A model of oxygen-glucose deprivation and reperfusion of aged SH-SY5Y cells and its construction method and application

Abstract

The present invention discloses an oxygen-glucose deprivation reperfusion model of aging SH-SY5Y cells and a construction method and application thereof, and relates to the field of biomedical technology. The construction method comprises the following steps: (1) using D-galactose to induce aging of SH-SY5Y cells, thereby constructing aging SH-SY5Y cells; (2) the aging SH-SY5Y cells are first subjected to hypoxia-sugar deprivation treatment, and then subjected to reoxygenation treatment, thereby obtaining the oxygen-glucose deprivation reperfusion model of aging SH-SY5Y cells. The present invention successfully constructs an oxygen-glucose deprivation reperfusion model of SH-SY5Y cells, which can simulate the pathophysiological process of aging brain ischemia-hypoxia injury, and lays a foundation for subsequent research on the neuroprotective effect of drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an oxygen-glucose deprivation-reperfusion model of aged SH-SY5Y cells and a construction method and application thereof. Background Art

[0002] The human neuroblastoma cell (SH-SY5Y) model damaged by oxygen glucose deprivation / reoxygenation (OGD / R) can simulate the pathological process of brain ischemia-reperfusion injury in vivo, and has become an important means to study the mechanism of brain ischemia injury and drug effects in vitro. By controlling the duration of OGD exposure, the pathological conditions at different time points can be observed; using various molecular biological techniques, the relationship between a functional molecule and drug action can be analyzed in a targeted manner.

[0003] Existing studies have shown that cerebral ischemia-reperfusion injury has age-related differences. Therefore, it is not appropriate to use the general SH-SY5Y cell OGD / R model to study the neuroprotective effect of drugs at the cellular level. Therefore, it is urgent to establish an oxygen-glucose deprivation-reperfusion model of aged SH-SY5Y cells to simulate the pathophysiological process of cerebral ischemia-hypoxia injury in the elderly.

[0004] D-galactose (D-gal) is a cell aging inducer. Normal concentrations of D-gal are converted into glucose in the liver, while higher concentrations of D-gal can be catalyzed by galactose oxidase to produce hydrogen peroxide and aldose, resulting in a large amount of ROS that can cause protein and lipid peroxidation, mitochondrial damage and dysfunction, energy metabolism disorders, and ultimately cell aging. Summary of the invention

[0005] The purpose of the present invention is to provide an oxygen-glucose deprivation / reperfusion model of aging SH-SY5Y cells and a construction method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention successfully constructs a aging cell OGD / R model, which can simulate the pathophysiological process of aging brain ischemia-hypoxia injury, laying a foundation for subsequent research on the neuroprotective effect of drugs.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for constructing an oxygen-glucose deprivation-reperfusion model of aged SH-SY5Y cells, comprising the following steps:

[0008] (1) Using D-galactose to induce senescence in SH-SY5Y cells, thus constructing senescent SH-SY5Y cells;

[0009] (2) The senescent SH-SY5Y cells are first subjected to glucose and oxygen deprivation treatment, and then subjected to reoxygenation treatment, thereby obtaining the oxygen-glucose deprivation and reperfusion model of senescent SH-SY5Y cells.

[0010] Furthermore, in step (1), the concentration of D-galactose used is 100 mM.

[0011] Furthermore, the aging time of the D-galactose is 48h.

[0012] Furthermore, in step (2), the glucose-and oxygen-deficiency treatment is specifically as follows: the aged SH-SY5Y cells obtained in step (1) are placed in a sugar-free DMEM culture medium, and 99.9% high-purity nitrogen is introduced and then sealed and cultured.

[0013] Furthermore, when 99.9% high-purity nitrogen was introduced, the flow rate was 1 L / min and the introduction time was 5 min.

[0014] Furthermore, the sealed culture time is 1 hour.

[0015] Furthermore, in step (2), the reoxygenation treatment time is 24 hours.

[0016] The present invention also provides an oxygen-glucose deprivation-reperfusion model of senescent SH-SY5Y cells constructed according to the above construction method.

[0017] The present invention also provides application of the above-mentioned aged SH-SY5Y cell oxygen-glucose deprivation reperfusion model in screening neuroprotective drugs.

