Application of enhanced mesenchymal stem cells and exosomes in chronic nephritis and renal failure

Abstract

The application provides an application of enhanced mesenchymal stem cells and exosomes in chronic nephritis renal failure, and belongs to the technical field of biological medicine manufacturing.The application significantly enhances the treatment potential of exosomes secreted by mesenchymal stem cells through the composite pretreatment of rhodioside and astragalus polysaccharide, provides a new idea for the intervention of chronic kidney disease, and covers various pathologies of chronic kidney disease.The application also combines a 3D microenvironment simulated by a fibrin gel constructed extracellular matrix in the body, is more suitable for the natural growth state of stem cells, promotes the directional differentiation and functional regulation of stem cells, and thus improves the biological activity and targeting of the exosomes.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine manufacturing, and in particular to application of enhanced mesenchymal stem cells and exosomes in chronic nephritis renal failure. BACKGROUND

[0002] As a kind of natural extracellular vesicle, exosomes show significant potential in the treatment of kidney diseases due to their low immunogenicity, high biocompatibility and ability to deliver bioactive molecules across the membrane. For example, the stem cell exosomes disclosed in Chinese patent CN115040544A can reduce kidney injury and slow down the progression of renal fibrosis, and can be used for treating chronic kidney disease.

[0003] However, current exosome-based kidney disease treatment still faces the following technical bottlenecks:

[0004] Existing researches mostly rely on exosomes secreted by untreated mesenchymal stem cells (MSCs), and the functions of the exosomes mainly depend on the inherent secretion characteristics of stem cells. Although such exosomes can delay renal fibrosis through mechanisms such as anti-inflammatory and promotion of angiogenesis, they lack precise regulation ability for specific pathological pathways and are difficult to cover the multi-factor pathological characteristics of chronic kidney disease (CKD) (such as renal tubular injury, immune inflammatory cascade reaction and excessive deposition of extracellular matrix, etc.).

[0005] Some studies attempt to pretreat MSCs with growth factors (such as VEGF, HGF) or chemical drugs (such as rapamycin) to enhance the function of exosomes, but such methods have the following defects: single-factor pretreatment only activates limited signal pathways (such as PI3K / Akt or mTOR), and it is difficult to synergistically regulate multi-dimensional pathological mechanisms such as anti-fibrosis, anti-inflammatory and renal tubular repair. High-concentration chemical drug pretreatment may induce stem cell toxicity or abnormal exosome components, affecting the safety of clinical translation. In addition, traditional methods generally use exosomes obtained in a 2D culture system, and the biological activity and targeting of the exosomes are limited by the difference between the in vitro culture environment and the in vivo microenvironment, and the targeting ability to the injury site is low.

[0006] Therefore, current exosome treatment technology still has significant technical gaps in terms of precise regulation of function, optimization of biological activity and safety of clinical translation. SUMMARY

[0007] The purpose of the present application is to provide an application of enhanced mesenchymal stem cells and exosomes in chronic nephritis renal failure, which has excellent effects.

[0008] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0009] The present invention provides a use of optimized exosomes in preparing a drug for treating chronic kidney disease, wherein the optimized exosomes are obtained by a preparation method comprising the following steps:

[0010] The mesenchymal stem cells are inoculated into a culture medium containing salidroside and astragalus polysaccharide for culture, thereby inducing the release of exosomes and obtaining cultured mesenchymal stem cells and a culture supernatant;

[0011] Extracting and purifying exosomes from the culture supernatant to obtain the optimized exosomes;

[0012] The weight ratio of salidroside to astragalus polysaccharide is 1:1.

