Muse cell-derived protein complex, preparation method therefor, and use thereof

Abstract

The present invention belongs to the field of biopharmaceuticals. Disclosed are a Muse cell-derived protein complex, a preparation method therefor, and use thereof. The composition of the protein complex is: a stem cell lysate of Muse cells after being cultured in a stress environment for a predetermined time, or a protein complex obtained by separating and purifying the stem cell lysate. The protein complex has a good cell damage repair effect, especially a strong ability to repair nerve cell damage, and is expected to be used for treating neurodegenerative diseases, cerebral stroke, cerebrovascular disease, arthritis, enteritis, recovery after trauma, autism, depression, and pulmonary fibrosis. 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).

Description

Muse cell-derived protein complexes, their preparation methods, and applications.

[0001] This disclosure claims priority to Chinese Patent Application No. 202411422168.1, filed on October 12, 2024, entitled "Muse Cell-Derived Protein Polymer and its Preparation Method and Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biopharmaceuticals, specifically to protein complexes derived from Muse cells, their preparation methods, and their applications in diseases such as nervous system disorders, arthritis, enteritis, and pulmonary fibrosis. Background Technology

[0003] Stem cells 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 not requiring matching, low infection rate, strong differentiation potential, high proliferation capacity, and convenient collection. Stem cells 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), participating in the regulation of 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. However, the content of stem cells in biological tissues is low, and they are generally difficult to obtain; for example, umbilical cord stem cells are limited to umbilical cord sources. Muse cells (multilineage differentiating stress enduring cells) are a new type of stem cell derived from various tissues and organs such as human skin and bone marrow. They are widely available and easier to obtain and prepare. Moreover, Muse cells are natural cells, controlled by the body's immune system, and are less prone to becoming cancerous.

[0004] The inventors discovered that stimulating Muse cells with stress conditions such as radiation or ultrasound, followed by cell lysis, yields a protein complex with physiological and pharmacological activity. Furthermore, the composition of the stress protein expressed within Muse cells varies depending on the stress conditions, resulting in different physiological activities of the protein complex during application. Therefore, it is essential to screen the stress conditions for Muse cell culture to explore the optimal conditions for Muse cell expression of stress proteins, thereby achieving stable and large-scale production of the protein complex. Summary of the Invention

[0005] The purpose of this invention is to overcome at least one deficiency of the prior art and provide a protein complex derived from Muse cells, obtained by screening Muse cells to express stress proteins under stress conditions.

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

[0007] A protein complex derived from Muse cells, the protein complex being composed of: stem cell lysate of Muse cells cultured under stress for a predetermined time, or a protein complex obtained by separating and purifying the stem cell lysate;

[0008] The stress environment includes at least one of the following stress conditions: ultraviolet radiation, infrared radiation, electromagnetic field, low temperature, low oxygen, high oxygen, oxidation, reduction, high pH value, low pH value, ultrasound, terahertz electromagnetic wave, X-ray, microwave, alpha rays, beta rays, gamma rays, electron beam, high CO2 concentration atmosphere and low CO2 concentration atmosphere.

[0009] The predetermined time is 0.5h to 96h.

[0010] In some protein complex embodiments, the ultraviolet radiation stress conditions satisfy at least one of the following conditions: irradiation time of 1 h to 96 h, and irradiation intensity of 1 μW / cm². 2 ~500μW / cm 2 The wavelength of ultraviolet light is 0.150μm to 0.380μm;

[0011] The infrared irradiation stress condition satisfies at least one of the following conditions:

[0012] The irradiation time ranges from 1 hour to 96 hours, and the irradiation intensity is 1 μW / cm². 2 ~500μW / cm 2 The infrared wavelength is 0.760μm~1000μm;

[0013] The low-temperature stress conditions are 2.0℃~25.0℃;

[0014] The hypoxic stress condition is characterized by an oxygen volume content of 3.0% to 16.0%.

[0015] The hyperoxia stress condition is characterized by an oxygen volume content of 22.0% to 100.0%.

[0016] The high pH stress condition is 7.6–10.0;

[0017] The low pH stress condition is 1.0–6.8;

[0018] The ultrasound intensity of the ultrasound stress condition does not exceed the ultrasound intensity that Muse cells can tolerate.

[0019] The terahertz electromagnetic wave frequency of the stress condition is 0.1THz to 10THz, and the wavelength range is 30μm to 3000μm.

