CN115040544A - Application of stem cell exosome in preparation of medicine for treating chronic kidney disease - Google Patents

Abstract

The invention relates to an application of a stem cell exosome in preparing a medicament for treating chronic kidney disease. The stem cell exosome can reduce kidney injury and slow down the progression of renal fibrosis, and can be used for treating chronic kidney disease.

Description

Application of stem cell exosome in preparation of medicine for treating chronic kidney disease

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a stem cell exosome in preparing a medicine for treating chronic kidney diseases.

Background

The exosome is a small-particle-size extracellular vesicle with the diameter of 30-150 nm, has stable property, low immunogenicity and no teratogenicity, and can pass through a blood brain barrier. Exosomes mature and are secreted extracellularly through three stages: firstly, endocytic vesicles are formed through the invagination of a plasma membrane; secondly, the endosomal membrane buds inwards to generate a multivesicular body; finally, exosomes are released extracellularly from the fusion of multivesicular bodies with the plasma membrane. Almost all types of cells in an organism can release exosomes and reside in large amounts in body fluids. Exosomes are rich in various bioactive molecules, carry information through substances such as proteins, lipids, nucleic acids and the like, mediate cell communication, proliferation and apoptosis, participate in angiogenesis and immune regulation, and can play an important role in epigenetic regulation. Exosomes are considered to be important pathways for stem cells to function via paracrine action, providing a new strategy for "cell-free" therapies while avoiding the risks associated with stem cell transplantation.

Disclosure of Invention

The research of the invention discovers a new application of the stem cell exosome, which can reduce inflammatory factors and slow down the process of renal fibrosis. Based on the above, the invention provides an application of a stem cell exosome in preparing a medicament for treating chronic kidney disease.

In one embodiment, the chronic kidney disease comprises one or more of the following diseases: glomerulonephritis, tubulointerstitial diseases, renal vascular diseases, diabetic nephropathy and hereditary renal diseases.

In one embodiment, the chronic kidney disease is characterized by at least one of the following features: renal fibrosis, renal resident cell proliferation and leukocyte infiltration.

In one embodiment, the stem cell exosomes comprise one or more of the following: embryonic stem cell exosomes, mesenchymal stem cell exosomes and induced pluripotent stem cell exosomes.

In one embodiment, the medicament comprises an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient is the stem cell exosome.

In one embodiment, the dosage form of the drug is a tablet, a capsule, a granule, a sustained release preparation, a powder, a pill, or an injection.

In one embodiment, the pharmaceutical is a tablet, and the pharmaceutically acceptable excipient comprises one or more of a diluent, a binder, a wetting agent, a disintegrant, and a lubricant.

In one embodiment, the pharmaceutically acceptable excipient satisfies one or more of the following conditions:

the diluent comprises one or more of starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate and calcium carbonate;

the adhesive comprises one or more of starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone and polyethylene glycol;

the disintegrating agent comprises one or more of starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch and polyoxyethylene;

the lubricant comprises one or more of talcum powder, silicon dioxide, stearate, liquid paraffin and polyethylene glycol;

and, the wetting agent comprises one or more of water, ethanol and isopropanol.

In one embodiment, the medicament is an injection, and the pharmaceutically acceptable excipients include one or more of a solubilizer, a pH regulator and an osmotic pressure regulator.

In one embodiment, the pharmaceutically acceptable excipient satisfies one or more of the following conditions:

the solubilizer comprises one or more of ethanol, isopropanol, propylene glycol, polyethylene glycol, poloxamer, lecithin and hydroxypropyl-beta-cyclodextrin;

the pH regulator comprises one or more of citrate, phosphate, carbonate, acetate, hydrochloric acid and hydroxide;

and the osmotic pressure regulator comprises one or more of sodium chloride, mannitol, glucose, phosphate, citrate and acetate.

Drawings

FIG. 1 is an electron micrograph of an embryonic stem cell exosome prepared in example 1;

FIG. 2 is a graph showing a distribution of particle sizes of exosomes of embryonic stem cells prepared in example 1;

FIG. 3 is the Western Blot results of the surface markers on the cell culture supernatants of the ES cell lysate, mES-exos and extraction of the cells in example 1;

FIG. 4 is the retention of dye-labeled exosomes of example 2 in the kidney over time;

FIG. 5 is a flowchart of the experiment for constructing a model of Dox chronic nephritis and intervention measures in example 3;

FIG. 6 is a graph of the change in body weight of mice on day 21 relative to day 0 after Dox induction in example 3;

FIG. 7 is a graph showing the change in serological indicators of mice on day 21 after Dox induction in example 3;

FIG. 8 is a graph showing the change in expression of genes associated with inflammation and renal injury in mice on day 21 after Dox induction in example 3;

