CN116875530A - NAME model simulating human brain immunity microenvironment and construction method thereof - Google Patents
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Abstract
The invention discloses a NAME model for simulating human brain immunity microenvironment and a construction method thereof, wherein the NAME model comprises an upper layer cell, a middle layer cell and a lower layer cell which are sleeved in sequence; the upper layer chamber is inoculated with human brain microvascular endothelial cells; the middle layer cell is inoculated with human astrocytes and human microglia; the lower chamber is seeded with human hippocampal neuronal cells. The invention establishes a model for highly simulating human brain microenvironment for the first time, and carries out oxygen glucose deprivation treatment on the NAME model to obtain an OGD-NAME brain model in order to obtain an in vitro model consistent with the pathological phenotype of ischemic cerebral apoplexy. The model of the invention can screen effective cells, vesicles and traditional Chinese medicine preparations for repairing in-vitro nerve injury according to the cell cycle, apoptosis condition and nerve microenvironment regulation condition of human hippocampus neurons, and evaluate the safety and effectiveness of various preparations.
Description
Technical Field
The invention belongs to the technical field of neurobiology, and particularly relates to a NAME model for simulating human brain immunity microenvironment and a construction method thereof.
Background
Ischemic stroke is mainly caused by nerve injury caused by ischemia reperfusion injury, and has the dual effects of oxidative stress and inflammatory response, so that irreversible damage is often caused to the nervous system, and the prognosis of the disease is extremely difficult. Previous researches show that the reactivity of microglial cells, astrocytes and the like in the process of ischemic cerebral apoplexy is activated, the permeability of the blood brain barrier is improved, the steady state of the neuroimmune microenvironment is unbalanced, the damage and necrosis of neurons are finally caused, and the functions of human body such as movement, feeling and the like are affected.
At present, biological experiments of single cells cannot completely simulate the in-vivo neuroimmune microenvironment state, the damage degree of neurons is estimated, and animal models have the problems of species barrier and the like. On the basis, an OGD oxygen glucose deprivation nerve microenvironment is constructed and used for simulating nerve injury in hypoxia-ischemic encephalopathy. According to the effect of different types of preparations on the model after treatment, the regulation effect of the cell/vesicle preparation, the traditional Chinese medicine preparation and the like on nerve cells and the regulation and control on immune microenvironment are evaluated, and the effects of the different types of preparations on the model under the condition of injury are evaluated.
Disclosure of Invention
The invention aims to provide a NAME model simulating human brain immunity microenvironment and a construction method thereof.
A NAME model for simulating human brain immunity microenvironment comprises an upper layer cell, a middle layer cell and a lower layer cell which are sequentially sleeved; the upper layer chamber is inoculated with human brain microvascular endothelial cells; the middle layer cell is inoculated with astrocytes and microglia; the lower chamber is seeded with human hippocampal neuronal cells.
The number ratio of the human brain microvascular endothelial cells, the astrocytes, the microglial cells and the human sea Ma Shenjing cells is 2:2.5:1:4.
the culture system in the NAME model cell is as follows: dmem+10% fbs+1% diabody (0.5% penicillin, 0.5% streptomycin).
After the NAME model is stable, the culture medium is replaced by EBSS, the NAME model is placed in an anoxic incubator for 10-14 hours, the NAME model is subjected to integral oxygen glucose deprivation, and the NAME model is placed in a conventional incubator for 20-28 hours after being taken out, so that the OGD-NAME brain model is obtained.
The culture condition in the anoxic incubator is 37 ℃ and 95 percent of N 2 ，5％CO 2 。
The construction method of the NAME model simulating the human brain immune microenvironment is carried out according to the following steps:
(1) Human brain microvascular endothelial cells are connected into an upper layer Transwell cell and placed into a conventional incubator for culture overnight;
(2) Inoculating astrocytes and microglial cells into a middle-layer Transwell chamber, inoculating human hippocampal neuron cells into a lower-layer chamber, and culturing in a conventional incubator overnight;
(3) Attaching cells cultured overnight to the surface of a Transwell cell membrane, replacing a culture medium with EBSS, placing the model in an anoxic incubator for 10-14h, carrying out integral oxygen glucose deprivation on a NAME model, taking out the NAME model, placing the NAME model in a conventional incubator for culturing for 20-28h to obtain an OGD-NAME brain model, and setting an unoxyglucose deprived group as a control group.
The conditions for culturing in the conventional incubator are as follows: 37 ℃,95% O 2 ，5％CO 2 。
The culture system in the step (1) and the step (2) is as follows: dmem+10% fbs+1% diabody (0.5% penicillin, 0.5% streptomycin).
