Proteins polymers and their use in the treatment of spinocerebellar ataxias

Abstract

A protein polymer and its application in treating spinocerebellar ataxia. The protein polymer is obtained by stimulating MSCs to split, and is separated and purified. The protein polymer has strong oxidative damage repair capacity, can relieve cell stress state by regulating cell gene expression pathway, and promotes cells to restore normal morphology and function.

Description

Protein polymers and their application in the treatment of spinocerebellar ataxia

[0001] This disclosure claims priority to Chinese Patent Application No. 202410944325.9, filed on July 15, 2024, entitled "A Protein Polymer and Its Production Process", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to a protein polymer and its application in the treatment of spinocerebellar ataxia (SCA). Background Technology

[0003] Mesenchymal stem cells (MSCs) possess self-replication and multi-lineage differentiation potential, and are widely found in tissues such as bone marrow, adipose tissue, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amnion, peripheral blood, muscle, and urine. They are characterized by their wide availability, lack of matching requirements, low infection rate, strong differentiation potential, strong proliferation capacity, and convenient collection. They 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), and interferon (IFN), which participate in regulating cell growth, apoptosis, cell differentiation, antiviral activity, and immune maturation. They can be used for immune regulation, tissue repair, and the treatment of diseases such as acute lung injury, severe pneumonia, and acute respiratory distress syndrome.

[0004] Mesenchymal stem cells (MSCs) can produce different stress proteins when cultured under various stimuli, and these stress protein polymers possess complex physiological activities. Developing MSCs to produce protein polymers with specific biological activities is a highly challenging task. Summary of the Invention

[0005] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a protein polymer and its production process.

[0006] The technical solution adopted in this invention is:

[0007] The first aspect of the present invention provides:

[0008] A protein polymer, the production process of which includes:

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

[0010] S2) Mesenchymal stem cells were lysed and purified to obtain protein polymers;

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

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

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

[0014] Preferably, it further includes 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 protein polymers, the duration of ultraviolet irradiation to stimulate mesenchymal stem cells is 1 hour to 30 hours, preferably 10 hours to 30 hours, and more preferably 6 hours to 18 hours.

[0016] Preferably, the intensity of the ultraviolet radiation stimulation is 10 μW / cm². 2 ~100μW / cm 2The wavelength of ultraviolet light is preferably 290nm to 340nm;

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

[0018] In a 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 300 nm to 316 nm.

[0020] Preferably, the culture medium used for ultraviolet irradiation stimulation is serum-free MSCs culture medium.

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

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

[0023] In some instances of protein polymers, the protein polymers are derived from cells.

[0024] In some instances of protein polymers, the protein polymers are derived from culture medium supernatants.

[0025] In some instances of protein polymers, the protein polymers are derived from culture medium supernatants and intracellular sources.

[0026] In some examples of protein polymers, the lysis is carried out using pure water.

[0027] These features can be combined arbitrarily as long as they do not conflict with each other.

[0028] A second aspect of the present invention provides:

[0029] A process for producing a protein polymer includes MSC amplification, stress treatment of MSCs in a culture medium by irradiating them with ultraviolet light, collection of the stress-treated MSCs for lysis treatment, and separation and purification of proteins to obtain a protein polymer.

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

[0031] Preferably, the intensity of the ultraviolet radiation stimulation is 10 μW / cm². 2 ~100μW / cm 2 The wavelength of ultraviolet light is preferably 290nm to 340nm;

[0032] Preferably, the culture medium used for ultraviolet irradiation stimulation is serum-free MSCs culture medium.

[0033] These features can be combined arbitrarily as long as they do not conflict with each other.

[0034] A third aspect of the present invention provides:

[0035] The first aspect of this invention relates to the application of the protein polymer, including its use in the preparation of medicaments 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 various types of spinocerebellar ataxia (SCA).

[0036] The beneficial effects of this invention are:

[0037] The protein polymers of some examples of the present invention have good cell damage repair effects and are expected to be used to treat 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 processes described in some examples of this invention can effectively overcome the differences between different batches of MSCs, resulting in more stable MSCs with smaller batch-to-batch variations, thus greatly ensuring the quality and yield of protein polymers.

