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

Abstract

The invention belongs to the field of biological medicines, and discloses a stress-induced mesenchymal stem cell extract and a preparation method and application thereof.The preparation method comprises the steps that S1, mesenchymal stem cells are cultured under the high-temperature condition, swelling and cracking are conducted on the mesenchymal stem cells, or supernate obtained after culture of the mesenchymal stem cells is taken and filtered, and filtrate is obtained; the temperature of the high temperature ranges from 37.0 DEG C to 42.0 DEG C; s2, purifying the mesenchymal stem cell extract by adopting a molecular sieve exclusion chromatography method or a reverse chromatography method. Specific high-temperature stimulation is adopted to induce the mesenchymal stem cells, a purification method is combined, protein expression is promoted, the purity of the mesenchymal stem cell extract is improved, the prepared and obtained purified Alibitumen is used in a cell oxidative damage model, and the cell oxidative damage rate is increased. It is found that all the Aribitua derived from high-temperature stress culture has high oxidative damage repairing capacity.

Description

Technical Field

[0001] The present application belongs to the field of biological medicines, and specifically relates to stress-induced mesenchymal stem cell extracts and preparation methods and applications thereof. Background Art

[0002] Mesenchymal stem cells are multipotent stem cells with the potential for self-renewal and multidirectional differentiation. In the late 1970s, Freidenstein et al. discovered a type of cell group in the bone marrow that resembled fibroblasts and was clonally proliferating. They first proposed the concept of MSCs. Subsequently, researchers isolated and prepared MSCs from various tissue sources, such as fat, umbilical cord, lung, liver, placenta, skin, synovium, amniotic fluid, and umbilical cord blood. A large number of preclinical and clinical studies have shown that MSCs are an effective means of treating blood diseases, diabetes, and local organ or tissue damage. They are considered to be a new medical approach after drug therapy and surgical treatment, with considerable medical value and application prospects.

[0003] Mesenchymal stem cells can stimulate differentiation and produce a variety of stress proteins under stress conditions. Common stress conditions include hypoxic pretreatment or oxidative stress. The method of oxidative stress is to culture umbilical cord mesenchymal stem cells and adipose mesenchymal stem cells, add different concentrations of hydrogen peroxide or different volume fractions of oxygen intervention, and detect cell morphology, proliferation and expression of active proteins. Generally, cells cultured under stress will produce benign stress proteins that resist the influence of adverse external environments. Lysis of stress-cultured cells, collection and purification of benign stress proteins can be used to treat various diseases. However, oxidative stress can lead to the activation of multiple signaling pathways, such as NF-κB and HIF-1α. These pathways induce the expression of inflammatory cytokines and chemokines, aggravate cell dysfunction, thereby reducing the expression of benign stress proteins or producing adverse stress proteins. Therefore, how to screen stress conditions for the culture of mesenchymal stem cells that are simple and effective and do not produce adverse stress proteins, so as to produce specific biologically active proteins using mesenchymal stem cells, is an urgent problem to be solved. Summary of the invention

[0004] Based on this, the purpose of this application is to provide a high temperature stress condition that is easy to operate, to cultivate mesenchymal stem cells to express stress proteins of a specific composition, and the mesenchymal stem cell extract obtained by high temperature stress induction, because it contains a variety of stress proteins with specific biological activities, is found to have significant cell repair function, especially in the field of neuronal cell damage repair.

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

[0006] On the one hand, a method for preparing a stress-induced mesenchymal stem cell extract is provided, the preparation method comprising the following steps:

[0007] 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, filtering, and obtaining the mesenchymal stem cell extract; the high temperature is 37.0° C.-42.0° C.;

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

[0009] Method 1: using an ultrafiltration concentration tube with a molecular weight cutoff of 3KD to filter and concentrate the mesenchymal stem cell extract, and purifying it by molecular sieve exclusion chromatography, taking the volume of the eluent as a reference, collecting the eluent with an elution volume of 10 mL to 40 mL 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 by reverse chromatography, and collect the eluate with the peak time at 10 min-50 min as the purified sample of the mesenchymal stem cell extract based on the peak time.

[0011] In the present application, specific high temperature conditions are used to stimulate the culture of mesenchymal stem cells to prepare mesenchymal stem cell extracts.

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

[0013] In some embodiments, the high temperature time is 0.5h-18h, such as 0.5h, 8h, 12h or 18h.

