CN115354027A

CN115354027A - Aging SH-SY5Y cell oxygen sugar deprivation reperfusion model and construction method and application thereof

Info

Publication number: CN115354027A
Application number: CN202211164977.8A
Authority: CN
Inventors: 蒋嫦月; 韦文芳; 乃坚业
Original assignee: Nanning Fourth People's Hospital
Current assignee: Nanning Fourth People's Hospital
Priority date: 2022-09-23
Filing date: 2022-09-23
Publication date: 2022-11-18

Abstract

The invention discloses an aging SH-SY5Y cell oxygen sugar deprivation reperfusion model and a construction method and application thereof, relating to the technical field of biological medicine. The construction method comprises the following steps: (1) D-galactose is used for causing the SH-SY5Y cell to be aged, and the aged SH-SY5Y cell is constructed; (2) And the aged SH-SY5Y cells are subjected to sugar-deficiency and oxygen-deficiency treatment and then to reoxygenation treatment to obtain the aged SH-SY5Y cell oxygen-sugar deprivation reperfusion model. The invention successfully constructs an SH-SY5Y cell oxygen sugar deprivation reperfusion model which can simulate the pathophysiological process of the ischemia and hypoxia injury process of the aged brain and lays a foundation for the subsequent study on the neuroprotective effect of the medicament.

Description

Aging SH-SY5Y cell oxygen sugar deprivation reperfusion model and construction method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an aged SH-SY5Y cell oxygen sugar deprivation reperfusion model and a construction method and application thereof.

Background

An Oxygen sugar deprivation/reoxygenation (OGD/R) damaged human neuroblastoma cell (SH-SY 5Y) model can simulate an in-vivo damage pathological process during cerebral ischemia reperfusion, and becomes an important means for researching a cerebral ischemia damage mechanism and a drug action at an isolated level. By controlling the duration of OGD exposure, pathological conditions at different time nodes can be observed; the relationship between a certain functional molecule and the action of the drug can be analyzed in a targeted way by applying various molecular biology techniques.

The existing research shows that cerebral ischemia reperfusion injury has age-increasing difference, so that the general OGD/R model of SH-SY5Y cells is not suitable for researching the neuroprotective effect of the medicament on the cellular level. Therefore, the establishment of an aging SH-SY5Y cell oxygen sugar deprivation reperfusion model is urgently needed to simulate the pathophysiological process of the ischemia and hypoxia injury process of the aged brain.

D-galactose (D-gal) is a cell senescence inducer. D-gal at normal concentration is converted into glucose in the liver in vivo, while D-gal at higher concentration can be catalyzed by galactose oxidase to produce hydrogen peroxide and aldose, so that a large amount of ROS further causes protein and lipid peroxidation, and can also cause mitochondrial damage and dysfunction to cause energy metabolism disorder, and finally cell aging.

Disclosure of Invention

The invention aims to provide an aged SH-SY5Y cell oxygen sugar deprivation reperfusion model and a construction method and application thereof, and aims to solve the problems in the prior art.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a construction method of an aging SH-SY5Y cell oxygen sugar deprivation reperfusion model, which comprises the following steps:

(1) D-galactose is used for causing the SH-SY5Y cell to be aged, and the aged SH-SY5Y cell is constructed;

(2) And the aged SH-SY5Y cells are subjected to sugar-deficient hypoxia treatment and then to reoxygenation treatment to obtain the aged SH-SY5Y cell oxygen sugar deprivation reperfusion model.

Further, in the step (1), the D-galactose is used at a concentration of 100mM.

Further, the D-galactose had a senescence-causing time of 48h.

Further, in the step (2), the sugar-deficiency and oxygen-deficiency treatment specifically comprises: and (2) placing the aged SH-SY5Y cells obtained in the step (1) into a sugar-free DMEM culture solution, introducing 99.9% high-purity nitrogen, and then sealing and culturing.

Further, when 99.9% high purity nitrogen gas was introduced, the flow rate was 1L/min and the introduction time was 5min.

Further, the time of the sealed culture is 1h.

Further, in the step (2), the time of the reoxygenation treatment is 24h.

The invention also provides an aging SH-SY5Y cell oxygen sugar deprivation reperfusion model constructed according to the construction method.