[0020] The intensity of X-ray, microwave, alpha, beta, gamma, and electron beam irradiation shall not exceed the intensity that Muse cells can tolerate.

[0021] The electromagnetic field stress condition is that the magnetic field strength does not exceed the strength that Muse cells can tolerate;

[0022] The high CO2 concentration atmospheric stress conditions are: CO2 content of 5.1% to 15.0%;

[0023] The low CO2 concentration atmospheric stress conditions are: CO2 content of 1.0% to 4.9%;

[0024] The oxidation is a stress condition containing an oxidant, the concentration of which is 10. -2 nM~10 6 nM;

[0025] The reduction is a stress condition containing a reducing agent, the concentration of which is 10. -2 nM~10 6 nM.

[0026] In some protein complex embodiments, the oxidant is one or more selected from hydrogen peroxide, hypochlorous acid, sulfate, dichromate, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, potassium iodate, potassium bromate, chlorate, potassium peroxide, magnesium peroxide, calcium peroxide, barium peroxide, nitrate, fluorine, chlorine, oxygen, bromine water, elemental iodine, elemental sulfur, elemental silicon, nitric acid, manganese oxide, and ferric chloride.

[0027] In some protein complex embodiments, the oxidant is hydrogen peroxide and / or hypochlorous acid.

[0028] In some protein complex embodiments, the reducing agent includes one or more of glutathione, ferrous salt, thiourea dioxide, thiourea, sulfide reducing agents, thioacetic acid, TCEP, DTT, and mercaptoethanol.

[0029] In some protein complex embodiments, the ultrasonic intensity condition of the ultrasonic stress condition is: ultrasonic frequency greater than 2 × 10⁻⁶. 4 Hz, ultrasonic intensity greater than 0.003 W / cm 2 .

[0030] In some protein complex embodiments, the Muse cells were cultured under stress conditions using at least one of the following methods: fixed stress conditioned culture, sequential stress conditioned culture, and gradient stress conditioned culture.

[0031] In some protein complex embodiments, the predetermined time is 1h to 72h or 2h to 48h.

[0032] In some protein complex embodiments, it includes at least one of the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens; 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-1chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-AOS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy 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 1OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS = Homo sapiens.

[0033] The amount of sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens accounts for 45% to 55% of the total mass of the protein complex, the amount of sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens accounts for 10% to 20% of the total mass of the protein complex, and the amount of sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens accounts for 0.1% to 0.5% of the total mass of the protein complex.

[0034] These features can be combined arbitrarily as long as they do not conflict with each other.

[0035] A second aspect of the present invention provides:

[0036] A method for preparing a Muse cell-derived protein complex, used to prepare the Muse cell-derived protein complex as described in any one of the above-mentioned methods, the method comprising:

[0037] S1: Add Muse cells to serum-free stem cell culture medium for cell expansion;

[0038] S2: Control the culture environment of Muse cells to at least one of the following stress environments: ultraviolet irradiation, infrared irradiation, electromagnetic field, low temperature, low oxygen, high oxygen, oxidation, reduction, high pH value, low pH value, ultrasound, terahertz electromagnetic wave, X-ray, microwave, alpha ray, beta ray, gamma ray, electron beam, high CO2 concentration atmosphere, and low CO2 concentration atmosphere, and set the predetermined culture time to 0.5 to 96 hours;

[0039] S3: Lyse the Muse cells after stress culture to obtain the protein complex as the lysate of the Muse cells, or further separate and purify the cell lysate to obtain the protein complex.

[0040] In some embodiments of the methods for preparing protein complexes, the ultraviolet irradiation stress conditions satisfy at least one of the following conditions: irradiation time of 1 h to 96 h, and irradiation intensity of 1 μW / cm². 2 ~500μW / cm 2 The wavelength of ultraviolet light is 0.150μm to 0.380μm;

[0041] The infrared irradiation stress conditions satisfy at least one of the following conditions: irradiation time is 1 h to 96 h, and irradiation intensity is 1 μW / cm². 2 ~500μW / cm 2The infrared wavelength is 0.760μm~1000μm;

[0042] The low-temperature stress conditions are 2.0℃~25.0℃;

[0043] The hypoxic stress condition is characterized by an oxygen volume content of 3.0% to 16.0%.

[0044] The hyperoxia stress condition is characterized by an oxygen volume content of 22.0% to 100.0%.

