Protein polymer derived from stem cell lysate of ultrasonic stress culture as well as preparation method and application of protein polymer
By culturing stem cells under ultrasound conditions and isolating and purifying protein polymers, the problem of poor stem cell growth under ultrasound conditions was solved, and protein polymers with significant therapeutic effects were obtained for the treatment of various diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2026-04-10
AI Technical Summary
In existing technologies, culturing stem cells in the presence of ultrasound can impair cell growth, resulting in poor growth and an inability to effectively utilize the pharmacological activity of stress proteins generated under ultrasound stress conditions.
Stem cells were cultured in an ultrasonic environment with an ultrasonic frequency of not less than 2×104Hz and an intensity of not less than 0.003W/cm². Subsequently, protein polymers were obtained by separation and purification using methods such as chromatography and spectroscopy. Specific proteins were distributed in the range of 11KD to 100KD.
The obtained protein polymers have good cell damage repair effects and can effectively treat neurodegenerative diseases, stroke, arthritis, post-traumatic recovery, autism, enteritis, depression and other diseases. In particular, they have significant effects on neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.
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Figure CN121824731A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and particularly relates to a protein polymer derived from stem cell lysate under ultrasonic stress culture, and a preparation method and application thereof. BACKGROUND
[0002] Stem cells (SCs) have self-replication and multi-directional differentiation potential, and widely exist in bone marrow, fat, synovial membrane, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amnion, peripheral blood, muscle, urine and other tissues, and have characteristics of wide source, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, 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), and are involved 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 and acute respiratory distress syndrome.
[0003] Ultrasound can be used to break cells, and is an external factor that has potential influence on cell culture and cell proliferation, and can have a direct influence on cell growth and metabolism. Generally, cell culture should not be carried out in the presence of ultrasound to ensure normal growth and reproduction of cells. Culturing cells in the presence of ultrasound can damage stem cells and cause poor growth and other problems. However, the present application finds that stem cells cultured under the stimulation of ultrasound can produce appropriate stress proteins, and the stress protein polymer has certain unexpected physiological and pharmacological activities after extraction. SUMMARY
[0004] The present application aims to overcome at least one deficiency of the prior art, and provides a protein polymer derived from stem cell lysate under ultrasonic stress culture, and a preparation method and application thereof.
[0005] The technical solution adopted by the present application is as follows: The present application provides a preparation method of a protein polymer derived from stem cell lysate under ultrasonic stress culture, characterized in that the preparation method comprises the following steps: S1) Culturing stem cells and using an ultrasonic environment to culture to create a stress environment; S2) Lysing the stem cells after ultrasonic stress culture, and separating and purifying to obtain the protein polymer; The frequency of the ultrasound is not less than 2x10 4 Hz, and the ultrasonic intensity is not less than 0.003 W / cm² and not more than the tolerance limit of the stem cells.
[0006] Preferably, the method for separating and purifying comprises at least one of chromatography, spectrometry, volumetry, dialysis, salting-out, precipitation, acid extraction, molecular sieve, base extraction, ultrafiltration, chromatography, electrophoresis, and centrifugation.
[0007] Preferably, the protein polymer satisfies the following properties: when detected by SDS-PAGE, the sample is detected using a 4%-20% precast gel, and the sample bands are mainly distributed in 11KD-100KD, wherein the molecular weight is from large to small, the first band is between 75KD-100KD; the second band is between 63KD-75KD, and the band between 63KD-75KD is the main band.
[0008] Preferably, it comprises 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-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homosapiens 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
[0009] Preferably, the amount of sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens is 0.5% to 8.0% of the total mass of the protein polymer, and the amount of sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens is 0.5% to 5.0% of the total mass of the protein polymer.
[0010] Preferably, at least one of the following conditions is met when the stem cells are cultured under ultrasound stress: The time for ultrasonic culture is 0.5 h to 48 h, preferably 1 h to 30 h, more preferably 1 h to 24 h; The ultrasonic frequency is 2 x 10 5 Hz to 2 x 10 7 Hz; The ultrasonic intensity is 0.003 W / cm2to 30 W / cm2; The culture medium used is a serum-free stem cell culture medium.
