Genetic engineering construction method and application of anti-apoptosis human mesenchymal stem cells

Abstract

The invention belongs to the technical field of gene engineering and cell transformation, and discloses a gene engineering construction method and application of anti-apoptosis human mesenchymal stem cells. Through HDAC1 K412 site specific lactic acid mutation, breakthrough improvements are realized: for example, CRISPR-Cas9-mediated accurate gene editing is utilized, only a single amino acid site is changed, that is, K412R mutation simulates persistent lactic acid, and whole genome apparent modification disturbance can be avoided; the modification does not depend on an exogenous metabolic substrate, and the negative feedback effect of traditional metabolic intervention is overcome; the mutant continuously inhibits a P53 apoptosis pathway, so that the oxidative stress mediated apoptosis resistance of the MSC is remarkably improved. According to the design concept of one target spot and one regulation, the core contradiction that long-term effectiveness and specificity cannot be considered in the prior art is fundamentally solved, and a brand new normal form is provided for stem cell treatment.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and cell modification technology, and to a method for constructing mesenchymal stem cells, specifically to a genetic engineering method for constructing and applying anti-apoptotic human mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs), pluripotent adult stem cells derived from the mesoderm, are widely distributed in various human tissues, including bone marrow, adipose tissue, umbilical cord blood, placental tissue, dental pulp, and synovium. When these stem cells are transplanted into their own tissues, strict HLA typing is not required, significantly reducing the risk of immune rejection. They can be used to improve the local microenvironment and promote the repair of damaged tissues. Currently, MSC-based cell therapies have made significant progress in several clinical fields, and this cell therapy has broad prospects in regenerative medicine. MSC apoptosis refers to the programmed cell death process of mesenchymal stem cells under physiological or pathological conditions, regulated by internal and external factors. Unlike necrosis, apoptosis is a highly ordered, active cell suicide mechanism, crucial for maintaining stem cell pool homeostasis and tissue balance. Under pathological conditions such as oxidative stress, MSC apoptosis increases abnormally, ultimately resulting in the loss of repair and regeneration capabilities. The development of anti-apoptotic human mesenchymal stem cells stems from the core bottlenecks in the anti-apoptotic applications of traditional mesenchymal stem cells, as well as the deepening understanding of the mechanisms of apoptosis.

[0003] Currently, the main technical approaches to enhance the anti-apoptotic capacity of mesenchymal stem cells (MSCs) include genetic engineering, pretreatment strategies, microenvironment engineering, and epigenetic regulation. In 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 capacity of MSCs, while modification of anti-apoptotic genes (such as Bcl-2, Bcl-xL) or inhibition of the p53 pathway can effectively reduce apoptosis. Non-gene-editing pretreatment strategies, such as hypoxic culture (1-5% O2), chemical treatment (melatonin, resveratrol), or cytokine treatment (TGF-β, HGF), can temporarily enhance the antioxidant capacity of MSCs by activating endogenous protective mechanisms. Microenvironment engineering utilizes selenium-containing nanomaterials, polydopamine coatings, or other antioxidant scaffolds, or directly transplants healthy mitochondria, to improve the survival microenvironment of MSCs. Epigenetic regulation can relieve transcriptional repression of antioxidant genes by using DNA methylation inhibitors (5-azacytidine) or histone deacetylase inhibitors (TSA), while exosome delivery of SOD mRNA or miRNA can achieve targeted antioxidant therapy. However, these existing technologies have limitations such as a lack of precise regulation of gene overexpression, short-lived pretreatment effects, strong material dependence, or nonspecific epigenetic regulation.

