CN116463289A - Construction method of in-vitro primary microglial cell model for central nervous system inflammation drug research - Google Patents
Construction method of in-vitro primary microglial cell model for central nervous system inflammation drug research Download PDFInfo
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Abstract
The invention belongs to the technical field of medicine research of central nervous system inflammation related diseases. The invention provides a construction method of an in vitro primary microglial cell model for researching a central nervous system inflammation drug. The method for constructing the in vitro primary microglial cell model comprises a cell separation method under the conditions of no endotoxin (LPS) and ice bath or low temperature (4-8 ℃), a cell short-time in vitro culture method after low-density plating, a state quality control method for in vitro cultured cells simulating the state of in vivo cell subsets, and a method for developing drug research by adopting a high-throughput quantitative technology and a corresponding biological analysis method.
Description
Technical Field
The invention belongs to the technical field of drug research of central nervous system inflammation related diseases, and particularly relates to a construction method and a downstream application method of an in-vitro primary microglial cell model.
Background
Central nervous system Inflammation (Inflammation of Central Nervous System, or CNS Inflammation) broadly refers to acute or chronic Inflammation of the central nervous immune system of the brain, which is activated by an internal or external factor, either as a localized injury or as part of a systemic immune response (Fung, a., et al Central nervous system Inflammation in disease related conditions: mechanical processes resin 2012.1446: p.144-55.). Inflammation of the central nervous system is one of the important pathological mechanisms of chronic neurodegenerative diseases (Neurodegeneration Diseases), and is particularly remarkable in Alzheimer's Disease (AD), multiple sclerosis (Multiple Sclerosis, MS), parkinson's Disease (PD) and other diseases (Chitnis, T.and H.L.Weiner, CNS ligation and neurogenesis.J Clin Invest,2017.127 (10): p.3577-3587.). The central nervous system inflammation can be induced by internal cerebral factors (intracellular stress, misfolded protein reaction, etc.), or by external factors such as acute external Zhou Sunshang (infection, sepsis, surgery, burns, organ injury, etc.), chronic peripheral diseases (obesity, diabetes, depression, rheumatoid arthritis, etc.), etc. Exogenous induction of central nervous system inflammation can exacerbate the pathological lesions of neurodegenerative diseases (Cuello, a.c., early and Late CNS Inflammation in Alzheimer's Disease: two Extremes of a ContinuumTrends Pharmacol Sci,2017.38 (11): p.956-966.). Central nervous system inflammation may also be regulated by other factors including age and estrogen, among others. Although the specific etiopathological mechanisms of central nervous system inflammation have not been fully revealed, they can be involved in driving the pathological course of the disease and are therefore important diagnostic and therapeutic targets for related diseases (Stephenson, j., et al, inflammation in CNS neurodegenerative diseases.immunology,2018.154 (2): p.204-219.).
Recent animal models of disease and clinical studies have shown that Microglia (Mg) of brain myeloid origin dominate early immune activation and expansion of central nervous system inflammation (Srinivasan, k., et al, untangling the brain's neuroinflammatory and neurodegenerative transcriptional responses. Nat Commun, 2016.7:p.11295.). Microglia differentiate into several cell subsets in the pathological course of central nervous system inflammation, including Proliferation-Related components (pro-Related Modules), interferon-Related components (Interferon-Related Modules), LPS endotoxin-Related components (LPS-Related Modules), neurodegenerative-Related components (neurogenic-Related Modules, also known as Disease Associated Microglia, DAM) four major subgroups (Friedman, B.A., et al, diverse Brain Myeloid Expression Profiles Reveal Distinct Microglial Activation States and Aspects of Alzheimer's Disease Not Evident in Mouse modules.cell Rep,2018.22 (3): p.832-847.). Blocking both the pro-inflammatory and proliferative sub-populations (interferon-and LPS endotoxin-related components) can inhibit central nervous system inflammation and partially alleviate pathological damage of the related disease. However, only the endotoxin related component of LPS (namely M1-like microglial cells) in the inflammatory cell subset of the central nervous system can be realized through LPS stimulation experiments of in vitro cultured cells, and other cell subsets can not be simulated in vitro. In addition, the commonly used in vitro passaged cell lines of microglia (e.g., murine BV2, human HMC3, etc.) are differentiated cell lines and are also unable to mimic the primary cell state in vivo (Rangajan, P., L.Eng-Ang, and S.T.Dhen, potential drugs targeting microglia: current knowledge and future proclects.CNS Neurol Disord Drug Targets,2013.12 (6): p.799-806.). The existing method for separating primary cells of brain usually adopts separation operation at Room Temperature (RT) or 37 ℃ and cannot keep the in-vivo state of the primary cells. The existing common in vitro primary cell culture methods also result in primary cells that cannot be maintained in vivo. Therefore, there is a need in the preclinical research field of central nervous system inflammatory drugs for an in vitro cell model capable of fully simulating the state of in vivo cell subsets.
In the research and development process of the new drug, the phenotype screening of the disease animal model can truly reflect the pharmacological effects of the drug in vivo, but the cost is high, the period is long, and the high-flux operation is difficult to realize. Therefore, the development of in vitro high throughput screening of a large number of candidate drugs is a common method for developing new drugs. The currently used in vitro High throughput drug screening methods include two major classes (Blay, V., et al, high-Throughput Screening: today's biochemical and Cell-based appacous. Drug discovery Today,2020.25 (10): p.1807-1821.) of biochemical level detection (Biochemical Assay) and cellular level detection (Cell-based Assay). Biochemical level detection can detect biological processes such as enzymatic reaction and receptor binding, but because of lack of cellular biological structural functions such as signal cascade transduction, pharmacological effects of candidate drugs in vivo cannot be studied. The cell level detection can partially reflect the pharmacological efficacy of the candidate drugs in vivo, and can realize the early research of the drugs with high efficiency and low cost by constructing a high-throughput cell screening model. In addition, the high throughput cell screening model has a remarkable advantage that drug targets, pharmacological effects, drug agents, and drug resistance mechanisms can be revealed by differential gene expression and pathway changes in addition to detection of conventional cell phenotypes (Paananen, j. And v. Fortino, an omics perspective on drug target discovery platforms. Brief bioenform, 2020.21 (6): p.1937-1953.).
