Establishment method of nerve cell senescence model for simulating in-vivo senescence nerve cell characteristics

Abstract

The invention discloses a method for establishing a nerve cell senescence model for simulating in-vivo senescence nerve cell characteristics, and belongs to the technical field of biology. The establishment method comprises the following steps: after overexpressing DPF2 in P19 cells, inducing the P19 cells to directionally differentiate into nerve cells by using retinoic acid, so as to obtain the nerve cell senescence model. The cell model can promote expression of inflammatory factors STAT3 and senescence marker protein P16, forms protein aggregates of DPF2, STAT3 and P16, and can simulate senescence characteristics of senescence nerve cells in vivo. The nerve cell senescence model established by the invention not only accelerates screening of drugs for targeted removal of senescence cells or anti-aging, but also can promote development of individualized anti-aging strategies, provides a transformation bridge for delaying senescence-related diseases (such as senile dementia and neurodegenerative diseases), and finally assists in realizing healthy aging.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for establishing a neural cell aging model that simulates the characteristics of aging neural cells in vivo. Background Technology

[0002] Globally, the accelerating aging of the population has become an undeniable social phenomenon, profoundly impacting various social sectors, including the economy, politics, and culture. China currently has the world's largest elderly population, and this trend continues to expand. With the increasing health needs of the elderly, the incidence of age-related diseases is rising simultaneously, placing a heavy burden on public finances. However, as aging is an irreversible natural process, exploring its molecular mechanisms is particularly urgent, as this will help develop effective interventions to mitigate the negative effects of aging.

[0003] Developing novel aging cell models is crucial for understanding aging mechanisms and developing intervention methods. Traditional models struggle to accurately simulate the complex characteristics of aging nerve cells in vivo. Currently, China lacks research technologies for nerve cell aging models, particularly reports on cell aging models that simulate the characteristics of aging nerve cells in vivo, and no similar patents have been published. Summary of the Invention

[0004] The purpose of this invention is to provide a method for establishing a neuronal aging model that simulates the characteristics of aging neuronal cells in vivo, in order to solve the problems existing in the prior art. This cell model can promote the expression of inflammatory factor STAT3 and aging marker protein P16, and form aggregates of DPF2, STAT3 and P16 proteins, which can simulate the aging characteristics of aging neuronal cells in vivo.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for establishing a neural cell aging model that simulates the characteristics of aging neural cells in vivo, comprising overexpressing DPF2 in P19 cells and then inducing the P19 cells to differentiate into neural cells with retinoic acid, thereby obtaining a neural cell aging model.

[0006] Preferably, the CDS nucleotide sequence of the DPF2 is shown in SEQ ID NO.1:

[0007] The amino acid sequence encoded by the DPF2 cDNA is shown in SEQ ID NO.2: MAAVVENVVKLLGEQYYKDAMEQCHNYNARLCAERSVRLPFLDSQTGVAQSNCYIWMEKRHRGPGLASGQLYSYPARRWRKKRRAHPPEDPRLSFPSIKPDTDQTLKKEGLISQDGSSLEALLRTDPLEKRGAPDPRVDDDSLGEFPVTNSRARKRILEPDDFLDDLDDEDYEEDTPKRRGKGKSKGKGVGSARKK LDASILEDRDKPYACDICGKRYKNRPGLSYHYAHSHLAEEEGEDKEDSQPPTPVSQRSEEQKSKKGPDGLALPNNYCDFCLGDSKINKKTGQPEELVSSCSDCGRSGHPSCLQFTPVMMAAVKTYRWQCIECKCCNICGTSENDDQLLFCDDCDRGYHMYCLTPSMSEPPEGSWSCHLCLDLLKEKASIYQNQNSS.

[0008] Preferably, the induction conditions are: 10 μM retinoic acid induction of the P19 cells for 3 days.

[0009] Preferably, overexpression of DPF2 upregulates the expression of inflammatory cytokine STAT3 and senescence marker protein P16 in retinoic acid-induced P19 cells.

[0010] Preferably, in the neural cell aging model, protein aggregates of DPF2 and STAT3, as well as protein aggregates of DPF2 and P16, are formed.

[0011] The present invention also provides a neural cell senescence model, established by the aforementioned method.

[0012] The present invention also provides the application of the aforementioned neural cell aging model in the study of simulated in vivo neural cell aging characteristics, the study being for the purpose of non-disease diagnosis and treatment.

[0013] The present invention also provides the application of the aforementioned neural cell senescence model in the study of aging-related diseases, the study being for the purpose of non-disease diagnosis and treatment.