[0018] The present invention discloses the following technical effects:

[0019] The present invention first uses the CCK8 method to determine the concentration and time of D-gal acting on SH-SY5Y cells to induce senescence, and uses cell viability, cell cycle, cell proliferation detection and SA-β-Gal staining to verify cell senescence. On this basis, an OGD / R model is established using D-gal to induce senescent SH-SY5Y cells, and the OGD and reoxygenation time of senescent cells are determined by the CCK8 method. Finally, it is determined that the OGD 1h and reoxygenation 24h mode after 100mM D-gal induces senescence for 48h is used in the construction of the OGD / R model of senescent SH-SY5Y cells. The present invention successfully constructs an SH-SY5Y cell oxygen-glucose deprivation reperfusion model, which can simulate the pathophysiological process of aging cerebral ischemia-hypoxia injury, and lays a foundation for subsequent research on the neuroprotective effect of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The morphological changes of SH-SY5Y cells after 48 hours of treatment with different concentrations of D-gal were observed under a microscope (100×); A: Control group, B: 25mM group, C: 50mM group, D: 100mM group, E: 200mM group, F: 400mM group. The black arrows in the figure indicate the widening of the interneuronal gap and the change of cell morphology; the scale bars in A-F are all 100μM;

[0022] Figure 2 Cell viability of SH-SY5Y cells treated with different D-gal concentration gradients for 48 h; **** indicates p < 0.001;

[0023] Figure 3 β-galactosidase staining under different D-gal concentration gradients (×400); AF are β-galactosidase staining images of the Control group, 25mM group, 50mM group, 100mM group, 200mM group and 400mM group, respectively; G is the positive rate of stained cells; *p<0.05; **p<0.01;

[0024] Figure 4 Edu staining images under different D-gal concentration gradients (×400); A represents cell proliferation images, and B represents the statistical proliferation cell ratio; *p<0.05; **p<0.01; the scale bars in A are all 100 μM;

[0025] Figure 5 Flow cytometry was used to detect the cell cycle under different D-gal concentration gradients;

[0026] Figure 6 Comparison of cell survival rates at different glucose and oxygen deprivation time, ***p<0.005, ****p<0.001. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1 Materials and methods

[0034] 1.1 Main reagents for the experiment

[0035] Table 1 Main experimental reagents

[0036]

[0037]

[0038] 1.2 Main instruments and equipment

[0039] Table 2 Main instruments and equipment

[0040]

[0041]

[0042] 1.3 Reagent configuration

[0043] (1) Preparation of D-gal mother solution: Accurately weigh D-gal powder and dissolve it in sterile ultrapure water to prepare a mother solution with a final concentration of 1 mol / L. Prepare it for immediate use and dilute it according to the experimental requirements.

[0044] (2) Preparation of complete culture medium: Add 1% (V / V) double antibody (penicillin / streptomycin solution) and 10% (V / V) fetal bovine serum to the basal culture medium (DMEM / F12).

[0045] 1.4 Experimental methods

[0046] 1.4.1 Cultivation of SH-SY5Y cells

[0047] (1) SH-SY5Y cells were cultured in DMEM medium containing 10% FBS in an incubator at 37°C with 95% O2 and 5% CO2.

[0048] (2) Cell recovery: Before the experiment, ultra-clean laminar flow cell operation table and centrifuge tubes were disinfected by ultraviolet irradiation for 30 minutes, and the operation table surface was wiped twice with a wet alcohol cotton ball. The constant temperature water bath was turned on in advance and the temperature was set to 40°C. The cell cryopreservation tube was quickly taken out from the -80°C ultra-low temperature refrigerator, quickly placed in the water bath to thaw, and gently shaken back and forth. After the liquid was dissolved, it was quickly transferred to an EP tube with 2mL of complete culture medium added in advance, centrifuged at 1000rpm for 5min, the supernatant was discarded and 1mL of complete culture medium was added. The cell suspension was transferred to a T25 culture flask with 3mL of complete culture medium added in advance, and continued to be cultured at 37°C in an incubator containing 95% O2 and 5% CO2. The complete culture medium was replaced every other day to promote better cell growth.