FIGS. 9 to 10 show the apoptosis of mouse kidney on day 21 after Dox induction in example 3;

FIG. 11 is a kidney tissue stained section of mice at day 21 after Dox induction in example 3;

FIG. 12 is a flowchart of the experiment for constructing renal fibrosis model and intervention measures by Dox induction in example 4;

FIG. 13 is a graph of body weight change of mice at week 5 after Dox induction versus day 0 in example 4;

FIG. 14 is a survival curve of mice after Dox induction in example 4;

FIG. 15 is the organ coefficient change of the left and right kidneys of mice at 5 weeks after Dox induction in example 4;

FIG. 16 is a graph showing the change in serological indicators of the mice at 5 weeks after Dox induction in example 4;

FIG. 17 is a kidney tissue stained section of mice at 5 weeks after Dox induction in example 4;

FIG. 18 is a flowchart of UUO model construction and intervention in example 5;

FIG. 19 is a graph showing the change in serological indicators of the mice at day 23 after Dox induction in example 5;

FIG. 20 is a graph showing the changes in expression of the mouse disease infiltration, vascular loss and fibrosis-associated genes at day 23 after Dox induction in example 5;

FIG. 21 is a stained section of mouse kidney tissue at day 23 after Dox induction in example 5 (H & E, Masson three color and Sirius Red staining);

FIG. 22 is the immunohistochemical staining of fibronectin and CD31 in the kidney of mice at day 23 after Dox induction in example 5.

Detailed Description

The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

It should be noted that the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. The term "and/or" includes any and all combinations of one or more of the associated listed items. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. "optionally" herein means by way of example.

According to the 2019 global disease burden report, Chronic Kidney Disease (CKD) has entered the first ten disease burden lines of the middle-aged and elderly population over 50 years of age, the disability of the population is significantly affected to regulate the life years, and there is a trend of increasing year by year. The risk of cardiovascular disease (CVD) in people with CKD will rise significantly, especially in heart failure, peripheral arterial disease and stroke, with the CVD mortality rate of dialysis patients increasing 10-30 times compared to general people. Most of the characterizing factors of CVD, such as diabetes, systolic hypertension, etc., are prevalent in the CKD population. Similarly, CKD is associated with metabolic acidosis, hyperkalemia, thrombosis, and other diseases. Therefore, the interest in CKD is increased, and the risk of complications such as CVD is reduced.

The term "chronic renal disease" also known as "chronic renal insufficiency and chronic renal failure" refers to chronic renal structure and dysfunction (history of renal damage greater than 3 months) caused by various causes, including normal and abnormal pathological damage of Glomerular Filtration Rate (GFR), abnormal blood or urine composition and abnormal imaging examination, or an unexplained decrease in GFR (see the description of the drawings, supra and infra)<60mL/min·1.73m ² ) For more than 3 months, CKD (viral kidney disease) is obtained. The main symptoms of chronic kidney disease are: edema, proteinuria, hematuria, metabolic acidosis, mineral bone metabolic disorder, etc., are accompanied by complications such as hypertension, anemia, malnutrition, digestive system diseases, cardiovascular diseases, etc.

The research of the invention discovers that the embryonic stem cell exosome can obviously reduce the uric acid content in serum of a chronic nephritis model, reduce the urea nitrogen/creatinine ratio and the albumin content, reduce the expression quantity of inflammatory factors IL-1 beta, IL-4, IL-13, IL-1 alpha, IL-6, IL-18 and IFNG in the kidney, reduce the expression of fibrosis related factors E-cadherin, alpha-SMA and Collagen1, relieve the obvious rise of neutrophil gelatinase related lipoprotein (NGAL) of a kidney injury indication factor, inhibit the apoptosis of kidney cells to increase the retention of the kidney parenchyma cells so as to protect the kidney function, improve the kidney interstitial inflammation and edema, slow down the kidney fibrosis and improve the survival rate. In addition, the embryonic stem cell exosome can also improve creatinine and urea nitrogen metabolism, improve the filtering effect of glomeruli and further restore the renal function.

Based on the above, one embodiment of the present application provides a use of a stem cell exosome in the preparation of a medicament for treating chronic kidney disease.

Optionally, the chronic kidney disease has one or more of the following characteristics: renal fibrosis, renal resident cell proliferation and leukocyte infiltration.

