Application of stem cell extract in lysosome and mitochondrial related diseases

Abstract

The invention belongs to the field of biological medicines, and discloses an application of a stem cell extract in lysosome and mitochondrial related diseases, a preparation method of the stem cell extract comprises the following steps: S1) culturing mesenchymal stem cells and creating stress conditions to stimulate the mesenchymal stem cells; and S2) separating and purifying the culture supernatant of the mesenchymal stem cells cultured in the step S1) to obtain the stem cell extract, wherein S21) the culture supernatant is filtered to obtain a filtrate; s22) performing 3KD ultrafiltration concentration on the filtrate to obtain a crude sample; s23) purifying the crude sample by using molecular sieve exclusion chromatography or reversed-phase chromatography to prepare a purified sample; in molecular sieve exclusion chromatography, by taking the volume of the eluent as a reference, collecting the 10th to 40th mL of eluent, namely a purified sample; and S24) taking the purified sample to form a preparation. According to the application, the culture supernatant containing stress stem cell secretions is taken through stress culture of stem cells, and a purification method is combined, so that the prepared preparation can be used for treating lysosome and mitochondrial related diseases.

Description

Technical Field

[0001] The present application belongs to the field of medicine, and specifically relates to the application of a stress-induced stem cell extract in lysosome and mitochondria-related diseases. Background Art

[0002] Lysosomes are organelles that play a key role in the degradation and recycling of substances inside and outside cells. They perform the function of degrading substances inside and outside cells. Changes or dysfunctions of lysosomes can destroy the original homeostasis of cells and organisms, and induce or worsen human diseases, among which they are closely related to cancer, autoimmune diseases, neurodegenerative diseases, and cardiovascular diseases. Mitochondria are important organelles for oxidative metabolism in eukaryotes, and are closely related to the occurrence and development of many diseases such as stroke, myocardial disease, diabetes, kidney damage, and cancer.

[0003] The current treatment options for diseases related to lysosomes and mitochondria usually use corresponding targeted drugs to inhibit adverse mutations of lysosomes and mitochondria, thereby slowing the progression of related diseases.

[0004] However, the inventors of this application have found that lysosome-related diseases and mitochondrial-related diseases have a certain correlation, and the characterization indicators of the two are also consistent in the course of some diseases. Therefore, it is necessary to develop a relatively broad-spectrum drug that can treat both lysosome-related and mitochondrial-related diseases. Furthermore, the inventors found in the specific implementation process that stem cells will be induced to differentiate and secrete a variety of stress proteins under stress conditions to resist the influence of adverse external environments. Such stress proteins have a universal repair-promoting effect on various types of cell damage. How to obtain stem cell extracts with universal therapeutic effects on lysosome- and mitochondrial-related diseases through appropriate purification methods is a problem that needs to be solved urgently. Summary of the invention

[0005] Based on this, the inventors of the present application discovered that after culturing mesenchymal stem cells under appropriate stress conditions, a variety of proteins can be isolated from their culture supernatant. The isolated proteins can simultaneously repair the damage to the lysosome and mitochondrial structure and function induced by external pressure, and can be universally applied to the treatment of diseases related to abnormal lysosome and mitochondrial structure and function.

[0006] The specific technical solution of this application is:

[0007] A use of a stem cell extract in lysosome and mitochondria-related diseases, wherein the stem cells are mesenchymal stem cells, and the preparation of the stem cell extract comprises the following steps:

[0008] S1) culturing the mesenchymal stem cells and creating a stress condition to stimulate the mesenchymal stem cells;

[0009] S2) isolating and purifying the culture supernatant of the mesenchymal stem cells cultured in S1) to obtain the stem cell extract;

[0010] The separation and purification of the culture supernatant comprises the following steps:

[0011] S21) filtering the culture supernatant using a 0.22 μm filter membrane to obtain a filtrate;

[0012] S22) filtering and concentrating the filtrate using an ultrafiltration concentration tube with a molecular weight cutoff of 3KD to obtain a crude sample of the stem cell extract;

[0013] S23) purifying the crude sample by molecular sieve exclusion chromatography or reverse phase chromatography to obtain a purified sample of the stem cell extract; wherein the purified sample also satisfies the following characteristics: in the molecular sieve exclusion chromatography, taking the volume of the eluent as a reference, collecting the 10 mL to 40 mL of the eluent as the purified sample; and S24) preparing the purified sample into a preset dosage form.

