A method for delaying cell aging in mesenchymal stem cells in vitro expansion and application

Abstract

This invention discloses a method and its application for delaying cellular senescence in the in vitro expansion of mesenchymal stem cells. The method includes delaying the replicative senescence of human mesenchymal stem cells by inhibiting N-cadherin-mediated cell adhesion during in vitro expansion. Inhibition of N-cadherin-mediated cell adhesion includes adding inhibitors or reducing the expression level of genes encoding N-cadherin. The method of this invention can effectively reduce cell adhesion, restore the collective migration ability and traction of cells, and significantly downregulate the expression of aging-related markers p53 and p21, as well as SA-β-galactosidase activity. The method of this invention is simple to operate, low in cost, and can effectively improve the quality and yield of stem cell expansion.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and regenerative medicine, specifically relating to a method and application for delaying cell senescence in in vitro expanded mesenchymal stem cells. Background Technology

[0002] Human mesenchymal stem cells (MSCs) hold great promise for applications in cell therapy and tissue engineering due to their multipotent differentiation potential and immunomodulatory capabilities. In clinical applications, obtaining 103 MSCs is typically required. 7 -10 9 The production of functional stem cells is on a massive scale, making large-scale in vitro expansion an indispensable and crucial step. However, current in vitro expansion processes generally employ a "high-density adherent culture—high-confluence passage" model. During continuous passage, stem cells inevitably undergo replicative senescence, manifested as decreased proliferative capacity, weakened migration ability, loss of differentiation potential, and upregulation of senescence-related markers such as p53 and p21, severely limiting the quality and stability of stem cell preparations.

[0003] Existing technologies for delaying stem cell senescence mainly focus on intrinsic molecular regulation, such as antioxidant treatment, telomerase activation, or optimization of culture medium composition. For example, patent application 202010411360.6 discloses the application of a secreted protein (MYDGF) in the preparation of telomerase expression and cell senescence regulators. Based on its function of maintaining telomerase activity in telomerase-positive cells, MYDGF is added to the culture medium for human mesenchymal stem cells in vitro to inhibit the downregulation of telomerase TERT transcriptional levels in replicative senescence. These methods are often costly and cannot fundamentally solve the senescence problems caused by the culture process itself under large-scale expansion conditions.

[0004] Therefore, there is an urgent need for a method to regulate cellular replicative senescence under large-scale in vitro expansion conditions in order to improve the quality and applicability of stem cell expansion. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a method and application for delaying cell senescence in the in vitro expansion of mesenchymal stem cells. This method is applicable to the large-scale in vitro expansion of human mesenchymal stem cells. It delays or reverses the replicative senescence of stem cells by inhibiting N-cadherin-mediated cell adhesion and weakening intercellular mechanotransmission under high cell density conditions.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] On the one hand, a method for delaying cell senescence in in vitro expansion of mesenchymal stem cells is provided, comprising: in the in vitro expansion of human mesenchymal stem cells, delaying the replicative senescence of the human mesenchymal stem cells by inhibiting N-cadherin-mediated intercellular adhesion; wherein the inhibition of N-cadherin-mediated intercellular adhesion includes adding an inhibitor or reducing the expression level of the gene encoding N-cadherin.

[0008] On the other hand, the method for delaying cell senescence in the above-mentioned in vitro expanded mesenchymal stem cells is provided for use in the preparation of drugs for delaying cell senescence.

[0009] Compared with the prior art, the present invention has the following advantages:

[0010] 1. This invention establishes N-cadherin-mediated intercellular mechanical adhesion as a direct target for delaying stem cell senescence for the first time. It provides a method for delaying the replicative senescence of human mesenchymal stem cells by inhibiting N-cadherin-mediated intercellular adhesion during in vitro expansion of human mesenchymal stem cells. The method of this invention can effectively restore the migration ability and mechanical force of senescent cells and significantly reduce the expression of core senescence markers such as p53 and p21.