[0039] The production processes described in some examples of this invention can better ensure the activity of umbilical cord-derived MSCs and BMSCs, which is beneficial to increasing the original yield of MSCs.

[0040] The production processes described in some examples of this invention result in high cryopreservation and thawing survival rates of MSCs.

[0041] The production processes described in some examples of this invention can effectively separate and purify protein polymers. Attached Figure Description

[0042] Figure 1 is a photograph of the growth status of MSCs after being cultured in Tangyi 3D medium in culture experiment 1-1.

[0043] Figure 2 is a photograph of the cell state after 8 hours of ultraviolet irradiation in culture experiment 1-1.

[0044] Figure 3 shows the SDS-PAGE results of the protein harvested from culture experiment 1-1.

[0045] Figure 4 shows the cell state of MSCs before UV irradiation in culture experiments 1-2.

[0046] Figure 5 shows the cell state of MSCs after 6 hours of weak ultraviolet irradiation in culture experiments 1-2.

[0047] Figure 6 shows the SDS-PAGE results of the proteins harvested from culture experiments 1-2.

[0048] Figure 7 shows the SDS-PAGE results of intracellular proteins and proteins in the culture supernatant harvested from culture experiments 1-3.

[0049] Figure 8 is a statistical bar chart showing the ability of protein polymers obtained from different treatment groups in Experiment 2 to repair damaged nerve cells.

[0050] Figure 9 is a bar chart showing the percentage of motor neurons forming stress granules (SG) after SA damage in samples obtained from different treatment groups in Experiment 3.

[0051] Figure 10 shows the effect of protein polymers obtained from different treatment groups in Experiment 4 on the secretion of the inflammatory factor IL-6 by RAW cells.

[0052] Figure 11 is a statistical bar chart showing the ability of protein polymers obtained from different treatment groups in Experiment 5 to repair damaged nerve cells.

[0053] Figure 12 shows the SDS-PAGE electrophoresis images of proteins in the cerebrospinal fluid of the subjects before and after protein polymer treatment. HLQ represents the sample after treatment, and HXS represents the sample before treatment.

[0054] Figure 13 is a Venn diagram of the upregulated and downregulated proteins and the proteins contained in the protein polymer in the cerebrospinal fluid of subjects before and after protein polymer treatment.

[0055] Figure 14 is a volcano diagram of differentially expressed genes in the cerebrospinal fluid of subjects before and after protein polymer therapy.

[0056] Figure 15 is a bar graph showing the enrichment results of upregulated genes in the KEGG_PATHWAY of differentially expressed genes in the cerebrospinal fluid of subjects before and after protein polymer treatment.

[0057] Figure 16 is a bar graph showing the enrichment results of downregulated genes in the KEGG_PATHWAY of differentially expressed genes in the cerebrospinal fluid of subjects before and after protein polymer treatment.

[0058] Figure 17 is a bar graph showing the enrichment results of upregulated genes in the cerebrospinal fluid of subjects before and after protein polymer therapy, based on Gene Ontology (GO) analysis.

[0059] Figure 18 is a bar graph showing the enrichment results of downregulated genes in the cerebrospinal fluid of subjects before and after protein polymer therapy, based on Gene Ontology (GO) analysis.

[0060] Figure 19 is a bar graph showing the upregulated genes in the enrichment results of differentially expressed genes in the cerebrospinal fluid of subjects before and after protein polymer therapy, obtained from the DISEASE database.

[0061] Figure 20 is a bar graph showing the downregulated genes in the enrichment results of differentially expressed genes in the cerebrospinal fluid of subjects before and after protein polymer therapy, obtained from the DISEASE database.

[0062] Figure 21 is a statistical graph of differentially expressed genes in the protective damage group in Experiment 7.

[0063] Figure 22 is a statistical graph of differentially expressed genes in the damage repair group of Experiment 7. Detailed Implementation

[0064] The technical solution of the present invention will be further illustrated below with reference to experimental embodiments.