[0014] The present invention discloses the following beneficial effects: (1) Highly simulates in vivo aging characteristics: This invention is the first to construct a cell model that can accurately simulate key characteristics of aging nerve cells in vivo. By overexpressing DPF2 and inducing neural differentiation, this model successfully reproduces the hallmark phenomena observed in aging tissues, namely the upregulation of the expression of the inflammatory factor STAT3 and the aging marker protein P16, and the formation of specific DPF2 / STAT3 / P16 cytoplasmic protein aggregates. This feature is highly consistent with the pathological changes observed in the spinal cord of aging mice, overcoming the limitations of traditional cell models in simulating complex aging phenotypes in vivo.

[0015] (2) The construction method is reliable and reproducible: The model is based on P19 cells. Through a clear molecular operation (transfection with GFP-DPF2) and differentiation induction procedure (RA treatment), a cell population with a stable senescence phenotype can be obtained in a short time. The method has clear steps, controllable conditions, and good reproducibility and operability, providing a stable and convenient tool platform for aging research.

[0016] (3) Providing new perspectives and tools for the study of aging mechanisms: This model not only simulates the morphology and molecular markers of aging, but its core mechanism also suggests that DPF2 may drive neuronal aging by activating the P16(INK4A)-STAT3 axis and is associated with neuroinflammation (IBA1 upregulation). This provides a novel research model and direct experimental evidence for a deeper understanding of the driving factors and signaling networks of neuronal aging, and helps to reveal the potential molecular mechanisms of aging and related diseases (such as neurodegenerative diseases).

[0017] (4) Significant application and translational potential: The novel neural cell aging model established in this invention, by integrating directed neural differentiation technology and transcriptomics technology, can reproduce the molecular phenotypes and characteristics related to neural cell aging in a short period of time, providing a dynamic and precise research platform for elucidating the aging-driving mechanisms. This not only accelerates the screening of drugs that target and eliminate senescent cells (Senolytics) or anti-aging, but also promotes the development of personalized anti-aging strategies, providing a translational bridge for delaying age-related diseases (such as Alzheimer's disease and neurodegenerative diseases), and ultimately contributing to the realization of healthy aging. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1The study aimed to investigate the elevated levels of DPF2 and STAT3 proteins in the spinal cord of aging mice. A: The expression levels of STAT3, DPF2, and P16 (INK4A) in spinal cord samples from 2-month-old, 13-month-old, and 21-month-old mice were detected by Western blotting. B: Statistical analysis was performed on the standardized relative expression levels of STAT3, DPF2, and P16 using GAPDH as an internal reference. P-values ​​were determined using one-way ANOVA. Figure 2 The expression and distribution of STAT3 and DPF2 in the spinal cord of mice of different ages were analyzed by immunofluorescence detection. A: Immunofluorescence detection was performed on spinal cord samples from mice of different ages (2, 13, and 21 months). BD: The expression levels of STAT3 (green) and DPF2 (red) in the spinal cord of 2-month-old (B), 13-month-old (C), and 21-month-old (D) mice were analyzed by immunofluorescence detection. Triangles marked the nuclear distribution of DPF2, and arrows marked the cytoplasmic distribution of DPF2 and STAT3. Scale bar = 50µm. EF: The number of cytoplasmic aggregated cells in the spinal cord of 2-, 13-, and 21-month-old mice was statistically analyzed. P-values ​​were determined using one-way ANOVA. G: Statistical analysis of the total number of cells in the cross-section of the spinal cord of 2M, 13M, and 21M-aged mice. P-values ​​were determined using one-way ANOVA. Figure 3 To analyze the expression and distribution of P16(INK4A) and DPF2 in the spinal cord of mice of different ages using immunofluorescence detection; AC: Expression levels of P16(INK4A) (green) and DPF2 (red) in the spinal cord of 2-month-old (A), 13-month-old (B), and 21-month-old (C) mice by immunofluorescence detection; arrows indicate the co-localization of DPF2 and P16(INK4A) in the cytoplasm; scale bar represents 50µm; DE: Statistical analysis results of the number of cells with cytoplasmic aggregates and the total number of cells in the spinal cord of 2-month-old, 13-month-old, and 21-month-old mice; P-values ​​were obtained by one-way ANOVA; F: Statistical analysis results of the total number of cells in the cross-section of the spinal cord of 2-month-old, 13-month-old, and 21-month-old mice; P-values ​​were obtained by one-way ANOVA. Figure 4To investigate the effect of DPF2 overexpression on STAT3 and P16(INK4A) expression in RA-treated P19 cells; AB: Fluorescence and Western blotting (IB) detection of P19 cells overexpressing GFP and GFP-labeled DPF2, showing the expression levels of GFP and GFP-labeled DPF2 in P19 cells (A) and the results of triple-repeat IB detection of labeled protein expression (B); CE: Statistical analysis results of STAT3, IBA-1, and P16(INK4A) expression, respectively; P-values ​​were determined using Student's t-test. FG: Fluorescence and IB detection of P19 cells overexpressing GFP and GFP-labeled DPF2 three days after RA treatment, showing the expression levels of GFP and GFP-labeled DPF2 in RA-treated P19 cells (F) and the expression level of target protein detected by triple-repeat Western blotting analysis (G); HJ: Statistical analysis results of STAT3, IBA-1, and P16(INK4A) expression, respectively; P-values ​​were determined using Student's t-test. Figure 5 To investigate the effect of DPF2 overexpression on STAT3 in RA-treated P19 cells; A: RA-induced STAT3 co-expression by GFP and GFP-labeled DPF2 in P19 cells, with fluorescence micrographs showing the distribution of GFP, GFP-labeled DPF2 (green), STAT3 (red), and DAPI (blue); scale bar = 50 µm; B: Statistical analysis of the proportion of P19-positive cells to the total cell count; p-values ​​were determined using STUDENT'S t-test; C: Statistical analysis of the proportion of GFP-positive aggregates to the total cell count; p-values ​​were determined using STUDENT'S t-test. Figure 6 To illustrate the effect of DPF2 overexpression on P16(INK4A) in RA-treated P19 cells; A: Schematic diagram of RA-induced co-expression of P16(INK4A) by GFP and GFP-labeled DPF2 in P19 cells, with fluorescence micrographs showing the co-localization of GFP, GFP-labeled DPF2 (green), P16(INK4A) (red), and DAPI (blue); B: Statistical analysis of the proportion of P19-positive cells in total cells; P-values ​​were determined using the STUDENT T-test; Figure 7 Cellular senescence atlas detected by KEGG in P19 cells treated with RA after DPF2 knockdown. The green background box represents the downregulated genes, mainly ARF and P16. Figure 8 This is a KEGG-based cellular senescence map of the spinal cord of 2-month-old and 21-month-old mice. The red boxes represent upregulated genes, primarily ARF and P16. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. 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.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1: Detection of abnormal protein aggregation in spinal cord nerve cells of aging mice 1.1 Laboratory Animals and Grouping The experimental animals were C57BL / 6 mice, divided into three groups: 2 months old (adult group), 13 months old (middle-aged group), and 21 months old (senior group). On the day of sampling, mice used for morphological analysis received cardiac perfusion with 4% paraformaldehyde (Sigma); mice used for molecular biological analysis received cardiac perfusion with saline. In the 2-month-old mice undergoing transcriptome sequencing, females weighed 16-18g and males weighed 23-24g; in the 21-month-old mice, females weighed 26-29g and males weighed 25-31g. The experimental animals were purchased from Jiangsu Wukong Biotechnology Co., Ltd. and Anhui Provincial Animal Resource Center.

