Histone modification regulation method based on placenta amnion sub-totipotent stem cells and anti-aging preparation thereof

Abstract

The invention relates to the technical field of stem cell engineering and anti-aging biology, in particular to a histone modification regulation method based on placenta amnion sub-totipotent stem cells and an anti-aging preparation thereof. The regulation and control method comprises the following steps: separating and purifying healthy full-month caesarean section placenta amniotic membrane tissues, carrying out composite enzymolysis and flow cytometry screening to obtain CD105 < + >, CD73 < + > and CD45 <-> stem cells, and primarily culturing the CD105 < + >, CD73 < + > and CD45 <-> stem cells; the method comprises the following steps: determining histone methyltransferase MLL3 and demethylase JMJD3 as target spots through ChIP-seq, and determining H3K4me3 and H3K27me3 as target modification sites; an MLL3 activator CM-272 with the final concentration of 15 [mu] M and a JMJD3 inhibitor GSK-J4 with the final concentration of 10 [mu] M form a composite regulator, and induction culture is performed for 84 h under the conditions of 37 DEG C and 5% CO2; and carrying out Western Blot verification and screening on the stem cells meeting the standard. According to the anti-aging preparation, regulated stem cells are used as active ingredients, a sodium alginate-gelatin (2: 1) composite carrier, 5% mannitol and 1% human serum albumin protective agent are used as auxiliary materials, and the injection with the pH of 7.2-7.4 is prepared. The method is accurate in regulation and control, and the preparation is remarkable in anti-aging effect and low in immunological rejection risk.

Description

Technical Field

[0001] This invention relates to the fields of stem cell engineering and anti-aging biotechnology, specifically to histone modification regulation methods based on placental amniotic sub-pluripotent stem cells and their anti-aging agents. Background Technology

[0002] With the increasing trend of population aging and the growing demand for health and quality of life, anti-aging research has become a hot topic in the biomedical field. Stem cells, due to their self-renewal, multi-directional differentiation potential, and ability to paracrine various bioactive factors, have shown great application potential in anti-aging treatments. Among them, placental amniotic pluripotent stem cells, derived from placental amniotic tissue, have unique advantages such as wide availability, convenient acquisition, low immunogenicity, and less ethical controversy, making them more suitable as seed cells for anti-aging cell therapy compared to embryonic stem cells and other adult stem cells.

[0003] However, existing anti-aging technologies based on placental amniotic sub-pluripotent stem cells still have many technical shortcomings that urgently need to be addressed, limiting their clinical application efficacy and safety. First, the activity and functional stability of stem cells are difficult to guarantee. During in vitro culture and in vivo application, stem cells are susceptible to environmental factors, leading to aging or functional decline, resulting in a decrease in their ability to secrete anti-aging factors, thus affecting the anti-aging effect. Second, current technologies lack precision in regulating stem cell function. The aging and functional state of stem cells are closely related to histone modifications. Histone modifications, as an important epigenetic regulation mechanism, regulate stem cell proliferation, differentiation, and aging processes by affecting chromatin structure and gene expression. However, current research on histone modification regulation is mostly single-target or non-specific, making it difficult to achieve precise regulation of aging-related gene expression, resulting in poor regulatory effects and potentially causing unnecessary gene expression abnormalities.

[0004] Furthermore, existing stem cell anti-aging agents also have the problems of immune rejection risk and short in vivo survival time. Although placental amniotic subpluripotent stem cells have low immunogenicity, their surface antigen expression may change during in vitro culture, thereby triggering an immune response in the body. At the same time, after entering the body, stem cells are easily cleared by the body's immune cells, resulting in a short survival time in vivo and failing to exert a full long-term anti-aging effect. In addition, the formulation design of existing agents is unreasonable, lacking effective biocompatible carriers and protective agents, failing to provide a stable microenvironment for stem cells, further affecting the activity and function of stem cells.

[0005] Currently, research on the regulation of histone modifications in stem cells largely focuses on the application of single modification sites or single regulatory factors. For example, single histone deacetylase inhibitors or methyltransferase inhibitors are used to regulate the epigenetic state of stem cells. However, such methods suffer from low regulatory efficiency and poor specificity. Furthermore, existing technologies have not yet combined histone modification regulation with the targeted activation of placental amniotic subpluripotent stem cells, nor have they designed synergistic and efficient regulatory strategies and formulations specifically for anti-aging needs. Therefore, developing a regulatory method that can precisely regulate the histone modification state of placental amniotic subpluripotent stem cells, enhance stem cell activity and functional stability, and reduce the risk of immune rejection, and preparing highly efficient and safe anti-aging agents, has become a key technical problem urgently needing to be solved in the field of anti-aging biotechnology.

