A method for constructing an in vitro model of aging astrocytes

Abstract

This invention discloses a method for constructing an in vitro model of senescent astrocytes, belonging to the interdisciplinary fields of cell biology, neuroscience, and biomechanics. The method includes: isolating and culturing primary astrocytes; performing adherent cell culture on the obtained primary astrocytes; and applying a compressive stress of 3-10 kPa to the adherent astrocytes for induced culture to obtain a model of senescent astrocytes. By simulating the changes in the mechanical microenvironment during brain tissue aging, the model successfully induces astrocytes into a senescent state using continuous vertical compressive stress. The constructed model can be used to screen anti-aging drugs or drugs for treating neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary fields of cell biology, neuroscience and biomechanics, and specifically relates to a method for constructing an in vitro model of aging astrocytes. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Astrocytes are the most numerous type of glial cell in the central nervous system, playing a crucial role in maintaining brain homeostasis, metabolic support, the blood-brain barrier, and the function of the brain's lymphoid system. Numerous studies have shown that astrocyte senescence is an important pathological basis and driving factor in the development of brain aging and neurodegenerative diseases such as Alzheimer's disease. Therefore, establishing stable and reliable in vitro models of senescent astrocytes is essential for studying the role of astrocyte senescence in neurological diseases and for screening interventional drugs.

[0004] Currently, common methods for inducing astrocyte senescence in vitro include replicative senescence models, genotoxic stress-induced models, and oxidative stress-induced models. While these methods can induce senescence-related phenotypes in astrocytes to some extent, the induction mechanisms differ from the pathological microenvironment of astrocytes during the in vivo senescence process. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for constructing an in vitro model of senescent astrocytes. This method can efficiently (senescence rate greater than 90%) and stably induce astrocytes into a senescent state within a short period (e.g., within 3 days), and the induced cells highly mimic the key characteristics of senescent astrocytes in vivo, including stable cell cycle arrest, typical senescence-related secretory phenotypes, and specific changes in astrocyte function-related proteins.

[0006] The present invention adopts the following technical solution: A first aspect of the present invention provides a method for constructing an in vitro model of aging astrocytes, the method comprising: Primary astrocytes were isolated and cultured; the obtained primary astrocytes were cultured in an adherent culture; the adherent astrocytes were induced to undergo culture by applying a compressive stress of 3-10 kPa to obtain a senescent astrocyte model.

[0007] In a second aspect, the present invention provides an aging astrocyte model, wherein the aging astrocyte model is prepared by the method described in the first aspect.

[0008] A third aspect of the present invention provides the application of the aging astrocyte model prepared by the method described in the first aspect or the aging astrocyte model described in the second aspect in any of the following: a1) Its application in the preparation and screening of anti-aging drugs; a2) Application in the preparation and screening of drugs for the treatment of neurodegenerative diseases.

[0009] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method for constructing an in vitro model of aging astrocytes. By simulating the stiffer mechanical microenvironment of brain tissue under aging or pathological conditions through pressure stimulation, it can more realistically reproduce the abnormal mechanical signals experienced by astrocytes in vivo. Compared to traditional methods using chemical reagents or oxidative stress, this model more closely approximates the cellular pathological changes induced by extracellular matrix hardening in brain aging and neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease) at the physical level. Research results obtained based on this model have higher physiological and pathological relevance, which is beneficial for revealing the true mechanisms by which the mechanical microenvironment affects glial cell aging.