[0026] 1.2 Mouse spinal cord sampling Before surgery, the mice were weighed and anesthetized with an intraperitoneal injection of 3% sodium pentobarbital (0.1 mL / 20 g body weight). They were then fixed in a supine position on the operating table, and their hearts were perfused with 20-50 mL of physiological saline or 4% paraformaldehyde fixative. After cardiac perfusion, the skin, fascia, and muscles adjacent to the spine were dissected with a scalpel to expose the spine. The T9-T11 vertebral segments were removed, and the spinal cord was isolated on ice. The extracted spinal cord was then stored at -80°C for later use.

[0027] 1.3 Preparation of mouse spinal cord paraffin blocks and sections 1.3.1 After the dissection was completed, the spinal cord tissue was fully immersed in 4% paraformaldehyde and fixed at room temperature for 48 h.

[0028] 1.3.2 The embedding cassette containing the spinal cord was placed in a tissue dehydrator for dehydration in the following order: 30% ethanol → 50% ethanol → 70% ethanol → 95% ethanol I → 95% ethanol II → anhydrous ethanol I → anhydrous ethanol II. Next, xylene was used to replace the alcohol to make the tissue transparent, in the following order: 50% ethanol + 50% xylene → xylene I → xylene II. The paraffin infiltration order was paraffin I → paraffin II → paraffin III, with the first infiltration lasting 15 minutes and the subsequent two lasting 30 minutes, while maintaining the temperature at approximately 58-60℃.

[0029] 1.3.3 The thickness of the embedded wax block slices was 4 µm.

[0030] 1.4 Tissue immunofluorescence Preheat paraffin sections to 37°C in a fan box overnight or to 60°C in an oven for 1 hour. Dewax in a fume hood in the following order: xylene I 15 min → xylene II 15 min. After dewaxing, hydrate the sections with anhydrous ethanol in the following order: anhydrous ethanol I 15 min → anhydrous ethanol II 15 min → 90% ethanol 15 min → 80% ethanol 10 min → 70% ethanol 5 min → 50% ethanol 5 min. After dehydration, drain any residual ethanol from the sections and rinse the surface with ultrapure water for 3-5 minutes before removing.