[0014] In some optional application schemes, the stress condition is ultraviolet radiation.

[0015] In some optional application schemes, the ultraviolet irradiation satisfies the following conditions:

[0016] a. Irradiation time is 1h-30h;

[0017] b. Irradiation intensity is 10μW / cm2-100μW / cm2;

[0018] c. The wavelength of ultraviolet light is 290nm-340nm.

[0019] In some optional application schemes, in the molecular sieve exclusion chromatography, the instrument used is AKTAexplorer, and the chromatography column is a superdex150 chromatography column; the operation steps are:

[0020] S2311) washing the chromatography column with 2CV of pure water, then balancing the chromatography column with 2CV of 1×PBS reagent, and returning the ultraviolet absorption value of the chromatography column at 280nm to zero; and

[0021] S2312) 300 μL-700 μL of the crude sample is loaded, the loading flow rate is 0.4 mL / min, the elution flow rate of 1×PBS reagent is 0.1-0.3 mL / min, and elution is performed to the peak. When the volume of the eluent reaches 10 mL, the separated elution liquid is received to obtain the purified sample.

[0022] In some optional application schemes, the purified sample of the stem cell extract also meets the following characteristics: a predetermined volume segment of the purified sample is collected for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and it is measured that the purified sample contains at least protein molecules with a molecular weight distribution of 11KD-100KD.

[0023] In some optional application schemes, the purified sample at least includes protein molecules with a molecular weight between 63KD and 75KD.

[0024] In some optional application schemes, in the reverse phase chromatography, the instrument used is a high performance liquid chromatograph, the chromatographic column is XBridge Protein BEH C4, and the pore size of the filler is The filler particle size is 3.5 μm, the inner diameter*length is 4.6 mm*150 mm, and the mobile phase is PBS; the operation steps are as follows:

[0025] S2321) adding PBS reagent to dilute the crude sample;

[0026] S2322) Load 80-120 μL of the diluted crude sample, control the column temperature of the chromatographic column to 20°C-30°C, the flow rate to 0.2 mL / min-0.8 mL / min, the detection wavelength to 260 nm and / or 280 nm, set the separation time to 48 min, collect the eluate at the peak time of 10 min-40 min, and obtain the purified sample.

[0027] In some optional application schemes, the culture medium used to culture the mesenchymal stem cells is a serum-free stem cell culture medium.

[0028] In some optional application schemes, the mesenchymal stem cells are selected from at least one of umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells and skin-derived mesenchymal stem cells.

[0029] In some optional application schemes, the preset dosage form is one of lyophilized powder, injection, gel, spray, pill, tablet, powder and ointment.

[0030] In some optional application schemes, the administration method of the preset preparation when used is selected from intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, and oral administration.

[0031] The beneficial effects of this application are:

[0032] The present application prepares stem cell extracts (denoted as alituo) by culturing stem cells under stress, taking culture supernatant containing secretions of stressed stem cells, and combining with purification methods. Alituo can be prepared into different dosage forms such as lyophilized powder, injection, gel, spray, pill, tablet, powder and paste by different administration methods such as intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, oral administration, etc., and relevant experiments have verified that alituo can correct and reverse the mutation of lysosomes and mitochondria, and the structural abnormalities of lysosomes and mitochondria can mark the body to a certain extent. The conditions and pathology of related diseases. When the lysosomes and mitochondria are reversed to a relatively normal state, it means that Alituo can reverse the body's related diseases to a certain extent, proving that Alituo can be used to treat lysosome and mitochondrial related diseases. Alituo can protect the effects of external damage on the structure and function of mitochondria and lysosomes, and play an important role in cell resistance to stress and protecting cells from inflammatory damage. It has the potential to treat neurodegenerative diseases, stroke, cerebrovascular disease, arthritis, enteritis, post-traumatic recovery, autism, depression, pulmonary fibrosis and other diseases related to lysosomes and mitochondria. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the description of the present application will be briefly introduced below.

[0034] Figure 1 Figure 1: SDS-PAGE results of stress-induced stem cell extracts.

[0035] Figure 2 Figure 4: The results of nerve cell staining imaging.