[0011] Preferably, the stem cells are at least one selected from the group consisting of embryonic stem cells, mesenchymal stem cells, umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, in vitro induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, and MUSE cells.
[0012] Preferably, the separation and purification method is a molecular sieve exclusion chromatography method, and the operation comprises: After equilibrating a superdex 150 molecular sieve 8x500 molecular sieve chromatography column, sample loading is performed, PBS is used for elution, the elution rate is preferably 0.2-0.4 mL / min, collection is started from 4 mAU of 280 nm ultraviolet absorption value, and 5 components with elution collection volumes of 12.0 mL-13.0 mL, 15.0 mL-17.0 mL, 17.5 mL-20.0 mL, 29.0 mL-31.0 mL, and 32.0 mL-34.0 mL are collected.
[0013] Preferably, the separation and purification method is a reverse-phase liquid chromatography column method, and the operation comprises: The mobile phase A is a 0.1% TFA aqueous solution, the mobile phase B is a 0.075% TFA 71.4% acetonitrile solution, and the chromatography conditions are as follows:
[0014] The above features can be combined arbitrarily without conflict.
[0015] The present application provides a protein polymer obtained by the preparation method according to any one of the above.
[0016] The present application provides a preparation comprising an excipient and an active ingredient, wherein the active ingredient comprises the protein polymer obtained by the preparation method according to any one of the above.
[0017] Other active ingredients can also be included in the preparation to obtain a better effect.
[0018] The present application provides the use of the protein polymer obtained by the preparation method according to any one of the above or the preparation according to the above, and the use comprises: Preparation of a drug for treating or improving a neurodegenerative disease, a stroke, arthritis, post-traumatic recovery, intestinal inflammation, autism, depression, or pulmonary fibrosis; Preparation of a cell damage repair agent for experiments; Preparation of a nerve cell protection agent for experiments.
[0019] Preferably, the neurodegenerative disease comprises Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), cerebrovascular disease, or different types of spinocerebellar ataxia (SCA).
[0020] The present application has the following beneficial effects: The protein polymer in the stem cell lysate under the stress culture under the ultrasonic condition in some examples of the present application has good cell damage repair effect, and is expected to be used for treating neurodegenerative diseases, stroke, arthritis, post-traumatic recovery, autism, enteritis, depression, pulmonary fibrosis and the like. In particular, the neurodegenerative diseases include but are not limited to Alzheimer's disease (AD), cerebrovascular disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA). BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 is the growth state photo of the stem cell after being cultured in the 3D culture medium in culture test 1-1.
[0022] Figure 2 is the cell state photo after being cultured under the ultrasonic condition for 18h in culture test 1-1.
[0023] Figure 3 is the SDS-PAGE result of the harvested protein in culture test 1-1.
[0024] Figure 4 is the cell state photo before being cultured under the ultrasonic condition in culture test 1-2.
[0025] Figure 5 is the cell state photo after being cultured under the ultrasonic condition for 6h in culture test 1-2.
[0026] Figure 6 is the SDS-PAGE result of the harvested protein in culture test.
[0027] Figure 7 is the pharmacodynamic test result of the protein polymer on the neuron regeneration.
[0028] Figure 8 is the SDS-PAGE electrophoresis chart of the purified sample in experiment 3.
[0029] Figure 9 is the influence of the pharmacodynamic test result of the protein polymer on the neuron regeneration in experiment 3.
[0030] Figure 10 is the bar graph of the nerve cell damage repair percentage in each group in experiment 4. DETAILED DESCRIPTION
[0031] The technical solutions of the present application are further illustrated below in combination with experimental examples.
[0032] In theory, any stem cell can be used to prepare the protein polymer using the stress induction method described in the present application. For the convenience of illustration, mesenchymal stem cells are used as an example in the examples, but in practical applications, it is not limited to mesenchymal stem cells, and other stem cells can also produce similar or better stress proteins after stress induction. Other stem cells that can be used include, but are not limited to, at least one of embryonic stem cells, in vitro induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, and MUSE cells.
