Use of stem cell extract in lysosome- and mitochondrion-associated diseases

Abstract

The use of a stem cell extract in lysosome- and mitochondrion-associated diseases. A method for preparing the stem cell extract comprises: S1) culturing mesenchymal stem cells and creating stress conditions (such as ultraviolet irradiation) to stimulate the mesenchymal stem cells; and S2) isolating and purifying a culture supernatant of the mesenchymal stem cells cultured in S1) to obtain a stem cell extract, wherein the isolation and purification method comprises: S21) filtering the culture supernatant to obtain a filtrate; S22) concentrating the filtrate by means of 3KD ultrafiltration to obtain a crude sample; S23) purifying the crude sample by means of size-exclusion chromatography or reversed-phase chromatography to prepare a purified sample, wherein in the size-exclusion chromatography, with the volume of an eluent as a reference, the eluate collected from 10 mL to 40 mL is taken as the purified sample; and S24) preparing the purified sample into a preparation. The stem cell extract preparation has the potential to treat lysosome- and mitochondrion-related diseases, such as cerebrovascular disease, arthritis, enteritis, post-traumatic recovery, depression and pulmonary fibrosis.

Description

Application of stem cell extracts in lysosomal and mitochondrial related diseases

[0001] This disclosure claims priority to Chinese Patent Application No. 202510043832.X, filed on January 10, 2025, entitled "Application of Stem Cell Extracts in Lysosomal and Mitochondrial Related Diseases", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of medicine, specifically relating to the application of a stress-induced stem cell extract in lysosomal and mitochondrial related diseases. Background Technology

[0003] Lysosomes are organelles that play a crucial role in the degradation and recycling of substances inside and outside cells. They perform the function of degrading substances within and outside the cell. Changes or dysfunction of lysosomes can disrupt the original homeostasis of cells and organisms, triggering or worsening human diseases, including cancer, autoimmune diseases, neurodegenerative diseases, and cardiovascular diseases. Mitochondria are important organelles for oxidative metabolism in eukaryotes and are closely related to the development and progression of various diseases such as stroke, myocardial disease, diabetes, kidney damage, and cancer.

[0004] Current treatment protocols for diseases related to lysosomes and mitochondria typically employ corresponding targeted drugs to inhibit undesirable mutations in lysosomes and mitochondria, thereby slowing the progression of the related diseases.

[0005] However, the inventors of this application discovered a certain correlation between lysosomal and mitochondrial related diseases, and their characteristic indicators tend to be consistent in the course of some diseases. Therefore, it is necessary to develop a relatively broad-spectrum drug that can treat both lysosomal and mitochondrial related diseases simultaneously. Furthermore, the inventors discovered during the implementation process that stem cells can be induced to differentiate and secrete various stress proteins under stress conditions to resist adverse environmental influences. These stress proteins have a universal pro-repair effect on various types of cell damage. How to obtain stem cell extracts with a universal therapeutic effect on lysosomal and mitochondrial related diseases through appropriate purification methods is a problem that urgently needs to be solved. Summary of the Invention

[0006] Based on this, the inventors of this 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 structure and function of lysosomes and mitochondria induced by external stress, and can be universally applied to the treatment of diseases related to abnormalities in the structure and function of lysosomes and mitochondria.

[0007] The specific technical solution of this application is as follows:

[0008] The use of a stem cell extract in the preparation of a drug for the prevention or treatment of lysosomal and mitochondrial-related diseases, wherein the stem cells are mesenchymal stem cells, and the preparation of the stem cell extract includes the following steps:

[0009] 1) Culture the mesenchymal stem cells and create stress conditions to stimulate the mesenchymal stem cells;

[0010] S2) The stem cell extract is obtained by separating and purifying the culture supernatant of the mesenchymal stem cells cultured in S1);

[0011] The separation and purification of the culture supernatant includes the following steps:

[0012] S21) The culture supernatant was filtered through a 0.22 μm filter membrane to obtain the filtrate;

[0013] S22) The filtrate was concentrated by filtration using an ultrafiltration tube with a molecular weight cutoff of 3KD to obtain a crude sample of the stem cell extract;

[0014] S23) The crude sample is purified using size exclusion chromatography or reversed-phase chromatography to obtain a purified sample of the stem cell extract; wherein the purified sample also satisfies the following characteristics: in the size exclusion chromatography, the eluent from 10 mL to 40 mL is collected as the purified sample, using the volume of the eluent as a reference; and

[0015] S24) Take the purified sample and prepare a preset dosage form.