[0065] Experiment 1: Effects of different treatments on protein expression levels:

[0066] 1-1 Cultivation Experiment 1

[0067] Human umbilical cord mesenchymal stem cells (HUC-MSCs) were cultured in 2L of HK-G050(PRF) 3D medium from Tangyi Huike Biotechnology, with a total cell count of approximately 5 × 10⁻⁶ cells. 8 Each microcarrier was divided into four T225 flasks. Cell staining observation showed that the microcarriers were basically confluent with cells. The results are shown in Figure 1.

[0068] LED ultraviolet light irradiation of cells, irradiation conditions: 60 μW / cm² 2 Samples were taken at 8h, 12h, 16h, 18h, 24h, and 30h to observe cell morphology and collect intracellular proteins to measure protein concentration.

[0069] Taking 8 hours of UV irradiation as an example, the cell state after irradiation is shown in Figure 2. Observing Figures 1 and 2, it can be seen that the microcarrier was basically fully colonized with cells at 0 hours. After a period of UV irradiation, cell morphology is affected, creating survival stress on the cells, which in turn allows the cells to produce stress proteins under this stress environment.

[0070] After irradiation, cells were harvested at different time points to obtain protein polymers. Specifically, 8 mL of culture was collected from seven groups at 0h, 8h, 12h, 16h, 18h, 24h, and 30h, and the following procedures were performed: the culture was filtered through a 300-mesh filter bag to retain microcarriers. The supernatant was centrifuged at 1200 rpm for 6 min and stored at 4℃. The cell pellet was washed three times with 150 mL of physiological saline, lysed with 14 mL of pure water, and filtered through a 0.22 μm filter membrane. The harvested protein was frozen at -80℃ (or stored at 4℃ for short-term use). The harvested protein was analyzed by gel electrophoresis, and the SDS-PAGE results are shown in Figure 3.

[0071] 1-2 Cultivation Experiment Two

[0072] One small cell line of P8 generation HUC-MSCs was revived and added to nine T25 culture flasks. 2.5 mL of Huakan mesenchymal stem cell serum-free medium was added to each flask, and the cell density in each flask was 1 × 10⁻⁶ cells / mL. 5 per mL.

[0073] Four flasks of cells were taken from each flask and irradiated with two different intensities of LED ultraviolet light, mainly UV B at around 300nm–316nm, for 6h, 12h, 18h, and 24h, respectively, under the conditions shown in the table below. The cells were carefully cleaned of supernatant, washed twice with 1mL of physiological saline, and lysed by repeatedly pipetting and refluxing with 660μL of pure water for 10min. Then, the cells were filtered through a 0.22μm filter membrane and stored at 4℃.

[0074] The ultraviolet irradiation conditions are as follows:

[0075] Figure 4 shows the cell state before UV irradiation. Figure 5 shows the cell state after irradiation, taking 6 hours of weak irradiation as an example. It can be seen that UV irradiation affects cell morphology, causing survival stress, which in turn leads to the production of stress proteins in the cells under this stress environment. The protein concentration was determined using conventional methods well-known to those skilled in the art, including the Bradford method, BCA method, Lowry method, UV spectrophotometry, and Kjeldahl method. In this invention, the BCA method was used to determine the protein concentration at each time point. The harvested protein concentration and volume are shown in Table 1.

[0076] Table 1

[0077] The SDS-PAGE results of the harvested proteins are shown in Figure 6. As can be seen from Figure 6, both strong and weak UV irradiation conditions can promote the expression of the target protein. Under strong UV irradiation, the target protein becomes purer with increasing irradiation time; around 18 hours of irradiation, the target protein exhibits high purity and concentration. Weak UV irradiation can effectively promote the expression of the target protein, but requires a longer irradiation time than strong UV irradiation.

[0078] 1-3 Cultivation Experiment Three

[0079] Resuscitate one small P8 generation HUC-MSC into a T25 culture flask, adding 2.5 mL of Huakan mesenchymal stem cell serum-free culture medium to each flask, with a cell seeding density of 1 × 10⁶ cells per flask. 5 per mL.

