a column chart.

[0093] 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 MSCs 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.

[0094] Experiment four: the protein products obtained after the mesenchymal stem cells are cultured under ultraviolet irradiation have anti-inflammatory efficacy

[0095] 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.

[0096] 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.

[0097] 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.

[0098] Experiment 5: Intracellular protein products and supernatant protein products of amniotic mesenchymal stem cells cultured under UV irradiation exhibit nerve cell repair function.

[0099] 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.

[0100] 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.

[0101] 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 with the intracellular protein obtained from mesenchymal stem cells cultured for 18 h without UV irradiation (i.e. 0 h group) at a concentration of 100 ng / mL, and each sample had 5 replicate wells. The model group and normal cell group were replaced with normal culture medium. The drug administration group 1 was the intracellular protein control group obtained from mesenchymal stem cells cultured for 18 h without UV irradiation (i.e. 0 h group); the drug administration group 2 was the intracellular protein group obtained from mesenchymal stem cells cultured for 18 h with UV irradiation; the drug administration group 3 was the supernatant protein control group obtained from mesenchymal stem cells cultured for 18 h without UV irradiation (i.e. 0 h group); and the drug administration group 4 was the supernatant protein group obtained from mesenchymal stem cells cultured for 18 h with UV irradiation. The SH-SY5Y nerve cells were continuously cultured for 72 h, and then the cell viability was detected by using CellTiter-glo luminescence method (CellTiter-Luminescence Cell Viability Assay Kit, Chemiluminescence method, detection of cell viability at a wavelength of 590 nm). TM II Cell Viability Assay Kit, Chemiluminescence method, detection of cell viability at a wavelength of 590 nm).

[0102] The experimental results are shown in Figure 11. The intracellular protein and supernatant protein of the stem cells cultured for 18 h without UV irradiation (i.e. 0 h group) had a certain nerve cell protection function, but the protection ability was weak. The intracellular protein and supernatant protein of the stem cells cultured for 18 h with UV irradiation had a strong nerve cell repair and protection ability.

[0103] Experiment Six: Evaluation of the Biological Effects of Protein Polymers

[0104] The protein polymers obtained from Experiment One (Cultivation Experiment Two) of the present application were used to further study their biological activity.

[0105] Experiment 6.1 Changes in the Concentrations of Biomarkers β-amyloid Aβ-40 and Aβ-42 after Administration of Protein Polymers

[0106] Experimental Design:

[0107] Screening: The concentrations of β-amyloid Aβ-40 and Aβ-42 in the plasma of the subjects were detected by using digital single molecule immunoarray analysis, and the ratio of Aβ-42 / Aβ-40 was calculated. Based on the existing reference interval, two subjects with abnormal β-amyloid, subject A and subject B, were screened.

[0108] Administration: Take the freeze-dried preparation of the protein polymer obtained in Experiment 1 of the application, prepare an injection solution, and administer the drug prepared from the protein polymer to the two subjects in the form of intravenous administration, with a frequency of once a day, 5 days a week, 2 days off, for a total of 2 weeks.

[0109] Biomarker monitoring: After the end of the medication period, the concentrations of β-amyloid Aβ-40 and Aβ-42 in the plasma of subjects A and B were detected, and the Aβ-42 / Aβ-40 ratio was calculated.

[0110] Data statistics:

[0111] Conclusion:

[0112] From the data before medication, the amyloid deposition of Alzheimer's disease in subject B was more serious than that in subject A, and after the administration of the protein polymer, the levels of amyloid Aβ-40 and Aβ-42 in both subjects decreased, and the decrease in subject B was greater than that in subject A, which indicated that the protein polymer prepared in the application could effectively inhibit the malignant development of amyloid deposition and had a positive effect on the clearance of β-amyloid. In addition, the Aβ-42 / Aβ-40 values of both subjects increased after medication, indicating a trend of recovery from abnormal to normal values.

[0113] Experiment 6.2 Changes in biomarker p-Tau after administration of protein polymer

[0114] 6.2.1 Purpose of the experiment

[0115] The effect of the protein polymer on p-Tau in damaged motor neurons (MN) was studied to explore the therapeutic effect of the protein polymer of the application on diseases caused by pathological elevation of p-Tau protein in neurons, including Alzheimer's disease and other neurodegenerative diseases.

[0116] 6.2.2 Experimental preparation

[0117] Experimental cells:

[0118] Mouse BV2 cells, primary MN-E13 cultured on February 6, 2025, DIV7 MN

[0119] Test samples:

[0120] Protein polymer dry powder: batch number 20240408, prepared to a concentration of 70 μg / mL sample.

[0121] Experimental reagents:

[0122] LPS lipopolysaccharide (Sigma, Cat# L2630, 1 mg / mL)

[0123] Experimental materials:

[0124] 1. Reagents:

[0125] 2. Consumables:

[0126] 3. Instruments and equipment

[0127] 6.2.3 Experimental method:

[0128] 6.2.3.1 Extraction of motor neurons (MN)

[0129] S11: Cover glass sterilization: Prepare a sterile twelve-well plate, take 24 cover glasses and soak them in anhydrous ethanol, light the alcohol lamp, and sterilize the cover glasses by passing them through the flame.