[0014] In the present application, the swelling and lysis method is ultrapure water swelling, and the amount of ultrapure water is generally conventional in the art. After swelling with water, it is combined with repeated blowing until lysis. Preferably, the swelling and lysis time is generally 5min-30min, and more preferably 10min-30min.

[0015] In the present application, the filtration is generally carried out using a 0.22 μm filter membrane.

[0016] In some embodiments, before adding ultrapure water to swell and lyse the cells, the method may further include removing the supernatant of the stem cell culture medium and washing the mesenchymal stem cells twice with physiological saline.

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

[0018] In some embodiments, when the molecular sieve exclusion chromatography method described in method 1 is used for purification, the operation steps are:

[0019] The chromatographic column was balanced, and the mesenchymal stem cell extract was loaded onto the 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. The column was eluted to the peak, and the eluate filtered from the column was collected from the time when the ultraviolet absorption value at 280 nm reached 4 mAU to obtain a purified sample.

[0020] In some embodiments, the method of balancing the chromatography column generally comprises washing the chromatography column with 2CV of purified water, then balancing the chromatography column with 2CV of phosphate buffered saline (PBS), and returning the ultraviolet absorption value of the chromatography column to zero at 280nm.

[0021] Among them, the instrument used is generally AKTAexplorer, and the chromatography column is a superdex150 chromatography column with a size of 8×500 and a column volume of 30 mL.

[0022] In some embodiments, the eluate is divided into five elution volume segments according to the order of elution, and the first component sample with an elution volume segment of 12.0 mL-13.0 mL, the second component sample with an elution volume segment of 15.0 mL-17.0 mL, the third component sample with an elution volume segment of 17.5 mL-20.0 mL, the fourth component sample with an elution volume segment of 29.0 mL-30.5 mL, and the fifth component sample with an elution volume segment of 31.0 mL-33.5 mL are collected respectively.

[0023] In some embodiments, after the purified sample is purified by the molecular sieve exclusion chromatography, the purified sample is subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and detection, and the purified sample bands are measured to be distributed between 11KD-100KD, wherein the molecular weight is from large to small, the first band is located between 75KD-100KD; 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 the reverse chromatography method described in the second method is used for purification, the following steps are included:

[0042] 60 μL-100 μL of the diluted mesenchymal stem cell extract was loaded, the flow rate of the HPLC column was 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, and the eluate with a peak time of 10 min-50 min was collected to obtain a purified sample.

[0043] In some embodiments, the column temperature of the HPLC column is generally controlled to be 25.0°C-40.0°C, the HPLC column model is XBridge Protein BEH C4, and the filler 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, during the reverse chromatography purification, the chromatographic conditions used during HPLC-SEC detection are:

[0045] Time (min) Mobile phase A (%) Mobile phase B (%) Flow rate (mL / min) 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] Wherein, mobile phase A is a 0.1% trifluoroacetic acid aqueous solution, mobile phase B is a 0.075% trifluoroacetic acid 71.4% acetonitrile solution, and the chromatographic column is a reverse phase liquid chromatography column.

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

[0048] The reverse phase chromatography method has ten characteristic peaks at 10min-50min, among which the peak time of characteristic peak 1# is 13.5min-14.5min, the peak time of characteristic peak 2# is 21.5min-22.0min, the peak time of characteristic peak 3# is 22.2min-22.6min; the peak time of characteristic peak 4# is 23.0min-24.0min, and the peak time of characteristic peak 5# is 26.5min-27.5min. n, the elution time of characteristic peak 6# is 27.8min-28.8min, the elution time of characteristic peak 7# is 29.0min-30min, the elution time of characteristic peak 8# is 30.1min-30.6min, the elution time of characteristic peak 9# is 31.5min-32.5min, and the elution time of characteristic peak 10# is 33.5min-34.5min; the eluates corresponding to the characteristic peaks 1#-10# are collected respectively as the purified samples.