[0011] 2. The method for delaying cell senescence in the in vitro expansion of mesenchymal stem cells of the present invention is preferably based on ADH-1 treatment and low-density culture, which is easy to integrate and implement in existing cell culture systems, has low cost, and is suitable for large-scale production.

[0012] 3. The method for delaying cell senescence in the in vitro expansion of mesenchymal stem cells of the present invention preferably involves adding an inhibitor to suppress N-cadherin-mediated cell adhesion, which has high safety. By regulating the culture microenvironment rather than directly modifying the core genetic network of cells, the potential risks are lower and it is easier to translate into clinical applications.

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 The results of establishing and identifying a replicative aging model of human mesenchymal stem cells during continuous in vitro passage and expansion are presented, in which... Figure 1 The left image shows the SA-β-galactosidase staining results for cells at different passage stages. Figure 1 The right-hand figure shows the corresponding positive rate statistics.

[0015] Figure 2 The results of the collective migration behavior analysis of human mesenchymal stem cell monolayers at different aging stages are shown, where each sub-image corresponds to the cell migration trajectory obtained by time-lapse imaging and the velocity field distribution obtained by particle image velocimetry (PIV) analysis.

[0016] Figure 3 The results of traction force microscopy (TFM) and monolayer stress microscopy (MSM) analysis of cells at different aging stages are shown. Each sub-figure corresponds to the distribution of cell-matrix traction force, monolayer stress distribution and their quantitative statistical results.

[0017] Figure 4 and Figure 5 The expression and localization of N-cadherin in cells at different aging stages are shown. Figure 4 This image shows the expression and localization of N-cadherin in human mesenchymal stem cells from young, intermediate, and aging groups. Each sub-image corresponds to immunofluorescence staining and Western blot images, along with relevant quantitative analysis results. Figure 5 This diagram illustrates the expression levels of N-cadherin and the expression of p53 and p21 in human mesenchymal stem cells from the young, intermediate, and aging groups.

[0018] Figures 6 to 8 Together, they demonstrate the effects of knocking down N-cadherin expression with small interfering RNA on collective migration behavior, traction, monolayer stress, and the activities of aging-related markers p53, p21, and SA-β-galactosidase in senescent cells. Figure 6 This is a schematic diagram showing the results of the migration rate analysis before and after N-cadherin gene knockdown. Figure 7 This is a schematic diagram showing the traction and single-layer stress analysis results before and after knockdown. Figure 8 A summary figure showing the changes in the expression of N-cadherin and aging-related proteins before and after CDH2 gene knockdown.

[0019] Figure 9 The experimental results of treating senescent cells with the N-cadherin function inhibitor ADH-1 are shown, with each sub-figure corresponding to immunofluorescence, Western blotting, and SA-β-galactosidase staining analysis.

[0020] Figure 10 This study demonstrates the effect of sustained inhibition of N-cadherin-mediated intercellular adhesion (ADH-1 treatment) on the delay of replicative senescence of human mesenchymal stem cells during long-term in vitro expansion and continuous multi-generational culture.

[0021] Statistical analysis was performed using GraphPad Prism 8 software. One-way ANOVA and Tukey's multiple comparison test (t-test) were used to analyze the experimental data. A p-value < 0.05 was considered statistically significant. Data are expressed as mean ± SD. Detailed Implementation

[0022] The technical solution will now be clearly and completely described with reference to the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0023] In the following description, the term "and / or" is used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural.

[0024] In the following description, the terms “including,” “containing,” “having,” and “containing” are open-ended terms, meaning that they include but are not limited to.