In summary, there is currently no patent at home and abroad for constructing a primary cell model based on a disease animal model of central nervous system inflammation for related drug research. Aiming at the development requirement of the preclinical medicine of the new medicine for central nervous system inflammation, according to the research progress of the latest pathology pharmacological mechanism of related diseases, the patent discloses a method for constructing an in vitro primary cell model of the central nervous system inflammation on the basis of an animal model of the disease of the central nervous system inflammation for developing medicine research. The method highly simulates the signal path and the cell biological characterization of the neuroinflammatory cells in the body, has the remarkable advantages of high efficiency and low cost, and makes up the technical method deficiency in the field, thereby having higher social and economic values and wide application prospects.
Disclosure of Invention
In a first aspect, the present invention provides a method for constructing an in vitro primary microglial cell (Mg) model based on an animal model of a disease of central nervous system inflammation, from which the in vitro primary microglial cell model can be used for carrying out pharmaceutical research of diseases related to central nervous system inflammation.
The construction method of the invention comprises the following steps under the conditions of no endotoxin (LPS) and ice bath or low temperature (4-8 ℃):
S1, respectively obtaining brain tissues of animal carcasses of a disease animal model and a control animal model of central nervous system inflammation;
s2, dissociating brain tissue to release cells;
s3, obtaining primary microglial cells by using a brain cell separation method.
In the method of the present invention, a control animal model is set to control the quality of the isolated cells of the disease animal model.
In some embodiments, the construction method of the present invention, the steps are performed in the absence of endotoxin (LPS) and in ice bath or low temperature (4-8 ℃).
In some embodiments, the construction method of the present invention comprises the steps of:
s1-1 brain tissue from cadavers of disease animal model and control animal model of central nervous system inflammation were isolated under endotoxin-free (LPS) and ice bath or low temperature (4-8deg.C) conditions, respectively. This step avoids false positive immune activation of the cells.
S1-2 eliminates meninges and blood vessels to remove peripheral blood in the absence of endotoxin (LPS) and in ice bath or low temperature (4-8 ℃). This step replaces the traditional pre-sacrifice cardiac perfusion method, avoiding false positive activation of the central nervous immune system by the perfusion process.
S2, the brain tissue is minced and dissociated under the conditions of no endotoxin (LPS) and ice bath or low temperature (4-8 ℃), and the brain tissue is digested and dissociated by using low-temperature proteolytic enzyme and low-temperature collagenase or low-temperature mixed tool enzyme. Instead of the traditional enzymatic digestion and dissociation at 37 ℃, false positive immune activation of cells is avoided.
S3, obtaining the primary microglial cells by using a brain cell separation method under the conditions of no endotoxin (LPS) and ice bath or low temperature (4-8 ℃). The step replaces the traditional technology of separating and enriching living cells at Room Temperature (RT) or 37 ℃, thereby avoiding false positive immune activation of cells.
In some embodiments, the low temperature proteolytic enzyme in the construction method S2 of the present invention refers to a generic term for enzymes that catalyze the hydrolysis of polypeptides or proteins at low temperatures, examples of which include, but are not limited to, low temperature serine endoproteases, psychrophilic bacterial proteases. The low-temperature collagenase refers to a specific protease class which can specifically hydrolyze the three-dimensional spiral structure of natural collagen under physiological pH and low-temperature conditions without damaging other proteins and tissues, and examples thereof include but are not limited to deep sea cold-tolerant salt-tolerant collagenase. The enzymes also include low temperature mixed tool enzymes, examples of which include, but are not limited to, actuase low temperature mixed enzymes.
Preferably, in the step S2, the Ackutase low-temperature mixed enzyme ice bath or low-temperature (4-8 ℃) digestion is adopted, and the effect is the best.
In some embodiments, in the construction method S3 of the present invention, the brain cell separation method is one or more selected from immunomagnetic bead cell separation (Immunomagnetic cell separation, magnetic Activated Cell Sorting (MACS)), fluorescence activated cell sorting (Fluorescence-activated cell sorting (FACS)), density gradient centrifugation (Density gradient centrifugation), immunodensity cell separation (Immunodensity cell isolation), microfluidic cell sorting (Microfluidic cell sorting). The improvement step is to adopt ice bath or low temperature (4-8 ℃) buffer solution or sheath solution without endotoxin (LPS), and the whole process is operated by ice bath or low temperature (4-8 ℃) without endotoxin (LPS).
Preferably, in the step S3, the purity of primary microglial cells obtained by immunomagnetic bead cell separation (MACS) or Fluorescence Activated Cell Sorting (FACS) is the highest.
In a further embodiment, the construction method of the invention comprises the steps of: from animal carcasses of disease animal models and control animal models of central nervous system inflammation, respectively, a portion or all of brain tissue is then removed, followed by placement in ice-cold Dulbecco's Phosphate-Buffered Saline (DPBS) buffer; removing meninges and blood vessels, stripping brain tissues required by pathological pharmacology research, and flushing and changing liquid for 2-3 times to remove peripheral blood; cutting brain tissue, and treating dissociated tissue with low temperature proteolytic enzyme and low temperature collagenase digestive juice for 15-30 min in ice bath or at low temperature (4-8deg.C); and then obtaining the primary microglial cells with high purity by adopting a living cell separation and enrichment method.
In some embodiments, the construction method of the present invention further comprises S4 inoculating the primary microglial cells obtained in S3 to a cell culture well plate at a low concentration of 10,000 to 20,000 cells per square centimeter, followed by a low-density adherent culture for no more than 48 hours. Under these conditions, the in vitro culture of primary microglial cells for a short period of time within 48 hours can still maintain the in vivo cell biological characteristics. The principle is as follows: the low-density culture obviously reduces the interaction between immune cells and the regulation and control of cytokines, and microglial cells are recovered to a resting state after the adherent culture, so that the signal path and the cell biological characterization of neuroinflammatory cells in vivo are highly simulated. Therefore, the pharmacological effect results of in vivo administration can be highly reflected by the drug study conducted within the 48-hour window. The specific culture steps are as follows: the isolated primary microglial cells were fully suspended and cell counted using DMEM high-sugar medium with the addition of 10% (v/v) heat-inactivated fetal bovine serum (Foetal Bovine Serum, FBS) and 1% (v/v) Penicillin-Streptomycin (PS) (Penicillin 100U/mL, streptomycin 100. Mu.g/mL), and then low-density culture was performed at a density of 10,000 cells per square centimeter by inoculating cell culture well plates at 5% CO 2 Culturing in a cell culture box at 37 ℃ for 12-16 hours to achieve cell attachment. The microglial cells after adherence can maintain in-vivo cell biological characterization within 48 hours of total culture time, and can be used for related drug research simulating in-vivo pharmacological efficacy during the time period.
