A genetic engineering construction method of an anti-apoptosis type human mesenchymal stem cell and application thereof

Abstract

The application belongs to the technical field of genetic engineering and cell modification, and discloses a genetic engineering construction method and application of an anti-apoptosis human mesenchymal stem cell. The application realizes a breakthrough improvement through specific lactic acidification mutation at the HDAC1 K412 site, for example, precise gene editing mediated by CRISPR-Cas9, only a single amino acid site is changed, that is, K412R mutation simulates continuous lactic acidification, and the whole genome epigenetic modification disturbance can be avoided; and the modification is not dependent on exogenous metabolic substrate, and the negative feedback effect of traditional metabolic intervention is overcome; the mutant continuously inhibits the P53 apoptosis pathway, and the ability of MSC to resist oxidative stress-mediated apoptosis is significantly improved. The design concept of "one target point and one regulation" fundamentally solves the core contradiction that the prior art cannot consider long-acting and specificity at the same time, and provides a new paradigm for stem cell therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and cell modification, and relates to a construction method of mesenchymal stem cells, in particular to a genetic engineering construction method of anti-apoptotic human mesenchymal stem cells and application thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are multipotent adult stem cells derived from mesoderm, which are widely distributed in bone marrow, adipose tissue, umbilical cord blood, placental tissue, dental pulp and synovial membrane and other human tissues. When this kind of stem cells are transplanted between different individuals, strict HLA matching is not required, which significantly reduces the risk of immune rejection; and can be used to improve the local microenvironment and promote the repair of damaged tissues. At present, cell therapy based on MSCs has made important progress in multiple clinical fields, and this cell therapy has broad prospects in the field of regenerative medicine. MSC apoptosis refers to the programmed cell death process of mesenchymal stem cells under physiological or pathological conditions, which is regulated by internal and external factors. Unlike necrosis, apoptosis is a highly ordered active cell suicide mechanism, which is crucial for maintaining stem cell pool homeostasis and tissue balance. Under pathological conditions such as oxidative stress, the abnormal increase of MSC apoptosis eventually leads to the loss of repair and regeneration capacity. The research and development of anti-apoptotic human mesenchymal stem cells is based on the core bottleneck of traditional mesenchymal stem cells in anti-apoptotic applications and the deepening of human understanding of the mechanism of apoptosis.

[0003] Current strategies to enhance the anti-apoptotic ability of mesenchymal stem cells (MSCs) mainly include genetic engineering, pretreatment strategies, microenvironment engineering, and epigenetic regulation. In terms of genetic engineering, overexpression of antioxidant enzymes (such as SOD, CAT, GPx) or regulation of oxidative stress-related pathways (such as Nrf2 / ARE, SIRT1 / SIRT3) can significantly enhance the ROS scavenging ability of MSCs, while modification of anti-apoptotic genes (such as Bcl-2, Bcl-xL) or inhibition of the P53 pathway can effectively reduce cell apoptosis. Non-genetic editing pretreatment strategies such as hypoxic culture (1-5% O2), chemical drugs (melatonin, resveratrol), or cytokine (TGF-β, HGF) treatment can temporarily enhance the antioxidant ability of MSCs by activating endogenous protective mechanisms. Microenvironment engineering uses antioxidant scaffolds containing selenium nanomaterials, polydopamine coatings, or direct transplantation of healthy mitochondria to improve the survival microenvironment of MSCs. Epigenetic regulation uses DNA methylation inhibitors (5-azacytidine) or histone deacetylase inhibitors (TSA) to release the transcriptional repression of antioxidant genes, while exosome delivery of SOD mRNA or miRNA can achieve targeted antioxidant therapy. However, these existing technologies have limitations such as lack of precise regulation of gene overexpression, short-term effect of pretreatment, strong material dependence, and non-specificity of epigenetic regulation.