The invention also provides application of the aging SH-SY5Y cell oxygen sugar deprivation reperfusion model in screening of neuroprotective drugs.

The invention discloses the following technical effects:

the invention firstly determines the concentration and time of D-Gal acting on SH-SY5Y cell to induce senescence by a CCK8 method, verifies cell senescence by cell viability, cell cycle, cell proliferation detection and an SA-beta-Gal staining method, establishes an OGD/R model by using the D-Gal senescence-inducing SH-SY5Y cell on the basis, determines the OGD and reoxygenation time of the senescence cell by the CCK8 method, and finally determines the OGD 1h and reoxygenation 24h modes after adopting 100mM D-Gal senescence for 48h in the construction of the OGD/R model of the senescence SH-SY5Y cell. The invention successfully constructs an SH-SY5Y cell oxygen sugar deprivation reperfusion model which can simulate the pathophysiological process of the ischemia and hypoxia injury process of the aged brain and lays a foundation for the subsequent study on the neuroprotective effect of the medicament.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the morphological changes (100X) of SH-SY5Y cells observed under a microscope after 48h of the action of different concentrations of D-gal; wherein, A: control group, B:25mM group, C:50mM group, D:100mM group, E:200mM group, F:400mM, with the increase in the gap between nerve cells and the change in cell morphology indicated by black arrows; the scale in A-F is 100 mu M;

FIG. 2 shows the cell viability of SH-SY5Y cells treated for 48h at different D-gal concentration gradients; * Denotes p <0.001;

FIG. 3 shows the staining (. Times.400) with β -galactosidase at different D-gal concentration gradients; wherein, A-F are beta-galactosidase staining patterns of a Control group, a 25mM group, a 50mM group, a 100mM group, a 200mM group and a 400mM group respectively, and G is the positive rate of data statistics staining cells; * p <0.05; * P <0.01;

FIG. 4 is a graph of Edu staining at different D-gal concentration gradients (x 400); wherein A represents a cell proliferation map, and B represents a statistical proliferation cell ratio; * p <0.05; * P <0.01; the scale in A is 100 mu M;

FIG. 5 shows the flow cytometry detection of the cell cycle at different D-gal concentration gradients;

figure 6 shows a comparison of cell viability at different times of hypoxia-induced hypoxia, p <0.005, p <0.001.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, 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 belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

1 materials and methods

1.1 Experimental Primary reagents

TABLE 1 Main test reagents

1.2 Main instrumentation

TABLE 2 Main instrumentation

1.3 preparation of reagents

(1) D-gal mother liquor preparation: d-gal powder is precisely weighed, dissolved by sterilized ultrapure water, prepared into mother liquor with the final concentration of 1mol/L, prepared for use at present, and then diluted according to the test requirements.

(2) Preparation of complete medium: 1% (V/V) of diabody (penicillin/streptomycin solution) and 10% (V/V) of fetal bovine serum were added to the basal medium (DMEM/F12).

1.4 Experimental methods

1.4.1SH-SY5Y cell culture

(1) SH-SY5Y cell culture was carried out using DMEM cell culture medium containing 10% FBS, at 37 deg.C, containing 95% ₂ 、5％CO ₂ Culturing in the incubator.

(2) Cell recovery: before the experiment, the ultra-clean laminar flow cell operation table and the centrifuge tube are disinfected for 30min by ultraviolet irradiation, and the table top of the operation table is wiped by a wet alcohol cotton ball for 2 times. The constant temperature water bath kettle is opened in advance, the temperature is set to be 40 ℃, the cell freezing tube is taken out from a-80 ℃ ultra-low temperature refrigerator quickly, put into the water bath kettle quickly for unfreezing, and gently shaken back and forth. After the solution was dissolved, the solution was quickly transferred to an EP tube previously charged with 2mL of complete medium, centrifuged at 1000rpm for 5min, the supernatant was aspirated off and 1mL of complete medium was added, the cell suspension was slowly and uniformly blown with a 1000mL pipette tip at about 120 minutes, and the cell suspension was transferred to a T25 flask previously charged with 3mL of complete medium at 37 ℃ with 95% O ₂ 、5％CO ₂ The incubator of (2) continues to cultivate. Complete medium was changed every other day to promote better cell growth.

