Stress-induced mesenchymal stem cell extract and preparation method and application thereof

Abstract

This application belongs to the field of biopharmaceuticals and discloses a stress-induced mesenchymal stem cell extract, its preparation method, and its application. The preparation method includes S1: culturing mesenchymal stem cells under high temperature conditions, swelling and lysing the mesenchymal stem cells, or taking the supernatant of the mesenchymal stem cell culture and filtering to obtain the mesenchymal stem cell extract. The high temperature is 37.0℃-42.0℃; S2: purifying the mesenchymal stem cell extract using molecular sieve exclusion chromatography or reverse chromatography. This application uses a specific high temperature to stimulate and induce mesenchymal stem cells, combined with a purification method, which is beneficial to promoting protein expression and improving the purity of the mesenchymal stem cell extract. By using the purified alituxin obtained from the preparation in a cell oxidative damage model, it was found that alituxin derived from high-temperature stress culture has a strong oxidative damage repair capacity.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceuticals, specifically relating to stress-induced mesenchymal stem cell extracts, their preparation methods, and applications. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewal and multi-lineage differentiation potential. In the late 1970s, Freidenstein et al. discovered a clonal proliferating cell population resembling fibroblasts in bone marrow, first proposing the concept of MSCs. Subsequently, researchers isolated and prepared MSCs from various tissue sources, such as adipose tissue, umbilical cord, lung, liver, placenta, skin, synovium, amniotic fluid, and umbilical cord blood. Numerous preclinical and clinical studies have demonstrated that MSCs are an effective treatment for hematological diseases, diabetes, and local organ or tissue damage, and are considered a new medical approach following drug and surgical treatments, possessing significant medical value and application prospects.

[0003] Mesenchymal stem cells (MSCs) can differentiate into various stress proteins under stress conditions. Common stress conditions include hypoxia pretreatment or oxidative stress. Oxidative stress is assessed by culturing umbilical cord MSCs and adipose-derived MSCs, adding different concentrations of hydrogen peroxide or different volume fractions of oxygen, and then detecting cell morphology, proliferation, and the expression of active proteins. Typically, stress-cultured cells produce benign stress proteins that protect against adverse environmental influences. Lysing stress-cultured cells allows for the collection and purification of benign stress proteins, which can be used to treat various diseases. However, oxidative stress leads to the activation of multiple signaling pathways, such as NF-κB and HIF-1α. These pathways induce the expression of inflammatory cytokines and chemokines, exacerbating cellular dysfunction and thus reducing the expression of benign stress proteins or producing adverse stress proteins. Therefore, how to screen for simple, effective stress conditions for MSC culture that do not produce adverse stress proteins, and thus utilize MSCs to produce proteins with specific biological activities, is a pressing problem to be solved. Summary of the Invention

[0004] Based on this, the purpose of this application is to provide a simple high-temperature stress condition to culture mesenchymal stem cells to express stress proteins with specific composition. The mesenchymal stem cell extract obtained through high-temperature stress induction contains a variety of stress proteins with specific biological activities and has been found to have significant cell repair functions, especially in the field of neuronal cell damage repair.

[0005] The technical solution adopted in this application is:

[0006] One aspect provides a method for preparing stress-induced mesenchymal stem cell extract, the preparation method comprising the following steps:

[0007] S1: Mesenchymal stem cells are cultured under high temperature conditions, and the mesenchymal stem cells are swollen and lysed, or the supernatant of the mesenchymal stem cell culture is taken, filtered, and the mesenchymal stem cell extract is obtained; the high temperature is 37.0℃-42.0℃;

[0008] S2: Purify the mesenchymal stem cell extract, wherein the purification step is any one of the following:

[0009] Method 1: The mesenchymal stem cell extract is concentrated by filtration using an ultrafiltration tube with a molecular weight cutoff of 3KD, and purified by molecular sieve exclusion chromatography. The eluent volume is used as a reference, and the eluent volume from the 10th to the 40th mL is collected as the purified sample of the mesenchymal stem cell extract.

[0010] Method 2: Dilute the mesenchymal stem cell extract to a concentration of 1 mg / mL with phosphate buffer at pH 7.2, purify it using reverse chromatography, and collect the eluent with the peak time between 10 min and 50 min as the purified sample of the mesenchymal stem cell extract, using the peak time as a reference.

[0011] In this application, mesenchymal stem cells are cultured under specific high-temperature conditions to prepare mesenchymal stem cell extracts.

[0012] In this application, the duration of the high-temperature period is generally 0.5h-48h.

[0013] In some implementations, the high temperature duration is 0.5h-18h, for example 0.5h, 8h, 12h or 18h.