[0025] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0026] Those skilled in the art will understand that the numerical ranges in the embodiments of this application should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] The technical principle employed in this invention is as follows: During the research process, the inventors discovered that during large-scale in vitro expansion, cell density continuously increases, and cell-to-cell contact significantly increases. The resulting cell-to-cell adhesion and mechanotransmission may be important but long-neglected factors triggering replicative senescence. Based on the correlation between cell mechanotransmission and replicative senescence, this invention targets the function or expression of N-cadherin. N-cadherin, as the main cell-to-cell adhesion molecule in mesenchymal stem cells, exhibits changes in expression and function under high-density culture conditions that inhibit cell traction and collective migration, inducing the activation of senescence pathways such as p53 and p21. By knocking down the encoding gene of N-cadherin or introducing inhibitors that suppress N-cadherin-mediated cell-to-cell adhesion, replicative senescence of mesenchymal stem cells can be delayed without altering the culture system.

[0029] On one hand, a method for delaying cell senescence in in vitro expansion of mesenchymal stem cells is provided, comprising: in the in vitro expansion of human mesenchymal stem cells, delaying the replicative senescence of the human mesenchymal stem cells by inhibiting N-cadherin-mediated intercellular adhesion; wherein the inhibition of N-cadherin-mediated intercellular adhesion includes adding an inhibitor or reducing the expression level of the gene encoding N-cadherin, wherein the inhibitor is a polypeptide inhibitor containing a HAV motif, the gene encoding N-cadherin is a CDH2 gene, and the reduction of the expression level of the CDH2 gene encoding N-cadherin is performed by small interfering RNA, shRNA, miRNA, antisense oligonucleotides, or CRISPR.

[0030] In some embodiments, the inhibition of N-cadherin-mediated intercellular adhesion includes inhibiting the homologous binding function of N-cadherin, which is achieved by specifically binding to the homodimerization interface of the extracellular region of N-cadherin.

[0031] The binding between the extracellular domains of N-cadherin is a polypeptide binding that is attributed to the HAV sequence, and is called homology binding.

[0032] In some preferred embodiments, the reduction of CDH2 gene expression level encoding N-cadherin is performed via small interfering RNA.

[0033] Small interfering RNA (SRNA), shRNA, miRNA, antisense oligonucleotides, and CRISPR are all gene regulation technologies at the nucleic acid level. They achieve gene silencing, knockdown, or editing by targeting nucleic acids. Among them, small interfering RNA achieves gene knockdown by temporarily inhibiting gene expression. It has high targeting and high knockdown efficiency in reducing the expression level of the CDH2 gene encoding N-cadherin.

[0034] In some embodiments, delaying the replicative senescence of the human mesenchymal stem cells by adding an inhibitor includes adding the inhibitor to the human mesenchymal stem cell in vitro cell culture system, with a final concentration of 50 nM to 1 μM; in some preferred embodiments, the inhibitor is ADH-1, with a final concentration of 500 nM.

[0035] This invention provides two methods for delaying the replicative senescence of human mesenchymal stem cells. One method involves introducing an inhibitor into the in vitro cell culture system of human mesenchymal stem cells. Specifically, ADH-1, a specific competitive inhibitor of N-cadherin, is added to the cell culture medium to block its homologous binding, thereby weakening cell adhesion. Preferably, the final concentration of ADH-1 is 500 nM. The other method is based on reducing the expression level of the gene encoding N-cadherin, thereby reducing the expression level of N-cadherin at its source.

[0036] ADH-1 (ADH-1trifluoroacetate) is a selective and competitive N-cadherin antagonist that specifically binds to the homodimerization interface of the extracellular region of N-cadherin. Through steric hindrance, it interferes with the interactions between N-cadherin molecules, directly blocking N-cadherin intercellular adhesion and achieving specific inhibition of N-cadherin-mediated cell adhesion. Its structural formula is as follows:

[0037] .

[0038] During the research, the inventors discovered that, compared to reducing the expression level of genes encoding N-cadherin by gene knockdown, knockout, or silencing, adding inhibitors can effectively delay the aging of in vitro expanded mesenchymal stem cells. SA-β-gal staining results showed that the positive cell rate was reduced by about 65%.