[0031] Add citrate buffer (Zhongshan Jinqiao, ZLI-9064) (pH 6.0) to a 1000 mL beaker. Place the tissue sections in the beaker, ensuring the antigen retrieval solution completely covers the top of the tissue sections. Place the beaker in a microwave oven and heat on medium-high heat for at least 15 minutes until boiling. After observing the antigen retrieval solution boiling, remove the beaker and allow it to cool at room temperature for 5 minutes. Then, return the beaker to the microwave and heat on low for another 5 minutes. Once small bubbles appear in the antigen retrieval solution, remove the beaker and allow it to cool at room temperature. After the tissue has completely cooled, wash the beaker containing the tissue sections three times with 1×PBS (White Shark, BL302A) on a shaker for 5 minutes each time. Next, prepare a 0.5% Triton X-100 permeabilization buffer using 1×PBS and Triton X-100 (Sangon Biotech, 9002-93-1) at the specified ratio. Drop the permeabilization buffer onto the tissue surface of the tissue sections. After adding the buffer, allow the sections to stand at room temperature for 40-60 minutes.

[0032] Gently shake off the permeabilization solution from the slides, wash the slides with 1×PBS, and place the slides on a shaker to shake slowly for 5 minutes × 3 times. At the same time, prepare the blocking solution, using 0.5% BSA (Solarbio, PC0001, prepared with 1×PBS), and add it dropwise to the surface of the tissue on the slides, ensuring complete coverage of the tissue. Finally, place the slides in a humidified chamber and block at room temperature for 60 minutes.

[0033] Primary antibody incubation: Remove the slides from the humidified chamber and discard the blocking solution. Prepare suitable antibodies: TAT3 (Proteintech, 60199-1-Ig, 1:200), P16 (INK4A) (Santa Cruz, SC1661, 1:200), DPF2 (Proteintech, 12111-1-AP, 1:200). Add the prepared antibodies to the tissue surface of the slides, ensuring complete coverage. Add 20 µL of antibody to each spinal cord tissue section. Place the slides in the humidified chamber and incubate overnight at 4°C.

[0034] Secondary antibody incubation: The next day, remove the incubation chamber from the 4°C freezer and allow it to warm to room temperature for 30 minutes. Discard the antibodies on the tissue, place the slides on a shaker and gently shake, then wash the tissues with 1×PBS for 5 minutes each time (3 times). Simultaneously, prepare the corresponding fluorescent secondary antibody (Rabbit Alexa Fluor R594 and Mouse Alexa Fluor M488, Invitrogen, A11008 / A11001) according to the species characteristics of the primary antibody, using 1×PBS, and ensuring complete darkness during preparation. Add the prepared fluorescent secondary antibody to the slides. Place the humidified chamber in a dark room and incubate at room temperature for 60 minutes, protected from light.

[0035] DAPI (Roche, 10236276001) staining and mounting: Remove the slides from the humidified chamber and wash them three times for 5 min each, protected from light. Simultaneously, prepare DAPI dye using 1×PBS at a dilution of 1:1000. Add DAPI to the tissue surface and incubate in the humidified chamber for 2-3 min in the dark. Gently shake the slides to remove the DAPI dye. Then wash the slides three times for 5 min each with 1×PBS. Using a pipette tip, add 15 µL of anti-fluorescence attenuation mounting medium around the tissue to mount the slides, ensuring air isolation. Allow the slides to air dry at room temperature in the dark before immediately acquiring images. Store the stained slides at 4°C. Finally, use an upright fluorescence microscope for imaging.

[0036] 2. Results 2.1 Upregulation of DPF2, P16(INK4A), and STAT3 expression in the spinal cord of aging mice First, to further investigate the expression of relevant molecules in the spinal cord of aging mice, this invention selected spinal cord tissues from mice aged 2 months (young group), 13 months (middle-aged group), and 21 months (old group) for immunoblotting analysis. Figure 1 The experimental results showed that the expression of STAT3, DPF2, and P16(INK4A) in the spinal cord tissue of 21-month-old aging mice was higher than that in 2-month-old and 13-month-old mice. This result suggests that these molecules may play an important role in the aging process and provides important clues for further research on aging-related molecular mechanisms.