[0014] In this application, the swelling and pyrolysis method is swelling with ultrapure water. The amount of ultrapure water is generally conventional in the art. After swelling with water, it is repeatedly blown until pyrolysis. Preferably, the swelling and pyrolysis time is generally 5 min-30 min, and more preferably 10 min-30 min.

[0015] In this application, the filtration generally uses a 0.22μm filter membrane.

[0016] In some embodiments, before adding ultrapure water to swell and lyse the cells, the mesenchymal stem cells may be washed twice with physiological saline to remove the supernatant of the stem cell culture medium.

[0017] In some embodiments, the culture includes adding the mesenchymal stem cells to a culture medium for cell expansion, wherein the culture medium is a serum-free mesenchymal stem cell culture medium.

[0018] In some embodiments, when purification is performed using the molecular sieve size exclusion chromatography method described in Method 1, the operation steps are as follows:

[0019] The column was equilibrated, and the mesenchymal stem cell extract was loaded onto the column at a flow rate of 0.2 mL / min to 0.4 mL / min. Phosphate buffer was used as the mobile phase at a flow rate of 0.1 mL / min to 0.3 mL / min. Elution was continued until the peak was reached. The eluent filtered from the column was collected starting from when the UV absorbance at 280 nm reached 4 mAU to obtain the purified sample.

[0020] In some implementations, the method for equilibrating the chromatography column generally includes rinsing the column with 2CV of purified water, then equilibrating the column with 2CV of phosphate-buffered saline (PBS) and bringing the column to zero UV absorbance at 280 nm.

[0021] The instrument used is generally an AKTA explorer, and the chromatography column is a Superdex 150 chromatography column with a size of 8×500 and a column volume of 30mL.

[0022] In some embodiments, the eluent is divided into five elution volume segments according to the order of elution, and the following fractions are collected: the first fraction sample (elution volume segment 12.0 mL - 13.0 mL), the second fraction sample (elution volume segment 15.0 mL - 17.0 mL), the third fraction sample (elution volume segment 17.5 mL - 20.0 mL), the fourth fraction sample (elution volume segment 29.0 mL - 30.5 mL), and the fifth fraction sample (elution volume segment 31.0 mL - 33.5 mL).

[0023] In some embodiments, after purifying the sample using the molecular sieve exclusion chromatography method, the purified sample is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the bands of the purified sample are found to be distributed in the range of 11KD-100KD, with the molecular weight decreasing from large to small. The first band is located between 75KD-100KD, and the second band is located between 63KD-75KD.

[0024] Furthermore, in some embodiments, it includes at least one of the following proteins:

[0025] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0026] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0027] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0028] sp|P62736|ACTA_HUMANActin,aortic smooth muscle OS=Homo sapiens;

[0029] sp|P01009|A1AT_HUMANAlpha-1-antitrypsin OS=Homo sapiens;

[0030] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0031] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0032] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0033] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1chain OS=Homo sapiens;

[0034] sp|P21333|FLNA_HUMAN Filamin-AOS=Homo sapiens;

[0035] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy chain OS=Homosapiens;

[0036] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0037] sp|P01023|A2MG_HUMANAlpha-2-macroglobulin OS=Homo sapiens;

[0038] sp|P60709|ACTB_HUMANActin,cytoplasmic 1OS=Homo sapiens;

[0039] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens;

[0040] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0041] In some embodiments, when purifying using the reverse chromatography method described in Method 2, the following steps are included:

[0042] Load 60-100 μL of the diluted mesenchymal stem cell extract onto the high-performance liquid chromatography column at a flow rate of 0.4 mL / min-1 mL / min. Use an aqueous solution of trifluoroacetic acid and / or an acetonitrile solution of trifluoroacetic acid as the mobile phase. Collect the eluent with peak elution times between 10 min and 50 min to obtain the purified sample.

[0043] In some embodiments, the column temperature of the high-performance liquid chromatography (HPLC) column is typically controlled between 25.0℃ and 40.0℃. The HPLC column is an XBridge Protein BEH C4, and the packing pore size is... The filler particle size is 3.5μm, the inner diameter * length is 4.6mm * 150mm, and the detection wavelength is 220nm.

[0044] In some embodiments, the chromatographic conditions used for HPLC-SEC detection during the reverse chromatography purification are as follows:

[0045] 0 65 35 1.0 3 65 35 1.0 13 50 50 1.0 27 43 57 1.0 51 20 80 1.0 53 0 100 1.0 53.1 65 35 1.0 60 65 35 1.0

[0046] The mobile phase A was an aqueous solution of 0.1% trifluoroacetic acid, the mobile phase B was a 71.4% acetonitrile solution of 0.075% trifluoroacetic acid, and the chromatographic column was a reversed liquid chromatography column.

