A stem cell exosome composition for treating sarcopenia and its application

Abstract

The present invention provides a stem cell exosome composition for treating sarcopenia and its application, comprising the following components at the following concentrations: exosome 1×10 8 ‑1×10 13 particles / mg protein, nicotinamide 100‑3000mg / ml, wolfberry polysaccharides 50‑900mg / ml, vitamin B6 10‑600mg / ml, calcium gluconate 0.1‑2g / ml, i‑inositol 15‑50mg / mL, pyridoxine hydrochloride 0.5‑5mg / mL, vitamin B12 0.005‑0.01mg / mL, IGF‑120‑100ug / ml, VEGF 1.0‑3.0mg / ml, IL‑15 10‑100μg / mL, IL‑2500‑2000U / ml, IL‑21 5‑50μg / mL, IL‑11 10‑100ug / ml, α‑galactosylceramide 1‑20μg / mL. The stem cell exosome composition for treating sarcopenia described in the present invention contains exosomes, nicotinamide and wolfberry polysaccharides as a breakthrough in the treatment of sarcopenia, and is used to prevent / treat sarcopenia, physical weakness, loss of lean muscle mass, loss of skeletal muscle strength / loss of skeletal muscle function, and improve skeletal muscle mass, lean muscle mass, skeletal muscle strength / skeletal muscle function.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a stem cell exosome composition for treating sarcopenia and an application thereof. Background Art

[0002] Sarcopenia, also known as "muscle loss", is a disease that gradually and universally loses muscle mass and function with age. It has always been an important public health issue faced by many countries and regions. According to statistics, the incidence of sarcopenia is very high in the elderly, with an incidence of 5-13% in people aged 60-70 and 50-60% in people over 80 years old. Because the older you are, the weaker your muscle growth ability is. A 70-year-old muscle cell is not the same as a 40-year-old muscle cell. The occurrence of sarcopenia is also related to the decline in sex hormone levels, mitochondrial function, cell apoptosis, neurodegenerative diseases, chronic diseases and inflammatory states related to aging. The treatment of sarcopenia is becoming more and more challenging. At present, research on sarcopenia is still in its infancy. At present, exercise is considered to be the most effective solution for preventing sarcopenia. Exercise-induced mechanical load has been shown to induce the production of IGF-1, VEGF and hepatocyte growth factor in the bone cell line, which may play a role in regulating muscle growth. Importantly, physical exercise can also induce the production of osteocalcin (OCN), an osteoblast-derived hormone that has been shown to promote exercise adaptation in mice. Finally, it was also found that mitochondrial oxidase activity and the expression of mitochondrial biogenesis genes such as PGC-1, NRF-1, and TFAM were increased in elderly people who exercise regularly, indicating that physical exercise can enhance or even restore mitochondrial function and homeostasis by directly or indirectly stimulating mitochondrial biogenesis. Studies have shown that Lycium barbarum polysaccharides can increase the expression of PGC-1, NRF-1, and TFAM.

[0003] Due to the different physical conditions, living habits, underlying diseases and other factors of each person, the manifestation and severity of sarcopenia are also different. Therefore, individual differences need to be considered when formulating treatment plans, which increases the complexity of treatment. At present, there are few specific drugs for sarcopenia. Although some drugs (such as growth hormone, testosterone, etc.) can improve muscle mass or strength to a certain extent, their effects are not significant and may have side effects. The treatment of sarcopenia usually requires long-term persistence, including nutritional intervention and exercise training. However, the elderly often find it difficult to adhere to these treatment measures for a long time due to physical conditions, psychological factors and other reasons, resulting in poor treatment effects. In summary, the treatment of sarcopenia faces many challenges, and multi-faceted efforts are needed to overcome these bottlenecks and improve the treatment effect. In short, sarcopenia is an age-related loss of muscle mass involving many attributable factors. Stem cell therapy may be a new therapeutic intervention to alleviate sarcopenia because it has the ability to regenerate and produce anti-inflammatory cytokines, which can change the microenvironment to an environment that promotes neural regeneration and regeneration. However, stem cell transplantation is limited by several factors, including ethics, rejection and production restrictions.