[0033] In the examples, umbilical cord mesenchymal stem cells are used as an example for illustration, and in practical applications, mesenchymal stem cell sources include, but are not limited to, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.
[0034] The lysis method for culturing stem cells under stress is any available method that can lyse stem cells.
[0035] For the convenience of illustration, chromatography is used in some examples of the present application, and in practical applications, it can also be any method that can separate and extract the target protein from the lysate, including but not limited to at least one of chromatography, spectroscopy, volumetry, dialysis, molecular sieving, salting out, organic solvent precipitation, acid extraction, alkali extraction, ultrafiltration, chromatography, electrophoresis, and centrifugation.
[0036] Experiment 1: Effect of different treatments on protein expression:
[0037] 1-1 Culture Test One
[0038] Human umbilical cord-derived mesenchymal stem cells (HUC-MSC) were cultured in a total of 2L of HK-G050 (PRF) 3D medium from Tang Yihui Biotechnology, with a total cell count of about 5×10 8 cells, and were divided into 4 T225 bottles. Cell staining observation showed that the results were as shown in Figure 1
[0039] The stem cells were cultured under the conditions of an ultrasonic frequency of 2×10 7 Hz, an ultrasonic intensity of 0.3 W / cm², a culture temperature of 37.0°C, and 5% CO2 for 18 hours, and the state of the cells after culture was as shown in Figure 2 The cells before stress culture starting at 0h or after stress culture ending at 18h were collected, and 20mL of pure water was added to swell and lyse the cells, then the protein filtrate was collected after passing through a 0.22μm filter. The intracellular proteins harvested were taken for gel electrophoresis analysis, and the SDS-PAGE results were as shown inFigure 3 The results are shown in Table 1.
[0040] 1-2 Culture test two
[0041] One small crystal P8 generation HUC-MSC was recovered into multiple T25 culture bottles, and 2.5 ml of mesenchymal stem cell serum-free culture medium was added to each bottle.
[0042] Each of the 4 bottles of cells was cultured under two different ultrasonic conditions for 1 h, 6 h, 12 h, 24 h, and 48 h, respectively. The ultrasonic culture conditions are shown in Table 1.
[0043] Table 1
[0044] After the culture was completed, the cells cultured at different time points were harvested to obtain protein polymers. The specific operation is as follows: the supernatant was carefully removed, washed twice with 2 mL of normal saline, 1 mL of pure water was added and repeatedly blown and lysed for 10 min, 0.22 μm filter membrane was used, and 4°C was used for preservation. In this experiment, the same stem cells cultured without ultrasonic stimulation were used as the control group, and the culture time was 12 h. After the culture was completed, the lysate of the stem cells cultured without ultrasonic stimulation was collected in the same way, and was used as the control group.
[0045] The cell state before ultrasonic culture is shown in Table 1. Figure 4 For example, the cell state after ultrasonic culture for 6 h is shown in Table 1. Figure 5 It can be seen that ultrasonic culture has a certain influence on cell morphology, and ultrasonic conditions cause survival stress to cells, and then cells can produce stress proteins in this stress environment. The protein concentration and volume collected are shown in Table 2.
[0046] Table 2
[0047] As can be seen from Table 2, ultrasonic can effectively promote the expression of target proteins, and high-intensity ultrasonic can more quickly stimulate cells to produce more protein polymers, but requires ultrasonic culture for a longer time. The situation of the proteins collected under each different condition in the SDS-PAGE diagram is shown in Table 1. Figure 6
[0048] Experiment 2: Molecular sieve purification and activity detection of protein polymers
[0049] Experiment 2-1 Molecular sieve purification of protein polymers Instrument: AKTA explorer; Chromatography column: Nanomicro superdex150 molecular sieve 8x500, column volume about 30 mL; Reagents: 0.1M NaOH, 20% ethanol, 1x PBS, purified water Equilibration of chromatography column: First, flush the chromatography column with 2 CV of purified water, then equilibrate the chromatography column with 2 CV of 1x PBS, and set the 280 nm UV absorbance value to zero.