[0047] In some embodiments, the purified sample of the mesenchymal stem cell extract meets the following characteristics:

[0048] The reversed-phase chromatography method yielded ten characteristic peaks eluting between 10 and 50 minutes. Specifically, characteristic peak 1# eluted at 13.5-14.5 minutes, characteristic peak 2# at 21.5-22.0 minutes, characteristic peak 3# at 22.2-22.6 minutes, characteristic peak 4# at 23.0-24.0 minutes, and characteristic peak 5# at 26.5-27.5 minutes. The elution times of characteristic peak 6# are 27.8 min-28.8 min, characteristic peak 7# is 29.0 min-30 min, characteristic peak 8# is 30.1 min-30.6 min, characteristic peak 9# is 31.5 min-32.5 min, and characteristic peak 10# is 33.5 min-34.5 min. The eluents corresponding to characteristic peaks 1#-10# are collected as the purified samples.

[0049] In this application, the mesenchymal stem cells include at least one of the following: umbilical cord-derived human mesenchymal stem cells, multilineage differentiated stress-resistant cells (MUSE cells), embryonic-derived 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, and pancreatic stem cells.

[0050] This application also provides a stress-induced mesenchymal stem cell extract, prepared using the aforementioned preparation method.

[0051] This application also provides the application of stress-induced mesenchymal stem cell extract in cell damage repair. The mesenchymal stem cell extract can be used to treat neurodegenerative diseases, stroke, cerebrovascular disease, arthritis, enteritis, post-traumatic recovery, autism, depression, and pulmonary fibrosis. The neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and different types of spinocerebellar ataxia.

[0052] The beneficial effects of this application are:

[0053] This application utilizes specific high-temperature stimulation to induce mesenchymal stem cell (MSC) culture to prepare MSC extract (denoted as Alitux). Strong high temperature (42.0℃), medium high temperature (38.5℃), and weak high temperature (37.5℃) all effectively promoted protein expression. Strong high temperature stress culture promoted the expression rate of the target protein more rapidly, while medium high temperature stress culture promoted a higher concentration of the target protein. The MSC extract was purified, and the purified Alitux was used in a cell oxidative damage model. It was found that purified Alitux derived from high-temperature stress culture exhibited strong oxidative damage repair capabilities. In most cases, purified Alitux demonstrated superior neuronal cell damage repair capabilities compared to unpurified Alitux. The purified Alitux cultured at 39.0℃ for 24 hours showed the best effect in repairing cell damage, indicating that high-purity MSC extract can significantly improve the effectiveness of cell damage repair. Attached Figure Description

[0054] Figure 1 These are cell morphology photos of mesenchymal stem cells from culture experiment 1-1 before high-temperature culture in 3D medium.

[0055] Figure 2 These are photographs of the cell states of mesenchymal stem cells after culture in 3D medium under different high-temperature stresses in culture experiment 1-1.

[0056] Figure 3 The results are SDS-PAGE of cell lysate extracts and cell culture supernatant protein extracts obtained after 12 hours of culture under different high-temperature stresses in culture experiment 1-1.

[0057] Figure 4 These are photographs of the cell state before high-temperature stress culture in culture experiments 1-2.

[0058] Figure 5 These are photos of the cell state after 8 hours of high-temperature culture at 40.0℃ in culture experiments 1-2.

[0059] Figure 6 This is a statistical bar chart showing the effects of mesenchymal stem cell extracts and purified mesenchymal stem cell extracts on P12 cell regeneration under different stress temperatures and durations in Experiment 2.

[0060] Figure 7 This is an SDS-PAGE electrophoresis image of the purified mesenchymal stem cell extract after 18 hours of stress culture at 38.5℃ in Experiment 2.

[0061] Figure 8 This is a statistical bar chart showing the ability of different isolated components of mesenchymal stem cell extracts cultured under high temperature stress in Experiment 3 and purified mesenchymal stem cell extracts to repair neuronal cell damage.

[0062] Figure 9 This is a statistical bar chart showing the neuronal cell damage repair capabilities of mesenchymal stem cell extracts cultured under high-temperature stress and purified mesenchymal stem cell extracts from Experiment 4. Detailed Implementation

[0063] The technical solution of this application will be further described below with reference to exemplary embodiments.

[0064] In this application, for ease of explanation, mesenchymal stem cells and multilineage stress-resistant cells (MUSE cells) are used as examples in the embodiments. However, in practical applications, they are not limited to mesenchymal stem cells. Other stem cells can also produce similar or better stress proteins after stress induction. Therefore, other usable stem cells 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, and pancreatic stem cells.