[0080] Cells were irradiated with UVB at wavelengths of 300 nm to 316 nm for 6 h, 12 h, 18 h, 24 h, and 30 h.

[0081] The ultraviolet irradiation conditions are as follows:

[0082] After irradiation, cells were harvested at different time points to obtain protein polymers. Specifically, the culture supernatant was collected at 6h, 12h, 18h, 24h, and 30h after irradiation and the following steps were performed: the supernatant was filtered through a 0.22μL filter membrane and stored at 4℃.

[0083] The remaining cells were then washed twice with 2 mL of physiological saline, and then 1 mL of pure water was added and repeatedly pipetted to lyse the cells from the bottom of the flask. The cells were then filtered through a 0.22 μm filter membrane and stored at 4 °C for later use.

[0084] The harvested proteins were analyzed by gel electrophoresis. The protein concentrations obtained under various culture conditions are shown in Table 2 below:

[0085] Table 2

[0086] The intracellular proteins and stem cell culture supernatant proteins obtained above were analyzed using SDS-PAGE, and the results are shown in Figure 7. As shown in Figure 7, with the extension of irradiation time, the content of the target protein in the intracellular proteins gradually increased. When the irradiation time was about 18 hours, there were very few impurities, and most of the bands were the target protein. The target protein band was also present in the supernatant. Mass spectrometry analysis was performed on the above protein polymer. Based on the mass spectrometry data and matching with known proteins, the protein polymer was confirmed to contain the following two proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0087] Serum albumin protein content is at least 38% of the total protein polymer content, and Serotransferrin protein content is at least 2% of the total protein polymer content.

[0088] Furthermore, in addition to the two proteins mentioned above, the protein polymer obtained in this 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.

[0089] Experiment 2: Pharmacodynamic Test of Protein Polymers in Repairing Neuronal Cell Damage

[0090] Cell modeling and detection: SH-SY5Y cells were seeded into plates for 24 hours and then divided into three groups: normal group, model group, and drug-treated group. The model group and drug-treated group were induced with 250 μM H2O2 for 30 min, and the supernatant was discarded. The drug-treated group was treated with protein at a concentration of 100 ng / mL. Each sample was tested in 5 replicates. The model group and normal cell group were treated with normal culture medium. Group 1 was the intracellular protein control group at 0h in Experiments 1-3; Group 2 was the intracellular protein group at 6h in Experiments 1-3; Group 3 was the intracellular protein group at 12h in Experiments 1-3; Group 4 was the intracellular protein group at 18h in Experiments 1-3; Group 5 was the intracellular protein group at 24h in Experiments 1-3; Group 6 was the intracellular protein group at 30h in Experiments 1-3; Group 7 was the supernatant protein control group at 0h in Experiments 1-3; Group 8 was the supernatant protein group at 6h in Experiments 1-3; Group 9 was the supernatant protein group at 12h in Experiments 1-3; Group 10 was the supernatant protein group at 18h in Experiments 1-3; Group 11 was the supernatant protein group at 24h in Experiments 1-3; and Group 12 was the supernatant protein group at 30h in Experiments 1-3. SH-SY5Y neural cells were cultured for another 72 hours, and cell viability was detected by CellTiter-glo chemiluminescence assay (using Beyotime CellTiter-Lμm). TM II. Cell Viability Assay Kit (Cell viability was detected by chemiluminescence at a wavelength of 590 nm).

[0091] The experimental results are shown in Figure 8. While intracellular proteins and supernatant proteins from stem cells without UV irradiation (0h) exhibit some neuroprotective function, their protective ability is weak. However, intracellular proteins and supernatant proteins from stem cells cultured for different times under UV irradiation conditions all possess strong neuroprotective capabilities for repair. In particular, the neuroprotective capabilities of intracellular proteins and secreted proteins in the supernatant from stem cells cultured for more than 12h under UV irradiation conditions are the strongest.