Tubulointerstitial fibrosis (renal fibrosis for short) is the ultimate route of progression in almost all chronic kidney diseases. During fibrosis, all structures in the kidney are affected, such as glomerulosclerosis and arteriosclerosis in the vasculature. Renal fibrosis is characterized by excessive deposition of extracellular matrix (ECM), and abnormal expansion of the space between glomerular basement membrane and peritubular capillary vessels. The most abundant matrix proteins in renal fibrosis are collagen i, and in addition, collagen types III, V, VI, VII, and XV, fibronectin, and the like. The current methods for treating and relieving the progression of renal fibrosis include inhibiting angiotensin converting enzyme, blocking angiotensin receptor, controlling blood pressure and relieving metabolic acidosis by using sodium bicarbonate, and the novel therapeutic drugs for renal fibrosis mainly include transforming growth factor beta (TGF-beta) and its inhibitor, Connective Tissue Growth Factor (CTGF) and its inhibitor, BMP-7 and its agonist, Galectin-3(Gal-3) and its inhibitor, CC motif chemokine 2(CCL2) and its inhibitor, angiotensin converting enzyme inhibitor (ACE-I) and Angiotensin Receptor Blocker (ARB), and anti-inflammatory therapies such as TNF immunomodulatory drugs are also used. However, the above drugs have very limited therapeutic effects on patients who have progressed to the stage of fibrosis.

Further, chronic kidney disease includes one or more of the following diseases: glomerulonephritis, tubulointerstitial diseases, renal vascular diseases, diabetic nephropathy and hereditary renal diseases.

Glomerulonephritis is a type of glomerulonephritis characterized by intrinsic cell proliferation and/or leukocyte infiltration of the kidney, accounting for approximately 20% of CKD cases. In a young population, glomerulonephritis is the most common end stage renal disease. Glomerulonephritis includes immune complex type glomerulonephritis (e.g., IgA nephropathy, lupus nephritis, infection-related nephritis and fibroid glomerulopathy with polyclonal immunoglobulin deposition), oligoimmune complex type glomerulonephritis (e.g., MPO-ANCA-related glomerulonephritis, PR 3-ANCA-related glomerulonephritis and ANCA-negative glomerulonephritis), anti-basement membrane glomerulonephritis (anti-GBM glomerulonephritis), monoclonal Ig type glomerulonephritis (e.g., monoclonal immunoglobulin deposition disease, proliferative glomerulonephritis with monoclonal immunoglobulin deposition, immunowhisker-like glomerulopathy and fibroid glomerulopathy with monoclonal immunoglobulin deposition), and C3 renal disease (e.g., C3 nephritis and dense deposition disease). Glomerulonephritis is characterized primarily clinically by proteinuria, hematuria, elevated serum creatinine concentrations, podocyte invagination and disappearance, edema, abnormal weight gain, and even hypertension and renal failure. The pathogenesis of glomerulonephritis is complex, and the glomerulonephritis relates to a plurality of aspects such as inflammatory reaction, antibody and immune cell mediated immune reaction. In addition, diseases such as obesity, hypertension, diabetes and the like and genetic factors also affect the pathological process of glomerulonephritis, and about two thirds of patients with primary glomerulonephritis are detected to have genetic defects. The existing treatment schemes mostly use corticosteroids, calcineurin inhibitors, renin-angiotensin system blockers and the like, and have the defects of unstable effect, high recurrence rate, obvious adverse reaction and year-by-year increase of the number of patients with steroid drug resistance.

Optionally, the medicament comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is a stem cell exosome. An active ingredient refers to any ingredient that provides pharmacological activity or other direct effect or affects the structure or any function of the human and other animal body in diagnosing, curing, palliating, treating, or preventing disease. Specifically, the active ingredient includes stem cell exosomes. Further, the stem cell exosomes include one or more of the following: embryonic stem cell exosomes, mesenchymal stem cell exosomes and induced pluripotent stem cell exosomes. In an alternative embodiment, the active ingredient is an embryonic stem cell exosome. Of course, the active ingredient is not limited to embryonic stem cell exosomes, but may be other stem cell exosomes and/or other ingredients effective for chronic kidney disease. In other embodiments, the active ingredient of the above medicament is a combination of stem cell exosomes and other active ingredients.

Pharmaceutically acceptable excipients refer to pharmaceutically acceptable auxiliary materials or carriers which are compatible with the other ingredients of the pharmaceutical formulation and which are suitable for use in contact with the tissue or organ of the recipient (e.g., human or animal). There are no or few complications of toxicity, irritation, allergic response, immunogenicity, or other problems with use.

Specifically, the above medicines can be oral preparations such as tablets, disintegrating tablets, capsules, granules, pills, oral liquids and the like, and also can be injection preparations such as injection, freeze-dried powder injection, infusion solutions, liposome injection, microsphere injection and the like; of course, the preparation can be a common preparation, and can also be a novel or special preparation such as a sustained-release preparation, a controlled-release preparation, a directional preparation, a targeting preparation, a nano preparation and the like.

In some embodiments, the drug is a tablet. In order to prepare the above-mentioned drugs into tablets, various excipients known in the art may be used as pharmaceutically acceptable excipients. Specifically, the medicament is a tablet, and the pharmaceutically acceptable auxiliary materials comprise one or more of a diluent, a binder, a wetting agent, a disintegrating agent and a lubricant.