The culture condition in the anoxic incubator is 37 ℃ and 95 percent of N 2 ，5％CO 2 。
The invention has the beneficial effects that: the invention establishes a model which is composed of human nerve cells (N), astrocytes (A), microglial cells (M) and vascular endothelial cells (E) and highly simulates the human brain microenvironment for the first time. To obtain an in vitro model consistent with the pathological basis and phenotype of ischemic stroke, the NAME model was subjected to oxygen glucose deprivation treatment to obtain an OGD-NAME brain model. The model of the invention can screen effective cells, vesicles and effective concentrations of traditional Chinese medicine preparations for repairing in vitro nerve injury according to cell cycle and apoptosis conditions of neurons and nerve microenvironment regulation conditions, and evaluate the safety and effectiveness of various preparations.
Drawings
Figure 1 is a schematic representation of an in vitro NAME model.
FIG. 2 is a schematic diagram of the morphology of a hippocampal neuron in the cell;
in the figure, control, i.e., morphological manifestations of subventricular hippocampal neurons (4 fold mirror, 40 fold mirror) without oxygen deprivation NAME model; the morphology of the lower cell hippocampal neurons after the OGD group is deprived of oxygen sugar (4-fold mirror, 40-fold mirror); the hUMSC group is the number and morphology of hippocampal neurons in the lower chamber after NAME model is treated by oxygen glucose deprivation and umbilical cord mesenchymal stem cells.
FIG. 3 shows the immunofluorescent staining of the various layers of the ventricular nerve cells (20X);
in the figure, A is upper-layer small-chamber human brain microvascular endothelial cells, ZO-1 is dyed by green fluorescence, and DAPI is blue; b is basal hippocampal neuronal cells, immunofluorescent staining NeuN; c is middle-layer cell astrocyte, and the GFAP is immunofluorescence stained; d is middle microglial cells, and IBA1 is stained by immunofluorescence.
FIG. 4 shows inflammatory factor mRNA expression by astrocytes, microglia, and neuronal cells.
FIG. 5 shows the results of the active oxygen assay.
FIG. 6 shows the lower hippocampal neuronal cell cycle of NAME model.
FIG. 7 shows the results of apoptosis of hippocampal neurons in the lower layer of NAME model.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1 construction of an in vitro NAME brain model and an OGD-NAME brain model
First, human brain microvascular endothelial cells (Vascular endothelial cell-HCMEC/D3, 1X 10) 5 ) Is placed in a 12-well plate Transwell chamber and placed in an incubator. The proportion of glial cells to neurons is 1.5:1, and astrocytes are the glial cells with the highest content and account for 40 percent of glial cells; microglial cells account for about 15% of total glial cells, and human brain microvascular endothelial cells (Vascular endothelial cell-HCMEC/D3,1×10) 5 ) In the middle layer 6-well Transwell chamber, astrocytes (Astrocyte-SVG P12, 1.2X10) 5 ) And Microglia (Microglia-HMC 3, 0.45X10) 5 ) The method comprises the steps of carrying out a first treatment on the surface of the Human hippocampal neurons (Neuron-HPPNCs, 2×10) were seeded in the lower chamber 5 ) According to the number of central nervous system nerve cells, construct 2:2.5:1:4 (FIG. 1), co-culture was performed using a DMEM+10% FBS+1% diabody culture system.
After the cell model was stabilized, cells were attached to the surface of a Transwell cell membrane and the cell model was placed in an anoxic incubator for 42h (37 ℃ C., 95% N) with the exchange medium being Earle's Balanced Salt Solution (EBSS) 2 ，5％CO 2 ) The NAME model was subjected to total oxygen glucose deprivation (oxygen and glucose deprivation, OGD), removed and placed in a conventional incubator for cultivation for 24 hours, and a group not subjected to oxygen glucose deprivation was set as a control group.
Example 2NAME brain model and detection index of OGD-NAME brain model
After NAME model stabilization, cells were attached to the cell surface, oxygen Glucose Deprivation (OGD) was performed for 42h, reoxygenation was performed for 24h, and 5 th generation umbilical cord mesenchymal stem cells (hUMSC) were added for treatment during reoxygenation. And the group not subjected to the oxygen glucose deprivation treatment was used as a control group.
The underlying small hippocampal neuronal cells in the NAME model were observed using a (Leica, DM 500) microscope. The results are shown in FIG. 2, where the number of lower-level ventricular hippocampal neurons is significantly reduced after oxygen glucose deprivation, and tight junctions are loose.
Neural cell morphology detection: immunofluorescence method is adopted to detect ZO-1 expression of upper layer human brain microvascular endothelial cells; GFAP markers astrocytes; IBA1 markers microglial cells; NEUN marks hippocampal neuronal cells. The results are shown in FIG. 3, stained with specific antibodies, and confirmed to be cells that meet the expression of the specific markers.
Detecting the expression conditions of inflammatory factors IL-1 beta, IL-6 and TNF-alpha of two glial cells and neurons and the expression condition of HIF-1 alpha (hypoxia inducible factor) by adopting a q-PCR method, respectively carrying out OGD treatment on astrocytes and microglial cells in a NAME model, and researching to find that the hypoxia inducible factor HIF-1 alpha is highly expressed (p < 0.01) and the inflammatory cell factor is highly expressed (p < 0.05); quantitative PCR detection of mRNA was performed on the lower hippocampal neurons, and hypoxia inducible factor hif-1α was highly expressed (p < 0.01), and the inflammatory factor expression level was elevated (p < 0.01) (FIG. 4).