[0049] (3) Cell passaging: When the cell confluence reaches 90% under a microscope, discard the original culture medium, wash twice with PBS and remove the residual liquid, add 500 μL of 0.25wt% trypsin solution to cover the bottom of the bottle and digest for 30 seconds. Under the microscope, cells can be seen to become round and detach from the wall. Shake the bottom of the bottle and the detached cells can be seen to move with the liquid in the form of quicksand. Immediately add 2 mL of complete culture medium to terminate digestion, use a Pasteur pipette to suck out the culture medium containing cells and transfer it to a 10 mL centrifuge tube. After repeated blowing and beating, centrifuge at 1000 rpm for 5 minutes, discard the upper layer of liquid, add 3 mL of complete culture medium, and repeatedly blow and beating to prepare a single cell suspension, which is then transferred to a new 25T cell culture bottle. Shake the culture bottle in an eight-shaped manner to evenly distribute the cells in the bottle, and then place it in an incubator at 37°C, containing 95% O2 and 5% CO2 for continued culture.

[0050] (4) Cell cryopreservation: After cell passaging, centrifuge and discard the supernatant, add 1 mL of cell freezing solution, and follow the gradient freezing method by placing the cell cryopreservation tubes in a 4°C refrigerator for 20 min, a -20°C refrigerator for 40 min, and then in a -80°C refrigerator.

[0051] (5) Cell counting: Wipe the counting plate and cover glass clean with a wet alcohol cotton ball, and after drying, cover the cover glass on the counting plate. After the cells are digested and centrifuged, repeatedly blow the culture medium to form a single cell suspension. Pipette 10μL of cell suspension and gently drip it onto the counting plate along one side of the cover glass. Be careful not to mix in bubbles and the cell fluid cannot overflow the cover glass. Place the counting plate under a microscope and count the number of cells in the four large squares (each large square is divided into 16 small squares). Only count complete cells. If they are clustered together, count them as one cell. If there are cells on the line, count the cells on the lower line but not the cells on the upper line, and count the cells on the left line but not the cells on the right line. The error of the second repeated counting shall not exceed ±5%. Number of cells in cell suspension / mL = total number of cells in 4 large squares / 4×10 4 .

[0052] 1.4.2 Establishment of OGD / R model of cell senescence

[0053] 1.4.2.1 Cell senescence experimental method

[0054] SH-SY5Y human neuroblastoma cells with good growth status were selected and inoculated into 6-well culture plates at the same density (2×10 cells) one day before D-gal administration. 4 / well), 5% CO2, 37 ℃ incubator overnight, added 25mM D-gal, 50mM D-gal, 100mM D-gal, 200mM D-gal, 400mM D-gal respectively, set up a control group without drug intervention, continue to culture in the incubator for 48h, and detect various indicators.

[0055] 1.4.2.2 Establishment of OGD / R model of senescent SH-SY5Y cells

[0056] ① After promoting cell senescence according to the cell senescence promotion experimental method in 1.4.2.1, take SH-SY5Y cells in good growth state, under sterile conditions, use a Pasteur pipette to remove the original culture medium, wash once with PBS preheated at 37℃, and then use a Pasteur pipette to remove all the liquid, add sugar-free DMEM culture medium preheated at 37℃, open the lid of the well plate and put it into the autoclaved hypoxic box, and evenly flow 99.9% high-purity nitrogen from the small hole on the top of the box. Keep the flow rate at 1L / min, stop and quickly seal the hypoxic box after 5 minutes, and transfer it to a 37℃ constant temperature box without CO2 to continue culturing. This process is called sugar-deficient and oxygen-deficient treatment.

[0057] ② According to the experimental design, after different time points of hypoxia, the cells were taken out of the sealed hypoxia box and replaced with normal DMEM medium (containing FBS, the volume fraction of FBS in DMEM medium was 10%) under sterile conditions using a Pasteur pipette, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. The preparation of the OGD / R model of senescent SH-SY5Y cells was completed.