[0037] 2.2 DPF2, STAT3, and P16 (INK4A) co-aggregate and lead to the loss of spinal cord nerve cells in aging mice. The expression and distribution of STAT3 and DPF2 in the spinal cord of 2-month-old, 13-month-old, and 21-month-old mice were analyzed by immunofluorescence (IF). In the spinal cord of aged mice, a large number of cytoplasmic aggregates of DPF2 and STAT3 with significant co-localization were observed. Figure 2 In addition, the number of cells containing cytoplasmic aggregates and positive for STAT3 or DPF2 was significantly increased (AD). Figure 2 In the middle EF), the total number of cells in aging mice was significantly reduced ( Figure 2 (G). Subsequently, the expression and distribution of P16 (INK4A) and DPF2 in the spinal cord of mice of different ages were analyzed by immunofluorescence detection. Figure 3 (AC). Surprisingly, cytoplasmic aggregates of DPF2 and P16 (INK4A) were detected in the spinal cord of 13-month-old and 21-month-old mice. Furthermore, the number of cells containing cytoplasmic aggregates was significantly increased in 21-month-old mice, while the total cell number was significantly decreased. Figure 3 (DF).

[0038] Example 2: Preparation of P19 cells overexpressing DPF2 P19 cell culture technology 1.1 P19 cell resuscitation The mouse teratoma P19 cell line (purchased from the Cell Bank of the Chinese Academy of Medical Sciences, Shanghai) was used. P19 cells were rapidly transferred from liquid nitrogen onto ice to a sterile 37°C water bath and gently thawed. Thawed P19 cells were then transferred to 15mL centrifuge tubes, and 2mL of high-glucose DMEM (Gibco, 6124392) medium containing 10% FBS (Vicente, 086150) and 1% penicillin-streptomycin (Beyotime, C0222) was added. The cells were centrifuged at 1000rpm / min for 5min. The supernatant was discarded, and 2-3mL of complete DMEM medium was pipetted to prepare a single-cell suspension. The single-cell suspension was placed in a large dish, and 7-8mL of complete DMEM medium was added. The P19 cells were then cultured in a 37°C incubator containing 5% CO2.

[0039] 1.2 Plasmid DNA Extraction The prepared LB solution [10g NaCl, 10g tryptone (Oxoid, LP0042), 5g yeast extract (Oxoid, LP0021) added to pure water to a final volume of 1000mL, shaken well and adjusted to pH 7.0] was autoclaved and stored in a refrigerator at 4℃. Before use, kanamycin sulfate (BBI, 0408) was added to the LB liquid medium to prepare a concentration of 50µg / mL.

[0040] Wild-type DPF2 cDNA (DPF2 gene accession number AF001433.1 in GenBank, CDS sequence shown in SEQ ID NO.1) was constructed using the following method: Using the pCMV-SPORT6-DPF2 vector (Open Biosystems, USA) as a template, PCR amplification was performed using primers 5'-CGGAATTCCCATGGAAGATGGCGGCTGTGGTGGAG-3' (SEQ ID NO.3) and 5'-ACGCGTCGACCAAGAGGAGTTCTGGTTCTGGTAGA-3' (SEQ ID NO.4). The resulting PCR product was subcloned and inserted into the pEGFP-N1 vector via EcoRI / SalI restriction sites. A suitable amount of *E. coli* containing pEGFP and pEGFP-DPF2 plasmids was picked and incubated overnight (14-16 h) in 10 mL LB medium containing kanamycin-resistant culture medium at 37°C using a shaker. The following day, the LB culture medium containing E. coli was centrifuged at 4000 g for 10 minutes, the supernatant was discarded, and the precipitate was collected.

[0041] The collected precipitate was used to extract plasmids according to the instructions of the plasmid extraction kit, and stored at -20°C for later use.

[0042] 1.3 Plasmid transfection of P19 cells Observe cell density under a microscope. Passage cells when they reach approximately 80%-90% confluence. Prepare a cell suspension and aspirate into a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant and add 10 mL of DMEM complete culture medium, mixing thoroughly. Pipette 10 µL of the cell suspension for cell counting. The optimal cell number for seeding in a six-well plate is 2.5 × 10⁶ cells / well. 5 Add 100 cells / well and 2 mL of DMEM complete culture medium to each well. Then incubate at 37°C.

[0043] 24 hours later, prepare the DNA according to the transfection requirements (2µg DNA / 2.5×10⁻⁶). 5 Add plasmid to each seeded cell. Discard the old culture medium and wash once with PBS. Add 2 µg DNA to 200 µL of jetPRIME (Polypuls, 0000003176) buffer, vortex for 10 s (in a pre-prepared EP tube), then add 4 µL of jetPRIME transfection reagent and incubate at room temperature for 10 min. Evenly drop 200 µL of the transfection mixture into each well. After 6 h, when changing the medium, wash once with PBS, add 2 mL of DMEM complete medium (containing antibiotics) to each well, and incubate at 37 °C.