[0004] Although existing technologies have developed various methods to enhance the anti-apoptotic capacity of mesenchymal stem cells (MSCs), these methods all have significant limitations. While gene overexpression technology can enhance the activity of antioxidant enzymes, its sustained high expression disrupts redox homeostasis; for example, SOD overexpression leads to H2O2 accumulation, which exacerbates oxidative damage, and viral vectors carry an off-target risk of approximately 5-15%. Pretreatment strategies such as hypoxia or drug induction are not timely enough, as effector proteins like HIF-1α have short half-lives (<30 minutes), failing to meet the needs of chronic disease treatment. Materials engineering methods are limited by their localized action area (<2 mm) and material degradation cycle (2-4 weeks), making them unsuitable for systemic diseases. Epigenetic regulation, due to its broad-spectrum influence on approximately 8% of acetylation sites across the genome, may unintentionally activate pro-apoptotic pathways, potentially leading to long-term genomic instability (a 3-fold increase in mutation rate). While metabolic interventions such as exogenous lactate treatment can induce modification, they also feedback-inhibit HK2 activity, reducing ATP production by 35%. These shortcomings essentially stem from inherent contradictions in the technical principles—either a lack of target specificity (epimal drugs), limitations due to short-lived effects (pretreatment), or difficulty in balancing efficacy and safety (gene overexpression). Therefore, when facing the problem of MSC apoptosis caused by high ROS levels in the in vivo environment after MSC infusion (apoptosis rate >60% at 72 hours post-infusion), existing technologies generally face three core problems: insufficient specificity, short-lived effects, and limited systemic effects.

[0005] Therefore, this invention provides a genetic engineering method for constructing and applying anti-apoptotic human mesenchymal stem cells. Summary of the Invention

[0006] This invention aims to provide a genetic engineering method for constructing and applying anti-apoptotic human mesenchymal stem cells (MSCs). Utilizing HDAC1 K412 site-specific lactation mutation technology, it precisely edits a single amino acid site to simulate endogenous lactation modification. Without disrupting cellular metabolic homeostasis, it can continuously inhibit the P53 pathway, reducing oxidative stress-induced apoptosis. This significantly enhances the ability of MSCs to resist oxidative stress-mediated apoptosis, offering advantages such as long-lasting action and precise regulation. It avoids genome-wide effects and achieves stable and long-lasting cell protection in a non-metabolic manner. Mechanistically, it overcomes multiple limitations of existing technologies, providing a breakthrough solution for stem cell therapy in vivo for diseases (osteoporosis, heart failure, and spinal cord injury, etc.).

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a method for genetically engineering the construction of anti-apoptotic human mesenchymal stem cells, comprising the following steps:

[0009] S1. Design specific sgRNAs based on the K412 site on HDAC1;

[0010] S2. The HiFi Cas9 protein and sgRNA were mixed and incubated at room temperature for 10-15 minutes in vitro to form an RNP complex, which was then transfected to obtain the HiFi Cas9-sgRNA RNP complex.

[0011] S3, Synthesize ssODN repair template;

[0012] S4. Low-passaged MSCs were co-transfected with the HiFi Cas9-sgRNA RNP complex and ssODN. After 48 hours, puromycin was used to screen for viable stem cells, and anti-apoptotic human mesenchymal stem cells were obtained by isolation and monoclonal culture.

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

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

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

[0016] Preferably, the ssODN repair template contains a 50-80bp homologous arm and a PAM region silencing mutation.

[0017] The present invention also provides anti-apoptotic human mesenchymal stem cells constructed using the above-described construction method.

[0018] The present invention also provides the application of the above-mentioned anti-apoptotic human mesenchymal stem cells in the preparation of drugs that promote the anti-apoptotic ability of mesenchymal stem cells.

[0019] Preferably, the drug further includes medical excipients, and the drug formulation is an injection, a lyophilized powder injection, or a compound formulation, wherein the compound formulation is microspheres or liposomes.

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

[0021] The present invention also provides the application of the above-mentioned anti-apoptotic human mesenchymal stem cells in the preparation of drugs for treating osteoporosis, heart failure or spinal cord injury.

[0022] The nucleotide sequence of the sgRNA (SED ID NO.1): 5'-GCCATGGAGATCAAGCTGCGG-3'; The nucleotide sequence of HDAC1 (SED ID NO.2): 5'-GACAAGTTCGTGGACATCCGGGGCTGG-3' The nucleotide sequence of K412E (SED ID NO.3) is 5'-GACGAGTTCGTGGACATCCGAGGCTGG-3'.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] This invention utilizes CRISPR-Cas9-mediated HDAC1 K412 site-specific lactation mutations to target epigenetic modifications and develop anti-apoptotic human mesenchymal stem cells (MSCs). Compared to existing technologies, this invention is the first to propose a strategy of precisely editing the HDAC1 K412 site to simulate persistent lactation modification, thereby enhancing the oxidative stress-mediated apoptosis resistance of MSCs. This strategy does not aim to simply overexpress a specific antioxidant gene, but rather targets the core mechanism mediating MSC treatment failure—oxidative stress-induced apoptosis—by targeting the epigenetic regulatory hub of HDAC1, thereby regulating the p53 apoptosis pathway. K412 site-mutant MSCs, by stably maintaining lactation modification, can continuously resist oxidative stress-mediated apoptosis in a high ROS environment in vivo, thus significantly improving the efficacy and durability of MSC infusion therapy. This innovative strategy not only solves the problem of existing technologies failing to simultaneously address cell survival and functional differentiation, but also provides a novel epigenetic editing paradigm for stem cell infusion therapy. Attached Figure Description