[0004] Exosomes (EXO) are rich in proteins, nucleic acids (such as mRNA, miRNA, etc.), lipids and other bioactive molecules. These components constitute a complex and sophisticated information network that can accurately transmit signals between cells, regulate and optimize the functional state of receptor cells, and achieve efficient communication and collaboration between cells. Studies have shown that EXO is closely related to sarcopenia and may be a beneficial means of weakening / preventing and treating it. Exosomes are rich in motility factors / cytokines, which not only participate in the interaction between cells and tissues of the body, but also mediate many pathological and physiological processes including skeletal muscle cell proliferation and differentiation. In addition, exercise can effectively improve sarcopenia by promoting the release of exosomes and regulating the expression of miRNAs and / or proteins carried by exosomes.

[0005] Therefore, using stem cell exosomes may be better than using stem cells directly. More importantly, many studies on sarcopenia so far have investigated the role of exercise, nutritional supplements, and protein supplements in alleviating sarcopenia, but none have investigated the effects of using stem cell exosome compositions. Summary of the invention

[0006] In view of this, the present invention aims to overcome the defects in the prior art and proposes a stem cell exosome composition and application thereof for treating sarcopenia.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0008] A stem cell exosome composition for treating sarcopenia, comprising components with the following concentrations:

[0009] Exosomes 1×10 8 -1×10 13 Particles / mg protein, nicotinamide 100-3000mg / ml, wolfberry polysaccharide 50-900mg / ml, vitamin B6 10-600mg / ml, calcium gluconate 0.1-2g / ml, i-inositol 15-50mg / mL, pyridoxine hydrochloride 0.5-5mg / mL, vitamin B12 0.005-0.01mg / mL, IGF-1 20-100ug / ml, VEGF 1.0-3.0mg / ml, IL-15 10-100μg / mL, IL-2 500-2000U / ml, IL-21 5-50μg / mL, IL-1110-100ug / ml, α-galactosylceramide 1-20μg / mL. Further, the particle size of the exosomes is 30-150nm.

[0010] Furthermore, the method for preparing exosomes comprises the following steps:

[0011] (1) The supernatant of P5 MSC cells was centrifuged at 300g and 4°C for 10 min to remove dead cells and larger cell debris;

[0012] (2) The supernatant was centrifuged at 2000 g and 4 °C for 20 min to further remove impurities such as cell debris;

[0013] (3) The supernatant was centrifuged at 10,000 g and 4°C for 30 min to further remove smaller cell debris and impurities;

[0014] (4) filtering the obtained supernatant with a 0.22 μm PES filter membrane to fully remove bacteria, viruses, residual microcellular fragments, etc.;

[0015] (5) centrifuging the obtained filtrate at 100,000 g and 4° C. for 90 minutes;

[0016] (6) Aspirate and discard the supernatant, resuspend with 20 ml of sterile PBS, make up to volume, and centrifuge at 100,000 g at 4 °C for 90 min;

[0017] (7) Discard the supernatant, resuspend with 2.5 mL of sterile PBS, and store the extracted exosomes at -80°C.

[0018] Furthermore, the method for preparing P5 generation MSC cells comprises the following steps:

[0019] (1) Placenta-derived mesenchymal stem cells were cultured at 4.5×10 3 viable cells / cm 2Inoculation was carried out at an inoculation density of

[0020] (2) When the cell confluence reaches 80%, the culture medium is discarded, serum-free basal medium is added, and the cells are placed in a 37°C, 5% CO2 incubator for further culturing for 72 h;

[0021] (3) After culturing the cells for 72 hours, aspirate the supernatant culture medium and freeze it at -80°C.