[0016] In some alternative applications, the stress condition is ultraviolet radiation.

[0017] In some optional applications, the ultraviolet irradiation satisfies the following conditions:

[0018] a. Irradiation time is 1 hour to 30 hours;

[0019] b. Irradiation intensity is 10 μW / cm 2 ~100μW / cm 2 ;

[0020] c. The wavelength of ultraviolet light is 290nm-340nm.

[0021] In some optional applications, the molecular sieve size exclusion chromatography uses an AKTA explorer instrument and a Superdex 150 column; the operating procedures are as follows:

[0022] S2311) Rinse the chromatography column with 2CV of purified water, then equilibrate the column with 2CV of 1×PBS reagent, and bring the UV absorbance of the column to zero at 280 nm; and

[0023] S2312) Load 300 μL to 700 μL of the crude sample at a flow rate of 0.4 mL / min. Elute with 1×PBS at a flow rate of 0.1 mL / min to 0.3 mL / min until the peak is reached. Start collecting the separated eluent when the volume of the eluent reaches 10 mL to obtain the purified sample.

[0024] In some optional applications, the purified sample of the stem cell extract also meets the following characteristics: a predetermined volume of the purified sample is collected for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the purified sample is found to contain at least protein molecules with a molecular weight distribution of 11KD to 100KD.

[0025] In some alternative applications, the purified sample includes at least protein molecules with a molecular weight between 63 kDa and 75 kDa.

[0026] In some alternative applications, the reversed-phase chromatography uses a high-performance liquid chromatograph with an XBridge Protein BEH C4 column and a packing pore size of [missing information]. The packing material has a particle size of 3.5 μm and an inner diameter * length of 4.6 mm * 150 mm. The mobile phase is PBS. The operating steps are as follows:

[0027] S2321) Dilute the crude sample with PBS reagent;

[0028] S2322) Load 80 μL to 120 μL of the diluted crude sample onto the chromatographic column, control the column temperature to 20℃ to 30℃, the flow rate to 0.2 mL / min to 0.8 mL / min, the detection wavelength to 260 nm and / or 280 nm, and set the separation time to 48 min. Collect the eluent when the peak time is between 10 min and 40 min to obtain the purified sample.

[0029] In some alternative applications, the culture medium used to culture the mesenchymal stem cells is a serum-free stem cell culture medium.

[0030] In some alternative applications, 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 placental-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0031] In some optional applications, the preset dosage form is one of the following: lyophilized powder, injection, gel, spray, pill, tablet, powder, and ointment.

[0032] In some optional application schemes, the administration method of the preset formulation is selected from one of the following: intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, and oral administration.

[0033] The beneficial effects of this application are:

[0034] This application describes the preparation of stem cell extracts (denoted as protein polymers) by culturing stem cells under stress and using the culture supernatant containing secretions from the stressed stem cells, combined with purification methods. These protein polymers can be formulated into various dosage forms, including lyophilized powders, injections, gels, sprays, pills, tablets, powders, and ointments, using different administration methods such as intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, and oral administration. Related experiments have verified that protein polymers can correct and reverse lysosomal and mitochondrial abnormalities. Structural abnormalities of lysosomes and mitochondria can, to some extent, indicate the condition and pathology of related diseases in the body. When lysosomes and mitochondria are reversed to a relatively normal state, it means that protein polymers can, to some extent, reverse related diseases in the body, proving that protein polymers can be used to treat lysosomal and mitochondrial-related diseases. Protein polymers can protect against the effects of external damage on the structure and function of mitochondria and lysosomes, and play an important role in cellular stress resistance and protecting cells from inflammatory damage. They possess the potential to treat cerebrovascular diseases, arthritis, enteritis, post-traumatic recovery, depression, and pulmonary fibrosis, among other diseases related to lysosomes and mitochondria. Attached Figure Description

[0035] To more clearly illustrate the technical solution of this application, the accompanying drawings used in the description of this application will be briefly introduced below.