[0013] The present invention also provides a use of mesenchymal stem cells secreting optimized exosomes in the preparation of a drug for treating chronic kidney disease, wherein the optimized exosomes are obtained by a preparation method comprising the following steps:

[0014] The mesenchymal stem cells are inoculated into a culture medium containing salidroside and astragalus polysaccharide for culture, thereby inducing the release of exosomes and obtaining cultured mesenchymal stem cells and a culture supernatant;

[0015] Extracting and purifying exosomes from the culture supernatant to obtain the optimized exosomes;

[0016] The weight ratio of salidroside to astragalus polysaccharide is 1:1;

[0017] The cultured mesenchymal stem cells are further used to culture and secrete optimized exosomes, and the optimized exosomes are used to prepare drugs for treating chronic kidney disease.

[0018] Preferably, the concentration of salidroside in the culture medium is 2-3 μM.

[0019] Preferably, the concentration of astragalus polysaccharide in the culture medium is 2-3 μM.

[0020] Preferably, the culture medium contains DMEM / F12 culture medium and a fibrin gel system, wherein the fibrin gel system is obtained by cross-linking a fibrin solution and thrombin, and the concentration of the fibrin solution is 3-5 mg / ml;

[0021] The concentration of the thrombin is 1-3 U / ml;

[0022] The volume ratio of the fibrin solution to the thrombin is 8-12:1.

[0023] Preferably, the culture time is 5 to 10 days;

[0024] The method used for extracting exosomes includes ultracentrifugation, density gradient centrifugation or ultrafiltration.

[0025] Preferably, the chronic kidney disease is chronic nephritis or chronic renal failure.

[0026] Beneficial effects of the present invention:

[0027] This invention significantly enhances the therapeutic potential of exosomes secreted by mesenchymal stem cells through combined pretreatment with salidroside and astragalus polysaccharide. The two active ingredients achieve synergistic therapeutic effects through multi-pathway regulation, including anti-fibrosis, anti-inflammation, and renal tubular repair. This overcomes the limitations of single-ingredient pretreatment and provides a new approach to the intervention of chronic kidney disease, covering various pathologies of chronic kidney disease. The invention also incorporates a 3D microenvironment constructed with fibrin gel to simulate the extracellular matrix in vivo, more closely resembling the natural growth state of stem cells, promoting their directional differentiation and functional regulation, thereby enhancing the biological activity and targeting of exosomes.

[0028] The method provided by this invention utilizes amniotic fluid-derived mesenchymal stem cells, which have low immunogenicity and high proliferation capacity. Combined with low drug toxicity concentrations and a standardized exosome extraction process, this method lays a safe and reproducible foundation for clinical translation. By pre-processing and optimizing the therapeutic function of exosomes, this technology can be applied not only in isolation for kidney diseases but also as a delivery vehicle and subsequently combined with other therapeutically active drugs, providing a wider range of treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Figure ① is a comparison of inflammatory factor detection results;

[0030] Figure 2 Figure ② is a comparison of inflammatory factor detection results. DETAILED DESCRIPTION

[0031] The present invention provides a use of optimized exosomes in the preparation of a drug for treating chronic kidney disease. The optimized exosomes are obtained by a preparation method comprising the following steps: inoculating mesenchymal stem cells into a culture medium containing salidroside and astragalus polysaccharide for culturing, inducing exosome release, and obtaining cultured mesenchymal stem cells and a culture supernatant; extracting and purifying exosomes from the culture supernatant to obtain the optimized exosomes; the weight ratio of salidroside to astragalus polysaccharide is 1:1.

[0032] Preferably, the concentration of rhodioloside in the culture medium is 2-3 μM. Preferably, the concentration of astragalus polysaccharide in the culture medium is 2-3 μM. Preferably, the culture medium contains DMEM / F12 medium and a fibrin gel system, which is obtained by cross-linking a fibrin solution and thrombin, the concentration of the fibrin solution is 3-5 mg / ml; the concentration of the thrombin is 1-3 U / ml; the volume ratio of the fibrin solution to the thrombin is 8-12:1. Preferably, the culture time is 5-10 days; the method for extracting the exosomes includes ultracentrifugation, density gradient centrifugation or ultrafiltration. Preferably, the chronic kidney disease is chronic nephritis or chronic renal failure.