[0025] Figure 1 These are staining images and quantitative analysis results of Annexin V / PI double staining to detect MSC apoptosis levels under oxidative stress;

[0026] Figure 2 This is a staining pattern and quantitative analysis results of TUNEL staining to detect the apoptosis level of MSCs under oxidative stress;

[0027] Figure 3 This is an immunoblotting image used to detect the lactation level of MSCs under oxidative stress using Western blotting.

[0028] Figure 4 This is a fluorescence image and quantitative analysis result of immunofluorescence detection of lactation level of MSCs under oxidative stress;

[0029] Figure 5 The results are from the Seahorse assay, which detected the extracellular acidification rate and oxygen consumption level of MSCs under oxidative stress.

[0030] Figure 6 This is a staining pattern and quantitative analysis result of Annexin V / PI double staining to detect the apoptosis level of MSCs under oxidative stress by exogenous sodium lactate;

[0031] Figure 7 This is a TUNEL staining image and quantitative analysis results of the apoptosis level of MSCs under oxidative stress detected by exogenous sodium lactate.

[0032] Figure 8 This is an immunofluorescence assay showing the fluorescence pattern and quantitative analysis results of the lactation level of MSCs under oxidative stress by exogenous sodium lactate.

[0033] Figure 9 This is a heatmap of the modification levels of the lactoproteinome of MSCs;

[0034] Figure 10 This is an immunoblot image obtained by Western blotting to detect 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 due to nonspecific binding; control indicates no stimulation of MSCs; H2O2 indicates stimulation of MSCs with hydrogen peroxide only; Nala indicates stimulation of MSCs with sodium lactate only; H2O...) 2+ Nala indicates that H2O2 and sodium lactate jointly stimulate MSCs, while Oxamate indicates that glycolysis inhibitors stimulate MSCs.

[0035] Figure 11 This is a staining map and quantitative analysis results of Annexin V / PI double staining to detect the effect of HDAC1-K412 lactation sites on the apoptosis level of oxidative stress MSCs (contol represents the control group without any treatment, Vector represents empty vector transfection, HDAC1WT represents wild-type HDAC1, HDAC1K412R represents lactation-inactivated HDAC1, and HDAC1K412E represents lactation-activated HDAC1).

[0036] Figure 12 This is a staining map and quantitative analysis results of immunofluorescence detection of the effect of HDAC1-K412 lactation site on the p53 pathway in oxidative stress MSCs;

[0037] Figure 13 The results are obtained by RT-qPCR detection of the effect of HDAC1-K412 lactation site on the expression level of downstream molecules of P53 in oxidative stress MSCs;

[0038] Figure 14 Here is a schematic diagram of the mechanism: Lactation modification can occur at the K412 site of the deacetylase HDAC1. This modification enhances the enzyme activity of HDAC1, promotes its binding to p53, thereby inhibiting p53 acetylation and downregulating the activation of its mediated apoptosis and oxidative stress signaling pathways. Detailed Implementation

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0042] Example 1: Construction of a hydrogen peroxide-induced oxidative stress cell model

[0043] 1. Experimental Methods

[0044] (1) Constructing a hydrogen peroxide-induced oxidative stress cell model

[0045] 1) Obtaining primary MSCs: 30 ml of bone marrow was extracted from young, healthy volunteers via the posterior superior iliac spine and anticoagulated with heparin. The bone marrow was mixed with PBS and centrifuged (1500 rpm, 5 min) to remove the supernatant and fat. The cell suspension was slowly added to the upper layer of lymphocyte separation medium (Percoll) and centrifuged (2000 rpm, 20 min); the white membrane layer containing MSCs was aspirated. The cells were washed twice with PBS, resuspended, and seeded in medium containing 15% FBS for culture.