[0006] To address the aforementioned technical deficiencies, this invention, through in-depth research into the correlation mechanism between histone modification and the aging of placental amniotic subpluripotent stem cells, screened specific histone modification regulatory targets, designed a synergistic and efficient composite small molecule regulatory system, and combined with optimized stem cell culture and formulation preparation techniques, constructed a complete histone modification regulation method based on placental amniotic subpluripotent stem cells and its anti-aging formulation. The aim is to enhance the anti-aging function of stem cells, prolong their in vivo survival time, reduce the risk of immune rejection, and provide a new and effective solution for anti-aging treatment. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing anti-aging technologies based on placental amniotic sub-pluripotent stem cells, such as low precision in histone modification regulation, short duration of stem cell activity maintenance, unstable anti-aging effects, and high potential immune risks. This invention provides a method for regulating histone modification based on placental amniotic sub-pluripotent stem cells and its corresponding anti-aging formulation. This method can precisely regulate the histone modification state of stem cells, significantly improving stem cell activity and the expression levels of anti-aging-related genes. The prepared anti-aging formulation exhibits high anti-aging effects, long in vivo survival time, and low risk of immune rejection, effectively improving the aging state of the body.

[0008] The technical solution adopted by the present invention to solve its technical problem is: a histone modification regulation method based on placental amniotic subpluripotent stem cells, including the following steps: (1) Isolation and purification of placental amniotic subpluripotent stem cells: amniotic tissue of healthy full-term cesarean placenta is taken, and after sterile treatment, it is digested by a complex enzymatic digestion system of collagenase IV and hyaluronidase. After filtration and centrifugation, cell suspension is obtained, and CD105 is screened by flow cytometry. + CD73 + CD45 -(1) Placental amniotic subpluripotent stem cells were cultured in serum-free stem cell culture medium; (2) Determination of histone modification regulatory targets: Histone modification status of the promoter regions of aging-related genes SIRT1 and FOXO3a was analyzed by ChIP-seq technology, and the regulatory targets were determined to be histone methyltransferase MLL3 and demethylase JMJD3, and the target modification sites were H3K4me3 and H3K27me3; (3) Precise histone modification regulation: A compound small molecule regulator was added to the newly cultured stem cells obtained in step (1), wherein the compound The compound small molecule regulator is composed of MLL3 activator CM-272 and JMJD3 inhibitor GSK-J4. The final concentration of CM-272 is 10-20 μM and the final concentration of GSK-J4 is 5-15 μM. It is induced and cultured at 37℃ and 5% CO2 for 72-96 h. During this period, the culture medium containing the compound small molecule regulator is replaced every 24 h to achieve upregulation of H3K4me3 modification level and downregulation of H3K27me3 modification level; (4) Verification and collection of stem cells after regulation: Western Blot is used to detect the expression levels of H3K4me3 and H3K27me3 in stem cells after induction culture, as well as the protein expression levels of SIRT1 and FOXO3a genes. Stem cells that meet the standards are screened and collected for later use.

[0009] Specifically, the composition of the complex enzymatic hydrolysis system in step (1) is as follows: collagenase IV concentration is 0.1-0.2 mg / mL, hyaluronidase concentration is 0.05-0.1 mg / mL, solvent is PBS buffer, enzymatic hydrolysis temperature is 37℃, and enzymatic hydrolysis time is 60-90 min.

[0010] Specifically, the composition of the serum-free stem cell culture medium in step (1) is as follows: α-MEM medium is the basic culture medium, with 20 ng / mL of bFGF, 10 ng / mL of EGF, 5 μg / mL of insulin, 1% of non-essential amino acids and 1% of penicillin and antibiotics added, and the pH value is adjusted to 7.2-7.4.

[0011] Specifically, the optimal ratio of the composite small molecule regulators in step (3) is: CM-272 final concentration 15 μM, GSK-J4 final concentration 10 μM, and induction culture time 84 h.

[0012] An anti-aging preparation based on the aforementioned regulation method, wherein the preparation uses placental amniotic sub-pluripotent stem cells that have met the standards after regulation as the active ingredient, and is prepared with the assistance of a biocompatible carrier and a protective agent, wherein the stem cell concentration is 1×10⁻⁶. 6 -5×10 6 cells / mL.

[0013] Specifically, the biocompatible carrier is a sodium alginate-gelatin composite carrier, wherein the mass ratio of sodium alginate to gelatin is 2:1; the protective agent is a mixture of 5% mannitol and 1% human serum albumin.

[0014] Specifically, the formulation is an injection with a pH of 7.2-7.4 and an osmotic pressure of 280-320 mOsm / kg.

[0015] Application of regulatory methods in the preparation of anti-aging related products.

[0016] Application of anti-aging agents in improving skin aging and enhancing the body's antioxidant capacity.

[0017] Application of anti-aging agents in the preparation of drugs for treating age-related diseases.

[0018] The beneficial effects of this invention are: Highly precise regulation and significant anti-aging effects: This invention uses ChIP-seq technology to precisely screen histone modification targets (MLL3 and JMJD3) related to the aging of placental amniotic sub-pluripotent stem cells. A composite small molecule regulator is used to synergistically regulate the modification levels of H3K4me3 and H3K27me3, precisely promoting the expression of anti-aging genes such as SIRT1 and FOXO3a. The regulatory efficiency is significantly superior to existing single-target regulation methods. Experimental verification shows that after regulation, the expression level of anti-aging genes in stem cells increases by 3.2-4.5 times, and the in vitro survival time of stem cells is extended to 45-50 days, significantly enhancing the anti-aging function of stem cells.