(3) Cell passage: observing cell confluency under microscope to 90%, discarding original culture solution, washing with PBS for 2 times, sucking residual liquid, adding 0.25wt% trypsin solution 500 μ L to cover the bottom of the bottle, digesting for 30 s, allowing cells to round and remove wall under microscope, shaking the bottom of the bottle to allow the dropped cells to move in sand state with the liquid flow, immediately adding 2mL complete culture medium to stop digestion, and pasteurizingThe cell-containing culture medium is sucked out by a suction pipe, transferred to a 10mL centrifuge tube, repeatedly blown and beaten at 1000rpm, centrifuged for 5min, the supernatant is removed, 3mL complete culture medium is added, repeatedly blown and beaten to prepare single cell suspension, and the single cell suspension is transferred to a new 25T cell culture bottle. Shaking the culture flask in eight characters to uniformly distribute the cells in the flask, subsequently placing at 37 deg.C, 95% ₂ 、5％CO ₂ The incubator of (2) continues to cultivate.

(4) Freezing and storing cells: after cell passage, centrifuging to remove supernatant, adding 1mL of cell cryopreservation solution, sequentially placing the cell cryopreservation tube into a 4 ℃ refrigerator for 20min and a-20 ℃ refrigerator for 40min, and then placing the cell cryopreservation tube into a-80 ℃ refrigerator for cryopreservation according to a gradient cryopreservation method.

(5) Cell counting: clean counting plate and cover glass are wiped with wet alcohol cotton balls, and after drying, the counting plate is covered with the cover glass. Repeatedly pumping the culture solution into single cell suspension after cell digestion and centrifugation. 10 μ L of cell suspension was aspirated and gently dropped onto the counting plate along one side of the cover slip, taking care that no air bubbles could be mixed and the cell fluid could not overflow the cover slip. The cells in the four-corner large squares (each large square is divided into 16 small squares) of the counting plate are counted under a microscope, only the whole cells are counted, and if the cells are aggregated, the cells are counted according to the number of the cells. If any cell is on line, counting the lower line cell without counting the upper line cell, and counting the left line cell without counting the right line cell. Errors in duplicate counts were not checked + -5%. Cell suspension cell number/mL =4 total large lattice cells/4 × 10 ⁴ 。

1.4.2 establishment of cellular senescence OGD/R model

1.4.2.1 Experimental methods for cell senescence promotion

SH-SY5Y human neuroblastoma cells in good growth state are taken, and the cells are inoculated in a 6-well culture plate once at the same density one day before D-gal administration (cell amount is 2 multiplied by 10) ⁴ Per well), 5% CO ₂ 25mM D-gal, 50mM D-gal, 100mM D-gal, 200mM D-gal and 400mM D-gal are respectively added into an incubator at 37 ℃ overnight, a Control group without drug intervention is additionally arranged, the incubator is arranged for continuous culture for 48h, and detection of various indexes is carried out.

1.4.2.2 establishment of OGD/R model of senescent SH-SY5Y cells

(1) After the cell attenuation is promoted according to the experimental method of 1.4.2.1 cell attenuation promotion, SH-SY5Y cells with good growth state are taken, original culture solution is sucked off by a Pasteur suction pipe under the aseptic condition, PBS preheated at 37 ℃ is washed for 1 time, then the liquid is sucked off by the Pasteur suction pipe, sugar-free DMEM culture solution preheated at 37 ℃ is added, a hole plate cover is opened and is placed into an oxygen deficiency box sterilized under high pressure, 99.9 percent high-purity nitrogen gas uniformly flows into small holes in the top of the box, the flow rate is kept at 1L/min, the oxygen deficiency box is stopped and quickly sealed after 5min, and the oxygen deficiency box is transferred to a CO-free oxygen deficiency box ₂ The culture is continued in an incubator at 37 ℃, and the process is called sugar-deficient and anoxic treatment.

(2) According to the experimental design, after different time points of hypoxia, the cells are taken out from the closed hypoxia chamber, and the sugar-free DMEM medium is changed into the normal DMEM medium (containing FBS, the volume fraction of FBS in the DMEM medium is 10%) by using a Pasteur pipette under the sterile condition, the temperature is 37 ℃, and the CO is 5% ₂ The incubator of (2) was continued for 24 hours. And finishing the preparation of the OGD/R model of the aged SH-SY5Y cells.

