CN116875530A - NAME model simulating human brain immunity microenvironment and construction method thereof - Google Patents

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

A NAME model that simulates the human brain immune microenvironment and its construction method

Technical field

The invention belongs to the technical field of neurobiology, and specifically relates to a NAME model that simulates the human brain immune microenvironment and a construction method thereof.

Background technique

The onset of ischemic stroke is mainly due to nerve damage caused by ischemia-reperfusion injury. The dual effects of oxidative stress and inflammatory response often cause irreversible damage to the nervous system, making the prognosis of the disease extremely difficult. Previous studies have shown that during ischemic stroke, microglia, astrocytes, etc. are reactively activated, the permeability of the blood-brain barrier is increased, and the homeostasis of the neuroimmune microenvironment is imbalanced, ultimately leading to neuronal damage and necrosis. , affecting the movement, feeling and other functions of the human body.

At present, biological experiments on individual cells cannot completely simulate the state of the neuroimmune microenvironment in the body to assess the degree of neuronal damage, and animal models have problems such as species barriers. Based on this background, the inventors constructed a highly simulated human brain The "NAME" model of the nervous system immune microenvironment fully simulates the immune microenvironment of nerve cells. On this basis, an OGD oxygen-glucose deprivation neural microenvironment was constructed to simulate nerve damage in hypoxic-ischemic encephalopathy. According to the effect of different types of preparations on this model after treatment, the regulatory effects of cell/vesicle preparations, traditional Chinese medicine preparations, etc. on nerve cells and the regulation of the immune microenvironment are evaluated to evaluate the effects of different types of preparations on the treatment of injuries. The efficacy of the model.

Contents of the invention

The purpose of the present invention is to provide a NAME model that simulates the human brain immune microenvironment and a construction method thereof.

A NAME model simulating the immune microenvironment of the human brain, including an upper chamber, a middle chamber and a lower chamber connected in sequence; the upper chamber is inoculated with human brain microvascular endothelial cells; the middle chamber is inoculated with astrocytes and microglia cells; the lower chamber is inoculated with human hippocampal neuron cells.

The number ratio of human brain microvascular endothelial cells, astrocytes, microglia, and human hippocampal neuron cells is 2:2.5:1:4.

The culture system in the NAME model chamber is: DMEM+10% FBS+1% double antibody (0.5% penicillin, 0.5% streptomycin).

After the NAME model is stable, replace the culture medium with EBSS and place it in an anoxic incubator for 10-14 hours. The NAME model is deprived of oxygen and sugar as a whole. After taking it out, it is placed in a conventional incubator and cultured for 20-28 hours to obtain OGD-NAME. Brain model.

The culture conditions in the anoxic incubator are 37°C, 95% N ₂ , and 5% CO ₂ .

The construction method of the NAME model that simulates the human brain immune microenvironment is carried out according to the following steps:

(1) Connect human brain microvascular endothelial cells to the upper Transwell chamber and culture them in a conventional incubator overnight;

(2) Inoculate astrocytes and microglia into the middle Transwell chamber, inoculate human hippocampal neuron cells into the lower chamber, and culture in a conventional incubator overnight;

(3) Attach the cells cultured overnight to the surface of the Transwell chamber membrane, replace the medium with EBSS, place the model in an anoxic incubator for 10-14 hours, perform overall oxygen and sugar deprivation on the NAME model, remove it and place it in the After culturing for 20-28 hours in a conventional incubator, the OGD-NAME brain model was obtained, and a group without oxygen and glucose deprivation was set as a control group.

The culture conditions in the conventional incubator are: 37°C, 95% O ₂ , 5% CO ₂ .

The culture system described in steps (1) and (2) is: DMEM+10% FBS+1% double antibody (0.5% penicillin, 0.5% streptomycin).

The culture conditions in the anoxic incubator are 37°C, 95% N ₂ , and 5% CO ₂ .

Beneficial effects of the present invention: For the first time, the present invention establishes a highly simulated human brain microorganism composed of human nerve cells (N), astrocytes (A), microglia (M), and vascular endothelial cells (E). Model of the environment. In order to obtain an in vitro model consistent with the pathological basis and phenotype of ischemic stroke, the NAME model was subjected to oxygen and glucose deprivation treatment to obtain the OGD-NAME brain model. The model of the present invention can screen the effective cells, vesicles and effective concentrations of traditional Chinese medicine preparations for in vitro nerve damage repair based on the cell cycle and apoptosis of neurons, as well as the regulation of the neural microenvironment, and evaluate the safety and effectiveness of various preparations. sex.

