Use of stem cell extract in preparation of drug for treating or preventing arthritis by regulating lysosomal and / or mitochondrial functions

Abstract

The present application belongs to the field of biopharmaceuticals. Disclosed is the use of a stem cell extract in the preparation of a drug for treating or preventing arthritis by regulating lysosomal and / or mitochondrial functions, wherein a method for preparing the stem cell extract comprises: S1) culturing mesenchymal stem cells and creating stress conditions 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 S2) 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; and S24) preparing the purified sample into a preparation. In the present application, the culture supernatant containing secretions of the stressed stem cells is obtained by means of stress culturing of the stem cells, and in combination with a purification method, the supernatant is prepared into a preparation which can be used for treating arthritis.

Description

Application of stem cell extracts in the preparation of drugs for the treatment or prevention of arthritis by regulating lysosomal and / or mitochondrial function

[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 pharmaceutical field, specifically relating to the use of a stress-induced stem cell extract in the preparation of a drug that treats or prevents arthritis by regulating lysosomal and mitochondrial function. Background Technology

[0003] Lysosomes are organelles that play a crucial role in the degradation and recycling of substances inside and outside cells. Changes or dysfunction in lysosomes can disrupt cellular and biological homeostasis, triggering or exacerbating diseases such as neurodegenerative diseases and arthritis. Mitochondria are vital organelles in eukaryotes for oxidative metabolism and energy provision, and are closely related to the development of various diseases, including stroke, myocardial disease, diabetes, kidney damage, and arthritis. Abnormal increases and aggregation of lysosomes can, in turn, affect mitochondrial function. By regulating the related functions of lysosomes and mitochondria, it is possible to influence the disease progression of conditions such as arthritis, and thus, to prevent or treat various diseases.

[0004] Previous studies (Nat Rev Mol Cell Biol, 2011, 12(1): 9-14, Chinese Journal of Reparative and Reconstructive Surgery, Jun. 2023, Vol. 37, No. 6) have shown that lysosomal / mitochondrial dysfunction is associated with arthritis, and regulating lysosomal and mitochondrial function may help prevent or treat arthritis. For example, osteoarthritis (OA) is a common joint disease that is prevalent in the elderly. Just as machines wear down over time, human articular cartilage also gradually degenerates due to prolonged use. This degeneration not only leads to joint pain and stiffness but may also cause loss of work capacity, seriously affecting quality of life. Chondrocytes in the joint are the main component of cartilage tissue. When joints degenerate or age and wear down, programmed cell death of articular chondrocytes occurs, reducing the number of chondrocytes. Chondrocytes cannot regenerate, eventually damaging the cartilage tissue. Therefore, the occurrence of OA is closely related to the survival ability of chondrocytes. Mitochondria are tiny structures within cells, acting as the cell's energy factories responsible for producing the energy the cell needs. Their functional state directly impacts chondrocyte function. Joint mechanical wear and tear, inflammation, aging, and metabolic disorders can all trigger cellular stress responses, leading to mitochondrial dysfunction and damage. Damaged mitochondria accelerate chondrocyte death, and since chondrocytes cannot regenerate, this further contributes to and exacerbates osteoarthritis (OA). Therefore, regulating mitochondrial function can be used to prevent or treat OA.

[0005] Furthermore, the inventors discovered that stem cells, under stress conditions, can differentiate and secrete various stress proteins that regulate the function of mitochondria and lysosomes. These stress proteins can promote repair of chondrocyte damage and other conditions by regulating the number and function of mitochondria and lysosomes, thereby playing a role in the prevention or treatment of arthritis. How to obtain stem cell extracts that regulate lysosomal and mitochondrial function and can then be used to prevent or treat arthritis is a pressing problem that 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 the supernatant after the stem cell stress culture. 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, especially arthritis.

[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] The lysosomal and mitochondrial related diseases mentioned are arthritis.