1.4.3CCK8 detection of cellular Activity

SH-SY5Y neuroblastoma cells in good growth state are taken, and the cells are inoculated into a 96-well culture plate once at the same density one day before D-gal administration (cell amount is 10) ⁴ Per well), 5% CO ₂ 37 ℃ incubator overnight, 96 hole plate periphery round add distilled water to relieve evaporation. Adding D-gal with the concentrations of 25mM, 50mM, 100mM, 200mM and 400mM and a Control group without drug intervention in the next day, placing the culture box for continuous culture for 48 hours, sucking out the original culture medium by a Pasteur pipette according to the instruction of CCK8 reagent, washing the culture medium for 1 time by PBS preheated at 37 ℃, adding 100 mu L of culture medium containing DMEM CCK8 solution (CCK 8 solution 10 mu L + sugar-free culture medium 90 mu L), and avoiding light in the operation process. Placing the mixture into an incubator at 37 ℃ for continuous culture for 1h, measuring the absorbance OD value of each hole at the wavelength of 450nm of an enzyme-labeling instrument, and repeating the experiment for 3 times.

1.4.4 cell senescence beta-galactosidase staining

After SH-SY5Y cells with good growth state are treated according to the 1.4.2 experimental steps, the original culture medium is sucked and discarded, PBS is washed for 1 time, 1mL of beta-galactosidase staining fixing solution is added, the fixation is carried out for 15min at room temperature, the fixing solution is sucked and discarded, PBS is washed for 3 times, and each hole is added withAdding 1mL of dyeing working solution, wrapping 6-well plate with tinfoil to keep out of the sun, and adding CO-free solution ₂ Incubated overnight in an incubator at 37 ℃ and observed under a normal optical microscope.

1.4.5 cell proliferation assay

The experiment adopts BeyoClick ^TM Edu-488 cell proliferation assay kit (Beyoclick) ^TM Edu Cell promotion Kit with Alexa Fluor 488), the experimental principle is based on the incorporation of thymidine (thymidine) analogue Edu (5-ethyl-2' -deoxyuridine) in the DNA synthesis process, and the Edu is marked by the Alexa Fluor 488 through the subsequent Click reaction (Click reaction), so that the Kit has the characteristics of simplicity, rapidness and high sensitivity. SH-SY5Y cells with good growth state are treated according to the 1.4.2 experimental steps, added with Edu working solution preheated at 37 ℃ and continuously incubated for 6 hours; completely sucking the culture solution, adding 1mL of stationary liquid into each hole, fixing for 15min, and removing the stationary liquid; washing with 1mL PBS for 3 times, adding 1mL permeation liquid chamber, and incubating for 15min; after removing the permeation solution, washing with 1mL PBS for 2 times, adding 0.5mL Click reaction solution, gently shaking the culture plate to ensure that the reaction mixture uniformly covers the sample, and incubating at room temperature for 30min; the Click reaction solution was aspirated and washed 3 times with PBS, 1mL of Hoechst33342 solution was added to each well, and washed 3 times with PBS, and immediately observed under a fluorescence microscope and photographed. Wherein the nucleus stained Hoechst is blue fluorescence and the proliferating cells are bright red fluorescence.

1.4.6 flow cytometry detection of cell cycle changes

After processing SH-SY5Y cells in good growth state according to 1.4.2 experimental procedures, the culture solution is aspirated, the culture well is washed with 500. Mu.L of PBS solution and aspirated and added to a 1.5mL centrifuge tube. The cells were digested with 500. Mu.L of 0.25wt% trypsin EDTA solution and collected in a 1.5mL centrifuge tube, centrifuged at 1000g for 3min, and the supernatant was aspirated off while the broth was retained to avoid aspiration of the cells. 1mL of ice-cold PBS buffer was added to resuspend the cells and re-centrifuge. Adding 500 mu L of ethanol water solution with volume fraction of 70 percent precooled on ice, and gently blowing and mixing the mixture until no cells agglomerate. Cells were fixed at 4 ℃ for more than 4h. Cells were pelleted by centrifugation at 1000g for 3 min. 1mL of ice-cold PBS buffer was added to resuspend the cells and re-centrifuge. After centrifugation, the resuspension operation was repeated once. The bottom of the centrifugal tube is flicked slightly to disperse the cells and avoid the cells from agglomerating. 0.5mL of propidium iodide staining solution was added to each tube of cell samples, the cells were gently resuspended and mixed well, and incubated at 37 ℃ in the dark for 30min. The stained liquid was filtered using a 5mL cell sieve and the filtrate was collected. The red fluorescence signal (488 nm excitation wavelength) was detected by flow cytometry and the light scattering data was recorded. Cell cycle analysis was performed using flowjo software and plotted using GraphPad Prism8 software.