[0039] In some embodiments, the sense strand sequence of the small interfering RNA is shown in SEQ ID NO.1, and the antisense strand sequence is shown in SEQ ID NO.2.

[0040] In some embodiments, inhibiting N-cadherin-mediated cell adhesion by adding inhibitors specifically includes: seeding human mesenchymal stem cells into α-MEM medium containing 10% fetal bovine serum and 1% PS, adhering to the culture medium at 37°C and 5% CO2, digesting and passaged the cells when they reach 80% confluence, and repeating passage to ≥10 generations.

[0041] The passaged cells that have been repeatedly passaged to ≥10 generations were seeded into α-MEM medium containing 10% fetal bovine serum, 1% PS and inhibitors, and cultured at 37°C and 5% CO2 for 24-48 hours.

[0042] In some embodiments, the seeding density of passaged cells that have been passaged to ≥10 generations is 8000 cells / cm². 2 The final concentration of the inhibitor in the system was 500 nM; the inhibitor was ADH-1.

[0043] In some embodiments, inhibiting N-cadherin-mediated intercellular adhesion by adding an inhibitor specifically includes: seeding human mesenchymal stem cells into α-MEM medium containing 10% fetal bovine serum and 1% PS, adhering to the culture medium at 37°C and 5% CO2, digesting and passaged the cells when they reach 80% confluence, and repeating passage to ≤4 generations.

[0044] The cells that have been passaged to ≤4 generations were seeded into α-MEM medium containing 10% fetal bovine serum, 1% PS and inhibitors, and cultured at 37°C and 5% CO2. When the cells reached 80% confluence, they were passaged.

[0045] In some embodiments, the seeding density of passaged cells (passed to ≤4 generations) is 4000 cells / cm². 2 The final concentration of the inhibitor in the culture system containing the inhibitor is 500 nM; the inhibitor is ADH-1.

[0046] On the other hand, an application of the above method in delaying cell aging is provided, the application including the preparation of drugs for delaying cell aging.

[0047] Prior to the application for this invention, a series of experiments were conducted. Some of the experimental results are listed below to provide a more detailed description of the invention. The following is a detailed description in conjunction with the embodiments.

[0048] Example 1

[0049] This embodiment provides a process for establishing a human mesenchymal stem cell replicative aging model, including:

[0050] Human mesenchymal stem cells from consistent sources were collected at a rate of 8000 cells / cm². 2 The cells were cultured at 37°C and 5% CO2 in α-MEM medium containing 10% fetal bovine serum (FBS) and 1% PS. When the cells reached 80% confluence, they were digested and passaged at a ratio of 1:2. The passages were repeated until about 15 generations (P15).

[0051] P1-P4 were defined as the young group, P5-P9 as the intermediate group, and P10-P15 as the senescent group. The cell morphology of each group is as follows: Figure 1As shown in the left figure, the cell morphology of different groups is significantly different. As the number of passages increases, the cell proliferation rate gradually decreases. When the number of passages reaches 10 or more, the cells gradually flatten from a slender spindle shape and the cell volume increases, showing a typical replicative senescence phenotype.

[0052] SA-β-galactosidase staining reagent was used to identify the senescence of cells at different passage stages. The staining method included: adding 500 μL of fixative to the cells and fixing them for 15 min; removing the fixative and rinsing with buffer; removing the buffer; adding 400 μL of staining working solution to each well; incubating overnight at 37°C; photographing; counting positive cells; and determining the proportion of SA-β-gal positive cells to assess the degree of senescence in the cell population. The results are as follows: Figure 1 As shown in the right figure, compared with the low-passage cells (young group), the proportion of SA-β-galactosidase positive cells in the high-passage cells (senescent group) is significantly increased, and the difference is statistically significant. This embodiment successfully established a replicative aging model.