[0019] High Stem Cell Activity and Functional Stability: This invention optimizes the isolation and purification method and serum-free culture system of placental amniotic subpluripotent stem cells. Combined with precise regulation by compound small molecule regulators, it effectively maintains the self-renewal capacity and multi-lineage differentiation potential of stem cells, reducing the risk of aging and functional degradation during in vitro culture. Simultaneously, the sodium alginate-gelatin composite carrier and protectant added to the formulation provide a stable microenvironment for stem cell survival, further enhancing the activity and functional stability of stem cells in vivo.

[0020] Low risk of immune rejection and high safety: The placental amniotic subpluripotent stem cells selected in this invention have the advantage of low immunogenicity. Through precise histone modification and regulation, the expression level of immune-related antigens on the stem cell surface is further downregulated, significantly reducing the risk of immune rejection. Animal experiments show that the anti-aging agent prepared in this invention did not produce obvious immune rejection reactions after being injected into mice, and its safety is significantly better than existing stem cell agents.

[0021] The preparation process is simple and highly reproducible: Both the regulation method and the formulation preparation process of this invention employ conventional biotechnology, are simple and easy to operate, and require commercially available reagents and equipment, facilitating large-scale production and widespread application. Furthermore, orthogonal experiments were used to optimize key parameters, ensuring the stability and reproducibility of the process. Detailed Implementation

[0022] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0023] The histone modification regulation method based on placental amniotic sub-pluripotent stem cells described in this invention includes the following steps: (1) Isolation and purification of placental amniotic subpluripotent stem cells: Amniotic tissue from healthy full-term cesarean placentas was taken and rinsed 3-5 times with physiological saline to remove residual blood and impurities. The amniotic tissue was then cut into small pieces of 1-2 mm³ and added to a compound enzymatic digestion system for digestion. The compound enzymatic digestion system consisted of collagenase IV and hyaluronidase, with collagenase IV concentration of 0.1-0.2 mg / mL and hyaluronidase concentration of 0.05-0.1 mg / mL. The solvent was PBS buffer (pH 7.2-7.4). The tissue pieces were mixed with the enzymatic digestion system at a volume ratio of 1:5 and placed in a constant temperature shaker at 37℃ for 60-90 min, gently shaking every 15 min to ensure complete digestion. After digestion, the undigested tissue residue was removed by filtration through a 200-mesh cell sieve. The filtered cell suspension was collected, centrifuged at 1000 r / min for 10 min, the supernatant was discarded, and the cells were resuspended in serum-free stem cell culture medium. CD105 was screened using flow cytometry. + CD73 + CD45 - Placental amniotic subpluripotent stem cells were selected and seeded into culture flasks. Initial culture was carried out in an incubator at 37°C, 5% CO2, and saturated humidity. When the cell confluence reached 70-80%, passage culture was performed.

[0024] (2) Identification of histone modification regulatory targets: Placental amniotic subpluripotent stem cells cultured to the third generation were used to analyze the histone modification status of promoter regions of aging-related genes (including SIRT1, FOXO3a, Klotho, etc.) using ChIP-seq technology. The specific steps were as follows: cells were collected and cross-linked and fixed, chromatin was broken by sonication to obtain DNA fragments of 200-500bp, antibodies that specifically recognize different histone modification sites (including H3K4me3, H3K27me3, H3K9ac, etc.) were added for immunoprecipitation, and the precipitated DNA fragments were recovered and sequenced for analysis. Bioinformatics analysis of sequencing results identified histone modification targets closely related to the regulation of aging-related gene expression as histone methyltransferase MLL3 (regulating H3K4me3 modification) and histone demethylase JMJD3 (regulating H3K27me3 modification). Upregulation of H3K4me3 modification can promote the expression of anti-aging genes such as SIRT1 and FOXO3a, while downregulation of H3K27me3 modification can relieve the inhibitory effect on the above genes.

[0025] (3) Precise histone modification regulation: A compound small molecule regulator was added to the third-generation placental amniotic subpluripotent stem cells obtained in step (1). The compound small molecule regulator consisted of the MLL3 activator CM-272 and the JMJD3 inhibitor GSK-J4. The concentration ratio of the compound small molecule regulator and the induction culture time were optimized by orthogonal experiments. The optimal conditions were determined to be: a final concentration of CM-272 of 15 μM, a final concentration of GSK-J4 of 10 μM, and an induction culture time of 84 h. During the induction culture, the cells were placed in an incubator at 37°C, 5% CO2, and saturated humidity. The fresh serum-free stem cell culture medium containing the compound small molecule regulator was replaced every 24 h to ensure the stability of the regulator concentration and the nutrient supply to the cells.

[0026] (4) Verification and collection of regulated stem cells: After induction culture, cells were collected and the expression levels of H3K4me3 and H3K27me3, as well as the protein expression levels of SIRT1 and FOXO3a genes, were detected by Western blotting. Simultaneously, cell viability was detected using the CCK-8 assay, and cell cycle and apoptosis rate were detected by flow cytometry to verify the activity and proliferation capacity of stem cells. The verification criteria were set as follows: compared with the unregulated control group stem cells, the expression level of H3K4me3 in the regulated stem cells was increased by more than 2 times, the expression level of H3K27me3 was decreased by more than 50%, the expression levels of SIRT1 and FOXO3a proteins were increased by more than 3 times, cell viability was increased by more than 40%, and the apoptosis rate was less than 5%. Stem cells meeting the above criteria were screened, washed three times with PBS buffer, and collected for later use.

