Preparation of cat adipose-derived mesenchymal stem cell exosome based on hypoxia pretreatment and application of cat adipose-derived mesenchymal stem cell exosome in treatment of cat acute kidney injury

Abstract

The invention discloses preparation of cat adipose tissue-derived mesenchymal stem cell exosomes based on hypoxia pretreatment and application of the cat adipose tissue-derived mesenchymal stem cell exosomes in treatment of cat acute kidney injury. An application method of the cat adipose tissue-derived mesenchymal stem cell exosomes comprises the following steps: pretreating cat adipose tissue-derived mesenchymal stem cells by using cobalt chloride, collecting the exosomes, performing related identification on the exosomes, and applying the exosomes to an animal model for treating the cat acute kidney injury. The research can provide a theoretical basis for clinical treatment of cat renal failure diseases, and provides valuable references and clues for transformational medicine research of treatment of human renal injury diseases.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedicine, stem cell therapy, and more particularly to an application of a mesenchymal stem cell pretreatment method in the treatment of chronic renal failure in cats. Background Art

[0002] Acute kidney injury, also known as acute renal failure, is a serious, rapid, and complex kidney damage disease. In the treatment of acute kidney injury, in addition to the previous conservative treatment (infusion therapy), many new therapies for acute kidney injury in animals have recently emerged in veterinary clinics, including dialysis techniques such as hemodialysis and peritoneal dialysis. However, due to the strict requirements of dialysis operations, the need for sophisticated equipment, and high costs, only a few hospitals have the relevant equipment and professionals, and the scope of application in veterinary clinics is limited.

[0003] In recent years, many studies have shown that cell exosomes from mesenchymal stem cells derived from bone marrow, umbilical cord or adipose tissue have a positive therapeutic effect on renal injury. Cell exosomes derived from mesenchymal stem cells can effectively treat prerenal and nephrogenic renal injury in mice and rats. In vivo, adipose mesenchymal stem cells exist in a 5-9% environment. At present, many experiments have shown that a hypoxic environment can improve the proliferation, migration and differentiation of mesenchymal stem cells. Mesenchymal stem cell exosomes prepared under hypoxic conditions can provide a rich and efficient combination of pro-angiogenic factors, which helps to improve local microcirculation. Secondly, hypoxic pretreated mesenchymal stem cell exosomes are rich in a variety of anti-inflammatory cytokines, especially IL-10 and TGF-β. IL-10 is an important anti-inflammatory cytokine that can inhibit the production of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β. The present invention collects exosomes by hypoxic pretreatment of cat adipose mesenchymal stem cells, and uses the exosomes to treat acute kidney injury in cats; using exosomes derived under hypoxic conditions can more efficiently treat acute kidney injury in cats.

[0004] In view of this, this invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to prepare cat adipose mesenchymal stem cell exosomes pretreated with hypoxia and to effectively treat cat acute kidney injury disease.

[0006] The present invention is achieved in that:

[0007] Feline adipose-derived mesenchymal stem cells were pretreated with cobalt chloride to collect exosomes, which were then identified and used to treat a feline acute kidney injury animal model.

[0008] The present invention has the following beneficial effects:

[0009] The creatinine and urea nitrogen values ​​per unit time of exosomes derived from cat adipose-derived mesenchymal stem cells under hypoxic pretreatment conditions were changed faster than those of exosomes derived from normoxic conditions. Secondly, the kidney HE-stained sections of hypoxic exosomes showed better repair after kidney injury than those in the normoxic treatment group, and the phenomenon of post-injury repair appeared. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 A. Establishment of ADMSCs hypoxia model and expression level of VEGF, a downstream gene of cells, under hypoxic conditions

[0011] Figure 2 A. Exosome size distribution B. Transmission electron microscopy to observe exosome morphology and size C. Western blotting to detect exosome surface marker proteins TSG101 and CD63

[0012] Figure 3 A. Changes in creatinine in different experimental cat groups during the model establishment process B. Changes in blood urea nitrogen in different experimental cat groups during the model creation stage

[0013] Figure 4 A. Line graph showing the dynamic reduction in the proportion of creatinine in the experimental cats in different treatment groups during the exosome treatment period. B. Line graph showing the dynamic reduction in the proportion of blood urea nitrogen in the experimental cats in different treatment groups during the exosome treatment period. DETAILED DESCRIPTION

[0015] The present invention is described in detail below in combination with the accompanying drawings, but the protection scope of the present invention is not limited to the described contents.

[0016] Preparation of complete culture medium for feline adipose-derived mesenchymal stem cells: α-MEM medium, 10% FBS, 1% P / S, 1% NEAA, 1% GlutaMax.

[0017] Cobalt chloride was used for hypoxia pretreatment, and the specific operation was as follows: First, cat ADMSCs cultured to the 4th generation were inoculated in a 6-well culture dish. When the cells grew to the 80%-90% confluence stage, complete culture medium with concentrations of 50μM, 100μM, 150μM, and 200μM CoCl2 were added, respectively, and the treatment time was set to 6 hours, 8 hours, 12 hours, and 24 hours. At different time points of treatment, cells were collected by digestion and RNA was extracted, which was then reverse transcribed into cDNA. The real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) technology was used to detect the expression level of the downstream gene VEGF of cells under hypoxic conditions to evaluate the hypoxia-induced effect. After cobalt chloride treatment, the expression level of the downstream gene vascular endothelial growth factor of cells under 6h hypoxic conditions was significantly increased (see Figure 1 ).