[0017] In this invention, the drug achieves the prevention or treatment of arthritis by simultaneously regulating the function of lysosomes and mitochondria. In some alternative applications, the drug achieves the prevention or treatment of arthritis by regulating the function of either lysosomes or mitochondria.

[0018] In some alternative applications, the stress condition is ultraviolet radiation.

[0019] In some optional applications, the ultraviolet irradiation satisfies the following conditions:

[0020] a. Irradiation time is 1 hour to 30 hours;

[0021] b. Irradiation intensity is 10 μW / cm 2 ~100μW / cm 2 ;

[0022] c. The wavelength of ultraviolet light is 290nm~340nm.

[0023] 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:

[0024] 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

[0025] 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.

[0026] 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.

[0027] In some alternative applications, the purified sample includes at least protein molecules with a molecular weight between 63 kDa and 75 kDa.

[0028] 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:

[0029] S2321) Dilute the crude sample with PBS reagent;

[0030] 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.

[0031] In some alternative applications, the culture medium used to culture the mesenchymal stem cells is a serum-free stem cell culture medium.

[0032] 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.

[0033] In some optional applications, the preset dosage form is one of the following: lyophilized powder, injection, gel, spray, pill, tablet, powder, and ointment.

[0034] 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.

[0035] The beneficial effects of this application are:

[0036] 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 these stress-affected stem cells, combined with purification methods. The protein polymers can be administered via various routes, including intrathecal, intravenous, combined intrathecal and intravenous, intracranial, nasal spray, mucosal, and oral administration, and can be formulated into different dosage forms such as lyophilized powders, injections, gels, sprays, pills, tablets, powders, and ointments. Experimental verification has shown that the protein polymers can correct and reverse variations in lysosomes and mitochondria, and structural abnormalities in lysosomes and mitochondria can, to some extent, indicate the pathology and condition of related diseases. When lysosomes and mitochondria are reversed to a relatively normal state, it means that the protein polymers can, to some extent, reverse related diseases, proving that the protein polymers can be used to treat lysosomal and mitochondrial-related diseases. The 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 protection against inflammatory damage, demonstrating the potential to prevent or treat diseases related to lysosomes and mitochondria, such as arthritis. Attached Figure Description

[0037] 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.

[0038] Figure 1 shows the SDS-PAGE results of the stress-induced stem cell extracts from culture experiment one.

[0039] Figure 2 shows the SDS-PAGE results of the stress-induced stem cell extracts from culture experiment 2.

[0040] Figure 3 shows the results of nerve cell staining imaging.

[0041] Figure 4 shows a morphological analysis of nerve cells and intracellular organelles.

[0042] Figure 5 shows the mitochondrial morphology identification and analysis diagram.

[0043] Figure 6 shows the results of mitochondrial morphology analysis.

[0044] Figure 7 shows the identification and analysis of lysosome morphology.

[0045] Figure 8 shows the results of lysosomal morphological analysis.

[0046] Figure 9 shows the morphological analysis and size statistics of lysosomes.

[0047] Figure 10 is a schematic diagram of the anti-inflammatory effects of protein polymers.

[0048] Figure 11 is a schematic diagram showing the results of protein polymers promoting chondrocyte proliferation. Detailed Implementation

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Example 1: Culture of human umbilical cord mesenchymal stem cells

[0057] 1-1 Culture Experiment 1

[0058] 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.

[0059] The cells were irradiated with a 300nm wavelength LED ultraviolet lamp for 6 hours. The ultraviolet irradiation conditions were as follows:

[0060] The supernatant from the cell stress culture was collected, filtered through a 0.22 μm filter membrane, and the filtrate (containing protein polymers) was collected and 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 polymers 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.

[0061] 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:

[0062] (1) The lysis buffer contains some impurities that are difficult to separate, such as large molecules such as DNA remaining in the host cell.

[0063] (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.

[0064] (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.