[0004] Despite the development of various strategies to enhance the anti-apoptotic ability of mesenchymal stem cells (MSCs), these methods have significant limitations. Gene overexpression technology can enhance antioxidant enzyme activity, but continuous high expression disrupts the redox balance homeostasis, such as SOD overexpression leading to H2O2 accumulation and exacerbating oxidative damage, and viral vectors have a 5-15% off-target risk; pretreatment strategies such as hypoxia or drug induction are not time-efficient, as the half-life of HIF-1α and other effect proteins is short (<30 minutes), which cannot meet the needs of chronic disease treatment; material engineering methods are limited by the local action range (<2mm) and material degradation period (2-4 weeks), making it difficult to address systemic diseases; epigenetic regulation affects about 8% of acetylation sites in the whole genome due to its broad-spectrum nature, which may accidentally activate pro-apoptotic pathways and even lead to genomic instability (3-fold increase in mutation rate) in the long term; and metabolic intervention such as exogenous lactic acid treatment can induce modification, but it will feedback inhibit HK2 activity, reducing ATP production by 35%. These defects are essentially due to the inherent contradictions of the technical principles—either lack of target specificity (epigenetic drugs), or limited by the short-term effect (pretreatment), or difficult to balance efficacy and safety (gene overexpression). Therefore, in the face of the problem of MSC apoptosis caused by high ROS levels in the body after MSC infusion (apoptosis rate >60% 72 hours after infusion), existing technologies generally face three core problems: lack of specificity, short-term effect, and limited systemic effect.

[0005] To this end, the application provides a genetic engineering construction method and application of an anti-apoptotic human mesenchymal stem cell. SUMMARY

[0006] The application aims to provide a genetic engineering construction method and application of an anti-apoptotic human mesenchymal stem cell, which adopts HDAC1 K412 site-specific lactylation mutation technology to simulate endogenous lactylation modification by precisely editing a single amino acid site, can continuously inhibit the P53 pathway to reduce apoptosis induced by oxidative stress under the premise of not destroying the metabolic homeostasis of the cell, significantly improves the ability of MSC to resist oxidative stress-mediated apoptosis, has the advantages of long-acting and precise regulation, avoids the whole genome effect, and realizes stable and long-acting cell protection in a non-metabolic dependent manner, breaks through the multiple limitations of the prior art in mechanism, and provides a breakthrough solution for stem cell therapy for stem cell infusion for treating diseases (osteoporosis, heart failure, and spinal cord injury, etc.).

[0007] To achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0008] The application provides a genetic engineering construction method of an anti-apoptotic human mesenchymal stem cell, comprising the following steps:

[0009] S1, designing a specific sgRNA according to the K412 site on HDAC1;

[0010] S2, mixing HiFi Cas9 protein and sgRNA in vitro to form an RNP complex by incubating at room temperature for 10-15 minutes to obtain a HiFi Cas9-sgRNA RNP complex;

[0011] S3, synthesizing an ssODN repair template;

[0012] S4, co-transfecting the HiFi Cas9-sgRNA RNP complex and the ssODN into low-passage MSCs, and after 48 hours, performing puromycin screening on the surviving stem cells to obtain an anti-apoptotic human mesenchymal stem cell by culturing a single clone.

[0013] Preferably, the nucleotide sequence of the sgRNA is shown as SED ID NO. 1.

[0014] Preferably, the nucleotide sequence of the HDAC1 is shown as SED ID NO. 2.

[0015] Preferably, the ssODN repair template is K412E, and the nucleotide sequence of the K412E is shown as SED ID NO. 3.

[0016] Preferably, the ssODN repair template comprises 50-80 bp homologous arms and PAM region silencing mutations.

[0017] The application also provides the anti-apoptotic human mesenchymal stem cells constructed by the above method.

[0018] The application also provides the use of the anti-apoptotic human mesenchymal stem cells in the preparation of a drug for promoting the anti-apoptotic ability of mesenchymal stem cells.

[0019] Preferably, the drug further comprises a medical excipient, and the preparation of the drug is an injection, a freeze-dried powder injection or a complex preparation, and the complex preparation is a microsphere or a liposome.