[0045] The high pH stress condition is 7.6–10.0;

[0046] The low pH stress condition is 1.0–6.8;

[0047] The ultrasound intensity of the ultrasound stress condition does not exceed the ultrasound intensity that Muse cells can tolerate.

[0048] The terahertz electromagnetic wave frequency of the stress condition is 0.1THz to 10THz, and the wavelength range is 30μm to 3000μm.

[0049] The intensity of X-ray, microwave, alpha, beta, gamma, and electron beam irradiation shall not exceed the intensity that Muse cells can tolerate.

[0050] The electromagnetic field stress condition is that the magnetic field strength does not exceed the strength that Muse cells can tolerate;

[0051] The high CO2 concentration atmospheric stress conditions are: CO2 content of 5.1% to 15.0%;

[0052] The low CO2 concentration atmospheric stress conditions are: CO2 content of 1.0% to 4.9%;

[0053] The oxidation is a stress condition containing an oxidant, the concentration of which is 10. -2 nM~10 6 nM;

[0054] The reduction is a stress condition containing a reducing agent, the concentration of which is 10. -2 nM~10 6 nM.

[0055] In some embodiments of the preparation method of protein complexes, the oxidant is one or more selected from hydrogen peroxide, hypochlorous acid, sulfate, dichromate, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, potassium iodate, potassium bromate, chlorate, potassium peroxide, magnesium peroxide, calcium peroxide, barium peroxide, nitrate, fluorine, chlorine, oxygen, bromine water, elemental iodine, elemental sulfur, elemental silicon, nitric acid, manganese oxide, and ferric chloride.

[0056] In some embodiments of the preparation method of protein complexes, the oxidant is hydrogen peroxide and / or hypochlorous acid.

[0057] In some embodiments of the preparation methods of protein complexes, the reducing agent includes one or more of glutathione, ferrous salt, thiourea dioxide, thiourea, sulfide reducing agents, thioacetic acid, TCEP, DTT, and mercaptoethanol.

[0058] In some embodiments of the protein complex preparation method, the ultrasonic intensity condition of the ultrasonic stress condition is: ultrasonic frequency greater than 2 × 10⁻⁶. 4 Hz, ultrasonic intensity greater than 0.003 W / cm 2 .

[0059] In some embodiments of the protein complex preparation method, the Muse cells were cultured under stress using at least one of the following methods: fixed stress culture, sequential stress culture, and gradient stress culture.

[0060] In some protein complex embodiments, the predetermined time is 1 hour to 72 hours.

[0061] In some protein complex embodiments, the predetermined time is 2h to 48h.

[0062] In some embodiments of the protein complex preparation method, the method of lysing the stress-cultured Muse cells includes at least one of ultrapure water swelling, ultrasonic lysis, and chemical lysis, wherein the chemical lysis further includes urea lysis and guanidine hydrochloride lysis.

[0063] These features can be combined arbitrarily as long as they do not conflict with each other.

[0064] A third aspect of the present invention provides:

[0065] The application of a Muse cell-derived protein complex, using the protein complex described in any one of the above claims or the protein complex prepared by any one of the above claims, in the preparation of medicaments for treating neurodegenerative diseases, stroke, cerebrovascular diseases, arthritis, enteritis, post-traumatic recovery, autism, depression, and pulmonary fibrosis, further wherein the neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and different types of spinocerebellar ataxia (SCA).

[0066] The effects of the invention

[0067] This invention provides protein complexes derived from Muse cells, which, under specific stress conditions, stimulate Muse cells to produce proteins with specific biological activities. Experimental verification has shown that the protein complexes provided by this invention have excellent cell damage repair effects, especially strong repair capabilities for nerve cell damage, and hold promise for the treatment of neurodegenerative diseases, stroke, cerebrovascular diseases, arthritis, enteritis, post-traumatic recovery, autism, depression, and pulmonary fibrosis. 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).

[0068] The protein complex preparation methods of some examples of the present invention use Muse cells with high biological stability for stress culture, which are less prone to gene mutation and can effectively overcome the differences between different batches of stem cells. The Muse cells after stress culture have small batch-to-batch differences and more stable characteristics, which greatly ensures the quality and yield of lysates and protein complexes separated and purified from lysates.

[0069] The protein complex preparation methods of some examples of the present invention can improve the quality and yield of protein complexes.