[0065] The examples used umbilical cord mesenchymal stem cells to illustrate the problem. In practical applications, the sources of mesenchymal stem cells include, but are not limited to, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0066] In this embodiment, ultrapure water swelling was used to lyse the stress-cultured stem cells, but in practice, any other feasible lysis method can be used. The lysis methods for mesenchymal stem cells cultured under high-temperature conditions include, but are not limited to, at least one of ultrapure water swelling, repeated freeze-thaw lysis, ultrasonic lysis, chemical lysis, urea lysis, and guanidine hydrochloride lysis.

[0067] Theoretically, any method that can be used to separate proteins from cell lysates can also be used to separate and purify the arituximab described in this application from stress-cultured stem cells. For ease of explanation, chromatography, electrophoresis, and volumetric methods (molecular sieves) are used in some embodiments of this application. 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.

[0068] The technical solution of this application will be further explained below with reference to experimental embodiments. It can be understood that the altoxin defined in this application is a collection of various stress proteins with specific biological activities expressed by mesenchymal stem cells under stress conditions. Cell extracts necessarily contain these stress proteins and are therefore also a type of protein polymer; and the separation and purification of cell extracts to remove non-protein substances also results in a protein polymer.

[0069] Experiment 1: Preparation of mesenchymal stem cell extract

[0070] 1-1 Experiment 1

[0071] Human umbilical cord-derived 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 One cell was dispensed into four T225 flasks. Cell morphology was observed before initiating high-temperature cell culture. Figure 1 As shown.

[0072] Cells were cultured at 37.0℃, 38.0℃, 40.0℃, and 42.0℃, and the cell states after 2h, 6h, 12h, and 24h of high-temperature stress culture were as follows: Figure 2 As shown.

[0073] Stem cells were cultured under high-temperature stress conditions of 37.0℃, 38.0℃, 40.0℃, and 42.0℃ for 12 hours, and the cells were harvested. An appropriate amount of ultrapure water was added to each cell to swell and lyse them. The filtrate was then collected after filtration through a 0.22μm filter membrane, yielding alitux. Alternatively, after culturing stem cells under high-temperature stress conditions of 40.0℃ for 12 hours, the supernatant of the cell culture was collected, filtered through a 0.22μm filter membrane, and the filtrate was collected, also yielding a mixed protein sample containing alitux.

[0074] Cell lysate extracts after culturing at 37.0℃, 38.0℃, 40.0℃, and 42.0℃ for 12 h, and protein extracts from cell culture supernatant after culturing at 40.0℃ for 12 h, were analyzed by gel electrophoresis. The SDS-PAGE results are shown below. Figure 3 As shown.

[0075] Experiment 2 (1-2)

[0076] One small P8 generation HUC-MSC was revived and added to nine T25 culture flasks, with 2.5 mL of Huakan mesenchymal stem cell serum-free culture medium added to each flask.

[0077] Four flasks of cells were cultured at three different high-temperature conditions (37.5℃, 38.5℃, and 42.0℃) for 0.5h, 8h, 18h, and 48h, respectively. After culture, the supernatant in the stem cell culture medium was removed, and the mesenchymal stem cells were washed twice with 1mL of physiological saline. Then, 660μL of pure water was added to the mesenchymal stem cells to swell them, and the stem cells were repeatedly lysed by pipetting for 10min. The solution was then filtered through a 0.22μm filter membrane, and a predetermined volume of filtrate was collected to obtain alitoxil, and the protein concentration was determined.

[0078] Cell state before high temperature stress culture, such as Figure 4 As shown, taking culturing at 42.0℃ for 8 hours as an example, the cell state after high-temperature culture is as follows: Figure 5 As shown in the figure, high-temperature culture affects cell morphology and creates survival stress on the cells, which in turn allows the cells to produce stress proteins under this stress environment. The protein concentration and volume of the prepared mesenchymal stem cell extract are shown in Table 1.

[0079] Table 1 Stress Training Conditions

[0080] Protein concentration (mg / mL) under 37.5℃ stress culture 0.023 0.139 0.378 0.171 Protein concentration (mg / mL) under 38.5℃ stress culture 0.070 0.259 0.776 0.298 Protein concentration (mg / mL) under 42.0℃ stress culture 0.125 0.511 0.211 0.121 Volume (μL) 500 500 500 500

[0081] Table 1 shows that high temperature (42.0℃), medium temperature (38.5℃), and low temperature (37.5℃) can all effectively promote protein expression. High temperature stress culture promotes the expression of the target protein more rapidly, while low temperature stress culture requires a longer stress culture time, and the protein polymers produced by low temperature stress have a relatively lower protein content.