[0033] The application also provides a use of the secretion-optimized mesenchymal stem cell-derived exosomes in the preparation of a medicament for treating chronic kidney disease, wherein the optimized exosomes are obtained by a preparation method comprising the following steps: inoculating mesenchymal stem cells into a culture medium containing rhodioloside and astragalus polysaccharide for culture, inducing exosome release, obtaining the cultured mesenchymal stem cells and culture supernatant; extracting and purifying the culture supernatant to obtain the optimized exosomes; the weight ratio of rhodioloside to astragalus polysaccharide is 1:1; the cultured mesenchymal stem cells are used for further culture of secretion-optimized exosomes, and the optimized exosomes are used for the preparation of a medicament for treating chronic kidney disease. Preferably, the chronic kidney disease is chronic nephritis or chronic renal failure.

[0034] In the application, the dosage form of the medicament can include injection, oral preparation or topical external preparation, and specifically covers one or more combinations of solution, suspension, lyophilized powder or sustained-release preparation. The medicament can also contain pharmaceutically acceptable excipients, such as stabilizers, preservatives, osmotic pressure regulators or thickening agents.

[0035] In the application, the pharmaceutical composition can further contain other active ingredients used in combination with the exosomes, mesenchymal stem cells or culture supernatant, such as anti-inflammatory compounds, antioxidants or immunomodulators, to enhance the therapeutic effect on kidney disease through synergistic effect.

[0036] In the application, the excipients can include but are not limited to: freeze-drying protectants (such as sugars or polyols) for maintaining the activity of exosomes, carrier materials (such as cyclodextrin derivatives or phospholipids) for improving bioavailability, and matrix materials (such as natural or synthetic high molecular polymers) for adjusting the release rate.

[0037] In the application, the medicament can be prepared as a ready-to-use preparation or a long-term storage preparation, including a pre-filled syringe, a single-dose sealed ampoule or a multi-dose divided bottle, and is suitable for cold storage or room temperature storage conditions.

[0038] In the present invention, the application form may also include a combination therapy with other drugs for treating kidney disease, such as co-formulating a compound preparation with angiotensin-converting enzyme inhibitors, diuretics or immunosuppressants, or achieving staged treatment through sequential administration.

[0039] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] Pre-culture of mesenchymal stem cells:

[0042] Extraction and expansion of amniotic fluid-derived mesenchymal stem cells

[0043] 1. Take an amniotic fluid sample (50 mL) from 16-20 weeks of gestation, centrifuge at 1500 rpm / min for 10 min, discard the supernatant, and wash three times with PBS.

[0044] 2. Resuspend the cells in DMEM / F12 medium containing 15% fetal bovine serum (FBS) and 1% penicillin-streptomycin, seed them into low-adhesion culture dishes, and culture them at 37°C and 5% CO2 for 48 h to remove non-adherent cells.

[0045] 3. When the cell confluence reaches 80%, digest with 0.25% trypsin-EDTA and passage to passage 4 (P4) at a ratio of 1:3 to ensure the stem cell phenotype (CD90⁺ / CD105⁺ / CD34⁻).

[0046] (2) Construction of 3D pre-culture system:

[0047] Preparation of fibrinogen solution and thrombin: Use sterile DMEM / F12 culture medium preheated to 37°C, dissolve fibrinogen powder to a final concentration of 4 mg / ml, and add salidroside (2.5 μM) and astragalus polysaccharide (2.5 μM) at the same time. This concentration and addition amount can enable the drugs to have an induction effect without affecting the cells. In theory, fine-tuning can also achieve the induction effect, which also falls within the scope of protection of the present invention.

[0048] The thrombin stock solution (500 U / ml) was diluted with 2+ Dilute with PBS to a working concentration of 2 U / ml.