[0020] Preferably, the drug promotes the ability of MSCs to resist apoptosis mediated by oxidative stress by weakening the acetylation of P53 and down-regulating the mRNA level of P53 downstream apoptosis-related genes.

[0021] The application also provides the use of the anti-apoptotic human mesenchymal stem cells in the preparation of a drug for treating osteoporosis, heart failure or spinal cord injury.

[0022] The nucleotide sequence of sgRNA (SED ID NO. 1) is 5'-GCCATGGAGATCAAGCTGCGG-3';

[0023] The nucleotide sequence of HDAC1 (SED ID NO. 2) is 5'-GACAAGTTCGTGGACATCCGGGGCTGG-3'

[0024] The nucleotide sequence of K412E (SED ID NO. 3) is 5'-GACGAGTTCGTGGACATCCGAGGCTGG-3'

[0025] Compared with the prior art, the application has the following beneficial effects:

[0026] The application develops an anti-apoptosis type human mesenchymal stem cell by regulating epigenetic modification as a target point through CRISPR-Cas9 mediated HDAC1 K412 site specific lactylation mutation. Compared with the prior art, the application first proposes a strategy of simulating persistent lactylation modification by precisely editing the HDAC1 K412 site to enhance the anti-oxidative stress mediated apoptosis ability of MSCs. The above strategy does not simply overexpress an antioxidant gene as the goal, but targets the core mechanism of MSCs treatment failure, i.e., oxidative stress induced apoptosis, and realizes the regulation of the P53 apoptosis pathway by targeting the HDAC1, an epigenetic regulation hub. The K412 site mutant MSCs can continuously resist oxidative stress mediated apoptosis in the high ROS environment in vivo by stably maintaining lactylation modification, thereby significantly improving the efficacy and persistence of MSCs infusion treatment. This innovative strategy not only solves the problem that the prior art cannot balance cell survival and functional differentiation, but also provides a new epigenetic editing paradigm for stem cell infusion treatment of diseases. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a staining diagram and quantitative analysis result of Annexin V / PI double staining for detecting the apoptosis level of MSCs under oxidative stress;

[0028] Figure 2 is a staining diagram and quantitative analysis result of TUNEL staining for detecting the apoptosis level of MSCs under oxidative stress;

[0029] Figure 3 is an immunoblotting diagram for detecting the lactylation level of MSCs under oxidative stress by Western blotting;

[0030] Figure 4 is a fluorescence diagram and quantitative analysis result of immunofluorescence for detecting the lactylation level of MSCs under oxidative stress;

[0031] Figure 5 is a result of extracellular acidification rate and oxygen consumption rate level of MSCs under oxidative stress detected by Seahorse experiment;

[0032] Figure 6 is a staining diagram and quantitative analysis result of Annexin V / PI double staining for detecting the apoptosis level of MSCs under oxidative stress;

[0033] Figure 7 is a staining diagram and quantitative analysis result of TUNEL staining for detecting the apoptosis level of MSCs under oxidative stress;

[0034] Figure 8 is a fluorescence diagram and quantitative analysis result of immunofluorescence for detecting the lactylation level of MSCs under oxidative stress;

[0035] Figure 9 is a heat map of the modification level of MSCs lactate proteome;

[0036] Figure 10 is a western blotting map of the effect of different HDAC1 mutation sites on the apoptosis level of oxidative stress MSCs (IgG represents immunoglobulin G, used to exclude false positive results of non-specific binding, control represents no stimulated MSCs, H2O2 represents only hydrogen peroxide stimulated MSCs, Nala represents only sodium lactate stimulated MSCs, H2O 2+ Nala represents H2O2 and sodium lactate co-stimulated MSCs, Oxamate represents glycolysis inhibitor stimulated MSCs);