[0082] Experiment 2: Molecular sieve purification and activity assay of mesenchymal stem cell extracts

[0083] 2-1 Molecular sieve purification of mesenchymal stem cell extract

[0084] Instrument: AKTAexplorer

[0085] Chromatography column: Nanomicro Superdex 150 molecular sieve 8×500, column volume approximately 30mL

[0086] Reagents: 0.1M NaOH, 20% ethanol, 1×PBS, purified water

[0087] Ultraviolet absorption wavelength: 280nm, 260nm as reference

[0088] Equilibrium chromatography column sequence:

[0089] First, rinse the chromatography column with 2CV purified water, then equilibrate the 2CV column with 1×PBS to zero the 280nm UV absorbance.

[0090] Sample preparation: Take approximately 20 mL of the arithmetic sample that has been cultured at 38.5℃ for 18 h in Experiment 2. Use an ultrafiltration concentrator with a molecular weight cutoff of 3 KD to reduce the 20 mL sample to approximately 600 μL to obtain the crude sample.

[0091] Experimental Procedure: After equilibrating the chromatography column, a 500 μL loading loop was used to load the sample at a flow rate of 0.4 mL / min. Elution with 1×PBS was performed at a flow rate of 0.2 mL / min until the peak was reached. The eluent filtered from the column was collected starting at a UV absorbance of 4 mAU at 280 nm. Five fractions were collected 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-30.5 mL, and 31.0 mL-33.5 mL. The collected protein aggregates constituted the purified sample. Figure 6 The purified sample was prepared from the above five components, all of which have the function of repairing nerve cell damage. In this embodiment, the five purified components were collected separately, mixed, and then prepared into a lyophilized sample for use in activity detection.

[0092] 2-2 Bioactivity assay of mesenchymal stem cell extracts

[0093] Day 1: Cell Plating: Dilute PC12 low-differentiation cells with complete culture medium (5% FBS + DMEM) and plate them at a density of 6000 cells / well (96-well plate). Incubate overnight at 37.0°C with 5% CO2.

[0094] Day 2: Dilute the purified lyophilized sample obtained in Experiment 2-1 and the unpurified sample obtained in Experiment 1-1 (approximately 800 μg / mL) with DMEM + 5% FBS medium.

[0095] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS.

[0096] Hydrogen peroxide treatment: Select several 96-well plates, remove the PC12 cell culture supernatant in the wells at a rate of 80 μL / well, and then add hydrogen peroxide to the wells at a rate of 50 μL / well. Incubate at room temperature for 25 min.

[0097] Untreated control: Several wells were selected, and the PC12 cell culture supernatant was removed from each well at a rate of 80 μL / well. Then, 50 μL / well of DMEM + 5% FBS medium was added to each of the 96 wells as a damage treatment control. Figure 6 "Pure PC12 cell control"

[0098] Sample testing: Diluted purified lyophilized sample and unpurified sample were added to the wells of the plate after hydrogen peroxide treatment at a rate of 50 μL / well.

[0099] Remove the supernatant from the wells of the untreated control and add 100 μL of complete culture medium per well as a cell growth control.

[0100] Incubate at 37.0℃ for 2 days.

[0101] Day 5: Select at least one well without PC12 cells as a blank control. Add 100 μL of complete culture medium per well to the blank control well, and then add 10 μL of CCK8 per well to each well. Incubate at 37.0℃ for 3.5 h, and record the OD using a microplate reader. 450 The readings were used as the culture medium blank value. The cell culture supernatant in the wells inoculated with PC12 cells was removed, and fresh complete culture medium was added at a rate of 100 μL / well. Then, CCK8 was added to each well at a rate of 10 μL / well. The plates were incubated at 37.0℃ for 3.5 h, and the readings were analyzed using a microplate reader. The OD value was recorded after subtracting the culture medium blank value. 450 data.

[0102] Experimental results are as follows Figure 6 As shown, in the PC12 cell oxidative damage model, all samples derived from stem cell lysates cultured under high-temperature stress exhibited strong oxidative damage repair capabilities. Furthermore, some samples demonstrated superior neuronal cell damage repair capabilities compared to unpurified samples.

[0103] The purified sample from Experiment 2-1 after alitux purification was analyzed by SDS-PAGE electrophoresis. The electrophoresis results are as follows: Figure 7 As shown in the figure, the sample bands are mainly distributed between 11KD and 100KD. The first band, from largest to smallest molecular weight, is between 75KD and 100KD; the second band is between 63KD and 75KD. Further analysis revealed that the purified protein polymer contains at least one of the following proteins:

[0104] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0105] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens.