[0092] Experiment 3: Effects of protein polymers on the formation of stress granules (SG) in motor neurons damaged by sodium arsenite (SA).

[0093] Using the same experimental method as in Experiment 2, intracellular proteins and supernatant proteins from cell lysates of mesenchymal stem cells cultured for 18 hours under normal conditions without UV irradiation were prepared. Simultaneously, intracellular proteins and supernatant proteins from cell lysates of mesenchymal stem cells irradiated with UV for 18 hours under the conditions described in Experiment 2 were prepared. Also, pure stem cell culture media (with and without UV irradiation) and albumin control samples were prepared. The sample information is shown in Table 3.

[0094] Table 3 Experimental Sample Information

[0095] The pharmacodynamic experimental procedures for testing the above samples are as follows:

[0096] Primary motor neurons (MNs) were cultured to day 7 (DIV7) and then subjected to sodium arsenite (SA) lesions according to the following protocol:

[0097] (1) Model group (SA group): After SA 400μM damage for 30 min, the culture medium was replaced with complete medium and cultured for 1.5 h;

[0098] (2) Drug administration group: After SA 400μM injury for 30 min, the drug administration group was cultured for 1.5 h in a 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.

[0099] (3) Normal control group (Ctr group): The normal culture medium was changed throughout the process and the operation was carried out in parallel.

[0100] After cell fixation, stress granules were labeled with immunofluorescence staining. Images were taken using a fluorescence microscope, and the number of G3BP1 fluorescent granules (stress granules) in NeuN-positive neurons was analyzed using ImageJ. The number of SG granules in random fields of view of neurons in different groups was counted, and the total score-percentage was calculated using Graphpad. The results were then compiled into a bar chart.

[0101] Experimental Results: As shown in Figure 9, compared with the normal control, SA stimulation treatment increased the content of stress granules in nerve cells. Intracellular proteins and supernatant proteins of stem cells irradiated with ultraviolet light could repair the increase in stress granules in neurons caused by SA. However, the samples from each control group showed no protective or repairing function for neurons. These results suggest that UV-irradiated MSCs produce specific proteins that can alleviate neuronal damage caused by pathological aggregation of SG, and have therapeutic potential for nerve cell damage or related neurodegenerative diseases.

[0102] The protein products obtained from mesenchymal stem cell culture under ultraviolet irradiation in Experiment 4 showed anti-inflammatory effects.

[0103] Following the experimental conditions and methods described in Experiments 1-3, human umbilical cord mesenchymal stem cells or human amniotic mesenchymal stem cells were cultured under UV B ultraviolet irradiation for 18 hours. The supernatant was then collected, filtered through a 0.22 μL filter membrane, and stored at 4°C. 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°C for later use.

[0104] 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.

[0105] 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.

[0106] Experiment 5: Intracellular protein products and supernatant protein products of amniotic mesenchymal stem cells cultured under UV irradiation exhibit nerve cell repair function.

[0107] Human amniotic mesenchymal stem cells were collected using standard methods. The simplified steps are as follows: Amniotic membrane 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.

[0108] 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.

[0109] Cell modeling and detection: 24 hours after seeding SH-SY5Y cells into plates, three groups were set up: normal group, model group, and drug-treated group. The model group and drug-treated group were induced with 250 μM H2O2 for 30 min, after which the supernatant was discarded. The drug-treated group was treated with a protein concentration of 100 ng / mL, with five replicates per sample. The model group and normal cell group were cultured in normal medium. Drug-treated group 1 was the intracellular protein control group obtained after 18 hours of mesenchymal stem cell culture without UV irradiation (i.e., 0h group); drug-treated group 2 was the intracellular protein group obtained after 18 hours of mesenchymal stem cell culture under UV irradiation; drug-treated group 3 was the supernatant protein control group obtained after 18 hours of mesenchymal stem cell culture without UV irradiation (i.e., 0h group); and drug-treated group 4 was the supernatant protein group obtained after 18 hours of mesenchymal stem cell culture under UV irradiation. SH-SY5Y neural cells were cultured for another 72 hours, and cell viability was detected using CellTiter-glo chemiluminescence assay (using Beyotime CellTiter-Lμm). TM II. Cell Viability Assay Kit (Cell viability was detected by chemiluminescence at a wavelength of 590 nm).