[0070] The protein complex preparation methods of some examples of the present invention can isolate protein complexes with higher purity. Attached Figure Description

[0071] Figure 1 is a photograph of the growth status of Muse cells after culturing in 3D medium in culture experiment 1-1 of Example 1.

[0072] Figure 2 is a photograph of the Muse cells after 96 hours of culture under UV irradiation stress in culture experiment 1-1 of Example 1.

[0073] Figure 3 shows the SDS-PAGE results of the protein complex harvested in culture experiment 1-1 of Example 1.

[0074] Figure 4 shows the cell state of Muse cells before ultraviolet irradiation stress in culture experiments 1-2 of Example 1.

[0075] Figure 5 is a photograph of the cell state of Muse cells after 48 hours of ultraviolet irradiation stress culture in culture experiments 1-2 of Example 1.

[0076] Figure 6 is a statistical bar chart showing the effect of protein complexes from Muse cell lysates cultured under UV stress on neuronal cell regeneration in Example 2.

[0077] Figure 7 is a statistical bar chart showing the ability of protein complexes in the lysate of Muse cells cultured under oxidative stress in Example 3 to repair neuronal cell damage.

[0078] Figure 8 is a statistical bar chart showing the ability of protein complexes in the lysate of Muse cells cultured under ultrasound stress in Example 4 to repair neuronal cell damage. Detailed Implementation

[0079] The technical solution of the present invention will be further described below with reference to exemplary embodiments.

[0080] For ease of explanation, the examples used ultrapure water swelling and repeated freeze-thaw cycles to lyse Muse cells after stress culture. However, in practical applications, any other feasible lysis method can be used. The lysis methods for stress-cultured Muse cells include, but are not limited to, ultrapure water swelling, repeated freeze-thaw cycles, ultrasonic lysis, urea lysis, and guanidine hydrochloride lysis.

[0081] Theoretically, any method that can be used to separate proteins from cell lysates can be used to isolate and purify the protein complex described in this invention from Muse cells cultured under stress. For ease of explanation, chromatography, electrophoresis, and volumetric methods (molecular sieves) are used in some embodiments of this invention. In practical applications, any method that can separate and extract the target protein from the lysate can be used, including but not limited to at least one of the following methods: chromatography, spectrometry, volumetric methods, dialysis, salting out, precipitation, acid extraction, alkaline extraction, ultrafiltration, chromatography, electrophoresis, and centrifugation. The technical solution of this invention is further illustrated below with reference to experimental examples.

[0082] For ease of explanation, some exemplary embodiments use conditioned stress culture of Muse cells under ultraviolet irradiation, oxidation, or ultrasound. In practical applications, other methods may include infrared irradiation, electromagnetic fields, low temperature, low oxygen, high oxygen, reduction, high pH, ​​low pH, ultrasound, terahertz electromagnetic waves, X-rays, microwaves, alpha rays, beta rays, gamma rays, electron beams, high CO2 concentration atmospheres, or low CO2 concentration atmospheres.

[0083] For ease of explanation, the wavelength of ultraviolet irradiation used in some exemplary embodiments is 0.280 μm. In practical applications, the ultraviolet irradiation intensity is 1 μW / cm². 2 ~500μW / cm 2 Ultraviolet light with wavelengths ranging from 0.150μm to 0.380μm can be used.

[0084] For ease of explanation, the ultrasonic frequency used in some exemplary embodiments is 2×10⁻⁶. 5 Hz, in practical applications, ultrasonic frequencies are greater than 2×10 Hz. 4 Hz, ultrasonic intensity >0.003W / cm 2 All stress conditions can be used to stimulate Muse cells.

[0085] For ease of explanation, hydrogen peroxide (also known as H2O2) was used as the oxidant in the examples to prepare oxidative stress conditions. In practical applications, any other oxidant capable of preparing oxidative conditions can be used. Oxidants include, but are not limited to, hydrogen peroxide, hypochlorous acid, sulfates, dichromates, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, potassium iodate, potassium bromate, chlorate, potassium peroxide, magnesium peroxide, calcium peroxide, barium peroxide, nitrates, fluorine, chlorine, oxygen, bromine water, elemental iodine, elemental sulfur, elemental silicon, nitric acid, manganese oxide, and ferric chloride.

[0086] Example 1: Effects of different UV irradiation stress culture conditions on protein expression levels

[0087] 1-1 Culture Experiment 1

[0088] Muse cells were cultured in serum-free stem cell culture medium, with a total cell count of approximately 5 × 10⁶. 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.