[0018] Exosome isolation and identification: The isolated ADMSCs were passed to the fourth generation to isolate cell exosomes. When the cells grew to 80% confluence, the hypoxia treatment group was treated with complete medium containing 150 μM CoCl2 for 6 h, while the normoxic treatment group was cultured and the cell supernatant was collected. The experiment used differential ultracentrifugation to extract extracellular vesicles. The centrifuge was precooled at 4°C, 300g for 10 min, and the supernatant was taken into a new centrifuge tube; 10000g for 30 min, and the supernatant was taken into a new centrifuge tube; 100000g for 90 min, the supernatant was poured out, and the precipitate was retained; 40ml PBS was added to the precipitate and continued centrifugation; 100000g for 90 min, the supernatant was poured out, and the precipitate was retained; 250ul PBS was added to the precipitate to resuspend the precipitate, and the obtained precipitate was exosomes. The average particle size of exosomes was analyzed by nanoparticle tracing, and it can be seen that the exosomes in each group were about 100-150nm. Electron microscopy observation showed that the exosomes showed a typical saucer-shaped sunken vesicle structure. Western blot was used to detect the expression of exosome marker proteins CD63 and TSG101 (see Figure 2 (as shown in A, B, and C).

[0019] Establishment of cat acute kidney injury model: The experimental animals were randomly divided into 3 groups, 3 in each group, including symptomatic treatment group, normoxic exosome treatment group and hypoxic exosome treatment group. The symptomatic treatment group only received targeted treatment for its symptoms, while the normoxic exosome treatment group and the hypoxic exosome treatment group received intravenous delivery of corresponding exosomes. Each group of animals was injected with gentamicin twice a day at a dose of 200 mg / kg / time. During this process, blood was collected from the cats to monitor changes in creatinine and urea nitrogen concentrations. When one of the three indicators exceeded the normal value or was 1.5 times the baseline, the injection of gentamicin was stopped and the model was successfully established (see Figure 3 A, B).

[0020] Exosome treatment of cat renal injury: cats were observed and recorded every day during GM injection. When the experimental cats showed polydipsia, polyuria, mental depression, lethargy and loss of appetite, creatinine and urea nitrogen tests were performed. When the creatinine and urea nitrogen indicators reached 1.5 times the normal baseline, intravenous delivery of exosomes was performed. The number of exosome particles was about 2.5*10 8 During the exosome treatment, cats with dehydration and refusal to eat were given fluid rehydration, including lactated Ringer's sodium solution, normal saline and 5% glucose saline, all at a dose of 20 mL / kg / d. Cats with vomiting were treated with ranitidine (2 mg / kg) and atropine (0.1 mL / kg) for antiemetic treatment. During the treatment, creatinine and urea nitrogen were tested. Compared with the symptomatic treatment group and the normoxic exosome treatment group, the cats in the hypoxic exosome treatment group showed a more sustained creatinine decrease trend, and the percentage of decrease remained at a high level throughout the treatment period (see Figure 4 A, B).

[0021] In summary, the present invention provides a feline adipose-derived mesenchymal stem cell exosome preparation based on hypoxia pretreatment and its application in the treatment of acute kidney injury in cats. It has been successfully verified that collecting exosomes after hypoxia pretreatment can effectively improve cat kidney injury, and its therapeutic effect is significantly better than other exosome groups.

[0022] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. Preparation of cat adipose-derived mesenchymal stem cell exosomes based on hypoxia pretreatment and its application in the treatment of acute kidney injury in cats, characterized in that: Creation of hypoxic environment for adipose-derived mesenchymal stem cells: Cobalt chloride was used for hypoxia pretreatment. The expression level of the downstream gene VEGF in cells under hypoxia was detected at different time points and concentrations to evaluate the hypoxia-induced effect. Isolation and identification of exosomes from mesenchymal stem cells under hypoxia: Exosomes were isolated from cells at the 4th generation. When the cells grew to 80% confluence, 150 μM CoCl2 in complete medium was added to the hypoxia-treated group and continued to be treated for 6 h, while the normoxic group continued to be cultured, the cell supernatant was collected, and the exosomes were extracted using differential high-speed centrifugation. Nano-ion tracking analyzer, transmission electron microscopy, and protein blotting were used for identification.

2. The pretreated cat adipose-derived mesenchymal stem cell exosome preparation according to claim 1, characterized in that: The centrifugation operation in step (2) is to collect the cell supernatant into a centrifuge tube, centrifuge at 2000 rpm for 10 min, and then centrifuge at 10000 rpm for 30 min to remove cells or cell debris.

3. The method for treating feline renal failure by hypoxia pretreatment of feline adipose-derived mesenchymal stem cell exosomes according to claim 1, characterized in that: The exosome injection solution is composed of exosomes and normal saline, and each milliliter of injection water contains 1x10 7 exosome particles.

4. The hypoxia pretreated feline adipose-derived mesenchymal stem cell exosome injection according to claim 2, characterized in that: Hypoxia-pretreated feline adipose-derived mesenchymal stem cell exosomes can treat acute kidney injury in cats, and the rate of change of creatinine and urea nitrogen values ​​per unit time is faster than that of normoxic exosomes.

5. The hypoxia pretreated feline adipose-derived mesenchymal stem cell exosome injection according to claim 2, characterized in that: Hypoxia pretreatment of cat adipose-derived mesenchymal stem cell exosomes in the treatment of acute kidney injury in cats was better than that of normoxic exosomes. The kidney HE staining sections of hypoxic exosomes showed better repair after kidney injury than those in the normoxic treatment group, and post-injury repair phenomenon occurred.

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