1.4.7CCK-8 method for determining the survival Rate of senescent cells at different hypoxia times

After SH-SY5Y cells with good growth state are treated according to the 1.4.2 experimental steps, a 96-well plate is inclined, the culture medium is carefully removed by suction, 100 mu L PBS is added into each well to clean the cells, the PBS is removed by suction, sugar-free DMEM preheated at 37 ℃ is added, the cells are subjected to anoxic and sugar-deficient treatment according to 1.4.2.2, and the sugar-deficient time is set to be 0.5h, 1h, 1.5h and 2h. Thereafter, the sugar-free DMEM was replaced with complete medium, 37 ℃ and 5% CO ₂ The incubator was continued for 24h. The medium was aspirated, PBS preheated at 37 ℃ was added to each well and washed 1 time, and 100. Mu.L of medium containing CCK8 solution (10. Mu.L of CCK8 solution + 90. Mu.L of sugar-free DMEM medium) was added, and the procedure was performed in the absence of light. Placing the mixture into an incubator at 37 ℃ for continuous culture for 1h, measuring the absorbance OD value of each hole at the wavelength of 450nm of an enzyme-labeling instrument, and repeating the experiment for 3 times.

1.5 statistical analysis

The experimental data are expressed by mean + standard deviation, the statistical analysis and the mapping are carried out by using Graphpad prism8.0 software, and the measurement data are expressed by mean +/-standard error. Two groups of mean values are compared by T test of independent samples, more than 3 groups of mean values are compared by adopting one-way ANOVA (one-way ANOVA) analysis, when the data variance is uniform, two groups of mean values are compared by selecting LSD test, and when the data variance is not uniform, a Dunnett's T3 method is selected. P <0.05 was considered statistically significant.

2 results of the experiment

2.1 Observation of morphological changes of cells under different D-gal concentrations under microscope

Taking SH-SY5Y neuroblastoma cells with good growth state, and inoculating the cells into 6-hole culture at the same density once one day before D-galIn the culture plate (cell amount 10) ⁵ Per well), 5% CO ₂ And (2) overnight in an incubator at 37 ℃, adding 1mL of D-gal complete culture medium containing 25mM, 50mM, 100mM, 200mM and 400mM of concentration when the adherence fusion degree of the cells reaches more than 80%, additionally arranging a Control group without drug intervention, placing the incubator for continuous culture for 48h, observing the cell morphology under an inverted phase contrast microscope as shown in figure 1, wherein along with the increase of the concentration of the D-gal, the number of adherent cells is reduced, some cells still have swelling and flat shapes, the arrangement of the cells is loose, the morphological variation, the cell bodies become small and round, the refractivity is enhanced, even the connection between cells disappears, and prompting that the influence of the concentration of the D-gal on the cells is small within a certain range, but the concentration is too large, so that the D-gal has great toxicity on the cells.

2.2CCK8 reagent for detecting cell activity

In order to screen the appropriate attenuation concentration of D-gal, CCK8 kit was selected to detect the change in cell viability after 48h of action time under different D-gal concentration gradients, and the results are shown in FIG. 2. The experiment shows that: under the action of D-gal with different concentrations, the cell survival rate gradually decreased, wherein the cell survival rate was 25mM (76.65 + -7)%, 50mM (63.45 + -9)%, 100mM (59.3 + -11)%, 200mM (48.2 + -11)%, and 400mM (30.6 + -6)%. Based on the IC50, a D-gal action concentration of 100mM was chosen.