[0022] The present invention also provides an application of a stem cell exosome composition, and application of the composition in the preparation of a drug for treating sarcopenia.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The stem cell exosome composition for treating sarcopenia of the present invention contains exosomes, nicotinamide, Lycium barbarum polysaccharide, biotin B6, calcium gluconate, i-inositol, pyridoxine hydrochloride, vitamin B12, IGF-1, VEGF, IL-15, IL-2, IL-21, IL-11 and α-galactosylceramide. With the breakthrough of sarcopenia treatment, it is used to prevent / treat sarcopenia, physical weakness, loss of lean muscle mass, loss of skeletal muscle strength / loss of skeletal muscle function, and improve skeletal muscle mass, lean muscle mass, skeletal muscle strength / skeletal muscle function. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The electron micrographs of the exosomes described in Example 3 of the present invention are shown in Figure A under normoxic culture conditions with serum, Figure B under normoxic culture conditions without serum, and Figure C under hypoxic culture conditions without serum;

[0026] Figure 2 This is a transmission electron microscopy image of the exosome suspension described in Example 4 of the present invention;

[0027] Figure 3 This is a graph showing the size analysis of exosome nanoparticles described in Example 4 of the present invention;

[0028] Figure 4 The surface marker expression flow cytometry graphs described in Example 4 of the present invention are shown in Figure A, where Figure A shows the total expression of CD63, Figure B shows the CD63 fluorescence positivity rate, Figure C shows the addition of CD9-PE, Figure D shows the CD9 fluorescence positivity rate, Figure E shows the addition of CD81-PE, and Figure F shows the CD81 positivity rate;

[0029] Figure 5 This is the quantitative analysis of exosome proteins described in Example 4 of the present invention. DETAILED DESCRIPTION

[0030] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0031] The present invention will be described in detail below with reference to the embodiments.

[0032] Example 1 Preparation of mesenchymal stem cell culture supernatant

[0033] 1. Placenta-derived mesenchymal stem cells were cultured at 4.5×10 3 viable cells / cm 2 The cells were seeded into T225 culture flasks at a seeding density of

[0034] 2. When the cell confluence reaches 80%, discard the BP complete medium, add 50 ml of serum-free basal medium (containing recombinant factors but not human platelet lysate) to the culture bottle, and place it in a 37°C, 5% CO2 incubator for another 72 h.

[0035] 3. After culturing the cells for 72 hours, take the culture flask out of the incubator, use a pipette to aspirate the supernatant culture medium into a 250 mL centrifuge tube, and freeze it at -80°C for three months.

[0036] Example 2 Preparation of exosomes

[0037] 1. Aspirate the supernatant of P5 MSC cells into several 50mL centrifuge tubes, place them in a 4℃ precooled centrifuge, centrifuge at 300g, 4℃ for 10 minutes to remove dead cells and larger cell debris, and then transfer the supernatant to several new 50ml centrifuge tubes.

[0038] 2. Place the centrifuge tube in a 4°C precooled centrifuge and centrifuge at 2000g for 20 minutes at 4°C to further remove cell debris and other impurities;

[0039] 3. Take the supernatant into a high-speed centrifuge tube; place the centrifuge tube in a high-speed centrifuge and centrifuge at 10,000 g at 4°C for 30 minutes to further remove smaller cell fragments and impurities;

[0040] 4. Filter the supernatant with a 0.22 μm PES filter membrane (millipore) to fully remove bacteria, viruses, residual tiny cell fragments, etc.;

[0041] 5. Place the supernatant back into several 39.2 mL ultracentrifuge tubes (Beckman Coulter), balance them, and place them in an ultracentrifuge. Centrifuge the supernatant at 100,000 g and 4 °C for 90 minutes.

[0042] 6. After centrifugation, discard the supernatant and resuspend with 20 ml sterile PBS. Repeatedly blow the bottom of the ultracentrifuge tube to resuspend the exosome precipitate at the bottom. After the volume is adjusted to 39 ml, place it in an ultracentrifuge and centrifuge it again at 100,000 g, 4 ° C, and 90 minutes to further purify the exosomes.

[0043] 7. After centrifugation, discard the supernatant, resuspend with 2.5 mL sterile PBS, repeatedly blow the bottom of the centrifuge tube, and then divide the extracted exosome suspension into 2 mL cryotubes at a volume of 500 ul / tube and store at -80°C.