[0053] Human mesenchymal stem cells from young, intermediate, and senescent groups were seeded onto culture dishes at a density of approximately 8000 cells / cm². 2 The cells were allowed to form a continuous monolayer within 24 hours. The culture system was placed in a live-cell imaging system with constant temperature, humidity, and 5% CO2 for time-lapse imaging. Continuous imaging of the cell monolayer was performed using an inverted microscope under bright-field conditions, with acquisition intervals of 15 minutes and a total acquisition time of no less than 12 hours. The obtained image sequences were preprocessed using ImageJ software and then imported into a particle image velocimetry (PIV) analysis program to calculate the velocity field of the cell monolayer. The average migration velocity distribution and directional characteristics of cells at different aging stages were obtained to analyze the differences in collective cell mobility among different groups. The results are as follows: Figure 2 As shown, the cell's collective migration ability decreases significantly with increasing cellular senescence.

[0054] To detect cellular mechanical behavior and activity, human mesenchymal stem cells from young, intermediate, and senescent groups were seeded onto the surface of an elastic polymer hydrogel matrix functionalized with RGD peptides. After 24 hours of cell culture, fluorescence images were acquired in both the presence and lysis / removal states. By comparing the positional changes of fluorescent microspheres in the two sets of images, the distribution of the traction force exerted by the cells on the matrix was calculated. Furthermore, based on the traction force data, the stress distribution within a single cell layer was calculated using a single-layer stress microscopy algorithm. The results are as follows: Figure 3As shown, the preparation method of the hydrogel matrix includes: dissolving 100 mg of PEGDA700 (Sigma) in 1 mL of PBS solution, then adding 20 μL of RGD solution with a concentration of 10 mmol / L (product number 007551, VPeptides Interactions) to obtain a precursor mixture; adding 15 μL of carboxylated polystyrene fluorescent microspheres (fluorescent red latex beads, L3280-1 mL, Thermo Fisher Scientific, USA) to 1 mL of this precursor mixture; adding 20 μL of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP, Thermo Fisher Scientific) with a concentration of 4 mg / mL; and then exposing to 365 nm UV light for 15 minutes to complete the polymerization process, obtaining the hydrogel matrix. The matrix surface is functionalized with RGD peptides to promote cell adhesion, and fluorescent microspheres are uniformly dispersed in the hydrogel as displacement markers. Figure 3 It is evident that as the degree of replicative senescence deepens, the traction force of cells on the matrix and the level of monolayer stress both decrease significantly.

[0055] Figure 4 This study describes the expression and localization of N-cadherin in human mesenchymal stem cells (hMSCs) from young, intermediate, and senescent groups. Each sub-image corresponds to immunofluorescence staining, Western blot images, and related quantitative analysis results. The testing method included: fixing the cells with 4% paraformaldehyde at 37°C for 15 minutes, then permeating with 0.25% Triton X-100 for 10 minutes, and blocking with PBS containing 1% bovine serum albumin (BSA) for 60 minutes at room temperature. Subsequently, hMSCs were incubated overnight at 4°C with diluted primary antibodies. The primary antibodies used included p21 (1:200, Cell Signaling Technology, product number: 2947S), p53 (1:200, Cell Signaling Technology, product number: 2524S), and N-cadherin (1:200, Cell Signaling Technology, product number: 2947S). After washing, cells were incubated at 4°C for 2 hours with fluorescent secondary antibody, DAPI (1:1000, Sigma, product number: D9542), and phalloidin (1:1000, Thermo Fisher Scientific, product number: A12379). All reagents were prepared in 1% BSA and stored in the dark. Fluorescence microscopy was performed using a DMi8 microscope (Leica, Germany). The results showed that N-cadherin was significantly enriched at intercellular junctions as aging progressed.