In a further embodiment, the construction method of the present invention may further comprise the step of performing Quality Control (QC) on the primary microglial cell culture obtained in S3 or S4 to determine that the in vitro cell culture still retains its in vivo cell biological characteristics. In some embodiments, the quality control step comprises: after 12-16 hours of adherence of primary microglial cells in vitro with cell culture well plates, three cell subsets of microglial cells in central nervous inflammation, namely an Interferon-Related component (Interferon-Related Modules), an LPS endotoxin-Related component (LPS-Related Modules), and mRNA markers of a neurodegenerative-Related component (Neurodelegation-Related Modules, also known as Disease Associated Microglia, DAM) were randomly sampled and quantitatively analyzed for expression amounts. The significant difference in mRNA marker expression (P < 0.05) in the in vitro primary microglial cell model from the disease animal model compared to the in vitro primary microglial cell model from the control animal model indicates that the in vitro microglial cell model maintains in vivo cell biological characterization.
In the present invention, the animal model of diseases of central nervous system inflammation refers to an animal model of diseases related to acute or chronic inflammation caused by activation of the central nervous system immune system of the brain due to the influence of internal or external factors, examples of which include, but are not limited to, the following animal models of various diseases having pathological characteristics of central nervous system inflammation: senile dementia Alzheimer's Disease (AD), parkinson's Disease (PD), frontotemporal dementia (Frontotemporal Dementia, FTD), amyotrophic lateral sclerosis (Amyotrophic Lateral Sclerosis, ALS), cerebral Ischemic Stroke (Ischemic Stroke, IS), ischemia reperfusion injury (Ischemia Reperfusion Injury, ISCH/REP), multiple sclerosis (Multiple Sclerosis, MS), bacterial acute encephalitis (Acute Bacterial Encephalitis), bacterial Meningitis (Bacterial Meningitis), viral Meningitis (visual media), prion diseases (Prion Disease, PRD), parasitic encephalopathy (Parasitic Encephalopathy), glioblastoma multiforme (Glioblastoma Multiform, GBM), aging (Ageing), depression (Depression), epilepsy (Epileopsy), schizophrenia (Schizophrena), bidirectional disorders (Biorder, BPD), systemic lupus erythematosus (Systemic Lupus Erythematosus, SLE), vascular system (Primary Angitis of Central Nervous System), clinical fever (35), pannice, 3, peripheral neuropathy (37) and Pain), peripheral neuropathy (37 P.38, 3 P.D), peripheral neuropathy (37 P.E.23, P.M. 3, P.E. (37 P.E.), peripheral neuropathy (37 P.E.37, 3), lymphosis (37 P.E.), peripheral neuropathy (37 P.E.P.P.E.), lymphopenia.),37), lymphosis (37, P.E.),37) and (P.E.), cerebral Stroke/Stroke (central Stroke), rheumatoid arthritis (Rheumatoid Arthritis, RA), diabetes (Diabetes Mellitus, DM). In one embodiment, the disease animal model of central nervous system inflammation of Alzheimer's disease is familial AD 5XFAD mouse animal model of C57BL/6J genetic background (animal strain genotype designation: B6.Cg-Tg (APPSwFlLon, PSEN 1X M146L X L286V)) 6799 Vas/Mjax. The control animal model is a C57BL/6J mouse animal model with the same genetic background. In another embodiment, a 2 month old (2 mo) C57BL/6J mouse animal model was subjected to LPS induction of peripheral and neuro-inflammation by a method of molding in reference (Leng, L., et al, menin Deficiency Leads to Depressive-like Behaviors in Mice by Modulating Astrocyte-Mediated neuroi-stabilization, neuron,2018.100 (3): p.551-563e 7.), thereby constructing a Major Depressive Disorder (MDD) mouse animal model.
The in vitro primary microglial cell model comprises all primary cell subsets of myeloid-derived microglial cells in an animal model of a disease of central nervous system inflammation.
The subject of the in vitro cell model comprises a subpopulation of primary cells enriched and purified from any age individual of an animal model of a disease of central nervous system inflammation.
Further, the method of constructing an in vitro primary microglial cell model also includes a modified in vitro primary cell culture method. The main steps of the improved method comprise low-density adherence culture after low-concentration inoculation, and short-time in-vitro culture within 48 hours so as to maintain in-vivo cell biological characteristics.
In a second aspect, the invention also provides an in vitro primary microglial cell model obtained by the above construction method.
In a third aspect, the invention also provides the use of the in vitro primary microglial cell model in drug research of diseases related to central nervous system inflammation.
The drug research refers to preclinical research of drugs for diseases related to central nervous system inflammation, and comprises one or more of pharmacology, drug effect, toxicology, drug substitution, drug target, candidate drug design and optimization and candidate drug screening.
The central nervous system inflammation related diseases are selected from one or more of the following diseases: senile dementia Alzheimer's Disease (AD), parkinson's Disease (PD), frontotemporal dementia (Frontotemporal Dementia, FTD), amyotrophic lateral sclerosis (Amyotrophic Lateral Sclerosis, ALS), cerebral Ischemic Stroke (Ischemic Stroke, IS), ischemia reperfusion injury (Ischemia Reperfusion Injury, ISCH/REP), multiple sclerosis (Multiple Sclerosis, MS), bacterial acute encephalitis (Acute Bacterial Encephalitis), bacterial Meningitis (Bacterial Meningitis), viral Meningitis (visual media), prion diseases (Prion Disease, PRD), parasitic encephalopathy (Parasitic Encephalopathy), glioblastoma multiforme (Glioblastoma Multiform, GBM), aging (Ageing), depression (Depression), epilepsy (Epileopsy), schizophrenia (Schizophrena), bidirectional disorders (Biorder, BPD), systemic lupus erythematosus (Systemic Lupus Erythematosus, SLE), vascular system (Primary Angitis of Central Nervous System), clinical fever (35), pannice, 3, peripheral neuropathy (37) and Pain), peripheral neuropathy (37 P.38, 3 P.D), peripheral neuropathy (37 P.E.23, P.M. 3, P.E. (37 P.E.), peripheral neuropathy (37 P.E.37, 3), lymphosis (37 P.E.), peripheral neuropathy (37 P.E.P.P.E.), lymphopenia.),37), lymphosis (37, P.E.),37) and (P.E.), stroke (central Stroke), rheumatoid arthritis (Rheumatoid Arthritis, RA), diabetes (Diabetes Mellitus, DM).