[0050] Sample preparation: Take about 20 mL of the sample from the stress culture at different times in Experiment 1. Concentrate the 20 mL sample to about 600 μL using an ultrafiltration concentration tube with a molecular weight cutoff of 3 KD.
[0051] Experimental procedure: After equilibrating the chromatography column, select 500 μL of the sample to be loaded onto the column at a flow rate of 0.4 mL / min. Elute the sample at a flow rate of 0.2 mL / min using 1x PBS until the peak is reached. Collect the protein, starting from an ultraviolet absorbance value of 4 mAU, and collect five fractions with elution volumes of 12.0 mL-13.0 mL, 15.0 mL-17.0 mL, 17.5 mL-20.0 mL, 29.0 mL-31.0 mL, and 32.0 mL-34.0 mL, respectively. Each fraction has a function of repairing nerve cell damage. In this embodiment, the five purified fractions are mixed and used to detect the activity of repairing nerve cell damage.
[0052] 2-2 Bioactivity detection of protein polymer
[0053] Day 1: Cell plating: Dilute the PC12 low differentiation cells in complete culture medium (5% FBS + DMEM) and plate 6000 cells per well (96-well plate). Incubate at 37°C with 5% CO2 overnight.
[0054] Day 2: Dilute the freeze-dried sample and the sample before purification obtained in Experiment 2-1 in DMEM + 5% FBS medium (the concentration after dilution is about 800 μg / mL).
[0055] Take 30% hydrogen peroxide and dilute it 15000 times in DMEM + 5% FBS.
[0056] Hydrogen peroxide treatment of cells: Discard 80 μL / well of culture supernatant, and add 50 μL / well of diluted hydrogen peroxide to the corresponding cultured cells, and place at room temperature for 25 min.
[0057] Untreated control: Add 50 μL / well of DMEM + 5% FBS medium as the damage treatment control.
[0058] In the PC12 cell oxidative damage model, add the diluted freeze-dried sample and the sample before purification (50 μL / well), and set up a control without adding protein in the PC12 cell oxidative damage model (control group without adding protein after oxidative damage).
[0059] 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).
[0060] Incubate at 37℃ for 2 days.
[0061] 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°C for 3.5 h. OD 450 Readings. Calculated after subtracting the culture medium blank.
[0062] The results are as follows Figure 7 It is evident that in the PC12 cell oxidative damage model, protein polymers obtained from the lysis of stem cells not cultured under ultrasound stress conditions lacked oxidative damage repair capabilities, while protein polymers obtained from cells cultured under ultrasound stress conditions all possessed oxidative damage repair capabilities. Figure 7 It is evident that protein polymers obtained from cell culture under ultrasonic stress conditions, regardless of whether they have been purified, possess oxidative damage repair capabilities. Furthermore, purified protein polymers exhibit even stronger oxidative damage repair capabilities.
[0063] SDS-PAGE electrophoresis analysis was performed on purified stem cell lysate samples cultured under low-intensity ultrasound stress for 12 hours. The electrophoresis results are as follows: Figure 8 As shown. By Figure 8 The sample bands were found to be mainly distributed between 11 KD and 100 KD, with multiple bands present, the most prominent being between 63 KD and 75 KD. Further analysis revealed that the purified protein polymer contained 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-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homosapiens ; 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.
[0064] The Vimentin content is 0.5% to 8.0% of the total mass of the protein polymer, and the Tropomyosin alpha-1 chain content is 0.5% to 5.0% of the total mass of the protein polymer.
[0065] Experiment 3: HPLC purification of the protein polymer and detection of the activity of the protein polymer
[0066] 3-1 Stem cell stress culture and preparation of stem cell lysate Recover a small crystal P8 generation of HUC-MSC into several T25 culture bottles, and add mesenchymal stem cell serum-free culture medium 2.5 mL to each bottle.
