A method for constructing an in vitro model of trigeminal neuralgia targeting fibrocyte and microglia interaction and a drug screening method thereof

Abstract

The present application relates to a trigeminal neuralgia in vitro drug screening model construction method technical field, specifically discloses a kind of targeted fibroblast and microglia cell interaction's trigeminal neuralgia in vitro drug screening model construction method and its drug screening method, comprising the following steps: a cell recovery and subculture: the BV2 microglial cells in logarithmic growth phase are digested, centrifuged and inoculated, and are cultured under the condition containing 5-7% CO2, 35-41 DEG C;B. Specific induction: when BV2 cell adheres and reaches the expected fusion degree, the original culture solution is discarded, and negative control medium and / or clusterin protein enriched specific conditioned medium are added to different holes respectively, and continuous incubation is carried out for 24-72 hours.The present application reduces arachnoid fibroblast to microglial cell pathological paracrine stimulation, gives the screening method high disease microenvironment specificity, can intervene real microenvironment target.

Description

Technical Field

[0001] This invention relates to the technical field of in vitro drug screening model construction methods for trigeminal neuralgia, and in particular to a method for constructing an in vitro model of trigeminal neuralgia targeting the interaction between fibroblasts and microglia and its drug screening method. Background Technology

[0002] Trigeminal neuralgia (TN) is a severe facial pain caused by abnormal electrical activity in the trigeminal nerve. Current clinical treatments for TN are limited. First-line anticonvulsants (such as carbamazepine) can inhibit nerve excitation, but they often come with serious adverse reactions such as liver damage. Microvascular decompression surgery is effective, but it still carries the risk of recurrence and complications, and some patients do not have clear vascular compression. Recent studies have revealed that pathological neuroinflammation, particularly the abnormal activation of specific cell populations in the arachnoid microenvironment, plays a crucial role in the pathogenesis and progression of TN. Among these, microglia, as myeloid innate immune cells of the central nervous system, are a core component of exacerbated neuropathic pain due to their persistent endoplasmic reticulum stress and pro-inflammatory activation.

[0003] For the development of drugs targeting neuroinflammation and trigeminal neuralgia, establishing an efficient and reliable in vitro drug screening system is crucial for discovering lead compounds. However, existing in vitro screening methods have significant drawbacks: most methods rely on adding broad-spectrum exogenous toxins (such as lipopolysaccharide LPS) to monocultured microglia to induce an inflammatory response. This traditional stimulation method lacks disease specificity and completely deviates from the actual pathological triggering mechanism of the arachnoid microenvironment during TN development. "Effective" anti-inflammatory compounds screened using such traditional methods often fail to translate into TN in vivo animals or clinical trials because they cannot intervene in the actual microenvironmental targets (such as CLU / TREM2 interaction or its downstream endoplasmic reticulum stress). Summary of the Invention

[0004] In order to overcome the shortcomings of existing technologies, traditional stimulation methods lack disease specificity, completely deviate from the real pathological triggering mechanism of the arachnoid microenvironment during TN occurrence, and fail to intervene in real microenvironment targets (such as CLU / TREM2 interaction or its downstream endoplasmic reticulum stress) and thus encounter transformation failure.

[0005] The present invention adopts the following technical solution: A method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia includes the following steps: a. Cell resuscitation and passage: BV2 microglia in the logarithmic growth phase were digested, centrifuged and seeded, and cultured at 35-41°C with 5-7% CO2. b. Specific induction: When BV2 cells adhere to the well and reach the expected degree of confluence, discard the original culture medium and add negative control medium and / or cluster protein-rich specific conditioned medium to different wells, and incubate continuously for 24 to 72 hours.

[0006] Optionally, the method for preparing the cluster protein-rich specific conditioned medium includes the following steps: 1) Construct a lentiviral vector carrying a cluster protein overexpression sequence; 2) Inoculate host cells in good in vitro culture conditions into the culture device, and after the cells adhere to the wall, transfect them with the constructed lentiviral vector; 3) Continue culturing the cells for 48–72 hours; 4) Collect the cell culture supernatant, remove cell debris by centrifugation and filter to obtain a specific conditioned medium rich in cluster protein.

[0007] Step 3) involves the following steps: After transfecting the lentiviral vector, when the lentiviral titer is 1×10⁻⁶... 8 When the TU / mL concentration is reached, add the virus solution and polybrene at the same time, with a final concentration of 4-8 μg / mL. Replace with fresh culture medium 6-12 hours after infection and continue culturing for 24-48 hours.

