CN116286649A - OGD model and its application in identifying the effectiveness of neural stem cells - Google Patents

Abstract

The invention discloses an OGD model, which is constructed by the following steps: stimulating PC12 cells with murine nerve growth factor NGF, inducing them into sympathogenic neuron-like cells; the OGD model was then constructed using glucose-deprivation with sugarless DMEM to simulate neuronal ischemic injury in vitro. The use of the OGD model to identify the effectiveness of neural stem cells for transplantation therapy. The OGD model constructed by the invention can identify the effectiveness and the neuroprotection function of the neural stem cell transplantation treatment, the OGD model and NSC are co-cultured, and the action mechanism of the NSC on the apoptotic PC12 cells are analyzed by observing the morphology of the PC12 cells and detecting the survival rate of the PC12 cells, so that corresponding experimental and theoretical basis is further provided for treating the neural system diseases by NSC transplantation, and effective basic basis is provided for clinically researching the neural stem cell transplantation treatment.

Description

OGD model and its application in identifying the effectiveness of neural stem cells

technical field

The invention relates to an OGD model for simulating neuron damage and its application in identifying the effectiveness of neural stem cells for transplantation treatment, belonging to the technical field of neural stem cells.

Background technique

Brain diseases, including brain tumors, neurodegenerative diseases, cerebrovascular diseases, and traumatic brain injuries, are major diseases affecting human health for which there are currently no effective treatments. Due to the low regeneration ability of neurons, the insufficient secretion of neurotrophic factors, and the aggravation of ischemia and hypoxia after nerve injury, irreversible functional neuron loss and nerve tissue damage will occur, and the central nervous system is difficult to repair and regenerate after injury. Neural stem cells are pluripotent stem cells that only exist in the central nervous system, they have good self-renewal potential and the ability to differentiate into neurons, astrocytes and oligodendrocytes, and improve the cellular microenvironment. Based on the regenerative ability of neural stem cells, neural stem cell transplantation methods have been used to treat various neurodegenerative diseases.

In order to study the effectiveness of treatments for brain diseases, it is necessary to construct lesions of the nervous system. The in vivo simulation of nervous system injury is subject to various conditions, and the results are prone to deviation due to the interference of many factors, so the reliability is questioned. Primary neuron culture in vitro can avoid the interference of multiple factors and improve the reliability of the experimental results, but the low efficiency of primary culture and the natural aging of neurons can not guarantee continuous passage of neurons, which has prompted a large number of in vitro ischemia models. design. These experimental models help to elucidate damage mechanisms at the molecular, cellular, and whole organism levels. In addition, these models are used to test whether compounds that target damaged neural networks reduce morbidity and nerve cell damage.

The commonly used in vitro model for neural stem cell transplantation is the OGD model, which can be constructed by oxygen-glucose deprivation (OGD) of neurons or neuron-like cells, which can simulate neuronal ischemic injury in vitro. PC12 cells are a clonal line that has been extensively characterized in molecular, cellular, functional and stress studies, and OGD models constructed with PC12 cells have been used to test the effectiveness of many compounds in clinical therapy. PC12 cells are derived from adult rat adrenal medullary pheochromocytoma, which can differentiate into sympathetic neurons after being stimulated by mouse nerve growth factor NGF in vitro, and the differentiated PC12 cells have neuron-like physiological and biochemical aspects. It is widely used in neurophysiological and neuropharmacological research. By studying the effect of neural stem cells on OGD, we can further provide corresponding experimental and theoretical basis for NSC transplantation to treat nervous system diseases.

Contents of the invention

In view of the above prior art, the present invention provides an OGD model for simulating neuron damage and its application in identifying the effectiveness of neural stem cells for transplantation therapy.

The present invention is achieved through the following technical solutions:

An OGD model, the construction method of which is as follows: firstly, using mouse nerve growth factor NGF to stimulate PC12 cells to induce them into sympathetic neuron-like cells; then, using sugar-free DMEM to perform oxygen-glucose deprivation to construct an OGD model, simulating nerve cells in vitro Meta-ischemic injury, including the following steps:

(1) PC12 cells were seeded in a 24-well plate at 20,000 cells per well;

(2) After 24 hours, add DMEM medium containing 100ng/mL rat NGFβ to induce differentiation;

(3) Change the differentiation medium every two days, stimulate continuously for 6 days, and induce neuron-like transformation of PC12 cells;

After 6 days, under the microscope, it can be seen that the cell shape changes, the cell body becomes round, the cell process is relatively thick, and interweaves to form a network; the neuronal markers DCX and MAP2 are stained to determine whether the induced differentiation is successful;

(4) The culture medium was replaced with sugar-free DMEM medium, placed in an anoxic cell incubator, and cultured for 3 hours under the conditions of 5% CO ₂ , 1% O ₂ , 94% N ₂ , and 37°C.