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

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

[0051] The present application also provides an application of a stress-induced mesenchymal stem cell extract in cell damage repair, wherein 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, wherein 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 uses a specific high temperature stimulation to induce mesenchymal stem cells to prepare mesenchymal stem cell extracts (denoted as Alito). Strong high temperature (42.0°C), medium high temperature (38.5°C) and weak high temperature (37.5°C) can effectively promote protein expression. Strong high temperature stress culture promotes the expression of the target protein at a faster rate, and medium high temperature stress culture promotes the target protein to a higher concentration. The mesenchymal stem cell extract is purified, and the purified Alito is used in a cell oxidative damage model. It is found that the purified Alito derived from high temperature stress culture has a strong oxidative damage repair ability, and in most cases, the neuronal cell damage repair ability of the purified Alito is better than that of the unpurified Alito. The purified Alito cultured at 39.0°C for 24 hours has the best effect on repairing cell damage, indicating that high-purity mesenchymal stem cell extracts can improve the effectiveness of cell damage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a photograph of the cell morphology of mesenchymal stem cells in culture experiment 1-1 before high-temperature culture in 3D culture medium.

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

[0056] Figure 3 These are the SDS-PAGE results of the cell lysate extracts and cell culture supernatant protein extracts obtained after culturing at different high temperature stresses for 12 h in culture experiment 1-1.

[0057] Figure 4 This is a photograph of the cell state before high temperature stress culture in culture experiment 1-2.

[0058] Figure 5 This is a photograph of the cell status after high temperature culture at 40.0°C for 8 hours in culture experiment 1-2.

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

[0060] Figure 7 This is the SDS-PAGE electrophoresis diagram of the purified mesenchymal stem cell extract cultured at 38.5°C for 18 hours in Experiment 2.

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

[0062] Fig. 9 This is a statistical bar graph of the ability of mesenchymal stem cell extracts cultured under high temperature stress and purified mesenchymal stem cell extracts to repair neuronal cell damage in Experiment 4. DETAILED DESCRIPTION

[0063] The technical solution of the present application is further explained below in conjunction with exemplary embodiments.

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

[0065] In the embodiments, umbilical cord mesenchymal stem cells are used as an example to illustrate the problem. In actual applications, sources of mesenchymal stem cells include but are not limited to umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0066] In the embodiment, the ultrapure water swelling method is used to lyse the stress cultured stem cells, but any other feasible lysis method can also be used in practical applications. The lysis method of mesenchymal stem cells cultured under high temperature conditions includes but is 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] The methods that can be used to separate proteins from cell lysates can theoretically be used to separate and purify the alituo described in this application from stress-cultured stem cells. For the convenience 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 also be used, including but not limited to at least one of chromatography, spectroscopy, volumetric method, dialysis, salting out, precipitation, acid extraction, alkali extraction, ultrafiltration, chromatography, electrophoresis, centrifugation and the like.

[0068] The technical solution of the present application is further described below in conjunction with the experimental examples. It is understandable that the alituo defined in the present application is a collection of multiple stress proteins with specific biological activities expressed by mesenchymal stem cells under stress conditions, and the cell extract must contain these stress proteins, so it is also a protein polymer; and the separation and purification of the cell extract to remove non-protein substances therein is also a protein polymer.

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

[0070] 1-1 Test 1

[0071] Human umbilical cord-derived mesenchymal stem cells (HUC-MSC) were cultured in 2L of HK-G050 (PRF) 3D medium from Tangyi Huike Biotechnology, with a total number of cells of approximately 5×10 8 Before starting the high temperature cell culture, observe the cell morphology. Figure 1 shown.

[0072] The cells were cultured at 37.0℃, 38.0℃, 40.0℃ and 42.0℃. The cell states after high temperature stress culture for 2h, 6h, 12h and 24h were as follows: Figure 2 shown.

[0073] After culturing stem cells at 37.0°C, 38.0°C, 40.0°C and 42.0°C for 12 hours, the cells were collected respectively, and appropriate amounts of ultrapure water were added to the cells to swell and lyse the cells, and then the filtrate was collected after filtering with a 0.22 μm filter membrane to obtain Alito. Alternatively, after culturing stem cells at 40.0°C for 12 hours, the cell culture supernatant was collected, and the filtrate was collected after filtering with a 0.22 μm filter membrane to obtain a mixed protein sample containing Alito.

[0074] The cell lysate extracts after high temperature culture at 37.0℃, 38.0℃, 40.0℃ and 42.0℃ for 12h and the protein extracts of the cell culture supernatant after high temperature culture at 40.0℃ for 12h were analyzed by gel electrophoresis. The SDS-PAGE results are as follows: Figure 3 shown.

[0075] 1-2 Test 2

[0076] Resuscitate a small crystal of P8 HUC-MSCs into 9 T25 culture flasks and add 2.5 mL of Huakai mesenchymal stem cell serum-free culture medium to each flask.

