Mesenchymal stem cell exosomes carrying Klotho fusion gene, preparation method and application thereof

Abstract

The present invention provides a mesenchymal stem cell exosome carrying a Klotho fusion gene, a preparation method and an application thereof, belonging to the field of genetic engineering technology, wherein the Klotho fusion gene is composed of secretory Klotho, self-cleaving polypeptide T2A, and FGF23 connected in series in sequence; the artificial nucleic acid sequence of the secretory Klotho is shown in SEQ ID NO.2. The present invention optimizes the nucleotide sequence of the Klotho gene, improves the expression level of Klotho mRNA and the expression level of Klotho protein in Klotho-FGF23 exosomes; in the hypoxic injury of H9C2 cardiomyocytes and the myocardial hypertrophy injury induced by AngII, the Klotho-FGF23 exosomes prepared after the sequence optimization of the present invention can effectively improve the effect of cardiomyocyte damage.

Description

Technical Field

[0001] The present invention relates to a mesenchymal stem cell exosome carrying a Klotho fusion gene, a preparation method and an application thereof, and belongs to the technical field of genetic engineering. Background Art

[0002] Cardiovascular disease (CVD) is a disease that is prevalent in the population and is the leading cause of death. Klotho protein is considered a key regulator in the development of CVD. In some clinical studies, a relationship has been observed between low levels of soluble Klotho protein and the occurrence and severity of cardiovascular disease and a reduction in cardiovascular risk at high levels. Klotho protein improves endothelial cell dysfunction by promoting the production of nitric oxide (NO), mediates anti-inflammatory and anti-aging effects such as inhibiting adhesion molecule expression and inhibiting Wnt signaling; in addition, the protein is associated with the reduction of vascular calcification and the prevention of myocardial hypertrophy. Therefore, Klotho protein is associated with CVD and plays a role in maintaining functional vascular integrity.

[0003] The Klotho gene contains 5 exons, encoding a type I single-pass transmembrane protein with a very short intracellular region of only 10 amino acids. The extracellular region consists of two weakly homologous domains, named KL1 and KL2. The Klotho gene is highly expressed in the distal tubules of the kidney and the choroid plexus of the brain. Klotho protein binds to multiple FGFRs to form a complex co-receptor, binds to FGF23, and participates in regulating phosphate balance. Secretory Klotho protein protects cells and tissues from oxidative stress.

[0004] Exosomes are membrane-bound vesicles with a diameter between 30-150nm that are released by cells into the extracellular space. Exosomes carry a variety of contents, including proteins, lipids, RNA and DNA. Exosomes can serve as carriers of intercellular communication. Exosomes play a vital role in intercellular communication. They carry various biological materials and participate in various physiological and pathological processes.

[0005] At present, the research on Klotho is mainly in the field of chronic kidney disease and anti-aging. CN117679530A discloses a GP-pKlotho nano drug and its preparation method and application, which mainly uses GP-pKlotho nano drugs to treat acute kidney injury (IR-AKI) caused by ischemia-reperfusion or chronic kidney disease (CKD). CN118059266A discloses a nano targeted drug for treating chronic kidney disease and its preparation method and application, which uses nano composite materials to encapsulate Klotho plasmids for the treatment of renal fibrosis, diabetic nephropathy, chronic kidney disease, acute kidney injury and other diseases. CN117797249A discloses an mRNA-based anti-aging and anti-Alzheimer's prevention and treatment vaccine, which uses an mRNA vaccine containing Klotho for the treatment of anti-aging and anti-Alzheimer's disease.