[0067] Under the condition of ultrasonic frequency of 2 x 10 7 Hz and ultrasonic intensity of 0.3 W / cm2, culture for 16 h. Carefully remove the supernatant, wash twice with 2 mL of normal saline, add 1 mL of pure water, repeatedly blow and use the method of rapid freezing and recovery to lyse the stem cells. After lysing the stem cells, pass through a 0.22 μm filter membrane and store at 4°C. After the above treatment, a total of 0.25 mg of protein was harvested. In this experiment, the lysate obtained after lysing the stem cells without culturing under ultrasonic stress was used as a control.
[0068] Based on the difference in hydrophobicity, chromatography can be used to separate the protein polymers obtained by stimulating the present application.
[0069] Test sample information: protein polymer stock solution prepared by the method of experiment 3-1.
[0070] 3-2 Solution preparation Mobile phase A (0.1% TFA in water): Take 1000 mL of ultrapure water, add 1 mL of trifluoroacetic acid, mix well and ultrasonic to obtain.
[0071] Mobile phase B (0.075% TFA in 71.4% acetonitrile solution): Take 286 mL of ultrapure water, add 714 mL of acetonitrile, 0.75 mL of trifluoroacetic acid, mix well and ultrasonic to obtain.
[0072] Protein polymer sample: weigh an appropriate amount of protein polymer sample, dilute with PBS pH 7.2 buffer, mix well, and prepare a concentration of 1 mg / mL.
[0073] 3-3 Chromatography conditions Chromatography conditions: the chromatography column used is XBridge Protein BEH C4, 300 Å, 3.5 μm, 4.6 mm*150 mm, column temperature 40°C, mobile phase A: 0.1% TFA in water; mobile phase B: 0.075% TFA in 71.4% acetonitrile solution, detector is 220 nm. The chromatography conditions are shown in Table 3. The main peak under the above chromatography conditions is the target protein.
[0074] Table 3
[0075] 3-3 Activity detection Test sample information: protein polymer products prepared by experiments 3-1 and 3-2.
[0076] Day 1: Cell plating: Dilute PC12 low differentiation cells with complete medium (5% FBS + DMEM) to 6000 cells per well (96-well plate). Incubate at 37°C, 5% CO2 overnight.
[0077] Day 2: Dilute the freeze-dried sample and the sample before purification (the concentration after dilution is about 800 μg / mL) obtained in the above experiment 3-3 with DMEM + 5% FBS medium.
[0078] Take 30% hydrogen peroxide and dilute it 15000 times with DMEM + 5% FBS.
[0079] Hydrogen peroxide treatment of cells: Discard 80 μL / well of culture supernatant, and add 50 μL / well of diluted hydrogen peroxide to the corresponding cultured cells, and place at room temperature for 25 min.
[0080] Untreated control: Add 50 μL / well of DMEM + 5% FBS medium as damage treatment control.
[0081] In the PC12 cell oxidative damage model, add the diluted purified sample and the unpurified sample (50 μL / well) respectively, and set up a control without adding protein in the PC12 cell oxidative damage model (no protein added after oxidative damage control group).
[0082] Discard the supernatant in the untreated well, and add 100 μL / well of complete medium as a cell growth control (pure PC12 cell control group).
[0083] Incubate at 37°C for 2 days.
[0084] Day 5: Discard the culture supernatant, add 100 μL / well of complete medium, and set up a medium blank control well. Add 10 μL / well of CCK8, and incubate at 37°C for 3.5 h. OD 450 Readings. Calculate after subtracting the medium blank.
[0085] The biological activity results are shown in Table 1. Figure 9 In the PC12 cell oxidative damage model, if no protein polymer is added, oxidative damage cannot be resisted. If the protein polymer in the stem cell lysate cultured without stress is added, oxidative damage cannot be repaired either. However, both the unpurified protein polymer and the purified protein polymer in the stem cell lysate obtained by ultrasonic stress culture have the function of repairing oxidative damage. Moreover, the purified sample is more effective.
[0086] Experiment 4: Protein polymer can repair nerve cell damage
[0087] 4-1 Stem cell stress culture and preparation of protein polymer After the HUC-MSCs were recovered, they were cultured in T25 culture flasks using Huagang mesenchymal stem cell serum-free culture medium.