[0077] Four bottles of cells were taken from each group and cultured at three different high temperature conditions of 37.5°C, 38.5°C and 42.0°C for 0.5h, 8h, 18h and 48h respectively. After the culture was completed, the supernatant in the stem cell culture medium was removed, and the mesenchymal stem cells were washed twice with 1mL of normal saline, and then 660μL of pure water was added to the mesenchymal stem cells to swell and repeatedly blow and lyse the stem cells for 10min, and then filtered through a 0.22μm filter membrane to collect a predetermined volume of filtrate to obtain alituo, and the protein concentration therein was determined.

[0078] Cell status before high temperature stress culture Figure 4 As shown in the figure, taking 42.0℃ culture for 8h as an example, the cell status after high temperature culture is as follows Figure 5 As shown, it can be seen that high temperature culture has an impact on cell morphology and creates survival stress on cells, and then cells can 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 culture condition settings

[0080] Stress culture time (h) 0.5 8 18 48 Protein concentration in 37.5℃ stress culture (mg / mL) 0.023 0.139 0.378 0.171 Protein concentration in 38.5℃ stress culture (mg / mL) 0.070 0.259 0.776 0.298 42.0℃ stress culture protein concentration (mg / mL) 0.125 0.511 0.211 0.121 Volume (μL) 500 500 500 500

[0081] As shown in Table 1, strong high temperature (42.0℃), medium high temperature (38.5℃) and weak high temperature (37.5℃) can effectively promote protein expression. Strong high temperature stress culture promotes the expression of target protein faster, weak high temperature promotes the expression of target protein requires longer stress culture, and the protein content of protein polymers produced by weak high temperature stress is relatively low.

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

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

[0084] Instrument: AKTAexplorer

[0085] Chromatographic column: Nano-micron superdex150 molecular sieve 8×500, column volume is about 30mL

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

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

[0088] Equilibration column sequence:

[0089] The column was first rinsed with 2CV of pure water, and then equilibrated with 1×PBS for 2CV, and the UV absorption value at 280nm was returned to zero.

[0090] Sample preparation: Take the alituo that was cultured at 38.5℃ for 18 hours in Experiment 2, with a volume of about 20mL. Use an ultrafiltration concentration tube with a molecular weight cutoff of 3KD to reduce the 20mL sample to about 600μL to obtain a crude sample.

[0091] Experimental process: After balancing the chromatography column, select a 500μL sample loop for sample loading, the sample loading flow rate is 0.4mL / min, the 1×PBS elution flow rate is 0.2mL / min, and the elution is carried out to the peak. When the ultraviolet absorption value of 280nm ultraviolet light reaches 4mAU, the eluate filtered from the chromatography column is collected, and five components with elution volumes of 12.0mL-13.0mL, 15.0mL-17.0mL, 17.5mL-20.0mL, 29.0mL-30.5mL, and 31.0mL-33.5mL are collected respectively. The collection of collected proteins is the purified sample, corresponding to Figure 6 The above five components all have the function of repairing damaged nerve cells. In this embodiment, the five components collected and purified were mixed and made into freeze-dried samples for activity detection.

[0092] 2-2 Biological Activity Detection of Mesenchymal Stem Cell Extracts

[0093] Day 1: Cell plating: Complete medium (5% FBS + DMEM) was used to dilute PC12 poorly differentiated cells, and the cells were plated at a density of 6,000 cells / well (96-well plate). The cells were incubated overnight in an incubator at 37.0°C and 5% CO2.

[0094] The next day: the purified lyophilized sample prepared in Experiment 2-1 and the unpurified sample prepared in Experiment 1-1 were diluted with DMEM+5% FBS medium (about 800 μg / mL).

[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 specification of 80 μL / well, then add hydrogen peroxide to the above 96 wells at a specification of 50 μL / well, and leave at room temperature for 25 minutes.

[0097] Untreated control: Select several wells, remove the PC12 cell culture supernatant in the wells at a specification of 80 μL / well, and then add DMEM + 5% FBS culture medium to the above 96 wells at a specification of 50 μL / well as the injury treatment control, that is, Figure 6 “Pure PC12 cell control”.

[0098] Sample detection: Add diluted purified lyophilized samples and unpurified samples to the wells of the hydrogen peroxide-treated plate at a specification of 50 μL / well.