[0089] LED ultraviolet light (310nm) irradiation of cells, irradiation conditions: 60μW / cm² 2 Samples were taken after 0 h of culture and after 96 h of irradiation. The cells were then added to 20 mL of pure water to swell for 10 min, and the lysate was obtained by filtering through a 0.22 μm filter membrane.

[0090] Taking 96 hours of ultraviolet irradiation as an example, the cell state after irradiation is shown in Figure 2.

[0091] The harvested Muse cell lysates were analyzed by gel electrophoresis, and the SDS-PAGE results are shown in Figure 3.

[0092] 1-2 Cultivation Experiment Two

[0093] Resuscitate Muse cells into multiple T25 culture flasks, adding 2.5 mL of serum-free stem cell culture medium to each flask.

[0094] Four flasks of cells were irradiated with 308nm LED ultraviolet light for 0.5h, 8h, 24h, and 48h, respectively. The cells were carefully cleaned of supernatant and washed twice with 1mL of physiological saline. Then, 900μL of pure water was added and the cells were repeatedly lysed by pipetting for 10min. After passing through a 0.22μm filter membrane, Muse cell lysate was obtained and stored at 4℃.

[0095] The ultraviolet irradiation conditions are as described in Table 1:

[0096] Table 1

[0097] The cell state before UV irradiation is shown in Figure 4. Taking irradiation for 24 hours as an example, the cell state after irradiation is shown in Figure 5. It can be seen that UV irradiation affects cell morphology and causes survival stress on cells. As a result, cells can produce stress proteins for self-protection under this stress environment. These proteins produced due to stress also have potential protective effects on other cells.

[0098] Muse cells were cultured under standard conditions without UV irradiation for 8 hours. The supernatant was then removed, and the cells were washed twice with 1 mL of physiological saline. Next, 900 μL of pure water was added, and the cells were repeatedly lysed by pipetting for 10 min. The lysate was then filtered through a 0.22 μm filter and stored at 4 °C. This sample served as a control.

[0099] The protein concentration and volume harvested are shown in Table 2.

[0100] Table 2

[0101] As shown in Table 2, protein production is better when exposed to ultraviolet light for about 8 to 24 hours.

[0102] Example 2: Purification of protein complexes from UV-irradiated stress-cultured Muse cells using molecular sieves and their activity testing.

[0103] 2-1 Molecular sieve purification of Muse cell lysates

[0104] Instrument: AKTA explorer;

[0105] Chromatography column: Nanomicro Superdex 150 molecular sieve 8×500, column volume (CV) approximately 30 mL;

[0106] Reagents: 0.1M NaOH, 20% ethanol, 1×PBS, purified water;

[0107] UV absorption wavelength: 280nm, 260nm is used as a reference to determine the protein sample elution;

[0108] Equilibrium chromatography column sequence:

[0109] First, rinse the chromatography column with 2CV purified water, then equilibrate the column with 2CV 1×PBS.

[0110] Sample preparation: Take approximately 10 mL of each sample after UV irradiation in Experiment 1 (i.e., Muse cell lysate). Use an ultrafiltration concentrator with a molecular weight cutoff of 3 KD to reduce the 20 mL sample to approximately 600 μL.

[0111] Experimental procedure: After equilibrating the chromatography column, load the sample using a 500 μL loop at a flow rate of 0.4 mL / min. Elute with 1×PBS at a flow rate of 0.2 mL / min until the peak is reached. Collect the protein starting from a UV absorbance of 4 mAU, eluting and collecting the fraction between 4.0 mL and 34.0 mL. This is the purified protein complex.

[0112] The purified 28h sample (i.e. protein complex) was analyzed by SDS-PAGE electrophoresis. The electrophoresis results showed that the sample bands were mainly distributed in the range of 11KD to 100KD. Among them, the molecular weight decreased from large to small. The first band was located between 75KD and 100KD, and the second band was located between 63KD and 75KD.

[0113] Further mass spectrometry analysis of the purified protein complex revealed that it contained at least one of the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens; 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-1chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy 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 1OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0114] The amount of sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens accounts for 45% to 55% of the total mass of the protein complex, the amount of sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens accounts for 10% to 20% of the total mass of the protein complex, and the amount of sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens accounts for 0.1% to 0.5% of the total mass of the protein complex.