[0037] Figure 11 is a staining map and quantitative analysis result of Annexin V / PI double staining for detecting the effect of HDAC1-K412 lactate site on the apoptosis level of oxidative stress MSCs (control represents the control group without any treatment, Vector represents empty vector transfection, HDAC1 WT represents wild type HDAC1, HDAC1 K412R represents lactate inactivated HDAC1, HDAC1 K412E represents lactate activated HDAC1);

[0038] Figure 12 is a staining map and quantitative analysis result of immunofluorescence for detecting the effect of HDAC1-K412 lactate site on the p53 pathway in oxidative stress MSCs;

[0039] Figure 13 is the result of RT-qPCR for detecting the effect of HDAC1-K412 lactate site on the expression level of P53 downstream molecules in oxidative stress MSCs;

[0040] Figure 14 is a mechanism schematic diagram: K412 site of deacetylase HDAC1 can be modified by lactate. The modification enhances the enzyme activity of HDAC1, promotes its binding with p53, thereby inhibits p53 acetylation, and down-regulates the apoptosis and oxidative stress signal pathway mediated by p53. DETAILED DESCRIPTION

[0041] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0042] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0043] In order to make the objects, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application in combination with specific examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0044] Example 1: Construction of hydrogen peroxide-induced oxidative stress cell model

[0045] 1. Experimental method

[0046] (1) Construction of hydrogen peroxide-induced oxidative stress cell model

[0047] 1) Obtain primary MSCs: Extract 30 ml of bone marrow from a young and healthy volunteer through the posterior superior iliac spine, and anticoagulate with heparin. Centrifuge (1500 rpm, 5 min) the bone marrow mixed with PBS to remove the supernatant and fat. Slowly add the cell suspension to the upper layer of lymphocyte separation medium (Percoll) and centrifuge (2000 rpm, 20 min); absorb the white membrane layer containing MSCs. Wash twice with PBS, resuspend and inoculate in culture medium containing 15% FBS for culture.

[0048] 2) Cell plating: Plate the primary MSCs in a 6-well plate at a density of 1×10 5 cells / well.

[0049] 3) Construction of oxidative stress cell model: Treat MSCs with 0-400 uM of H2O2 (0, 100 uM, 200 uM and 400 uM) for 24 h.

[0050] (2) Determine the indicators in the oxidative stress cell model MSCs constructed in (1)

[0051] 1) Detect the lactic acidification level of MSCs by Western blotting: Lyse and quantify the proteins of the treated MSCs, and after electrophoresis by SDS-PAGE and membrane transfer, incubate with anti-lactic acidification antibody (such as pan-Kla) and internal reference antibody (β-actin / GAPDH), and analyze the lactic acidification protein level by ECL.

[0052] 2) Detect the lactic acidification level of MSCs by immunofluorescence: Plate the MSCs on a slide, fix with 4% paraformaldehyde, permeabilize with 0.2% Triton X-100, block with 1% BSA, and then incubate with anti-lactic acidification primary antibody (such as pan-Kla, 1:200) and fluorescent secondary antibody, respectively, stain the nucleus with DAPI, and finally mount the slide with anti-quenching agent, and observe under a confocal microscope.

[0053] 3) Detecting the level of MSC apoptosis by Tunel staining: After MSCs treated by H2O2 were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and added with TUNEL reaction mixture (containing fluorescently labeled dUTP and terminal deoxynucleotidyl transferase), incubated at 37°C for 1 hour in the dark, and then counterstained with DAPI before mounting, the apoptosis of MSCs was observed under a fluorescence microscope. Key points: ① Set positive control (treated with DNase I) and negative control (without enzyme); ② Optimize the permeation time (5-15 minutes) and TUNEL reaction time (30-90 minutes); ③ When quantifying, calculate the proportion of apoptotic cells (TUNEL-positive cells / DAPI-positive cells).

[0054] 4) Detecting the level of MSC apoptosis by Annexin V / PI: After MSCs treated by H2O2 were collected, washed with pre-cooled PBS, and resuspended in 1x Binding Buffer, 5 μL Annexin V-FITC (Annexin V is a protein that can bind to phosphatidylserine) and 5 μL PI (propidium iodide, a nuclear dye) were added in turn, and incubated at room temperature for 15 minutes in the dark. The apoptosis of MSCs was detected by flow cytometry (FITC: Ex 488 nm / Em 530 nm; PI: Ex 488 nm / Em 610 nm).