[0065] (4) The supernatant contains fewer types of proteins than the lysate, making it easier to purify and enrich them.

[0066] 1-2 Cultivation Experiment Two

[0067] Resuscitate one small P8 generation HUC-MSC into a T25 culture flask, adding 2.5 mL of Huakan mesenchymal stem cell serum-free culture medium to each flask, with a cell seeding density of 1 × 10⁶ cells per flask. 5 per mL.

[0068] Cells were irradiated with UVB at wavelengths of 300 nm to 316 nm for 6 h, 12 h, 18 h, 24 h, and 30 h.

[0069] The ultraviolet irradiation conditions are as follows:

[0070] After irradiation, cells were harvested at different time points to obtain protein polymers. Specifically, the culture products were collected at 6h, 12h, 18h, 24h, and 30h after irradiation. The culture products were separated into supernatant and cells, and the following procedures were performed: the precipitated cells were washed twice with 2mL of physiological saline, then 1mL of pure water was added and the cells were repeatedly pipetted down from the bottom of the flask. Cell lysis was achieved by repeated pipetting for about 6 minutes, followed by filtering through a 0.22μm filter membrane and storing at 4℃ for later use.

[0071] After filtering the supernatant through a 0.22 μL filter membrane, store it at 4°C for later use.

[0072] The harvested proteins were analyzed by gel electrophoresis. The protein concentrations obtained under various culture conditions are shown in the table below:

[0073] The intracellular proteins and stem cell culture supernatant proteins obtained above were analyzed using SDS-PAGE, and the results are shown in Figure 2. As shown in Figure 2, protein polymers were present in both the intracellular and supernatant, but the protein concentration in the supernatant was significantly higher.

[0074] Example 2: Molecular sieve purification of mesenchymal stem cell supernatant

[0075] 2-1 Molecular sieve purification of the supernatant after mesenchymal stem cell culture:

[0076] Instrument: AKTA explorer;

[0077] Chromatography column: Nanomicro Superdex 150 molecular sieve 8×500, column volume approximately 30mL;

[0078] Reagents: 0.1M NaOH, 20% ethanol, 1×PBS, purified water;

[0079] Ultraviolet absorption wavelength: 280nm, 260nm for reference;

[0080] Equilibrium chromatography column sequence:

[0081] 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.

[0082] 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.

[0083] 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.

[0084] Further analysis revealed that the purified protein polymer contained at least one of the following proteins:

[0085] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0086] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0087] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0088] sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens;

[0089] sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens;

[0090] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0091] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0092] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0093] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens;

[0094] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens;

[0095] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens;

[0096] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0097] sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens;

[0098] sp|P60709|ACTB_HUMAN Actin,cytoplasmic 1 OS=Homo sapiens;

[0099] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens;

[0100] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0101] 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.

[0102] Example 3: Evaluation of the biological effects of protein polymers

[0103] 3-1 Primary Cell Culture

[0104] S1. Pregnant mice on day 18 of the embryonic period (E18) were anesthetized with a mixture of ether and isoflurane gas;

[0105] S2. Quickly rupture the abdomen of the fetus on ice and remove it;

[0106] S3. Cut off the head of the fetal mouse and place it in a separation solution at 0℃~4℃;

[0107] 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;

[0108] S5. Remove the meninges of the brain and cerebellum, and cut the brain and cerebellum tissue into small pieces in a petri dish;

[0109] S6. Aspirate the separation fluid containing tissue fragments of the brain and cerebellum into a 5 mL ep tube;

[0110] S7. After standing for a period of time until the tissue fragments settle to the bottom, discard the supernatant in the ep tube;

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] S13. Centrifuge the ep tube from step S12 for 5 min to 6 min and aspirate the supernatant;

[0117] 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;

[0118] 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.

[0119] S16. Take an 8-well high-walled chamber 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;

[0120] 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.

[0121] 3-2 Live Cell Imaging

[0122] 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;

[0123] 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.