Further, the diluent comprises one or more of starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate and calcium carbonate. The humectant comprises one or more of water, ethanol and isopropanol. The binder comprises one or more of starch slurry, dextrin, syrup, Mel, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone and polyethylene glycol. The disintegrant comprises one or more of starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, polyoxyethylene, sorbitol, fatty acid ester and sodium dodecyl sulfate. The lubricant comprises one or more of talc, silica, stearate, tartaric acid, liquid paraffin and polyethylene glycol. Of course, the tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.

In some embodiments, the above-mentioned medicine is capsule or soft capsule, oriented capsule, instant capsule, etc. In order to prepare the medicine into special capsule preparations such as capsules or soft capsules, directional capsules, instant capsules and the like, the stem cell exosomes can be mixed with diluents, glidants and the like, and then the mixture obtained after mixing is directly placed into hard capsules, soft capsules or special capsules. Of course, in other embodiments, the stem cell exosomes may be mixed with one or more of a diluent, an adhesive and a disintegrant, and then made into granules, pellets, microspheres, liposomes, and the like, and then placed into hard capsules, soft capsules or special capsules.

In some embodiments, the medicament is an injection. In order to prepare the medicine into injection, water, ethanol, isopropanol, propylene glycol, polyethylene glycol or a mixture of the water, the ethanol, the isopropanol, the propylene glycol and the polyethylene glycol can be used as a solvent, and a proper amount of pharmaceutically acceptable auxiliary materials commonly used in the field are added and mixed for use.

Specifically, the medicament is an injection, and the pharmaceutically acceptable auxiliary materials comprise one or more of a solubilizer, a pH regulator and an osmotic pressure regulator. Further, the solubilizer comprises one or more of ethanol, isopropanol, propylene glycol, polyethylene glycol, poloxamer, lecithin and hydroxypropyl-beta-cyclodextrin. The pH regulator comprises one or more of citrate, phosphate, carbonate, acetate, hydrochloric acid and hydroxide. The osmotic pressure regulator comprises one or more of sodium chloride, mannitol, glucose, phosphate, citrate and acetate. For example, mannitol and glucose can be added as proppant for preparing lyophilized powder for injection. Further, additives such as coloring agents, preservatives, flavors, and flavoring agents may be added to the pharmaceutical preparation, if necessary.

The medicine comprises stem cell exosomes, and has a good effect of treating chronic kidney diseases.

In addition, an embodiment of the present application also provides a method for treating chronic kidney disease using the stem cell exosomes.

Optionally, the chronic kidney disease has one or more of the following characteristics: renal fibrosis, renal resident cell proliferation and leukocyte infiltration.

In some embodiments, the stem cell exosomes comprise one or more of the following: embryonic stem cell exosomes, mesenchymal stem cell exosomes and induced pluripotent stem cell exosomes.

In some embodiments, chronic kidney disease includes one or more of the following diseases: glomerulonephritis, tubulointerstitial diseases, renal vascular diseases, diabetic nephropathy and hereditary renal diseases.

In some embodiments, the stem cell exosomes are administered to the patient intravenously.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer. The mice in examples 3 and 4 were 8-week-old Balb/C male mice, and the mice in example 5 were 8-week-old C57BL/6 male mice.

Example 1

Preparation and identification of mouse embryonic stem cell exosome (hereinafter referred to as "embryonic stem cell exosome" or "mES-exos")

(1) Culturing the mouse embryonic stem cells: 20% mitomycin C treated fibroblasts were incubated in gelatin coated dishes for 2 hours, after which the medium was removed and the cells were washed 3 times with PBS, resulting in dishes with feeder layer; next, mouse embryonic stem cells (Jackson Laboratory) were seeded in a culture dish with feeder cells and cultured in H-DMEM medium containing 15% fetal bovine serum (2mM of L-glutamine, 0.1mM of MEM NEAA (i.e., MEM nonessential amino acid solution), 0.1mM of β -mercaptoethanol, 0.1mM of sodium pyruvate, 1000U/mL of LIF (leukemia inhibitory factor), 50. mu.g/mL of penicillin, and 50. mu.g/mL of streptomycin).