[0049] Construction of cell-gel composite system

[0050] P4 amniotic fluid mesenchymal stem cells (density 1×10 6cells / ml) and the fibrinogen solution to ensure uniform distribution of cells. The mixture was inoculated into a low-adsorption 24-well plate. After adding 100 μl of the mixture to each well, 10 μl of thrombin solution (fibrin concentration 4 mg / ml, thrombin concentration 2 U / ml, volume ratio 10:1 to achieve cross-linking and solidification) was immediately added dropwise. The gel was allowed to stand at 37°C for 10-15 minutes to complete gel solidification and cultured under conventional conditions for 7 days.

[0051] (3) Exosome extraction and purification:

[0052] 1. Supernatant Collection: Collect the culture supernatant and centrifuge at 300 × g for 10 minutes to remove floating cells. Then, centrifuge at 2000 × g for 20 minutes to remove dead cell debris and any gel fragments. 2. Ultracentrifugation: Filter the supernatant through a 0.22 μm filter and ultracentrifuge at 100,000 × g for 70 minutes at 4°C. Resuspend the pellet in PBS and repeat the centrifugation once to improve purity. 3. Place an appropriate amount of the exosome resuspension on a copper grid and allow it to stand at room temperature for 5 minutes to allow the exosomes to adsorb. Gently remove excess liquid with filter paper and add 2% phosphotungstic acid solution for negative staining. Stain at room temperature for 5 minutes. Remove excess stain again with filter paper. Allow the grid to air dry before observing exosome morphology under a transmission electron microscope. Results showed that the exosomes exhibited typical exosome characteristics of flat, disc-shaped, and biconcave surfaces. Dynamic light scattering (DLS) was used to determine the exosome size, which ranged from 70 to 130 nm.

[0053] Comparative Example 1

[0054] Compared with Example 1, salidroside (2.5 μM) and astragalus polysaccharide (2.5 μM) in the fibrinogen solution were replaced with salidroside (5 μM).

[0055] Comparative Example 2

[0056] Compared with Example 1, salidroside (2.5 μM) and astragalus polysaccharide (2.5 μM) in the fibrinogen solution were replaced with astragalus polysaccharide (5 μM).

[0057] Experimental example

[0058] 1. Materials and Methods

[0059] 1. Experimental Animal Grouping and Model Construction

[0060] 1.1 Experimental Animal Grouping

[0061] Healthy male Sprague-Dawley rats (weighing 180–220 g), SPF grade, were randomly divided into the following 6 groups (n = 10 per group):

[0062] Normal control group (NC group): no intervention, fed with conventional feed.

[0063] Chronic renal failure model group (CRF group): Chronic renal failure was induced by adenine combined with high-phosphorus feed.

[0064] Chronic nephritis model group (C-BSA group): Chronic nephritis was induced by cationized bovine serum albumin (C-BSA).

[0065] Treatment groups: The CRF and C-BSA model groups were divided into three groups, and exosomes with different pretreatments were injected into the tail vein (groups A, B, and C).

[0066] 1.2 Model Building

[0067] CRF model establishment: Adenine powder was dissolved in 0.5% sodium carboxymethylcellulose solution. Animals were gavaged daily with adenine solution (150 mg / kg) for four weeks, while simultaneously being fed a high-phosphorus diet (1.2%). Successful model establishment was considered achieved if serum creatinine (Scr) >150 μmol / L and blood urea nitrogen (BUN) >20 mmol / L at week four.

[0068] C-BSA model establishment: Cationized bovine serum albumin (isoelectric point pI = 8.5) was used using the ethylenediamine method. The initial injection dose was 10 mg / kg (1 mL of a 2 mg / mL solution for a 200 g rat), and the dose was subsequently increased to 12.5 mg / kg (1.25 mL / injection) for 4 weeks. A 24-hour urine protein count >150 mg at week 4 was considered a successful model.

[0069] 1.3 Dosage Regimen

[0070] The exosome preparation method is shown in Example 1, Comparative Example 1 and Comparative Example 2, which are correspondingly recorded as Group A, Group B and Group C as follows in this experiment:

[0071] Group A: Mesenchymal stem cells were pre-cultured in 3D fibrin gel with a composite reagent (salidroside + astragalus polysaccharide), and exosomes were extracted by ultracentrifugation.