Description of the drawings

Figure 1 is a schematic diagram of the in vitro NAME model.

Figure 2 is a schematic diagram of the morphology of intraventricular hippocampal neurons;

In the figure, Control is the NAME model without oxygen and sugar deprivation, and the morphological manifestations of hippocampal neurons in the lower compartment (4x and 40x); the OGD group is the morphology of hippocampal neurons in the lower compartment after oxygen and sugar deprivation. Performance (4x magnification, 40x magnification); hUMSC group is the NAME model. After oxygen and sugar deprivation and umbilical cord mesenchymal stem cell treatment, the number and morphology of hippocampal neurons in the lower compartment.

Figure 3 shows immunofluorescence staining of nerve cells in each layer of chambers (20X);

In the picture, A is the human brain microvascular endothelial cells in the upper chamber, with green fluorescent staining ZO-1, and blue is the nucleus DAPI; B is the bottom hippocampal neuron cells, immunofluorescence staining NeuN; C is the astrocytes in the middle chamber, immune Fluorescent staining for GFAP; D is the mid-layer microglia, and immunofluorescence staining for IBA1.

Figure 4 shows the mRNA expression of inflammatory factors in astrocytes, microglia, and neuron cells.

Figure 5 shows the results of active oxygen detection.

Figure 6 shows the cell cycle of hippocampal neurons in the lower layer of the NAME model.

Figure 7 shows the apoptosis results of lower hippocampal neurons in the NAME model.

Detailed ways

In order to facilitate an understanding of the invention, the invention will be described more fully below. However, the invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough understanding of the present disclosure will be provided.

Example 1 Construction of in vitro NAME brain model and OGD-NAME brain model

First, human brain microvascular endothelial cells (Vascular endothelial cell-HCMEC/D3, 1×10 ⁵ ) were connected to a 12-well plate Transwell chamber 72 hours in advance and placed in an incubator. The ratio of glial cells to neurons is 1.5:1. Astrocytes are the most abundant glial cells, accounting for 40% of glial cells; microglia account for about 15% of the total glial cells, in the upper layer The chamber was inoculated with human brain microvascular endothelial cells (Vascular endothelial cell-HCMEC/D3, 1×10 ⁵ ), and the middle 6-well Transwell chamber was inoculated with astrocytes (Astrocyte-SVG P12, 1.2×10 ⁵ ) and microglia. cells (Microglia-HMC3, 0.45×10 ⁵ ); inoculate human hippocampal neurons (Neuron-HPPNCs, 2×10 ⁵ ) in the lower chamber according to the number of central nervous system nerve cells to construct a cell ratio model of 2:2.5:1:4 (Figure 1), the culture system of DMEM+10% FBS+1% double antibody was used for co-culture.

After the cell model is stable, the cells adhere to the surface of the Transwell chamber membrane, replace the culture medium with Earle's Balanced Salt Solution (EBSS), and place the model in an anoxic incubator for 42 hours (37°C, 95% N ₂ , 5% CO ₂ ), the NAME model was subjected to overall oxygen and glucose deprivation (OGD), taken out and cultured in a conventional incubator for 24 hours, and a group without oxygen and glucose deprivation was set as a control group.

Example 2 Detection indicators of NAME brain model and OGD-NAME brain model

After the NAME model was stabilized, the cells were attached to the surface of the chamber, oxygen-glucose deprived (OGD) for 42 hours, and reoxygenated for 24 hours. During the reoxygenation treatment, fifth-generation umbilical cord mesenchymal stem cells (hUMSC) were added for treatment. The group without oxygen and glucose deprivation was used as the control group.

A (Leica, DM500) microscope was used to observe hippocampal neuron cells in the lower compartment of the NAME model. The results are shown in Figure 2. After oxygen and sugar deprivation, the number of hippocampal neurons in the lower compartment was significantly reduced, and tight connections were loosened.

Nerve cell morphology detection: Immunofluorescence method was used to detect the expression of ZO-1 in upper human brain microvascular endothelial cells; GFAP marked astrocytes; IBA1 marked microglia; NEUN marked hippocampal neuronal cells. The results are shown in Figure 3 and were stained with specific antibodies, confirming that they were all cells that expressed specific markers.

The q-PCR method was used to detect the expression of inflammatory factors IL-1β, IL-6, TNF-α and HIF-1α (hypoxia-inducible factor) in two glial cells and neurons. Glial cells and microglia were treated with OGD respectively. The study found that the hypoxia-inducible factor hif-1α was highly expressed (p<0.01) and inflammatory cytokines were highly expressed (p<0.05); the lower hippocampal neuron cells were Quantitative PCR detection of mRNA showed high expression of hypoxia-inducible factor hif-1α (p<0.01), and increased expression levels of inflammatory factors (p<0.01) (Figure 4).