[0036] Figure 1 shows the SDS-PAGE results of stress-induced stem cell extracts.

[0037] Figure 2 shows the results of nerve cell staining imaging.

[0038] Figure 3 shows the morphological analysis of nerve cells and intracellular organelles.

[0039] Figure 4 shows the mitochondrial morphology identification and analysis diagram.

[0040] Figure 5 shows the results of mitochondrial morphology analysis.

[0041] Figure 6 shows the identification and analysis of lysosome morphology.

[0042] Figure 7 shows the results of lysosomal morphological analysis.

[0043] Figure 8 shows the morphological analysis and size statistics of lysosomes. Detailed Implementation

[0044] The embodiments of this implementation are described in detail below. These embodiments are only used to explain this implementation and should not be construed as limiting this implementation.

[0045] In this application, for ease of explanation, mesenchymal stem cells are used as an example in the embodiments. However, in practical applications, the use is not limited to mesenchymal stem cells. Other stem cells can also produce similar 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, pancreatic stem cells, and MUSE cells.

[0046] Mesenchymal stem cells (MSCs) can theoretically produce corresponding stress proteins after undergoing stress treatment. For ease of explanation, ultraviolet irradiation was used as the stress condition in this example to prepare a MSC 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, hyperxia, oxidation (under conditions containing oxidants such as hydrogen peroxide or hypochlorous acid), high pH, ​​low pH, ultrasound, terahertz electromagnetic waves, X-rays, microwaves, radiation, ion beams, high CO2, and low CO2. Various stress treatments can be performed sequentially or simultaneously if they do not conflict.

[0047] The examples used umbilical cord mesenchymal stem cells as an example to illustrate the problem. In practical applications, the sources of mesenchymal stem cells also include, but are not limited to, bone marrow-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0048] Theoretically, any method that can be used to separate proteins from cell culture supernatant can also be used to separate and purify the stem cell extract 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 filtrate can also be used, including but not limited to at least one of the following methods: chromatography, spectroscopy, volumetric methods, dialysis, salting out, precipitation, acid extraction, alkaline extraction, ultrafiltration, chromatography, electrophoresis, and centrifugation.

[0049] Spectroscopic methods utilize the absorbance of target proteins to specific wavelengths of light to screen for target extracts; dialysis involves exchanging solutions between a dialysis bag and a buffer solution to obtain a purified and concentrated target extract; organic solvent precipitation involves adding organic solvents to the culture supernatant to achieve a precipitation effect similar to high-concentration salt, with the precipitate being the target extract; acid extraction involves adding concentrated hydrochloric acid to obtain the target extract; alkaline extraction involves treating the culture supernatant with sodium hydroxide or potassium hydroxide to obtain the target extract; ultrafiltration involves adding the cell culture supernatant to an ultrafiltration column, continuously centrifuging, adding buffer, and repeating ultrafiltration multiple times to finally concentrate the target extract; chromatography includes ion exchange chromatography, gel filtration chromatography, and affinity chromatography, with different chromatography methods selected based on the characteristics of the target extract; electrophoresis separates protein molecules in a dispersion matrix using electrophoresis to obtain the target extract; and centrifugation involves centrifuging the supernatant to obtain the target extract.

[0050] The technical solution of this application will be further explained below with reference to experimental embodiments. It can be understood that the protein polymer defined in this application is a collection of various stress proteins with specific biological activities expressed by mesenchymal stem cells under stress conditions, and cell extracts necessarily contain these stress proteins; and the separation and purification of cell extracts to remove non-protein substances also results in protein polymers.

[0051] Example 1: Culture of human umbilical cord mesenchymal stem cells

[0052] Resuscitate one small P8 generation HUC-MSC (human umbilical cord-derived mesenchymal stem cell) into a T25 culture flask and add 2.5 mL of Huakan mesenchymal stem cell serum-free culture medium.

[0053] The cells were irradiated with a 300nm wavelength LED ultraviolet lamp for 6 hours. The ultraviolet irradiation conditions were as follows:

[0054] The supernatant from the cell stress culture was collected, filtered through a 0.22 μm filter membrane, and the filtrate (also denoted as protein polymer) was stored at 4°C for later use. The protein concentration of the protein polymer was measured to be 0.655 mg / mL, and the volume was 500 μL. The SDS-PAGE results of the harvested protein polymer are shown in Figure 1. As can be seen from the figure, the sample bands are distributed between 11 KD and 100 KD, especially concentrated between 63 KD and 75 KD.