[0006] Currently, no results have been retrieved for transfecting klotho fusion gene into mesenchymal stem cells to prepare exosomes for the treatment of cardiovascular diseases. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a mesenchymal stem cell exosome carrying a Klotho fusion gene, a preparation method and an application thereof, to achieve the following invention objectives: to increase the expression levels of Klotho mRNA and Klotho protein in exosomes carrying a Klotho fusion gene, and to improve the therapeutic effect of exosomes carrying a Klotho fusion gene on myocardial cell hypoxia injury and myocardial hypertrophy injury.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A mesenchymal stem cell exosome carrying a Klotho fusion gene, wherein the Klotho fusion gene is composed of secretory Klotho, self-cleaving polypeptide T2A, and FGF23 connected in series in sequence; the artificial nucleic acid sequence of the secretory Klotho is shown in SEQ ID NO.2.

[0010] The artificial nucleic acid sequence of the self-cleaving polypeptide T2A is shown in SEQ ID NO.3; the artificial nucleic acid sequence of FGF23 is shown in SEQ ID NO.4.

[0011] The method for preparing the exosomes is to construct the Klotho fusion gene into a recombinant expression vector, then package it into an adeno-associated virus, and then infect mesenchymal stem cells, so that the mesenchymal stem cells secrete exosomes.

[0012] The exosomes are used in the preparation of a drug for treating myocardial cell hypoxia damage and / or myocardial hypertrophy damage.

[0013] Compared with the prior art, the present invention achieves the following beneficial effects:

[0014] (1) The present invention optimizes the nucleotide sequence of the Klotho gene to increase the expression level of Klotho mRNA and the expression level of Klotho protein in Klotho-FGF23 exosomes;

[0015] (2) For the H2O2-induced myocardial cell inflammatory response, the Klotho-FGF23 exosomes prepared after sequence optimization of the present invention can reduce the expression level of pro-inflammatory factor mRNA and increase the expression level of anti-inflammatory factor mRNA, and have an anti-inflammatory effect. In the hypoxic injury of H9C2 myocardial cells and the myocardial hypertrophy injury induced by AngII, the Klotho-FGF23 exosomes prepared after sequence optimization of the present invention can effectively improve the damage of myocardial cells, make the morphology of damaged myocardial cells tend to normal, and have the effect of treating myocardial hypoxic injury and myocardial hypertrophy injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a microscope image of P4 mesenchymal stem cells;

[0017] Figure 2 Fluorescence images of MSCs infected with three types of adeno-associated viruses;

[0018] Figure 3 It is a bar graph of the relative levels of Klotho mRNA in MSCs infected with three types of adeno-associated viruses;

[0019] Figure 4 It is a bar graph of the relative levels of Klotho mRNA and Klotho protein in the three exosomes;

[0020] A is a bar graph of the relative levels of Klotho mRNA in the three exosomes, and B is a bar graph of the relative levels of Klotho protein in the three exosomes;

[0021] Figure 5 It is a bar graph of the relative levels of M1 / M2 inflammatory factor mRNA in H9C2 cells in Example 5;

[0022] Among them, (a) is a bar graph of the relative level of IL-1β mRNA in H9C2 cells; (b) is a bar graph of the relative level of TNF-α mRNA in H9C2 cells; (c) is a bar graph of the relative level of IL-6 mRNA in H9C2 cells; (d) is a bar graph of the relative level of IL-4 mRNA in H9C2 cells; (e) is a bar graph of the relative level of IL-10 mRNA in H9C2 cells; (f) is a bar graph of the relative level of Arg-1 mRNA in H9C2 cells;

[0023] Figure 6 The western blot and bar graph of the expression of IL-4, IL-10, and Arg-1 proteins in H9C2 cells in Example 5;

[0024] Among them, (a) is a western blot diagram of the expression of IL-4, IL-10, and Arg-1 proteins in H9C2 cells; (b) is a bar graph of the relative expression of IL-4 protein; (c) is a bar graph of the relative expression of IL-10 protein; (d) is a bar graph of the relative expression of Arg-1 protein;

[0025] Figure 7 The figure is a bar graph showing the cell morphology of the H9C2 cardiomyocyte hypoxia model and the relative level of HIF-1α mRNA in cells under different hypoxia time;