[0046] 2) Cell seeding: Primary MSCs were seeded at a ratio of 1×10⁻⁶. 5 / The density of the wells was seeded in a 6-well plate.

[0047] 3) Construction of oxidative stress cell model: MSCs were treated with 0-400 uM H2O2 (0, 100 uM, 200 uM and 400 uM) for 24 h.

[0048] (2) The various indicators in the oxidative stress cell model MSCs constructed in (1) were measured.

[0049] 1) Detection of MSC lactation level by Western blotting: After treatment, MSCs were lysed and proteins were quantified. After SDS-PAGE electrophoresis and membrane transfer, they were incubated with anti-lactation antibody (such as pan-Kla) and internal control antibody (β-actin / GAPDH). ECL imaging was used to analyze the lactation protein level.

[0050] 2) Detection of MSC lactation level by immunofluorescence: MSCs were seeded on a slide, treated with H2O2, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 1% BSA, and then incubated sequentially with anti-lactation primary antibody (e.g., pan-Kla, 1:200) and fluorescent secondary antibody, stained with DAPI, and finally mounted with anti-quenching agent and observed under a confocal microscope.

[0051] 3) Detection of MSC apoptosis levels by TUNEL staining: MSCs treated with H2O2 were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then incubated with TUNEL reaction mixture (containing fluorescently labeled dUTP and terminal deoxynucleotidyl transferase) at 37°C in the dark for 1 hour. After counterstaining the nuclei with DAPI, the slides were mounted and observed under a fluorescence microscope. Key points: ① Set up a positive control (DNase I treatment) and a negative control (no enzyme added); ② Optimize the permeabilization time (5-15 minutes) and TUNEL reaction time (30-90 minutes); ③ Quantify the apoptotic cell ratio (TUNEL-positive cells / DAPI-positive cells).

[0052] 4) Detection of MSC apoptosis levels by Annexin V / PI: Collect MSCs treated with H2O2, wash with pre-cooled PBS and resuspend in 1×Binding Buffer, add 5 μL Annexin V-FITC (Annexin V is a protein that can bind to phosphatidylserine) and 5 μL PI (propidium iodide, a nuclear dye) in sequence, incubate at room temperature in the dark for 15 minutes, and detect by flow cytometry (FITC: Ex 488 nm / Em 530 nm; PI: Ex 488 nm / Em 610 nm).

[0053] 5) Detecting the levels of glycolysis and oxidative phosphorylation in MSCs by extracellular acidification rate and oxygen consumption rate: MSCs treated with H2O2 were placed at the optimal density (e.g., 2 × 10⁻⁶). 4 ( / well) cells were seeded into Seahorse XF96 cell culture plates and incubated overnight at 37°C. After attachment, the medium was replaced with unbuffered medium (containing 2 mM GlutaMAX and 1 mM sodium pyruvate). 10 mM glucose (glycolytic stress test), 1 μM oligomycin (ATP coupling inhibitor), 50 mM 2-DG (glycolytic inhibitor) or 1.5 μM FCCP (mitochondrial uncoupling agent), and 0.5 μM rotenone / antimycin A (ETC inhibitor) were injected sequentially into the Seahorse XF analyzer. Changes in extracellular acidification rate (mpH / min) and oxygen consumption rate (pmol / min) were monitored in real time.

[0054] 2. Experimental Results

[0055] 2.1 Flow cytometry detection of MSC apoptosis levels under oxidative stress

[0056] Using the Annexin V / PI method, it was found that after 24 hours of treatment with different concentrations of H2O2, the apoptosis rate of MSCs (Q2+Q3, the ratio of the second quadrant to the third quadrant) gradually increased and was positively correlated with the H2O2 concentration, indicating that H2O2 can promote MSC apoptosis (see Annexin V / PI method). Figure 1 ).

[0057] 2.2 TUNEL staining to detect MSC apoptosis levels under oxidative stress

[0058] TUNEL staining refers to the deoxyribonucleotide terminal transferase-mediated dUTP nick-end labeling method. TUNEL staining revealed that after 24 hours of treatment with different concentrations of H2O2, the TUNEL staining level of MSCs gradually increased, indicating a gradual increase in apoptosis levels, and this increase was positively correlated with H2O2 concentration. H2O2 can promote MSC apoptosis (see...). Figure 2 ).