[0056] Figure 5This diagram illustrates the expression levels of N-cadherin and p53 and p21 in human mesenchymal stem cells from young, intermediate, and aging groups. The results were validated using Western blotting (WB) experiments. The assay method included: seeding cells into six-well plates, extracting total protein using RIPA buffer (APEXBIO, USA) with the addition of 1 mM benzyl sulfonyl fluoride (PMSF); determining protein concentration using a BCA protein assay kit (Betaine Biotechnology, China); and then loading 20 μg of total protein into an 8%... Electrophoresis was performed on a 12% SDS-PAGE gel (GenScript, USA). The gel was then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) at 200 mA for 90 minutes in transfer buffer. Specific primary antibodies against N-cadherin (Cell Signaling Technologies, Product No. 13116S), p21 (Cell Signaling Technologies, Product No. 2947S), and p53 (Cell Signaling Technologies, Product No. 2524S) were diluted 1:2000. After three 10-minute BST washes, the membrane was incubated with an appropriate secondary antibody (diluted 1:5000) at room temperature for 2 hours. The immunoreaction bands were visualized using Western luminescence assay (4A Biotech). The results showed that the expression of N-cadherin, aging molecules p53, and p21 were significantly upregulated with the progression of aging.

[0057] Example 2

[0058] This embodiment provides a method for examining the effect of inhibiting N-cadherin function on the phenotype of senescent cells. CDH2 is a specific gene encoding N-cadherin. This embodiment examines the effect on senescent cells by knocking down the CDH2 gene. The specific operation is as follows:

[0059] Human mesenchymal stem cells from the aging group in Example 1 were used to target and silence the CDH2 gene encoding N-cadherin using small interfering RNA (siRNA). The siRNA was transfected into the human mesenchymal stem cells of the aging group. 24 hours after transfection, the downregulation effect of N-cadherin expression was verified by immunofluorescence staining and Western blotting.

[0060] The small interfering RNA sequence:

[0061] SEQ ID NO.1: sense: 5-CAGUCAACUGCAACCGUGUdTdT-3′;

[0062] SEQ ID NO.1: antisense: 5′-ACACGGUUGCAGUUGACUGdTdT-3′;

[0063] The specific process of small interfering RNA targeting CDH2 is as follows: using Lipofectamine™ RNAiMAX (ThermoFisher Scientific), cells seeded in cell culture plates are transfected with 36 μmol / L small interfering siRNA to target CDH2, and knockdown experiments are performed according to the instructions.

[0064] Subsequently, cell senescence-related indicators and mechanical behavior were detected. The testing methods were basically the same as those in Example 1, and the results are as follows: Figures 6-8 As shown. Figure 6 This is a schematic diagram of the migration velocity analysis results before and after CDH2 gene knockdown. The scale bar is 200 μm. siNC represents the negative control group of the transfection reagent. The black dashed line represents the peak velocity of siNC. The upper right figure is before knockdown, and the lower right figure is after knockdown. It can be seen that the cell migration velocity increased after CDH2 knockdown, with the peak velocity increasing from 0.8 μm / h to 1.1 μm / h.

[0065] Figure 7 This is a schematic diagram showing the traction and single-layer stress analysis results before and after knockdown. Figure 7 The left image shows the bright field image of the velocity field region in the traction and single-layer stress areas, with a scale bar of 50 μm. Figure 7 The right figure shows the traction force quantification (n = 3) and single-layer tension quantification (n = 3). Figure 7 The results showed that CDH2 knockdown enhanced traction and monolayer stress, with the mean cellular traction force increasing from 420 Pa to 630 Pa and the monolayer stress increasing from 0.012 N / m to 0.014 N / m.

[0066] Figure 8 This is a summary diagram of the changes in N-cadherin and aging-related protein expression before and after CDH2 gene knockdown. It includes fluorescence imaging and intensity diagrams of aging-related proteins before and after knockdown, Western blot (Wb) and quantitative statistical diagrams of N-cadherin and aging-related proteins before and after knockdown, and SA-β-gal staining images and calculation results of the percentage of positive cells before and after knockdown. It can be seen that after knockdown, the fluorescence intensity of aging-related proteins P21 and P53 is significantly reduced, with P21 fluorescence intensity decreasing by 46.4% and P53 fluorescence intensity decreasing by 45.7%. Western blot results show that after knockdown, N-cadherin decreased by 76.1%, p21 decreased by 29.5%, and p53 decreased by 48.6%. SA-β-gal staining results show that after knockdown, the percentage of SA-β-gal positive cells decreased by 62.1%.