[0026] Among them, (a) is the cell morphology of the H9C2 control group; (b) is the cell morphology of the H9C2 cardiomyocyte hypoxia model; (c) is a bar graph of the relative level of HIF-1α mRNA in cells under different hypoxia time;

[0027] Figure 8 The figure is a bar graph showing the cell morphology of the H9C2 cardiac hypertrophy injury model and the relative mRNA levels of ANP, BNP and β-MHC in cells treated with different concentrations of AngII;

[0028] Among them, (a) is the cell morphology of the H9C2 cell control group; (b) is the cell morphology of the H9C2 cardiac hypertrophy injury model induced by 4 μM AngII; (c) is a bar graph of the relative mRNA levels of ANP in cells after treatment with different concentrations of AngII; (d) is a bar graph of the relative mRNA levels of BNP in cells after treatment with different concentrations of AngII; (e) is a bar graph of the relative mRNA levels of β-MHC in cells after treatment with different concentrations of AngII;

[0029] Fig. 9 The western blot and bar graph of the expression level of HIF-1α protein in the treatment of myocardial cell hypoxia injury by exosomes in Example 6;

[0030] Among them, (a) is a western blot diagram of the expression level of HIF-1α protein in the treatment of cardiomyocyte hypoxia injury by exosomes; (b) is a bar graph of the relative level of HIF-1α protein in the treatment of cardiomyocyte hypoxia injury by exosomes;

[0031] Fig.10 Western blot and bar graph of the protein expression levels of ANP and BNP in the treatment of myocardial hypertrophy injury by exosomes in Example 6;

[0032] Among them, (a) is a western blot diagram of the expression levels of ANP and BNP proteins in exosomes for the treatment of myocardial hypertrophic injury; (b) is a bar graph of the relative level of ANP protein in exosomes for the treatment of myocardial hypertrophic injury; (c) is a bar graph of the relative level of BNP protein in exosomes for the treatment of myocardial hypertrophic injury. DETAILED DESCRIPTION

[0033] Example 1 Construction of Klotho fusion gene recombinant expression vector

[0034] When expressing Klotho protein in vitro, the present invention improves the efficiency of foreign protein expression through codon optimization, thereby inducing the body to produce more protein.

[0035] The optimized artificial nucleic acid sequence of the Klotho fusion gene is shown in SEQ ID NO. 1. The fusion gene is composed of secretory Klotho, self-cleaving polypeptide T2A, and FGF23 connected in series; wherein the optimized artificial nucleic acid sequence of the secretory Klotho is shown in SEQ ID NO. 2; the artificial nucleic acid sequence of the self-cleaving polypeptide T2A is shown in SEQ ID NO. 3; the artificial nucleic acid sequence of FGF23 is shown in SEQ ID NO. 4 (CDS sequence in NM_020638.3).

[0036] According to the extracellular domain and signal peptide of Klotho, the CDS sequence in the XM_047430776.1 sequence was selected as the artificial nucleic acid sequence of Klotho before optimization, and the artificial nucleic acid sequence of Klotho before optimization and the artificial nucleic acid sequences of the self-cleaving polypeptide T2A and FGF23 were sequentially connected in series to obtain the artificial nucleic acid sequence of the Klotho fusion gene of the control group, as shown in SEQ ID NO.5.

[0037] The artificial nucleic acid sequences of the Klotho fusion gene and the control group Klotho fusion gene were commissioned to Beijing Bomade Gene Technology Co., Ltd. for synthesis, and respectively inserted into the pAAV-EF1α-GFP vector and transformed into E. coli (TOP10). After correct sequencing, the plasmid was extracted and purified using the plasmid extraction kit of OMEGA Company to obtain recombinant plasmids, which were named pAAV-Klotho-FGF23 and pAAV-Klotho-FGF23 control group, respectively. The concentrations of both plasmids were adjusted to 1 μg / mL.

[0038] At the same time, prepare the empty pAAV plasmid and adjust the plasmid concentration to 1 μg / mL.