[0010] This invention can induce a definite senescent state in over 94% of astrocytes within 3 days (e.g., low expression of lamin B1, increased expression of cyclin-dependent kinase inhibitor 2A (CDKN2A or p16) and γ-H2A.X variant histone, γH2A.X), whereas traditional replicative senescence models typically require several weeks or even longer of continuous passage to obtain a sufficient number of senescent cells. This highly efficient modeling method significantly shortens the experimental cycle and reduces the risk of genetic drift and phenotypic drift that may occur during long-term in vitro cell culture. Therefore, this model is particularly suitable for downstream experiments requiring rapid acquisition of large numbers of senescent cells, such as omics analysis, high-throughput drug screening, and co-culture studies. Attached Figure Description

[0011] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0012] Figure 1 A schematic diagram illustrating the process of constructing a model of senescent astrocytes using the method of this invention; Figure 2This is an immunofluorescence staining image of glial fibrillary acidic protein (GFAP), a specific marker for astrocytes. In the image, blue fluorescence (DAPI) marks the cell nucleus, and green fluorescence marks glial fibrillary acidic protein (GFAP). Merge indicates a merged group, which means that images from different fluorescence channels are combined into one image. Figure 3 The percentage chart for astrocyte purity reflects the relative purity of astrocytes by statistically analyzing the proportion of GFAP-positive cells (i.e., astrocyte-specific markers) among DAPI-positive cells (i.e., all nuclear markers). The percentages shown in the chart are calculated as follows: (Number of GFAP-positive cells ÷ Total number of DAPI-positive cells) × 100%. Figure 4 Immunofluorescence staining images of astrocytes cultured at 5 kPa for 36 h, 48 h, and 72 h are shown. In the figures, Control represents the normal control group, red fluorescence indicates Lamin B1, green fluorescence indicates glial fibrillary acidic protein (GFAP), and blue fluorescence indicates the cell nucleus. Merge indicates the merged group, which refers to images from different fluorescence channels being combined into one image. The scale bar in the figures is 50 μm. Figure 5 The graph shows the statistical analysis of the immunofluorescence staining Lamin B1 fluorescence intensity of astrocytes after culturing at 5 kPa for 36 h, 48 h, and 72 h. In the graph, Control represents the normal control group. Figure 6 The graph shows the percentage of astrocyte viability detected by the CCK-8 method. In the graph, Control represents the normal control group, and the percentage is calculated based on the control group. Figure 7 Immunofluorescence staining images of astrocytes cultured at 8 kPa for 36 h, 48 h, and 72 h are shown. In the figure, Control represents the normal control group, red fluorescence labels Lamin B1, green fluorescence labels glial fibrillary acidic protein (GFAP), blue fluorescence labels cell nuclei, and Merge represents the merged group, which means that images from different fluorescence channels are combined into one image. The scale bar in the figure is 50 μm. Figure 8 The graph shows the statistical analysis of the immunofluorescence staining intensity of Lamin B1 (left), GFAP (middle), and senescent astrocytes (cells with low Lamin B1 expression and positive GFAP) after culturing astrocytes at 8 kPa for 36 h, 48 h, and 72 h. In the graph, Control represents the normal control group. Figure 9This is a comparative graph showing the expression levels of p16 and γH2A.X proteins detected by Western blotting. Lanes 1 and 2: normal control group; lanes 3 and 4: group cultured at 8 kPa for 72 h. Figure 10 This is a statistical plot of the p16 and γH2A.X protein bands in Western Blot. Detailed Implementation

[0013] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0014] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0015] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0016] As described in the background section, current common methods for inducing astrocyte senescence in vitro have the following shortcomings: 1. Replicative senescence: Cells are brought to growth arrest through multiple passages. However, this method is time-consuming (weeks to months), and primary astrocytes have limited proliferative capacity, making it difficult to obtain a large number of homogeneous senescent cells. This method is limited to primary astrocytes with limited proliferative capacity and is not applicable to traditional astrocyte lines such as the C8-D1A mouse astrocyte line.

[0017] 2. Genotoxic stress induction: such as using drugs like etoposide and bleomycin. However, this method may cause severe DNA damage stress, and the induced aging phenotype may differ from physiological aging. Furthermore, it results in a high cell death rate, which is not conducive to mechanistic studies and drug screening.