[0099] The supernatant in the untreated control wells was removed, and complete culture medium was added at 100 μL / well as a cell growth control.

[0100] Incubate at 37.0°C for 2 days.

[0101] Day 5: Select at least one plate well without PC12 cells as a blank control, add complete medium to the plate well as a blank control at a specification of 100 μL / well, and then add CCK8 to each plate well at a specification of 10 μL / well. Incubate at 37.0℃ for 3.5h, and record the OD value with a microplate reader. 450 The reading is taken as the blank value of the culture medium. Remove the cell culture supernatant in the wells inoculated with PC12 cells, add new complete culture medium at a specification of 100 μL / well, and then add CCK8 at a specification of 10 μL / well to each well of the plate, incubate at 37.0℃ for 3.5h, send it to the microplate reader for detection, and record the OD after deducting the blank value of the culture medium. 450 data.

[0102] The experimental results are as follows Figure 6 As shown in the figure, in the PC12 cell oxidative damage model, each sample of stem cell lysate derived from high temperature stress culture has a strong ability to repair oxidative damage. Moreover, the neuronal cell damage repair ability of some samples is better than that of unpurified samples.

[0103] The purified sample of Alito purified in Experiment 2-1 was subjected to SDS-PAGE electrophoresis analysis. The electrophoresis results are as follows: Figure 7 As shown in the figure, the sample bands are mainly distributed between 11KD and 100KD, and the first band is between 75KD and 100KD, and the second band is between 63KD and 75KD. Further analysis shows that the purified protein polymer includes 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 extracts and detection of the nerve damage repair function of the purified mesenchymal stem cell extracts

[0121] 3.1 Preparation of MUSE stem cell extracts from high temperature stress culture

[0122] MUSE cells are a type of stem cell. MUSE cells were cultured in T25 culture flasks, and 2.5 mL of stem cell serum-free medium was added to each flask. They were cultured at 39.0°C for 16 hours. After the culture was completed, the stem cells were carefully cleared of the supernatant and washed twice with 1 mL of normal saline. Then 1 mL of ultrapure water was added to the cells to swell and the stem cells were repeatedly blown and lysed for 10 minutes. The filtrate was then filtered through a 0.22 μm filter membrane and the filtrate was collected 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, reverse phase chromatography is 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 is low, the protein polymer has a strong hydrophobic effect on the stationary phase, and is almost completely adsorbed by the stationary phase; when the organic components in the mobile phase reach a specific concentration, the protein polymer is completely eluted from the stationary phase and no longer interacts with the stationary phase. Therefore, a very slight change in the organic components of the mobile phase can significantly affect the reverse phase retention behavior of the protein polymer. It can be understood that the MUSE stem cell extract obtained by this method and its purified sample are both a type of Alito.

[0125] 3.2.1 Preparation of chromatographic mobile phase solution

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

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

[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 XBridge Protein BEH C4, 3.5μm, 4.6mm*150mm, column temperature 40.0℃, mobile phase A: 0.1% TFA in water; mobile phase B: 0.075% TFA in 71.4% acetonitrile, detector 220nm; the test sample was the MUSE stem cell extract product prepared by the method of Experiment 3.1, and the injection volume was 80μL.

[0131] Table 2. Chromatographic conditions used to separate and purify protein aggregates

[0132] Time (min) Mobile phase A (%) Mobile phase B (%) Flow rate (mL / min) 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: 1# 13.5min-14.5min; 2# 21.5min-22.0min; 3# 22.2min-22.6min; 4# 23.0min-24.0min; 5# 26.5min-27.5min; 6# 27.8min-28.8min; 7# 29.0min-30.0min; 8# 30.1min-30.6min; 9# 31.5min-32.5min; 10# 33.5min-34.5min.

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

[0136] 3.3 Detection of the neural injury repair function of MUSE stem cell extract

[0137] S1. Cell seeding plate

[0138] SH-SY5Y cells were adjusted to 10 5 The density of cells / mL was set at 100 μL / well and inoculated into a 96-well plate.

[0139] S2. Cell modeling and drug intervention

[0140] The model was established 24 hours after the cells were plated. The modeling process was as follows:

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

[0142] Group design: normal cell group, model control group, sample intervention group, 4 replicate wells in each group.

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

[0144] The supernatant of the complete cell culture medium in all wells of the well plate was aspirated, and the 200 μM hydrogen peroxide treated as above was added to the model control group and the sample intervention group at a specification of 100 μL / well. The cells were damaged for 30 minutes, and the cell modeling was completed.