[0106] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0107] sp|P62736|ACTA_HUMANActin,aortic smooth muscle OS=Homo sapiens;

[0108] sp|P01009|A1AT_HUMANAlpha-1-antitrypsin OS=Homo sapiens;

[0109] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0110] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0111] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0112] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1chain OS=Homo sapiens;

[0113] sp|P21333|FLNA_HUMAN Filamin-AOS=Homo sapiens;

[0114] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy chain OS=Homosapiens;

[0115] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0116] sp|P01023|A2MG_HUMANAlpha-2-macroglobulin OS=Homo sapiens;

[0117] sp|P60709|ACTB_HUMANActin,cytoplasmic 1OS=Homo sapiens;

[0118] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens;

[0119] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0120] Experiment 3: HPLC-SEC purification of mesenchymal stem cell extract and detection of the nerve damage repair function of the purified mesenchymal stem cell extract.

[0121] 3.1 Preparation of MUSE stem cell extract derived from high-temperature stress culture

[0122] MUSE cells are a type of stem cell. MUSE cells were cultured in T25 culture flasks, with 2.5 mL of serum-free stem cell culture medium added to each flask. The flasks were cultured at 39.0 °C for 16 h. After culturing, 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 filtrate was then collected after filtration through a 0.22 μm filter membrane to obtain the MUSE stem cell extract.

[0123] 3.2 HPLC-SEC purification of MUSE stem cell extract

[0124] Based on the difference in hydrophobicity, reversed-phase chromatography was subsequently used to separate the mesenchymal stem cell extract obtained by stimulation in this application. Under initial conditions, the concentration of organic components in the mobile phase was low, and the protein polymers exhibited strong hydrophobic interactions with the stationary phase, resulting in almost complete adsorption. When the organic component concentration in the mobile phase reached a specific level, the protein polymers were completely eluted from the stationary phase and ceased to interact with it. Therefore, even minute changes in the organic component of the mobile phase can significantly affect the reversed-phase retention behavior of the protein polymers. It is understood that the MUSE stem cell extract and its purified sample obtained by this method are both forms of altrol.

[0125] 3.2.1 Preparation of chromatographic mobile phase solution

[0126] Mobile phase A (0.1% TFA aqueous solution): Take 1000 mL of ultrapure water, add 1 mL of trifluoroacetic acid, mix well and sonicate to obtain the mobile phase A.

[0127] Mobile phase B (71.4% acetonitrile solution of 0.075% TFA): Take 286 mL of ultrapure water, add 714 mL of acetonitrile and 0.75 mL of trifluoroacetic acid, mix well and sonicate to obtain the mobile phase B.

[0128] MUSE stem cell extract sample: Weigh an appropriate amount of MUSE stem cell extract sample, dilute with PBS pH 7.2 buffer, mix well, and prepare a concentration of 1 mg / mL.

[0129] 3.2.2 Chromatographic conditions

[0130] Chromatographic conditions (see Table 2): The chromatographic column used was an XBridge Protein BEH C4. 3.5 μm, 4.6 mm * 150 mm, column temperature 40.0 ℃, mobile phase A: 0.1% TFA aqueous solution; mobile phase B: 0.075% TFA in 71.4% acetonitrile solution, detector 220 nm; the test sample was the MUSE stem cell extract product prepared by the method in Experiment 3.1, and the injection volume was 80 μL.

[0131] Table 2. Chromatographic conditions used for the separation and purification of protein polymers

[0132] 0 65 35 1.0 3 65 35 1.0 13 50 50 1.0 27 43 57 1.0 51 20 80 1.0 53 0 100 1.0 53.1 65 35 1.0 60 65 35 1.0

[0133] 3.2.3 Analysis Results

[0134] The collection time range is approximately as follows: #1 13.5 min - 14.5 min; #2 21.5 min - 22.0 min; #3 22.2 min - 22.6 min; #4 23.0 min - 24.0 min; #5 26.5 min - 27.5 min; #6 27.8 min - 28.8 min; #7 29.0 min - 30.0 min; #8 30.1 min - 30.6 min; #9 31.5 min - 32.5 min; #10 33.5 min - 34.5 min.

[0135] Purified samples of MUSE stem cell extracts from the time ranges 1 to 10 were collected for subsequent nerve damage repair function testing.

[0136] 3.3 Detection of the nerve damage repair function of MUSE stem cell extract

[0137] S1, cell seeding plate

[0138] 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.

[0139] S2, cell modeling and drug intervention

[0140] Cells were seeded in plates for 24 hours before modeling was performed. The modeling process is as follows:

[0141] Hydrogen peroxide treatment: Take 1M hydrogen peroxide of theoretical concentration and dilute it to 200μM with complete cell culture medium. Prepare fresh each time you use it.

[0142] Group design: normal cell group, model control group, sample intervention group, with 4 replicates per group.