[0059] 2.3 Western blot analysis of lactation levels in MSCs under oxidative stress

[0060] Western blotting revealed that after 24 hours of treatment with different concentrations of H2O2, the level of lactation modification in MSCs gradually increased and was positively correlated with the H2O2 concentration, indicating that H2O2 can promote lactation modification of MSCs (see...). Figure 3 ).

[0061] 2.4 Immunofluorescence detection of lactation level in MSCs under oxidative stress

[0062] Immunofluorescence revealed that after 24 hours of treatment with different concentrations of H2O2, the level of lactation modification in MSCs gradually increased and was positively correlated with the H2O2 concentration, indicating that H2O2 can promote lactation modification of MSCs (see...). Figure 4 ).

[0063] 2.5 Seahorse assay to detect extracellular acidification rate and oxygen consumption level of MSCs under oxidative stress

[0064] The Seahorse assay is an experimental method for real-time measurement of cellular metabolic function, primarily assessing mitochondrial respiration (aerobic metabolism) and glycolysis (anaerobic metabolism). It is widely used in metabolic research, oncology, immunology, cardiovascular disease, and drug development.

[0065] Seahorse experiments revealed that after 24 hours of treatment with different concentrations of H2O2, the extracellular acidification rate of MSCs gradually increased and was positively correlated with H2O2 concentration; while the oxygen consumption rate of MSCs gradually decreased and was negatively correlated with H2O2 concentration, indicating that H2O2 can promote glycolysis activity and inhibit oxidative phosphorylation activity of MSCs (see...). Figure 5 ).

[0066] Example 2: Effects of exogenous sodium lactate on oxidative stress-mediated MSCs

[0067] This embodiment verifies the effect of exogenous sodium lactate on oxidative stress-mediated apoptosis of MSCs. The specific scheme is as follows:

[0068] MSCs were distributed at a rate of 1×10 5 MSCs were seeded at a density of 1 / 2 well in 6-well plates. After pretreatment with sodium lactate at different concentrations (0-20 mM) for 24 hours, each group of MSCs was treated with 200 μM H2O2 for 24 hours. Finally, the lactation level of MSCs was detected by immunofluorescence, and the apoptosis level of MSCs was detected by TUNEL staining and Annexin V / PI (the specific detection methods here are the same as those in Example 1).

[0069] Annexin V / PI assay results showed that after 24 hours of pretreatment with different concentrations of sodium lactate, followed by 24 hours of treatment with 200 μM H2O2, the apoptosis rate of MSCs (Q2+Q3) gradually decreased, and was negatively correlated with sodium lactate concentration. Sodium lactate significantly improved H2O2-induced MSC apoptosis (see Annexin V / PI assay results). Figure 6 Tunel staining results showed that the Tunel staining level of MSCs gradually decreased, indicating a gradual decrease in apoptosis levels, and this was negatively correlated with sodium lactate concentration. Sodium lactate significantly improved H2O2-induced MSC apoptosis (see...). Figure 7 Immunofluorescence assays showed that the level of lactation modification in MSCs gradually increased and was positively correlated with sodium lactate concentration, indicating that sodium lactate can promote lactation modification of MSCs (see [link to study]). Figure 8 ).

[0070] In summary, exogenous sodium lactate can significantly improve H2O2-induced MSC apoptosis and promote MSC lactation modification.

[0071] Example 3: Lactation of HDAC1 can alleviate oxidative stress-mediated apoptosis in MSCs.

[0072] This embodiment first investigates the protective effect of sodium lactate pretreatment on the lactation modification profile of human mesenchymal stem cells (MSCs) under hydrogen peroxide (H2O2)-induced oxidative stress by constructing a model. The experiment included three groups: a blank control, an H2O2 model, and a sodium lactate intervention group. After 12 hours of 20 mM sodium lactate pretreatment, an oxidative damage model was established by stimulation with 500 μM H2O2 for 6 hours. High-quality protein samples were prepared by on-ice lysis and BCA quantification, and then lactation modification proteomics analysis was performed using anti-lactation antibody magnetic bead enrichment combined with liquid chromatography-tandem mass spectrometry. Bioinformatics analysis showed that the lactation modification levels of lysines at positions 412 and 444 (K412, K444) of the HDAC1 protein in the sodium lactate intervention group were 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 were validated three times independently by biological means, and statistical analysis was performed using one-way ANOVA (screening criteria: fold change > 1.5, P < 0.05).