(2) Mouse embryonic stem cell exosome preparation: H-DMEM medium containing 15% fetal calf serum at 1X 10 ⁵ g. Centrifuging at 4 deg.C for 12 hr, and removing precipitate (to remove exosomes from H-DMEM medium containing 15% fetal calf serum); washing the mouse embryonic stem cells of step (1) with PBS for three times, adding centrifuged H-DMEM medium containing 15% fetal bovine serum, and placing in an incubator (37 ℃, 5% CO) ₂ ) Culturing for 24 hours; then transferring the culture medium containing the mouse embryonic stem cells after the culture is finished into a centrifuge tube, and centrifuging the centrifuge tube at 2000g and 4 ℃ for 20 minutes to precipitate the mouse embryonic stem cells; the centrifuged supernatant was then aspirated into another centrifuge tube and centrifuged at 1X 10 ⁴ g. Centrifuging at 4 ℃ for 30 minutes; the supernatant was aspirated into a high-speed centrifuge tube (1X 10) ⁵ g. Centrifuging at 4 ℃ for 70 minutes; the supernatant was discarded completely and resuspended in 1mL of PBS; then, 1 × 10 ⁵ g. Centrifuging at 4 ℃ for 90 minutes; the supernatant was discarded, resuspended in 500. mu.L PBS and filtered through a filter with a 0.22 μm mesh to obtain mouse embryonic stem cell exosomes, which were stored at-80 ℃.

(3) And (3) identifying an embryo stem cell exosome:

and (3) after negative staining is carried out on the embryonic stem cell exosomes, observing the morphology of the embryonic stem cell exosomes by using a transmission electron microscope. In the experiment, after the copper mesh for negative dyeing is subjected to vacuum static elimination for 2min, 5 mu L of diluted sample is dropwise added on the surface of the copper mesh, and the sample is sucked and discarded after 5 s. The solution was pre-dyed by dropping 4. mu.L of dye solution for 1min and then discarded, residual dye was removed by blotting with filter paper, 4. mu.L of dye solution was dropped twice, and after 1min, the solution was discarded, dried at room temperature and observed by electron microscopy, and the results are shown in FIG. 1.

Embryonic stem cell exosomes were analyzed for particle size using dynamic light scattering. In the case of pure water and PBS as controls, 10. mu.L of the diluted sample was added to the detection tube and observed, and the results are shown in FIG. 2.

Surface markers CD9, CD63 and TSG101 of the embryonic stem cell exosomes were detected by Western Blotting, and the results are shown in FIG. 3.

As can be seen from fig. 1 to 3, the transmission electron microscope showed that the extracted embryonic stem cell exosomes are typical bilayer membrane vesicles and have a clear cup-like structure (fig. 1). Dynamic light scattering showed a median particle size of 150.6nm (n-4) for mES-exos, consistent with the embryonic stem cell exosome particle size characteristics (fig. 2). Western blot showed that mES-exos and ES cell lysates expressed embryonic stem cell exosome surface markers CD9, TSG101 and CD63, while cell lysates simultaneously expressed β -actin (β -actin), whereas cell culture supernatants after extraction of embryonic stem cell exosomes hardly expressed the above markers (fig. 3). Therefore, it was confirmed that the mouse embryonic stem cell exosomes obtained in step (2) were embryonic stem cell exosomes.

Example 2

Embryonic stem cell exosome labeling and in vivo biodistribution

Mouse embryonic stem cell exosomes were labeled with the dye PKH67 to study the distribution and residence time of mES-exos in the kidney. The labeled embryonic stem cell exosomes were injected into the tail vein of mice, and organs were imaged ex vivo using a small animal in-vivo imager at 24 hours, 48 hours, and 72 hours after injection, with the results shown in fig. 4 (in fig. 4, "Control" means a Control group, and "exo" means an embryonic stem cell exosome group).

As can be seen from the image of FIG. 4, at 72 hours, the retention of embryonic stem cell exosomes in the kidney was significantly increased and spread from the renal pelvis to the renal medulla and cortex.

Example 3

Therapeutic Effect of mES-exos on Chronic nephritis

1. Preparation of mouse chronic nephritis model and intervention of mES-exos

Mouse chronic nephritis is induced by Doxorubicin (Doxorubicin, Dox for short). The Doxorubicin-induced nephropathy is a classic rodent model of chronic nephritis, and is a model simulating human chronic nephritis. The severity of tissue damage is positively correlated with mortality and weight loss, and triggers a persistent inflammatory response one week after drug administration. Therefore, Balb/c male mice of 8 weeks old were randomly divided into three groups, which were designated as a blank Control group (Control), a model group (Doxorubicin), and a treatment group (Doxorubicin + exo), wherein the model group and the treatment group were intraperitoneally injected with 10mg/kg of Doxorubicin (abbreviated as "Dox") according to the body weight of the mice, and the treatment group was injected with 20 μ g of the embryonic stem cell exosome prepared in example 1 starting from the tail vein on day 8 on the basis of the Dox injection for 4 times, specifically, referring to fig. 5, and the blank Control group was injected with the same amount of PBS.

2. Detection indexes are as follows:

(1) weight change

The change in body weight of the mice was observed with respect to day 0 and counted, and the results are shown in fig. 6. In fig. 6 ". star" means p < 0.001.