[0058] 1.4.3CCK8 detection of cell activity

[0059] SH-SY5Y neuroblastoma cells with good growth status were taken and the cells were seeded once in a 96-well culture plate at the same density (cell volume 10 4 / well), 5% CO2, 37 ℃ incubator overnight, distilled water was added to the outer circle of the 96-well plate to ease evaporation. The next day, 25mM, 50mM, 100mM, 200mM, and 400mM concentrations of D-gal were added, and the control group without drug intervention was placed in the incubator for 48 hours. According to the instructions of the CCK8 reagent, the original culture medium was sucked out with a Pasteur pipette, and washed once with PBS preheated at 37 ℃, and 100μL of culture medium containing CCK8 solution (10μL of CCK8 solution + 90μL of sugar-free DMEM culture medium) was added. The operation process needs to be protected from light. Place in a 37 ℃ incubator and continue to culture for 1h, and measure the absorbance OD value of each well at a wavelength of 450nm on an enzyme reader. The experiment was repeated 3 times.

[0060] 1.4.4 Cell senescence β-galactosidase staining

[0061] After the SH-SY5Y cells with good growth status were treated according to the experimental steps in 1.4.2, the original culture medium was discarded, the cells were washed once with PBS, 1 mL of β-galactosidase staining fixative was added, the cells were fixed at room temperature for 15 min, the fixative was discarded, the cells were washed three times with PBS, 1 mL of staining working solution was added to each well, the 6-well plate was wrapped with tin foil to avoid light, and the plate was placed in a CO2-free 37°C incubator for overnight incubation and observed under an ordinary optical microscope.

[0062] 1.4.5 Cell proliferation assay

[0063] Experiment using BeyoClick TM Edu-488 Cell Proliferation Detection Kit (BeyoClick TMThe Edu Cell Proliferation Kit with Alexa Fluor 488 is used for detection. The experimental principle is based on the incorporation of thymidine analogue Edu (5-ethynyl-2'-deoxyuridine) during DNA synthesis, and the subsequent click reaction allows Edu to be labeled with Alexa Fluor 488. It is simple, rapid and highly sensitive. After the SH-SY5Y cells with good growth status were treated according to the experimental steps in 1.4.2, the Edu working solution preheated at 37℃ was added and incubated for 6 hours; the culture medium was aspirated, 1mL of fixative solution was added to each well, fixed for 15min, and the fixative solution was removed; after washing 3 times with 1mL PBS, 1mL of permeabilization solution was added and incubated at room temperature for 15min; after removing the permeabilization solution, 1mL of PBS was removed and washed 2 times, 0.5mL of Click reaction solution was added, and the culture plate was gently shaken to ensure that the reaction mixture evenly covered the sample, and incubated at room temperature for 30min; the Click reaction solution was aspirated, washed 3 times with PBS, 1mL of Hoechst33342 solution was added to each well, washed 3 times with PBS, and immediately observed and photographed under a fluorescence microscope. The cell nucleus stained with Hoechst showed blue fluorescence, and the proliferating cells showed bright red fluorescence.

[0064] 1.4.6 Flow cytometry to detect changes in the cell cycle

[0065] After the SH-SY5Y cells with good growth status were treated according to the experimental steps in 1.4.2, the culture medium was aspirated, and the culture wells were washed with 500μL PBS solution, then aspirated and added to a 1.5mL centrifuge tube. The cells were digested with 500μL 0.25wt% trypsin EDTA solution and collected in a 1.5mL centrifuge tube. Centrifuged at 1000g for 3min, the supernatant was aspirated and a trace amount of culture medium was retained to avoid aspirating the cells. 1mL of ice-cold PBS buffer was added to resuspend the cells and centrifuged again. 500μL of ice-cold 70% ethanol aqueous solution was added and gently pipetted to mix until there were no clumping cells. Fix the cells at 4℃ for more than 4h. Centrifuge at 1000g for 3min to precipitate the cells. 1mL of ice-cold PBS buffer was added to resuspend the cells and centrifuged again. Repeat the resuspension operation after centrifugation. Gently flick the bottom of the centrifuge tube to disperse the cells to avoid cell clumping. Add 0.5 mL of propidium iodide staining solution to each tube of cell sample, gently resuspend the cells to mix them thoroughly, and incubate them at 37°C in the dark for 30 minutes. Filter the stained liquid using a 5 mL cell sieve tube and collect the filtrate. Flow cytometry was used to detect red fluorescence signals (488 nm excitation wavelength) and record light scattering data. Cell cycle analysis was performed using flowjo software and graphed using GraphPad Prism 8 software.