[0044] 1.4 Retinoic acid (RA) induces neural differentiation of P19 cells P19 cells that had been transfected with plasmids in the previous step were added to complete medium containing RA (Sigma, 10µM) for induction culture 24h after transfection. The medium was changed every other day until RA induction was performed for 3 days, after which the cells were collected for use.

[0045] 1.5 Western Blot Experiment 1.5.1 Extraction of total protein from tissues and cells (1) Solution preparation: RIPA buffer: 0.87 g NaCl solid, 1 mL Triton-100, 5 mL 1 M Tris-HCl (pH 8.0), 1 mL 10% SDS, 1 mL NP-40, 0.05 g Sodium deoxycholate, 0.8 mL 0.5 M EDTA (pH 8.0), 1 mL PMSF (Sigma, P7626, add to 1 mM immediately before use), 1 mL PIC (Roche, 04693116001, add to 1× immediately before use), add deionized water to 100 mL, store at 4 °C.

[0046] PMSF (100mM): Weigh 174mg of PMSF powder and dissolve it in 10mL of isopropanol. This is the 100× storage concentration. After dispensing, store at -20℃. Dilute to 1× before use.

[0047] PIC stock solution: Dissolve one PIC tablet in 2 mL of sterile ultrapure water to obtain a 25× stock concentration. After aliquoting, store at -20°C. Dilute to 1× before use.

[0048] (2) Tissue protein extraction: First, take the spinal cord tissue out of the -80℃ freezer and thaw it on ice. After weighing, cut the tissue into small pieces with scissors and transfer it to a grinder (pre-cooled). Add protein lysis buffer (1 mL of lysis buffer per 0.1 g of tissue). Weigh and balance the tissue and place the appropriate size grinding beads according to the instructions of the grinder. Grind for 45 min to 1 h. Finally, transfer the grinding buffer into an EP tube and centrifuge at 20000 g for 15 min in a 4℃ centrifuge. Repeat the centrifugation once more. After the centrifugation, carefully aspirate the supernatant into a new EP tube to obtain the protein stock solution.

[0049] (3) Cell protein extraction: After cell collection, centrifuge at 1000 rpm / min for 5 min, discard the supernatant, and place the precipitate on ice. The protein lysis buffer is prepared in the same manner as for tissue lysis. Add 200-400 µL of cell lysis buffer to each well of a six-well plate and lyse on ice for 30 min. To ensure complete cell lysis, vortex the cells every 5 min. After 30 min, centrifuge at 12000 g for 15 min, and use a pipette to aspirate the supernatant and discard the precipitate. The retained supernatant is the protein stock solution.

[0050] 1.5.2 Protein Quantification and Denaturation After determining the protein concentration using the BCA (Thermo) protein assay kit according to the instructions, the sample with the lowest concentration was selected as the standard. Protein lysis buffer was then used to balance all samples to the same protein concentration level. The 6×SDS loading buffer was removed from the -20°C freezer and thawed on ice beforehand. The protein loading buffer was then added to the protein sample at a 1:5 ratio. The sample was then placed in a 100°C water bath for 5-10 minutes to ensure complete protein denaturation.

[0051] 1.5.3 SDS-PAGE electrophoresis Select the appropriate concentration of separating gel according to the molecular weight of the target protein. Prepare a 12% separating gel according to the gel preparation kit instructions: ddH2O 4.9mL, 30% Acr-Bis 6mL, 1M Tris (Solarbio, T8060) pH 8.8 3.8mL, 10% SDS (Solarbio, S8010) 0.15mL, 10% ammonium persulfate (AP) 0.15mL, TEMED (BIOSHARP) 0.006mL. Prepare a 5% stacking gel: ddH2O 4.1mL, 30% acrylamide-methylenebisacrylamide (Acr-Bis) 1mL, 1M Tris pH 6.8 0.75mL, 10% SDS 0.06mL, 10% AP 0.06mL, TEMED 0.006mL.

[0052] The electroporation conditions were 200mA for 120min.