[0036] Figure 3 For: Morphological analysis of nerve cells and intracellular organelles.

[0037] Figure 4 For: Diagram of mitochondrial morphology identification and analysis.

[0038] Figure 5 Figure 2: Mitochondrial morphology analysis results.

[0039] Figure 6 For: Lysosome morphology identification and analysis diagram.

[0040] Figure 7 Figure 2: Lysosome morphology analysis results.

[0041] Figure 8 For: Lysosome morphology analysis and size statistics results. DETAILED DESCRIPTION

[0042] Examples of the present embodiment are described in detail below. The examples are only used to explain the present embodiment and are not to be construed as limiting the present embodiment.

[0043] In this application, for the convenience of explanation, mesenchymal stem 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 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, pancreatic stem cells, and MUSE cells.

[0044] After undergoing stress conditions, mesenchymal stem cells can theoretically induce the production of corresponding stress proteins. For the convenience of explanation, ultraviolet irradiation is used as a stress condition in the embodiment to prepare a mesenchymal stem cell extract containing stress proteins that can be used to treat lysosomes and mitochondria. In practical applications, other stress conditions can achieve similar effects. Other stress conditions include, but are not limited to, at least one of the following stress methods: infrared irradiation, electromagnetic field, high temperature, low temperature, hypoxia, hyperoxia, oxidation (under conditions containing oxidants such as hydrogen peroxide or hypochlorous acid), high pH, ​​low pH, ultrasound, terahertz electromagnetic waves, X-rays, microwaves, rays, rays, rays, ion beams, high CO2, and low CO2. Various stress treatments can be performed one after another, or simultaneously when they do not conflict.

[0045] In the examples, umbilical cord mesenchymal stem cells are used as an example to illustrate the problem. In actual applications, the sources of mesenchymal stem cells also include but are not limited to bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0046] Theoretically, any method that can be used to separate proteins from cell culture supernatants can be used to separate and purify the stem cell extract 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 filtrate 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.

[0047] Spectroscopic method, using the absorbance of the target protein under a specific wavelength of light to screen out the target extract; dialysis method is to use a dialysis bag to exchange solutions with a buffer solution, and finally obtain a purified and concentrated target extract; organic solvent precipitation method, by adding an organic solvent to the culture supernatant, to achieve a precipitation effect similar to that of high-concentration salt, and filter out the precipitate, which is the target extract; acid extraction method, by adding concentrated hydrochloric acid to obtain the target extract; alkaline extraction method, by adding sodium hydroxide or potassium hydroxide to treat the culture supernatant to obtain the target extract; ultrafiltration method, adding the supernatant after cell culture to an ultrafiltration column, continuously centrifuging, adding buffer, and ultrafiltration filtration multiple times, and finally concentrating to obtain the target extract; chromatography method, including ion exchange chromatography, gel filtration chromatography, affinity chromatography, different chromatography methods are selected according to the characteristics of the target extract; electrophoresis method, by applying electricity to separate protein molecules in a dispersed matrix, thereby obtaining the target extract; centrifugation method, by centrifuging the supernatant, filter out the target extract.

[0048] 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.

[0049] Stage 1: Cultivation of human umbilical cord mesenchymal stem cells

[0050] Resuscitate a small crystal of P8 HUC-MSC (human umbilical cord-derived mesenchymal stem cells) into a T25 culture flask and add 2.5 mL of Huakai mesenchymal stem cell serum-free culture medium.

[0051] The cells were irradiated under a 300 nm wavelength LED UV lamp for 6 h. The UV irradiation conditions were as follows:

[0052] Intermediate medium T25 bottle <![CDATA[UVA(μW / cm 2 )]]> 11.71 <![CDATA[UVB(μW / cm 2 )]]> 26.81 Peak wavelength (nm) 320.4

[0053] The supernatant after cell stress culture was aspirated, and the filtrate was collected after passing through a 0.22 μm filter membrane (also referred to as ALEETO stock solution, abbreviated as ALEETO stock solution or ALT stock solution), and stored at 4°C for later use. The protein concentration of the ALEETO stock solution was measured to be 0.655 mg / mL, and the volume was 500 μl. The SDS-PAGE test results of the harvested ALEETO stock solution are as follows: Figure 1 As shown in the figure, the sample bands are distributed between 11KD and 100KD, especially concentrated between 63KD and 75KD.