[0072] Group B: Mesenchymal stem cells were pre-cultured in 3D fibrin gel mixed with salidroside, and exosomes were extracted.

[0073] Group C: Mesenchymal stem cells were pre-cultured in 3D culture medium with astragalus polysaccharide mixed with fibrin gel, and exosomes were extracted.

[0074] Dosage regimen: After successful model establishment, each group received an injection of exosomes (200 μg / time, dissolved in 0.5 mL PBS) via the tail vein once a week for 4 weeks.

[0075] 2. Detection indicators and methods

[0076] 2.1 Serum biochemical index detection

[0077] Sample collection: After treatment, the subjects were fasted for 12 hours, and blood was collected from the heart under anesthesia. Serum was separated by centrifugation at 3000 rpm for 10 minutes.

[0078] Test items:

[0079] Scr and BUN were detected by picric acid method (kit) and measured by automatic biochemical analyzer (model: Cobas c311).

[0080] 24-hour urine protein quantification: Urine was collected in a metabolic cage and the absorbance was detected using the Coomassie Brilliant Blue method (kit). The absorbance was measured using a microplate reader (wavelength 595 nm) and the concentration was calculated using a standard curve.

[0081] 2.2 Renal histopathological analysis

[0082] Sample processing: The left kidney was removed, fixed with 10% neutral formaldehyde for 24 hours, dehydrated with graded ethanol, embedded in paraffin, and 4 μm sections were prepared.

[0083] Hematoxylin and eosin (HE) staining: Sections were dewaxed and stained with hematoxylin for 5 minutes, with the water turning blue. Sections were then incubated with 1% hydrochloric acid-ethanol for 10 seconds and stained with eosin for 2 minutes. Sections were mounted with neutral gum and examined under a light microscope (200×) to reveal glomerular structure, inflammatory cell infiltration, and tubular damage. Glomerulosclerosis was defined as capillary collapse and hyalinization. Images were analyzed directly using Motic Med 6.0.

[0084] Masson trichrome staining: Sections were sequentially immersed in Ponceau-Fuchsin solution (10 minutes), phosphomolybdic acid solution (5 minutes), and aniline blue solution (5 minutes). After dehydration and mounting, the percentage of renal interstitial collagen deposition area was analyzed using ImageJ software.

[0085] 2.3 Immunohistochemistry (IHC)

[0086] Sections were immersed in citrate buffer (pH 6.0) and autoclaved for 20 minutes. Incubated in 3% H₂O₂ for 10 minutes at room temperature in the dark, and rinsed three times with PBS (5 minutes each). Blocked with 5% BSA for 1 hour at room temperature.

[0087] Primary antibody incubation: add α-SMA (1:100), E-cadherin (1:100), and TGF-β1 (1:200) primary antibodies and incubate at 4°C overnight.

[0088] Secondary antibody incubation: Incubate with HRP-conjugated secondary antibody (1:200) at room temperature for 30 minutes, and then rinse three times with PBS.

[0089] The slides were stained with DAB colorimetric reagent (time was controlled under a microscope), counterstained with hematoxylin, and sealed with neutral gum.

[0090] Image-Pro Plus 6.0 software was used to calculate the average optical density of the positive area (IOD / Area).

[0091] 2.4 Western Blot Detection of Fibrosis-Related Proteins

[0092] Renal cortical tissue was obtained and homogenized on ice in RIPA lysis buffer (containing protease inhibitors). The cells were centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected. The cells were subjected to 10% SDS-PAGE electrophoresis (80 V for 30 minutes and 120 V for 60 minutes) and wet-transferred to a PVDF membrane (300 mA for 90 minutes).