[0044] Example 3 Purity Assessment of Exosomes

[0045] A batch of working bank seed cells was selected, and in the Wharton's jelly mesenchymal stem cell culture of the P2-P5 working bank, they were cultured to the P5 generation using a serum culture system and a serum-free normoxic and hypoxic culture system, respectively, 72 hours before the cell supernatant was harvested.

[0046] The cell incubation conditions in the serum-free culture system were normal oxygen partial pressure culture: 21% O2 concentration and low oxygen partial pressure culture, 2% O2 concentration. Then the cell supernatant was obtained and the exosomes were extracted. The process development was evaluated from three aspects: transmission electron microscopy, nano-force analysis, and total protein content.

[0047] like Figure 1 As shown in the figure, under 200nm electron microscopy, the exosomes obtained from the serum culture system and the serum-free culture system are both complete in morphology and have exosome membrane structure, but the number of exosomes in the serum-free culture system is significantly more and denser than that in the serum culture system. There is no significant difference in the microscopic observation of exosomes in the serum-free culture system under normal oxygen partial pressure and low oxygen partial pressure.

[0048] As shown in Table 1, the average particle size of exosomes under normoxic culture conditions with serum was 127 ± 0.9 nm, and the concentration of exosomes after purification was 1.24 × 10 9 / mL; under serum-free normoxic culture conditions, the average particle size of exosomes was 116±0.4nm, and the concentration of exosomes after purification was 4.21×10 10 / mL; under serum-free hypoxic culture conditions, the average particle size of exosomes was 125±0.5nm, and the concentration of exosomes after purification was 3.07×10 10 / mL.

[0049] Table 1 Exosome particle size results

[0050] Culture conditions Average particle size Exosome concentration Under normoxic conditions 127±0.9nm <![CDATA[1.24×10 9 / mL]]> Serum-free normoxic conditions 116±0.4nm <![CDATA[4.21×10 10 / mL]]> Serum-free hypoxic conditions 125±0.5nm <![CDATA[3.07×10 10 / mL]]>

[0051] The final concentration of exosome protein extracted by serum-containing normoxic cell culture process was 0.036 mg / mL; the final concentration of exosome protein extracted by serum-free normoxic cell culture process was 0.068 mg / mL; the final concentration of exosome protein extracted by serum-free hypoxic cell culture process was 0.062 mg / mL. The exosome protein content in serum-free culture process was significantly higher than that in serum-containing culture process, while there was no significant difference in the exosome protein content obtained by serum-free culture process under normoxic and hypoxic conditions.

[0052] Based on the above results, the exosome extraction process was carried out using a serum-free normoxic partial pressure culture system.

[0053] Example 4 Exosome Quality Detection

[0054] 1. Electron microscope detection

[0055] After the extracted exosome stock solution was diluted 100×, the morphology of the exosomes was observed using a transmission electron microscope. At 200nm and 500nm scales, it was found that the exosomes had a typical double-layer membrane structure and a "tray"-like morphology. The surface membrane structure of the exosomes was intact, and there were few background impurities (such as Figure 2 shown).

[0056] 2. Particle size detection

[0057] After the extracted exosome stock solution was diluted 100×, the particle size / concentration of the exosomes was detected using a nanoparticle size analyzer. The results showed that the average particle size of the exosomes was between 30-200nm, and there was a single particle size peak at 110nm. The average particle size of the exosomes was 116nm, and the content of exosomes with a particle size higher than 200nm was very small. The exosome concentration was 4.18×10 10 particles / mL (such as Figure 3 shown).

[0058] 3. Surface marker detection

[0059] Exosomes were labeled using an exosome-human CD63 separation / detection kit (Thermofisher, 10606D). After the exosomes were combined with CD63 magnetic beads, they were incubated and rinsed. Exosomes without fluorescent antibody labeling were used as negative controls to set gates. Exosomes were labeled with CD9-PE and CD81-PE fluorescent antibodies for flow cytometry detection of surface markers. CD63+CD9+CD81+≥80% (e.g. Figure 4 shown).