[0145] The dosing process is as follows:

[0146] After the cell modeling was completed, the supernatant of the complete cell culture medium was immediately aspirated as clean as possible. The sample intervention group was administered with the purified samples of 1# to 10# MUSE stem cell extracts isolated in the aforementioned experiment 3.2 and the unpurified MUSE stem cell extracts in experiment 3.1 at a specification of 100 μL / well. The normal cell group and the model control group were added with equal volumes of complete cell culture medium and continued to incubate in an incubator at 37.0°C and 5% CO2 for 72 hours.

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

[0148] NFL is a cytoskeletal protein expressed in neurons. It is a type of neurofilament protein (NF). It is mainly expressed in the axons and axons of neurons, and plays a role in maintaining the stability of axonal morphology and ensuring neuronal signal conduction. Under physiological conditions, axons release a small amount 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 damage. Abnormally elevated NFL protein levels indicate neurodegeneration. Because the detection of NFL content in the supernatant can be used to assess the ability of nerve cells to repair damage.

[0149] Refer to the instruction manual of Jianglai Bio-Kit to prepare the standard curve. 450 The sample concentration was calculated based on the absorbance value. The content of NFL in the sample was calculated based on the fitted standard curve, and the repair efficiency was calculated. NFL repair rate = (content in the model group - content in the drug-added group) / content in the model group, expressed as a percentage.

[0150] Depend on Figure 8It can be seen that in the cell oxidative damage model, all samples have the ability to repair nerve cell damage. Among them, samples 1, 2, 3, 6, 7, 8, and 10 all have strong oxidative damage repair capabilities. Their repair capabilities are better than those of unpurified MUSE stem cell extracts. The repair capabilities of sample No. 4, sample No. 5, and purified sample No. 9 are lower than those of unpurified MUSE stem cell extracts. Therefore, in the specific implementation process, the sample components with separation times of 23min-27.5min and 31.5min-32.5min can be further eliminated through separation technology, which can further improve the ability of protein polymer products to repair cell damage.

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

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

[0153] The MUSE stem cells were cultured at 37.5°C, 39.0°C and 42.0°C for 24 hours. After the culture was completed, the supernatant of the stem cell culture medium was removed and washed twice with 1 mL of saline. Then, 660 μL of pure water was added to the cells to swell and repeatedly blow and lyse the MUSE stem cells for 10 minutes. The filtrate was then collected after filtering 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, and the detailed steps were the same as above.

[0155] First, wash the column with 2CV of pure water, then balance the column with 1×PBS for 2CV, and return the UV absorption value at 280nm to zero. Take each sample obtained in the experiment, with a volume of about 20mL.

[0156] The ultrafiltration concentration tube with a molecular weight cutoff of 3KD can reduce 20mL of sample to about 600μL.

[0157] After balancing the chromatography column, select a 500 μL sample loop for sample loading, the sample loading flow rate is 0.4 mL / min, the 1×PBS elution flow rate is 0.2 mL / min, and the elution is eluted to the peak. Collect the protein, and collect the eluate filtered from the chromatography column from the time the ultraviolet absorption value reaches 4 mAU. The elution volume is 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. 5 components. Each component has the function of repairing nerve cell damage. In this embodiment, the five purified components are collected and mixed for use in the activity detection of repairing nerve cell damage. The activity detection method of the obtained sample is the same as that of Experiment 3.3.

[0158] The experimental results are as follows Fig. 9 As shown, it can be understood that the unpurified sample refers to the filtrate collected after MUSE cell lysis and filtration through a 0.22 μm filter membrane, that is, the MUSE stem cell extract, and the purified sample refers to the purified sample of the MUSE stem cell extract after purification by chromatography, that is, the mixture of the five components collected above, and the purified samples of the MUSE stem cell extract obtained after lysis and purification of stem cells cultured under three high-temperature stress conditions all have the effect of repairing fluid cell damage, among which the purified sample of the MUSE stem cell extract purified after 39.0°C high-temperature stress culture has the best effect.

[0159] The above is a further detailed description of the present application, which shall not be regarded as a limitation on the specific implementation of the present application. For ordinary technicians in the technical field to which the present application belongs, simple deduction or replacement without departing from the concept of the present application shall fall within the protection scope of the present application.