2.3 cell senescence beta-galactosidase staining

To detect cellular senescence 48h after D-gal induction, cells were stained with the β -galactosidase kit, which is the most commonly used marker of cellular senescence. It can catalyze substrate X-Gal to generate dark blue product, and SA-beta-Gal staining method is one of the most effective detection methods for cell senescence and is also the gold standard for detecting senescence. Blue-stained cells under an inverted microscope are senescent cells, while unstained are normal cells. The experimental results are shown in fig. 3, the staining positive rate of beta-galactosidase is obviously increased after the induction of D-gal, and the staining positive rate of beta-galactosidase is increased along with the increase of the concentration, the staining positive rate of blank control group is (5.47% +/-1.09)%, the staining positive rate of 25mM D-gal is (7.11 + -1.08)%, the staining positive rate of 50mM is (7.74 + -3.35)%, the staining positive rate of 100mM is (13.9 + -4.65%), the staining positive rate of 200mM is (19.88 + 1.453)%, and the staining positive rate of 400mM is (56.43 + 11.43)%. Notably, the blue-staining positive rate was significantly reduced in the 100mM group, with statistical differences occurring (P < 0.05). In combination with cell viability and SA- β -gal staining results, 100mM was chosen as the final molding concentration.

2.4 cell proliferation assay

An Edu cell proliferation experiment is adopted to evaluate the cell proliferation condition, in the DNA synthesis process, edu replaces thymidine to be doped into new DNA, edu ethynyl and a fluorescent-labeled small-molecule azide probe are catalyzed by monovalent copper ions to generate covalent reaction to form a stable triazole ring, the proliferating cells show red fluorescence under a microscope, and cell nuclei show blue. Data were processed in accordance with the Control group, and as a result, as shown in FIG. 4, the proliferation rate in the 25mM group was (94.89. + -. 9.48)%, the proliferation rate in the 50mM group was (80.8. + -. 18.67)%, the proliferation rate in the 100mM group was (72.24. + -. 22.81%), the proliferation rate in the 200mM group was (64.97. + -. 3.779)%, and the staining positive rate in the 400mM group was (38.37. + -. 15.81)%. It can be seen that the cell proliferation rate decreased with increasing D-gal concentration, and statistical differences began to appear at 100mM.

2.5 changes in the cell cycle

One common feature of senescent cells is irreversible cell cycle arrest, where the S phase is the DNA synthesis phase. Cell cycle assays were performed on cells treated for 48 hours at different concentrations, and the results (FIG. 5) showed that the S-phase cell fraction was different among the groups, including Control group (28.63. + -. 0.41)%, 50mM group (34.33. + -. 3.6)%, 100mM group (26.8. + -. 0.55%), 200mM group (25. + -. 0.79)%, and 400mM group (19.13. + -. 9.9)%. It follows that 100mM caused S phase cell cycle arrest and was statistically significant (P < 0.05). The G0/G1 was analyzed by Graphpad prism8.0 statistical software, the proportion of cells increased gradually with increasing concentration, and particularly the percentage of cells in the G0/G1 phase (57.033 + -0.153)% in the 100mM group cell cycle was significantly higher than that in the Control group (49.467 + -2.4)%, and was statistically significant (P < 0.05). Meanwhile, G2/M is analyzed by utilizing Graphpad prism8.0 statistical software, cells in the G2/M phase are gradually reduced, the percentage (12.96 +/-0.35)% of cells in the G2 phase in the cell cycle of a 100mM group is obviously lower than that of cells in a Control group (19.57 +/-0.7)%, and the statistical significance is achieved (P < 0.05).

2.6 detection of cell viability at different hypoxia-hypoglycaemia times

SH-SY5Y cells which are promoted to age and have good growth states are inoculated in a 96-well plate, 6 multiple wells are arranged in each group, and cell survival rate detection is carried out after hypoxia for 0.5h, 1h, 1.5h and 2h and reoxygenation for 24h according to a modeling experiment operation method of 1.4.2.2. The results of cell viability at different hypoxic time points calculated according to the CCK8 cell activity assay instructions are shown in fig. 6, where: the cell survival rate of 0.5h hypoxia is (94.1 +/-2.8)%, the cell survival rate of 1h hypoxia is (53 +/-1.6)%, the cell survival rate of 1.5h is (41.8% +/-4.5)%, and the cell survival rate of 2h is (23.5 +/-1.5)%. Compared with the Control group, the survival rate is gradually reduced along with the prolonging of the hypoxia time, and the survival rate has statistical significance, and the time period of about 50 percent of the cell survival rate is generally considered as the optimal time of the drug treatment, so that hypoxia and glucose deprivation are selected as follow-up research.