[0055] It should be noted that this application collects the supernatant after cell stress culture, not cell lysis buffer, for one or more of the following reasons:

[0056] (1) The lysis buffer contains some impurities that are difficult to separate, such as large molecules such as DNA remaining in the host cell.

[0057] (2) The proteins in the supernatant have different molecular weights and structures compared to the proteins in the lysate, meaning that the proteins extracted from the supernatant are more suitable for use in lysosomal and mitochondrial related diseases.

[0058] (3) Protein molecules secreted from the cell in the supernatant may be able to penetrate the cell membrane more effectively, which is beneficial for them to enter abnormal cells and exert their effects in subsequent applications.

[0059] (4) The supernatant contains fewer types of proteins than the lysate, making it easier to purify and enrich them.

[0060] Example 2: Molecular sieve purification of mesenchymal stem cell supernatant

[0061] 2-1 Molecular sieve purification of the supernatant after mesenchymal stem cell culture:

[0062] Instrument: AKTA explorer;

[0063] Chromatography column: Nanomicro Superdex 150 molecular sieve 8×500, column volume approximately 30mL;

[0064] Reagents: 0.1M NaOH, 20% ethanol, 1×PBS, purified water;

[0065] Ultraviolet absorption wavelength: 280nm, 260nm for reference;

[0066] Equilibrium chromatography column sequence:

[0067] First, rinse the chromatography column with 2CV purified water, then equilibrate the column with 2CV 1×PBS reagent, and bring the UV absorbance of the column to zero at 280nm.

[0068] Sample preparation: Following the method in Example 1, collect 20 mL of protein polymer stock solution. Use an ultrafiltration concentrator with a molecular weight cutoff of 3 KD to reduce the 20 mL protein polymer stock solution to approximately 600 μL, obtaining a crude sample.

[0069] 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 was collected starting at a UV absorbance of 4 mAU. The collected eluent was then lyophilized into a stem cell extract and stored for later use.

[0070] Further analysis revealed that the purified protein polymer contained at least one of the following proteins:

[0071] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0072] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0073] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0074] sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens;

[0075] sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens;

[0076] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0077] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0078] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0079] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1chain OS=Homo sapiens;

[0080] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens;

[0081] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy chain OS=Homo sapiens;

[0082] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0083] sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens;

[0084] sp|P60709|ACTB_HUMAN Actin,cytoplasmic 1OS=Homo sapiens;

[0085] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens;

[0086] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0087] 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.

[0088] Example 3: Evaluation of the biological effects of protein polymers

[0089] 3-1 Primary Cell Culture

[0090] S1. Pregnant mice on day 18 of the embryonic period (E18) were anesthetized with a mixture of ether and isoflurane gas;

[0091] S2. Quickly rupture the abdomen of the fetus on ice and remove it;

[0092] S3. Cut off the head of the fetal mouse and place it in a separation solution at 0℃~4℃;

[0093] S4. Remove the skin and skull of the fetal mouse head, remove the brain and cerebellum together and place them in another petri dish containing separation solution at 0℃~4℃, and place the petri dish on ice;

[0094] S5. Remove the meninges of the brain and cerebellum, and cut the brain and cerebellum tissue into small pieces in a petri dish;

[0095] S6. Aspirate the separation fluid containing tissue fragments of the brain and cerebellum into a 5 mL ep tube;

[0096] S7. After standing for a period of time until the tissue fragments settle to the bottom, discard the supernatant in the ep tube;

[0097] S8. Take a new 5mL EP tube, add pancreatin, DNase and digestion solution in a volume ratio of 1:1:3 and mix. After filtering, add the mixture to the EP tube containing tissue fragments in S7.

[0098] S9. Place the ep tube containing tissue fragments and mixed liquid from S8 at 37°C and digest for 10 minutes, wherein the ep tube is mixed every 2 minutes.