[0073] This embodiment, through a heatmap of the modification levels of the lactated proteome, revealed that lactation at the K412 and K444 sites of HDAC1 was higher in the H2O2 / Nala group, indicating that sodium lactate may promote lactation modification at the K412 and K444 sites of HDAC1 in MSCs (see...). Figure 9 HDAC1 lactation can alleviate oxidative stress-mediated apoptosis in MSCs by inhibiting the P53 signaling pathway.

[0074] Example 4: Verifying the effect of site mutations mimicking lactation on oxidative stress-mediated apoptosis in MSCs.

[0075] In this embodiment, a simulated lactation mutation (K412E) at the HDAC1 K412 site was introduced into MSCs using gene editing technology (CRISPR-Cas9) to verify the effect of the simulated lactation site mutation on the resistance of mesenchymal stem cells to oxidative stress-mediated apoptosis.

[0076] 1. Experimental Methods

[0077] (1) CRISPR-Cas9-mediated HDAC1 K412 site-specific lactation mutation

[0078] First, based on the NCBI HDAC1 gene sequence (Gene ID: 3065), highly specific sgRNAs (such as 5'-GCCATGGAGATCAAGCTGCGG-3') targeting the K412 site were designed, and off-target risks were assessed using CRISPOR. Two ssODN repair templates were synthesized: K412E (AAG→GAG, simulating persistent lactation) and K412R (AAG→CGG, simulating delactation), both containing 50-80 bp homologous arms and PAM region silencing mutations. The HiFi... The Cas9-sgRNARNP complex was co-transfected with two types of ssODN into low-passaged MSCs, with an untransfected control group. 48 hours post-transfection, puromycin (1-2 μg / mL) was used for selection. 72 hours later, editing efficiency was verified by T7E1 digestion and Sanger sequencing (covering 100 bp upstream and downstream of the mutation site). Single clones were isolated using limiting dilution, and after 2-3 weeks of culture, genotypes were confirmed by Sanger sequencing. Differences in K412E / R mutation and lactation modification status were verified by Western blotting (anti-HDAC1 antibody) and mass spectrometry. RS-1 (5 μM) was added to improve HDR efficiency and the cell cycle was synchronized. Whole-genome sequencing was used to rule out off-target effects.

[0079] (2) Cell seeding: MSCs were seeded at a ratio of 1×10⁻⁶. 5 / well density seeding in 6-well plates

[0080] (3) Construct a hydrogen peroxide-induced oxidative stress cell model: treat each group of MSCs with 200 uM H2O2 for 24 h.

[0081] (4) The lactation 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. The specific experimental methods were the same as those in Example 1.

[0082] (5) Detection of p53 pathway activation level in MSCs by RT-qPCR: Total RNA was extracted from MSCs in the H2O2 treatment group and the control group and reverse transcribed into cDNA. RT-qPCR was performed using the SYBR Green method to detect the expression changes of key genes in the p53 pathway (such as CDKN1A, BAX, and PUMA). GAPDH or β-actin was used as internal references. The relative expression level was calculated by the 2-ΔΔCt method to verify the activation effect of oxidative stress on the p53 pathway.

[0083] (6) Detection of P53 pathway activation level in MSCs by immunofluorescence: H2O2-treated MSCs were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, labeled with anti-P53-K379Ac specific antibody (1:200) and corresponding fluorescent secondary antibody (such as Alexa Fluor 594), and the cell nuclei were counterstained with DAPI. The intranuclear fluorescence intensity of K379 acetylated P53 was quantitatively analyzed under a confocal microscope to verify the activation level of P53 pathway by oxidative stress (isotype control and untreated control group were set up).

[0084] 2. Experimental Results

[0085] 2.1 Western blot analysis showed that HDAC1-K412 lactation was significantly enhanced in oxidative stress MSCs: Western blot analysis revealed that stimulation with H2O2 alone or simultaneously with sodium lactate promoted HDAC1 lactation modification, while stimulation with the glycolysis inhibitor Oxamate inhibited HDAC1 lactation modification; overexpression of wild-type HDAC1 (HDAC1WT) and site-mutant HDAC1 (HDAC1K412R and HDAC1K444R) showed that the K412 site mutation inhibited HDAC1 lactation modification, while the K444 site mutation had no significant effect, indicating that lactation modification exists at the K412 site of HDAC1 (see...). Figure 10 ).