As can be seen in fig. 6, it was found that there was a sustained decrease in body weight in mice following Dox induction, but that treatment with embryonic stem cell exosomes alleviated this.

(2) Serological index changes

Because various physiological indexes in blood change stably, nephropathy is usually diagnosed by blood examination in clinic. Therefore, we examined the ratio of urea nitrogen (BUN) to Creatinine (CREA), Uric Acid (UA) and Albumin (ALB) levels in the serum of mice at day 21 after Dox induction to determine the progression of kidney disease in mice, and the results are shown in fig. 7. In fig. 7, a is a statistical result of uric acid contents in sera of the blank Control group (Control), the model group (Doxorubicin), and the treatment group (Doxorubicin + Exo), B is a statistical result of a ratio of BUN to CREA in sera of the blank Control group, the model group, and the treatment group, and C is a statistical result of albumin contents in sera of the blank Control group, the model group, and the treatment group; "+" indicates p <0.05, "+" indicates p <0.01, "+" indicates p <0.001 (the same applies hereinafter).

As can be seen from FIG. 7, on day 21 after Dox induction, the uric acid, urea nitrogen/creatinine ratio and albumin expression levels in the serum of the model group were significantly higher than those of the control group, and this phenomenon was significantly alleviated after the treatment with the embryonic stem cell exosomes.

(3) Changes in expression of inflammation and kidney injury related genes at the mRNA level:

the expression of inflammation and kidney injury related genes at the mRNA level after day 21 after Dox induction was examined using qPCR. The results show that after Dox induction, the expression levels of inflammatory factors IL-1 beta, IL-4, IL-13 and IFNG in the kidney of the mouse are obviously increased, and the Neutrophil Gelatinase Associated Lipoprotein (NGAL) which is a factor for indicating kidney injury is also obviously increased, and the increase is obviously relieved by the exosome of the embryonic stem cell.

(4) Apoptosis of mouse kidney on day 21 after Dox induction:

in order to examine the effect of the exosomes of embryonic stem cells on the retention rate of renal parenchymal cells, the change in the expression level of B-cell lymphoma 2(Bcl2) -associated X protein (Bax) was examined by Western Blot, and the results are shown in fig. 9 to 10. Bax is a pro-apoptotic factor and belongs to the Bcl2 subfamily. As is apparent from fig. 9 to 10. The expression level of Bax is obviously increased in the kidney of the mouse on day 21 after Dox induction, and the expression level is obviously reduced after the treatment of the embryonic stem cell exosome, which indicates that the embryonic stem cell exosome increases the retention of renal parenchymal cells by inhibiting apoptosis so as to protect the renal function.

(5) Interstitial inflammation and edema of the kidneys in mice on day 21 after Dox induction

Chronic inflammation induced by Dox gradually accumulates in the renal interstitium over time and initiates interstitial edema and shedding of renal tubular epithelial cells. Therefore, we prepared kidney tissue sections and performed pathological staining, from which histological observation was performed, and the results are shown in fig. 11. In fig. 11, H & E stained arrows indicate inflammatory cell infiltration, and five stars indicate vacuolization; arrows in PAS staining indicate glomerular basement membrane; scale bar 100 μm. It was found by H & E and periodic acid-schiff (PAS) staining that on day 21 after Dox induction, there was some structural change in the mouse kidney, inflammatory cells began to infiltrate into the renal interstitium, renal tubules were edematous, epithelial cell exfoliation, slight vacuolization and thickening of glomerular basement membrane, and these symptoms were significantly improved in mice treated with embryonic stem cell exosomes.

Example 4

Therapeutic effect of mES-exos on renal fibrosis

(1) Change in basal index of mice at 5 weeks after Dox induction

Since the kidney of the mouse begins to generate fibrosis about 4 weeks after the induction of Dox, the induction time is prolonged to 5 weeks to observe the effect of the embryonic stem cell exosome stem on the kidney fibrosis induced by Dox, and the experimental flow is shown in fig. 12. We observed the change in body weight of mice (fig. 13) and plotted the survival curve according to survival rate (fig. 14). After 5 weeks, the mice were euthanized and the organ coefficient changes of the left and right kidneys were observed (fig. 15). It was found that mice show significant weight loss after Dox induction, and this sustained weight loss is alleviated after the prognosis of embryonic stem cell exosome stem. From the survival curves, it can be seen that the mice in the model group (Doxorubicin) began to die at day 9 after Dox induction, while the initial death time of the embryonic stem cell exosome group was delayed to day 29, and by the end of the experiment, the mortality rate of the model group was 44.4% and the mortality rate of the embryonic stem cell exosome group was 22.2%, which also indicates that the embryonic stem cell exosomes reduced the mortality rate of the mice. In addition, as can be seen from the weight change of the mouse kidney at the end point of the experiment, after Dox induction, the left and right kidney coefficients (kidney weight/body weight) of the mouse are obviously increased, which is probably caused by the deposition of kidney edema and fibrosis, and the symptom is relieved by the intervention of the exosomes of the embryonic stem cells.