[0093] Antibody incubation: Primary antibodies: TGF-β1 (1:1000), α-SMA (1:800), COL1A1 (1:500), incubated overnight at 4°C. Secondary antibody: HRP-conjugated anti-rabbit IgG (1:5000), incubated for 1 hour at room temperature. Development: ECL chemiluminescence reagent, grayscale analysis with ImageJ.

[0094] 2.5 Detection of inflammatory factors

[0095] ELISA method: Serum IL-6 and TNF-α levels were detected (kit), absorbance was read using a microplate reader (450 nm wavelength), and standard curve was used for quantification.

[0096] 2.6 Data processing: Data are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) and Tukey's post hoc test were used for comparison among groups. *P < 0.05 compared with the model group, **P < 0.01 compared with the model group, and #P < 0.05 compared with the NC group.

[0097] Since the models focus on different indicators, the “-” in the following results means that the data was not detected, and the results are displayed together.

[0098] 2. Experimental results Table 1 Results of serum biochemical indicators

[0099]

[0100] Note: CRF model detects Scr and BUN, and C-BSA model detects urine protein (“-” means not tested);

[0101] #P<0.01: NC group, *P<0.05: model group.

[0102] Table 2 Results of renal pathology analysis

[0103] Group Glomerular sclerosis rate (CRF model, %) Interstitial fibrosis area (CRF model, %) Inflammatory cell infiltration score (C-BSA model, 0-4 points) NC group 0 1.2 ± 0.1 0.5 ± 0.1 CRF model group 38.5 ± 1.2## 34.6 ± 2.5## - Exosome A group (CRF) 12.1 ± 1.1* 11.8 ± 1.2* - Exosome B group (CRF) 18.3 ± 1.1* 17.5 ± 2.3* - Exosome C group (CRF) 24.6 ± 2.3* 19.8 ± 1.1* - C-BSA model group - - 3.2 ± 0.2## Exosome A group (C-BSA) - - 1.1 ± 0.1* Exosome B group (C-BSA) - - 1.8 ± 0.1* Exosome C group (C-BSA) - - 2.5 ± 0.1*

[0104] ##P<0.01: NC group, *P<0.05: model group.

[0105] Table 3 Immunohistochemistry and Western Blot results

[0106] Group α-SMA (CRF model, IOD / Area) TGF-β1 (CRF model, gray value) E-cadherin (C-BSA model, IOD / Area) NC group 0.12 ± 0.03 0.38 ± 0.05 0.85 ± 0.10 CRF model group 0.65 ± 0.10## 1.42 ± 0.16## - Exosome A group (CRF) 0.25 ± 0.05* 0.67 ± 0.09* - Exosome B group (CRF) 0.41 ± 0.07* 0.92 ± 0.12* - Exosome C group (CRF) 0.56 ± 0.09* 1.18 ± 0.15* - C-BSA model group - - 0.48 ± 0.05## Exosome A group (C-BSA) - - 0.75 ± 0.06* Exosome B group (C-BSA) - - 0.63 ± 0.07* Exosome C group (C-BSA) - - 0.55 ± 0.06*

[0107] Note: The CRF model detects fibrosis markers (α-SMA, TGF-β1), and the C-BSA model detects renal tubular epithelial integrity (E-cadherin);

[0108] ##P<0.01: NC group, *P<0.05: model group.

[0109] Table 4 Inflammatory factor detection results

[0110] Group IL-6 (pg / mL) TNF-α (pg / mL) NC group 13.45 ± 1.51 16.78 ± 1.66 C-BSA model group 85.62 ± 7.12## 92.36 ± 8.40## Exosome A group (C-BSA) 32.44 ± 3.45* 38.65 ± 4.12* Exosome B group (C-BSA) 58.34 ± 2.27* 69.42 ± 6.88* Exosome C group (C-BSA) 75.89 ± 7.12* 86.34 ± 8.45*

[0111] Note: The C-BSA model specifically detects inflammatory factors (not detected in the CRF model);

[0112] ##P<0.01 compared with the NC group, *P<0.05 compared with the C-BSA model group.

