Small extracellular vesicles containing Klotho protein and / or mRNA as well as preparation method and application of small extracellular vesicles

Abstract

The invention relates to a small extracellular vesicle containing Klotho protein and / or mRNA as well as a preparation method and application of the small extracellular vesicle. The preparation method of the small extracellular vesicle comprises the following steps: S1, constructing a recombinant expression vector containing a Klotho protein coding gene; s2, introducing the recombinant expression vector into recipient cells to obtain a receptor cell strain with stable expression of the Klotho gene; and S3, culturing the receptor cell strain, collecting a culture supernatant, and extracting the small extracellular vesicles from the culture supernatant. The small extracellular vesicles containing the Klotho protein and / or mRNA can regulate and control the expression of endogenous Klotho, the expression profile of bioactive molecules in the small extracellular vesicles is also changed, the small extracellular vesicles are rich in various kidney protective proteins, miRNA and other bioactive molecules, and the treatment effect on AKI and AD is remarkably enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to small extracellular vesicles and their use in treating diseases, and specifically to small extracellular vesicles containing Klotho protein and / or mRNA and a preparation method and application thereof. Background Art

[0002] Extracellular vesicles (EV) refer to a class of tiny vesicles with a lipid bilayer membrane structure secreted by cells, including exosomes (Exo) and microvesicles, which differ in source and particle size. Exosomes are EVs with a diameter of 30-150nm that are released by the fusion of multivesicular bodies with cell membranes. EVs not only contain many bioactive molecules, such as nucleic acids (DNA, mRNA, miRNA, etc.), proteins, lipids, etc., but also give it the characteristics of being a micro-drug delivery system, so that it can specifically carry exogenous bioactive molecules. Due to the current constraints of exosome purification technology, we cannot completely separate exosomes from microvesicles. Therefore, the International Society for Extracellular Vesicles recommends that extracellular vesicles with a diameter of less than 200nm be collectively referred to as small extracellular vesicles (sEV).

[0003] Since sEV contains many bioactive molecules, it has certain therapeutic functions. For example, sEV derived from mesenchymal stem cells (MSC) (MSC-sEV) inherits the immunomodulation, tissue regeneration and repair potential of mesenchymal stem cells, and shows the ability to prevent and treat a variety of diseases, including but not limited to stroke, neurodegenerative diseases Parkinson's syndrome and Alzheimer's disease (AD), burns, respiratory diseases asthma and allergic rhinitis, deafness, acute kidney injury (AKI) and chronic kidney disease (CKD). In addition, sEV can be used as a carrier to carry effective molecules, nucleic acids or genes to play a therapeutic role in diseases.

[0004] However, sEV has the disadvantages of being expensive to prepare, difficult to prepare on a large scale, and having limited sources. Therefore, reducing the amount and increasing the efficiency is a major strategy to make up for the shortcomings of sEV and enhance its application value. However, there is currently a lack of effective sEV reduction and efficiency solutions. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide small extracellular vesicles containing Klotho protein and / or mRNA and a preparation method and application thereof. The preparation method can successfully prepare small extracellular vesicles rich in Klotho protein and mRNA, and the expression profile of bioactive molecules in the small extracellular vesicles has changed, and the therapeutic effect on AKI and AD has been significantly improved, which can achieve reduced dosage and increased efficacy.

[0006] The first aspect of the present invention is to provide a method for preparing small extracellular vesicles containing Klotho protein and / or mRNA, comprising the following steps: S1, constructing a recombinant expression vector containing a Klotho protein encoding gene; S2, introducing the recombinant expression vector into a receptor cell to obtain a receptor cell line with stably expressed Klotho gene; S3, culturing the receptor cell line, collecting the culture supernatant, and extracting small extracellular vesicles therefrom.

[0007] In some embodiments, the recombinant expression vector in step S1 is one or more of a recombinant lentiviral vector, a recombinant adenoviral vector, a recombinant retroviral vector, a recombinant liposome and a recombinant plasmid expression vector.

[0008] In some embodiments, the recipient cell in step S2 is a stem cell, HEK293T cell, HEK293F cell, Chinese hamster ovary cell (CHO), Sf9 cell, Sf21 cell or Hi5 cell.

[0009] In some embodiments, the stem cells are mesenchymal stem cells.

[0010] In some embodiments, the method for introducing the recombinant expression vector into the recipient cell in step S2 is viral infection, liposome transfection or electroporation transfection.

[0011] In some embodiments, the method of introducing the recombinant expression vector into the recipient cell in step S2 is a viral infection method;

[0012] Preferably, the recipient cells are infected with the virus twice.

[0013] In some embodiments, step S3 is: culturing the recipient cell line at 37±1°C, then transferring to 42±1°C for 1h to 6h, continuing to culture at 37±1°C, collecting the culture supernatant, and extracting small extracellular vesicles therefrom; preferably, transferring to 42±1°C for 1h to 4h; more preferably, transferring to 42±1°C for 1h to 3h.

[0014] In some embodiments, the Klotho protein comprises the amino acid sequence shown in SEQ ID NO.2.

[0015] In some embodiments, the Klotho protein encoding gene comprises the nucleotide sequence shown in SEQ ID NO.1.

[0016] The second aspect of the present invention is to provide small extracellular vesicles containing Klotho protein and / or mRNA prepared by the preparation method as described above.

[0017] The third aspect of the present invention is to provide the use of the small extracellular vesicles containing Klotho protein and / or mRNA as described above in the preparation of a drug for treating acute kidney injury.

[0018] The fourth aspect of the present invention is to provide the use of the small extracellular vesicles containing Klotho protein and / or mRNA as described above in the preparation of a drug for treating Alzheimer's disease.

[0019] In some embodiments, the small extracellular vesicles containing Klotho protein and / or mRNA are small extracellular vesicles derived from stem cells containing Klotho protein and / or mRNA; preferably small extracellular vesicles derived from mesenchymal stem cells containing Klotho protein and / or mRNA.

[0020] The fifth aspect of the present invention is to provide a drug for treating acute kidney injury, which comprises an active ingredient and a pharmaceutically acceptable excipient; the active ingredient includes the small extracellular vesicles containing Klotho protein and / or mRNA as described above.

[0021] The sixth aspect of the present invention is to provide a drug for treating Alzheimer's disease, the drug comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient includes the small extracellular vesicles containing Klotho protein and / or mRNA as described above.

[0022] The present invention has the following beneficial effects:

[0023] The present invention successfully stably and highly expresses the Klotho gene in the receptor cells, and obtains small extracellular vesicles (Klotho-sEV) rich in Klotho protein and / or mRNA secreted by the receptor cells. In addition, by stimulating and culturing the receptor cells at 42±1°C for a suitable time during the culture process, the yield of the Klotho-sEV can be effectively increased, and the Klotho-sEV can be produced efficiently.

[0024] The Klotho-sEV of the present invention can not only regulate the expression of endogenous Klotho, but also change the expression spectrum of bioactive molecules therein, enriching with a variety of kidney protective proteins and bioactive molecules such as miRNA, which can activate phosphorylation signaling pathways including mTOR and MEK1 / 2, thereby significantly enhancing the therapeutic effect on AKI. In addition, the efficacy of Klotho-sEV on AD is further improved compared with sEV, which may be because the expression spectrum of bioactive molecules related to AD treatment has also changed.

[0025] The expression profile of bioactive molecules in the small extracellular vesicles prepared by the present invention undergoes changes that are beneficial to the treatment of certain diseases, thereby enhancing the therapeutic effect on diseases including AKI and AD, and has high biosafety, thereby achieving a reduction in the amount of sEV and increasing its efficacy, thereby improving its clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the plasmid structure and the extraction of Klotho-sEV from the culture supernatant using anion chromatography.

[0027] Figure 2 The results of preparation and identification of MSCs overexpressing Klotho (LV-KL-MSCs).

[0028] Figure 3 The results of Klotho level detection in LV-KL-MSC cells.

[0029] Figure 4 These are the results of MSC marker molecule identification of LV-KL-MSC cells.

[0030] Figure 5 This is the identification result of Klotho-sEV.

[0031] Figure 6 This is the detection result of Klotho enrichment in Klotho-sEV.

[0032] Figure 7 These are the safety test results of Klotho-sEV.

[0033] Figure 8 Klotho-sEV alleviates myoglobin-induced HK2 death.

[0034] Fig. 9 Klotho-sEV significantly alleviated the symptoms of AKI in mice.

[0035] Fig.10 Klotho-sEV promotes kidney cell proliferation and reduces the expression of damage and inflammatory molecules.

[0036] Fig.11 Kloth-sEV treatment can upregulate the expression of endogenous Klotho in mice.

[0037] Fig.12 Proteomic mass spectrometry analysis of renal tissue.

[0038] Fig.13 Kloth-sEV treatment activates the mTOR and MEK1 / 2 phosphorylation signaling pathways.

[0039] Fig.14 These are the results of sEV / Kloth-sEV proteomic sequencing.

[0040] Fig.15 These are the sequencing results of sEV / Kloth-sEV small RNA.