[0088] Different samples in each group were cultured under different conditions, i.e., without any ultrasound, under ultrasound conditions with an ultrasound intensity of 0.3 W / cm², under ultrasound conditions with an ultrasound intensity of 0.003 W / cm², and under ultrasound conditions with an ultrasound intensity of 30 W / cm², for 4 h. After the culture was completed, the culture supernatant was aspirated and filtered through a 0.22 μm filter membrane, and stored at 4°C. Among them, a total of 0.25 mg of protein was harvested after the above-mentioned treatment. In this experiment, the supernatant sample cultured without ultrasound stress was used as a control. Moreover, Serum albumin (i.e., human albumin), Serotransferrin (i.e., transferrin), Vimentin, and Tropomyosin alpha-1 chain were mixed in a mass ratio of 77.5:15:5:2.5, and then incubated under ultrasound conditions with an ultrasound intensity of 0.3 W / cm² for 4 h, as a mixed protein control.
[0089] 4-2 Study on the repair and protection of nerve cells by protein polymers The cells used were SHSY-5Y cells, and the culture medium was DMEM medium containing 12% FBS (GIBCO Brazilian serum).
[0090] 4-2-1 Preparation: (1) Washing and sterilization of cell slides: 50 mL of chromic acid washing solution was added to a 150 mL reagent bottle, and the slides were completely immersed in the washing solution at room temperature overnight. The chromic acid washing solution was recovered, and then washed with pure water, ultrapure water, 75% ethanol, and anhydrous ethanol, respectively, and sterilized by high-pressure steam and dried in an oven.
[0091] (2) PDL coating of cell slides: sterile cell slides were taken out of the 12-well plate, and the working concentration of PDL (100 ng / mL) 1 mL / well was taken, so that the PDL solution immersed the cell slides, and the PDL was coated at 37°C overnight.
[0092] (3) Washing the slides: the PDL solution was recovered, and then an appropriate amount of sterile ultrapure water was aspirated with a disposable sterile Pasteur pipette, added to the well plate along the wall of the well plate, and the well plate and the slides in the well were gently shaken horizontally to wash away the residual PDL solution, and the washing was repeated twice. The water after the third washing was reserved in the well plate for use.
[0093] 4-2-2 Cell plating: (1) The original culture solution was aspirated, PBS was added to rinse the cells, and the PBS was discarded.
[0094] (2) Add trypsin, gently shake the culture bottle to infiltrate all cells, incubator digestion for 1 min, microscopic observation can see the cell layer off, that is, digestion is complete.
[0095] (3) Add preheated complete medium to terminate digestion, gently shake the cell bottle to detach, use an electric pipette to gently blow the cells to make them as single-cell suspension as possible.
[0096] (4) Collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 800 rpm / min for 3 min, aspirate the supernatant and discard.
[0097] (5) Add fresh culture medium to resuspend the cell pellet, gently blow the cells to make them as single-cell suspension as possible, take out 10 μL of cell suspension to the cell counting plate for counting.
[0098] (6) Dilute the cell suspension to 500,000 cells / mL, and plate 1 mL / well into a 12-well plate (containing PDL-coated cell slides in the wells), gently shake the plate to ensure uniform cell density distribution on the slides, and incubate in the incubator (37°C, 5% CO2). Cells are cultured for 42 h (80% confluence), and the next step "SA stress and drug administration" is performed.
[0099] 4-2-3 SA stress and drug administration: After the plated cells are cultured for 42 h (80% confluence), the following SA (NaAsO2, molecular weight 129.91, concentration 0.5M) stress and drug administration steps are performed: (1) SA grouping: different groups are set according to Table 4 to verify the nerve cell damage repair function of the protein polymer.
[0100] Table 4
[0101] (2) Stress administration steps: 1) Observation: Take out the cells in the 12-well plate from the incubator (42 h after plating), observe the cell confluence (80%) on the cell slides under a microscope, and the cells are evenly distributed without a large number of aggregates or suspended cells. Then, put the cells back into the incubator for further culture.