[0099] S10. Take a new 5mL ep tube, add pancreatic enzyme inhibitor and separation solution at a volume ratio of 1:4 to prepare pancreatic enzyme inhibitor mixture. Take the ep tube containing digested tissue fragments obtained in step S9 out of 37°C, centrifuge, remove excess liquid, add pancreatic enzyme inhibitor mixture to stop digestion, and let stand at 37°C for 2 to 3 minutes.

[0100] 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 obtained by digestion in step S10, and add it to the ep tube in step S10.

[0101] S12. Transfer the subsequent experiments to ice operation, let the ep tube from step S11 stand until the tissue fragments settle to the bottom or centrifuge until the tissue fragments settle to the bottom, then aspirate the supernatant, add the DNA enzyme diluted with the separation solution, and pipette the tissue fragments.

[0102] S13. Centrifuge the ep tube from step S12 for 5 min to 6 min and aspirate the supernatant;

[0103] S14. Add 2 mL to 3 mL of separation solution to the ep tube in step S13, and use a pipette or pipette to disperse the tissue block;

[0104] S15. Take 10 μL of the suspension in the ep tube from step S14, add 90 μL of trypan blue to dilute it 10 times, mix thoroughly, and count the live cells.

[0105] S16. Take an 8-well high-walled glass slide (ibidiμ-Slide 8 Well high) pre-coated with matrix gel as a culture dish, and take 1 × 10⁶ live cells from step S15 at a ratio of 1 × 10⁶ cells per well. 5 Number of cells for plating;

[0106] S17. Place the culture dish from step S16 in a 37°C, 5% CO2 incubator, change the culture medium every 3.5 days, and continue culturing until day 28.

[0107] 3-2 Live Cell Imaging

[0108] 1. Cell grouping: Using cells cultured in 3-1, establish blank groups (Control, CTR): cultured in blank medium; lipopolysaccharide group: cultured in lipopolysaccharide-containing medium; drug blank group: cultured in protein polymer-containing medium; drug treatment group: first cultured in lipopolysaccharide (LPS)-containing medium and then in protein polymer-containing medium;

[0109] 2. On day 27, LPS was added to the culture medium of the lipopolysaccharide group and the drug treatment group to adjust the LPS concentration in the culture medium to 10 μg / mL. After 1 hour, the drug blank group and the drug treatment group were transferred to the culture medium with protein polymer added to adjust the protein polymer concentration in the culture medium to 50 μg / mL and cultured for 24 hours.

[0110] 3. Prepare a culture medium containing 500 nM lysosomal dye (SiR-lysosome kit) and 250 nM mitochondrial dye (PK Mito 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 min;

[0111] 4. Imaging using Zeiss structured light illumination microscopy.

[0112] 3-3. Experimental Results

[0113] As shown in Figure 2, the pink spherical structures represent lysosomes, and the green filamentous structures represent mitochondria. Comparing the four groups, compared to the untreated control group (Control, CTR), nerve cells treated with endotoxin LPS showed a large accumulation and enlargement of lysosomes (pink spherical shapes) in certain regions, leading to a significant reduction or even disappearance of mitochondria (green filamentous structures) in these areas. Nerve cells in the control group treated solely with protein polymers, or in the treatment groups treated with LPS and protein polymers respectively, showed similar results to the control group. This indicates that protein polymers have a repairing and reversing effect on nerve cell damage caused by lysosomal or mitochondrial mutations. This data demonstrates that protein polymers can effectively regulate lysosomal and mitochondrial abnormalities, thereby regulating nerve cell function.

[0114] As shown in Figure 3, the white spherical structures represent lysosomes, and the white rod-shaped (filamentous) structures represent mitochondria. Comparing the four groups, the control group showed normal nerve cell morphology, fewer lysosomes, and regularly arranged mitochondria. In contrast, nerve cells treated with LPS exhibited disordered cell morphology, with mitochondria becoming disorganized. The nerve cells in the control group treated solely with protein polymers, or in the treatment group treated with LPS followed by protein polymers, showed similar normal cell morphology to the control group. This indicates that protein polymers can reverse the abnormal nerve cell morphology caused by the endotoxin LPS.