[0041] Fig.16 The results of the mouse water maze experiment.

[0042] Fig.17 These are the results of the expression level detection of NLRP3, Pre-caspase1, Caspase1 and IL-1β in the mouse brain.

[0043] Fig.18 This is the identification result of Klotho-293T-sEV. DETAILED DESCRIPTION

[0044] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0045] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0046] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0047] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0048] Klotho was first discovered by Kuro-o et al. in 1997. Mice with Klotho gene (KL) defects will show phenomena similar to human aging, mainly manifested as premature aging characteristics such as shortened lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis, etc. The protein encoded by Klotho is a well-known anti-aging molecule.

[0049] Some embodiments of the present invention relate to a method for preparing Klotho-sEV, comprising the following steps: S1, constructing a recombinant expression vector containing a Klotho protein encoding gene; S2, introducing the recombinant expression vector into a receptor cell to obtain a receptor cell line in which the Klotho gene is stably expressed; S3, culturing the receptor cell line, collecting the culture supernatant, and extracting small extracellular vesicles therefrom.

[0050] In some embodiments, the recombinant expression vector is a vector that can mediate the expression of an exogenous Klotho protein encoding gene in a recipient cell, for example, a recombinant lentiviral vector, a recombinant adenoviral vector, a recombinant retroviral vector, a recombinant liposome, and a recombinant plasmid expression vector, etc. The recombinant expression vector can be introduced into the recipient cell by methods known in the art, such as viral infection, liposome transfection, electroporation transfection, etc.

[0051] In some preferred embodiments, the recombinant expression vector is a recombinant lentiviral expression vector, and the recombinant lentiviral expression vector is introduced into the recipient cell by viral infection.

[0052] In some preferred embodiments, the method for preparing Klotho-sEV comprises the following steps: S1, constructing a recombinant lentiviral vector containing a Klotho protein encoding gene; S2, transfecting a tool cell with the recombinant lentiviral vector to obtain a recombinant lentivirus; using the recombinant lentivirus to infect a receptor cell to obtain a receptor cell line in which the Klotho gene is stably expressed; S3, culturing the receptor cell line, collecting the culture supernatant, and extracting small extracellular vesicles therefrom.

[0053] More preferably, the preparation method of Klotho-sEV comprises the following steps: S1, constructing a recombinant lentiviral vector containing a Klotho protein encoding gene; S2, transfecting the recombinant lentiviral vector into tool cells to obtain a recombinant lentivirus; using the recombinant lentivirus to infect receptor cells, screening positive receptor cells for secondary viral infection, and obtaining a receptor cell line with stable expression of the Klotho gene; S3, culturing the receptor cell line, collecting the culture supernatant, and extracting small extracellular vesicles therefrom. It is generally believed in the art that the target gene can be effectively integrated into the receptor cell through a single viral infection. However, the inventors found that the infection efficiency of the recombinant lentiviral vector of the present invention is low in a single viral infection, and a secondary infection can significantly increase the viral infection rate.

[0054] In some embodiments, step S3 is: culturing the receptor cell line at 37±1°C, then transferring to 42±1°C for 1h to 6h, continuing to culture at 37±1°C, collecting the culture supernatant, and extracting small extracellular vesicles therefrom; preferably, transferring to 42±1°C for 1h to 4h; more preferably, transferring to 42±1°C for 1h to 3h. The inventors have found through research that stimulating and culturing the receptor cells with stably expressed Klotho gene at 42±1°C for a suitable time during the culture process can effectively increase the yield of Klotho-sEV and efficiently produce the Klotho-sEV.

[0055] In some embodiments, the culture is continued at 37±1° C. for 36 h to 56 h, and the culture supernatant is collected; preferably, the culture is continued at 37±1° C. for 42 h to 48 h, and the culture supernatant is collected.

[0056] In some preferred embodiments, the recombinant lentiviral vector is LV-EF1a-3XFlag-Klotho-P2A-PGK-Puro-WPRE.

[0057] In some preferred embodiments, the recipient cells are stem cells, preferably MSCs. MSCs are a type of multipotent stem cells derived from the mesoderm, which have multiple functions such as self-renewal, tissue replacement, immune regulation, and anti-fibrosis. These cells have the advantages of low immunogenicity and can be transplanted by a third party. Currently, their usage rate in clinical trials is second only to blood stem cells.

[0058] In some preferred embodiments, the culture supernatant is a serum-free culture supernatant.

[0059] In some preferred embodiments, small extracellular vesicles are extracted from the culture supernatant using conventional techniques in the art, such as anion chromatography, ultracentrifugation, size exclusion chromatography, immunoprecipitation, and the like.

[0060] In some preferred embodiments, small extracellular vesicles are extracted from the culture supernatant by anion chromatography, and the anion chromatography comprises the following steps: 1) adding an equilibrium solution to the chromatography column to add the gel to the chromatography column, and after the gel is evenly precipitated, the equilibrium solution is drained, and then the equilibrium solution is added to wash the buffer gel; 2) adding the collected culture supernatant, and then adding a washing solution to remove protein impurities not derived from extracellular vesicles; 3) adding an eluent to the chromatography column, and collecting the eluent into a sample collection tube, repeating the above steps to obtain an eluent; 4) identifying the sEV concentration of the eluent by the protein concentration in the sample; 5) according to the result of step 4), determining the eluent rich in sEV, mixing it and dialyzing it against PBS to obtain Klotho-sEV.

[0061] In some preferred embodiments, the amino acid sequence of the Klotho protein is shown in SEQ ID NO. 2, and the nucleotide sequence of the gene encoding the Klotho protein is shown in SEQ ID NO. 1. The present invention successfully integrates the CDS sequence of NM 004795.4 (as shown in SEQ ID NO. 1) into the genome of the recipient cell for the first time and achieves stable high expression, and makes the sEV secreted by the recipient cell rich in Klotho protein and / or mRNA, and successfully prepares Klotho-sEV.

[0062]

[0063]

[0064] In some embodiments, the Klotho protein encoding gene can be co-transfected with exosome-related genes to increase its expression in sEVs.

[0065] Some embodiments of the present invention involve a Klotho-sEV, which is prepared by the above-mentioned preparation method. The Klotho-sEV is rich in Klotho protein and mRNA, which can not only regulate the expression of endogenous Klotho, but also change the expression spectrum of biologically active molecules therein, especially rich in a variety of kidney protective proteins and miRNA, which can activate phosphorylation signaling pathways including mTOR and MEK1 / 2.

[0066] Some embodiments of the present invention relate to the use of the above-mentioned Klothho-sEV in the preparation of drugs for the treatment of acute kidney injury.

[0067] AKI is a common clinical syndrome characterized by a sudden, rapid, and sustained decline in renal function within a few hours or days, which causes elevated serum creatinine and urea, accumulation of toxic substances in the body, and imbalances in water, electrolytes, and acid-base levels. If AKI is not identified, intervened, and treated in a timely manner, it will seriously or even irreversibly damage kidney function, and may progress to chronic kidney disease, or even lead to death. AKI is extremely harmful, and is mainly treated symptomatically. The clinical demand for more effective, accurate, and specific treatments for AKI has not yet been met and is in urgent need of being met.

[0068] The Klotho-sEV of the present invention can restore endogenous Klotho expression and may improve acute kidney injury by activating the mTOR and MEK1 / 2 signaling pathways. We performed proteomic sequencing on Klotho-sEV and MSC-sEV, and detected a total of 322 differentially expressed proteins, most of which were upregulated in Klotho-sEV. These differentially expressed proteins include molecules related to anti-inflammatory effects, involvement in damaged kidney repair and maintenance of renal function. This indicates that the protein expression profile in Klotho-sEV has changed, producing a variety of proteins that can participate in the repair of acute kidney injury.

[0069] In addition to proteins, small RNA is the main nucleic acid component of sEVs, among which miRNA, a type of non-coding single-stranded RNA, mainly plays a role through RNA post-transcriptional regulation, thereby participating in sEV-mediated intercellular signal transduction. In the small RNA sequencing of Klotho-sEVs and MSC-sEVs, we detected a total of 1007 miRNAs, of which 51 were differentially expressed miRNAs, and these differential miRNAs included molecules related to anti-inflammation and tissue repair.

[0070] In addition, we found that Klotho mRNA was also enriched in Klotho-sEVs. Previous studies have shown that sEVs can exert their effects through mRNA, and sEVs can restore skeletal muscle regeneration and the biological functions of aging cells through Klotho mRNA.

[0071] Some of the embodiments involve the use of the above-mentioned Klotho-sEV in the preparation of drugs for treating Alzheimer's disease.

[0072] As mentioned above, the expression profile of bioactive molecules in Klotho-sEV has changed. In addition to being enriched with more bioactive molecules with kidney-protective effects, the therapeutic effect of Klotho-sEV on Alzheimer's disease is also further enhanced compared with MSC-sEV, including enhancing the cognitive function of model animals and reducing neuroinflammatory responses in the brain.

[0073] Some of the embodiments involve a drug for treating acute kidney injury, comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient includes the Klotho-sEV as described above.