[0102] 2) Preparation of stress culture solution and preheating: Prepare the stress culture solution (the experiment is carried out in a 12-well plate, the culture volume is 1 mL / well, and two parallel wells / group are set for each group). First, combine 28 mL of culture medium + 16.8 μL of 0.5M SA, mix well, then take out 2.5 mL from each, and then add the sample to the working concentration. After the solution is prepared, preheat the stress culture solution in a 37°C water bath for 10 min, and mix well after preheating.
[0103] 3) Start stress dosing: Take out the cells in the incubator, label the groups, aspirate the culture medium in one well, replace the culture medium in each group well and duplicate well at 1 mL / well, and then return to the incubator for 1 h.
[0104] 4) Prepare recovery culture solution and preheat: Prepare the recovery culture solution as shown in Table 5 (the experiment is carried out in a 12-well plate, the culture volume is 1 mL / well, and two parallel wells / group are set for each group): Table 5
[0105] After the solution is prepared, preheat the recovery culture solution in a 37°C water bath for 10 min, and mix well after preheating; 5) Replace the culture medium after 1 h of stress: Take out the cells from the incubator, aspirate the residual stress solution in each well, replace the stress culture solution in each group well and duplicate well at 1 mL / well, and then return the cells to the incubator for 2 h of culture.
[0106] Then perform immunofluorescence staining and take pictures for statistics In the design of this experimental scheme, the antibody NeuN is used as the marker antibody (Marker) of SH-SY5Y cells, the G3BP1 antibody is used as the antibody for marking stress granules (Stress granule, SG), and DAPI marks the cell nucleus.
[0107] After the cells are replaced with culture medium and cultured for 2 h, perform immunofluorescence staining operation, and the specific steps are as follows: 5-1: Preheat the FA in a water bath at 37°C before use.
[0108] 5-2 Fixation: Take out the cells, aspirate the culture medium in the well, add 500 μL / well of preheated 4% PFA, and fix at room temperature for 30 min.
[0109] 5-3 Washing and permeabilization: Add 500 μL / well of PBST along the wall, shake at 75 rpm for 5 min / time, and wash for 3 times.
[0110] 5-4 Blocking: 500 µL / well of blocking solution was added slowly along the wall, 75 rpm on a shaker for 1 h.
[0111] 5-5 Incubation of primary antibody: Antibody dilution dilutes G3BP1 (1:1000) and NeuN (1:750), and the appropriate size of the parafilm is cut and placed in the wet box, and the antibody is added dropwise on the parafilm. The cell slides are clamped with tweezers, and the cell side is attached to the antibody droplet on the parafilm. Incubate overnight in the refrigerator at 4°C. After incubation, the slides are placed back into the 12-well plate for washing.
[0112] 5-6 Washing of primary antibody: PBST is added slowly along the wall, and washed 3 times for 5 min each time.
[0113] 5-7 Incubation of secondary antibody: Antibody dilution dilutes mouse 488 fluorescent secondary antibody (1:1000) and rabbit 594 fluorescent secondary antibody (1:1000), and the incubation of secondary antibody is the same as step 5-5. Incubate for 2 h at room temperature in the dark. After incubation, the slides are placed back into the 12-well plate for DAPI staining and washing.
[0114] 5-8 DAPI staining: Discard the liquid in the well, take DAPI staining solution (1 µg / mL DAPI solution), and stain for 5 min.
[0115] 5-9 Washing of secondary antibody: PBST is added slowly along the wall, 80 rpm on a shaker for 5 min each time, and washed 2 times.
[0116] 5-10 Label the slides and add anti-fluorescence quencher on the slides, place the cell side of the slides on the anti-fluorescence quencher to prevent air bubbles, and prepare the slides after the anti-fluorescence quencher is dry.
[0117] 5-11 Mounting: Add nail polish slowly along the edge of the cell slides, and air dry for 6 h ~8 h at room temperature in the dark.