The central nervous system inflammation may be any of mild (early), moderate (mid) or severe (late).
In a fourth aspect, the present invention also provides a method for evaluating the effectiveness of a constructed in vitro primary microglial cell model using high throughput quantification techniques and corresponding biological analysis methods, characterized in that it comprises the step of using the constructed in vitro primary microglial cell model of the invention. In some embodiments, the method comprises the steps of: after the in vitro primary microglial cell model from the disease animal model of central nervous system inflammation obtained by using the construction method of the invention is adhered to the cell culture pore plate of the in vitro primary microglial cell model from the control animal model for 12-16 hours, mRNA markers of three cell subsets of microglial cells in central nervous inflammation, namely an Interferon-Related component (Interferon-Related Modules), an LPS endotoxin-Related component (LPS-Related Modules) and a neurodegenerative-Related component (Neurodeletion-Related Modules, also known as Disease Associated Microglia, DAM) are randomly sampled and quantitatively analyzed. The in vitro primary microglial cell model from the disease animal model has a significant difference in the expression level of the mRNA markers (P < 0.05) compared with the in vitro primary microglial cell model from the control animal model, which indicates that the in vitro microglial cell model maintains the in vivo cell biological characteristics and can be used for the next preclinical drug study.
Wherein the control animal model refers to an animal model with negative pathological phenotype of the related disease, and examples thereof include, but are not limited to, a Wild Type (Wild-Type) animal model and a non-transgenic (non tg) animal model. In the present invention, the methods for constructing two in vitro primary microglial cell models of a control animal model and a disease animal model of central nervous system inflammation are the same.
In a fifth aspect, the present invention also provides a method for evaluating the pharmacological efficacy of a drug candidate using high throughput quantification techniques and corresponding biological assays, characterized in that it comprises the step of using the in vitro primary microglial cell model constructed in accordance with the present invention. In some embodiments, the method comprises the steps of: the method comprises the steps of culturing an in-vitro primary microglial cell model in vitro laid cell culture pore plate of an animal model of a disease from central nervous system inflammation obtained by using the construction method of the invention for 12-16 hours, respectively adding candidate drugs with proper concentration according to the requirement, setting proper negative and positive control drugs, and treating for proper time according to the requirement, wherein the total treatment time is not more than 48 hours after primary cell inoculationLong; after reaching the candidate drug treatment time point, a proper method is adopted to collect a biological sample of a required type, a high-throughput technology is used for quantitatively detecting the pathology-related pathway, and the high-throughput technology is compared with a control drug so as to relatively quantitatively evaluate the activation or blocking effect of the candidate drug. In one embodiment, an in vitro primary microglial cell model derived from a 13 month old (13 mo) AD 5XFAD (strain designation: b6.Cg-Tg (APPSwFlLon, PSEN1 x M146L x L286V) 6799Vas/mm jax) mouse animal model is treated with a plurality of immune blockers or inhibitors. Nanjing New grid Biotechnology Co High throughput drug screening RNA library construction kit user manual (96-Well version) constructs a second generation sequencing library, entrusts the south-genipin medical examination laboratory limited to use PE150 sequencing strategy on Illumina NovaSeq sequencer platform, and each sample (each Well in cell culture Well plate) acquires 3G sequencing data. In another embodiment, a 2 month old (2 mo) C57BL/6J mouse model is constructed from a major depressive disorder (Major Depression Disorder, MDD) mouse animal model by peritoneal injection of LPS-induced peripheral inflammation, and then an in vitro primary microglial cell model derived from the MDD mouse animal model is treated with various immune blockers or inhibitors. Di-Feng from Nanjing New grid biotechnology Co>High throughput drug screening RNA library construction kit user handbook (96-Well version) constructs a second generation sequencing library, entrusts Nanjing Tongyuan medical examination laboratory Co., ltd. To adopt PE150 sequencing strategy on an Illumina Novaseq sequencer platform, and each sample (cell culture Well plate) acquires 3G sequencing data.
In the fourth and fifth aspects described above, the high throughput quantification techniques include quantitative PCR (qPCR and qRT-PCR), digital quantitative PCR (Digital qPCR), immunoblotting (Immunoblotting) gene chip (Genechip), microarray chip (Microarray), genomics (Genomics) method, transcriptomics (Transcriptomics) method, proteomics (Proteomics) method, epigenomics (Epigenomics) method, metabonomics (Metabonomics) method, and glycogenomics (glycogenomics).
The corresponding biological analysis methods of the high-throughput quantification technology comprise Genomics (Genomics) analysis methods, transcriptomics (Transcriptomics) analysis methods, proteomics (Proteomics) analysis methods, epigenomics (Epigenomics) analysis methods, metabonomics (Metabonomics) analysis methods and glycosylation (glycogenomics) analysis methods. The genomic analysis method comprises: QC analysis (sequencing alignment related quality control, indel region re-alignment, base quality correction, genetic relationship analysis, hubert balance analysis, heterozygous quality control, population stratification correction), common variation analysis (genotype interpolation, genotype phenotype association (GWAS) analysis, association analysis result quality control), post-GWAS analysis (mutation site function annotation, site gene association analysis, polygenic risk scoring, co-localization analysis, mendel randomization), rare variation analysis based on populations (load check, variance component check, rare variation association test, confounding factor influence analysis and control), rare variation analysis based on families (composite heterozygote screening, candidate gene screening, de novo variation discovery, validation linkage analysis), genomic structure variation analysis (sequencing depth quality control and correction, exclusion based on capture and repeat regions, copy number variation analysis, insertion analysis, inversion analysis, ectopic analysis, deletion analysis, interruption end analysis, transposon annotation, repetitive element annotation), enrichment analysis (function enrichment analysis, reflection enrichment, KEGG pathway enrichment analysis). The (II) transcriptomic analysis method comprises: QC analysis (sequencing alignment related quality control, transcript level RNA integrity analysis, sequencing saturation analysis, batch effect analysis and correction), transcript quantification (gene level quantification, transcript level quantification, absolute quantification), principal component PCA analysis, differential gene analysis (differential assay, assay result correction), cluster analysis, enrichment analysis (GO function enrichment analysis, reactiome enrichment analysis, KEGG pathway enrichment analysis, protein interaction network enrichment analysis, transcription factor target enrichment analysis, cell type marker enrichment analysis, disease type enrichment analysis), co-expression analysis (co-expression network construction, expression correlation analysis, core module discovery), deconvolution analysis (cell composition analysis, ligand receptor annotation), new transcript discovery (new transcript de novo assembly, with reference new transcript discovery), endogenous retrovirus analysis (endogenous retrovirus quantification, endogenous retrovirus annotation). The proteomic