[0018] 3. Oxidative stress induction: such as using hydrogen peroxide (H2O2). The aging state induced by this method is often unstable, cells are prone to apoptosis, and it is difficult to precisely control the degree of induction. Furthermore, the induction method tends to be acute injury or toxic stimulation, making it difficult to simulate the long-term, mild microenvironmental changes during brain aging.

[0019] In addition, existing technologies also have the following problems and shortcomings: 1. Insufficient model realism: The key characteristics of senescent cells induced by existing methods, such as senescence-related secretory phenotypes and metabolic changes, differ from those of senescent astrocytes in vivo.

[0020] 2. Poor efficiency and stability: long induction cycle, low success rate, and short duration of aging state.

[0021] 3. Low reproducibility: Due to the lack of standardization of induction conditions (such as drug concentration and reaction time), there are large differences in results between different laboratories.

[0022] Existing research suggests that the mechanical properties of brain tissue change during aging and related diseases, manifested as an increase in Young's modulus and a higher degree of "stiffness." However, current technologies do not consider this change in brain tissue mechanical properties, nor do they provide methods for constructing in vitro models to induce astrocyte aging through mechanical means.

[0023] Based on this, the present invention provides a method for constructing an in vitro model of senescent astrocytes.

[0024] A typical embodiment of the present invention provides a method for constructing an in vitro model of aging astrocytes, the method comprising: Primary astrocytes were isolated and cultured; the obtained primary astrocytes were cultured in an adherent culture; the adherent astrocytes were induced to undergo culture by applying a compressive stress of 3-10 kPa to obtain a senescent astrocyte model.

[0025] During aging and neurodegenerative diseases, the biomechanical microenvironment of brain tissue changes, manifesting as increased local tissue stiffness. Astrocytes are highly sensitive to mechanical stimuli, and prolonged exposure to a stiffer mechanical environment can induce cellular dysfunction and promote aging. This invention utilizes an in vitro cell compression culture system to apply a stable and controllable continuous vertical compressive stress of 3-10 kPa to cultured astrocytes under controlled conditions. This simulates the stiffer biomechanical microenvironment of brain tissue, thereby inducing aging-related phenotypes in astrocytes.

[0026] In a specific embodiment of the present invention, the compressive stress includes continuous constant loading.

[0027] In a specific embodiment of the present invention, the compressive stress is 5-9 kPa. Preferably, the compressive stress can be 5 kPa, 6 kPa, 7 kPa, 8 kPa, or 9 kPa.

[0028] In a specific embodiment of the present invention, the induction culture conditions are 37°C and 5% CO2.

[0029] In a specific embodiment of the present invention, the induction culture time is 12-96 hours, preferably 24-72 hours. More preferably, the induction culture time can be 24 hours, 36 hours, 48 ​​hours, or 72 hours.

[0030] In this invention, the magnitude of the applied compressive stress, the frequency of application, and the total duration can all be precisely set via the device, achieving standardized control of the mechanical stimulation parameters. Strictly consistent conditions can be maintained between different batches of experiments, significantly reducing the impact of operator differences or environmental fluctuations on the modeling results. This high degree of controllability and good reproducibility provides a technical foundation for large-scale, multi-center in vitro studies, and is particularly suitable for drug screening and mechanism verification experiments requiring high-throughput, standardized operations.

[0031] In a specific embodiment of the present invention, when the cells adhere to the culture vessel and reach a confluence of 50%-80%, they are induced to undergo further culture.

[0032] In a specific embodiment of the present invention, the primary astrocytes are derived from mammalian brain tissue.