[0124] 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;

[0125] 4. Imaging using Zeiss structured light illumination microscopy.

[0126] 3-3. Experimental Results

[0127] As shown in Figure 3, 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. However, 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.

[0128] Figure 4 shows the morphological analysis of nerve cells. The white spheres 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 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 morphology to the control group. This indicates that protein polymers can reverse the morphological abnormalities of nerve cells caused by the endotoxin LPS.

[0129] As shown in Figure 5, the mitochondrial morphology identification analysis results indicate that the long 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 demonstrates that protein polymers can reverse the mitochondrial morphological abnormalities caused by the endotoxin LPS.

[0130] As shown in Figure 6, 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.

[0131] As shown in Figure 7, 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.

[0132] Referring to the morphological analysis results of lysosome shape in Figure 8, 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.

[0133] Referring to the morphological analysis of lysosome size in Figure 9, 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.

[0134] 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.

[0135] 3-4. Summary and Discussion

[0136] 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.

[0137] Example 4: Protein products obtained from mesenchymal stem cell culture under ultraviolet irradiation have anti-inflammatory effects.

[0138] The protein polymer-containing sample obtained in Experiment 2 of Example 1 above was used for an experiment on anti-inflammatory efficacy.

[0139] One of the main characteristics of arthritis is the inflammatory response at the joint site. RAW cells are a common inflammatory cell model. This experiment used RAW cells as the inflammatory model cells. RAW cells were seeded at a rate of 20,000 cells / well in a 96-well plate. The inflammatory cell model was established by stimulating the RAW cells with LPS (500 ng / mL) for 24 hours. The LPS supernatant was then discarded, and samples (corresponding to samples with 500 ng / mL protein content) were added. Fresh culture medium was added to the model group. The cell supernatant was collected after 24 hours. The supernatant was diluted 15-20 times and the IL-6 content was measured according to the ELISA kit instructions.

[0140] IL-6 is the most common inflammatory factor. During an inflammatory response, IL-6 levels rise, and the ability to reduce IL-6 levels indicates anti-inflammatory function. The experimental results are shown in Figure 10. The protein polymers obtained from umbilical cord mesenchymal stem cells or amniotic mesenchymal stem cells cultured under ultraviolet irradiation stress for a certain period of time, as described in this invention, can inhibit inflammatory responses in model cells, exhibiting anti-inflammatory function. Since inflammation is one of the main characteristics of arthritis, this demonstrates that the stress-cultured stem cell extract described in this invention has the potential to treat arthritis.

[0141] Example 5: Protein products from stem cell culture after UV irradiation have the function of promoting chondrocyte proliferation.

[0142] Human umbilical cord mesenchymal stem cells or human amniotic mesenchymal stem cells were cultured under UV B ultraviolet irradiation for 18 hours under the conditions of Experiment 2 in Example 1 above. The supernatant was then collected, filtered through a 0.22 μL filter membrane, and stored at 4°C. The remaining cells were then washed twice with 2 mL of physiological saline, followed by repeated pipetting with 1 mL of saline solution to lyse the cells from the bottom of the flask for approximately 6 minutes. The lysed cells were then filtered through a 0.22 μm filter membrane and stored at 4°C for later use.

[0143] In subsequent experiments, intracellular proteins and supernatant proteins from human umbilical cord mesenchymal stem cells cultured under normal conditions without ultraviolet irradiation were used as controls. Human albumin samples that underwent 18 hours of ultraviolet irradiation and those that did not were also used as controls.