[0115] As shown in Figure 4, the white or colored rod-shaped (filamentous) structures are mitochondria. In the control group, the mitochondrial morphology in the cells was normal, while in the lipopolysaccharide group treated with LPS, the mitochondrial morphology in the nerve cells was disordered and disorganized. The nerve cells in the drug control group treated only with protein polymers, or the drug treatment group treated with LPS first and then protein polymers, were similar to the control group, with normal mitochondrial morphology. This indicates that protein polymers can reverse the mitochondrial morphological abnormalities caused by the endotoxin LPS.

[0116] As shown in Figure 5, measurements and calculations showed that the ratio of the long to short axis of mitochondria in the cells of the control group was approximately 3, and the calculated eccentricity was within the normal range. In contrast, the ratio of the long to short axis of mitochondria in the cells of the lipopolysaccharide (LPS) group was approximately 2.4, and the calculated eccentricity deviated from the normal value, indicating that nerve cells exhibited a transformation from a filamentous mitochondrial structure to a short, thick rod-like structure. The ratio of the long to short axis and the eccentricity of mitochondria in the cells of the drug control and drug treatment groups were similar to those of the control group, both within the normal range, indicating that the protein polymer can effectively reverse the mitochondrial morphological changes induced by LPS.

[0117] As shown in Figure 6, the white or colored spherical structures are lysosomes. In the control group, the cells contained smaller and fewer lysosomes, while in the lipopolysaccharide group, the lysosomes were significantly larger and more numerous. Based on the experimental results of the drug treatment group, treatment with protein polymers significantly reversed the trend of lysosome enlargement and increase, restoring the number and size of lysosomes to normal. This indicates that protein polymers can reverse the abnormal changes in the number and morphology of lysosomes caused by endotoxin LPS stimulation.

[0118] Referring to the morphological analysis results of lysosome shape in Figure 7, measurements showed that the ratio of the long to short axis of lysosomes in the blank control group was approximately 1.5, with eccentricity within the normal range. In contrast, the ratio of the long to short axis of lysosomes in the lipopolysaccharide group was approximately 1.7, indicating abnormal eccentricity. The drug treatment group, after being cultured in a medium containing protein polymers, showed a significant reversal of the changes in the long and short axes and eccentricity of lysosomes, restoring them to normal. This further demonstrates that protein polymers can reverse the trend of variation in lysosome number and morphology.

[0119] Referring to the morphological analysis of lysosome size in Figure 8, the lysosomes in the lipopolysaccharide group were significantly larger than those in the control group. Furthermore, the drug treatment group, after being cultured in a medium containing protein polymers, showed a return to normal lysosome size. This further demonstrates that protein polymers can be used to reverse lysosomal variations.

[0120] In summary, the experimental results show that LPS-induced cellular stress is mainly manifested in: 1) enlarged lysosomes that aggregate around the cell nucleus, and this aggregation and enlargement further damages mitochondria; 2) mitochondria break down, shorten, and thicken, with the disappearance of their reticular and filamentous structures. However, after 24 hours of protein polymer treatment, subsequent cell staining and imaging of nerve cells showed a reversal of the abnormalities in lysosomes and mitochondria: mitochondria remained unchanged, and lysosomes did not show significant enlargement or aggregation. This demonstrates that protein polymers can effectively alleviate the cellular stress and lysosomal and mitochondrial abnormalities caused by the endotoxin LPS, and that protein polymers provide organelle-level protection for nerve cells.