Use of the OGD model to identify the effectiveness of neural stem cells for transplantation therapy. The OGD model was co-cultured with NSCs, and by studying the effect of neural stem cells on OGD cells, it will further provide corresponding experimental and theoretical basis for NSC transplantation to treat nervous system diseases.

Specifically, the method for identifying the effectiveness of neural stem cells for transplantation therapy using the above-mentioned OGD model is:

(1) Add the NSC supernatant to the OGD model, and place it in an incubator at 37°C, 5% CO ₂ and saturated humidity for 2 days;

The NSC supernatant is prepared by the following method: culture the NSC cells in DMEM/F12 medium for 3 days, centrifuge, take the supernatant, obtain it, subpackage and freeze it, and perform cell counting;

(2) After culturing for 2 days, discard the supernatant, add DMEM/F12 medium containing 10% CCK-8, and culture in a cell culture incubator for 1 hour; detect the OD value at a wavelength of 450nm, and the detection results are used for evaluation and identification Protective effect of neural stem cells against nerve injury.

The invention continuously induces rat adrenal pheochromocytoma cells for 6 days, and achieves a neuron induction success rate of more than 90%, so as to better approach the physiological structure of the nervous system. The induced rat adrenal pheochromocytoma cells were successfully induced to construct a neuron ischemic injury model through oxygen glucose deprivation/reoxygenation injury, which provides a tool for screening drugs that protect against cerebral ischemic injury. The invention co-cultivates the rat adrenal pheochromocytoma cell oxygen-glucose deprivation injury model and neural stem cells, and determines the optimal co-cultivation mode and cell dosage. The effectiveness of neural stem cell transplantation therapy can be identified by the degree of recovery of rat adrenal pheochromocytoma cells after co-culture, and the difference in the effectiveness of different batches of neural stem cells can be compared.

The OGD model constructed by the present invention can identify the effectiveness and neuroprotective function of neural stem cell transplantation. The present invention co-cultures the OGD model with NSC, and analyzes the effect of NSC on apoptosis by observing the morphology of PC12 cells and detecting the survival rate of PC12 cells. The function and mechanism of PC12 cells can further provide corresponding experimental and theoretical basis for NSC transplantation to treat nervous system diseases, and provide an effective basis for clinical research on neural stem cell transplantation.

Description of drawings

Figure 1: White light acquisition of rat adrenal pheochromocytoma cells induced and differentiated for 6 days.

Figure 2: DCX staining of rat adrenal pheochromocytoma cells after induction and differentiation for 6 days.

Figure 3: MAP2 staining of rat adrenal pheochromocytoma cells induced to differentiate for 6 days.

Figure 4: Schematic diagram of the protective effect of NSC indirect co-culture with different cell numbers and NSC supernatant on the OGD model.

Figure 5: Schematic diagram of the protective effect of NSC supernatants with different cell numbers on the OGD model.

Figure 6: Schematic diagram of the protective effect of different batches of NSC on the OGD model.

Detailed ways

The present invention will be further described below in conjunction with embodiment. However, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are conventional instruments, reagents, and materials in the prior art, and can be obtained through formal commercial channels. The experimental methods and detection methods involved in the following examples are conventional experimental methods and detection methods in the prior art unless otherwise specified.

The Chinese meanings of some English professional terms involved in the present invention:

OGD: oxygen-glucose deprivation, oxygen-glucose deprivation;

OGD/R: oxygen-glucose deprivation/Reperfusion, oxygen-glucose deprivation/reoxygenation;

PC12: adult rat adrenal medulla pheochromocytoma cells;

NGF: Nerve growth factor, mouse nerve growth factor;

MAP2: microtubule-associated protein 2, microtubule-associated protein 2;

DCX: Doublecortin, doublecortin antigen;

NSC: neural stem cell, neural stem cell.

Reagent sources: Mouse NGF 2.5S Native Protein (Thermo Fisher, 13257-019), Cellcounting kit-8 (MCE, HY-K0301), MAP2 antibody (Abcam, ab5392), DCX antibody (Santa Cruz, SC-271390), goat Anti-mouse secondary antibody-Cy3 (Jackson, 115-165-068), goat anti-chicken secondary antibody (Jackson, 103-545-155).

Experiment 1: Inducing neuron-like cells in vitro to construct an oxygen-glucose deprivation/reoxygenation (OGD/R) injury model

Proceed as follows:

(1) PC12 cells were seeded in a 24-well plate at 20,000 cells per well;

(2) After 24 hours, add DMEM medium containing 100ng/mL rat NGFβ to induce differentiation;

(3) Change the differentiation medium every two days, stimulate continuously for 6 days, and induce neuron-like transformation of PC12 cells;

After 6 days, under the microscope, it can be seen that the cell shape changes, the cell body becomes round, the cell process is relatively thick, and intertwined to form a network (as shown in Figure 1); the neuronal markers DCX and MAP2 staining (results in Figure 2, Figure 3 Shown), confirm that the induction of differentiation is successful;

(4) Replace the medium with sugar-free DMEM medium, place it in a hypoxic cell incubator, and culture it for 3 hours under the conditions of 5% CO ₂ , 1% O ₂ , 94% N ₂ , and 37°C to obtain the OGD model .