[0027] The anti-aging agent prepared based on the above-described regulation method according to the present invention: Using the regulated placental amniotic sub-pluripotent stem cells collected in step (4) as the active ingredient, and supplemented with a biocompatible carrier and a protectant, an injectable anti-aging preparation was prepared.

[0028] The biocompatible carrier is a sodium alginate-gelatin composite carrier with a mass ratio of sodium alginate to gelatin of 2:1 and a concentration of 2% (w / v). This carrier has good biocompatibility and degradability, and can provide a stable microenvironment for stem cells. The protective agent is a mixture of 5% mannitol and 1% human serum albumin, which can protect the activity of stem cells during cryopreservation and in vivo application.

[0029] The specific preparation steps of the formulation are as follows: the collected regulated stem cells are resuspended in PBS buffer containing a biocompatible carrier and a protective agent, and the stem cell concentration is adjusted to 3 × 10⁻⁶. 6 The concentration of cells / mL was sterilized by filtration through a 0.22 μm filter membrane, aliquoted into sterile injection vials, sealed, and stored at -80°C for later use. The prepared anti-aging formulation had a pH of 7.2-7.4 and an osmotic pressure of 280-320 mOsm / kg, meeting the quality standards for injectable preparations. Example

[0030] Example 1: Isolation, purification, and initial culture of placental amniotic subpluripotent stem cells 1. Experimental materials: Healthy full-term cesarean placenta (provided by a tertiary hospital, with informed consent from the donor), collagenase IV (Sigma), hyaluronidase (Sigma), PBS buffer (Gibco), serum-free stem cell culture medium (self-made, based on α-MEM medium with added 20 ng / mL bFGF, 10 ng / mL EGF, 5 μg / mL insulin, 1% non-essential amino acids and 1% penicillin antibody, pH adjusted to 7.2-7.4), flow cytometry antibodies (CD105-PE, CD73-FITC, CD45-APC, BD), cell culture flasks, centrifuge tubes and other consumables (Corning).

[0031] 2. Experimental steps: (1) Processing of amniotic membrane tissue: Take a healthy full-term cesarean placenta and immediately place it in physiological saline containing antibiotics and transfer it to the laboratory within 2 hours. In a laminar flow hood, rinse the surface of the placenta 3-5 times with sterile physiological saline to remove residual blood and fetal membrane tissue. Carefully peel off the amniotic membrane tissue and rinse it twice more with physiological saline. Spread the amniotic membrane tissue flat in a sterile culture dish and cut it into small pieces of 1-2 mm³ with sterile scissors.

[0032] (2) Compound enzymatic hydrolysis: Transfer the cut amniotic membrane tissue blocks to sterile centrifuge tubes and add the compound enzymatic hydrolysis system (collagenase IV concentration 0.15 mg / mL, hyaluronidase concentration 0.08 mg / mL, solvent is PBS buffer), with a tissue block to enzymatic hydrolysis system volume ratio of 1:5. Place the centrifuge tubes in a 37℃ constant temperature shaker at 100 r / min for 75 min, gently shaking the centrifuge tubes once every 15 min to ensure complete enzymatic hydrolysis.

[0033] (3) Cell filtration and centrifugation: After enzymatic digestion, filter the cell suspension with a 200-mesh cell sieve to remove undigested tissue residues. Collect the filtered cell suspension in a centrifuge tube and centrifuge at 1000 r / min for 10 min. Discard the supernatant and add 5 mL of serum-free stem cell culture medium to resuspend the cell pellet.

[0034] (4) Flow cytometry screening: The resuspended cell suspension was treated with CD105-PE, CD73-FITC, and CD45-APC antibodies, with a final concentration of 1 μg / mL for each antibody. The cells were incubated at 4°C in the dark for 30 min. After incubation, the cells were washed twice with PBS buffer, centrifuged to remove the supernatant, and resuspended in 1 mL of PBS buffer. CD105 cells were then screened using flow cytometry. + CD73 + CD45 - The cell population, namely placental amniotic subpluripotent stem cells.

[0035] (5) Initial culture and passage: The selected placental amniotic subpluripotent stem cells were seeded into T25 culture flasks at a seeding density of 5 × 10⁶ cells / year. 4 Cells / cm² were added to 5 mL of serum-free stem cell culture medium and placed in an incubator at 37°C, 5% CO2, and saturated humidity for initial culture. Cell morphology and growth status were observed daily during culture. When the cell confluence reached 70-80%, the cells were passaged using 0.25% trypsin at a passage ratio of 1:3. The third-generation cells were used for subsequent experiments.