(2) Doxorubicin induces changes in serological indices in 5-week mice

In order to observe the change in kidney injury in mice at 5 weeks after Dox induction, they were also serologically examined, and the results are shown in fig. 16. As can be seen from FIG. 16, the serum contents of BUN, CREA and UA of the model group mice increased significantly over 5 weeks. After the treatment of the embryonic stem cell exosome, the treatment rate is obviously reduced except for uric acid.

(3) Renal interstitial inflammation and fibrosis in mice at week 5 after Dox induction

In order to observe the development of inflammation and the fibrotic deposition in the kidney of mice at 5 weeks after Dox induction, kidney tissue sections were prepared for pathological observation and the fibrotic area was quantified, and the results are shown in fig. 17 (the scale bar in fig. 17 is 100 μm). As can be seen from fig. 17, the deposition of interstitial inflammation between the model groups was greatly increased compared to the 21-day group by H & E staining and Masson trichrome staining, and the glomeruli were proliferated to some extent, and the renal interstitium began to be deposited with fibrosis (blue-stained area in the color image), and was mainly concentrated around the blood vessels. After the embryonic stem cell exosome is dried, the interstitial inflammation of the kidney is obviously relieved. Also, we found that collagen deposition was significantly reduced in the embryonic stem cell exosome group compared to the model group after quantitative analysis by Masson trichrome staining.

Example 5

Therapeutic Effect of mES-exos on UUO-induced Kidney injury

1. Mouse UUO model preparation and intervention of mES-exos

The Unilateral Ureteral Obstruction (UUO) model is also one of the classic models of renal fibrosis, which induces inflammatory infiltration and fibrosis of the kidney primarily by increasing renal pressure, inducing hydronephrosis and reducing nephrons. Therefore, we constructed a UUO model and observed the effect of embryonic stem cell exosome stem prediction on kidney injury induced by this model. Mouse renal fibrosis was induced using unilateral ureteral ligation (UUO). Mice of 8 weeks old were randomly divided into three groups, which were designated as Sham (Sham), model (UUO) and treatment (UUO + exo), and the left and right ureters of the left kidney of the mice were ligated with 1/3 each, using 6-0 nylon thread, and the ureters were cut off from the middle of the two nylon threads. The treatment group was injected with 20 μ g of the embryonic stem cell exosome prepared in example 1 in tail vein starting on day 15 for 4 times, specifically, see fig. 18, and the blank control group was injected with the same amount of control solution.

2. Detection of

(1) Changes in serum and urine indices.

Serum and urine tests are the basis for the diagnosis of renal injury, particularly with the elevation of creatinine as the gold standard. Therefore, we examined the levels of CREA, BUN and UA in the serum and CREA in the urine of mice at day 23 after Dox induction, and the results are shown in fig. 19. In FIG. 19, A is the amount of CREA expression in mouse serum; b is the expression level of BUN in mouse serum; c is the UA expression level in mouse serum; d amount of CREA expression in mouse urine.

As can be seen in FIG. 19, the CREA, BUN and UA levels in UUO mice were significantly increased compared to control mice, while the intervention of embryonic stem cell exosomes significantly reversed the increase in serum BUN and urine CREA, with some remission of the remaining criteria. Therefore, ESC-exos alleviated kidney injury in UUO mice.

(2) Changes in expression of inflammation-and kidney injury-associated genes at mRNA level

To further observe the pathological course of mice, we examined the expression level of neutrophil gelatinase-associated lipocalin (NGAL) and the expression of fibrosis and inflammation-associated genes, another kidney injury marker, by qPCR, as shown in FIG. 20 (in FIG. 20, A is the expression level of mRNA of NGAL; B to D are the expression levels of mRNA of fibrosis-associated factors E-cadherin, alpha-SMA and Collagen 1; E to H are the expression levels of mRNA of inflammation factors IL-1 alpha, IL-1 beta, IL-6 and IL-18 in the left kidney).

As can be seen in figure 20, NGAL in UUO mice increased significantly and decreased significantly after ESC-exos intervention. UUO mouse fibrosis related gene E-cadherin is obviously reduced, and alpha-SMA and Collagen1 are up-regulated; the changes in the first two genes caused by UUO could be significantly reversed by ESC-exos treatment, but the decrease in Collagen1 was not significant. Furthermore, IL-1 α, IL-1 β, IL-6 and IL-18 inflammatory cytokine levels were significantly elevated in the kidney of UUO mice, while ESC-exos treatment significantly reduced these inflammatory cytokines in addition to IL-1 β, with a decrease in IL-1 β. This suggests that embryonic stem cell exosomes may delay the progression of renal fibrosis by alleviating inflammatory cell infiltration, inhibiting activation of inflammatory pathways, suggesting that ESC-exos reduce inflammatory infiltration, vascular loss and fibrosis of the mouse kidney.