[0143] The model control group did not receive any treatment or intervention; the sample intervention group contained samples, including the purified sample of MUSE stem cell extract from Experiment 3.2 and the unpurified MUSE stem cell extract from Experiment 3.1, each prepared as a mesenchymal stem cell extract sample with a protein concentration of 100 ng / mL, diluted with complete cell culture medium, and each mesenchymal stem cell extract sample was used to conduct a sample intervention group experiment.

[0144] The supernatant of complete cell culture medium in all wells of the plate was aspirated. 200 μM hydrogen peroxide was added to the model control group and the sample intervention group at a concentration of 100 μL / well. The cell model was established after 30 min of damage.

[0145] The dosing process is as follows:

[0146] Immediately after cell modeling, the supernatant of the complete cell culture medium was aspirated, and the sample intervention group was administered the purified samples of MUSE stem cell extracts 1# to 10# separated in Experiment 3.2 and the unpurified MUSE stem cell extract in Experiment 3.1 at a rate of 100 μL / well. The normal cell group and the model control group were added to the complete cell culture medium in equal volumes and incubated in an incubator at 37.0℃ and 5% CO2 concentration for 72 h.

[0147] S3. Detection of NFL (Neurofilament Light Protein) content in supernatant

[0148] 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 stability 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 the NFL content in the supernatant can be used to assess the neuronal cell repair capacity.

[0149] 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 is used to calculate the sample concentration. The NFL content in the sample is calculated based on the fitted standard curve, and the regeneration efficiency is calculated. NFL regeneration rate = (model group content - drug-treated group content) / model group content, expressed as a percentage.

[0150] Depend on Figure 8It was found that all samples in the cellular oxidative damage model possessed the ability to repair nerve cell damage. Samples 1, 2, 3, 6, 7, 8, and 10 exhibited strong oxidative damage repair capabilities, superior to those of the unpurified MUSE stem cell extract. Samples 4, 5, and 9, being purified, showed lower repair capabilities than the unpurified MUSE stem cell extract. Therefore, in practical implementation, further separation techniques can be used to remove sample components separated at times between 23-27.5 min and 31.5-32.5 min, which can further enhance the cell damage repair capabilities of the protein polymer product.

[0151] Experiment 4: Activity test of mesenchymal stem cell extracts cultured under high temperature stress and their purified form.

[0152] MUSE stem cells were cultured in T25 culture flasks, with 2.5 mL of serum-free stem cell culture medium added to each flask.

[0153] MUSE stem cells were cultured at 37.5℃, 39.0℃, and 42.0℃ for 24 h, respectively. After culture, the supernatant of the stem cell culture medium was removed, and the cells were washed twice with 1 mL of physiological saline. Then, 660 μL of pure water was added to the cells to swell them, and the MUSE stem cells were repeatedly lysed by pipetting for 10 min. The filtrate was then collected after filtration through a 0.22 μm filter membrane to obtain the stress-cultured MUSE stem cell extract.

[0154] The purified sample of MUSE stem cell extract was further separated from the MUSE stem cell extract using chromatography, with the detailed steps as above.

[0155] First, rinse the chromatography column with 2CV purified water, then equilibrate the column with 1×PBS to zero the 280nm UV absorbance. Take approximately 20mL of each sample obtained from the experiment.

[0156] An ultrafiltration concentrator with a molecular weight cutoff of 3 kDa reduced a 20 mL sample to approximately 600 μL.

[0157] After equilibrating the chromatography column, a 500 μL loading loop was used to load the sample at a flow rate of 0.4 mL / min. Elution with 1×PBS was performed at a flow rate of 0.2 mL / min until the peak was reached. Proteins were collected, starting from a UV absorbance of 4 mAU. Five fractions were collected in elution volumes: 12.0 mL-13.0 mL, 15.0 mL-17.0 mL, 17.5 mL-20.0 mL, 29.0 mL-30.5 mL, and 31.0 mL-33.5 mL. Each fraction has the function of repairing nerve cell damage. In this example, the five purified fractions were collected separately and mixed for use in detecting their activity in repairing nerve cell damage. The activity detection method for the obtained samples was the same as in Experiment 3.3.

[0158] Experimental results are as follows Figure 9 As shown, it can be understood that the unpurified sample refers to the filtrate collected after MUSE cells are lysed and filtered through a 0.22μm filter membrane, which is the MUSE stem cell extract. The purified sample refers to the purified sample of the MUSE stem cell extract after being purified by chromatography, which is the mixture of the five components collected above. The purified samples of MUSE stem cell extract obtained after lysing and purifying stem cells cultured under three high-temperature stress conditions all have the effect of repairing cell damage caused by the solution. Among them, the purified sample of MUSE stem cell extract obtained after high-temperature stress culture at 39.0℃ has the best effect.

[0159] The above is a further detailed description of this application and should not be considered as a limitation on the specific implementation of this application. For those skilled in the art to which this application pertains, simple deductions or substitutions without departing from the concept of this application are all within the protection scope of this application.