[0008] Optionally, in step 3), the cell supernatant transfected with empty lentivirus can be collected simultaneously as a negative control culture medium.

[0009] Preferably, the host cell is a HEK293 cell or a HEK293T cell.

[0010] Optionally, the method for preparing the cluster protein-rich specific conditioned medium includes the following steps: 1) A rat model of trigeminal neuralgia was established by infraorbital nerve ligation; 2) Dissect the arachnoid tissue or related skull base arachnoid tissue from TN model rats, prepare small tissue blocks in pre-cooled PBS or HBSS, add tissue digestion solution, digest, rinse with PBS, filter, and prepare a single-cell suspension; 3) Resuspend the cells in PBS and adjust the cell concentration to 1×10⁻⁶. 6 ~1×10 7 Cells / mL were incubated with fluorescently labeled antibodies against FN1, COL1A2, PDGFR, and α-SMA at 3–7°C in the dark for 30–45 minutes. After washing with PBS, FN1 cells were screened using flow cytometry. + COL1A2 + PDGFR + and α-SMA + Co-expressed fibroblast subsets; 4) After sorting, the cells are seeded in a culture device. After the cells reach 70% to 90% confluence, they are washed several times with PBS and cultured for 24 to 48 hours with serum-free or fetal bovine serum at a concentration of 0.5% to 10%. 5) After collecting the culture supernatant, the cells and cell debris were removed by centrifugation multiple times, filtered, and fibroblast conditioned medium was obtained. The continuous expression of CLU protein in the fibroblast conditioned medium was confirmed by ELISA.

[0011] Preferably, the tissue digestion solution contains 1–2 mg / mL Collagenase IV, 1–2 mg / mL Dispase II, and 50–100 μg / mL DNase I.

[0012] This invention discloses a drug screening method. The method uses an in vitro drug screening model for trigeminal neuralgia constructed based on the aforementioned method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia. The method includes the following steps: 1) The culture equipment inoculated with BV2 microglia should be divided into at least a normal control group, a model control group, and a drug screening group. The normal control group is cultured with negative control medium, the model control group is induced with cluster protein-rich specific conditioned medium for 24-72 hours, and the drug screening group is conditioned with cluster protein-rich specific conditioned medium and the candidate drug or inhibitor to be tested. 2) After 24–48 hours of drug treatment, extract cell proteins / nucleic acids or collect culture supernatant for quantitative detection and evaluation of molecular and phenotypic indicators.

[0013] Optional quantitative detection and assessment of molecular and phenotypic indicators include one or more of the following: transmembrane receptor and kinase pathway assessment, transcriptional repression and ubiquitination axis assessment, and endoplasmic reticulum stress and inflammation endpoint assessment.

[0014] Optionally, the transmembrane receptor and kinase pathway assessment includes the following steps: extracting total cellular protein, detecting the phosphorylation level of DAP12, a downstream protein of TREM2 activation, and the activation status of the RAS / MEK / ERK pro-inflammatory signaling pathway by Western blotting.

[0015] Optionally, the assessment of the transcriptional repression and ubiquitination axis includes the following operations: detecting the expression of the intracellular deubiquitinase OTULIN, the linear ubiquitination and phosphorylation levels of STAT1 by IP-WB or WB, and detecting the mRNA and protein expression levels of the α subunit of the nascent polypeptide-associated complex by RT-qPCR and WB.

[0016] Optionally, the assessment of endoplasmic reticulum stress and inflammation endpoints includes the following procedures: detecting the phosphorylation level of endoplasmic reticulum stress sensor proteins by Western blotting, detecting the expression intensity of inducible nitric oxide synthase in cells by immunofluorescence, and detecting nitric oxide and tumor necrosis factor-α in cell culture supernatant by Griess assay or ELISA kit.

[0017] The beneficial effects of the present invention include at least the following: 1) This invention creatively proposes using conditioned medium containing a high concentration of clustering protein (CLU) as a specific stimulant. Specifically, this invention provides two precise sources for preparing the culture medium: on the one hand, high-titer culture supernatant (CLU-CM) collected from host cells overexpressing the CLU sequence (such as HEK293 cells); on the other hand, conditioned medium containing pathogenic Mgp_Fibroblast fibroblast subsets sorted and cultured in high-throughput from arachnoid tissue of a TN pathological model. These two sources fundamentally replicate the pathological paracrine stimulation of microglia by arachnoid fibroblasts, endowing this screening method with extremely high specificity for the disease microenvironment, enabling intervention at real microenvironmental targets.