Flow cytometry was used to detect ROS reactive oxygen species in the lower hippocampal neurons. The results are shown in Figure 5. Compared with the OGD group, the average fluorescence intensity of ROS in the lower hippocampal neurons was significantly increased, which was about 3.5 times that of Control (p<0.01). , however, after adding umbilical cord mesenchymal stem cells (hUMSC) intervention, the fluorescence intensity of ROS in the lower hippocampal neurons significantly decreased, and the oxidative stress damage was improved (p<0.05).

Using the PI cell cycle detection kit, the cell cycle results were obtained by flow cytometry. As shown in Figure 6, after adding hUMSC to the upper chamber, the cell cycle of the lower hippocampal neurons changed significantly. The cell cycle of the lower hippocampal neurons There was a significant decrease in the G0/G1 phase (p<0.0001), a significant increase in the S phase (p<0.001), and a significant increase in the G2 and M phases (p<0.001), indicating that the hippocampus in the lower layer of the model Neurons enter a phase of replication and proliferation.

The AV/PI apoptosis detection kit was used to obtain the cell apoptosis results through flow cytometry. The results are shown in Figure 7. After adding hUMSC to the upper chamber, the apoptosis detection of the lower hippocampal neuron cells also changed significantly. , after NAME model was treated with OGD, early apoptosis and late apoptosis were significantly increased (p<0.001). However, after hUMSC treatment, the proportion of early and late apoptosis in hippocampal neurons was significantly reduced (p<0.001). ).

The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.

Claims (9)

1. A NAME model simulating the immune microenvironment of the human brain, characterized in that it includes an upper chamber, a middle chamber and a lower chamber connected in sequence; the upper chamber is inoculated with human brain microvascular endothelial cells; the middle chamber is inoculated with astrocytes plasma cells and microglia; the lower chamber is inoculated with human hippocampal neuron cells. 2. The simulated human brain immune microenvironment NAME model according to claim 1, characterized in that the number ratio of human brain microvascular endothelial cells, human astrocytes, microglia, and human hippocampal neuron cells is :2:2.5:1:4. 3. The NAME model simulating the human brain immune microenvironment according to claim 1, characterized in that the culture system in the NAME model chamber is: DMEM+10% FBS+1% double antibody. 4. The simulated human brain immune microenvironment NAME model according to claim 1, characterized in that, after the NAME model is stabilized, the culture medium is replaced with EBSS, placed in an anoxic incubator for 36-48h, and the NAME model is overall Oxygen and sugar deprivation were taken out and placed in a conventional incubator for 24 hours to obtain the OGD-NAME brain model. 5. The simulated human brain immune microenvironment NAME model according to claim 4, characterized in that the culture conditions in the anoxic incubator are 37°C, 95% N ₂ and 5% CO ₂ . 6. The construction method of the simulated human brain immune microenvironment NAME model according to claim 1, characterized in that it is carried out according to the following steps: (1) Connect human brain microvascular endothelial cells to the upper Transwell chamber 72 hours in advance and place them in a conventional incubator until the fusion rate reaches more than 90% and a tight junction is formed; (2) Inoculate astrocytes and microglia in proportion to the middle Transwell chamber 24 hours in advance, inoculate human hippocampal neuron cells in the lower chamber, and culture them in a conventional incubator overnight; (3) Replace the culture medium in the model with EBSS and place it in a hypoxic incubator for 36-48 hours. Perform overall oxygen and sugar deprivation on the NAME model. After taking it out, replace the culture medium with DMEM+10% FBS+1% double antibody. , placed in a conventional incubator and cultured for 24 hours to obtain the OGD-NAME brain model, and a group without oxygen and glucose deprivation was set as a control group. 7. The method for constructing the human brain immune microenvironment NAME model according to claim 6, characterized in that the culture conditions in the conventional incubator are: 37°C, 95% O ₂ , 5% CO ₂ . 8. The method for constructing the human brain immune microenvironment NAME model according to claim 6, characterized in that the culture system described in steps (1) and (2) is: DMEM+10% FBS+1% double antibody. 9. The method for constructing the human brain immune microenvironment NAME model according to claim 6, characterized in that the culture conditions in the anoxic incubator are 37°C, 95% N ₂ and 5% CO ₂ .