D-gal-induced senescence models are commonly used in senescence studies. When the concentration of D-gal in cells is too high to be catalyzed and metabolized, resulting in cellular penetration, swelling and high ROS, changes beyond the scavenging capacity of the oxidative defense system can lead to metabolic disturbances and ultimately to aging. Because the invention needs to perform OGD/R secondary striking on the cells subsequently, the proper concentration needs to be selected according to the experiment requirement, and the conventional experiment concentration cannot be referred. The invention discovers that the cell activity is reduced along with the increase of the D-gal concentration, particularly when the D-gal concentration is 200mM or more, the cell activity is rapidly reduced, obvious cell nucleus reduction, cell arrangement sparseness, cell rounding or cell wall separation and even cell death occur. The present invention uses D-gal at 100mM corresponding to approximately 50% cell viability as the subsequent cell senescence concentration.

During senescence, dividing cells gradually lose proliferative capacity, with unique morphological changes and altered expression of senescence-specific markers. The recognition or quantification of cellular senescence is mainly reflected by changes in cell morphology, increased SA- β -Gal activity, growth inhibition, cell cycle arrest, and the like. Morphological features of senescent cells are generally characterized by increased volume, increased nuclear size and irregular shape. The observation is convenient and is used as an inspection index. The experiment of the invention finds that the cell number is gradually reduced along with the increase of the concentration of D-gal for 48 hours, and particularly, the cell morphology is obviously distributed and scattered when the concentration exceeds 100mM, and even cell death occurs. Senescent cells express highly active beta-galactosidase (SA-beta-gal) at pH6.0 and appear as blue-stained precipitates, so the senescent trend can be assessed by analyzing the percentage of positive staining of the cells. In the present invention, the staining rate of SA- β -Gal was significantly increased and dose-dependent by treating 48h cells with different doses of D-Gal (25, 50, 100, 200, 400) mM. Although SA- β -Gal activity has been widely used as a classical marker of senescence, studies have shown that its activity may also be elevated under conditions independent of senescence. Thus, the present invention further analyzed whether other senescence markers could be observed in D-gal induced SH-SY5Y cells. The cell cycle is the process from the completion of the last division to the completion of the next division of a continuously dividing cell and is considered to be an important mechanism for controlling normal cell proliferation and biological senescence. Senescent cells stagnate at G1/G0 stage and fail to enter S stage smoothly, which is manifested as decreased S-stage cells. The cell cycle results in the present invention show that cells are arrested more and more in the G1 phase with increasing concentrations of D-gal. At 100mM S phase, a turn occurred, indicating that the number of cells entering the replication phase was greatly reduced and had statistical differences. In the Edu experiment, the proportion of the proliferated cells is obviously seen through the proliferated cells/cell nucleuses, and the statistical difference is found at the beginning of 100mM, so that the cell cycle is obviously inhibited, and the result is compounded with the cell cycle result measured by a flow cytometer. Therefore, the invention adopts 100mM D-gal to act for 48h as subsequent experimental study.