[0054] It should be noted that in this application, the supernatant after cell stress culture is collected instead of the cell lysate, for one or more of the following reasons:

[0055] (1) There are some impurities in the lysate that are difficult to separate, for example, the lysate may contain macromolecules such as DNA remaining from host cells;

[0056] (2) The molecular weight and structure of proteins in the supernatant are different from those in the lysate, which means that proteins extracted from the supernatant are more suitable for applications in lysosome- and mitochondrial-related diseases;

[0057] (3) The protein molecules secreted from the cells in the supernatant may be able to penetrate the cell membrane more effectively, which is beneficial for entering abnormal cells and exerting drug effects in subsequent applications.

[0058] (4) The supernatant contains fewer types of proteins than the lysate, making it easier to purify and enrich.

[0059] Stage 2: Molecular sieve purification of mesenchymal stem cell supernatant

[0060] 2-1 Molecular sieve purification of the supernatant after mesenchymal stem cell culture:

[0061] Instrument: AKTA explorer

[0062] Chromatographic column: Nano-micron superdex150 molecular sieve 8×500, column volume is about 30mL

[0063] Reagents: 0.1M NaOH, 20% ethanol, 1× PBS, purified water

[0064] UV absorption wavelength: 280nm, 260nm as reference

[0065] Equilibration column sequence:

[0066] The chromatography column was first rinsed with 2CV of pure water, and then equilibrated with 1×PBS reagent for 2CV, and the ultraviolet absorption value of the chromatography column at 280nm was returned to zero.

[0067] Sample preparation: Collect 20 mL of the stock solution of Alito according to the method in Example 1. Use an ultrafiltration concentration tube with a molecular weight cutoff of 3KD to reduce the 20 mL of the stock solution of Alito to about 600 μl to obtain a crude sample.

[0068] 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 eluted to the peak. The separated elution liquid is collected from the ultraviolet absorption value of 4mAU, and the collected eluate is prepared into a freeze-dried stem cell extract (also recorded as ALEETO, abbreviated as ALEETO or ALT), which is stored for later use.

[0069] Further analysis showed that the purified alitu also contained at least one of the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0070] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0071] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0072] sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens;

[0073] sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens;

[0074] sp|P07951|TPM2_HUMAN Tropomyosinbeta chain OS=Homo sapiens;

[0075] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0076] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0077] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1chain OS=Homo sapiens;

[0078] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens;

[0079] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy chain OS=Homosapiens;

[0080] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0081] sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens;

[0082] sp|P60709|ACTB_HUMAN Actin,cytoplasmic 1OS=Homo sapiens;

[0083] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens;

[0084] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0085] Furthermore, the sum of the masses of the sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens protein and the sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens protein accounts for no less than 40% of the total mass of the stem cell extract.

[0086] Phase 3 Evaluation of the biological effects of alitox

[0087] 3-1 Primary cell culture

[0088] S1. Pregnant mice at embryonic day 18 (E18) were anesthetized with a mixture of ether and isoflurane;

[0089] S2. Rapidly perform abdominal incision on ice to remove the fetus;

[0090] S3. Cut off the head of the fetal mouse and place it in a separation solution at 0℃-4℃;

[0091] S4. Peel off the skin tissue and skull of the fetal mouse head, take out the brain and cerebellum together and place them in another culture dish filled with 0-4℃ separation solution, and place the culture dish on ice;

[0092] S5. Peel off the meninges of the brain and cerebellum, and cut the brain and cerebellum tissues into small pieces in a culture dish;

[0093] S6. Pipette the separation solution containing the brain and cerebellum tissue fragments into a 5 mL eppendorf tube;

[0094] S7. Let it stand for a while, and discard the supernatant in the ep tube after the tissue fragments settle to the bottom;

[0095] S8. Take a new 5 mL eppendorf tube, add trypsin, DNase, and digestion solution in a volume ratio of 1:1:3 and mix them. After filtering with a filter, add the mixture to the eppendorf tube containing tissue fragments in S7.