[0039] Example 2 Resuscitation of umbilical cord mesenchymal stem cells

[0040] Take out a tube of P3 umbilical cord mesenchymal stem cells from the -80℃ refrigerator, quickly revive them in a 40℃ water bath, disinfect the cryopreserved tubes with alcohol, and put them in a clean bench. Take out the revive cells with a pipette, add physiological saline to 50mL, and use it to wash the cryopreserved solution in the cells. After centrifugation, remove the supernatant, and inoculate the cells into a culture bottle. The cell inoculation amount is 1.05×10 6 Each bottle was cultured with 25 mL of mesenchymal stem cell culture medium (HMSC Culture Medium, purchased from Tianjin Simpson Life Science Co., Ltd.) and observed under a microscope every day. When the mesenchymal stem cells covered 80% of the bottom of the bottle, they were passaged and used in experiments at the P4 generation (see Figure 1 ).

[0041] Example 3 Adeno-associated virus infection of mesenchymal stem cells and identification

[0042] 1. Transfection of recombinant plasmid into 293T cells and packaging into adeno-associated virus

[0043] 293T cells were revived according to the conventional method, and after one passage, they were used for transfection when the cells covered 80% of the bottom of the bottle. The recombinant plasmid pAAV-Klotho-FGF23 (pAAV-Klotho-FGF23 control group) and the adeno-associated virus packaging plasmid pAAV-RC8 and pHelper were transfected into 293T cells using LipoFiter transfection reagent, and 293T cells were transfected with empty pAAV plasmid at the same time.

[0044] After 48 hours, the fluorescence expression of 293T cells after transfection was observed under a microscope. After 72 hours, all supernatants and cells were collected and transferred to centrifuge tubes, centrifuged at 4000g for 30 min, the supernatant was removed, PBS was added to mix, and the tubes were placed at -80°C and frozen and thawed repeatedly for 5 times to fully lyse the cells. The cells were purified and concentrated using an ultracentrifuge, and resuspended in PBS to obtain adeno-associated viruses containing pAAV-Klotho-FGF23, adeno-associated viruses containing a control group of pAAV-Klotho-FGF23, and adeno-associated viruses containing empty pAAV, and the titer of the adeno-associated viruses was measured.

[0045] The virus titer of the adeno-associated virus containing pAAV-Klotho-FGF23 in the present invention is 1.06×10 9 TU / mL, and the virus titer of the adeno-associated virus containing pAAV-Klotho-FGF23 control group was 1.11×10 9 TU / mL, the virus titer of adeno-associated virus containing empty pAAV was 1.23×10 9 TU / mL.

[0046] (II) Adeno-associated virus infection of umbilical cord mesenchymal stem cells and collection of exosomes

[0047] The number of umbilical cord mesenchymal stem cells (MSCs) at P4 was counted, and the number of MSCs seeded was 6×10 6 / T175 flask, placed in the incubator for 2 hours, and used for adeno-associated virus transfection after the cells adhered to the wall. The prepared adeno-associated virus containing pAAV-Klotho-FGF23, the adeno-associated virus containing the pAAV-Klotho-FGF23 control group, and the adeno-associated virus containing the empty pAAV were added at MOI=100 (calculated according to the number of MSCs initially inoculated), and cultured in a 37°C, 5% CO2 incubator for 24 hours. After centrifugation at 400g for 5 minutes, the supernatant was removed, and fresh mesenchymal stem cell culture medium was added. After culturing for 3 days, two types of stem cells expressing the Klotho fusion gene and the empty transfected MSC were collected and named Klotho-FGF23-MSC, Klotho-FGF23 control group-MSC and pAAV-MSC; the expression of GFP in MSC cells was observed under a microscope (such as Figure 2 ), Figure 2 It showed that the adeno-associated virus was successfully transfected into the cells and Klotho-FGF23 fusion protein was secreted.

[0048] The expression of Klotho at the mRNA level in Klotho-FGF23-MSC, Klotho-FGF23 control group-MSC, and pAAV-MSC was detected.