[0110] The experimental results are shown in Figure 11. While the intracellular proteins and supernatant proteins of stem cells cultured without UV irradiation (group cultured for 18 hours without UV irradiation) possess some neuroprotective function, their protective ability is very weak. In contrast, the intracellular proteins and supernatant proteins of stem cells cultured under UV irradiation conditions exhibit strong neuroprotective and neurorepair capabilities.

[0111] Experiment 6: Differential Protein Analysis of Cerebrospinal Fluid Samples Before and After SCA Drug Administration in Subjects

[0112] S61: Collect cerebrospinal fluid samples (HXS) from SCA subjects before administration of protein polymers (1-2 culture experiment 2) and cerebrospinal fluid samples (HLQ) after administration.

[0113] S62: Extraction of proteins from cerebrospinal fluid samples:

[0114] S621: Add 200 μL of rinsing buffer to an EP tube containing BioRAD proteominer beads, invert to mix and wash, centrifuge at 4℃, 5000 rpm for 5 min, discard the supernatant, and wash three times.

[0115] S622: Take 500 μL of each sample, concentrate by ultrafiltration, wash 3 times with PBS, collect upside down, about 200 μL, add the sample to the washed microbeads, mix at room temperature for 2 hours;

[0116] S623: Centrifuge at 4℃, 5000rpm for 5min, discard the supernatant, and wash the beads 3 times with rinsing buffer;

[0117] S624: Add 200 μL of 1% trifluoroacetic acid (TFA), shake to mix for 10 min, centrifuge and collect the supernatant, repeat twice;

[0118] S625: Combine the supernatants from both processes and freeze-dry.

[0119] S63: The lyophilized sample was reconstituted with 50 μL of 8M uric acid solution UA, and the concentration of the extracted protein was determined by the BCA method.

[0120] S64: LC-MS / MS mass spectrometry analysis:

[0121] S641: Mass spectrometry setup:

[0122] Prepare mobile phases A (100% water, 0.1% formic acid) and B (80% acetonitrile, 0.1% formic acid). Dissolve the lyophilized powder from step S2625 in 10 μL of mobile phase A, centrifuge at 14000g for 20 min at 4°C, and inject 1 μg of the supernatant as sample for LC-MS analysis. The elution conditions for LC-MS are shown in the table below:

[0123] Using Orbitrap Exploris TM 480 mass spectrometer, optional FAIMS Pro TM Interface, compensation voltage CV switches between -45 and -65 every 1 second, Nanospray Flex TM The NSI ion source was set to an ion spray voltage of 2.0 kV and an ion transmission tube temperature of 320 °C. Mass spectrometry was performed in a data-dependent acquisition mode with a full scan range of 350-1500 m / z. The primary mass spectrometry resolution was set to 120,000 (200 m / z), AGC was 300%, and the maximum C-trap injection time was 50 ms. Secondary mass spectrometry detection was performed in "Top Speed" mode with a resolution of 15,000 (200 m / z), AGC of 75%, a maximum injection time of 22 ms, and a peptide fragmentation collision energy of 33%.

[0124] S642: Generate raw mass spectrometry detection data (.raw).

[0125] S65: Using the Homo sapiens SP database, employ UNIPROT to search for gene functions and analyze the roles of genes in the dataset:

[0126] S651: Use Proteome Discoverer 2.4 software for database searching. See the table below for parameter settings:

[0127] S652: The statistical analysis results of the global identification of proteins in cerebrospinal fluid samples are shown in the table below:

[0128] S653: Screening of differentially expressed proteins, statistical visualization of protein polymers and up- and down-regulated proteins:

[0129] SDS-PAGE: See Figure 12. Proteins before and after medication were analyzed by loading samples. The differentially expressed proteins that were significantly downregulated after treatment were screened from the SDS-PAGE gel images and protein expression levels.