[0074] Some of the embodiments involve a drug for treating Alzheimer's disease, comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient includes the Klotho-sEV as described above.

[0075] In some preferred embodiments, the drug may use Klotho-sEV as a single active ingredient, or may use Klotho-sEV in combination with other substances as active ingredients.

[0076] In some preferred embodiments, the pharmaceutically acceptable excipients are not particularly limited and may be conventional excipients in the art. Appropriate excipients may be selected according to the pharmaceutical dosage form to be prepared and the requirements.

[0077] The present invention is further described in detail below with reference to specific embodiments.

[0078] In the following embodiments:

[0079] (1) Source of experimental animals

[0080] 4-6 weeks old C57BL / 6 strain SPF female mice were bred and distributed by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. and raised and conducted in the SPF barrier environment of Area F of the Animal Experiment Center of Sun Yat-sen University North Campus. All animal operations in this experiment were strictly carried out in accordance with the requirements of experimental animal welfare and ethics of Sun Yat-sen University, and the pain caused to animals by experimental operations was minimized as much as possible. The approval number for the use of experimental animals and ethical review is SYSU-IACUC-2022-000827.

[0081] (2) Schematic drawing

[0082] The schematic diagram was created using BioRender and Adobe Photoshop.

[0083] (3) Statistical methods

[0084] The experimental data were statistically analyzed using GraphPad Prism 10.1.2 software. The differences between two groups of data were compared using t-test, and the differences between multiple groups of data were compared using one-way analysis of variance. Each group of data was presented as mean ± SEM, and P < 0.05 indicated statistical significance.

[0085] Example 1 Preparation and identification of Klotho-sEV

[0086] 1. Experimental Methods

[0087] 1.1 Preparation of Klotho-overexpressing mesenchymal stem cell line (LV-KL-MSC):

[0088] First, a lentivirus overexpressing Klotho was constructed, and then frozen iPSC-MSC (hereinafter referred to as MSC) was revived and infected with lentivirus to construct a LV-KL-MSC cell line stably overexpressing Klotho. MSC was cultured in N1 medium (as shown in Table 1). The specific operation process is as follows:

[0089] Table 1 N1 medium formula

[0090]

[0091] 1) First, a LV-EF1a-3XFlag-Klotho-P2A-EGFP-PGK-Puro-WPRE lentiviral plasmid vector was constructed (LV-EF1a-3XFlag-P2A-EGFP-PGK-Puro-WPRE and LV-EF1a-3XFlag-Klotho-P2A-EGFP-PGK-Puro-WPRE were purchased from Wuhan Shumi Brain Science Technology Co., Ltd., and the plasmid structure is as follows Figure 1 As shown in A). This time, a three-plasmid system was used to transfect 293T cells to package lentiviral particles, separate Gag / Pol from the vector, and use the membrane protein of VSV-G. The packaged lentivirus has a wide host range and is an ideal tool for gene transduction.

[0092] 2) The above plasmid vector was transfected into 293T cells, and the supernatant was extracted, concentrated and titrated.

[0093] 3)1.5× 10 4 MSCs were plated into 24-well culture plates.

[0094] 4) Calculate the amount of virus to be added based on the inoculation density and MOI 30-50. Prepare for infection when the cell confluence is 30%-40%. Preheat the culture medium in advance before infection and change the medium half an hour in advance.

[0095] 5) Take out the human Klotho lentivirus, put it on ice to melt, and centrifuge it in a small desktop centrifuge to make the virus liquid accumulate at the bottom of the tube.

[0096] 6) Add the virus and polybrene with a final concentration of 2μg / mL to 10μg / mL according to the calculated amount of virus, and gently shake the plate to mix. The virus can be diluted with culture medium in advance according to the experimental situation.

[0097] 7) 24 hours after the first virus infection, replace the culture medium with new one and culture for 24 hours. Then, subculture the cells to two wells of a 24-well plate and screen them with puromycin after the cells adhere to the plate.

[0098] 8) After one round of drug screening, virus infection is performed again under the same conditions, followed by drug screening.

[0099] 9) After 2-4 days, the fluorescence expression can be observed and related tests can be performed, and the cells can be kept as seeds.

[0100] 1.2 MSC / LV-KL-MSC identification:

[0101] 1.2.1 Detection of GFP positive rate to confirm cell infection rate

[0102] 1) Cell slides: MSC / LV-KL-MSC cells at 5000 / cm 2 The cells were inoculated into a culture plate with a glass slide added, and after the cells grew to an appropriate density, the nuclei were stained with DAPI, the glass slide was removed, and the GFP-positive rate of the cells was observed under a fluorescence microscope.

[0103] 2) Flow cytometry: MSC / LV-KL-MSC cells were taken for flow cytometry analysis to detect the GFP positivity rate.

[0104] 1.2.2 Determination of KL gene expression in MSC / LV-KL-MSC cells

[0105] 1) Note that RNA is unstable. Unless otherwise specified, all operations should use RNase-free consumables and reagents and should be performed on ice.

[0106] 2) Add 1 mL of Trizol reagent to the EP tube containing MSC / LV-KL-MSC cells (operate in a fume hood), oscillate on a vortex oscillator at maximum amplitude for 15 seconds, and then let stand at room temperature for 5 minutes.

[0107] 3) Add 200 μL of chloroform to the EP tube (operate in a fume hood), oscillate on a vortex oscillator at maximum amplitude for 15 seconds, and then let stand at room temperature for 2-3 minutes.

[0108] 4) Centrifuge at 12000g, 4℃ for 15-20min. The liquid will be divided into 3 layers after centrifugation. During centrifugation, label a new RNase-free EP tube for later use.

[0109] 5) Carefully pipette the colorless liquid on the upper layer of the EP tube into a new EP tube, then add 500 μL of pre-cooled isopropanol into the tube and gently invert it upside down to mix the liquid. This step should be performed in a fume hood.

[0110] 6) Centrifuge at 12000R, 4°C for 15 min. White precipitate can be seen at the bottom of the EP tube after centrifugation.

[0111] 7) Discard the liquid in the EP tube, add 1 mL of pre-cooled 75% ethanol, gently invert upside down to mix the liquid, and then centrifuge at 7500g, 4°C for 5 minutes.

[0112] 8) Repeat step (7) once.

[0113] 9) Discard the liquid in the EP tube and dry it on ice in a fume hood (about 7 minutes). Then add an appropriate amount of RNase-free water to resuspend the RNA. Measure the RNA concentration and quality using Nanodrop. Reverse the extracted RNA into cDNA or store it in a -80°C refrigerator.

[0114] 10) Take 1000 ng RNA, add 4 μL reverse transcription Master Mix, quantify to 20 μL with RNase-free water, and then perform RNA reverse transcription according to the conditions in Table 2; dilute the reversed cDNA to 5 ng / μL with ddH2O.

[0115] Table 2 RNA reversal conditions

[0116]

[0117] 11) Take 20 ng cDNA for real-time quantitative polymerase chain reaction (RT-PCR), and use the ΔΔt method for data analysis. The RT-PCR reaction system is shown in Table 3, and the primer information is shown in Table 4.

[0118] Table 3 RT-PCR system

[0119]

[0120] Table 4 PCR primers

[0121]

[0122]

[0123] 1.2.3 Flow cytometry analysis to detect the expression of surface markers of MSC / LV-KL-MSC

[0124] MSC / LV-KL-MSC cells were taken and incubated with anti-HLA-DR-APC / Cyanine7, anti-CD14-PE-CYN7, anti-CD73-APC, anti-CD 105-APC, anti-CD90-APC, anti-CD34-PE, anti.CD45.APC, anti-CD19-PerCP-Cyanine5.5 and other flow cytometry antibodies at 4°C in the dark for 30 minutes. After washing with PBS, the cells were detected by Beckman flow cytometry.

[0125] 1.3 Preparation of sEV / Klotho-sEV

[0126] 1.3.1 Obtaining culture supernatant in serum-free culture system

[0127] 1) Thaw frozen MSC / LV-KL-MSC cells to 150cm 2 The culture dishes were used for culture and subculture at 37°C, 5% CO2, and N1 medium.

[0128] 2) When the cells grow to a density of about 85%-90%, the culture medium is replaced with 15 mL of serum-free CDPF medium (configuration as shown in Table 5) and divided into two groups for the experiment: Group 1 continues to be cultured at 37°C for 3 h, and Group 2 is transferred to 42°C for 3 h; both Group 1 and Group 2 discard the culture medium and replace it with 30 mL of CDPF medium and continue to be cultured at 37°C for 42 h.

[0129] Table 5 CDPF medium preparation

[0130]

[0131] 3) Obtain the culture supernatant enriched with extracellular vesicles: Precool the centrifuge to 4°C, pipette the CDPF culture medium from the culture dishes of group 1 and group 2 into 50 mL centrifuge tubes, centrifuge at 4°C, 2650g for 20 min. If extraction is not performed immediately, store at -20°C.