[0049] The results are as follows Figure 3 As shown, compared with pAAV-MSC, the expression intensity of Klotho-FGF23-MSC and Klotho-FGF23 control group-MSC at the mRNA level was 1326 times that of pAAV-MSC, and the expression intensity of Klotho-FGF23 control group-MSC at the mRNA level was 967 times that of pAAV-MSC; compared with Klotho-FGF23 control group-MSC, the expression intensity of Klotho mRNA level in Klotho-FGF23-MSC was significantly increased, indicating that the codon-optimized Klotho-FGF23 can increase the transcription level of Klotho mRNA.

[0050] All cell culture supernatants were collected and ultracentrifuged to obtain exosomes, which were named Klotho-FGF23 Exo, Klotho-FGF23 control group Exo, and pAAV Exo. Transmission electron microscopy and Nanosight particle tracking analysis showed that there was no significant difference in the morphology, particle size distribution, and yield of pAAV Exo, Klotho-FGF23 Exo, and Klotho-FGF23 control group Exo. There was no significant difference in the concentration of pAAV Exo, Klotho-FGF23 Exo, and Klotho-FGF23 control group Exo. The concentration of pAAV Exo was (1.24±0.02)×10 8 particles / mL (n=4), the concentration of Klotho-FGF23 Exo was (1.21±0.04)×10 8 particles / mL (n=4), the concentration of Exo in the Klotho-FGF23 control group was (1.27±0.05)×10 8 Particles / mL (n=4).

[0051] Example 4 Identification of Klotho-FGF23 Exo

[0052] (I) qRT-PCR detection of Klotho mRNA expression level in exosomes

[0053] qRT-PCR was used to detect the expression levels of Klotho RNA in the three exosomes, including Klotho-FGF23 Exo harvested from the culture supernatant of MSCs transfected with pAAV-Klotho-FGF23, Klotho-FGF23 control group Exo collected from the culture supernatant of MSCs transfected with pAAV-Klotho-FGF23 control group, and pAAV Exo collected from the culture supernatant of MSCs transfected with empty pAAV.

[0054] The results are as follows Figure 4 Middle A shows that compared with Klotho mRNA in pAAV Exo, the expression levels of Klotho mRNA in Klotho-FGF23Exo and Klotho-FGF23 control group Exo were significantly increased, and the expression level of Klotho mRNA in Klotho-FGF23 Exo was higher than that in Klotho-FGF23 control group Exo.

[0055] (II) ELISA detection of Klotho protein expression level in exosomes

[0056] The ELISA method (reagents purchased from Wuhan Huamei Bioengineering Co., Ltd.) was used to detect the expression levels of Klotho protein in the three exosomes, including Klotho-FGF23 Exo harvested from the culture supernatant of MSCs transfected with pAAV-Klotho-FGF23, Klotho-FGF23 control group Exo collected from the culture supernatant of MSCs transfected with pAAV-Klotho-FGF23 control group, and pAAV Exo collected from the culture supernatant of MSCs transfected with empty pAAV.

[0057] The results are as follows Figure 4 Middle B shows that compared with the Klotho protein in pAAV Exo, the expression level of Klotho protein in Klotho-FGF23 Exo and Klotho-FGF23 control group Exo was significantly increased, and the expression level of Klotho protein in Klotho-FGF23 Exo was higher than that in Klotho-FGF23 control group Exo.

[0058] Example 5 Klotho-FGF23 Exo inhibits H2O2-induced inflammatory response in cardiomyocytes

[0059] H9C2 cells in logarithmic growth phase were collected and plated at 3×10 5 The cells were inoculated into a six-well plate at a density of cells / mL, 2 mL of cell suspension was added to each well, and the cells were divided into 5 groups. The specific groups are as follows:

[0060] A: blank control group;

[0061] B: H2O2 model group;

[0062] C: H2O2+ pAAV Exo group;

[0063] D: H2O2+ Klotho-FGF23 control group Exo group;

[0064] E: H2O2+Klotho-FGF23 Exo group;