As a result of detecting ROS reactive oxygen species of lower hippocampal neurons by flow cytometry, as shown in fig. 5, the mean fluorescence intensity of ROS of lower hippocampal neurons is significantly improved by about 3.5 times (p < 0.01) compared with OGD group, however, after intervention with umbilical cord mesenchymal stem cells (hmscs), the fluorescence intensity of ROS of lower hippocampal neurons is significantly reduced and oxidative stress damage is improved (p < 0.05).
Cell cycle results were obtained by flow cytometry using PI cell cycle detection kit, as shown in fig. 6, after addition of hmscs in the upper chamber, the cell cycle of the lower hippocampal neurons was significantly changed, the cell cycle of the lower hippocampal neurons was significantly decreased (p < 0.0001) in G0/G1 phase, significantly increased (p < 0.001) in S phase, and simultaneously significantly increased (p < 0.001) in G2 phase and M phase, indicating that the lower hippocampal neurons entered replication and proliferation phase in the model.
The apoptosis results were obtained by flow cytometry using the AV/PI apoptosis detection kit, and the results are shown in fig. 7, wherein after the hUMSC was added into the upper chamber, the apoptosis detection of the lower hippocampal neurons was also significantly changed, and after the OGD treatment, the early apoptosis and the late apoptosis were both significantly improved (p < 0.001), respectively, in the NAME model, whereas after the hUMSC treatment, the proportion of early and late apoptosis was significantly reduced (p < 0.001) in the hippocampal neurons.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (9)
1. The NAME model for simulating the human brain immunity microenvironment is characterized by comprising an upper layer cell, a middle layer cell and a lower layer cell which are sequentially sleeved; the upper layer chamber is inoculated with human brain microvascular endothelial cells; the middle layer cell is inoculated with astrocytes and microglia; the lower chamber is seeded with human hippocampal neuronal cells.
2. The simulated human brain immune microenvironment NAME model of claim 1, wherein the ratio of the number of human brain microvascular endothelial cells, human astrocytes, microglial cells, human sea Ma Shenjing cells is 2:2.5:1:4.
3. the simulated human brain immune microenvironment NAME model of claim 1, wherein the culture system within the NAME model cell is: dmem+10% fbs+1% diabody.
4. The simulated human brain immune microenvironment NAME model of claim 1, wherein after the NAME model is stable, the medium is replaced by EBSS, the NAME model is placed in an anoxic incubator for 36-48 hours, the NAME model is subjected to overall oxygen glucose deprivation, and the NAME model is placed in a conventional incubator for 24 hours after being taken out, so as to obtain the OGD-NAME brain model.
5. The simulated human brain immune micro-environment NAME model of claim 4, wherein said anoxic incubator has a culture condition of 37 ℃ and 95%N 2 ，5％CO 2 。
6. The method for constructing a NAME model simulating a human brain immune microenvironment according to claim 1, which is characterized by comprising the following steps:
(1) The human brain microvascular endothelial cells are grafted into an upper layer Transwell cell and placed into a conventional incubator until the fusion rate reaches more than 90% in advance for 72 hours, so that tight connection is formed;
(2) Inoculating astrocytes and microglial cells in the middle-layer Transwell chamber in proportion 24h in advance, inoculating human hippocampal neuron cells in the lower-layer chamber, and culturing overnight in a conventional incubator;
(3) And replacing the culture medium in the model with EBSS, placing the culture medium in an anoxic incubator for 36-48 hours, carrying out overall oxygen glucose deprivation on the NAME model, replacing the culture medium of DMEM+10% FBS+1% double antibody after the NAME model is taken out, placing the NAME model in a conventional incubator for culturing for 24 hours, obtaining an OGD-NAME brain model, and setting an unoxyglucose deprived group as a control group.
7. The method for constructing a NAME model in a human brain immune microenvironment according to claim 6, wherein the conditions for culturing in the conventional incubator are as follows: 37 ℃,95% O 2 ，5％CO 2 。
8. The method for constructing a NAME model in a human brain immune microenvironment according to claim 6, wherein the culture system in the step (1) and the culture system in the step (2) are as follows: dmem+10% fbs+1% diabody.
9. The method for constructing a NAME model in a human brain immune microenvironment according to claim 6, wherein the culture condition in the anoxic incubator is 37 ℃ and 95% N 2 ，5％CO 2 。
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|---|---|---|---|---|
| CN117535242A (en) * | 2023-11-09 | 2024-02-09 | 威海海高园明睿智琳生物科技有限公司 | Biological ink, 3D printing Alzheimer's disease brain-like model, method and application |
| CN117535242B (en) * | 2023-11-09 | 2024-04-09 | 威海明睿智琳生物科技有限公司 | Biological ink, 3D printing Alzheimer's disease brain-like model, method and application |
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