[0055] 5) Detecting the levels of glycolysis and oxidative phosphorylation of MSCs by extracellular acidification rate and oxygen consumption rate: MSCs treated by H2O2 were seeded in Seahorse XF96 cell culture plates at an optimal density (e.g., 2x10 4 per well), and after overnight attachment at 37°C, the culture medium was replaced with unbuffered medium (containing 2 mM GlutaMAX and 1 mM sodium pyruvate). In the Seahorse XF analyzer, 10 mM glucose (glycolysis stress test), 1 μM oligomycin (ATP coupling inhibitor), 50 mM 2-DG (glycolysis inhibitor), or 1.5 μM FCCP (mitochondrial uncoupler), 0.5 μM rotenone / antimycin A (ETC inhibitor) were injected in turn, and the changes in extracellular acidification rate (mpH / min) and oxygen consumption rate (pmol / min) were monitored in real time.

[0056] 2. Experimental results

[0057] 2.1 Flow cytometry detection of the level of MSC apoptosis under oxidative stress

[0058] It was found by Annexin V / PI method that after being treated with different concentrations of H2O2 for 24 hours, the apoptosis rate (Q2+Q3, the proportion of the second and third quadrants) of MSCs gradually increased and was positively correlated with the concentration of H2O2, indicating that H2O2 could promote the apoptosis of MSCs (seeFigure 1 ).

[0059] 2.2 TUNEL staining to detect the level of MSCs apoptosis under oxidative stress

[0060] TUNEL staining refers to the dUTP gap end labeling method mediated by deoxyribonucleotide terminal transferase. Through Tunel staining, it was found that after 24 hours of treatment with different concentrations of H2O2, the Tunel staining level of MSCs gradually increased, that is, the apoptosis level gradually increased, and was positively correlated with the concentration of H2O2. H2O2 can promote MSC apoptosis (see Figure 2 ).

[0061] 2.3 Western blotting to detect the level of MSCs lactate modification under oxidative stress

[0062] It was found by Western blotting that after 24 hours of treatment with different concentrations of H2O2, the lactate modification level of MSCs gradually increased, and was positively correlated with the concentration of H2O2, indicating that H2O2 can promote MSC lactate modification (see Figure 3 ).

[0063] 2.4 Immunofluorescence to detect the level of MSCs lactate modification under oxidative stress

[0064] It was found by immunofluorescence that after 24 hours of treatment with different concentrations of H2O2, the lactate modification level of MSCs gradually increased, and was positively correlated with the concentration of H2O2, indicating that H2O2 can promote MSC lactate modification (see Figure 4 ).

[0065] 2.5 Seahorse experiment to detect the level of extracellular acidification rate and oxygen consumption rate of MSCs under oxidative stress

[0066] Seahorse experiment is an experimental method for real-time measurement of cell metabolic function, mainly evaluating the ability of mitochondrial respiration (aerobic metabolism) and glycolysis (anaerobic metabolism). It is widely used in the fields of metabolism research, oncology, immunology, cardiovascular disease and drug development.

[0067] It was found by Seahorse experiment that after 24 hours of treatment with different concentrations of H2O2, the extracellular acidification rate level of MSCs gradually increased, and was positively correlated with the concentration of H2O2; the oxygen consumption rate level of MSCs gradually decreased, and was negatively correlated with the concentration of H2O2, indicating that H2O2 can promote the glycolytic activity of MSCs and inhibit the oxidative phosphorylation activity (see Figure 5 ).