[0096] S9. Place the ep tube containing the tissue fragments and the mixed solution in S8 at 37°C for 10 minutes, wherein the ep tube is mixed every 2 minutes;

[0097] S10. Take a new 5 mL eppendorf tube, add trypsin inhibitor and separation solution in a volume ratio of 1:4 to prepare a trypsin inhibitor mixture, take out the eppendorf tube containing the digested tissue fragments obtained in step S9 from 37°C, centrifuge, remove the excess liquid on it, add the trypsin inhibitor mixture to terminate the digestion, and let it stand at 37°C for 2min-3min;

[0098] S11. Add a certain amount of 5% FBS (fetal bovine serum) + Neurobasal medium + additive B27 to the pre-coated dish as the culture medium for the primary cells digested in step S10, and add it to the ep tube in step S10;

[0099] S12. Transfer the subsequent experiments to ice, keep the eppendorf tube in step S11 still, wait for the tissue fragments to settle to the bottom or centrifuge to make the tissue fragments settle to the bottom, aspirate the supernatant, then add the DNase diluted with the separation solution, and blow the tissue fragments;

[0100] S13. Centrifuge the ep tube in step S12 for 5 min-6 min, and aspirate the supernatant;

[0101] S14. Add 2 mL to 3 mL of separation solution to the ep tube in step S13, and use a pipette or a pipette to disperse the tissue blocks;

[0102] S15. Take 10 μL of the suspension in the ep tube in step S14, add 90 μL of trypan blue to dilute 10 times, mix well, and count the live cells;

[0103] S16. Take an 8-well high-wall chamber slide (ibidiμ-Slide 8Well high) coated with matrix gel in advance as a culture dish, take the living cells in step S15 at a rate of 1×10 per well 5 Number of cells plated;

[0104] S17. Place the culture dish in step S16 in a 37°C, 5% CO2 incubator, replace the culture medium every 3.5 days, and continue culturing until the 28th day.

[0105] 3-2 Live Cell Imaging

[0106] 1. Cell grouping: Use the cells cultured in 3-1 to establish a blank group (Control, CTR):

[0107] Blank culture medium; lipopolysaccharide group (LPS): cultured in culture medium containing lipopolysaccharide (LPS); drug blank group (ALT): cultured in culture medium containing alituo; drug treatment group (LPS+ALT): first cultured in culture medium containing lipopolysaccharide (LPS) and then cultured in culture medium containing alituo;

[0108] 2. On the 27th day, LPS was added to the culture medium of the lipopolysaccharide group (LPS) and the drug treatment group (LPS+ALT), and the LPS concentration in the culture medium was adjusted to 10 μg / mL. One hour later, the drug blank group (ALT) and the drug treatment group (LPS+ALT) were transferred to the culture medium supplemented with ALEETO (ALT), and the ALT concentration in the culture medium was adjusted to 50 μg / mL, and cultured for 24 hours;

[0109] 3. Prepare a culture medium containing 500 nM lysosome dye (SiR-lysosome kit) and 250 nM mitochondrial dye (PKMito Red), add it to the cell culture medium of the blank group, lipopolysaccharide group, drug blank group, and drug treatment group, and stain for 10 minutes;

[0110] 4.Zeiss structured light illumination microscope captures images.

[0111] 3-3. Experimental results

[0112] like Figure 2 As shown in the figure, the pink spheres are lysosomes, and the green filaments are mitochondria. From the comparison of the results of the four groups, compared with the blank groups (Control, CTR) that have not undergone any treatment, a large number of lysosomes (vermicelli spheres) in the nerve cells treated with endotoxin LPS gathered and enlarged in certain areas, causing the mitochondria (green filaments) to be significantly reduced or even disappeared in this area. The nerve cells in the drug blank group treated with Alituo alone or the drug treatment group treated with LPS and Alituo before and after were similar to the blank group, indicating that Alituo has the effect of repairing and reversing nerve cell damage caused by lysosomal or mitochondrial mutations. This data shows that Alituo can effectively regulate lysosomal and mitochondrial abnormalities, thereby regulating nerve cell function.