Experiment 2 OGD model co-cultured with NSC in different ways

The OGD model obtained in Experiment 1 was divided into three groups: OGD model group, NSC co-culture group, and NSC supernatant group.

The OGD model group was added with DMEM/F12 medium, and placed in an incubator at 37°C, 5% CO ₂ and saturated humidity for 2 days.

In the NSC co-culture group, 1×10 ³ , 2×10 ³ , and 3×10 ³ NSC cells were added in a transwell manner, and placed in an incubator at 37°C, 5% CO ₂ and saturated humidity for 2 days.

The supernatants corresponding to 1×10 ³ , 2×10 ³ , and 3×10 ³ NSCs were added to the NSC supernatant group respectively, and placed in an incubator at 37° C., 5% CO ₂ and saturated humidity for 2 days. The NSC supernatant is prepared by the following method: NSC cells grown for 7 days are placed in DMEM/F12 medium and cultured for 3 days, centrifuged, and the supernatant is obtained, which is obtained, subpackaged and frozen, and cell count.

After culturing for 2 days, the supernatant was discarded, and 400 μL of DMEM/F12 medium containing 10% CCK-8 was added to each well, and cultured in a normal cell culture incubator for 1 h; the OD value was detected at a wavelength of 450 nm.

The OGD model group represents the degree of OGD damage, and the NSC group has a significant difference compared with it, indicating the protective effect of NSC on nerve damage.

Comparing the co-culture group with the same number of NSC cells and the supernatant group, it was found that the protective effect of the NSC supernatant on OGD was better than that of the direct co-culture of NSC (as shown in Figure 4), so the NSC supernatant was selected as the co-culture of NSC and OGD cells. The way of cultivation.

Repeat the experiment, add 1×10 ³ , 2×10 ³ , 3×10 ³ , 5×10 ³ , 8×10 ³ , 1×10 ⁴ , 2×10 ⁴ , 5×10 ⁴ NSC corresponding to the NSC supernatant group The results are shown in Figure 5. The results show that the supernatant corresponding to 5×10 ⁴ NSCs has the best protective effect on OGD, so 5×10 ⁴ NSCs are selected when NSCs and OGD cells are co-cultured The corresponding supernatant is appropriate.

Experiment 3 Using the OGD model to detect the effectiveness of different batches of neural stem cells

Different batches of neural stem cells were used for the experiment, and the experimental method was the same as that in Experiment 2. The results are shown in Figure 6. The results show that: DSN019-1 cells have the best protective effect on nerve injury, DSN024P18-A-1, DSN024P19-A-1, etc. also have obvious neuroprotective effects, DSN024P19-B-1, DSN024P20 -A-3 etc. did not show neuroprotective effect.

The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments and not to limit the scope of the disclosure herein. Modifications obvious to those skilled in the art are intended to be within the scope of the appended claims.

Claims (5)

1. A construction method of OGD model, it is characterized in that: first, utilize mouse nerve growth factor NGF to stimulate PC12 cell, it is induced into sympathetic neuron sample cell; Then, utilize sugar-free DMEM to carry out oxygen-glucose deprivation and construct OGD model , to simulate neuronal ischemic injury in vitro, comprising the following steps: (1) PC12 cells were seeded in a 24-well plate at 20,000 cells per well; (2) After 24 hours, add DMEM medium containing 100ng/mL rat NGFβ to induce differentiation; (3) Change the differentiation medium every two days, stimulate continuously for 6 days, and induce neuron-like transformation of PC12 cells; (4) The culture medium was replaced with sugar-free DMEM medium, placed in an anoxic cell incubator, and cultured for 3 hours under the conditions of 5% CO ₂ , 1% O ₂ , 94% N ₂ , and 37°C. 2. The construction method of OGD model according to claim 1, it is characterized in that: after step (3) contact stimulation 6 days, observe cell morphology under the microscope, and carry out neuronal marker DCX and MAP2 staining, to determine whether to induce differentiation success. 3. utilize the OGD model that the construction method described in claim 1 or 2 obtains. 4. the application of the OGD model described in claim 3 in the validity of identification neural stem cell. 5. application according to claim 4, is characterized in that, utilizes the method for the validity of OGD model identification neural stem cell to be: (1) Add the NSC supernatant to the OGD model, and place it in an incubator at 37°C, 5% CO ₂ and saturated humidity for 2 days; The NSC supernatant is prepared by the following method: culture the NSC cells in DMEM/F12 medium for 3 days, centrifuge, and take the supernatant; (2) After culturing for 2 days, discard the supernatant, add DMEM/F12 medium containing 10% CCK-8, and cultivate in a cell culture incubator for 1 hour; detect the OD value at a wavelength of 450nm, and the detection results are used for evaluation and identification Protective effect of neural stem cells against nerve injury.

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