[0066] 1.4.7 CCK-8 method to detect the survival rate of senescent cells at different hypoxia time

[0067] After the SH-SY5Y cells with good growth status were treated according to the experimental steps of 1.4.2, the 96-well plate was tilted, the culture medium was carefully aspirated, 100 μL PBS was added to each well to wash the cells, the PBS was aspirated, and the sugar-free DMEM preheated at 37°C was added. The cells were treated with oxygen and glucose deprivation according to 1.4.2.2, and the glucose deprivation time was set to 0.5h, 1h, 1.5h, and 2h. After that, the sugar-free DMEM was replaced with complete culture medium, and cultured for 24h in a 37°C, 5% CO2 incubator. The culture medium was aspirated, each well was washed once with PBS preheated at 37°C, and 100 μL of culture medium containing CCK8 solution (10 μL CCK8 solution + 90 μL sugar-free DMEM culture medium) was added. The operation process should be protected from light. Place in a 37°C incubator and continue to culture for 1h, and measure the absorbance OD value of each well at a wavelength of 450nm on an ELISA reader. The experiment was repeated 3 times.

[0068] 1.5 Statistical analysis

[0069] The experimental data were expressed as mean + standard deviation, and Graphpad prism8.0 software was used for statistical analysis and drawing. The measurement data were expressed as mean ± standard error. The independent sample t test was used to compare the means of two groups, and the one-way ANOVA was used to compare the means of more than three groups. When the data variance was equal, the LSD test was used for pairwise comparisons between multiple groups, and the Dunnett's T3 method was used when the variance was unequal. P < 0.05 was considered statistically significant.

[0070] 2 Experimental results

[0071] 2.1 Observation of cell morphology under different D-gal concentrations under microscope

[0072] SH-SY5Y neuroblastoma cells with good growth status were taken and the cells were seeded once in a 6-well culture plate at the same density (cell volume 10 5 / well), 5% CO2, 37 ℃ incubator overnight, until the cell adhesion and fusion degree reached more than 80%, add 1mL of complete medium containing 25mM, 50mM, 100mM, 200mM, 400mM D-gal, and set up a control group without drug intervention, and continue to culture in the incubator for 48h. The cell morphology was observed under an inverted phase contrast microscope. Figure 1As shown in the figure, with the increase of D-gal concentration, the number of attached cells decreased, some cells were still attached but swollen and flat, the cells were sparsely arranged, the morphology was abnormal, the cell body became smaller and rounder, the refractive index was enhanced, and some cell connections even disappeared, indicating that D-gal concentration had little effect on cells within a certain range, but if the concentration was too high, it would be highly toxic to cells.

[0073] 2.2 CCK8 reagent to detect cell activity

[0074] In order to screen the appropriate concentration of D-gal to cause senescence, the CCK8 kit was used to detect the changes in cell viability after 48 hours of action under different D-gal concentration gradients. The results are shown in Figure 2 The experiment showed that under the action of different concentrations of D-gal, the cell survival rate gradually decreased, among which the cell survival rate of the 25mM group was (76.65±7)%, the cell survival rate of the 50mM group was (63.45±9)%, the cell survival rate of the 100mM group was (59.3±11)%, the cell survival rate of the 200mM group was (48.2±11)%, and the cell survival rate of the 400mM group was (30.6±6)%. According to IC50, 100mM D-gal concentration was selected.