[0132] 1.3.2 Extraction of sEV / Klotho-sEV from culture supernatant using anion chromatography:

[0133] The extraction process is as follows Figure 1 As shown in B, the specific steps are as follows:

[0134] 1) Add 4 mL of equilibration solution to the chromatography column. Add the gel (packed in 4 mL solid phase) to the chromatography column. After the gel is evenly precipitated, slowly drain the equilibration solution. Then gently add 12 mL of equilibration solution to wash the buffer gel.

[0135] 2) Slowly add 150 mL of culture supernatant, and then slowly add 40 mL of washing solution to remove non-extracellular vesicle-derived protein impurities.

[0136] 3) Slowly add 1 mL of eluent to the chromatography column and collect the eluent into a sample collection tube. Repeat the above steps 8 times to obtain 8 mL of eluent.

[0137] 4) Identify the concentration of sEV / Klotho-sEV in the eluate by the protein concentration in the sample.

[0138] 5) Based on the results of step 4, determine the eluate rich in sEV / Klotho-sEV, mix them and dialyze them against PBS.

[0139] 6) Collect sEV samples after dialysis for further analysis, use Coomassie Brilliant Blue method and BCA method to detect protein concentration and Nanosight to detect the concentration of purified sEV / Klotho-sEV samples. sEV / Klotho-sEV are packaged and stored in a -80℃ refrigerator.

[0140] The equilibration, washing and elution solution formulations are as follows:

[0141] Balance solution: 25mL Phosphate Buffer (1M) + 5.0mL NaCl (5.0M) + 470mL H2O;

[0142] Washing solution: 25 mL Phosphate Buffer (1 M) + 10.0 mL NaCl (5.0 M) + 465 mL H2O;

[0143] Elution solution: 25 mL Phosphate Buffffer (1 M) + 100.0 mL NaCl (5.0 M) + 375 mL H2O.

[0144] 1.4 sEV / Klotho-sEV identification

[0145] 1.4.1 Nanosight Identification of sEV / Klotho-sEV Particle Characteristics:

[0146] The prepared sEV / Klotho-sEV samples were diluted with vesicle-free PBS at a ratio of 1:100 or 1:200. 1 mL of the diluted sample was loaded onto the instrument and tested using the Nanosight NS300 instrument. Each sample was tested 3 times, each time for 30 seconds, and the particle concentration and particle size distribution of sEV / Klotho-sEV were recorded.

[0147] 1.4.2 Transmission electron microscopy to identify sEV / Klotho-sEV vesicle morphology:

[0148] The prepared sEV / Klotho-sEV samples were fixed with 2% glutaraldehyde for 30 min, 10 μL of the fixed samples were pipetted onto the copper mesh, 3% phosphotungstic acid was added to the copper mesh, ddH2O was added after 2 min, and the samples were dried for 5-10 min before observation and photography under an electron microscope.

[0149] 1.4.3 Identification of sEV / Klotho-sEV exosome marker molecules by Western Blot:

[0150] 1) Protein sample preparation: After RIPA lysis of MSC, LV-KL-MSC, sEV, and Klotho-sEV samples, protein was determined by the BCA method. Samples were then prepared to a protein concentration of 1 μg / μL, denatured (treated at 95°C for 10 min), aliquoted, and subjected to western blotting experiments or stored in a -80°C refrigerator.

[0151] 2) Electrophoresis: Prepare a PAGE gel with a separation gel concentration of 10%, add equal amounts of protein samples to the PAGE gel, and then perform electrophoresis at a constant voltage of 80 V for 20 min, and then at 120 V for 60 min-80 min.

[0152] 3) Transfer: Assemble the sandwich clamp, transfer the PAGE gel, and transfer the membrane at a constant current of 250mA for 150min.

[0153] 4) Blocking: Prepare skim milk powder with TBST as solvent to make a 5% milk powder liquid, wash the PVDF membrane with TBST for 5 minutes, repeat the washing for 3 times, and then block with 5% milk powder at room temperature for 1-2 hours.

[0154] 5) Incubation with primary antibody: Primary antibody preparation: Use 5% milk to prepare primary antibody, and the antibody dilution ratio is Calnexin (1:1000), GM130 (1:1000), CD9 (1:2000), CD63 (1:2000), CD81 (1:2000), Alix (1:5000), TSG101 (1:1000) and Klotho (1:1000). Wash the blocked PVDF membrane with TBST for 10 minutes, repeat the washing for 3 times, and then incubate with the corresponding antibody at 4°C overnight.

[0155] 6) Incubation with secondary antibody: Secondary antibody preparation: Use 5% milk to prepare secondary antibody, and the antibody dilution ratio is 1:2000. Wash the PVDF membrane with TBST for 10 minutes, repeat the washing for 3 times, and then incubate with secondary antibody at room temperature for 1 hour.

[0156] 7) Development: The PVDF membrane was washed with TBST for 10 min, and the washing was repeated 3 times; then the PVDF membrane was reacted with the luminescent solution and developed on a chemiluminescence instrument.

[0157] 2. Experimental results

[0158] 2.1 Successful preparation of MSC cell line overexpressing Klotho (LV-KL-MSC)

[0159] First, we constructed a LV-EF1a-3XFlag-Klotho-P2A-EGFP-PGK-Puro-WPRE recombinant lentiviral plasmid vector and used 293T cell lines to complete lentiviral packaging. Then, lentivirus infected MSC cells to obtain a stable cell line ( Figure 2 The cells infected by the lentivirus spontaneously emitted green fluorescence (GFP). Immunofluorescence showed that the LV-KL-MSC cells were GFP positive and had indeed completed the lentivirus infection. Flow cytometry further confirmed that the LV-KL-MSC virus infection rate reached 74% ( Figure 2 Middle B~ Figure 2 Middle C), the infection rate is higher.

[0160] After the first lentiviral infection of MSC cells, we used fluorescence microscopy to observe the expression of marker GFP. Figure 2 Middle D) shows that compared with the control LV-C treatment group, the LV-KL treatment group has a small proportion of GFP-positive cells and weak GFP fluorescence intensity, a low infection rate, and a single viral infection failed to efficiently integrate the Klotho protein encoding gene into the recipient cell MSC as expected. This may be because the number of bases of the Klotho protein encoding gene connected in the recombinant lentiviral vector is too large and is not easy to integrate into the recipient cell MSC. To this end, we conducted a large number of experiments and found that repeated secondary viral infection of drug-screened positive recipient cells after a single viral infection can effectively improve the infection efficiency of the recombinant lentiviral vector.

[0161] To confirm that lentiviral infection had successfully integrated the Klotho gene into MSC cells, we performed RT-PCR experiments, and the results showed that the Klotho mRNA level in LV-KL-MSCs was significantly increased compared with MSCs, up to 150 times ( Figure 3 Subsequently, we performed a Western Blot experiment, and the results showed that LV-KL-MSC Klotho protein was also overexpressed ( Figure 3 (middle B).

[0162] As a type of pluripotent stem cell, MSCs express multiple biomarkers, specifically CD73, CD90, and CD105 positive, and CD14, CD19, CD34, CD45, and HLA-DR negative. In order to verify whether overexpression of Klotho changes the basic properties of MSCs, we performed flow cytometry analysis on LV-KL-MSCs, and the results showed that overexpression of Klotho did not change the expression of MSC surface biomarkers ( Figure 4 ).

[0163] The above results suggest that we successfully constructed a Klotho-overexpressing MSC stable cell line LV-KL-MSC using lentiviral infection.

[0164] 2.2 Preparation and identification of Klotho-sEV

[0165] At present, conventional methods for extracting sEV, such as ultracentrifugation, size exclusion chromatography, and immunoprecipitation, all have their own limitations, such as time-consuming, instrument-dependent, low extraction efficiency, and inability to scale up. In order to achieve better clinical transformation in the future, our research group has established a scalable and efficient method system for preparing sEV using anion chromatography technology, and successfully and efficiently obtained sEV derived from mesenchymal stem cells. Nanosight detection showed that the average particle size of sEV was 163.5nm, and the average particle size of Klotho-sEV was 166.71nm ( Figure 5 Middle A), sEV and Klotho-sEV both have a double-layer membrane structure under transmission electron microscopy ( Figure 5 Middle B), Western Blot analysis showed that both sEV and Klotho-sEV expressed sEV marker molecules CD9, CD63, CD81, Alix, and TSG101, but did not express cell markers Ca1nexin and GM130 ( Figure 5 These results suggest that anion chromatography can successfully purify sEVs from culture supernatants and that Klotho-sEVs derived from engineered LV-KL-MSCs possess the basic properties of sEVs.

[0166] In order to further increase the production of Klotho-sEV secreted by LV-KL-MSC cells, we conducted a lot of research and optimization. The results showed that transferring LV-KL-MSC cells to 42°C for a suitable stimulation culture time (1h~6h, preferably 1h~4h, more preferably 1h~3h) during the culture process can increase the production of Klotho-sEV and effectively increase the amount of Klotho-sEV secreted by them. Taking the example of transferring LV-KL-MSC cells to 42°C for 3h during the culture process, Figure 5 As shown in D, the concentration of sEV in the supernatant of group 2 (treated at 42°C) was significantly increased compared with group 1 (conventional culture). In group 1, LV-KL-MSC cells were cultured at 37°C, while in group 2, LV-KL-MSC cells were transferred to 42°C for stimulation culture for 3h, and then continued to be cultured at 37°C, and finally the culture supernatant was collected to extract Klotho-sEV. After 42°C stimulation culture, the amount of Klotho-sEV secreted into the culture supernatant by LV-KL-MSC cells increased significantly.