[0115] Bioactivity assay of the 2-2 protein complex

[0116] Day 1: Cell Plating: Dilute PC12 low-differentiation cells with complete culture medium (5% FBS + DMEM) and plate 6000 cells / well (96-well plate). Incubate overnight at 37°C with 5% CO2.

[0117] Day 2: Dilute the unpurified and purified samples obtained in the above experiment with DMEM + 5% FBS medium (the concentration after dilution is approximately 800 μg / mL).

[0118] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS.

[0119] Hydrogen peroxide treatment of cells: Discard 80 μL / well of cell culture supernatant, add 50 μL / well of diluted hydrogen peroxide to the corresponding cultured cells, and incubate at room temperature for 25 min.

[0120] Untreated control: 50 μL / well of DMEM + 5% FBS medium was added as a damage treatment control.

[0121] In the PC12 cell oxidative damage model, diluted unpurified samples and purified samples (50 μL / well) were added, respectively. At the same time, a control group without protein was set up in the PC12 cell oxidative damage model (the control group without protein after oxidative damage).

[0122] Discard the supernatant from the untreated wells and add 100 μL of complete culture medium per well as a cell growth control (pure PC12 cell control group).

[0123] Incubate at 37℃ for 2 days.

[0124] Day 5: Discard the culture supernatant, add 100 μL / well of complete culture medium, and set up blank control wells. Add 10 μL / well of CCK8 and incubate at 37℃ for 3.5 h. OD 450 Readings. Calculated after subtracting the culture medium blank.

[0125] The experimental results are shown in Figure 6. In the PC12 cell oxidative damage model, all samples derived from Muse cell lysates cultured under UV stress exhibited oxidative damage repair capabilities. Moreover, the protein complexes prepared by UV stress treatment for 8–24 h showed better activity, and the purified samples demonstrated superior neuronal cell damage repair capabilities compared to the unpurified samples.

[0126] Example 3: Preparation and activity testing of Muse stem cell lysates derived from oxidative stress culture

[0127] 3.1 Preparation of protein complexes

[0128] Muse cells were cultured in T25 culture flasks, with 2.5 mL of serum-free stem cell culture medium added to each flask. The cells were cultured in serum-free stem cell culture medium containing 1 mM H2O2 for 16 h. After culture, the supernatant was carefully removed from the stem cells, and they were washed twice with 1 mL of physiological saline. Then, 1 mL of ultrapure water was added to the cells to swell them, and the stem cells were repeatedly lysed by pipetting for 10 min. The lysate was then collected after filtration through a 0.22 μm filter membrane, yielding the Muse stem cell lysate.

[0129] 3.2 Purification of protein complexes

[0130] The obtained unpurified protein complex sample was diluted to 300 μg / mL with PBS, and then purified using Agilent AdvanceBio SEC 2.7 μm. The column was 7.8 × 300 mm, and the injection volume was 500 μL. 150 mmol / L PB was used as the mobile phase, the time was 40 min, the flow rate was 0.35 mL / min, the column temperature was 30 ℃, and the detection wavelength was 280 nm UV. The components in the main peak from 21 min to 23 min were collected, which were the protein complex components.

[0131] 3.3 Detection of the neural repair activity of protein complexes

[0132] S1, cell seeding plate

[0133] Adjust SH-SY5Y cells to 10 5 At a density of 100 μL / well, 100 μL of the solution was seeded into a 96-well plate.

[0134] S2, cell modeling and drug intervention

[0135] The cell model was established 24 hours after cell seeding. The modeling process is as follows:

[0136] Dilute hydrogen peroxide (1M concentration) to 200μM with complete culture medium. Prepare fresh solution each time you use it.

[0137] The experiment was divided into three groups: normal cell group, model control group, and sample intervention group, with five replicates in each group.

[0138] The samples added to the sample intervention group were divided into purified samples, unpurified samples, and mixed protein groups (a mixture of three proteins: Serum albumin, Serotransferrin, and Actin-aortic smooth muscle, with a mass ratio of 10:4:1). The protein concentration of the added samples was uniformly 100 ng / mL, diluted with culture medium. One sample group was set up for each sample. The unpurified samples were the protein components of Muse cell lysates, and the purified samples were the purified protein components.

[0139] Aspirate the culture supernatant from all wells. Add 200 μM hydrogen peroxide (100 μL / well) to the model control group and the sample intervention group. After about 30 minutes of damage, the cell modeling is complete.