[0130] S12: Glass slide coating with polylysine PDL: Take 500 μL of 100 μg / mL PDL solution, cover the cover glasses, and gently move them to the 37°C incubator for overnight coating.

[0131] S13: Glass slide coating with laminin: Take 21 μL of laminin solution and dissolve it in 6 mL of HBSS buffer. Wash the PDL-coated glass slides with sterile ultrapure water three times, discard the liquid, and add 500 μL of laminin solution to each glass slide. Incubate at 37°C, and wash with sterile ultrapure water three times before use.

[0132] S14: Isolation of spinal cord tissue: Take the mouse embryo, remove the thin sheath on the back skin, pierce and lift the spinal cord with tweezers, transfer the spinal cord to a new dish containing HBSS, open the central channel along the dorsal side of the spinal cord, remove the sheath from the dorsal root ganglion (DRG) on the cervical side, transfer the remaining spinal cord tissue to a new dish containing HBSS, discard most of the liquid and retain a small amount of HBSS, and cut the spinal cord tissue.

[0133] S15: Papain digestion of cells: Take 25 μL of papain and add it to 5 mL of Neurobasal medium, transfer the spinal cord tissue fragments to the enzyme solution, add 25 μL of Dnase enzyme (25 ng / mL), invert and shake well, and incubate in a 37°C incubator for 30 min, with 5-6 shakes in between.

[0134] S16: Collect cells: After digestion, centrifuge briefly, discard supernatant, add 6 mL Neurobasal medium, gently blow 10 times with a pipette, stand for 1 min, take 3 mL of the upper liquid through a 40 μm filter to collect in a new 15 mL centrifuge tube; add 3 mL of new Neurobasal medium, gently blow 10 times, stand for 1 min, repeat the above filtering and collecting process 2 times, a total of 9 mL of cell suspension is collected; centrifuge at 1000 rpm for 5 min, discard the supernatant and collect the cell precipitate.

[0135] S17: Purification of motor neurons (MN) by density gradient centrifugation: The cell precipitate is suspended with 1 mL of HBSS, gently spread on the surface of 6 mL of 1.06 g / mL Optiprep solution, centrifuged at 400 x G for 5 min.

[0136] S18: MN culture: Take the uppermost 1 mL of cell solution, which is the purified MN, mix with 9 mL of Neurobasal, centrifuge at 1200 rpm for 10 min, discard the supernatant, and gently suspend the precipitate with complete medium, count with a cell counting plate, resuspend at 550,000 / well and transfer to a cover glass in a laminin-coated dish, and culture in a 37°C incubator; after the MN adhere and grow for 1 h, discard the supernatant medium and non-adherent cells, and replace with fresh 2 mL of complete medium for continuous culture.

[0137] The composition of the complete medium is: Neurobasal + 10% horse serum + 1% Glutamax I + 1% B27.

[0138] S19: MN medium replacement and continuous culture, replace the medium half every 2 days and observe the cell growth.

[0139] 6.2.3.2 Preparation of conditioned medium (CM)

[0140] S21: Grouping: control group (Ctr) and model group (LPS)

[0141] S22: BV2 cells are digested and plated into T25 bottles at 800,000 / bottle for culture;

[0142] S23: After 24 h of culture: the Ctr control group cells are replaced with normal medium 5 mL for continuous culture for 24 h; the LPS model group cells are replaced with medium containing 500 ng / mL LPS (5 mL of normal medium + 2.5 μL of LPS) for continuous stimulation and culture for 24 h;

[0143] S24: Collect the culture medium supernatant of Ctr control group and LPS model group cells, centrifuge at 1000 rpm for 5 min, collect the supernatant as CM-LPS and CM-Ctr, discard the cell precipitate.

[0144] 6.2.3.3 CM injury and administration of MN

[0145] The primary MNs were cultured to the 7th day (obtained DIV7 MNs), and the CM replacement injury and culture were performed according to the following scheme.

[0146] Grouping and injury conditions

[0147] Control group (Ctr group): DIV7 MNs were transferred to CM-Ctr injury half liquid (500 μL CM-Ctr medium + 500 μL complete medium), and cultured for 24 h;

[0148] Model group (LPS group): DIV7 MNs were transferred to CM-LPS injury half liquid (500 μL CM-LPS medium + 500 μL complete medium), and cultured for 24 h;

[0149] Protein polymer 300 ng / mL group (LPS+protein polymer 300): DIV7 MNs were transferred to CM-LPS injury half liquid containing 300 ng / mL protein polymer (500 μL CM-LPS medium + 500 μL complete medium + 4.3 μL protein polymer), and cultured for 24 h;

[0150] Protein polymer 600 ng / mL group (LPS+protein polymer 600): DIV7 MNs were transferred to CM-LPS injury half liquid containing 600 ng / mL protein polymer (500 μL CM-LPS medium + 500 μL complete medium + 8.6 μL protein polymer), and cultured for 24 h.