Claims

1. A method for preparing a stress-induced mesenchymal stem cell extract, characterized in that: The steps include: 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, wherein the high temperature is 37.0° C.-42.0° C.; S2: Purifying the mesenchymal stem cell extract, wherein the purification step is any one of the following: Method 1: using an ultrafiltration concentration tube with a molecular weight cutoff of 3KD to filter and concentrate the mesenchymal stem cell extract, and purifying it by molecular sieve exclusion chromatography, taking the volume of the eluent as a reference, collecting the eluent with an elution volume of 10 mL to 40 mL as the purified sample of the mesenchymal stem cell extract; Method 2: Add pH 7.2 phosphate buffer to dilute the mesenchymal stem cell extract to a concentration of 1 mg / mL, and purify it by reverse chromatography. Taking the peak time as a reference, the eluate at the peak time of 10 min-50 min is collected as the purified sample of the mesenchymal stem cell extract.

2. The preparation method according to claim 1, characterized in that: Meet one or more of the following conditions: a. The high temperature time is 0.5h-48h; b. The swelling and cracking method is ultrapure water swelling, and the swelling and cracking time is 5min-30min; c. The filtration is carried out using a 0.22 μm filter membrane.

3. The preparation method according to claim 1, characterized in that: In the first embodiment, when the molecular sieve exclusion chromatography is used for purification, the following steps are included: The mesenchymal stem cell extract was loaded onto a balanced 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, and eluted to the peak. The eluate filtered from the chromatography column was collected from the time when the ultraviolet absorption value at 280 nm reached 4 mAU to obtain the purified sample.

4. The preparation method according to claim 3, characterized in that: The purified sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and detection, and the bands of the purified sample were measured to be distributed between 11KD and 100KD, wherein the molecular weight from large to small, the first band was located between 75KD and 100KD; the second band was located between 63KD and 75KD.

5. The preparation method according to claim 4, characterized in that: In the first embodiment, the purified sample satisfies the following characteristics: According to the order of elution, the eluate was divided into five elution volume segments, and the first component sample with an elution volume segment of 12.0mL-13.0mL, the second component sample with an elution volume segment of 15.0mL-17.0mL, the third component sample with an elution volume segment of 17.5mL-20.0mL, the fourth component sample with an elution volume segment of 29.0mL-30.5mL, and the fifth component sample with an elution volume segment of 31.0mL-33.5mL were collected respectively.

6. The preparation method according to claim 1, characterized in that: In the second method, when the reverse chromatography method is used for purification, the following steps are included: 60 μL-100 μL of the diluted mesenchymal stem cell extract was loaded, the flow rate of the HPLC column was 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, and the eluate with a peak time of 10 min-50 min was collected to obtain the purified sample.

7. The preparation method according to claim 6, characterized in that: In the second method, the purified sample meets the following characteristics: The reverse phase chromatography method has ten characteristic peaks at 10min-50min, among which the peak time of characteristic peak 1# is 13.5min-14.5min, the peak time of characteristic peak 2# is 21.5min-22.0min, the peak time of characteristic peak 3# is 22.2min-22.6min; the peak time of characteristic peak 4# is 23.0min-24.0min, and the peak time of characteristic peak 5# is 26.5min-27.5min. The elution time of characteristic peak 6# is 27.8min-28.8min, the elution time of characteristic peak 7# is 29.0min-30min, the elution time of characteristic peak 8# is 30.1min-30.6min, the elution time of characteristic peak 9# is 31.5min-32.5min, and the elution time of characteristic peak 10# is 33.5min-34.5min; the eluates corresponding to the characteristic peaks 1#-10# are collected respectively as the purified samples.

8. The preparation method according to any one of claims 1-2, characterized in that: The culturing comprises adding the mesenchymal stem cells into a culture medium to perform cell expansion, and the culture medium is a serum-free mesenchymal stem cell culture medium.

9. The preparation method according to any one of claims 1 to 2, characterized in that: The mesenchymal stem cells include at least one of umbilical cord-derived human mesenchymal stem cells, multilineage differentiated stress-resistant cells, embryo-derived 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 and pancreatic stem cells.

10. A stress-induced mesenchymal stem cell extract, characterized in that: The method is prepared by the method according to any one of claims 1 to 9.

11. A use of the stress-induced mesenchymal stem cell extract according to claim 10 in cell damage repair, characterized in that: The mesenchymal stem cell extract is used for treating 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 spinocerebellar ataxia.

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