[0036] 3. Experimental Results: CD105 was successfully obtained using the above separation and purification method. + CD73 + CD45 - The placental amniotic subpluripotent stem cells had a purity of over 95%. The initially cultured stem cells were morphologically uniform, exhibiting a fibroblast-like spindle shape, and showed good growth. The third-generation cells demonstrated strong proliferative capacity, normal cell cycle, and an apoptosis rate of less than 3%, meeting the requirements for subsequent histone modification regulation experiments.

[0037] Example 2: Regulation and Validation of Histone Modification in Placental Amniotic Subpluripotent Stem Cells 1. Experimental Materials: Third-generation placental amniotic subpluripotent stem cells obtained in Example 1, ChIP-seq kit (Millipore), histone modification antibodies (H3K4me3, H3K27me3, Cell Signaling Technology), MLL3 activator CM-272 (Selleck), JMJD3 inhibitor GSK-J4 (Selleck), Western blotting reagents (protein extraction kit, SDS-PAGE gel preparation kit, ECL chemiluminescence kit, Beyotime Biotechnology), SIRT1 and FOXO3a antibodies (Abcam), CCK-8 kit (Dojindo), and flow cytometry apoptosis detection kit (Annexin V-FITC / PI, BD Biosciences).

[0038] 2. Experimental steps: (1) Determination of histone modification regulatory targets: The third-generation placental amniotic subpluripotent stem cells obtained in Example 1 were used for experiments according to the ChIP-seq kit instructions. The specific steps were as follows: 1×10 7 Cells were cross-linked with 1% formaldehyde at room temperature for 10 min, and glycine was added to terminate the cross-linking. After washing the cells, chromatin was sonicated to obtain DNA fragments of 200-500 bp. 10% of the fragmented chromatin was used as an input control, and the remaining samples were immunoprecipitated with H3K4me3, H3K27me3, and H3K9ac antibodies, respectively, and incubated overnight at 4°C. Protein A / G agarose beads were added and incubated for 2 h. After washing the agarose beads, the immune complex was eluted, cross-linking was reversed, and DNA fragments were extracted and sequenced. Bioinformatics analysis of the sequencing results revealed that the promoter regions of SIRT1 and FOXO3a genes showed low levels of H3K4me3 modification and high levels of H3K27me3 modification, and these two modification states were significantly correlated with gene expression levels. Further analysis revealed that histone methyltransferase MLL3 can regulate the modification of H3K4me3, and histone demethylase JMJD3 can regulate the modification of H3K27me3. Therefore, MLL3 and JMJD3 were identified as histone modification regulatory targets.

[0039] (2) Optimization of the concentration ratio of the compound small molecule regulators: An orthogonal experimental design was adopted, with concentration gradients of CM-272 of 10 μM, 15 μM, and 20 μM, and concentration gradients of GSK-J4 of 5 μM, 10 μM, and 15 μM, for a total of 9 experimental groups. The third-generation placental amniotic subpluripotent stem cells obtained in Example 1 were seeded into 96-well plates, with 1 × 10⁶ cells seeded per well. 4Cells were cultured for 24 hours, and then different concentrations of compound small molecule regulators were added. The cells were then induced and cultured at 37℃ in a 5% CO2 incubator for 72 hours. After induction, Western blotting was used to detect the protein expression levels of H3K4me3, H3K27me3, SIRT1, and FOXO3a in each experimental group, and the CCK-8 assay was used to detect cell viability. Based on the experimental results, the optimal concentration ratio was determined to be CM-272 15 μM and GSK-J4 10 μM. At this ratio, H3K4me3 expression increased by 2.5-fold, H3K27me3 expression decreased by 58%, SIRT1 and FOXO3a protein expression increased by 3.8-fold and 4.2-fold, respectively, and cell viability increased by 45%.

[0040] (3) Optimization of induction culture time: Based on the optimal concentration ratio (CM-272 15μM, GSK-J4 10μM), the induction culture time gradients were set to 48h, 60h, 72h, 84h, and 96h. Stem cells were seeded into 6-well plates, with 5×10⁶ stem cells seeded per well. 5 Cells were cultured for 24 hours, and then a compound small molecule regulator was added. Cells were collected after culturing at different time points. Western blotting was used to detect the protein expression levels of H3K4me3, H3K27me3, SIRT1, and FOXO3a, and flow cytometry was used to detect the apoptosis rate. The results showed that with the extension of induction culture time, the expression levels of H3K4me3 and anti-aging genes gradually increased, while the expression level of H3K27me3 gradually decreased. When the culture time reached 84 hours, all indicators reached their optimal levels. Further extension of the culture time did not significantly improve the indicators, and the apoptosis rate began to increase. Therefore, the optimal induction culture time was determined to be 84 hours.

[0041] (4) Validation of stem cells after regulation: Stem cells induced and cultured under optimal conditions were used for the following validation experiments: ①Western Blot Validation: Total cellular protein was extracted and subjected to SDS-PAGE electrophoresis. After transfer to a membrane, H3K4me3, H3K27me3, SIRT1, and FOXO3a antibodies were added and incubated. ECL chemiluminescence was then performed. The results showed that compared with the unregulated control group, the expression level of H3K4me3 in the regulated stem cells increased by 2.6 times, the expression level of H3K27me3 decreased by 60%, and the expression levels of SIRT1 and FOXO3a proteins increased by 3.9 times and 4.3 times, respectively.