[0075] 2.3 Cell senescence β-galactosidase staining

[0076] In order to detect cell senescence after 48 hours of D-gal induction, the cells were stained with a β-galactosidase kit. β-galactosidase is the most commonly used marker of cell senescence. It can catalyze the substrate X-Gal to generate a dark blue product. SA-β-Gal staining is one of the most effective methods for detecting cell senescence and is also the gold standard for detecting senescence. Cells that are stained blue under an inverted microscope are senescent cells, and those that are not stained are ordinary cells. The experimental results are as follows Figure 3 As shown, D-gal induction significantly increased the positive rate of β-galactosidase staining, and with the increase of concentration, the positive rate of β-galactosidase staining increased. The positive rate of blank control group was (5.47% ± 1.09)%, the positive rate of 25mM D-gal staining was (7.11 ± 1.08)%, the positive rate of 50mM staining was (7.74 ± 3.35)%, the positive rate of 100mM staining was (13.9 ± 4.65%), the positive rate of 200mM staining was (19.88 + 1.453)%, and the positive rate of 400mM staining was (56.43 + 11.43)%. It is worth noting that the blue staining positive rate of 100mM group was significantly reduced, and there was a statistical difference (P < 0.05). Combined with the cell viability and SA-β-gal staining results, 100mM was selected as the final modeling concentration.

[0077] 2.4 Cell proliferation assay

[0078] The Edu cell proliferation assay was used to evaluate cell proliferation. During DNA synthesis, Edu was incorporated into new DNA instead of thymidine. The Edu acetylene group and the fluorescently labeled small molecule azide probe reacted covalently with each other through the catalysis of monovalent copper ions to form a stable triazole ring. Proliferating cells showed red fluorescence under a microscope, and the cell nucleus showed blue. Data processing was based on the Control group. The results are shown in Figure 2. Figure 4 As shown, the proliferation rate of the 25mM group was (94.89±9.48)%, the proliferation rate of the 50mM group was (80.8±18.67)%, the proliferation rate of the 100mM group was (72.24±22.81%), the proliferation rate of the 200mM group was (64.97±3.779)%, and the staining positive rate of the 400mM group was (38.37±15.81)%. It can be seen that with the increase of D-gal concentration, the cell proliferation rate decreased, and statistical differences began to appear at 100mM.

[0079] 2.5 Changes in the cell cycle

[0080] A common feature of senescent cells is irreversible cell cycle arrest, in which the S phase is the DNA synthesis phase. We performed cell cycle detection on cells treated with different concentrations for 48 hours. The results were ( Figure 5 ) showed that the proportion of cells in the S phase of each group was different, including the Control group (28.63±0.41)%, the 50mM group (34.33±3.6)%, the 100mM group (26.8±0.55%), the 200mM group (25±0.79)%, and the 400mM group (19.13±9.9)%. It can be seen that 100mM can cause cell cycle arrest in the S phase, and the specific statistical significance (P<0.05). Graphpad prism8.0 statistical software was used to analyze G0 / G1. With the increase of concentration, the proportion of cells gradually increased, especially the percentage of cells in the G0 / G1 phase of the cell cycle in the 100mM group (57.033±0.153)% was significantly higher than that in the Control group (49.467±2.4)%, and it was statistically significant (P<0.05). At the same time, Graphpad prism 8.0 statistical software was used to analyze G2 / M. The number of cells in the G2 / M phase gradually decreased, and the percentage of cells in the G2 phase of the cell cycle in the 100mM group (12.96±0.35)% was significantly lower than that in the Control group (19.57%±0.7)%, and it was statistically significant (P<0.05).

[0081] 2.6 Detection of cell survival rate at different times of hypoxia and glucose deprivation

[0082] Take the SH-SY5Y cells that have been promoted to be in good growth state and inoculate them in a 96-well plate. Set up 6 replicate wells for each group. According to the modeling experiment operation method in 1.4.2.2, after 0.5h, 1h, 1.5h, and 2h of hypoxia, reoxygenate for 24h, and then perform cell survival rate detection. According to the CCK8 cell activity detection manual, the cell survival rate results at different hypoxia time points are calculated. Figure 6 , among which: the cell survival rate of 0.5h hypoxia was (94.1±2.8)%, the cell survival rate of 1h hypoxia was (53±1.6)%, the cell survival rate of 1.5h hypoxia was (41.8%±4.5)%, and the cell survival rate of 2h was (23.5±1.5)%. Compared with the Control group, the survival rate gradually decreased with the extension of hypoxia time, and all of them were statistically significant. It is generally believed that the time period when the cell survival rate is around 50% is the best time for drug treatment. Therefore, 1h hypoxia and glucose deprivation was selected as a follow-up study.