[0067] based on Figures 6-8In summary, CDH2 knockdown reduced N-cadherin expression, significantly weakened intercellular adhesion, decreased p53 and p21 expression levels, reduced the proportion of SA-β-galactosidase positive cells, and significantly improved cell migration and traction. This indicates that inhibiting N-cadherin-mediated intercellular adhesion can restore the mechanical behavior of senescent cells and reverse their senescent phenotype.

[0068] Example 3

[0069] This embodiment provides a method for delaying aging by reducing N-cadherin expression through drug intervention, including:

[0070] Human mesenchymal stem cells from the aging group (aged group, passage number P10 or higher) in Example 1 were used at a concentration of 8000 cells / cm². 2 The culture medium was densely seeded into petri dishes and cultured at 37°C and 5% CO2 in MEM-alpha medium containing 10% FBS, 1% PS and ADH-1, a competitive functional inhibitor of N-cadherin. The final concentration of ADH-1 in the medium was 500 nM and the culture time was 24 hours. During the culture process, the temperature, gas environment and medium composition of the culture system were kept constant to eliminate interference from non-specific factors.

[0071] The cells obtained in this example were subjected to immunofluorescence staining and Western blot analysis to detect the expression of N-cadherin, p53, and p21. The results are as follows: Figure 9 The diagram shows fluorescence imaging and intensity of aging-related proteins before and after ADH-1 intervention, Western blot (Wb) and quantitative statistical analysis of N-cadherin and aging-related proteins before and after interference, and SA-β-gal staining images and calculation results of the percentage of positive cells before and after interference. It is evident that after ADH-1 intervention, the expression of aging-related proteins decreased. Fluorescence results showed a 32.8% decrease in P21 fluorescence intensity and a 43.5% decrease in P53 fluorescence intensity. Western blot results showed a 31.3% decrease in p21 and a 50.2% decrease in p53 after interference. The percentage of SA-β-gal positive cells decreased by 64.8% after interference. Furthermore, SA-β-galactosidase staining and mass migration and mechanical analysis were performed on the treated cells. The results indicated that ADH-1 treatment effectively weakened cell adhesion, reduced the expression of aging markers, and partially restored cell migration ability and traction levels without significantly altering the overall expression level of N-cadherin.

[0072] Example 4

[0073] This embodiment provides a method for inhibiting intercellular adhesion to delay replicative senescence during long-term in vitro expansion, including:

[0074] Human mesenchymal stem cells from the young group (young group, passages P1-P4) in Example 1 were collected at a concentration of 4000 cells / cm². 2 The culture medium was densely inoculated into petri dishes and cultured at 37°C and 5% CO2 in MEM-alpha medium containing 10% FBS, 1% PS and ADH-1; the final concentration of ADH-1 in the medium was 500 nM.

[0075] Once the cells reach approximately 80% confluence, passage is performed, and this process is repeated three times.

[0076] After passage, cells were seeded into MEM-alpha medium containing 10% FBS and 1% PS without ADH-1 and cultured at 37°C and 5% CO2 until confluence reached 30%, which served as the experimental group.

[0077] The culture medium in the control group did not contain ADH-1.