[0113] like Figure 3As shown in the neuron morphology analysis diagram, the white spheres in the figure are lysosomes, and the white long rods (long filaments) are mitochondria. Comparing the results of the four groups, the neuron morphology in the blank group was normal, with fewer lysosomes and regular arrangement of mitochondria, while the cell morphology of the neuron cells treated with LPS stimulation was disordered, and the mitochondria became disorganized. The neuron cells in the drug blank group treated with only alituo or the drug treatment group treated with LPS first and then alituo were similar to the blank group, and the cell morphology was normal. This shows that alituo can reverse the abnormal neuron morphology caused by endotoxin LPS.

[0114] like Figure 4 As shown in the results of mitochondrial morphological identification analysis, white or colored long rod-shaped (long filamentous) structures are mitochondria. The mitochondrial morphology in the cells of the blank group was normal, while the mitochondrial morphology in the nerve cells of the lipopolysaccharide group treated with LPS was disordered and chaotic; the nerve cells of the drug blank group treated with only alituo or the drug treatment group treated with LPS first and then alituo were similar to the blank group, and the mitochondrial morphology in the cells was normal. This shows that alituo can reverse the abnormal mitochondrial morphology caused by endotoxin LPS.

[0115] like Figure 5 As shown, after measurement and calculation, the ratio of the long and short axes of mitochondria in the cells of the blank group was about 3, and the calculated eccentricity was within the normal range. The ratio of the long and short axes of mitochondria in the cells of the lipopolysaccharide group was about 2.4, and the calculated eccentricity deviated from the normal value, indicating that the nerve cells showed a transformation from mitochondrial filamentous structure to a short and thick rod-like structure. The ratio of the long and short axes of mitochondria and the eccentricity of the cells in the drug blank group and the drug treatment group were similar to those of the blank group, and were all within the normal range, indicating that Alitol can effectively reverse the changes in mitochondrial morphology caused by LPS.

[0116] like Figure 6 The results showed that the white or colored spherical structures were lysosomes. The lysosomes in the blank group were smaller and fewer, while the lysosomes in the lipopolysaccharide group were significantly larger and more numerous. According to the experimental results of the drug treatment group, the cells were significantly reversed after being treated with ALT, and the number and size of lysosomes were restored to normal. This shows that ALT can reverse the abnormal changes in the number and morphology of lysosomes caused by endotoxin LPS stimulation.

[0117] Reference Figure 7The morphological analysis results of the lysosome shape showed that the ratio of the long and short axes of the lysosomes in the cells of the blank group was about 1.5, and the eccentricity was within the normal range. The ratio of the long and short axes of the lysosomes in the cells of the lipopolysaccharide group was about 1.7, and the eccentricity was abnormal. After the drug treatment group was added to the culture medium containing Alito, the changes in the long and short axes of the lysosomes and the variation in the eccentricity were significantly reversed and restored to normal. This further shows that Alito can reverse the variation trend of the number and morphology of lysosomes.

[0118] Reference Figure 8 Morphological analysis of the size of lysosomes showed that the lysosomes in the cells of the lipopolysaccharide group were significantly larger than those in the blank group. After the drug treatment group was added to the culture medium containing alituo, the size of the lysosomes in the cells returned to normal. This further proves that alituo can be used to reverse the mutation of lysosomes.

[0119] In summary, the experimental results show that LPS-induced cell stress is mainly manifested in: 1) lysosomes become larger and aggregate around the nucleus, and the aggregation and enlargement of lysosomes will further cause mitochondrial damage; 2) mitochondria break and become shorter and thicker, and the reticular filamentous structure disappears. After 24 hours of alituo treatment, the nerve cells were stained and imaged, and it can be seen that the abnormal conditions of lysosomes and mitochondria were reversed: mitochondria can remain the same, and lysosomes have not obviously enlarged or aggregated, proving that alituo can effectively alleviate the cell stress and lysosomal and mitochondrial abnormalities caused by endotoxin LPS, and alituo has a protective effect on nerve cells at the organelle level.