[0065] After culturing H9C2 cells in each group for 12 hours, 100 μg of exosome pAAV Exo was added to group C, 100 μg of exosome Klotho-FGF23 control group Exo was added to group D, and 100 μg of exosome Klotho-FGF23 Exo was added to group E. After pretreatment for 24 hours, the medium was changed; then, except for the blank control group, 100 pmol / L (final concentration) of H2O2 was added to the remaining groups and cultured for 6 hours. Cells in each group were collected, RNA was extracted, and the expression of M1 / M2 inflammatory factor mRNA in cells was detected by qPCR. At the same time, total cell protein was extracted, and the content of anti-inflammatory factor protein in cells was detected by Western blot.

[0066] like Figure 5 As shown in the figure, the expression of inflammatory factor mRNA detected by qPCR showed that compared with group A, the mRNA expression levels of M1 pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) in group B and group C cells were significantly increased, and the mRNA expression levels of M2 anti-inflammatory cytokines (IL-4, IL-10, Arg-1) were significantly decreased. Compared with group B, the mRNA expression levels of M1 pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) in group D and group E cells were significantly decreased, and the mRNA expression levels of M2 anti-inflammatory cytokines (IL-4, IL-10, Arg-1) were significantly increased. However, the effect of group E was better than that of group D in both pro-inflammatory and anti-inflammatory factors.

[0067] like Figure 6 As shown in the figure, the expression of anti-inflammatory factor proteins detected by Western blot showed that the expression of IL-4, IL-10, and Arg-1 proteins in groups B and C was significantly reduced compared with group A. Compared with group B, the expression of IL-4, IL-10, and Arg-1 proteins in cells of groups D and E was significantly increased, but the protein expression level shown in group E was significantly higher than that in group D.

[0068] Example 6 Effects of Klotho-FGF23 Exo on myocardial cell hypoxia injury and myocardial hypertrophy injury.

[0069] (1) Establishment of myocardial cell hypoxia and myocardial hypertrophy model

[0070] ①H9C2 cardiomyocytes were seeded in 6-well plates and cultured in a 95% N2, 5% CO2 incubator for 2h, 4h, and 6h of hypoxia, respectively. Total RNA was extracted from the H9C2 cell samples cultured under hypoxia and normal conditions, and the expression level of HIF-1α mRNA under hypoxia was detected to determine the hypoxia condition. After 6h of hypoxia treatment, the H9C2 cardiomyocytes were observed under an inverted microscope and found to have obvious changes in cell morphology, shrinkage, poor wall adhesion, etc.

[0071] ② When 0, 1 and 4 μM angiotensin II (AngII) were added to H9C2 cells, the cells became hypertrophic. The cells were collected and total RNA was extracted to prepare cDNA samples, and the mRNA expression levels of ANP, BNP and β-MHC were detected.

[0072] Results Analysis: In the myocardial cell hypoxia model, Figure 7 As shown in Figure 2, as the hypoxia treatment time increases, the mRNA of hypoxia-inducible factor HIF-1α increases. At the same time, after 6 hours of hypoxia, the cardiomyocytes become wrinkled and irregular in shape, indicating that hypoxia treatment causes cardiomyocyte damage. In the cardiomyocyte hypertrophy model, Figure 8 As shown, the relative levels of mRNA of myocardial hypertrophy-related factors were significantly increased after H9C2 cardiomyocytes were treated with 4 μM AngII. In terms of cell morphology, compared with the control group, the cells treated with 4 μM AngII became significantly enlarged, leading to myocardial hypertrophy damage, indicating that the model was successfully constructed.