[0068] Example 2 Effect of exogenous sodium lactate on oxidative stress-mediated MSCs

[0069] This example verifies the effect of exogenous sodium lactate on MSC apoptosis mediated by oxidative stress, and the specific scheme is as follows:

[0070] MSCs were seeded in 6-well plates at a density of 1 x 10 5 After 24 hours of pretreatment with different concentrations (0-20 mM) of sodium lactate, each group of MSCs was treated with 200 uM H2O2 for 24 hours. Finally, the level of MSC lactylation was detected by immunofluorescence, and the level of MSC apoptosis was detected by Tunel staining and Annexin V / PI (the specific detection methods are the same as those in Example 1).

[0071] The results of Annexin V / PI detection showed that after 24 hours of pretreatment with different concentrations of sodium lactate and 24 hours of treatment with 200 uM H2O2, the apoptosis rate (Q2+Q3) of MSCs gradually decreased, and was negatively correlated with the concentration of sodium lactate. Sodium lactate can significantly improve H2O2-induced MSC apoptosis (see Figure 6 ). The Tunel staining results showed that the Tunel staining level of MSCs gradually decreased, i.e., the apoptosis level gradually decreased, and was negatively correlated with the concentration of sodium lactate. Sodium lactate can significantly improve H2O2-induced MSC apoptosis (see Figure 7 ). The results of immunofluorescence detection showed that the level of lactylation pan-modification of MSCs gradually increased, and was positively correlated with the concentration of sodium lactate, indicating that sodium lactate can promote the lactylation modification of MSCs (see Figure 8 ).

[0072] In summary, exogenous sodium lactate can significantly improve H2O2-induced MSC apoptosis and promote the lactylation modification of MSCs.

[0073] Example 3 Lactylation of HDAC1 can reduce oxidative stress-mediated apoptosis of MSCs

[0074] The present embodiment first constructs a hydrogen peroxide (H2O2)-induced oxidative stress human mesenchymal stem cell (MSCs) model to explore the protective effect of sodium lactate pretreatment on the cell lactate modification spectrum. The experiment sets up three groups of blank control, H2O2 model and sodium lactate intervention, and after 12 hours of 20 mM sodium lactate pretreatment, 500 μM H2O2 is used to stimulate for 6 hours to establish an oxidative damage model. High-quality protein samples are prepared by ice lysis and BCA quantification, and then lactate modification proteomics analysis is carried out by using anti-lactate antibody magnetic beads enrichment combined with liquid chromatography-tandem mass spectrometry technology. Bioinformatics analysis results show that the lactate modification levels of K412 and K444 of HDAC1 protein in the sodium lactate intervention group are significantly higher than those in the model group, indicating that sodium lactate may enhance the oxidative stress resistance of MSCs by specifically regulating the epigenetic modification of key proteins. All data are verified by three independent biological repeats, and statistical processing is carried out by one-way analysis of variance (screening standard: change fold>1.5 times, P<0.05).

[0075] In this embodiment, it is found by the modification level heat map of lactate proteome that the lactate modification of K412 and K444 sites of HDAC1 is higher in the H2O2 / Nala group, indicating that sodium lactate may promote the lactate modification of K412 and K444 sites of HDAC1 of MSC (see Figure 9 ). And the lactate modification of HDAC1 can reduce the oxidative stress-mediated apoptosis of MSC by inhibiting the P53 signaling pathway.

[0076] Example 4 Verification of the effect of simulated lactate modification site mutation on oxidative stress-mediated apoptosis of MSC

[0077] In this embodiment, the simulated lactate modification mutation (K412E) of HDAC1 K412 site is introduced into MSCs by gene editing technology (CRISPR-Cas9) to verify the effect of simulated lactate modification site mutation on the resistance of mesenchymal stem cells to oxidative stress-mediated apoptosis.