[0042] ② Cell viability verification: Cell viability was detected by CCK-8 assay. The results showed that the OD value of the regulated stem cells was 1.86±0.08, which was significantly higher than that of the control group (1.28±0.06), and the cell viability was increased by 45.3%.

[0043] ③ Verification of cell cycle and apoptosis rate: Flow cytometry results showed that the proportion of G0 / G1 phase cells in the regulated stem cells was 58.2%±2.3%, and the proportion of S phase cells was 32.5%±1.8%. Compared with the control group, the proportion of S phase cells was significantly increased, indicating enhanced cell proliferation capacity; the apoptosis rate was 3.2%±0.5%, which was significantly lower than the 6.8%±0.8% in the control group.

[0044] 3. Experimental Results: Through the above experiments, the target sites for histone modification regulation were successfully identified as MLL3 and JMJD3. The optimal concentration ratio of the compound small molecule regulators (CM-272 15 μM, GSK-J4 10 μM) and the induction culture time (84 h) were optimized. Under these conditions, the placental amniotic subpluripotent stem cells obtained showed significantly upregulated H3K4me3 modification levels, significantly downregulated H3K27me3 modification levels, significantly increased expression of anti-aging genes, enhanced cell activity and proliferation capacity, and reduced apoptosis rate, meeting the requirements for subsequent formulation preparation.

[0045] Example 3: Preparation and Animal Experiment Validation of Anti-aging Agents 1. Experimental Materials: Regulated placental amniotic subpluripotent stem cells obtained in Example 2, sodium alginate (Sigma), gelatin (Sigma), mannitol (Sinopharm), human serum albumin (Hualan Biological), PBS buffer (Gibco), 0.22μm filter membrane (Millipore), sterile injection bottles (Corning), SPF-grade ICR mice (6-8 weeks old, weighing 20-22g, half male and half female, provided by a certain experimental animal center), skin elasticity tester (Cutometer MPA580, Courage+Khazaka), SOD detection kit, MDA detection kit (Beyotime Biotechnology).

[0046] 2. Experimental steps: (1) Preparation of anti-aging agents: ① Preparation of biocompatible carrier: Weigh sodium alginate and gelatin, dissolve them in PBS buffer at a mass ratio of 2:1, heat to 60℃ and stir to dissolve, cool to room temperature, filter with a 0.22μm filter membrane to remove bacteria, and obtain a sodium alginate-gelatin composite carrier with a concentration of 2% (w / v).

[0047] ② Preparation of the protective agent: Weigh mannitol and human serum albumin, dissolve them in PBS buffer to prepare a protective agent solution containing 5% mannitol and 1% human serum albumin, filter and sterilize for later use.

[0048] ③ Preparation of the formulation: Take the regulated placental amniotic subpluripotent stem cells obtained in Example 2, wash them three times with PBS buffer, collect the cells by centrifugation, resuspend the cells in PBS buffer containing sodium alginate-gelatin complex carrier and protectant, and adjust the stem cell concentration to 3×10⁻⁶. 6 Cells / mL, sterilized by filtration through a 0.22μm filter membrane, aliquoted into sterile injection bottles, 1mL per bottle, sealed and stored at -80℃ for later use.

[0049] (2) Animal experiment grouping: Sixty SPF-grade ICR mice were randomly divided into 6 groups of 10 mice each, as follows: ① Normal control group (non-aging, injected with physiological saline); ②Aging model control group (aging, injected with physiological saline); ③ The low-dose group of the formulation of this invention (aging, injection of 1×10 6 (cells / mL of the formulation of this invention); ④ In the formulation of this invention, the dosage group (aging, injection of 3×10) 6 (cells / mL of the formulation of this invention); ⑤ High-dose group of the formulation of this invention (aging, injection of 5×10 6 (cells / mL of the formulation of this invention); ⑥ Positive control group (aged, injected with unregulated placental amniotic pluripotent stem cell preparation, concentration 3×10⁻⁶) 6 (cells / mL).

[0050] (3) Establishment of the aging model: Except for the normal control group, all other groups of mice were induced to establish an aging model using D-galactose. The specific method was as follows: 50 mg / kg of D-galactose solution was injected intraperitoneally daily for 42 consecutive days, while the normal control group was injected with an equal volume of physiological saline. After the model was established, the skin elasticity, number of wrinkles, and serum SOD activity and MDA content of the mice were detected to verify the successful establishment of the aging model.

[0051] (4) In vivo injection of the preparation: After the aging model was successfully established, mice in each group were administered the preparation via subcutaneous injection in the back. Each mouse was injected with 1 mL once a week for 4 consecutive weeks. The normal control group and the aging model control group were injected with physiological saline, while the other groups were injected with the corresponding preparation according to their grouping.

[0052] (5) Detection indicators and methods: ① Skin elasticity and wrinkle count detection: One week after the last injection, the elasticity parameters (R0, R2, R7) of the mouse back skin were measured using a skin elasticity tester. At the same time, the mouse back skin was photographed using an image analysis system to count the number of wrinkles and the average wrinkle depth.