analysis method (III) comprises the following steps: QC analysis (normalization, coefficient of variation analysis, batch effect analysis and correction), protein identification (reference spectrum construction, protein characterization, protein quantification), principal component PCA analysis, differential protein analysis (differential assay, assay result correction), cluster analysis, enrichment analysis (GO function enrichment analysis, reactiome enrichment analysis, KEGG pathway enrichment analysis, COG function annotation), network analysis (protein interaction network, co-expression network construction, core module discovery). The apparent histology analysis method (IV) includes two types. Wherein methylation histology analysis of apparent histology comprises: QC analysis (sequencing alignment related quality control, common single nucleotide polymorphism site filtration, batch effect correction, methylation level quantification), differential methylation analysis (differential methylation site analysis, differential methylation region annotation), enrichment analysis (site/region and gene association analysis, GO function enrichment analysis, reactiome enrichment analysis, KEGG pathway enrichment analysis), cell composition analysis (deconvolution analysis), methyl site related sequence analysis (Motif analysis, transcription factor binding analysis). In addition, the apparent serological ATAC-seq chromatin patency analysis included: QC analysis (sequencing alignment correlation quality control, non-redundant score, batch effect correction, over amplification analysis), open area signal identification (peak identification, nucleosome free area identification), differential open area analysis (differential open area identification, differential open area annotation), mutation and openness correlation analysis (study of the effect of a specific mutation on the chromatin openness of the area), enrichment analysis (area and gene association analysis, GO function enrichment analysis, reactiome enrichment analysis, KEGG pathway enrichment analysis), regulatory area analysis (Motif analysis, transcription factor binding analysis). The metabonomic analysis method comprises the following steps: QC analysis (noise reduction, baseline calibration, batch effect analysis and correction), substance identification (deconvolution, peak alignment, peak identification, peak feature extraction, normalization, metabolite characterization, metabolite quantification), cluster analysis (clustering of metabolites), differential metabolite analysis (differential assay, assay result correction), enrichment analysis (KEGG pathway enrichment analysis, differential metabolic pathway analysis, cell type enrichment analysis), network analysis (protein-metabolite interaction network, metabolite co-expression network construction, metabolite module conservation analysis). The method for analyzing the glycosylation group comprises the following steps: QC analysis (normalization, coefficient of variation analysis, batch effect analysis and correction), glycopeptide identification (peptide fragment identification, variable modification search, offset search, glycopeptide quantification, glycosylation site localization, sugar chain annotation), differential glycosylation analysis (differential glycopeptide abundance analysis, differential glycosylation site analysis, correction of assay results), structural and functional analysis (prediction of the influence of glycosylation on protein structure, annotation of the influence of glycosylation on protein structure).
Advantageous effects
Compared with the existing in-vitro passage cell model, the in-vitro primary microglial cell model obtained by the method can highly simulate the signal path and the cell biological characterization of in-vivo neuroinflammatory cells, in particular to four large cell subsets of microglial cells (Mg) related to various central nervous inflammation newly defined by the latest research. Compared with the existing disease animal model, the invention can greatly reduce the consumption of the disease animal model, and microglial cells obtained by each animal model can be used for cell culture pore plate plating culture, thereby greatly improving the utilization efficiency of experimental animals, reducing the experimental cost and shortening the experimental period. Compared with the conventional research method for screening the existing drug targets and pharmacological effects, the invention fully utilizes the advantages of the high-throughput quantitative technology and the biological analysis method, can more comprehensively reveal the drug targets, pharmacological effects, drug agents, drug resistance mechanisms and the like, and is helpful for discovering the drug off-target effect, potential risks and the like.
Drawings
FIG. 1 is a schematic flow chart of the sorting of brain microglial cells (Mg) of the AD 5XFAD mouse animal model using Fluorescence Activated Cell Sorting (FACS) in example 1. (A) First order round robin (Gating) selects FSC cell entities (Lymphocytes) and removes cell debris. (B) Second round selection (Gating) Hoechst 33342 stain positive cells (FSC-A, DAPI-A subset) were selected to remove small amounts of multicellular adherents. (3) Third order round selection (Gating) selects and sorts CD11b positive myeloid cells (FSC-se:Sub>A, CD11b subset), mainly brain Microglise:Sub>A (MG).
FIG. 2 shows the quantitative RT-PCR (qRT-PCR) method used in example 2 to detect the regulation of the expression level of molecular markers of cell subsets after immune blocker treatment of microglial cells (Mg) sorted from the brains of AD 5XFAD mice animal model and C57BL/6J (co-bred batch of non Tg mice model) control mice. (A-B) mRNA markers Irf7 and Isg15 of the Interferon-Related component (Interferon-Related Modules) were expressed in significantly higher amounts in 5XFAD mouse model sorted Mg than in non Tg control mice sorted Mg. The cells are treated by using Sting covalent blocker C-178 to block the activation of Sting signal channels, so that the gene expression levels of markers Irf7 and Isg15 in the sorted Mg of the 5XFAD mouse animal model are obviously reduced. (C-D) mRNA markers C3 and Tspo of LPS-Related components (LPS-Related Modules) were expressed in significantly higher amounts in 5XFAD mouse model sorted Mg than in non Tg control mice sorted Mg. By treating cells with Myd88 inhibitor TJ-M2010-5, myd88 signaling pathway activation is inhibited, and the gene expression levels of markers C3 and Tspo in 5XFAD mouse animal model sorted Mg are significantly reduced. (E-F) mRNA markers Cst7 and Itgax of neurodegenerative Related component (neurogenesis-Related Modules) were expressed in significantly higher amounts in 5XFAD mouse model sorted Mg than in non Tg control mice sorted Mg. By treating cells with an mTOR inhibitor Rapamycin (AY-22989), the inhibition of mTOR signaling pathway activation significantly down-regulates the gene expression levels of markers Cst7 and Itgax in 5XFAD mouse animal models sorted Mg. In this qRT-PCR assay, 3 biological replicates were set per drug treatment. (P <0.05, < P <0.01, < P < 0.001)
Figure 3 shows that the various immune blockers or inhibitors of example 3 significantly reduced activation of the inflammatory immune signaling pathway of the AD central nervous system in an in vitro primary microglial cell model derived from a 13 month old (13 mo) AD 5XFAD mouse animal model. (A) Principal component analysis (Principal Component Analysis, PCA) cluster analysis plots demonstrate the overall differences between the different groups caused by the different blocking treatments. Where D is the DMSO vehicle control and the others are individual blocking treated samples. (B) Random clustering (Hierarchical clustering) heatmaps (Heat map) of differentially expressed genes (Differentially expressed genes, DEGs) demonstrate the overall difference that individual blocking treatments affect gene expression. (C) Pathway enrichment analysis (Pathway enrichment analysis) demonstrates a comparative analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes, KEGG) pathway enrichment of down-regulated DEGs of individual blocking treated samples.