Compared with animal experiments, the cells have the advantages of uniform research sample, contribution to observing single factor, artificial controllable research conditions and the like in the culture process. At present, the establishment of the in vitro cell sugar and sugar deficiency model is mainly divided into two types of physical and chemical oxygen deficiency, wherein the chemical oxygen deficiency is that certain chemical substances, such as sodium hydrosulfite and cyanide, are added into a cell culture solution to damage the oxygen utilization capability of cells or exhaust the culture solutionOxygen causes the cells to lack oxygen. Chemical hypoxia has the defects of damage to cells, difficult control of experimental operation and the like. The physical hypoxia model is a model simulating hypoxia experiment by reducing the proportion of oxygen in a culture environment by using a mixed gas culture method, and generally comprises a liquid level closed model, a self-made simple sealed model and a three-gas culture box model. There are a wide range of techniques currently used to create hypoxic conditions, including anaerobic incubators, anoxic chamber glove boxes, or home-made anoxic devices. Nitrogen is an inert gas, and a nitrogen/carbon dioxide mixed gas is usually used for regulating O ₂ And (4) horizontal. According to the invention, the sugar-free culture solution is added into the culture dish, the culture dish is placed into the anoxic box, nitrogen is filled as fast as possible, and the time and the airflow magnitude of each perfusion are kept consistent. When the whole anoxic box is filled with nitrogen, stopping filling the nitrogen, sealing and placing the anoxic box into the constant-temperature incubator to achieve the effect of cell OGD. In the experiment, OGD damage and reoxygenation are two stages, the OGD is firstly given to cells from a cell layer to simulate the state of nerve damage after cerebral ischemia and hypoxia, the reoxygenation is a process simulating reperfusion, on one hand, the changes of the cells in quantity, structure and characteristic phenotype can be observed morphologically, and on the other hand, the pathological changes and the drug action mechanism of the cerebral ischemia reperfusion damage can be researched biochemically and on the protein level. OGD/R duration and intensity are key parameters for the evaluation of neuroprotective potential drugs. Cell death rates between 40% and 60% are the best choice for the study of neuroprotective drugs, since significant neuroprotection is difficult to achieve at higher cell death values.

Human studies have shown a 25% -40% reduction in cerebral blood flow and oxygen consumption between the ages of 30 and 89 years. The neurovascular units in the elderly brain are particularly vulnerable to hypoxia, where neuronal cells are the most fragile and die rapidly. In the experiment, the aged cells induced by D-gal also show the same characteristics, and the cell viability is greatly reduced after OGD/R. In CIRI, the brain undergoes a series of "ischemic cascades" events, and the reduction of blood flow to the brain reduces the availability of nutrients, oxygen and energy, and the viability of nerve cells is lost. The degree of cell damage depends on the duration and severity of ischemia and hypoxia, so that the OGD model of aging cells needs to be optimized, and the SH-SY5Y cell activity is gradually reduced along with the prolongation of the OGD time. Referring to an OGD/R model of the SH-SY5Y cells, the OGD is subjected to unified reoxygenation for 24h, and experiments show that the OGD acts on the SH-SY5Y cells for 1h for reoxygenation for 24h, and the cell survival rate is (53 +/-1.6)%. In the experiment of the invention, the cell activity of 2h under hypoxia is only 23.5%. It can be seen that aged cells are more significantly damaged after OGD/R, therefore, hypoxia for 1h was used for subsequent experimental study.

In conclusion, the invention firstly determines the concentration and time of aging induction of SH-SY5Y cells by D-Gal through a CCK8 method, verifies the aging of the cells through cell activity, cell cycle, cell proliferation detection and SA-beta-Gal staining methods, establishes an OGD/R model by using the SH-SY5Y cells induced by D-Gal on the basis, determines the OGD and reoxygenation time of the aged cells through the CCK8 method, and finally determines the OGD 1h and reoxygenation 24h modes after 48h of aging induced by 100mM D-Gal in the construction of the OGD/R model of the aged SH-SY5Y cells.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A construction method of an aged SH-SY5Y cell oxygen sugar deprivation reperfusion model is characterized by comprising the following steps:

(1) Using D-galactose to make SH-SY5Y cells senesce, and constructing the senescing SH-SY5Y cells;

2. The method of constructing according to claim 1, wherein the D-galactose is used at a concentration of 100mM in step (1).

3. The method according to claim 2, wherein the D-galactose has an aging time of 48 hours.

4. The construction method according to claim 1, wherein in the step (2), the sugar-deficient anoxic treatment is specifically: and (2) placing the aged SH-SY5Y cells obtained in the step (1) into a sugar-free DMEM culture solution, introducing 99.9% high-purity nitrogen, and then sealing and culturing.

5. The method according to claim 4, wherein the flow rate of the high-purity nitrogen gas of 99.9% is 1L/min and the flow time is 5min.

6. The method of claim 4, wherein the sealed culture time is 1h.

7. The constructing method according to claim 1, wherein in the step (2), the time of the reoxygenation treatment is 24 hours.

8. An aged SH-SY5Y cell oxygen sugar deprivation reperfusion model constructed by the construction method according to any one of claims 1 to 7.

9. Use of the aged SH-SY5Y cell oxygen deprivation reperfusion model of claim 8 in screening for neuroprotective drugs.