[0060] 4. Western Blot Protein Detection

[0061] Western blot was used to quantitatively analyze the protein in the extracted exosome suspension, and calnexin was selected as the negative control protein. The intracellular proteins Alix and Flot1, and the surface proteins CD63, CD9, and CD81 of the exosomes were detected. The results showed that the above proteins were significantly expressed in the exosomes (such as Figure 5 shown).

[0062] Example 5 Study on the effectiveness of stem cell exosome composition

[0063] Sixty 3-month-old male SAMP8 mice and 10 SAMR1 control mice of the same age were enrolled for the efficacy test, and were divided into blank control group (No. 1), model group (No. 2), low-dose exosome group (No. 3), high-dose exosome group (No. 4), low-dose exosome combination group (No. 5), high-dose exosome combination group (No. 6), and combination control group (No. 7), a total of 7 groups, 10 mice / group. Among them, the model group was injected with an equal amount of PBS, and the low-dose MSC-EXO group and the high-dose MSC-EXO group were injected with different concentrations of the test products, among which the low-dose exosome group: containing a concentration of 1×10 10 The high-dose exosome group contained exosomes with a concentration of 1×10 13 The low-dose exosome composition group contained: a concentration of 1×10 10 The exosomes contained 1000 particles / mg protein, 600 mg / ml nicotinamide, 500 mg / ml wolfberry polysaccharide, 100 mg / ml vitamin B6, 0.5 g / ml calcium gluconate, 30 mg / mL i-inositol, 2 mg / mL pyridoxine hydrochloride, 0.1 mg / mL vitamin B12, 50 ug / ml IGF-1, 1.8 mg / ml VEGF, 20 μg / mL IL-15, 1000 U / ml IL-2, 20 μg / mL IL-21, 50 ug / ml IL-11, and 5 μg / mL α-galactosylceramide; the high-dose exosome composition group contained: a concentration of 1×10 13Exosomes with 1000 particles / mg protein, nicotinamide 600mg / ml, wolfberry polysaccharide 500mg / ml, vitamin B6 100mg / ml, calcium gluconate 0.5g / ml, i-inositol 30mg / mL, pyridoxine hydrochloride 2mg / mL, vitamin B12 0.1mg / mL, IGF-1 50ug / ml, VEGF 1.8mg / ml, IL-15 20μg / mL, IL-2 1000U / ml, IL-21 20μg / mL, IL-11 50ug / ml, α-galactosylceramide Amine; the control group of the composition contained: nicotinamide 600mg / ml, wolfberry polysaccharide 500mg / ml, vitamin B6 100mg / ml, calcium gluconate 0.5g / ml, i-inositol 30mg / mL, pyridoxine hydrochloride 2mg / mL, vitamin B12 0.1mg / mL, IGF-1 50ug / ml, VEGF 1.8mg / ml, IL-15 20μg / mL, IL-2 1000U / ml, IL-21 20μg / mL, IL-11 50ug / ml, α-galactosylceramide 5μg / mL;

[0064] The injection route was tail vein injection, and the dosage volume was 100ul / mouse; a total of 16 injections were given once a week. The weight and movement of the mice were tested to see if there was a slowing effect.

[0065] 1. Changes in mouse weight

[0066] The body weight changes of all mice after 8 and 16 weeks of drug administration are shown in Table 2.

[0067] Table 2 Changes in mouse body weight

[0068] Group N 0W 8W 16W No. 1 10 35.94±23.82 49.83±3.21 69.2±2.04 No. 2 10 29.92±3.26 35.64±1.28 38.59±1.08 No.3 10 31.67±2.27 41.23±3.17 47.28±3.65 No. 4 10 30.70±2.42 44.96±4.10 49.98±3.42 No. 5 10 31.31±3.12 47.97±1.52 59.09±3.15 No. 6 10 30.48±1.94 49.16±2.42 68.64±2.01 No.7 10 30.91±1.85 41.05±3.16 44.62±4.27

[0069] After the administration, the average weight of the mice in the model group was significantly reduced compared with the mice in the blank control group, and there was a significant difference (P≤0.05); after 8 weeks of intervention with the exosome composition, the average weight of the mice in the low-dose and high-dose exosome composition groups was not significantly different from that of the rats in the model group (P≥0.05). After 16 weeks, there was a significant difference compared with the rats in the model group (P≤0.05), and the body weight of the high-dose exosome composition group had reached the level of the blank control group; there was no significant difference between the low-dose exosome group and the high-dose exosome group compared with the model group at 8 weeks after administration, and was significantly higher than the model group at 16 weeks after administration, but significantly lower than the blank control group; the composition control group was slightly higher than the model group, with no significant difference. The conclusion was that the weight of the mice in the high-dose group recovered best, and there was no difference from the blank control group.