[0140] Add protein complex: Immediately after modeling, aspirate the supernatant as cleanly as possible. The sample intervention group is administered the drug at the above concentration, 100 μL per well. The model control group and normal cell group are replaced with 100 μL of normal culture medium per well. Continue to incubate at 37°C and 5% CO2 concentration in an incubator for 72 h.

[0141] S3. Detection of NFL (Neurofilament Light Protein) content in supernatant

[0142] NFL is a cytoskeletal protein expressed in neurons, belonging to the neurofilament protein (NF) family. It is primarily expressed in neuronal axons and axons, playing a role in maintaining axonal morphology and ensuring neuronal signal transduction. Under physiological conditions, axons release small amounts of NFL protein, but under pathological conditions, such as axonal injury, the release of NFL protein increases significantly. This characteristic makes NFL protein a key biomarker for assessing neuronal axonal injury. Abnormally elevated NFL protein levels suggest neurodegeneration. Therefore, detecting NFL content in the supernatant can be used to assess the neuronal cell repair capacity.

[0143] According to the instructions for use of Jianglai Biotechnology reagent kit, a standard curve was prepared, and the OD values ​​of the samples were analyzed. 450 The absorbance value was used to calculate the sample concentration. The NFL content in the sample was calculated based on the fitted standard curve, and the repair efficiency was calculated. NFL repair rate = (model group content - intervention group content) / model group content, expressed as a percentage. The NFL repair rates for each group are shown in Figure 7.

[0144] As shown in Figure 7, the protein complex sample possessed the ability to repair nerve cell damage in the cellular oxidative damage model. Furthermore, the purified sample showed better results. The control group, consisting of mixed proteins, exhibited no cell damage repair ability.

[0145] Example 4: Activity test of protein complexes obtained from Muse cell lysates cultured under ultrasound stress and purified from them.

[0146] Muse cells were cultured in T25 culture flasks, with 2.5 mL of serum-free stem cell culture medium added to each flask. Muse cells were cultured at 0.3 W / cm². 2 Muse cells were cultured under ultrasonic intensity for 0.5 h, 24 h, and 96 h, respectively. After culture, the supernatant was carefully removed from the stem cells, and they were washed twice with 1 mL of physiological saline. Then, 1 mL of pure water was added to the cells to swell them, and the cells were repeatedly pipetted and lysed for 10 min. The lysate was then filtered through a 0.22 μm filter membrane and collected to obtain the stress-cultured Muse cell lysate.

[0147] The obtained unpurified protein complex sample was diluted to 300 μg / mL with PBS, and then purified using an Agilent AdvanceBio SEC 2.7 μm filter. The column was 7.8 × 300 mm, and the injection volume was 500 μL. 150 mmol / L PB was used as the mobile phase, the time was 40 min, the flow rate was 0.35 mL / min, the column temperature was 30 ℃, and the detection wavelength was 280 nm UV. The components in the main peak from 21 min to 23 min were collected, which were the protein complex components.

[0148] The purified and unpurified protein complexes were subjected to activity testing, following the same procedure as in section 3.3 of Example 3. The activity test results for each group in repairing nerve cell damage are shown in Figure 8.

[0149] As shown in Figure 8, the stress-induced protein complexes have cell repair activity. Among them, the protein complexes obtained by culturing under ultrasound stress for 24 hours have the best effect, and the repair effect of purified samples is better than that of unpurified samples.

[0150] The embodiments described in this invention use intracellular proteins, but in practical applications, protein complexes secreted into the supernatant outside the cell can also be used.

[0151] 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 method for preparing a protein complex derived from Muse cells, characterized in that, The preparation method includes: S1: Add Muse cells to serum-free stem cell culture medium for cell expansion; S2: Control the culture environment of Muse cells to at least one of the following stress environments: ultraviolet irradiation, infrared irradiation, electromagnetic field, low temperature, low oxygen, high oxygen, oxidation, reduction, high pH value, low pH value, ultrasound, terahertz electromagnetic wave, X-ray, microwave, alpha ray, beta ray, gamma ray, electron beam, high CO2 concentration atmosphere, and low CO2 concentration atmosphere, and set the predetermined culture time to 0.5h to 96h; S3: Lyse the Muse cells after stress culture to obtain the protein complex as the lysate of the Muse cells, or further separate and purify the cell lysate to obtain the protein complex.