[0130] Venn plot creation: Refer to Figure 13. From the file containing all original protein expression data, use the T-test to perform differential analysis. Based on the changes in expression levels, identify the differentially regulated and downregulated proteins. Statistically analyze and use Rstudio software to create a Venn plot to display the number and inclusion relationship of differentially regulated proteins and drug proteins.

[0131] Differential protein screening: The molecular weight of downregulated proteins was determined based on SDS-PAGE electrophoresis. Proteins with a molecular weight of 15 kDa that showed a significant decrease after drug administration were screened based on the P-value. Based on the corresponding protein molecular weight and changes, the genes HBB / HBD / HBA1 corresponding to proteins with a significantly downregulated molecular weight of around 15 kDa were matched.

[0132] S654: Differential Gene Display:

[0133] Referring to Figure 14, a volcano plot was created based on the selected differentially expressed genes, using different shades of gray to represent their up- and down-regulation. Figure 14 contains two parallel dashed lines separating three regions: the left side corresponds to down-regulated genes, the middle side to normal genes, and the right side to up-regulated genes. It can be seen that genes such as HBD, HBB, HBA, CA2, and CA1 deviate significantly from the baseline, indicating significant differences in their expression among down-regulated proteins.

[0134] S66: Analyze using the GSEA database:

[0135] GSEA analysis was performed on the mass spectrometry data of proteins in S652 to determine the up- and down-regulation of specific biological pathways or functional gene sets reflected by the differentially expressed proteins, as shown in the table below:

[0136] Analysis yielded the following results:

[0137] Upregulated pathways include those involved in caspase-mediated programmed cell death, epithelial-mesenchymal transition, encoding components of the coagulation system, and encoding components of the apical linker complex, suggesting a positive regulatory role in apoptosis, skeletal muscle development, epithelial-mesenchymal transition, and wound healing.

[0138] The downregulated pathways include heme metabolism, the il2-stat5 signaling pathway, xenobiotic metabolism, mtorc1 signaling, and components of the complement system.

[0139] It is evident that after administering the protein polymer to the subjects, it was able to regulate the more pronounced abnormalities in SCA disease progression, including metabolism of oxygen and foreign matter, glycolysis, and other abnormalities.

[0140] S67: KEGG_PATHWAY gene enrichment analysis of differentially expressed proteins:

[0141] Pathway analysis: Referring to Figures 15 and 16, the enrichment results of KEGG_PATHWAY show that the upregulated differentially expressed genes (Figure 15) are mainly enriched in lysosomes, sphingolipid glycosphingolipid metabolism and synthesis, axon formation, etc.; the downregulated differentially expressed genes (Figure 16) are mainly enriched in metabolic pathways such as nitrogen metabolism, HIF-1 signaling pathway, etc.

[0142] It is evident that, after administering the protein polymer to the subjects, on the one hand, the axon formation in the subjects' nerve cells is enhanced, allowing the nerve cells to maintain normal cell morphology and restore their biological functions; on the other hand, downregulation of metabolic pathways such as nitrogen metabolism and the HIF-1 signaling pathway can alleviate the abnormal energy metabolism caused by SCA, while actively clearing erroneous proteins.

[0143] S68: Gene Ontology (GO) enrichment analysis of differentially expressed proteins:

[0144] Referring to Figures 17 and 18, the enrichment results of GO show that the upregulated differentially expressed genes (Figure 17) are mainly enriched in processes such as nervous system development and axonal structure maintenance; while the downregulated differentially expressed genes (Figure 18) are mainly enriched in processes such as redox reactions and glycolysis.

[0145] It is evident that after administering the protein polymer to the subject, axon formation in the subject's nerve cells was enhanced, allowing the nerve cells to maintain normal cell morphology and restore their biological function.