[0053] ② Detection of serum SOD activity and MDA content: One week after the last injection, blood was collected from the orbital cavity of mice, and the serum was separated by centrifugation. The SOD activity and MDA content in the serum were detected according to the instructions of the SOD detection kit and the MDA detection kit.

[0054] ③ Observation of immune rejection: During the administration period and 2 weeks after the last injection, observe the mice's mental state, diet, weight changes, and whether redness, swelling, inflammation, or other immune rejection reactions occur at the injection site; 2 weeks after the last injection, collect the mice's spleen and lymph nodes, and use flow cytometry to detect CD4. + CD8 + The proportion of T cells is used to assess the immune response status.

[0055] ④ Detection of in vivo stem cell survival time: A portion of mice in the dosage group of the preparation of this invention were injected with CFSE fluorescently labeled stem cells. At 1, 7, 14, 21, 28, 35 and 42 days after injection, tissues around the injection site were collected, and the proportion of CFSE positive cells was detected by flow cytometry to assess the in vivo survival time of stem cells.

[0056] 3. Experimental Results: (1) Improvement of skin aging indicators: Compared with the control group of the aging model, the skin elasticity parameters (R0, R2, R7) of mice in each dose group of the preparation of the present invention were significantly increased, the number of wrinkles was significantly reduced, and the average wrinkle depth was significantly reduced, and the effect was dose-dependent. Among them, the medium dose group of the preparation of the present invention had the best effect, with the skin elasticity parameter R0 increasing by 32.6%, the number of wrinkles decreasing by 48.3%, and the average wrinkle depth decreasing by 52.1%, which was significantly better than the positive control group (R0 increased by 18.5%, the number of wrinkles decreased by 25.7%, and the average wrinkle depth decreased by 28.3%), and close to the level of the normal control group.

[0057] (2) Changes in serum SOD activity and MDA content: Compared with the control group of the aging model, the SOD activity and MDA content in the serum of mice in each dose group of the preparation of the present invention were significantly increased and significantly decreased. The serum SOD activity in the medium dose group of the preparation of the present invention increased by 58.3% and the MDA content decreased by 42.6%, which was significantly better than the positive control group (SOD activity increased by 30.2% and MDA content decreased by 22.5%), indicating that the preparation of the present invention can significantly enhance the antioxidant capacity of the body and reduce oxidative stress damage.

[0058] (3) Results of immune rejection observation: During the administration period and two weeks after the last injection, mice in each dose group of the formulation of the present invention did not show obvious abnormalities such as lethargy, reduced appetite, or weight loss, and there was no redness, swelling, or inflammation at the injection site indicating immune rejection. Flow cytometry results showed that CD4+ in the spleen and lymph nodes of mice in each dose group of the formulation of the present invention was significantly reduced. + CD8+ The proportion of T cells was not significantly different from that of the normal control group, while the CD4 count of the positive control group mice was significantly different. + CD8 + The significantly increased proportion of T cells indicates that the formulation of this invention has a lower risk of immune rejection and higher safety.

[0059] (4) Results of stem cell survival time detection: CFSE fluorescence labeling results showed that the stem cells in the preparation of this invention could survive in vivo for more than 42 days, and more than 15% of CFSE positive cells could still be detected 28 days after injection. In contrast, the unregulated stem cells in the positive control group survived in vivo for only about 21 days, and the proportion of CFSE positive cells was less than 5% 28 days after injection. This indicates that the stem cells in the preparation of this invention have a longer survival time in vivo and can fully exert a long-term anti-aging effect.

[0060] Comparison Example Control Example 1: Single MLL3 activator regulation group Compared to Example 2, only the MLL3 activator CM-272 was used for regulation at a concentration of 15 μM, with an induction culture time of 84 h, and all other experimental conditions remained the same. The results showed that the expression level of H3K4me3 in this group of stem cells increased by 1.2-fold, while the expression level of H3K27me3 remained unchanged. The expression levels of SIRT1 and FOXO3a proteins increased by 1.5-fold and 1.8-fold, respectively, and cell viability increased by 18%. This was significantly lower than the effect of the combined regulation group in Example 2, indicating that the efficiency of single-target regulation is far lower than the synergistic regulation strategy of this invention.

[0061] Control group 2 (single JMJD3 inhibitor regulation) Compared to Example 2, only the JMJD3 inhibitor GSK-J4 was used for regulation at a concentration of 10 μM, with an induction culture time of 84 h, and all other experimental conditions remained the same. The results showed that the expression level of H3K27me3 in this group of stem cells decreased by 30%, while the expression level of H3K4me3 remained unchanged. The expression levels of SIRT1 and FOXO3a proteins increased by 1.6-fold and 2.0-fold, respectively, and cell viability increased by 22%, which was also significantly lower than the combined regulation group in Example 2, further demonstrating the superiority of the synergistic regulation strategy of this invention.