[0033] In a specific embodiment of the present invention, the isolation and culture of primary astrocytes specifically includes the following steps: Brain tissue was removed under sterile conditions, placed in a buffer solution, and after the meninges and blood vessels were removed, it was cut into small pieces. The brain tissue was digested using a combination of mechanical blowing and trypsin digestion to obtain a single-cell suspension. After digestion, undigested tissue fragments were removed by terminating digestion, filtration, and centrifugation. The obtained cell pellet was resuspended in culture medium and seeded into cell culture containers for culture. During the culture process, the culture medium was changed regularly to remove non-adherent neurons and other cell types, and astrocytes were gradually enriched. After primary astrocytes are cultured to a certain degree of confluence, microglia and a small number of residual oligodendrocyte precursors are removed to obtain high-purity primary astrocytes. In a specific embodiment of the present invention, the digestion treatment conditions are digestion for 5 minutes at an incubator temperature of 37°C.

[0034] In a specific embodiment of the present invention, the culture temperature is 37°C and the culture gas environment is an air environment containing 5% CO2.

[0035] In a specific embodiment of the present invention, the method for removing microglia and a small amount of residual oligodendrocyte precursors includes one of shaking, differential adhesion, or immunoadsorption.

[0036] In a specific embodiment of the present invention, the method for cell adherent culture includes the following steps: Primary astrocytes in the logarithmic growth phase were digested and counted, and then seeded at a predetermined density into appropriate culture plates for adherent culture.

[0037] In a specific embodiment of the present invention, the establishment of an aging astrocyte model is confirmed by detecting the expression levels of classic aging markers. These aging markers include, but are not limited to, Lamin B1, p16, and γH2A.X. Generally, low expression of Lamin B1 and / or elevated expression of p16 and / or γH2A.X indicate that the astrocytes have entered a senescent state.

[0038] Existing technologies often rely on exogenous chemicals (such as hydrogen peroxide and bleomycin) or strong oxidants to induce cellular senescence. These substances may cause non-specific DNA damage, mitochondrial dysfunction, and cytotoxic reactions, interfering with the explanation of the essential mechanisms of aging. This invention employs purely physical pressure stimulation without introducing any chemical inducers, thus eliminating background interference from chemical toxicity. This makes the constructed aging model "cleaner," more accurately reflecting the aging phenotype of astrocytes under purely mechanical conditions, and improving the reliability and interpretability of experimental data.

[0039] In another embodiment of the present invention, an aging astrocyte model is provided, which is prepared by the above-described method.

[0040] A third embodiment of the present invention provides an application of the senescent astrocyte model prepared by the above method or the senescent astrocyte model in any of the following: a1) Its application in the preparation and screening of anti-aging drugs; a2) Application in the preparation and screening of drugs for the treatment of neurodegenerative diseases.

[0041] In specific embodiments of the present invention, the neurodegenerative diseases include, but are not limited to, Alzheimer's disease and Parkinson's disease.

[0042] The aging astrocyte model constructed using this invention can be directly used to analyze how mechanical signals regulate the aging process of glial cells, filling the research gap in mechanical signal transduction that traditional chemically induced models cannot address. Simultaneously, this model can serve as an in vitro platform to rapidly evaluate the reversal or alleviation effects of anti-brain aging or neuroprotective candidate drugs on mechanically induced aging phenotypes. Because the model eliminates chemical interference, drug screening results better reflect the true efficacy of drugs against the mechanical pathological microenvironment, exhibiting higher clinical translational potential.

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0045] Materials: C57BL / 6 suckling mice aged 1-3 days, DMEM complete culture medium (Thermo Fisher Scientific, catalog number A5669701) containing 10% fetal bovine serum (Thermo Fisher Scientific, catalog number A5669701), 1% penicillin-streptomycin double antibody solution (Solepro, catalog number 32.P7630), the pressure stimulation induction device of this invention (bionic cell pressure culture system, Shenzhen Ruicheng Technology Co., Ltd.), anti-Lamin B1 (Abcam, catalog number Ab16048), p16 (Abcam, catalog number Ab54210), γH2A.X (Proteintech, catalog number 29380-1-ap), and GFAP (Millpore, catalog number MAB360), etc.