[0083] The D-gal-induced aging model is often used in aging research. When the D-gal concentration in the cell is too high to be catalyzed and metabolized, it will cause cell infiltration, swelling and high ROS. When these changes exceed the clearance capacity of the oxidative defense system, it will cause metabolic disorders and ultimately lead to aging. Because the present invention needs to perform a secondary OGD / R attack on the cells later, it is necessary to select a suitable concentration according to the experimental requirements and cannot refer to the conventional experimental concentration. The present invention found that cell viability decreased with the increase of D-gal concentration, especially when the D-gal concentration was 200mM and above, the cell viability dropped sharply, and there was an obvious reduction in the nucleus, sparse cell arrangement, rounding or separation of the cells from the wall, or even death. The present invention uses 100mM D-gal corresponding to a cell viability of nearly 50% as the subsequent cell aging concentration.

[0084] During the aging process, dividing cells gradually lose their ability to proliferate, accompanied by unique morphological changes and changes in the expression of aging-specific markers. The identification or quantification of cell aging is mainly reflected by changes in cell morphology, increased SA-β-Gal activity, growth inhibition, cell cycle arrest, etc. The morphological characteristics of senescent cells are generally manifested as increased volume, enlarged nuclei and irregular shapes. It is very convenient to observe and is used as an inspection indicator. The present invention experiment found that as the concentration of D-gal increased for 48 hours, the number of cells gradually decreased, especially when it exceeded 100mM, the cell morphology was obviously sparsely distributed, and even cell death occurred. Senescent cells express β-galactosidase (SA-β-gal) with high enzyme activity at pH 6.0, and blue staining precipitates appear, so the aging trend can be evaluated by analyzing the percentage of positive staining of cells. In the present invention, cells were treated with different doses of D-gal (25, 50, 100, 200, 400) mM for 48 hours, and the SA-β-Gal staining rate increased significantly, and was dose-dependent. Although SA-β-Gal activity has been widely used as a classic marker of aging, studies have shown that its activity may also increase under conditions independent of aging. Therefore, the present invention further analyzes whether other aging markers can be observed in D-gal-induced SH-SY5Y cells. The cell cycle is the process of continuously dividing cells from the completion of the last division to the completion of the next division, and is considered to be an important mechanism for controlling normal cell proliferation and biological aging. Senescent cells stagnate in the G1 / G0 phase and cannot smoothly enter the S phase, which is manifested as a decrease in S phase cells. The cell cycle results in the present invention show that as the concentration of D-gal increases, more and more cells are blocked in the G1 phase. At 100mM in the S phase, a turning point occurs, representing that the number of cells entering the replication phase is greatly reduced, and there is a statistical difference. In the Edu experiment, the present invention clearly sees the proportion of proliferating cells through proliferating cells / nuclei, and also finds that statistical differences begin to appear at 100mM, indicating that the cell cycle has been significantly inhibited, and the results are compounded with the cell cycle results measured by flow cytometry. Therefore, the present invention uses 100mM D-gal for 48h as a subsequent experimental study.

[0085] Compared with animal experiments, cells have the advantages of uniform research samples, easy observation of single factors, and artificial controllable research conditions during the culture process. At present, the establishment of in vitro cell hypoxia and hypoxia models is generally divided into physical and chemical hypoxia. Chemical hypoxia is the addition of certain chemicals, such as sodium dithionite and cyanide, to the cell culture fluid, which damages the ability of cells to use oxygen or depletes the oxygen in the culture fluid to cause cell hypoxia. Chemical hypoxia has the disadvantages of causing damage to cells and difficult experimental operations. The physical hypoxia model uses a mixed gas culture method to reduce the proportion of oxygen in the culture environment, thereby simulating the hypoxia experimental model, usually with a liquid surface closed model, a homemade simple sealing model, and a three-gas incubator model. At present, there are a wide range of technologies for producing hypoxic conditions, including anaerobic incubators, hypoxic chamber glove boxes, or homemade hypoxia devices. Nitrogen is an inert gas, and nitrogen / carbon dioxide mixed gas is usually used to adjust the O2 level. The present invention adds sugar-free culture fluid to the culture dish and puts the culture dish into the hypoxic box, and perfuses nitrogen as quickly as possible, and the time and airflow size of each perfusion are kept consistent. When the nitrogen fills the entire hypoxia box, stop filling and seal it and put it into a constant temperature incubator to achieve the effect of cell OGD. In the experiment, OGD injury and reoxygenation are two stages. OGD is first given to the cells at the cellular level to simulate the state of neural damage after cerebral ischemia and hypoxia. Reoxygenation is a process that simulates reperfusion. On the one hand, the changes in cell quantity, structure and characteristic phenotype can be observed morphologically. On the other hand, the pathological changes and drug action mechanisms of cerebral ischemia-reperfusion injury can be explored at the biochemical and protein levels. The duration and intensity of OGD / R are key parameters for evaluating drugs with neuroprotective potential. A cell death rate between 40% and 60% is the best choice for studying neuroprotective drugs, because it is difficult to obtain significant neuroprotection at higher cell death values.