[0146] S69: DISEASE gene enrichment analysis of differentially expressed proteins:

[0147] Referring to Figures 19 and 20, the DISEASE database can integrate disease information and discover the association between genes and diseases. According to the enrichment results of DISEASE, the upregulated differentially expressed genes (Figure 19) are mainly enriched in neurodegeneration, spinocerebellar ataxia, and thrombotic tendency, which correspond to the indications used in this application.

[0148] It is evident that protein polymers have a certain regulatory effect on genes related to SCA.

[0149] By integrating key differentially expressed proteins and matching them with key genes across different databases, statistical analysis based on scoring weights revealed that key differentially expressed genes are mainly divided into four categories. The first category primarily includes amyloid protein APLP1, whose main functions are related to axonal growth and synapsis. The second category mainly includes glycosylhydrolase HEXA and ceramidinase ASAH1. The former breaks down glycolipids and glycoproteins, maintaining cellular function and metabolism, and corresponds to genes related to glycolysis; the latter is involved in cell proliferation, differentiation, and apoptosis. The third category mainly includes increased hemoglobin subunits of HBB, HBA1, and BPGM, improving intracellular oxygen transport efficiency. The fourth category mainly includes γ-enolase ENO2, which has neurotrophic and neuroprotective properties on a broad spectrum of central nervous system (CNS) neurons and also plays an important role in the nervous system.

[0150] Experiment 7: In vitro damage simulation experiment of SH-SY5Y cells

[0151] S71: Experimental Grouping and Processing

[0152] Normal group: Untreated SH-SY5Y cells, used for baseline gene expression analysis, corresponding to groups 1-N;

[0153] Injury group: The group that used stress agents to induce nerve injury is group 1-M;

[0154] Damage repair group: Damage was induced first, and then different doses (10ng / mL, 100ng / mL, 1000ng / mL) of the protein polymer obtained in Experiment 1 were added to the damaged SH-SY5Y cells, corresponding to groups 1-10 (10ng / mL group), 1-100 (100ng / mL group), and 1-1000 (1000ng / mL group).

[0155] The protective damage group: Different doses (10ng / mL, 100ng / mL, 1000ng / mL) of the protein polymer were first added to SH-SY5Y cells, and then the cells were placed in stress reagents of corresponding concentrations to induce damage, corresponding to groups 2-10 (10ng / mL group), 2-100 (100ng / mL group), and 2-1000 (1000ng / mL group).

[0156] S72: RNA extraction and qPCR experiment:

[0157] S721: Total RNA was extracted from cells in each experimental group using the TRIzol method;

[0158] S722: Reverse transcription of the above RNA to synthesize cDNA;

[0159] S723: qPCR program: amplify the cDNA.

[0160] S73: Data Analysis: Comparative analysis of gene expression levels in the normal group, damaged group, damaged repair group (Figure 21), and protected damaged group (Figure 22) was conducted. The relative changes in gene expression were calculated using the ΔΔCt method. See Figures 21 and 22 for specific results.

[0161] As can be seen from Figures 21 and 22, among the differentially expressed genes, the downregulated genes include HBB, ENO2, and BPGM; the upregulated genes include ASAH1 and HEXA.

[0162] Further analysis revealed that key genes such as HEXA, HBB, and ENO2 exhibited significant effects in promoting neurite and axon growth and maintaining normal protein structure. Some genes were downregulated during the disease course to maintain redox balance and efficiently transport oxygen via hemoglobin subunits. Consequently, genes performing these functions were upregulated during the disease progression. After the addition of protein polymers, SCA symptoms were alleviated, and the body's energy metabolism and respiratory function recovered, leading to a corresponding downregulation of the expression levels of related genes.

[0163] In summary, the above analysis and the symptom relief observed in the subjects indicate that protein polymers play a regulatory role in the symptoms of SCA patients. Our extraction and analysis of differentially expressed proteins, along with enrichment data from databases such as KEGG and GO, revealed the potential for further research in areas such as protein modification and processing, apoptosis, and cell structure.

[0164] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

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, and separating and purifying 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 neurodegenerative diseases, and further, the neurodegenerative diseases comprise spinocerebellar ataxia (SCA).

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