[0167] Next, we validated Klotho-sEVs derived from engineered LV-KL-MSC cells. Western Blot results showed that Klotho protein was significantly enriched in Klotho-sEVs compared with unengineered sEVs ( Figure 6RT-PCR showed that Klotho mRNA was not detected in the unengineered sEVs, but was detected in Klotho-sEVs ( Figure 6 These results suggest that Klotho-sEVs derived from engineered LV-KL-MSC cells not only achieve Klotho protein enrichment but also successfully carry Klotho mRNA.

[0168] Based on the above results, we successfully constructed Klotho-sEVs enriched with Klotho, and found that Klotho-sEVs enriched with Klotho did not affect the particle size characteristics, vesicle morphology and expression of sEV marker molecules.

[0169] Example 2 Biosafety Assessment of Klotho-sEV

[0170] Biosafety is related to life and health, and is also the starting step of drug clinical trials. In order to promote the clinical transformation of Klotho-sEV, we first need to evaluate the biosafety of Klotho-sEV in animals. sEV / Klotho-sEV in vivo safety testing:

[0171] 1. Experimental Methods

[0172] C57BL / 6 strain SPF female mice were raised and the experiments were conducted in an SPF barrier environment. Figure 7 As shown in A. On day 0 and day 1, 100 μg sEV / Klotho-sEV or an equal volume of PBS were injected into the tail vein of mice, and the weight of mice was recorded on days 0, 1, 3, 5, 7, 9, 11, and 13. On day 13, the mice were killed and samples were collected for detection.

[0173] 1.1 Serum creatinine and urea test:

[0174] 1) Blood was collected from the mouse eyeball and placed in a clean EP tube and allowed to stand at room temperature for 1-2 hours.

[0175] 2) Centrifuge at 3000R, 4℃ for 15-20min, aspirate the upper serum into a new EP tube, and test immediately or store in a -80℃ refrigerator.

[0176] 3) The levels of serum creatinine and urea were detected by automatic biochemical analyzer to evaluate the renal function of mice.

[0177] 1.2 Hematoxylin-eosin staining (HE staining) detection:

[0178] 1) After perfusion of mouse heart with PBS, the brain, heart, liver, spleen, lung and kidney were removed and fixed with 4% paraformaldehyde at room temperature. After dehydration, they were embedded in paraffin and cut into slices.

[0179] 2) HE staining of sections: The sections were dewaxed - stained with hematoxylin - washed with water - 1% hydrochloric acid ethanol for 30 seconds - washed with water - eosin staining - washed with water - dehydrated - sealed with neutral resin.

[0180] 3) Observe and record the condition of each organ under a microscope.

[0181] 2. Experimental results

[0182] We designed a safety assessment experiment using C57BL6 mice. We divided the animals into three groups: PBS group (control group), sEV group, and Klotho-sEV group. On day 0 and day 1, 100 μg sEV / Klotho-sEV or an equal volume of PBS were injected into the tail vein of the mice, and the body weights of the mice were recorded on days 0, 1, 3, 5, 7, 9, 11, and 13. The mice were killed on day 13 and samples were collected for testing ( Figure 7 We found that sEV and Klotho-sEV treatment had no significant effect on the body weight and renal function of mice compared with the control group ( Figure 7 Middle B~ Figure 7 In addition, HE staining results showed that sEV and Klotho-sEV treatment had no significant effect on mouse brain, heart, liver, spleen, lung, kidney and other tissues ( Figure 7 These results suggest that sEV and Klotho-sEV have high biological safety and have no obvious toxic side effects on mice.

[0183] Example 3 K10tho-sEV alleviates HK2 death caused by myoprotein

[0184] 1. Experimental Methods

[0185] 1.1 HK2 cell experiments

[0186] HK2 cells were generously donated by Professor Zhou Yi from the Department of Nephrology, the First Affiliated Hospital of Sun Yat-sen University. HK2 cells were cultured in complete DMEM / F12 medium composed of 90% DMEM / F12, 10% FBS, and 1% double antibody.

[0187] 1.2 Myoglobin-induced cytotoxicity model

[0188] 1) This experiment was divided into 4 groups: control group (PBS), model group (4 mg / mL Myoglobin + PBS), model + sEV group (4 mg / mL Myoglobin + 25 μg / mL sEV), model + Klotho-sEV (4 mg / mL Myoglobin + 25 μg / mL Klotho-sEV).

[0189] 2) HK2 cells at 5000 / cm 2 The cells were inoculated into culture plates, and 25 μg / mL sEV / Klotho-sEV or an equal volume of PBS was added to the culture medium and co-cultured with HK2 cells for 24 h.

[0190] 3) 240 μM Myoglobin and 2.40 mM L-ascorbic acid were mixed in advance; the corresponding groups were treated according to the experimental design, and 4 mg / mL Myoglobin was treated for 20-24 hours.

[0191] 4) Digest with EDTA-free trypsin and collect the cells by centrifugation at 300g and 4°C for 5 min.

[0192] 5) Wash the cells with pre-cooled PBS and centrifuge at 300g and 4°C for 5 min; repeat this step once.

[0193] 6) Take 1-5x10 5 Discard PBS, add 100 μL 1× Binding Buffer to resuspend the cells, then add 5 μL Annexin V-FITC and 10 μL PI Staining Solution, and mix gently.

[0194] 7) Incubate at room temperature in the dark for 10-15 min, add 400 μL 1× Binding Buffer, mix well and place on ice. Detect the sample by flow cytometry within 1 hour.

[0195] 2. Experimental results

[0196] Rhabdomyolysis can cause the release of muscle cell contents (such as enzymes, potassium, phosphate, creatinine and myoglobin) into the extracellular fluid and blood circulation, which in turn causes tubular obstruction, necrosis and cast formation, leading to AKI. In order to explore the effect of Klotho-sEV on AKI, we treated HK2 cells with 4 mg / mL myoglobin to induce cell apoptosis. Figure 8 As shown, we found that myoglobin treatment can cause apoptosis in 30% of HK2 cells. Compared with the model group, Klotho-sEV can reduce the proportion of apoptotic cells to the level of the PBS control group and maintain the proportion of live cells at the same level as the PBS control group. Although sEV treatment has an effect trend, there is no statistical difference. These results show that Klotho-sEV can promote the proliferation of damaged HK2 cells and weaken the killing effect of myoglobin on HK2 cells compared with sEV. Among them, A. Typical flow cytometry images of HK2 cells stained with Annexin-V and PI; B. Statistical graph of apoptotic cells; C. Statistical graph of live cells. *P<0.05, ns, no statistical difference.

[0197] Example 4 Klotho-sEV can alleviate renal injury in AKI mice

[0198] 1. Experimental Methods

[0199] Induction of AKI mice and the effects of sEV / Klotho-sEV on AKI

[0200] C57BL6 mice were deprived of water 18-24 h in advance as described above, and 50% hypertonic glycerol was injected intramuscularly at 8 mg / kg into the hind limbs. 24 h after intramuscular injection of hypertonic glycerol, 100 μg sEV / Klotho-sEV or an equal volume of saline was injected into the tail vein, and the mice were killed at the end of the experiment to obtain samples for subsequent experiments.

[0201] 1.1 Serum creatinine and urea test:

[0202] 1) Blood was collected from the mouse eyeball and placed in a clean EP tube and allowed to stand at room temperature for 1-2 hours.

[0203] 2) Centrifuge at 3000R, 4℃ for 15-20min, aspirate the upper serum into a new EP tube, and test immediately or store in a -80℃ refrigerator.

[0204] 3) The levels of serum creatinine and urea were detected by automatic biochemical analyzer to evaluate the renal function of mice.

[0205] 1.2 Hematoxylin-eosin staining (HE staining) detection:

[0206] 1) After perfusion of mouse heart with PBS, kidneys were removed and fixed with 4% paraformaldehyde at room temperature, dehydrated, embedded in paraffin and cut into slices.

[0207] 2) HE staining of sections: The sections were dewaxed - stained with hematoxylin - washed with water - 1% hydrochloric acid ethanol for 30 seconds - washed with water - eosin staining - washed with water - dehydrated - sealed with neutral resin.

[0208] 3) Observe and record the casts and tubular necrosis under a microscope.

[0209] 1.3 Periodic acid Schiff staining (PAS staining)

[0210] 1) After perfusion of mouse heart with PBS, kidneys were removed and fixed with 4% paraformaldehyde at room temperature, dehydrated, embedded in paraffin and cut into slices.

[0211] 2) PAS staining of sections: dewax paraffin sections to water - wash with distilled water - rinse with periodic acid solution - rinse with tap water - Schiff's solution - rinse with running water - stain the nucleus with hematoxylin - rinse with running water - routine dehydration, transparentization and sealing.