[0118] 6) Take pictures and count the results: The results of the immunofluorescence sections are photographed under a fluorescence inverted microscope, 40X magnification, and uniform exposure time and gain value information are set. Randomly take pictures of 30 fields of view in each group, and capture the fluorescence of NeuN, G3BP1, and DAPI channels in each field of view at the same time. Organize the field of view pictures of each group, and calculate the number of all cells (Cell) and the number of stress granules (Count of SG) in each field of view of each group using the ImageJ program to calculate the repair percentage.
[0119] The experimental results are as follows Figure 10The results showed that protein polymers prepared from stem cells cultured under ultrasound stress at an intensity of 0.3 W / cm² exhibited better neuronal repair effects than those under ultrasound conditions of 0.003 W / cm² and 30 W / cm². Compared with the control group, the protein polymers obtained from stem cells cultured under ultrasound stress showed significantly enhanced neuronal repair function. These results confirm that ultrasound stimulation can promote the production of protein polymers, especially under specific ultrasound conditions, resulting in high concentrations of protein polymers with excellent neuronal repair capabilities.
[0120] 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.
[0121] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.
Claims
1. A method for preparing a protein polymer derived from stem cell lysates cultured under ultrasound stress, characterized in that, The preparation method includes the following steps: S1) Culture stem cells and use an ultrasound environment to create a stress environment; S2) The stem cells cultured under ultrasound stress are lysed and purified to obtain the protein polymer; The frequency of the ultrasound is not less than 2×10. 4 The ultrasonic intensity is not less than 0.003 W / cm², and does not exceed the tolerance limit of stem cells.
2. The preparation method according to claim 1, characterized in that, The separation and purification methods include at least one of the following: chromatography, spectroscopy, volumetric method, dialysis, salting out, precipitation, acid extraction, molecular sieving, alkaline extraction, ultrafiltration, chromatography, electrophoresis, and centrifugation.
3. The preparation method according to claim 1, characterized in that, The protein polymer satisfies the following characteristics: when using SDS-PAGE for detection, 4%~20% precast gel is used for sample detection, and the sample bands are mainly distributed in the range of 11KD~100KD. Among them, the molecular weight decreases from large to small. The first band is located between 75KD~100KD; the second band is located between 63KD~75KD, and the band located between 63KD~75KD is the main band.
4. The preparation method according to claim 1, characterized in that, 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-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.
5. The preparation method according to claim 4, characterized in that, The amount of sp|P08670|VIME_HUMANVimentin OS=Homo sapiens accounts for 0.5% to 8.0% of the total mass of the protein polymer, and the amount of sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens accounts for 0.5% to 5.0% of the total mass of the protein polymer.
6. The preparation method according to claim 1, characterized in that, During ultrasound stress culture, at least one of the following conditions must be met: The ultrasonic incubation time is 0.5h~48h, preferably 1h~30h, and more preferably 1h~24h; The ultrasonic frequency is 2×10 5 Hz~2×10 7 Hz; The ultrasonic intensity is 0.003 W / cm² to 30 W / cm². The culture medium used was serum-free stem cell culture medium.
7. The preparation method according to claim 1, characterized in that, The stem cells are selected from at least one of the following: embryonic stem cells, mesenchymal stem cells, umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, human placental-derived mesenchymal stem cells, in vitro induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, and MUSE cells.
8. A protein polymer, characterized in that, Obtained by the preparation method according to any one of claims 1 to 7.
9. A formulation comprising excipients and an active ingredient, characterized in that, The active ingredient includes the protein polymer of claim 8.
10. The application of the protein polymer obtained by the preparation method according to any one of claims 1 to 7, or the protein polymer according to claim 8, or the formulation according to claim 9, wherein the application includes: Prepare drugs for the treatment or improvement of neurodegenerative diseases, stroke, arthritis, post-traumatic recovery, enteritis, autism, depression, and pulmonary fibrosis, wherein the neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), cerebrovascular disease, and different types of spinocerebellar ataxia (SCA). Preparation of experimental cell damage repair agents; Prepare experimental neuroprotective agents.