(3) Fibrosis of fiber

Renal fibrosis due to UUO induction will begin to develop on day 7 and continue to accumulate over time. We performed histopathological analysis of kidney sections by H & E, Masson trichrome and Sirius Red staining and quantified the collagen deposition area by Masson trichrome staining, as shown in figure 21 (scale bar 100 μm; statistical plots of H & E staining, Masson trichrome and Sirius Red staining of kidney tissue sections; b. Masson trichrome staining).

As can be seen in fig. 21, UUO surgery induced significant renal vacuolization, tubular atrophy and inflammatory cell infiltration as evidenced by H & E staining, Masson trichrome staining and sirius red staining; also, the loss of a large number of renal parenchymal cells and interstitial collagen deposition suggest the appearance of our fibrosis. Injection of ESC-exos, however, significantly attenuated tubular atrophy, interstitial leukocyte infiltration and collagen deposition in UUO mice.

To further examine mouse kidney damage and fibrosis, we performed immunohistochemical staining for fibronectin and platelet-endothelial cell adhesion molecule (CD31), with results shown in fig. 22. In fig. 22, a is an immunohistochemical staining of fibronectin and CD 31; b is a statistical plot of fibronectin expression levels; c is a statistical plot of CD31 expression levels; scale bar 100 μm.

As can be seen in FIG. 22, ESC-exos injection significantly reduced fibronectin expression in the kidney of UUO mice, further demonstrating that ESC-exos reduced fibrosis. In addition, the glomerular filtration rate is significantly affected by the blood flow of the kidney. Our results using immunohistochemical staining to detect CD31 expression showed that ESC-exos significantly reversed the down-regulation of CD31 in the kidney of UUO mice. Therefore, the embryonic stem cell exosome can effectively promote the regeneration of blood vessels, improve the metabolism of creatinine and urea nitrogen, improve the filtration effect of glomeruli and further restore the renal function.

The technical features of the embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the combinations should be considered as the scope of the description in the present specification.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. It should be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.

Claims (10)

1. Use of a stem cell exosome in the preparation of a medicament for the treatment of chronic kidney disease.

2. The use according to claim 1, wherein the chronic kidney disease comprises one or more of the following diseases: glomerulonephritis, tubulointerstitial diseases, renal vascular diseases, diabetic nephropathy and hereditary renal diseases.

3. The use according to claim 1, wherein the chronic kidney disease is characterized by at least one of the following features: renal fibrosis, renal resident cell proliferation and leukocyte infiltration.

4. The use according to claim 1, wherein the stem cell exosomes comprise one or more of: embryonic stem cell exosomes, mesenchymal stem cell exosomes and induced pluripotent stem cell exosomes.

5. The use according to any one of claims 1 to 4, wherein the medicament comprises an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is the stem cell exosome.

6. The use according to claim 5, wherein the medicament is in the form of tablets, capsules, granules, sustained release formulations, powders, pellets or injections.

7. The use of claim 6, wherein the medicament is a tablet and the pharmaceutically acceptable excipient comprises one or more of a diluent, a binder, a wetting agent, a disintegrant, and a lubricant.

8. The use according to claim 7, wherein the pharmaceutically acceptable excipient meets one or more of the following conditions:

the diluent comprises one or more of starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate and calcium carbonate;

the adhesive comprises one or more of starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone and polyethylene glycol;

the disintegrating agent comprises one or more of starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch and polyoxyethylene;

the lubricant comprises one or more of talcum powder, silicon dioxide, stearate, liquid paraffin and polyethylene glycol;

and, the wetting agent comprises one or more of water, ethanol and isopropanol.

9. The use of claim 8, wherein the medicament is an injection, and the pharmaceutically acceptable excipient comprises one or more of a solubilizer, a pH regulator and an osmotic pressure regulator.

10. The use according to claim 9, wherein the pharmaceutically acceptable excipient meets one or more of the following conditions:

the solubilizer comprises one or more of ethanol, isopropanol, propylene glycol, polyethylene glycol, poloxamer, lecithin and hydroxypropyl-beta-cyclodextrin;

the pH regulator comprises one or more of citrate, phosphate, carbonate, acetate, hydrochloric acid and hydroxide;

and the osmotic pressure regulator comprises one or more of sodium chloride, mannitol, glucose, phosphate, citrate and acetate.

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