Claims

1. A method for preparing a stress-induced mesenchymal stem cell extract, characterized by, Includes the following steps: S1: Mesenchymal stem cells are cultured under high temperature conditions, and the mesenchymal stem cells are swollen and lysed, or the supernatant of the mesenchymal stem cell culture is taken, filtered, and the mesenchymal stem cell extract is obtained. The high temperature is 39°C, and the high temperature time is 0.5 h-48 h. The culture includes adding the mesenchymal stem cells to a culture medium for cell expansion, wherein the culture medium is a serum-free mesenchymal stem cell culture medium, and the mesenchymal stem cells are MUSE stem cells; S2: Purify the mesenchymal stem cell extract, wherein the purification step is any one of the following: Method 1: The mesenchymal stem cell extract is concentrated by filtration using an ultrafiltration tube with a molecular weight cutoff of 3 KD, and purified by molecular sieve exclusion chromatography. The eluent volume is used as a reference, and the eluent volume from the 10th to the 40th mL is collected as the purified sample of the mesenchymal stem cell extract. Method 2: Dilute the mesenchymal stem cell extract to a concentration of 1 mg / mL with phosphate buffer at pH 7.2, purify it by reversed-phase chromatography, and collect the eluent with the peak time between 10 min and 50 min as the purified sample of the mesenchymal stem cell extract, using the peak time as a reference. In Method 1, the purification using the molecular sieve exclusion chromatography method includes the following steps: The mesenchymal stem cell extract was loaded onto the equilibrated chromatography column at a flow rate of 0.2 mL / min-0.4 mL / min. Phosphate buffer was used as the mobile phase at a flow rate of 0.1 mL / min-0.3 mL / min. Elution was continued until the peak was reached. The eluent filtered from the chromatography column was collected starting from when the UV absorbance at 280 nm reached 4 mAU to obtain the purified sample. In method one, the purified sample satisfies the following characteristics: Based on the order of elution, the eluent was divided into five elution volume segments, and the following fractions were collected: the first fraction sample (elution volume segment 12.0 mL - 13.0 mL), the second fraction sample (elution volume segment 15.0 mL - 17.0 mL), the third fraction sample (elution volume segment 17.5 mL - 20.0 mL), the fourth fraction sample (elution volume segment 29.0 mL - 30.5 mL), and the fifth fraction sample (elution volume segment 31.0 mL - 33.5 mL). In the second method, the purification using the reversed-phase chromatography method includes the following steps: 60 µL-100 µL of the diluted mesenchymal stem cell extract was loaded onto the high-performance liquid chromatography (HPLC) column at a flow rate of 0.4 mL / min-1 mL / min. The mobile phase was an aqueous solution of trifluoroacetic acid and / or an acetonitrile solution of trifluoroacetic acid. The eluent with peak elution at 10 min-50 min was collected to obtain the purified sample. The chromatographic column was an XBridge Protein BEHC4. In method two, the purified sample satisfies the following characteristics: The reversed-phase chromatography method yielded ten characteristic peaks eluting between 10 and 50 minutes. Specifically, characteristic peak 1# eluted at 13.5-14.5 minutes; characteristic peak 2# at 21.5-22.0 minutes; characteristic peak 3# at 22.2-22.6 minutes; characteristic peak 4# at 23.0-24.0 minutes; characteristic peak 5# at 26.5-27.5 minutes; characteristic peak 6# at 27.8-28.8 minutes; characteristic peak 7# at 29.0-30 minutes; characteristic peak 8# at 30.1-30.6 minutes; characteristic peak 9# at 31.5-32.5 minutes; and characteristic peak 10# at 33.5-34.5 minutes. min; collect the eluent corresponding to the characteristic peaks 1#, 2#, 3#, 6#, 7#, 8#, and 10# respectively as the purified samples.

2. The production method according to claim 1, characterized by, One or more of the following conditions must be met: The swelling and pyrolysis method is ultrapure water swelling, and the swelling and pyrolysis time is 5 min-30 min; The filtration process uses a 0.22 μm filter membrane.

3. The preparation method according to claim 1, characterized in that, The purified sample was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the band distribution of the purified sample was found to be between 11 KD and 100 KD, with the molecular weight decreasing from large to small. The first band was between 75 KD and 100 KD, and the second band was between 63 KD and 75 KD.

4. The preparation method according to any one of claims 1-2, characterized in that, The mesenchymal stem cells include human mesenchymal stem cells derived from umbilical cord and / or mesenchymal stem cells derived from bone marrow.

5. A stress-induced mesenchymal stem cell extract, characterized in that, It is prepared by the preparation method according to any one of claims 1-4.

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