[0121] 3-4. Summary and Discussion

[0122] Experimental results demonstrate that protein polymers can protect against the effects of external damage on the structure and function of mitochondria and lysosomes. Since structural abnormalities in lysosomes and mitochondria can, to some extent, indicate the pathology and condition of related diseases, the ability of protein polymers to correct and reverse these variations suggests that they can, to some extent, reverse the associated diseases. The above experiments verify that protein polymers play an important role in nerve cell stress resistance and protecting nerve cells from inflammatory damage. Therefore, protein polymers have potential in the treatment of cerebrovascular diseases, arthritis, enteritis, post-traumatic recovery, depression, and pulmonary fibrosis. Because lipopolysaccharide-induced cell damage is universal and exhibits essentially the same symptoms as other types of damage, such as hydrogen peroxide damage and protein aggregation damage, it indicates that the protective effect of protein polymers on nerve cells is also somewhat universal and applicable to other stress-induced diseases, especially those involving mitochondrial or lysosomal damage.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. The use of a stem cell extract in the preparation of a medicament for the prevention or treatment of lysosomal and mitochondrial-related diseases, characterized in that, The stem cells are mesenchymal stem cells, and the preparation of the stem cell extract includes the following steps: S1) Culture the mesenchymal stem cells and create stress conditions to stimulate the mesenchymal stem cells; S2) The stem cell extract is obtained by separating and purifying the culture supernatant of the mesenchymal stem cells cultured in S1); The separation and purification of the culture supernatant includes the following steps: S21) The culture supernatant was filtered through a 0.22 μm filter membrane to obtain the filtrate; S22) The filtrate was concentrated by filtration using an ultrafiltration tube with a molecular weight cutoff of 3KD to obtain a crude sample of the stem cell extract; S23) The crude sample is purified using size exclusion chromatography or reversed-phase chromatography to obtain a purified sample of the stem cell extract; wherein the purified sample also satisfies the following characteristics: in the size exclusion chromatography, the eluent from 10 mL to 40 mL is collected as the purified sample, using the volume of the eluent as a reference; and S24) Take the purified sample and prepare a preset dosage form.

2. The application according to claim 1, characterized in that, The stress condition is ultraviolet radiation; Preferably, the ultraviolet irradiation satisfies the following conditions: a. Irradiation time is 1 hour to 30 hours; b. Irradiation intensity is 10 μW / cm 2 ~100μW / cm 2 ; c. The wavelength of ultraviolet light is 290nm~340nm.

3. The application according to claim 1, characterized in that, In the molecular sieve size exclusion chromatography, the instrument used was an AKTA explorer, and the chromatography column was a Superdex 150 chromatography column; the operating steps were as follows: S2311) Rinse the chromatography column with 2CV of purified water, then equilibrate the chromatography column with 2CV of 1×PBS reagent, and bring the UV absorbance of the chromatography column to zero at 280nm. as well as S2312) Load 300 μL to 700 μL of the crude sample at a flow rate of 0.4 mL / min. Elute with 1×PBS at a flow rate of 0.1 mL / min to 0.3 mL / min until the peak is reached. Start collecting the separated eluent when the volume of the eluent reaches 10 mL to obtain the purified sample.

4. The application according to claim 3, characterized in that, The purified sample of the stem cell extract also meets the following characteristics: a predetermined volume of the purified sample is collected for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the purified sample is found to contain at least protein molecules with a molecular weight distribution of 11KD to 100KD.

5. The application according to claim 4, characterized in that, in, The purified sample includes at least protein molecules with a molecular weight between 63KD and 75KD.

6. The application according to claim 1, characterized in that, In the reversed-phase chromatography, a high-performance liquid chromatograph was used, and the chromatographic column was an XBridge Protein BEH C4, with a packing pore size of [missing information]. The packing material has a particle size of 3.5 μm and an inner diameter * length of 4.6 mm * 150 mm. The mobile phase is PBS. The operating steps are as follows: S2321) Dilute the crude sample with PBS reagent; S2322) Load 80 μL to 120 μL of the diluted crude sample onto the chromatographic column, control the column temperature to 20℃ to 30℃, the flow rate to 0.2 mL / min to 0.8 mL / min, the detection wavelength to 260 nm and / or 280 nm, and set the separation time to 48 min. Collect the eluent when the peak time is between 10 min and 40 min to obtain the purified sample.

7. The application according to claim 1, characterized in that, The culture medium used to culture the mesenchymal stem cells is a serum-free stem cell culture medium.

8. The application according to claim 1, characterized in that, The mesenchymal stem cells are selected from at least one of the following: human mesenchymal stem cells derived from umbilical cord, mesenchymal stem cells derived from bone marrow, mesenchymal stem cells derived from human placenta, mesenchymal stem cells derived from adipose tissue, and mesenchymal stem cells derived from skin.

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

10. The application according to claim 9, characterized in that, The administration method of the preset formulation is selected from one of the following: intrathecal administration, intravenous administration, combined intrathecal and intravenous administration, intracranial administration, nasal spray administration, mucosal administration, and oral administration.

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