[0078] 1. Experimental method

[0079] (1) CRISPR-Cas9-mediated HDAC1 K412 site-specific lactate modification mutation

[0080] Firstly, based on the NCBI HDAC1 gene sequence (Gene ID: 3065), a high-specificity sgRNA was designed for K412 site (such as 5'-GCCATGGAGATCAAGCTGCGG-3') and the off-target risk was evaluated by CRISPOR; two kinds of ssODN repair templates were synthesized: K412E (AAG→GAG, simulating sustained lactate state) and K412R (AAG→CGG, simulating de-lactate state), both of which contain 50-80 bp homologous arms and PAM region silencing mutations; the HiFi Cas9-sgRNA NPs complex was co-transfected with the two kinds of ssODN into low-passage MSCs using the Neon electroporation system, and an untransfected control group was set up; 48 hours after transfection, puromycin (1-2 μg / mL) screening was performed, and 72 hours later, the editing efficiency was verified by T7E1 enzyme digestion and Sanger sequencing (covering 100 bp upstream and downstream of the mutation site); single clones were isolated by limiting dilution, and the genotype was confirmed by Sanger sequencing after 2-3 weeks of culture, and the K412E / R mutation and lactate modification state difference were verified by Western blotting (anti-HDAC1 antibody) and mass spectrometry; to improve the HDR efficiency, RS-1 (5 μM) was added and the cell cycle was synchronized, and whole genome sequencing was used to exclude off-target effects.

[0081] (2) Cell plating: MSCs were seeded in 6-well plates at a density of 1×10 5 / well.

[0082] (3) Construction of hydrogen peroxide-induced oxidative stress cell model: each group of MSCs was treated with 200 uM of H2O2 for 24 h.

[0083] (4) The lactate level of MSCs was detected by Western blotting and immunofluorescence, and the apoptosis level of MSCs was detected by Tunel staining and Annexin V / PI, and the specific experimental methods were the same as those in Example 1.

[0084] (5) The activation level of P53 pathway of MSCs was detected by RT-qPCR: after extracting the total RNA of H2O2 treatment group and control group MSCs and reverse transcribing into cDNA, RT-qPCR was performed using SYBR Green method, the expression changes of key genes (such as CDKN1A, BAX, PUMA) of p53 pathway were detected, GAPDH or β-actin was used as internal reference, the relative expression was calculated by 2-ΔΔCt method, and the activation effect of oxidative stress on p53 pathway was verified.

[0085] (6) Detection of the activation level of P53 pathway in MSCs by immunofluorescence: MSCs treated with H2O2 were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and then labeled with anti-P53-K379Ac specific antibody (1:200) and corresponding fluorescent secondary antibody (e.g. Alexa Fluor 594). The nucleus was counterstained with DAPI. The intranuclear fluorescence intensity of acetylated P53 at K379 site was quantitatively analyzed under confocal microscope to verify the activation level of P53 pathway by oxidative stress (with isotype control and untreated control groups).

[0086] 2. Experimental results

[0087] 2.1 Western blotting showed that HDAC1-K412 lactylation was significantly enhanced in oxidative stress MSCs: Western blotting showed that stimulation with H2O2 and sodium lactate alone or simultaneously promoted the lactylation modification of HDAC1, and stimulation with glycolysis inhibitor Oxamate inhibited the lactylation modification of HDAC1. Overexpression of wild-type HDAC1 (HDAC1WT), site-mutated HDAC1 (HDAC1K412R and HDAC1K444R) showed that K412 site mutation inhibited the lactylation modification of HDAC1, while K444 site mutation had no obvious effect, indicating that there was lactylation modification at K412 site of HDAC1 (see Figure 10 ).

[0088] 2.2 Flow cytometry showed that HDAC1-K412 lactylation inhibited oxidative stress-mediated MSC apoptosis: Flow cytometry showed that overexpression of wild-type HDAC1 (HDAC1WT), lactylation-inactivated HDAC1 (HDAC1K412R), and lactylation-activated HDAC1 (HDAC1K412E) in MSCs stimulated with 200 uM H2O2 for 24 hours found that lactylation-inactivated HDAC1 (HDAC1K412R) promoted H2O2-induced MSC apoptosis, while lactylation-activated HDAC1 (HDAC1K412E) inhibited H2O2-induced MSC apoptosis, indicating that K412 lactylation of HDAC1 could protect MSCs from oxidative stress-induced apoptosis (see Figure 11 ).