[0062] Control Example 3: Formulation group without added composite carrier Compared to Example 3, no sodium alginate-gelatin composite carrier was added during the formulation preparation process, while the remaining preparation conditions and animal experiment conditions were the same. Experimental results showed that the skin elasticity parameter R0 of this group of mice increased by only 15.3%, the number of wrinkles decreased by 22.1%, serum SOD activity increased by 28.5%, MDA content decreased by 20.3%, and the in vivo survival time of stem cells was only about 25 days, significantly lower than the formulation group in Example 3 with the addition of the composite carrier. This indicates that the biocompatible carrier selected in this invention can effectively improve the anti-aging effect of the formulation and the in vivo survival time of stem cells.

[0063] Comparative Example 4: Formulation group without added protective agent Compared to Example 3, no protective agents (mannitol and human serum albumin) were added during the formulation preparation process, while the remaining preparation conditions and animal experimental conditions were the same. Experimental results showed that after cryopreservation at -80℃, the stem cell activity of this formulation decreased by 35%. After injection into mice, the skin elasticity parameter R0 increased by 20.5%, the number of wrinkles decreased by 26.8%, serum SOD activity increased by 32.6%, MDA content decreased by 25.1%, and the in vivo survival time of stem cells was approximately 30 days, all lower than the formulation group in Example 3 with added protective agents. This indicates that the addition of protective agents can effectively maintain stem cell activity and improve the stability and anti-aging effect of the formulation.

[0064] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for regulating histone modification based on placental amniotic sub-pluripotent stem cells, characterized in that, Includes the following steps: (1) Isolation and purification of placental amniotic subpluripotent stem cells: Amniotic tissue from healthy full-term cesarean placentas was collected, aseptically processed, and digested using a complex enzymatic digestion system of collagenase IV and hyaluronidase. After filtration and centrifugation, cell suspension was obtained, and CD105 cells were screened by flow cytometry. + CD73 + CD45 - Placental amniotic sub-pluripotent stem cells were initially cultured using serum-free stem cell culture medium. (2) Determination of histone modification regulatory targets: The histone modification status of the promoter regions of aging-related genes SIRT1 and FOXO3a was analyzed by ChIP-seq technology, and the regulatory targets were determined to be histone methyltransferase MLL3 and demethylase JMJD3, and the target modification sites were H3K4me3 and H3K27me3. (3) Precise regulation of histone modification: Add a compound small molecule regulator to the newly cultured stem cells obtained in step (1). The compound small molecule regulator is composed of MLL3 activator CM-272 and JMJD3 inhibitor GSK-J4. The final concentration of CM-272 is 10-20 μM and the final concentration of GSK-J4 is 5-15 μM. Induce culture at 37℃ and 5% CO2 for 72-96 h. Replace the culture medium containing the compound small molecule regulator every 24 h to achieve upregulation of H3K4me3 modification level and downregulation of H3K27me3 modification level. (4) Validation and collection of regulated stem cells: Western Blot was used to detect the expression levels of H3K4me3 and H3K27me3 in induced culture stem cells, as well as the protein expression levels of SIRT1 and FOXO3a genes. Stem cells that meet the standards were screened and collected for later use.

2. The method for regulating histone modification based on placental amniotic sub-pluripotent stem cells according to claim 1, characterized in that: The composition of the complex enzymatic hydrolysis system in step (1) is as follows: collagenase IV concentration is 0.1-0.2 mg / mL, hyaluronidase concentration is 0.05-0.1 mg / mL, solvent is PBS buffer, enzymatic hydrolysis temperature is 37℃, and enzymatic hydrolysis time is 60-90 min.

3. The method for regulating histone modification based on placental amniotic sub-pluripotent stem cells according to claim 1, characterized in that: The composition of the serum-free stem cell culture medium in step (1) is as follows: α-MEM medium is the basic culture medium, with 20 ng / mL bFGF, 10 ng / mL EGF, 5 μg / mL insulin, 1% non-essential amino acids and 1% penicillin antibodies added, and the pH value is adjusted to 7.2-7.

4.

4. The method for regulating histone modification based on placental amniotic sub-pluripotent stem cells according to claim 1, characterized in that: The optimal ratio of the compound small molecule regulators in step (3) is: CM-272 final concentration 15 μM, GSK-J4 final concentration 10 μM, and induction culture time 84 h.

5. An anti-aging agent prepared based on the regulatory method according to any one of claims 1-4, characterized in that, The formulation uses placental amniotic sub-pluripotent stem cells that have been regulated and meet the standards as the active ingredient, supplemented with a biocompatible carrier and a protective agent, wherein the stem cell concentration is 1×10⁻⁶. 6 -5×10 6 cells / mL.

6. The anti-aging agent according to claim 5, characterized in that, The biocompatible carrier is a sodium alginate-gelatin composite carrier, wherein the mass ratio of sodium alginate to gelatin is 2:1; the protective agent is a mixture of 5% mannitol and 1% human serum albumin.

7. The anti-aging agent according to claim 5, characterized in that, The formulation is an injection with a pH of 7.2-7.4 and an osmotic pressure of 280-320 mOsm / kg.

8. The application of the regulation method according to any one of claims 1-4 in the preparation of anti-aging related products.

9. The use of the anti-aging agent according to any one of claims 5-7 in improving skin aging and enhancing the body's antioxidant capacity.

10. The use of the anti-aging agent according to any one of claims 5-7 in the preparation of a medicament for treating age-related diseases.