[0078] Senescence markers in cells from different treatment groups were detected, and the results are as follows: Figure 10 As shown, Figure 10 This diagram illustrates the fluorescence imaging and intensity of senescence-related proteins in cells after three passages under continuous ADH-1 intervention, along with Western blotting (WB) results and calculations of these proteins, SA-β-gal staining images, and statistical results of the percentage of positive cells. It is evident that continuous ADH-1 intervention can delay the senescence process of stem cells. Fluorescence results show a 48.2% decrease in P21 fluorescence intensity and a 56.8% decrease in P53 fluorescence intensity. WB results show a 47.3% decrease in p21 and a 44.9% decrease in p53 after continuous intervention. SA-β-gal staining results show a 70.2% decrease in the percentage of positive cells after continuous intervention. Compared to the conventionally cultured control group, cells with continuously inhibited N-cadherin-mediated intercellular adhesion maintained lower p53 and p21 expression levels and SA-β-galactosidase activity even after multiple passages of expansion. This indicates that the method of this invention can significantly delay the replicative senescence of human mesenchymal stem cells during large-scale in vitro expansion, demonstrating stability and feasibility in practical expansion applications.

Claims

1. A method for delaying cell senescence in in vitro expanded mesenchymal stem cells, characterized in that, include: In the in vitro expansion of human mesenchymal stem cells, the replicative senescence of the human mesenchymal stem cells is delayed by inhibiting N-cadherin-mediated intercellular adhesion; the inhibition of N-cadherin-mediated intercellular adhesion includes adding an inhibitor or reducing the expression level of the gene encoding N-cadherin.

2. The method for delaying cell senescence in in vitro expanded mesenchymal stem cells according to claim 1, characterized in that, The inhibitor is a polypeptide inhibitor containing the HAV motif, and the gene encoding N-cadherin is the CDH2 gene. The expression level of the CDH2 gene encoding N-cadherin is reduced by small interfering RNA, shRNA, miRNA, antisense oligonucleotides, or CRISPR.

3. The method for delaying cell senescence in in vitro expanded mesenchymal stem cells according to claim 2, characterized in that, The reduction in CDH2 gene expression level encoding N-cadherin was achieved via small interfering RNA.

4. The method for delaying cell senescence in in vitro expanded mesenchymal stem cells according to claim 3, characterized in that, The sense strand sequence of the small interfering RNA is shown in SEQ ID NO.1, and the antisense strand sequence is shown in SEQ ID NO.

2.

5. The method for delaying cell senescence in in vitro expanded mesenchymal stem cells according to claim 3, characterized in that, The inhibition of N-cadherin-mediated intercellular adhesion includes inhibiting the homologous binding function of N-cadherin, which is achieved by specifically binding to the homodimerization interface of the extracellular region of N-cadherin.

6. The method for delaying cell senescence in in vitro expanded mesenchymal stem cells according to claim 1, characterized in that, Delaying the replicative senescence of the human mesenchymal stem cells by adding an inhibitor includes adding the inhibitor to the in vitro cell culture system of the human mesenchymal stem cells, with a final concentration of the inhibitor of 50 nM to 1 μM.

7. The method for delaying cell senescence in in vitro expanded mesenchymal stem cells according to claim 1, characterized in that, The final concentration of the inhibitor was 500 nM.

8. The method for delaying cell senescence in in vitro expanded mesenchymal stem cells according to claim 1, characterized in that, The inhibitor is ADH-1.

9. The method for delaying cell senescence in in vitro expanded mesenchymal stem cells according to claim 1, characterized in that, Delaying the replicative senescence of human mesenchymal stem cells by adding inhibitors includes: seeding human mesenchymal stem cells into α-MEM medium containing 10% fetal bovine serum and 1% PS, and culturing them adherently at 37°C and 5% CO2. When the cells reach 80% confluence, they are digested and passaged, and the passages are repeated for ≥10 generations. The passaged cells that have been passaged for ≥10 generations are then seeded into α-MEM medium containing 10% fetal bovine serum, 1% PS and inhibitors, and cultured at 37°C and 5% CO2 for 24-48 hours.

10. The application of the method for delaying cell senescence in in vitro expanded mesenchymal stem cells as described in claim 1 in the preparation of drugs for delaying cell senescence.

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