[0073] (2) Effects of Klotho-FGF23 Exo on myocardial cell hypoxia injury and myocardial hypertrophy injury

[0074] ① H9C2 cells were seeded in 6-well plates (7.5×10 5 cells), and after 24 hours, the cell coverage rate was about 60%. The experiment was divided into 5 groups:

[0075] A: H9C2 control group;

[0076] B: Hypoxia model group;

[0077] C: hypoxia+pAAV Exo group;

[0078] D: Hypoxia + Klotho-FGF23 control group Exo group;

[0079] E: Hypoxia + Klotho-FGF23 Exo group;

[0080] 100 μg of exosome pAAV Exo was added to group C, 100 μg of exosome Klotho-FGF23 control group Exo was added to group D, and 100 μg of exosome Klotho-FGF23 Exo was added to group E. Then, except for group A, the other four groups were cultured in a 95% N2, 5% CO2 incubator for 6 h and then transferred to a normoxic incubator. After 48 h of normoxic culture, the morphological changes of the cells were observed under an inverted microscope, the cells were collected, and the whole cell lysate was prepared. The protein levels of HIF-1α and Actin in the cells were detected by Western blot. The results are shown in Fig. 9 .

[0081] ②H9C2 cells were seeded in 6-well plates (7.5×10 5 cells), and the cell coverage rate was about 60% after 24 hours. The experiment was divided into 5 groups:

[0082] A: H9C2 control group;

[0083] B: AngII 4 μM model group;

[0084] C: AngII 4μM+pAAV Exo group;

[0085] D: AngII 4μM+Klotho-FGF23 control group Exo group;

[0086] E: AngII 4μM+Klotho-FGF23 Exo group;

[0087] 100 μg of exosome pAAV Exo was added to group C, 100 μg of exosome Klotho-FGF23 control group Exo was added to group D, and 100 μg of exosome Klotho-FGF23 Exo was added to group E. At the same time, AngII with a final concentration of 4 μM was added to groups B, C, D, and E, respectively. After 48 hours, the morphological changes of cells were observed under an inverted microscope, and the expression levels of ANP and BNP proteins, the myocardial hypertrophy indicators, in the cells of each group were detected by Western blot.

[0088] Result analysis:

[0089] Through cell morphological observation, it is clear that whether it is hypoxic injury of H9C2 cardiomyocytes or AngII-induced myocardial hypertrophy injury, the addition of Klotho exosomes can effectively improve the damage of cardiomyocytes and make the morphology of damaged cardiomyocytes return to normal.

[0090] Western blot results showed that ( Fig. 9 ), in the myocardial hypoxia injury experiment, compared with group C, groups D and E were able to significantly increase the expression level of HIF-1α protein, but group E increased the expression level of HIF-1α protein higher than group D, indicating that the codon-optimized Klotho-FGF23 Exo has a better effect and can reduce the damage caused by myocardial hypoxia.

[0091] In the myocardial hypertrophy injury experiment ( Fig.10 ), compared with group C, groups D and E could significantly inhibit the expression of ANP and BNP proteins, but the efficiency of group E in inhibiting ANP and BNP proteins was higher than that of group D, indicating that the codon-optimized Klotho-FGF23Exo has better effect and can treat myocardial hypertrophy.

Claims

1. An umbilical cord mesenchymal stem cell exosome carrying a Klotho fusion gene, characterized in that: The Klotho fusion gene is composed of secretory Klotho, self-cleaving polypeptide T2A, and FGF23 in series; the artificial nucleic acid sequence of the secretory Klotho is shown in SEQ ID NO.

2.

2. The umbilical cord mesenchymal stem cell exosomes carrying the Klotho fusion gene according to claim 1, characterized in that: The artificial nucleic acid sequence of the self-cleaving polypeptide T2A is shown in SEQ ID NO.3; the artificial nucleic acid sequence of FGF23 is shown in SEQ ID NO.

4.

3. The method for preparing umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that: The method for preparing the exosomes is to construct the Klotho fusion gene into a recombinant expression vector, then package it into an adeno-associated virus, and then infect mesenchymal stem cells, so that the mesenchymal stem cells secrete exosomes.

4. Use of the exosomes according to any one of claims 1 to 2 in the preparation of a drug for treating myocardial cell hypoxia injury and / or myocardial hypertrophy injury.

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