[0144] This experiment used primary mouse chondrocytes to study the effect of protein polymers on chondrocyte proliferation. First, 8-week-old C57 mice were euthanized by cervical dislocation and immersed in a beaker containing 75% ethanol for 5 minutes. Then, the leg bones of the mice were removed and placed in a culture dish containing 75% ethanol for 2 minutes, followed by a culture dish containing PBS. Next, in a laminar flow hood, hyaline cartilage from the knee joint was separated into 15 mL centrifuge tubes using sterilized surgical scissors. The cartilage was rinsed twice with an appropriate amount of PBS. Then, the washed cartilage was cut into small pieces using sterilized surgical scissors and placed in six-well plates. 0.2% mouse type II collagenase solution (10 mg type II collagenase dissolved in 5 mL DMEM basal medium) was added until the cartilage pieces were just submerged. The plates were incubated in cell culture for 1 hour. During this time, the six-well plates were removed approximately every 15 minutes and gently pipetted to ensure sufficient contact between the collagenase solution and the cartilage pieces, facilitating digestion into single cells. Then, add approximately 6 mL of DMEM complete medium to terminate the digestion. Pipette the solution and filter through a 200-mesh screen. Collect the filtrate in a 50 mL centrifuge tube and centrifuge at 1500 rpm for 5 min. Discard the supernatant, resuspend the solution in 6 mL of DMEM complete medium, and centrifuge again at 400 rpm for 5 min. Collect the supernatant and culture it in 2 mL / well of a six-well plate. Incubate the plate statically for 24 h, then change the medium. Collect the cartilage fragments remaining on the filter and the precipitate from the previous centrifugation step. Place the plate in a six-well plate, add type II collagenase solution to submerge the cartilage fragments, and incubate for another 1 h. Gently pipette the solution every 15 min to ensure adequate contact between the cartilage fragments and the collagenase solution. Then, add 6 mL of DMEM complete medium to terminate the digestion, filter again, collect the filtrate in a centrifuge tube, centrifuge at 1500 rpm for 5 min, and discard the supernatant. Resuspend the cells in 6 mL of DMEM complete medium, centrifuge at 400 rpm for 5 min, collect the supernatant and culture 2 mL / well in a 6-well plate. Resuspend the remaining precipitate in 2 mL of DMEM complete medium and culture in 6-well plates. Transfer the plates to cell culture flasks and incubate for 24 h, then change the medium. Observe cell growth under a microscope. When the cell growth area reaches 80%–90% of the bottom area of ​​the cell culture flask, seed the cells into 96-well plates. Third-generation mouse chondrocytes were used in this experiment. Seedlings of approximately 10,000 cells per well were placed in 96-well plates with 100 μL / well of DMEM complete medium and incubated overnight. The next day, 100 μL of DMEM complete medium containing 100 ng / mL protein sample was added to each well, and the plates were incubated for 7 days. Then, add 10 μL of CCK8 solution to each well, gently tap the edge of the 96-well plate to mix it evenly, and then incubate it in an incubator for about 4 hours. Then, use a microplate reader to detect the absorbance at 450 nm.Perform the corresponding calculations according to the CCK8 kit instructions.

[0145] A sample containing only chondrocytes was used as a negative control. Medical chitosan is a currently marketed product for treating osteoarthritis; therefore, medical chitosan (3 mg / mL) was used as a positive control.

[0146] The experimental results are shown in Figure 11. The protein polymer obtained from the umbilical cord mesenchymal stem cells or amniotic mesenchymal stem cells described in this invention after being cultured under ultraviolet irradiation stress for a certain period of time can increase the content of chondrocytes.

[0147] Some embodiments of this invention used unpurified mixed protein samples containing protein polymers for experiments. In practical applications, appropriately purified protein polymer samples can also be used. Purification methods include, but are not limited to, common protein purification methods such as HPLC, SEC, ultrafiltration, filtration, and centrifugation.

[0148] 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. Use of a stem cell extract for the preparation of a medicament for the prevention or treatment of arthritis by modulating lysosomal and / or mitochondrial function, 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 one or more of 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. Use 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 of 10 μW / cm 2 ~ 100 μW / cm 2 ; c. The wavelength of ultraviolet light is 290nm~340nm.

3. Use 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. Use 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. Use 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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