Figure 4 shows that treatment of the peripheral inflammation of LPS derived from 2 months of peritoneal injection (2 mo) with various immune blockers or inhibitors of example 3 resulted in an in vitro primary microglial cell model of MDD mouse animal model, significantly reducing activation of MDD central nervous system inflammatory immune signaling pathway. (A) Principal component analysis (Principal Component Analysis, PCA) cluster analysis plots showed overall differences between the different groups resulting from the different blocking treatments. Where D is the DMSO vehicle control and the others are individual blocking treated samples. (B) Random clustering (Hierarchical clustering) heatmaps (Heat map) of differentially expressed genes (Differentially expressed genes, DEGs) show the overall differences that individual blocking treatments affect gene expression. (C) Pathway enrichment analysis (Pathway enrichment analysis) demonstrates a comparative analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes, KEGG) pathway enrichment of down-regulated DEGs of individual blocking treated samples.
Detailed Description
Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.
The experimental methods used in the examples below are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1: primary microglial cells (Mg) were enriched and sorted from the brains of the AD mouse animal model using immunomagnetic bead cell separation and purification (MACS) and Fluorescence Activated Cell Sorting (FACS).
Familial AD 5XFAD mouse animal models of bg genetic background and C57BL/6J genetic background (strain name: b6.Cg-Tg (APPSwFlLon, PSEN1 x M146L x L286V) 6799 Vas/Mmjax) and C57BL/6J mouse animal models as control animal models (the same breeding lot of non Tg mouse models) were purchased from the model biotechnology company, inc. Both AD disease animal models and C57BL/6J control animal models were bred in SPF-class animal houses.
The immunomagnetic bead cell separation and purification Method (MACS) was optimized and improved with reference to the Isolation and cultivation of microglia from adult mouse or rat brain experimental procedure from Miltenyi Biotec, germany. First, the mice were sacrificed immediately after removal of intact brain tissue and immediately after placement in ice-cold DPBS (Gibco, 14190144). The meninges and blood vessels of the brain tissues of the mice are removed under an dissecting microscope, cerebral cortex and hippocampus are peeled off, and the peripheral blood is removed by flushing and changing liquid for 2-3 times. After brain tissue was minced, the dissociated tissue released cells were treated with Actuase digest (Innovative Cell Technologies, NC 9839010) at 4℃for 15 minutes. Cell debris and myelin were then cleared using Debris Removal Solution debris clearing solution (Miltenyi Biotec, 130-109-398). Suspension cells were blocked with DPBS+0.5% (v/v) FBS and washed 2 times, labeled with CD11b (Microglia) MicroBeads immunomagnetic beads (Miltenyi Biotec,130-093-634,130-093-636) and purified by column. Purified microglial cells (Mg) were cultured in an adherent manner in 24-well cell culture well plates. The DMEM medium (Gibco, 11965092) used contained 10% (v/v) heat-inactivated FBS (Gibco, 16140089) and 1% (v/v) Penicillin-Streptomycin (Gibco, 15140122). All tissue and cell experiments were performed on ice or in endotoxin free (LPS) solutions or reagents at 4 ℃ to avoid contamination and false positive activation.
Fluorescence Activated Cell Sorting (FACS) methods were optimized and improved with reference to the references (Srinivasan, k., et al Untangling the brain's neuroinflammatory and neurodegenerative transcriptional responses. First, whole brain tissue was removed after sacrifice, and placed immediately in ice-cold DPBS (Gibco, 14190144). The meninges and blood vessels of the brain tissues of the mice are removed under an dissecting microscope, cerebral cortex and hippocampus are peeled off, and the peripheral blood is removed by flushing and changing liquid for 2-3 times. After brain tissue was minced, the dissociated tissue was treated with Accutase digest (Innovative Cell Technologies, NC 9839010) at 4 ℃ for 15 minutes. Cell debris and myelin were then cleared using Debris Removal Solution debris clearing solution (Miltenyi Biotec, 130-109-398). Suspension cells were blocked with DPBS+0.5% (v/v) FBS and washed 2 times, then labeled with APC-conjugated anti-CD11b (BD Biosciences,561690, 1:250) antibody, hoechst 33342 (Invitrogen, H21492,5 ng/mL) on ice for 20 minutes, and finally CD11b positive microglial cells (Mg) were sorted using BD FACSAria flow cytometer. All tissue and cell experiments were performed in ice bath or low temperature (4-8 ℃) and endotoxin (LPS) free solutions or reagents to avoid contamination and false positive activation. Raw data from FACS sorting was analyzed using FlowJo v10.6.1 software and a schematic flow chart is shown in fig. 1.
Example 2: quantitative RT-PCR (qRT-PCR) method is adopted to detect various immune blockers and inhibitors to regulate and control the gene expression of mRNA markers of microglial cell (Mg) cell subtype of the AD mouse animal model.
Primary microglial cells (Mg) were isolated and purified by MACS method and inoculated into 24-well cell culture well plates with 20000 cells per well, referring to the experimental procedure of example 1, from brain tissue of AD 5XFAD mouse animal model and C57BL/6J control mouse animal model (experimental and control animal model sources were the same as in example 1), respectively. The cells were incubated in a 5% CO2,37℃cell incubator for 24 hours with medium containing 10% (v/v) heat-inactivated fetal bovine serum (Gibco, 670086) and 1% (v/v) Penicillin-Streptomycin (Gibco, 15140122). Cells were treated with Sting antagonist C-178 (Selleck, S6667) at a final concentration of 1. Mu.M/mL, myd88 inhibitor TJ-M2010-5 (MCE, HY-139397) at a final concentration of 1. Mu.M/mL, and Rapamycin (AY-22989) at a final concentration of 1. Mu.M/mL (MCE, HY-10219) for 8 hours, and control cells were treated with an equal amount of DMSO solvent for 8 hours. The final DMSO concentration in both the treatment and control groups was 0.1% (v/v).