[0212] 3) Observe and record the casts and tubular necrosis under a microscope.

[0213] 1.4 Proliferating cell nuclear antigen (PCNA) immunohistochemical staining

[0214] 1) After perfusion of mouse heart with PBS, kidneys were removed and fixed with 4% paraformaldehyde at room temperature, dehydrated, embedded in paraffin and sliced.

[0215] 2) Dewax and rehydrate the sections according to conventional procedures.

[0216] 3) Antigen retrieval: The sections were boiled in citric acid retrieval solution at 95-100°C for 15 min, and then washed with TBS for 5 min, twice.

[0217] 4) Inactivation of endogenous peroxidase: Wash the sections with 3% H2O2 at room temperature for 10 min, then with TBS for 5 min, three times.

[0218] 5) Block with 5% goat serum at room temperature for 1 h.

[0219] 6) Primary antibody incubation: PCNA 1:400 dilution, incubate overnight at 4°C. Then wash with TBS for 3-5 minutes, 3 times.

[0220] 7) Secondary antibody incubation: incubate at room temperature for 30 min, then wash with TBS for 3-5 min, 3 times.

[0221] 8) BAD color development for 40-50 seconds, then washed with water.

[0222] 9) Hematoxylin staining for 5 minutes.

[0223] 10) The sections were then dehydrated according to conventional procedures.

[0224] 11) Sealing: After air drying, seal the slides with neutral resin.

[0225] 12) Observe under a microscope.

[0226] 1.5RT-PCR determination of the expression of inflammatory factors and pathological molecules in kidney tissue

[0227] 1) Take an appropriate amount of chopped kidney tissue and put it into an RNase-free EP tube with a locker, add an appropriate amount of TRIzol reagent and steel beads (tissue: steel beads: TRIzol volume = 1:1:2), oscillate at speed 8 in a homogenizer for 1 min, transfer the EP tube to crushed ice for 5 min, add TRIzol reagent to 1 mL, centrifuge briefly at 7500R for 1 min, and aspirate the supernatant into a new EP tube.

[0228] 2) The subsequent RNA extraction steps and RT-PCR method were the same as above. The RT-PCR primer sequences are shown in Table 6.

[0229] Table 6 RT-PCR primers for kidney injury molecules and inflammatory factors

[0230]

[0231]

[0232] 2. Experimental results

[0233] In order to further explore the therapeutic effect of Klotho-sEV on AKI in vivo, we designed an AKI model caused by rhabdomyolysis, which is simple to operate and relatively stable. The specific operation is: C57BL6 mice were deprived of water for 18-24 hours and then injected with 50% hypertonic glycerol at 8 mg / kg intramuscularly. We administered sEV / Klotho-sEV once through the tail vein 24 hours after AKI modeling ( Fig. 9 We found that after intramuscular injection of 50% hypertonic glycerol, serum creatinine and urea in the Vehicle group mice were significantly increased (more than 1.5 times) compared with the control PBS group (Control group), indicating that our model was successfully constructed. Klotho-sEV treatment can significantly reduce serum creatinine and urea levels compared with the Vehicle group. It is worth mentioning that in our experimental system, sEV also has a tendency to reduce serum creatinine and urea, but there is no statistical difference ( Fig. 9 Middle B~ Fig. 9 Tubular necrosis and tubular cast formation are the renal histopathological features of AKI caused by rhabdomyolysis. We found that the number of tubular casts and tubular necrosis were obvious in the renal HE-stained sections of the mice in the Vehicle group injected with 50% hypertonic glycerol. Compared with the Vehicle group, Klotho-sEV treatment could significantly reduce the formation of tubular casts and tubular necrosis ( Fig. 9 At the same time, we also performed PAS staining of mouse kidneys. Consistent with HE staining, the number of casts in the Klotho-sEV group was significantly reduced and tubular necrosis was significantly alleviated ( Fig. 9 Middle E).

[0234] We further explored the effect of Klotho-sEV on renal cell proliferation. PCNA immunohistochemical staining results showed that Klotho-sEV could significantly enhance renal cell proliferation compared with the Vehicle group ( Fig.10Middle A). Studies have shown that the levels of inflammatory factors and injury molecules in renal tissues are increased in the AKI state. To explore the role of Klotho-sEV, we extracted RNA from renal tissues to detect differences in transcriptional levels of related molecules. Consistent with previous research results, 50% hypertonic glycerol treatment can significantly increase the levels of renal injury molecules PAI, NGAL, and SOX9. We found that Klotho-sEV treatment can stably reduce PAI and NGAL expression, and SOX9 expression also has a downward trend, although there is no statistical difference. We found that sEV has a trend to reduce injury indicators, but there is no statistical difference ( Fig.10 At the same time, we found that the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the Vehicle group were significantly increased, which is similar to the results of previous studies. Compared with the Vehicle group, Klotho-sEV treatment can steadily reduce the expression of IL-1beta, IL-6, and TNF-alpha, while sEV treatment also has a downward trend but no statistical difference ( Fig.10 Middle C).

[0235] In summary, Klotho-sEV can significantly and stably relieve the AKI symptoms caused by rhabdomyolysis in mice, while sEV has a tendency to relieve the symptoms but there is no statistical difference. Klotho-sEV is more effective than sEV.

[0236] Example 5 Analysis of Klotho-sEV biologically active components

[0237] 1. Experimental Methods

[0238] 1.1 Proteomic sequencing of renal tissue

[0239] Proteomic sequencing of renal tissue was commissioned by Myvi Metabolism. This test used four-dimensional data-independent acquisition (4D-DIA) proteomic sequencing. The screening criteria for differential proteins: Fc>1.5 or Fc<0.6667, and P value<0.05 were defined as significantly differential proteins.

[0240] The specific steps are as follows:

[0241] 1.1.1 Sample processing

[0242] 1) Take an appropriate amount of frozen kidney tissue from the -80℃ freezer, grind it into powder in liquid nitrogen, and transfer the tissue to a centrifuge tube.

[0243] 2) Add an appropriate amount of protein lysis buffer to the crushed tissue, mix well and incubate on ice for 5 minutes.

[0244] 3) Add dithiothreitol to a final concentration of 10 mM, sonicate on ice for 5-15 min, then centrifuge at 13000 g at 4°C for 20 min, and transfer the supernatant to a new centrifuge tube.

[0245] 4) Add 4 times the volume of cold acetone, let stand at -20℃ for 2h, centrifuge and discard the supernatant; add 1mL of cold acetone to the precipitate, vortex and let stand at -20℃ for 30min; centrifuge at 13000g at 4℃ and discard the supernatant, air-dry the protein precipitate; add 8M urea / 100mM tetraethylammonium bromide solution to re-dissolve the protein.

[0246] 5) Add dithiothreitol to a medium concentration of 10 mM, and incubate at 56°C in a water bath for 30 min for reduction reaction; add iodoacetamide to a final concentration of 55 mM, and place in the dark at room temperature for 30 min for alkylation reaction.

[0247] 6) Add 4 volumes of cold acetone to a final concentration of 10 mM dithiothreitol, precipitate at -20°C for 2 h, then centrifuge at 13,000 g for 20 min at 4°C, discard the supernatant, and air-dry the protein precipitate.

[0248] 7) Add 8M urea / 100mM tetraethylammonium bromide solution to re-dissolve the protein

[0249] 8) Determine the protein concentration of the sample using the BCA method. If the next experiment is not performed immediately, store the protein sample at -80℃.

[0250] 1.1.2 nanoLC-MS / MS detection

[0251] 1) NanoElute high performance liquid chromatography separates the samples. The samples are separated using the NanoElute system with a nanoliter flow rate.

[0252] 2) Data acquisition by timesTOF Pro2 mass spectrometer: After chromatographic separation, the mixed sample was first collected using the ddaPASEF mode of the timesTOF Pro mass spectrometer to establish a suitable acquisition window for the diaPASEF acquisition method.

[0253] 1.1.3 Mass spectrometry data analysis

[0254] The library search software used for DIA mass spectrometry data in this project is ['DIA-NN (v1.8.1)'], and the library search is performed using the Libraryfree method. The search parameters are as follows: the database is uniprot-proteome_UP000000589_Mus musculus.fastadatabase (a total of 55,319 sequences), and the deep learning-based parameters are checked to predict a spectral library; MBR is checked to generate a spectral library using DIA data, and the spectral library is used to reanalyze the DIA data to obtain protein quantification; the FDR of both the precursor ion and protein levels are filtered at 1%, and the filtered data can be used for subsequent bioinformatics analysis.

[0255] 1.2 Preparation of kidney tissue homogenate

[0256] 1) Preparation: Pre-cool the tissue grinder.

[0257] 2) Cut an appropriate amount of kidney tissue, put it into a 2 mL EP tube with a locker, and quickly freeze it in liquid nitrogen. Then add an appropriate amount of steel beads (the tissue is under the steel beads) and place the sample in liquid nitrogen for later use.

[0258] 3) Cryo-grinding: Place the EP tube containing the tissue into the grinder and run it at 50 Hz for 2 min to break the tissue.