[0046] Example 1 according to Figure 1 The flowchart shown illustrates the construction of a model of senescent astrocytes. Specific methods include: 1.1 Isolation and Culture of Primary Astrocytes Under aseptic conditions, the hippocampus of newborn mice was harvested and placed in pre-cooled buffer solution. After removing the meninges and blood vessels, the tissue was cut into small pieces. Following mechanical dissociation by pipetting and trypsin digestion, the pieces were inoculated into poly-L-lysine-coated culture flasks. Digestion was performed at 37°C for 5 minutes to obtain a single-cell suspension. After digestion, undigested tissue fragments were removed by terminating digestion, filtration, and centrifugation.

[0047] The obtained cell pellet was resuspended in culture medium and seeded into cell culture vessels for culture under constant temperature and humidity conditions. During the culture process, the culture medium was changed regularly to remove non-adherent neurons and other cell types, thereby gradually enriching astrocytes.

[0048] Primary astrocytes were cultured at 37°C and 5% CO2 until they reached a certain confluence. Microglia and a small amount of residual oligodendrocyte precursors were then removed using methods such as shaking, differential adhesion, or immunoadsorption to obtain high-purity (>90%) primary astrocytes. Figure 2 , 3 As shown, immunofluorescence revealed that the expression of the astrocyte marker protein GFAP was above 93%, indicating that the cultured primary astrocytes were of sufficient purity for subsequent experiments.

[0049] The purified astrocytes were cultured in a medium containing 10% fetal bovine serum until the cells were stable and morphologically uniform. The number of passages was controlled during culture, with low passage numbers being preferred to preserve the cells' biological characteristics.

[0050] 1.2 Model Processing After digesting and counting the purified astrocytes from generations 2 and 3, they were processed at a concentration of 4 × 10⁻⁶. 5 Seeds were planted at a density of 2 × 10⁶ cells / well in 6-well plates for Western blotting analysis. 4 Cells were seeded at a density of [number] cells / well in 24-well plates for immunofluorescence detection. When cells adhered and grew to approximately 65% ​​confluence, the complete culture medium was replaced once in both the control and experimental groups. The experimental group cells were placed in a pressure-stimulated culture device, which was then placed in a cell culture incubator for continuous induction for 36 h, 48 h, and 72 h, respectively. Control group cells were cultured normally in a cell culture incubator.

[0051] 1.3 Induced aging and model identification Purified astrocytes from passages 2 and 3 were stimulated with a stable vertical compressive stress of 5 kPa. Western blot analysis showed that, compared with astrocytes cultured under normal conditions (without pressure), the expression level of Lamin B1 protein in the cells was significantly reduced after 72 h of stimulation with 5 kPa. Lamin B1, as an important component of the nuclear lamina, is widely recognized as a reliable marker of cellular senescence due to its downregulation. These results indicate that continuous 5 kPa pressure stimulation for 72 h can successfully induce astrocytes into a senescent state. Figure 4 , 5 ).

[0052] Example 2 2.1 Isolation and Culture of Primary Astrocytes Primary astrocytes were isolated and cultured according to the method in Example 1.

[0053] 2.2 Model Processing The model was treated according to the method in Example 1, except that when the cells adhered and grew to approximately 75% confluence, the complete culture medium was replaced once in both the control and experimental groups. The experimental group's well plates were placed in a pressure-stimulated culture device, which was then placed in a cell culture incubator for continuous induction for 36 h, 48 h, and 72 h, respectively. The control group cells were cultured normally in a cell culture incubator.

[0054] 2.3 Induced aging and model identification To optimize the pressure stimulation conditions and clarify the dose-response relationship between mechanical intensity and aging degree, this embodiment increases the pressure to 8 kPa and stimulates astrocytes for 36 h, 48 h and 72 h respectively.