Cells were collected by TRizol (Invitrogen, 15596018) lysis, total RNA was purified by ZYMO Direct-zol RNA Microprep Kits (ZYMO, R2063), and cDNA was synthesized by TaKaRa PrimeScript RT Master Mix (TaKaRa, RR 036A). qPCR reactions were then performed on a Bio-Rad CFX Connect Real-Time PCR detection system using PowerUp SYBR Green Master Mix (Applied Biosystems, A25742) and ROX internal controls (Invitrogen, 12223012).
The quantitative RT-PCR detection targets and the primers used are as follows: mouse Gapdh internal standard gene (qmGAPDh-1F/R: GACTTCAACAGCAACTCCCAC (SEQ ID NO: 1), TCCACCACCCTGTTGCTGTA (SEQ ID NO: 2)). The mouse Irf7 gene (qmIrf 7-F/R: CAATTCAGGGGATCCAGTTG (SEQ ID NO: 3), AGCATTGCTGAGGCTCACTT (SEQ ID NO: 4)). The mouse Isg15 gene (qmIsg 15-F/R: CTAGAGCTAGAGCCTGCAG (SEQ ID NO: 5), AGTTAGTCACGGACACCAG (SEQ ID NO: 6)). The mouse C3 gene (qmC 3-F/R: CCAGCTCCCCATTAGCTCTG (SEQ ID NO: 7), GCACTTGCCTCTTTAGGAAGTC (SEQ ID NO: 8)). The mouse Tspo gene (qmTspo-F/R: GCCTACTTTGTACGTGGCGAG (SEQ ID NO: 9), CCTCCCAGCTCTTTCCAGAC (SEQ ID NO: 10)). The mouse Cst7 gene (qmCst 7-F/R: GGAGCTGTACTTGCCGAGC (SEQ ID NO: 11), CATGGGTGTCAGAAGTTAGGC (SEQ ID NO: 12)). Mouse Itgax gene (qmItgax-F/R: CTGGATAGCCTTTCTTCTGCTG (SEQ ID NO: 13), GCACACTGTGTCCGAACTCA (SEQ ID NO: 14)). Wherein Irf7 and Isg15 are mRNA markers of an Interferon-Related component (Interferon-Related Modules), C3 and Tspo are mRNA markers of an LPS endotoxin-Related component (LPS-Related Modules), and Cst7 and Itgax are mRNA markers of a neurodegenerative-Related component (Neurode-Related Modules, also known as Disease Associated Microglia, DAM). The difference in expression level of these 6 mRNA markers was significant in the in vitro primary microglial cell model from the 5XFAD disease animal model versus the in vitro primary microglial cell model from the C57BL/6J control animal model (P < 0.05), indicating that the in vitro microglial cell model maintained in vivo cell biology characterization.
All RNA experiments were performed in an RNase-free/DNase-free clean environment to avoid RNA degradation and DNA contamination. 3 replicates Kong Shengwu were set per treatment, data were analyzed for significance using Student's t-Test statistical methods in Microsoft EXCEL software, and plots were generated by GraphPad Prism 9 software with experimental results shown in FIG. 2.
Example 3: various immune blockers or inhibitors were used to treat in vitro primary microglial cell models (strain name: b6.Cg-Tg (APPSwFlLon, PSEN x 146L x L286V) 6799 Vas/Mmjax) derived from 13 month old (13 mo) Alzheimer's Disease (AD) 5 xfas mouse animal models (control animal models are C57BL/6J mouse animal models of the same genetic background), or to treat 2 month old (2 mo) C57BL/6J peritoneal injection LPS-induced peripheral inflammatory Major Depressive Disorder (MDD) mouse animal models (see references (Leng, L., et al Menin Deficiency Leads to Depressive-like Behaviors in Mice by Modulating Astrocyte-Mediated neuroimaging, 2018.100 (3): p.551-563e 7.) primary depressive mouse models were constructed by peritoneal injection of LPS-induced peripheral inflammatory and neuroinflammatory disorders in C57BL/6J mouse models of 2 months. Wherein the isolation, culture and administration methods of primary microglial cells in vitro of the AD and MDD mouse animal models all adopt the isolation, culture and administration method of primary microglial cells in vitro based on the immunomagnetic bead cell isolation and purification Method (MACS) of the AD mouse animal model in example 1. And finally, collecting a total RNA sample, and quantitatively detecting the activation level of the microglial inflammatory immune signal pathway by adopting a high-throughput transcriptome RNAseq sequencing technology.
Specifically, the sample preparation procedure for high throughput transcriptome RNAseq second generation sequencing detection is the same as the cell culture and sample collection method of example 2. The basic steps of the RNAseq experiment of the AD in vitro primary microglial cell model are as follows: total RNA was purified using ZYMO Direct-zol RNA Microprep Kits (ZYMO, R2063) and then RNAseq library was prepared directly using TaKaRa SMARTer Stranded Total RNA-seq Kit v2-Pico Input Mammalian Kit (TaKaRa, 634412). The total RNA and RNAseq library were quantified using a Qubit 3.0 fluorometer. RNA integrity and cDNA fragments were analyzed using an AATI fragment analyzer. Finally, these sequencing libraries were mixed in equal amounts, high throughput sequencing was performed on an Illumina NovaSeq6000 sequencer using a PE150 double-ended sequencing mode, each library producing 3G sequencing data. The basic steps of RNAseq experiment of MDD in vitro primary microglial cell model are: nanjing New grid Biotechnology CoHigh throughput drug screening RNA library construction kit user handbook (96-Well version) constructs a second generation sequencing library, entrusts Nanjing Tongyuan medical examination laboratory Co., ltd on an Illumina Novaseq6000 sequencer platform, adopts PE150 sequencing strategy, and each sample (each Well in a cell culture Well plate) acquires 3G sequencing data. Read length of RNAseq was annotated with mouse UCSC mm10 (RefSeq gene annotation) using STAR alignment software. And differential gene expression analysis was performed by the rlog switching function of DESeq 2. The significance of gene regulation was assessed using one-way ANOVA and Bonferroni test was performed. Selection of significant regulatory genes (adj.P.Val) <0.05 Performing an analysis: first, the overall differences between the different groups were measured using principal component analysis (Principal Component Analysis, PCA), which was performed by the R function prcomp, and the results were visualized using ggplot (v.3.4.0). (II) random cluster (Hierarchical clustering) heatmap (Heat map) of differentially expressed genes (Differentially expressed genes, DEGs) was generated from R-packet pheeatmap (v.1.0.12), and the gene expression amounts were scaled in units of rows. (III) pathway enrichment analysis (Pathway enrichment analysis) was performed by R package clusterifier (v.4.6.0), and the results of the enrichment analysis of the down-regulated differential genes were visualized using ggplot (v.3.4.0). The quantitative and relative quantitative analysis of the gene number and statistical scoring (pvalue) of the enrichment pathways of each blocker sample reflects the blocking pathways and blocking capacity of each blocker qualitatively and relatively. (IV) marker analysis of microglial cell subsets (DAM, neurodegenerative related, interferon related, proliferation related, lipopolysaccharide related) was generated according to the references (Friedman, B.A., et al, diverse Brain Myeloid Expression Profiles Reveal Distinct Microglial Activation States and Aspects of Alzheimer's Disease Not Evident in Mouse models.cell Rep,2018.22 (3): p.832-847.). In this study, 3 replicate wells of biological replicates were placed per blocker blocking treatment. The experimental results of the blocking effect of the AD neuroinflammation microglial immune signal pathway are shown in FIG. 3, and the blocking effect of the MDD neuroinflammation microglial immune signal pathway is shown in the specification The results of the experiments are shown in FIG. 4. />
Claims (10)
1. A method for constructing an in vitro primary microglial cell model, comprising the following steps under the conditions of no endotoxin (LPS) and ice bath or low temperature (4-8 ℃):
s1, respectively obtaining brain tissues of animal carcasses of a disease animal model and a control animal model of central nervous system inflammation;
s2, dissociating brain tissue to release cells;
s3, obtaining primary microglial cells by using a brain cell separation method.