[0086] Human studies have shown that cerebral blood flow and oxygen consumption decrease by 25%-40% between the ages of 30 and 89. Therefore, the neurovascular units in the elderly brain are particularly susceptible to hypoxia, among which neurons are the most vulnerable and will die rapidly. In the experiment, it can be seen that senescent cells induced by D-gal also exhibit the same characteristics, and cell viability is greatly reduced after OGD / R. In CIRI, the brain undergoes a series of "ischemic cascade" events, and the reduction in blood flow to the brain reduces the availability of nutrients, oxygen and energy, and depletes the vitality of nerve cells. The degree of cell damage depends on the duration and severity of ischemia and hypoxia, so the OGD model of senescent cells needs to be optimized. In the present invention, the viability of SH-SY5Y cells gradually decreases with the extension of OGD time. Referring to the SH-SY5Y cell OGD / R model of the present invention, the uniform reoxygenation is 24h. It is found that the cell survival rate is (53±1.6)% when OGD acts on SH-SY5Y cells for 1h and reoxygenates for 24h. In the experiment of the present invention, it was found that the cell viability of hypoxia for 2h was only 23.5%. It can be seen that aged cells are more obviously damaged after OGD / R, so the experiment used 1h of hypoxia as a follow-up experimental study.

[0087] In summary, the present invention first uses the CCK8 method to determine the concentration and time of D-gal inducing senescence of SH-SY5Y cells, and verifies cell senescence by cell viability, cell cycle, cell proliferation detection and SA-β-Gal staining. On this basis, the OGD / R model is established by using D-gal to induce senescent SH-SY5Y cells, and the OGD and reoxygenation time of senescent cells are determined by the CCK8 method. Finally, it is determined that the OGD 1h and reoxygenation 24h mode after 100mM D-gal inducing senescence for 48h is adopted in the construction of the OGD / R model of senescent SH-SY5Y cells.

[0088] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing an oxygen-glucose deprivation-reperfusion model of aged SH-SY5Y cells, characterized in that: The following steps are involved: (1) Using D-galactose to induce senescence in SH-SY5Y cells, thus constructing senescent SH-SY5Y cells; (2) The senescent SH-SY5Y cells are first subjected to glucose and oxygen deprivation treatment, and then subjected to reoxygenation treatment, to obtain the senescent SH-SY5Y cell oxygen-glucose deprivation and reperfusion model; In step (1), the concentration of D-galactose is 100 mM; The aging time of the D-galactose is 48h; In step (2), the glucose-and oxygen-deficiency treatment is specifically as follows: placing the aged SH-SY5Y cells obtained in step (1) in a sugar-free DMEM culture medium, introducing 99.9% high-purity nitrogen gas, and then sealing and culturing; The sealed culture time is 1h; In step (2), the reoxygenation treatment time is 24 hours.

2. The construction method according to claim 1, characterized in that: When 99.9% high-purity nitrogen was introduced, the flow rate was 1 L / min and the introduction time was 5 min.

3. An oxygen-glucose deprivation-reperfusion model of senescent SH-SY5Y cells constructed according to the construction method according to any one of claims 1-2.

4. Use of the oxygen-glucose deprivation-reperfusion model of senescent SH-SY5Y cells as claimed in claim 3 in screening neuroprotective drugs.

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