2. The preparation method according to claim 1, characterized in that, The ultraviolet radiation stress conditions satisfy at least one of the following conditions: irradiation time is 1 h to 96 h, and irradiation intensity is 1 μW / cm². 2 ~500μW / cm 2 The wavelength of ultraviolet light is 0.150μm to 0.380μm; The infrared irradiation stress conditions satisfy at least one of the following conditions: irradiation time is 1 h to 96 h, and irradiation intensity is 1 μW / cm². 2 ~500μW / cm 2 The infrared wavelength is 0.760μm~1000μm; The low-temperature stress conditions are 2.0℃~25.0℃; The hypoxic stress condition is characterized by an oxygen volume content of 3.0% to 16.0%. The hyperoxia stress condition is characterized by an oxygen volume content of 22.0% to 100.0%. The high pH stress condition is 7.6–10.0; The low pH stress condition is 1.0–6.8; The ultrasound intensity of the ultrasound stress condition does not exceed the ultrasound intensity that Muse cells can tolerate. The terahertz electromagnetic wave frequency of the stress condition is 0.1THz to 10THz, and the wavelength range is 30μm to 3000μm. The intensity of X-ray, microwave, alpha, beta, gamma, and electron beam irradiation shall not exceed the intensity that Muse cells can tolerate. The electromagnetic field stress condition is that the magnetic field strength does not exceed the strength that Muse cells can tolerate; The high CO2 concentration atmospheric stress conditions are: CO2 content of 5.1% to 15.0%; The low CO2 concentration atmospheric stress conditions are: CO2 content of 1.0% to 4.9%; The oxidation is a stress condition containing an oxidant, the concentration of which is 10. -2 nM~10 6 nM; The reduction is a stress condition containing a reducing agent, the concentration of which is 10. -2 nM~10 6 nM.

3. The preparation method according to claim 2, characterized in that, The oxidant is one or more of the following: hydrogen peroxide, hypochlorous acid, sulfate, dichromate, peracetic acid, chromic acid, ammonium persulfate, sodium hypochlorite, sodium percarbonate, sodium perborate, potassium perborate, perchlorate, permanganate, sodium peroxide, potassium iodate, potassium bromate, chlorate, potassium peroxide, magnesium peroxide, calcium peroxide, barium peroxide, nitrate, fluorine, chlorine, oxygen, bromine water, elemental iodine, elemental sulfur, elemental silicon, nitric acid, manganese oxide, and ferric chloride; preferably hydrogen peroxide and / or hypochlorous acid.

4. The preparation method according to claim 2, characterized in that, The reducing agent includes one or more of glutathione, ferrous salt, thiourea dioxide, thiourea, sulfide reducing agent, thioacetic acid, TCEP, DTT, and mercaptoethanol.

5. The preparation method according to claim 2, characterized in that, The ultrasonic intensity condition for the ultrasonic stress condition is: ultrasonic frequency greater than 2 × 10⁻⁶. 4 Hz, ultrasonic intensity greater than 0.003 W / cm 2 .

6. The preparation method according to claim 1, characterized in that, The Muse cells were cultured under stress conditions using at least one of the following methods: fixed stress conditioned culture, sequential stress conditioned culture, and gradient stress conditioned culture.

7. The preparation method according to claim 1, characterized in that, Methods for lysing the stressed Muse cells include at least one of ultrapure water swelling, ultrasonic lysis, and chemical lysis. Preferably, the chemical pyrolysis includes urea pyrolysis and guanidine hydrochloride pyrolysis.

8. A protein complex obtained by the preparation method according to any one of claims 1 to 7.

9. The protein complex according to claim 8, characterized in that, The protein complex includes at least one of the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens; 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-1chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy 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 1OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens; The amount of sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens accounts for 45% to 55% of the total mass of the protein complex, the amount of sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens accounts for 10% to 20% of the total mass of the protein complex, and the amount of sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens accounts for 0.1% to 0.5% of the total mass of the protein complex.

10. The use of the protein complex obtained by the preparation method according to any one of claims 1 to 7 or the protein complex according to claim 8 or 9 in the preparation of medicaments for treating neurodegenerative diseases, stroke, cerebrovascular diseases, arthritis, enteritis, post-traumatic recovery, autism, depression and pulmonary fibrosis; Preferably, the neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and different types of spinocerebellar ataxia (SCA).

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