[0259] 4) Add RIPA lysis buffer containing phosphatase inhibitors, PMSF, and protease inhibitors to the EP tube. The amount of lysis buffer added is in the ratio of lysis buffer: tissue = 10:1.

[0260] 5) Mix the homogenate with a vortexer, centrifuge briefly, and then transfer the supernatant to a new EP tube.

[0261] 6) Lyse the mixture of tissue homogenate and lysis buffer on crushed ice for 30 minutes, mixing several times during the process (it can also be done on a shaker).

[0262] 7) Centrifuge at 14000g for 10 min at 4°C, aspirate the supernatant, and obtain kidney tissue protein.

[0263] 8) Then perform protein determination, protein denaturation, and Western Blot experiments according to the methods in Chapter 2.

[0264] 1.3 Determination of endogenous Klotho expression in mouse kidney tissue

[0265] Western Blot assay was used to determine the endogenous Klotho protein level in mouse kidney tissue. The experimental method was the same as above. Note: The instruction manual of the anti-Klotho antibody (Abcam) indicates that the Klotho protein detected by this antibody is approximately 130KDa for mouse Klotho protein and approximately 65KDa for human Klotho protein.

[0266] 1.4 Klotho-sEV and sEV proteomics and small RNA sequencing

[0267] Klotho-sEV and sEV proteomics and small RNA sequencing were commissioned to Shenzhen BGI Genomics Co., Ltd.

[0268] 1.4.1 Klotho-sEV and sEV small RNA detection

[0269] Differentially expressed miRNAs (microRNAs) in small RNA sequencing were screened by |log2Fc|>0 and Qvalue≤0.05.

[0270] 1.4.2 Klotho-sEV and sEV proteomics detection

[0271] Protein spectra were detected by isotope tags for relative and absolute quantification (iTRAQ), and significantly differentially expressed proteins were screened under the conditions of Q value ≤ 0.05 and |log2Fc| ≥ 0.26303. The specific operation is as follows:

[0272] 1) Add 30 μL of 5% SDC to the sample, vortex and mix, and place in a 95°C metal bath for 10 minutes; take out the sample, wait for the sample to return to room temperature, vortex and mix, centrifuge at 2000g for 10 minutes, take the supernatant and transfer it to a new centrifuge tube. This supernatant is the exosome protein solution. Take 2.5 μL of protein solution, dilute it 10 times with pure water to 25 μL for quantification, and take 2.5 μL of protein solution for SDS-PAGE running.

[0273] 2) Proteolysis: Add enzyme solution at the ratio of Trypsin enzyme (μg): substrate protein (μg) = 1:20, vortex and shake, centrifuge at low speed for 1 min, and incubate at 37°C for 4 h; take out the digested peptide solution for desalting, and then freeze-dry the peptide solution obtained after desalting.

[0274] 3) Peptide labeling: according to the number of samples, take out a certain amount of iTRAQ label reagent; after the reagent returns to room temperature, add 50μL of isopropanol to each tube of reagent, vortex and centrifuge at low speed; dissolve the peptide sample with 0.5M tetraethylammonium bromide and add it to the corresponding iTRAQ label reagent. Different iTRAQ labels are used for different sample peptides; let it stand at room temperature for 2 hours.

[0275] 4) Peptide separation: Shimadzu LC-20AD liquid phase system was used to take 20 μg of the mixed sample and perform liquid phase separation on the sample using a 5 μm 20 cm x 180 μm Gemini C18 column.

[0276] 5) HPLC: The dried peptide samples were reconstituted with mobile phase A (2% CAN, 0.1% FA) and centrifuged at 20,000 g for 10 min, and the supernatant was injected and separated using Thermo UltiMate 3000 UHPLC.

[0277] 6) Mass spectrometry detection: The peptides separated by liquid phase are ionized by nanoESI source and then enter into tandem mass spectrometer Q-Exactive HF X for DDA (Data Dependent Acquisition) mode detection.

[0278] 2. Experimental results

[0279] 2.1 Administration of Klotho-sEV can upregulate the expression of endogenous Klotho in mice

[0280] We detected endogenous Klotho in mouse kidney tissue. The results showed that compared with the healthy control group, the endogenous Klotho level in the AKI group was significantly reduced, while Klotho-sEV treatment increased the expression of Klotho ( Fig.11 ). These results not only indirectly confirmed that Klotho-sEV treatment improved renal injury, but also suggested that Klotho-sEV could play a role by regulating endogenous Klotho expression.

[0281] 2.2 Proteomic sequencing results of renal tissue

[0282] In order to further explore the mechanism of action of Klotho-sEV in acute kidney injury, we took kidney tissues of AKI model mice for 4D-DIA proteomics sequencing, and a total of 10,970 proteins were detected. KEGG analysis found that the differential proteins in kidney tissues of healthy mice and AKI model mice were mainly concentrated in metabolism, mainly including metabolism-related signaling pathways, carbon metabolism, valine, leucine and isoleucine degradation oxidative phosphorylation and other pathways; mainly affecting the Rap1 signaling pathway, PI3K-AKT signaling pathway, and mTOR signaling pathway. The results of KEGG analysis of the healthy group and the AKI group suggested that AKI mainly affects renal metabolism, and signaling pathways such as Rap1, PI3K-AKT, and mTOR play an important role in it ( Fig.12 Then, we performed KEGG analysis on the healthy control group Control, model group Vehicle, and Klotho-sEV group. We found that the differentially expressed proteins were still concentrated in metabolism. As before, the first two were also metabolism-related signaling pathways and carbon metabolism, followed by cofactor biosynthesis and valine, leucine, and isoleucine degradation oxidative phosphorylation; the main impact was on the PI3K-AKT signaling pathway, Rap1 signaling pathway, MAPK signaling pathway, and mTOR signaling pathway ( Fig.12 (middle B).

[0283] 2.3 Klotho-sEV can activate mTOR and MEK1 / 2 phosphorylation signaling pathways and promote endogenous expression of Klotho

[0284] Klotho is an important component of the endocrine fibroblast growth factor (FGF) receptor complex and is required for high-affinity binding of FGF to the homologous FGF receptor. Previous studies have shown that FGF and Klotho affect MEK1 / 2 and mTOR signals. Combined with the previous renal proteomics test results, we performed Western Blot detection and verification of AKT, mTOR, and MEK1 / 2 signals. Fig.13 As shown in the figure, we found that compared with the healthy group, AKT signaling was activated after AKI, pAKT protein increased, and AKT was significantly phosphorylated; compared with the Vehicle group, pmTOR protein was upregulated in the Klotho-sEV treatment group; compared with the healthy group, MEK1 / 2 signaling was activated after AKI. It is worth noting that compared with the Vehicle group, p-MEK1 / 2 protein increased and MEK1 / 2 was significantly phosphorylated. The above results suggest that the occurrence, development, and recovery of AKI are regulated by multiple proteins and signaling pathways, and mTOR and MEK1 / 2 phosphorylation signaling pathways may play an important role in the treatment of acute kidney injury with Klotho-sEV.

[0285] 2.4 Klotho-sEV contains multiple kidney-protective proteins

[0286] The lipid bilayer membrane structure allows sEV to contain a variety of bioactive molecules, including proteins, nucleic acids, lipids, etc. To further explore the mechanism of action of Klotho-sEV on AKI, we then performed proteomic sequencing on Klotho-sEV and sEV. iTRAQ detected a total of 4347 proteins in Klotho-sEV and sEV, of which 40.9% (1776) were proteins that had been detected in the vesiclepedia database ( Fig.14 A total of 322 differentially expressed proteins (DEPs) were detected, of which 212 proteins were highly expressed in Klotho-sEVs (Fc>1.2) and 110 proteins were lowly expressed in Klotho-sEVs (Fc<0.8333) ( Fig.14 The top ten differentially expressed proteins included CC126, PHKG2, RMD1, LIPA3, LRP8, TPRX1, TBCA, Klotho, MYO9B and RIPK2, and the top ten differentially underexpressed proteins included QCR1, ACSA, DKK1, PDK2, PKHA5, PEBP1, T2EA, CHID1, PTPRJ and KCC2G, which once again confirmed that Klotho was enriched in Klotho-sEVs. In addition, we found that compared with sEVs, three proteins, LRP8 (Fc 2.46), ARF5 (Fc 1.98), and FRAS1 (Fc 1.66), were also highly expressed in Klotho-sEVs ( Fig.14 Middle C), previous studies have shown that LRP8 mainly plays an anti-inflammatory role, ARF5 can activate mTOR signaling, and FRAS1 is involved in kidney development and glomerular formation, which provides evidence that Klotho-sEV can activate mTOR signaling and has a better therapeutic effect on AKI than sEV. iTRAQ protein detection suggests that Klotho-sEV contains a variety of kidney protective molecules in addition to Klotho protein.