[0053] 1.5.4 Antibody Incubation Block the PVDF (Millipore) membrane with 5%-10% skim milk at room temperature for 2 hours. After blocking, use 1×TBST (10×TBS buffer preparation: weigh 30.28g Tris-base and 87.66g NaCl on weighing paper, add ultrapure water to fully dissolve and bring the volume to 1000mL, adjust the pH to 8.0 with NaOH, and store at room temperature). Take 100mL of 10×TBS buffer, add ultrapure water to bring the volume to 1000mL, then add 1mL of Tween-20 and mix thoroughly. Wash the PVDF membrane for 3-5 minutes, repeating the washing 5 times. Subsequently, the antibodies were diluted according to the concentrations recommended in the antibody instructions (1×TBST dilution). The antibodies used in this invention were PAX6 (Proteintech, 12323-1-AP, 1:1000), STAT3 (1:1000), DPF2 (1:1000), P16(INK4A) (1:1000), TUJ1 (Proteintech, 66375-1-Ig, 1:1000), GFAP (Proteintech, 60190-1-Ig, 1:1000), IBA1 (Sigma, SAB2702364, 1:1000), GFP (Proteintech, 66002-1-Ig, 1:10000), and GAPDH (Proteintech, 60004-1-lg, 1:10000). The PVDF membrane was incubated overnight at 4°C in the primary antibody dilution buffer. The next day, the antibody incubation cassette was removed from the refrigerator and allowed to warm to room temperature for half an hour. The membrane was washed 8 times for 5 minutes each with 1×TBST. Secondary antibodies of the corresponding species were selected based on the primary antibody: mouse antibody 1:10000, rabbit antibody 1:5000. After incubation at room temperature for 1 hour, the membrane was washed 8 times for 5 minutes each with 1×TBST. The membrane was then developed using ECL (Advansta, D20362), and images with clear bands were selected for grayscale analysis using ImageJ software.

[0054] 2. Results 2.1 DPF2 upregulates the expression of PAX6, P16(INK4A), STAT3 and IBA1 in RA-induced P19 cells. To determine whether DPF2 directly induces STAT3 expression, the expression levels of STAT3 in P19 cells overexpressing GFP and those labeled with GFP (GFP-DPF2) were measured under retinoic acid-free (RA) conditions. Figure 4 (A). The results showed that overexpression of GFP-tagged DPF2 (rather than GFP itself) led to a decrease in STAT3 expression in P19 cells. Figure 4(Central BC). It is speculated that DPF2-induced STAT3 may have neuronal specificity. Subsequent detection of selected neuronal marker expression revealed that GFP-labeled DPF2 had no effect on the expression of PAX6 (neural stem cells), TUJ1 (neurons), and GFAP (astrocytes). Figure 4 (B), while increasing the expression of IBA1 and P16(INK4A) (B). Figure 4 (D and E in the middle).

[0055] To further investigate the potential role of DPF2 in regulating P16(INK4A) expression in nerve cells, plasmids encoding GFP and GFP-labeled DPF2 were transfected into P19 cells, followed by culturing with retinoic acid (RA) for 3 days. Figure 4 Cells were then lysed and subjected to Western blotting to determine the expression levels of PAX6, STAT3, TUJ1, GFAP, IBA1, and P16(INK4A). As expected, overexpression of GFP-labeled DPF2 in the presence of RA significantly increased STAT3 expression (F). Figure 4 GH). PAX6 expression was significantly upregulated, while TUJ1 and GFAP expression were unaffected by RA treatment. Furthermore, GFP-labeled DPF2 also significantly enhanced the expression of IBA1 and P16(INK4A). Figure 4 (I and J).

[0056] 2.2 In P19 cells, RA-induced co-localization of DPF2, STAT3, and P16 (INK4A) was significant. Furthermore, GFP-labeled DPF2 induced the conversion of DPF2 and STAT3 in the cytoplasm. Figure 5 (as indicated by the arrow) or P16(INK4A) ( Figure 6 The aggregation of STAT3 and P16(INK4A) (indicated by the arrow) suggests that DPF2 may directly regulate the senescence process of P19 cells by enhancing the expression and aggregation of STAT3 and P16(INK4A). This indicates that DPF2 may promote neuronal senescence by activating the P16(INK4A)-STAT3 axis. Given that IBA1 has long been used as a microglial marker mediating neuroinflammation, these results suggest that DPF2 may activate neuroinflammation and regulate neuronal senescence. This is consistent with the findings of this invention in mouse spinal cord immunoblotting experiments.

[0057] Example 3: DPF2-induced upregulation of P16 (INK4A) gene expression in P19-derived neurons is associated with upregulation of P16 (INK4A) gene expression in the spinal cord of aging mice. method 1.1 siRNA interference in P19 cells 1.1.1 Cell Seeding P19 cells in optimal growth condition were digested with trypsin and suspended in DMEM complete medium to form a cell suspension. The suspension was then prepared at a concentration of 3.5 × 10⁶ cells per well. 5 One cell was seeded into a 6-well culture plate and cultured for 24 hours.

[0058] 1.1.2 Transfection Before use, centrifuge siRNA (GenePharma, Shanghai, China) at 4000 rpm for 3 min and follow the instructions. For the negative control (NC) siRNA (2 OD): add 62.5 µL of DEPC water to each EP tube and mix well.