[0089] 2.3 Immunofluorescence shows that HDAC1-K412 lactylation inhibits the activation of the P53 pathway: By immunofluorescence, it was found that after 24 hours of stimulation with 200 uM of H2O2 in MSCs overexpressing wild-type HDAC1 (HDAC1WT), lactylation-inactivated HDAC1 (HDAC1K412R) and lactylation-activated HDAC1 (HDAC1K412E), the lactylation-inactivated HDAC1 (HDAC1K412R) activated the p53 pathway (the level of p53-K379Ac increased), while the lactylation-activated HDAC1 (HDAC1K412E) inhibited the p53 pathway (the level of p53-K379Ac decreased), indicating that the K412 lactylation of HDAC1 can inhibit the p53 pathway (see Figure 12 ).

[0090] 2.4 RT-qPCR shows that HDAC1-K412 lactylation inhibits the expression of P53 pathway downstream genes Noxa, Puma, Bax and Apaf1: By RT-qPCR, it was found that after 24 hours of stimulation with 200 uM of H2O2 in MSCs overexpressing wild-type HDAC1 (HDAC1WT), lactylation-inactivated HDAC1 (HDAC1K412R) and lactylation-activated HDAC1 (HDAC1K412E), the lactylation-inactivated HDAC1 (HDAC1K412R) promoted the expression of p53 pathway downstream molecules, while the lactylation-activated HDAC1 (HDAC1K412E) inhibited the expression of p53 pathway downstream molecules, indicating that the K412 lactylation of HDAC1 can inhibit the p53 pathway (see Figure 13 ).

[0091] In summary, after the K412E simulated lactylation mutation was made to the HDAC1-K412 site, the level of oxidative stress-mediated apoptosis of MSCs was significantly reduced, the acetylation of P53 was weakened, and the mRNA level of P53 downstream apoptosis-related genes was decreased, proving that the lactylation-activated HDAC1 can significantly promote the ability of MSCs to resist oxidative stress-mediated apoptosis.

[0092] The specific embodiments are only an explanation of the present application, which is not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A genetic engineering construction method of an anti-apoptotic type of human mesenchymal stem cell, characterized by, The method comprises the following steps: S1. Designing sgRNA according to K412 site on HDAC1 as shown in the nucleotide sequence of SED ID NO. 2, wherein the sgRNA is shown in the nucleotide sequence of SED ID NO. 1; S2. Transfecting RNP complex formed by mixing HiFi Cas9 protein and sgRNA in vitro to obtain HiFi Cas9-sgRNA RNP complex; S3. Synthesizing ssODN repair template, wherein the ssODN repair template is K412E as shown in the nucleotide sequence of SED ID NO. 3; S4. Co-transfecting HiFi Cas9-sgRNA RNP complex and ssODN into low-passage MSCs, and after 48 hours, performing puromycin screening on surviving stem cells, and obtaining anti-apoptosis type human mesenchymal stem cells by isolating and culturing single clones.

2. The construction method according to claim 1, characterized in that, The ssODN repair template comprises 50-80 bp homologous arms and PAM region silencing mutation.

3. Anti-apoptosis type human mesenchymal stem cells obtained by the construction method of claim 1 or 2.

4. Application of the anti-apoptosis type human mesenchymal stem cells of claim 3 in the preparation of a drug for treating osteoporosis, heart failure or spinal cord injury.

5. Use according to claim 4, characterized in that, The drug further comprises a medical excipient.

6. Use according to claim 4, characterized in that, The preparation of the drug is an injection, a freeze-dried powder injection or a complex preparation.

7. Use according to claim 6, characterized in that, The complex preparation is a microsphere or a liposome.

8. Use according to claim 4, characterized in that, The drug promotes the ability of MSCs to resist apoptosis mediated by oxidative stress by weakening the acetylation of P53 and down-regulating the mRNA level of P53 downstream apoptosis-related genes.

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