[0086] 2.2 Flow cytometry showed that HDAC1-K412 lactation inhibited oxidative stress-mediated MSC apoptosis: After stimulating wild-type HDAC1 (HDAC1WT), lactation-inactivated HDAC1 (HDAC1K412R), and lactation-activated HDAC1 (HDAC1K412E) MSCs with 200 μM H2O2 for 24 hours using Annexin V / PI, it was found that lactation-inactivated HDAC1 (HDAC1K412R) promoted H2O2-induced MSC apoptosis, while lactation-activated HDAC1 (HDAC1K412E) inhibited H2O2-induced MSC apoptosis. This indicates that K412 lactation of HDAC1 can protect MSCs from oxidative stress-induced apoptosis (see Annexin V / PI). Figure 11 ).

[0087] 2.3 Immunofluorescence showed that HDAC1-K412 lactation inhibited p53 pathway activation: Immunofluorescence was used to stimulate wild-type HDAC1 (HDAC1WT), lactation-inactivated HDAC1 (HDAC1K412R), and lactation-activated HDAC1 (HDAC1K412E) MSCs with 200 μM H2O2 for 24 hours. The results showed that lactation-inactivated HDAC1 (HDAC1K412R) activated the p53 pathway (increased p53-K379Ac levels), while lactation-activated HDAC1 (HDAC1K412E) inhibited the p53 pathway (decreased p53-K379Ac levels). This indicates that K412 lactation of HDAC1 can inhibit the p53 pathway (see [link to study]. Figure 12 ).

[0088] 2.4 RT-qPCR showed that HDAC1-K412 lactation inhibited the expression of downstream genes Noxa, Puma, Bax, and Apaf1 in the p53 pathway: RT-qPCR was performed on overexpressing wild-type HDAC1 (HDAC1WT), lactation-inactivated HDAC1 (HDAC1K412R), and lactation-activated HDAC1 (HDAC1K412E) MSCs after stimulation with 200 μM H2O2 for 24 hours. The results showed that lactation-inactivated HDAC1 (HDAC1K412R) promoted the expression of downstream molecules in the p53 pathway, while lactation-activated HDAC1 (HDAC1K412E) inhibited the expression of downstream molecules in the p53 pathway. This indicates that K412 lactation of HDAC1 can inhibit the p53 pathway (see [link to RT-qPCR]). Figure 13 ).

[0089] In summary, the oxidative stress-mediated apoptosis level of MSCs was significantly downregulated, P53 acetylation was weakened, and the mRNA levels of downstream apoptosis-related genes of P53 decreased after the HDAC1-K412 site was mutated to mimic lactation (K412E). This demonstrates that lactation-activated HDAC1 can significantly promote the ability of MSCs to resist oxidative stress-mediated apoptosis.

[0090] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for genetically engineering the construction of anti-apoptotic human mesenchymal stem cells, characterized in that, Includes the following steps: S1. Design sgRNA with nucleotide sequences as shown in SEDID NO.1 based on the K412 site on HDAC1 as shown in SED ID NO.

2. S2. The HiFi Cas9-sgRNA RNP complex was obtained by transfecting the RNP complex formed by incubating HiFi Cas9 protein and sgRNA in vitro. S3. Synthesize the ssODN repair template, wherein the ssODN repair template is a nucleotide sequence as shown in SED ID NO.3, K412E; S4. Low-passaged MSCs were co-transfected with the HiFi Cas9-sgRNA RNP complex and ssODN. After 48 hours, puromycin was used to screen for viable stem cells, and anti-apoptotic human mesenchymal stem cells were obtained by isolation and monoclonal culture.

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

3. Anti-apoptotic human mesenchymal stem cells constructed using the construction method described in claim 1 or 2.

4. The use of the anti-apoptotic human mesenchymal stem cells as described in claim 3 in the preparation of a drug that promotes the anti-apoptotic ability of mesenchymal stem cells.

5. The application according to claim 4, characterized in that, The drug also includes medical excipients.

6. The application according to claim 4, characterized in that, The drug is formulated as an injection, a lyophilized powder injection, or a compound preparation.

7. The application according to claim 6, characterized in that, The composite formulation is a microsphere or liposome.

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

9. The use of the anti-apoptotic human mesenchymal stem cells according to claim 4 in the preparation of a medicament for treating osteoporosis, heart failure or spinal cord injury.

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