2. The construction method according to claim 1, comprising the steps of:
s1-1, separating brain tissues of animal carcasses from a disease animal model and a control animal model of central nervous system inflammation under endotoxin-free (LPS) and ice bath or low temperature (4-8 ℃) conditions, respectively;
s1-2, removing meninges and blood vessels under endotoxin-free (LPS) and ice bath or low temperature (4-8 ℃) conditions to remove peripheral blood;
s2, the brain tissue is minced and dissociated under the conditions of no endotoxin (LPS) and ice bath or low temperature (4-8 ℃), and the brain tissue is digested and dissociated by using low-temperature proteolytic enzyme and low-temperature collagenase or low-temperature mixed tool enzyme.
S3, obtaining the primary microglial cells by using a brain cell separation method under the conditions of no endotoxin (LPS) and ice bath or low temperature (4-8 ℃).
3. The construction method according to claim 1 or 2, wherein,
In S2, the low temperature proteolytic enzyme is selected from the group consisting of low temperature serine endoprotease, psychrophilic bacterial protease; the low-temperature collagenase is deep sea cold-fit salt-tolerant collagenase; the low temperature mixed tool enzyme is Ackutase low temperature mixed enzyme, preferably, in S2, ackutase low temperature mixed enzyme ice bath or low temperature (4-8deg.C) digestion is adopted, and/or
In S3, the brain cell separation method is one or more selected from immunomagnetic bead cell separation, fluorescence activated cell separation, density gradient centrifugation, immunodensity cell separation and microfluidic cell classification, and preferably, in S3, primary microglial cells obtained by immunomagnetic bead cell separation or fluorescence activated cell separation method are adopted.
4. The construction method according to claim 1 or 2, comprising the steps of: from animal carcasses of disease animal models and control animal models of central nervous system inflammation, respectively, followed by removal of part or all of brain tissue followed by placement in ice-cold Du's phosphate buffered saline buffer; removing meninges and blood vessels, stripping brain tissues required by pathological pharmacology research, and flushing and changing liquid for 2-3 times to remove peripheral blood; cutting brain tissue, and treating dissociated tissue with low temperature proteolytic enzyme and low temperature collagenase digestive juice for 15-30 min in ice bath or at low temperature (4-8deg.C); and then obtaining the primary microglial cells with high purity by adopting a living cell separation and enrichment method.
5. The construction method according to claim 1 or 2, further comprising S4 inoculating the primary microglial cells obtained in S3 to a cell culture well plate at a low concentration of 10,000 to 20,000 cells per square centimeter, followed by a low-density adherent culture for not more than 48 hours.
6. The construction method according to claim 1 or 5, further comprising the step of quality control of the primary microglial cell culture obtained in S3 or S4.
7. An in vitro primary microglial cell model obtained by the construction method of any one of claims 1-6.
8. Use of the in vitro primary microglial cell model according to claim 7 in the pharmaceutical research of diseases related to central nervous system inflammation, preferably selected from one or more of the following diseases: senile dementia Alzheimer's disease, parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, cerebral ischemic stroke, ischemia reperfusion injury, multiple sclerosis, bacterial acute encephalitis, bacterial meningitis, viral meningitis, prion diseases, parasitic encephalopathy, glioblastoma multiforme, aging, depression, epilepsy, schizophrenia, bidirectional disorders, systemic lupus erythematosus, central nervous system vasculitis, cerebral amyloid angiopathy, inflammatory demyelinating diseases, uremic encephalopathy, traumatic brain injury, chronic alcoholism neuropathy, thermal jet diseases, peripheral inflammatory pain, neuromyelitis optica, neuropathic pain, stroke, rheumatoid arthritis, diabetes.
9. A method for evaluating the effectiveness of a constructed in vitro primary microglial cell model using high throughput quantification techniques and corresponding biological analysis methods, comprising the step of using the in vitro primary microglial cell model of claim 7.
10. A method of evaluating the pharmacological efficacy of a candidate drug using high throughput quantification techniques and corresponding biological analysis methods, comprising the step of using the in vitro primary microglial cell model of claim 7, wherein the drug is used to prevent or treat a central nervous system inflammation-related disorder, preferably selected from one or more of the following disorders: senile dementia Alzheimer's disease, parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, cerebral ischemic stroke, ischemia reperfusion injury, multiple sclerosis, bacterial acute encephalitis, bacterial meningitis, viral meningitis, prion diseases, parasitic encephalopathy, glioblastoma multiforme, aging, depression, epilepsy, schizophrenia, bidirectional disorders, systemic lupus erythematosus, central nervous system vasculitis, cerebral amyloid angiopathy, inflammatory demyelinating diseases, uremic encephalopathy, traumatic brain injury, chronic alcoholism neuropathy, thermal jet diseases, peripheral inflammatory pain, neuromyelitis optica, neuropathic pain, stroke, rheumatoid arthritis, diabetes.
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