[0120] 3-4. Summary and discussion

[0121] The experimental results prove that Alituo can protect the effects of external damage on the structure and function of mitochondria and lysosomes. Since the structural abnormalities of lysosomes and mitochondria can mark the condition and pathology of related diseases in the body to a certain extent, when Alituo can correct and reverse the mutations of lysosomes and mitochondria, it means that Alituo can reverse the related diseases of the body to a certain extent. The above experiments verify that Alituo can play an important role in the process of nerve cells resisting stress and protecting nerve cells from inflammatory damage. Therefore, Alituo has certain potential in the treatment of neurodegenerative diseases, stroke, cerebrovascular disease, arthritis, enteritis, post-traumatic recovery, autism, depression and pulmonary fibrosis. Since the cell damage caused by lipopolysaccharide is universal, it is basically the same as other injuries, such as hydrogen peroxide damage, protein aggregation damage, etc., which shows that Alituo's protective effect on nerve cells is also universal and can be applied to other diseases caused by different stresses, especially those involving mitochondrial damage or lysosomal damage.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A use of a stem cell extract in lysosome and mitochondria-related diseases, characterized in that: The stem cells are mesenchymal stem cells, and the preparation of the stem cell extract comprises the following steps: S1) culturing the mesenchymal stem cells and creating a stress condition to stimulate the mesenchymal stem cells; S2) isolating and purifying the culture supernatant of the mesenchymal stem cells cultured in S1) to obtain the stem cell extract; The separation and purification of the culture supernatant comprises the following steps: S21) filtering the culture supernatant using a 0.22 μm filter membrane to obtain a filtrate; S22) filtering and concentrating the filtrate using an ultrafiltration concentration tube with a molecular weight cutoff of 3KD to obtain a crude sample of the stem cell extract; S23) purifying the crude sample by molecular sieve exclusion chromatography or reverse phase chromatography to obtain a purified sample of the stem cell extract; wherein the purified sample further satisfies the following characteristics: in the molecular sieve exclusion chromatography, taking the volume of the eluent as a reference, collecting the 10 mL to 40 mL of the eluent as the purified sample; and S24) taking the purified sample to prepare a preset dosage form.

2. The use according to claim 1, characterized in that: The stress condition is ultraviolet irradiation.

3. The use according to claim 2, characterized in that: The ultraviolet irradiation meets the following conditions: a. Irradiation time is 1h-30h; b. Irradiation intensity is 10μW / cm 2 -100μW / cm 2 ; c. The wavelength of ultraviolet light is 290nm-340nm.

4. The use according to claim 1, characterized in that: In the molecular sieve exclusion chromatography, the instrument used is AKTAexplorer, and the chromatography column is a superdex150 chromatography column; the operation steps are: S2311) washing the chromatography column with 2CV of pure water, then balancing the chromatography column with 2CV of 1×PBS reagent, and returning the ultraviolet absorption value of the chromatography column at 280nm to zero; as well as S2312) 300 μL-700 μL of the crude sample is loaded, the loading flow rate is 0.4 mL / min, the elution flow rate of 1×PBS reagent is 0.1-0.3 mL / min, and elution is performed to the peak. When the volume of the eluent reaches 10 mL, the separated elution liquid is received to obtain the purified sample.

5. The use according to claim 4, characterized in that: The purified sample of the stem cell extract also meets the following characteristics: a predetermined volume segment of the purified sample is collected for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and it is measured that the purified sample at least contains protein molecules with a molecular weight distribution of 11KD-100KD.

6. The use according to claim 5, characterized in that: in, The purified sample at least includes protein molecules with a molecular weight between 63KD and 75KD.

7. The use according to claim 1, characterized in that: In the reverse phase chromatography, the instrument used is a high performance liquid chromatograph, the chromatographic column 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 mobile phase is PBS; the operation steps are as follows: S2321) adding PBS reagent to dilute the crude sample; S2322) Load 80-120 μL of the diluted crude sample, control the column temperature of the chromatographic column to 20°C-30°C, the flow rate to 0.2 mL / min-0.8 mL / min, the detection wavelength to 260 nm and / or 280 nm, set the separation time to 48 min, collect the eluate at the peak time of 10 min-40 min, and obtain the purified sample.

8. The use according to claim 1, characterized in that: The culture medium used for culturing the mesenchymal stem cells is a serum-free stem cell culture medium.

9. The use according to claim 1, characterized in that: The mesenchymal stem cells are selected from at least one of umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells and skin-derived mesenchymal stem cells.

10. The use according to claim 1, characterized in that: The preset dosage form is one of lyophilized powder, injection, gel, spray, pill, tablet, powder and ointment.

11. The use according to claim 10, characterized in that: The administration method of the preset preparation when used is selected from one of intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, and oral administration.

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