[0287] 2.5 Klotho-sEV contains multiple anti-inflammatory and kidney-protective miRNAs

[0288] Nucleic acids are also the main components of extracellular vesicles (sEVs). Small RNA (sRNA), including miRNA, tsRNA, piRNA, snoRNA, etc., are indispensable regulatory molecules for important biological processes such as gene expression and regulation, RNA processing and splicing, and protein translation. They are also the main nucleic acid components of sEVs. We have previously analyzed the proteomics of Klotho-sEVs and sEVs. Next, we will further clarify the small RNA profiles of Klotho-sEVs and sEVs. A total of 1007 miRNAs were detected by RNA sequencing. We found that 9 of the 10 most highly expressed miRNAs in Klotho-sEV and sEV overlapped, including hsa-let-7a-5p, hsa-miR-1246, hsa-miR-146a-5p, novel-hsa-miR264-3p, hsa-let-7f-5p, hsa-let-7i-5p, hsa-miR-127-3p, hsa-miR-100-5p, and hsa-miR-181a-5p. A total of 51 differentially expressed miRNAs (|log2Fc|≥0, q value≤0.05) were detected, of which 30 miRNAs were lowly expressed in Klotho-sEV and 21 miRNAs were highly expressed in Klotho-sEV ( Fig.15 (A) Cluster analysis of sEV and Kloth-sEV miRNAs Fig.15 As shown in B. Among these 51 differential miRNAs, hsa-miR-141-3p, hsa-miR-375-3p, novel-hsa-miR245-5p, novel-hsa-miR62-5p, hsa-miR-3651, hsa-miR-182-5p, hsa-let-7b-5p, hsa-miR-1246, novel-hsa-miR289-5p, and hsa-let-7d-5p were significantly upregulated in Klotho-sEV. , novel-hsa-miR30-3p, novel-hsa-miR148-3p, hsa-miR-130b-5p, hsa-miR-7704, hsa-miR-487a-5p, novel-hsa-miR293-5p, hsa-miR-10a-5p, novel-hsa-miR294-3p, hsa-miR-337-5p, and hsa-miR-369-3p were significantly downregulated in Klotho-sEVs ( Fig.15C). Previous studies have reported that the let-7 family and miR-1246 play an anti-inflammatory role. While miR-1246 and multiple let-7 family miRNAs are highly expressed in sEVs, their expression is significantly increased in Klotho-sEVs ( Fig.15 This also provides data support for the better anti-inflammatory and repair effects of Klotho-sEV compared with sEV. The results of KEGG analysis of differential miRNA target prediction showed that miRNA also affects MAPK signaling and mTOR signaling.

[0289] In summary, we found that Klotho-sEVs are enriched with a variety of renal protective proteins and miRNAs in addition to Klotho. Most of these molecules are upregulated in Klotho-sEVs, which may also be part of the reason why Klotho-sEVs show better AKI therapeutic effects than sEVs.

[0290] Example 6 Protective effect of Klotho-sEV on AD model mice

[0291] 1. Experimental Methods

[0292] 1.1 AD model construction

[0293] Transgenic AD mice were bred by introducing five familial AD mutation sites (K670N / M671L, I716V, V717I, M146L and L286V) of the human gene into APP / PS1 transgenic model mice (purchased from Jackson lab in the United States and bred by ourselves after purchase).

[0294] 1.2 Drug handling and testing

[0295] Ten-month-old 5×FAD mice were injected with 100 μg sEV or Klotho-sEV via the tail vein, and the control group was given an equal amount of PBS. The mice were given the drug once every 2 weeks. After 4 doses, the cognitive function and brain inflammatory response of the mice were detected.

[0296] 2. Experimental results

[0297] 2.1 Cognitive function testing

[0298] The water maze experiment was used to detect the cognitive function of model mice. Fig.16As shown in the figure, compared with MSC-sEV (EV), Klotho-sEV (KL-EV) treatment can better improve the cognitive ability of model mice. Among them, A is a representative water maze trajectory diagram of mice in each group, the circle is the platform position, the quadrant where it is located is the target quadrant, the red dot is the initial position of the mouse, and the blue dot is the final position of the mouse; B is the escape latency trend diagram of mice in each group on days 1-5; C is the number of times each group of mice crossed the original platform position; D is the movement time of mice in each group in the target quadrant; E is the movement distance of mice in each group in the target quadrant. * p<0.05, ** p<0.01.

[0299] 2.2 Brain inflammation

[0300] Western blot was used to detect the expression levels of NLRP3, Pre-Caspase1, Caspase1 and IL-1β in the brain of model mice to reflect the level of inflammatory response in the brain of model mice. Fig.17 As shown in the figure, Klotho-sEV treatment can improve the inflammatory response in the brain of model mice and reduce the expression level of related inflammatory factors. A is a Western blot showing the expression of NLRP3, Pre-Caspase1, Caspase1 and IL-1β in the mouse brain; B is a comparative statistical chart of NLRP3, Pre-Caspase-1, Caspase-1 and IL-1β between different groups; *p<0.05.

[0301] The above results show that compared with sEV, Klotho-sEV of the present invention can better improve the cognitive function of AD model mice, reduce the inflammatory response in the brain, and has a better therapeutic effect on AD.

[0302] Example 7 Preparation and Identification of Klotho-293T-sEV

[0303] In this example, 293T cells were used as recipient cells to prepare small extracellular vesicles (Klotho-293T-sEV) containing Klotho protein and / or mRNA. 293T cells were infected with a lentivirus overexpressing Klotho to construct a KL-293T cell line that stably overexpressed Klotho. The specific operation method was the same as Example 1 except that 293T cells were used as recipient cells.

[0304] The culture medium of 293T cells is: 90% DMEM high glucose medium + 10% BSA + 1% Glucose + 1% double antibody, and 0.25% trypsin is used for digestion.

[0305] The preparation and identification methods of KL-293T-sEV are the same as those in Example 1.

[0306] The results are as follows Fig.18 As shown, Fig.18 In the figure, A is the GFP positivity of cells observed by fluorescence microscopy, and the spontaneous GFP fluorescence of cells infected with lentivirus, with a scale of 50μm; B is the flow cytometry detection of the FITC positivity of KL-293T cells; C is the particle characteristics of 293T-sEV / KL-293T-sEV detected by Nanosight; D is the Western Blot detection of 293T-sEV / KL-293T-sEV exosome marker proteins (positive marker proteins CD9, CD63, TSG101, negative marker protein Calnexin); E is the Western Blot detection of Klotho protein enrichment in 293T-sEV / KL-293T-sEV. The results show that we have successfully prepared LV-KL-293T cells. Under a fluorescence microscope, we observed that the cells infected with lentivirus spontaneously expressed GFP ( Fig.18 A), flow cytometry showed that the infection rate of 293T cells was 85.2% ( Fig.18 NTA showed that the particle size of sEVs from the two sources was similar, with an average particle size of about 150 nm ( Fig.18 Western Blot results showed that sEVs from both sources expressed exosome marker proteins ( Fig.18 D), with similar exosome marker expression profiles. We also confirmed that engineering modification also successfully achieved Klotho enrichment in 293T-sEV ( Fig.18 Middle E).

[0307] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing small extracellular vesicles containing Klotho protein and / or mRNA, characterized in that: The following steps are involved: S1. constructing a recombinant expression vector containing a gene encoding a Klotho protein; S2. introducing the recombinant expression vector into a receptor cell to obtain a receptor cell line stably expressing the Klotho gene; S3. culturing the receptor cell line, collecting the culture supernatant, and extracting small extracellular vesicles therefrom.

2. The preparation method according to claim 1, characterized in that The recombinant expression vector in step S1 is one or more of a recombinant lentiviral vector, a recombinant adenoviral vector, a recombinant retroviral vector, a recombinant liposome and a recombinant plasmid expression vector; and / or, The recipient cells in step S2 are stem cells, HEK293T cells, HEK293F cells, CHO cells, Sf9 cells, Sf21 cells or Hi5 cells.

3. The preparation method according to claim 1 or 2, characterized in that: The method for introducing the recombinant expression vector into the recipient cell is virus infection, liposome transfection or electroporation transfection.

4. The preparation method according to claim 3, characterized in that: The method for introducing the recombinant expression vector into the recipient cells in step S2 is a viral infection method; preferably, the recipient cells are infected with the virus twice.

5. The preparation method according to claim 1, characterized in that: Step S3 is: culturing the recipient cell line at 37±1°C, then transferring to 42±1°C for 1h to 6h, continuing to culture at 37±1°C, collecting the culture supernatant, and extracting small extracellular vesicles therefrom.

6. The preparation method according to claim 1, characterized in that: The Klotho protein comprises the amino acid sequence shown in SEQ ID NO.

2.

7. The preparation method according to claim 6, characterized in that: The gene encoding the Klotho protein comprises the nucleotide sequence shown in SEQ ID NO.

1.

8. Small extracellular vesicles containing Klotho protein and / or mRNA obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the small extracellular vesicles containing Klotho protein and / or mRNA as claimed in claim 8 in the preparation of a medicament for treating acute kidney injury or Alzheimer's disease.

10. A drug for treating acute kidney injury or Alzheimer's disease, characterized in that: The drug comprises an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises the small extracellular vesicles containing Klotho protein and / or mRNA as claimed in claim 8.

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