[0113] Comparison of IL-6 test results in each group Figure 1 As shown in the figure, the comparison of TNF-α detection results is shown in Figure 2 shown.

[0114] in conclusion:

[0115] This study demonstrated that mesenchymal stem cell exosomes pretreated with a combination of salidroside and astragalus polysaccharide (Group A) exhibited optimal therapeutic efficacy in both chronic renal failure (CRF) and chronic nephritis (C-BSA) models. In the CRF model, Group A exosomes significantly reduced serum creatinine and urea nitrogen levels and effectively inhibited the progression of glomerular sclerosis and interstitial fibrosis, demonstrating significantly superior effects compared to Groups B (salidroside alone) and C (astragalus polysaccharide alone), which were pretreated with either component alone. In the C-BSA-induced chronic nephritis model, Group A exosomes not only significantly reduced 24-hour urinary protein excretion but also significantly inhibited inflammatory cell infiltration and the release of inflammatory factors (IL-6 and TNF-α). Furthermore, they promoted the expression of the renal tubular epithelial marker E-cadherin, suggesting a positive effect on tubular structural repair.

[0116] The results of the two models jointly showed that the synergistic pretreatment of salidroside and astragalus polysaccharide can enhance the therapeutic potential of exosomes secreted by mesenchymal stem cells, especially in improving renal function, inhibiting fibrosis and inflammatory response, providing a new potential strategy for the treatment of chronic kidney disease.

[0117] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. Use of optimized exosomes in the preparation of a drug for treating chronic kidney disease, characterized in that: The optimized exosomes are obtained by a preparation method comprising the following steps: The amniotic fluid mesenchymal stem cells were inoculated into a culture medium containing salidroside and astragalus polysaccharide for culture, thereby inducing the release of exosomes and obtaining cultured mesenchymal stem cells and culture supernatant; Extracting and purifying exosomes from the culture supernatant to obtain the optimized exosomes; The weight ratio of salidroside to astragalus polysaccharide is 1:1; The concentration of salidroside in the culture medium is 2-3 μM; The concentration of astragalus polysaccharide in the culture medium is 2-3 μM; The culture medium contains DMEM / F12 culture medium and a fibrin gel system, wherein the fibrin gel system is obtained by cross-linking a fibrin solution and thrombin, and the concentration of the fibrin solution is 3-5 mg / ml; The concentration of the thrombin is 1-3 U / ml; The volume ratio of the fibrin solution to the thrombin is 8-12:1; The chronic kidney disease is chronic nephritis or chronic renal failure.

2. The use according to claim 1, characterized in that The culture time is 5 to 10 days; The method used for extracting exosomes includes ultracentrifugation, density gradient centrifugation or ultrafiltration.

3. Use of mesenchymal stem cells secreting optimized exosomes in the preparation of a drug for treating chronic kidney disease, characterized in that: The optimized exosomes are obtained by a preparation method comprising the following steps: The amniotic fluid mesenchymal stem cells were inoculated into a culture medium containing salidroside and astragalus polysaccharide for culture, thereby inducing the release of exosomes and obtaining cultured mesenchymal stem cells and culture supernatant; Extracting and purifying exosomes from the culture supernatant to obtain the optimized exosomes; The weight ratio of salidroside to astragalus polysaccharide is 1:1; The cultured mesenchymal stem cells are further used to culture and secrete optimized exosomes, and the optimized exosomes are used to prepare a drug for treating chronic kidney disease; The chronic kidney disease is chronic nephritis or chronic renal failure; The concentration of salidroside in the culture medium is 2.5 μM; The concentration of astragalus polysaccharide in the culture medium is 2.5 μM; The culture medium contains DMEM / F12 culture medium and a fibrin gel system, wherein the fibrin gel system is obtained by cross-linking a fibrin solution and thrombin, and the concentration of the fibrin solution is 4 mg / ml; The concentration of the thrombin is 2 U / ml; The volume ratio of the fibrin solution to the thrombin is 10:1.

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