[0070] 2. Mouse grip test

[0071] After 8 and 16 weeks of drug administration, all mice were tested for limb grip strength using a grip strength tester. The changes in grip strength are shown in Table 3.

[0072] Table 3 Changes in the gripping strength of mice

[0073]

[0074]

[0075] After the administration, the average gripping force of the blank control group mice was significantly reduced compared with the positive control group and the experimental mice, and there was a significant difference (P≤0.05); after 8 weeks of intervention in the exosome composition group, the average gripping force of the low-dose and high-dose exosome composition group mice was significantly different from that of the model group rats (P>0.05), and the high-dose exosome composition group basically recovered to the level of the blank control group 16 weeks after administration. There was no significant difference between the low-dose exosome group and the high-dose exosome group and the model group at 8 and 16 weeks after administration, and was slightly higher than the model group at 16 weeks after administration, but there was no significant difference, and significantly lower than the blank control group; there was no significant difference between the composition control group and the model group. Conclusion: The high-dose group mice recovered the best gripping force, which basically recovered to the level of the normal group.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A stem cell exosome composition for treating sarcopenia, characterized in that: The following concentrations of components are included: Exosomes 1×10 8 -1×10 13 particles / mg protein, nicotinamide 100-3000mg / ml, wolfberry polysaccharide 50-900mg / ml, vitamin B6 10-600mg / ml, calcium gluconate 0.1-2g / ml, i-inositol 15-50mg / mL, pyridoxine hydrochloride 0.5-5mg / mL, vitamin B12 0.005-0.01mg / mL, IGF-1 20-100ug / ml, VEGF 1.0-3.0mg / ml, IL-15 10-100μg / mL, IL-2 500-2000U / ml, IL-21 5-50μg / mL, IL-1110-100ug / ml, α-galactosylceramide 1-20μg / mL.

2. The stem cell exosome composition for treating sarcopenia according to claim 1, characterized in that: The particle size of the exosomes is 30-150nm.

3. The stem cell exosome composition for treating sarcopenia according to claim 1, characterized in that: The method for preparing exosomes comprises the following steps: (1) Centrifuge the P5 MSC cell supernatant at 300g and 4°C for 10 min; (2) Centrifuge the supernatant at 2000g and 4°C for 20 min; (3) Centrifuge the supernatant at 10,000 g and 4 °C for 30 min; (4) filtering the obtained supernatant using a 0.22 μm PES filter membrane; (5) centrifuging the obtained filtrate at 100,000 g and 4° C. for 90 minutes; (6) Aspirate the supernatant, resuspend with 20 ml of sterile PBS, make up to volume, and centrifuge at 100,000 g at 4 °C for 90 min; (7) Discard the supernatant, resuspend with 2.5 mL of sterile PBS, and store the extracted exosomes at -80°C.

4. The stem cell exosome composition for treating sarcopenia according to claim 3, characterized in that: The method for preparing P5 generation MSC cells comprises the following steps: (1) Placenta-derived mesenchymal stem cells were cultured at 4.5×10 3 viable cells / cm 2 Inoculate at an inoculation density of (2) When the cell confluence reaches 80%, the culture medium is discarded, serum-free basal medium is added, and the cells are placed in a 37°C, 5% CO2 incubator for further culturing for 72 h; (3) After culturing the cells for 72 hours, aspirate the supernatant culture medium and freeze it at -80°C.

5. The use of the stem cell exosome composition according to any one of claims 1 to 4, characterized in that: The application of the composition in preparing a medicine for treating sarcopenia.

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