[0055] First, the cells treated with 8 kPa for the longest time (72 h) were subjected to CCK8 assay to assess their cell viability. Figure 6 As shown, there was no significant difference in cell viability between cells stimulated with 8 kPa pressure for 72 h and normally cultured cells (Control group vs. 8 kPa treatment group for 72 h, 100% vs. 101%). Immunofluorescence results are shown below. Figure 7 , 8 As shown, compared with the normal culture control group, 8 kPa pressure stimulation for 36 h induced a significant decrease in Lamin B1 levels in astrocytes, indicating that cellular senescence occurred at an earlier time point. With the extension of pressure stimulation time to 48 h and 72 h, Lamin B1 expression levels further decreased, and the senescence phenotype showed a time-dependent increasing trend. This result indicates that 8 kPa pressure stimulation can not only rapidly initiate the cellular senescence process, but also significantly enhance the degree of senescence with prolonged stimulation time. Furthermore, by detecting the upregulation of the astrocyte-specific marker GFAP and reactive astrocyte characteristics such as cell hypertrophy, the results showed that the fluorescence intensity of GFAP significantly increased after 36 h of 8 kPa pressure stimulation, and further increased with prolonged stimulation time. Further analysis of the number of senescent cells (Lamin B1 low expression and GFAP positive cells) to quantify the degree of senescent astrocyte induction showed that after 36 h of stimulation with 8 kPa pressure, the proportion of senescent astrocytes reached 77.38%; after 48 h, it increased to 91.82%; and after 72 h, it further increased to 94.78%.

[0056] To further validate the cellular senescence phenotype induced by 8 kPa pressure stimulation from multiple dimensions, Western blotting was used to detect the protein expression levels of classic aging-related biomarkers. Astrocytes were stimulated with 8 kPa pressure for 72 h, and the expression levels of p16 and γH2A.X were then examined. The results showed that compared with normally cultured astrocytes, the expression levels of p16 protein were significantly increased in the 8 kPa pressure stimulation group for 72 h, and the expression levels of γH2A.X protein were also significantly upregulated. Figure 9 , 10p16 is a cell cycle-dependent kinase inhibitor, and its high expression is a key driver of cell cycle arrest and senescence. γH2A.X is a marker of DNA damage response, and its upregulation reflects the accumulated DNA damage response in senescent cells. Based on these results, astrocytes stimulated with 8 kPa pressure for 72 h simultaneously exhibited typical senescent molecular characteristics, including decreased Lamin B1, increased p16, and increased γH2A.X. Therefore, it can be considered a reliable model of senescent astrocytes.

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing an in vitro model of aging astrocytes, characterized in that, The method includes: Primary astrocytes were isolated and cultured; the obtained primary astrocytes were cultured in an adherent culture; the adherent astrocytes were induced to undergo culture by applying a compressive stress of 3-10 kPa to obtain a senescent astrocyte model.

2. The method as described in claim 1, characterized in that, The primary astrocytes mentioned above are derived from mammalian brain tissue.

3. The method as described in claim 2, characterized in that, The mammalian brain tissue includes the cerebral cortex or hippocampus.

4. The method as described in claim 1, characterized in that, The compressive stress includes continuous constant loading.

5. The method as described in claim 1, characterized in that, The induction culture conditions were 37°C and 5% CO2.

6. The method as described in claim 1, characterized in that, The induction culture time is 12-96 hours.

7. The method as described in claim 1, characterized in that, When the cells adhere to the culture vessel and reach a confluence of 50%-80%, they are then induced to undergo further culture.

8. A model of aging astrocytes, characterized in that, The aging astrocyte model is prepared by the method described in any one of claims 1-7.

9. The application of the aging astrocyte model according to claim 8, characterized in that, The application includes any two of the following: a1) Its application in the preparation and screening of anti-aging drugs; a2) Application in the preparation and screening of drugs for the treatment of neurodegenerative diseases.

10. The application as described in claim 9, characterized in that, The neurodegenerative diseases mentioned include, but are not limited to, Alzheimer's disease and Parkinson's disease.

Images