[0151] 6.2.3.4 Immunofluorescence staining

[0152] S41: Cell fixation: Take the culture plate, add the four groups of cells in 6.2.3.3 to the corresponding plate holes, preheat 4% PFA to room temperature, add 1 mL of 4% PFA per hole, and fix at room temperature for 20 min.

[0153] S42: Washing and permeabilization: Wash the above culture plate with PBS containing 0.2% Triton (PBST), and send to the shaker for shaking washing at 80 rpm, 5 min each time, and repeat for 3 times.

[0154] S43: Sealing: Add 5% sheep serum (PBS containing 0.2% Triton, with 1‰ Proclean bacteriostatic agent) into the plate hole of the culture plate, and send it to the shaker for 1h of shaking cleaning at 80rpm.

[0155] S44: Incubation of primary antibody: Transfer the culture plate to the wet box, and place the sealing film. Take 25μL of diluted antibody solution (mouse anti-Tuj1 (Bioss, 1:250) and rabbit anti-p-Tau (HuaBio, 1:250) and 1‰ Proclean bacteriostatic agent) on the sealing film, and incubate overnight at 4℃.

[0156] S45: Incubation of secondary antibody: After mixing the rabbit 488 fluorescent secondary antibody (1:1000) and the mouse 594 fluorescent secondary antibody (1:300) with the secondary antibody diluent, add 1‰ Proclean bacteriostatic agent, and take 25μL of the secondary antibody diluent on the sealing film. Incubate in the dark at room temperature for 2h in the wet box.

[0157] S46: DAPI staining: Take DAPI (1μg / mL) staining solution on the sealing film, and shake for 8min.

[0158] S47: Cleaning: Clean the culture plate with PBS containing 0.2% Triton (PBST), and send it to the shaker for 5min of shaking cleaning each time, and repeat for 2 times.

[0159] S48: Transfer the stained cells to the glass slide, add 30μL of anti-fluorescence quencher, and then place the cover glass on the glass slide.

[0160] S49: Mounting: Nail polish mounting.

[0161] 6.2.3.5 Photographing and counting the results

[0162] Take pictures of the immunofluorescence sections using a fluorescence inverted microscope (see Figure 12). Randomly take pictures of 40 fields of view for each group, calculate the mean fluorescence intensity (Mean) of p-Tau on motor neurons in each field of view for each group using the ImageJ program, and use Graphpad to perform statistical analysis of significant differences, and arrange the results in a column chart (see Figure 13). The detailed data is shown in the table below:

[0163] 6.2.3.6 Experimental results

[0164] By statistically analyzing the average fluorescence intensity of p-Tau of motor neurons in 40 random fields of each group of immunofluorescence sections and performing significant difference analysis, it is found that the condition medium (CM-LPS) of LPS-stimulated BV2 cells can cause abnormal increase of p-Tau on neurons after damaging MN, and the protein polymer-300ng / mL and the protein polymer-600ng / mL have obvious inhibitory effect on abnormal phosphorylation of Tau protein, reduce the increase of p-Tau on motor neurons caused by neuroinflammation, inhibit the spread of inflammation in the nervous system, and suppress the formation of neurofibrillary tangles, and have obvious therapeutic effect on Alzheimer's disease and other neurodegenerative diseases caused by pathological increase of p-Tau protein on neurons.

[0165] Specific results can be seen in FIGS. 12 and 13. Compared with the Ctr control group, the average fluorescence intensity of p-Tau on MN in the LPS group is significantly increased by 16.4% (P #### <0.0001), indicating that the p-Tau on MN damaged by the condition medium (CM-LPS) of LPS-stimulated BV2 cells is significantly increased; compared with the LPS model group, the average fluorescence intensity of p-Tau on damaged MN in the protein polymer-300ng / mL (protein polymer-300 group) and the protein polymer-600ng / mL (protein polymer-600 group) is significantly decreased by 28.1% and 31.6% (P **** <0.0001), indicating that the protein polymer of the present application can significantly reduce the over-phosphorylated Tau protein (p-Tau) on damaged MN, and has the effect of reversing the phosphorylation trend of Tau protein.

[0166] The above is a further detailed description of the present application, which cannot be regarded as a limitation on the specific implementation of the present application. For ordinary skilled persons in the technical field 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 1-30 hours, preferably 10-30 hours, and more preferably 6-18 hours. 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, and separating and purifying proteins 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 1-30 hours, preferably 10-30 hours, and more preferably 6-18 hours. 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. Use of the protein polymer according to any one of claims 1-4, wherein the use comprises preparing a medicament for treating neurodegenerative diseases, and further, the neurodegenerative diseases comprise Alzheimer's disease (AD).

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