[0059] DPF2 siRNA (4 OD) (sense strand 5′-GCUUUCUUUCCCAUCGAUUTT-3′, SEQ ID NO.5 and antisense strand 5′-AAUCGAUGGGAAAGAAAGCTT-3′, SEQ ID NO.6): Add 125µL DEPC water to each EP tube and mix well. Transfection reagent preparation: (6µL Lipofectamine 2000 (Invitrogen) + 10µL siRNA + 100µL opti-MEM (Invitrogen)) / well, incubate at room temperature for 20 min to form the transfection complex. Transfection procedure: Before transfection, wash cells twice with serum-free DMEM medium, then add 1mL of serum-free DMEM medium. Add the transfection complex to the cells, mix gently, and incubate at 37℃ in a 5% CO2 incubator. After 6 h, discard the culture medium and replace with serum-containing complete medium and incubate for 12-24 h to allow cells to express the exogenous gene. The cells were then induced and treated with a medium containing 2 μM RA for 3 days, and the cells were harvested for subsequent related experiments.

[0060] 1.2 RNA-seq bioinformatics analysis 1.2.1 Sample Processing From NC siRNA and DPF2 P19 cells containing siRNA genomes (3 biological replicates per genome) were treated with TRIzol (Invitrogen) and mRNA was sequenced according to Novogene's protocol. In short, RNA purity was determined using a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), and RNA integrity was assessed using the RNA Nano 6000 assay kit from a Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

[0061] 1.2.2 Library sequencing and data acquisition According to the manufacturer's instructions, the indexed samples were clustered on the cBot cluster generation system using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina). After clustering, the library was sequenced on the Illumina NovaSeq platform, generating 150bp end reads. Clean reads were obtained by deleting reads containing adapters, reads containing poly-N, and low-quality reads of the original data.

[0062] 1.2.3 Establishment of the Reference Genome Index An internal reference genome index was constructed using Hisat2 v2.0.5, and the terminal cleanreads were sequenced using Hisat2 v2.0.5.

[0063] 1.2.4 Differential Gene Expression Analysis Gene expression level feature counting v1.5.0-p3 was used to count the reads for each gene. The FPKM for each gene was then calculated based on gene length and read count. For DESeq2 with biological replicates, differential expression analysis was performed on two conditions / groups (3 biological replicates per condition / group) using the DESeq2 R package (1.16.1). The resulting p-values ​​were adjusted using the Benjamini and Hochberg method to control for false detection rates. Genes with p-values ​​<0.05 detected by DESeq2 were considered differentially expressed.

[0064] 1.2.5 Gene enrichment analysis processing This invention uses the clusterProfiler R package to detect the statistical enrichment of differentially expressed genes in the KEGG (Kyoto encyclopedia of genes and genomes) pathway. Gene set enrichment analysis (GSEA) is used to determine whether a predefined gene set can show significant concordance differences between two biological states. A local version of the GSEA tool was used: ( http: / / www.broadinstitute.org / gsea / index.jsp GSEA uses the KEGG dataset.

[0065] 2. Results This invention uses KEGG detection and analysis to reveal the cell senescence profile of P19 cells after DPF2 knockdown and RA treatment. The results indicate that the P16 gene (green fill) is significantly downregulated, suggesting that DPF2 can upregulate P16 (INK4A). Figure 7 This result is closely related to the significant upregulation of the p16 gene (red fill) expression in the cell senescence atlas of aging mouse spinal cord cells. Figure 8 ).

[0066] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for establishing a neural cell aging model that simulates the characteristics of aging neural cells in vivo, characterized in that, This includes overexpressing DPF2 in P19 cells and then inducing the P19 cells to differentiate into nerve cells using retinoic acid, thus obtaining a nerve cell senescence model.

2. The method for establishing as described in claim 1, characterized in that, The nucleotide sequence of the DPF2 is shown in SEQ ID NO.

1.

3. The method for establishing as described in claim 1, characterized in that, The induction conditions were: 10 μM retinoic acid was used to induce the P19 cells for 3 days.

4. The method for establishing as described in claim 1, characterized in that, Overexpression of DPF2 upregulates the expression of inflammatory cytokine STAT3 and senescence marker protein P16 in retinoic acid-induced P19 cells.

5. The method for establishing as described in claim 1, characterized in that, In the described neuronal aging model, protein aggregates of DPF2 and STAT3, as well as protein aggregates of DPF2 and P16, were formed.

6. A model of neural cell aging, characterized in that, It is established by the method described in any one of claims 1-5.

7. The application of the neural cell aging model as described in claim 6 in the study of simulated in vivo aging neural cell aging characteristics, characterized in that, The study was conducted for purposes other than disease diagnosis and treatment.

8. The application of the neural cell senescence model as described in claim 6 in the study of aging-related diseases, characterized in that, The study was conducted for purposes other than disease diagnosis and treatment.

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