[0018] 2) The screening method provided by this invention is based on specific CLU medium induction, which can accurately activate specific cascade pathways in microglia; and by quantitatively detecting the activation of TREM2 receptor, phosphorylation level of endoplasmic reticulum stress sensor proteins (such as PERK / IRE1α), and expression rescue of core transcription factor NACA in microglia after the addition of candidate drugs, a multi-dimensional and mechanism-clear efficacy evaluation is achieved, which greatly eliminates the false positive interference of non-specific anti-inflammatory compounds.

[0019] 3) This invention successfully transforms the complex in vivo pathological interaction mechanism (which requires extremely high cost and a long period of TN rat model construction and behavioral assessment) into a standardized, mass-producible in vitro culture medium preparation and cell-level screening system. The drug screening method is simple to operate and has high batch-to-batch stability, making it possible to conduct high-throughput primary screening of novel trigeminal neuralgia targeted drugs that target "fibroblast-microglia microenvironment interaction" and "relief of microglia endoplasmic reticulum stress". This significantly reduces R&D costs and accelerates the new drug discovery process. Attached Figure Description

[0020] Figure 1 This is a fluorescence diagram illustrating the specific binding of CLU in the culture medium to the microglial cell membrane receptor TREM2, as confirmed by immunofluorescence in Example 1 of this invention. Figure 2 This is a scatter plot of flow cytometry in Embodiment 3 of the present invention; Figure 3This is a histogram of the flow cytometry in Embodiment 3 of the present invention; Figure 4 These are comparative surgical procedures of the rat trigeminal neuralgia animal model (infraorbital nerve CCI model) in Example 5 of this invention; Figure 5 This is a graph showing the CLU concentration detection results in a rat trigeminal neuralgia animal model according to Example 5 of the present invention; Figure 6 This is a diagram showing the ECL chemiluminescence imaging results in the evaluation of transmembrane receptor and kinase pathways in Example 5 of this invention. Figure 7 This is a graph showing the detection results of the ubiquitination level of the STAT1 protein in Example 5 of the present invention; Figure 8 This is a graph showing the detection results of the expression of the ubiquitinase OTULIN in Example 5 of the present invention; Figure 9 This is a graph showing the detection results of the protein expression level of the α subunit (NACA) of the neonatal polypeptide-associated complex in Example 5 of the present invention; Figure 10 This is a graph showing the detection results of phosphorylation levels of endoplasmic reticulum stress sensor proteins (IRE1α, PERK, ATF6) by Western blot in Example 5 of the present invention. Figure 11 This is a graph showing the results of immunofluorescence detection of the expression intensity of inducible nitric oxide synthase (iNOS) in cells according to Example 5 of the present invention. Figure 12 This is a graph showing the detection results of nitric oxide (NO) in cell culture supernatant by the Griess method in Example 5 of the present invention; Figure 13 This is a graph showing the detection results of tumor necrosis factor-α (TNF-α) in cell culture supernatant using either an ELISA kit or Example 5 of the present invention. Detailed Implementation

[0021] The present invention will now be further described with reference to the accompanying drawings and specific embodiments.

[0022] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0023] Unless otherwise specified, the raw materials and equipment used in this invention are all conventional raw materials and equipment in the art and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in this invention are all conventional methods in the art.

[0024] As used in this invention, unless otherwise clearly stated, the singular forms “a,” “an,” and “the” include embodiments with plural indicators.

[0025] As used in the specification and appended claims of this invention, unless otherwise clearly stated, the term "or" is generally used in its meaning as including "and / or".

[0026] Unless otherwise indicated, all figures expressing characteristic magnitudes, quantities, and physical properties will be understood to be modified by the term “about”, regardless of whether the term “about” is immediately present. Therefore, unless indicated to the contrary, the numerical parameters presented are approximations that may vary depending on the desired properties sought by those skilled in the art using the teachings disclosed herein.

[0027] The ELISA assay of this invention, formally known as enzyme-linked immunosorbent assay, is a highly sensitive immunological detection technique that primarily detects trace substances in body fluids through antigen-antibody reaction combined with enzyme color development.

[0028] The BV2 cells used in this invention are immortalized cell lines obtained from microglia. Specifically, products from Wuhan Shangen Biotechnology Co., Ltd. can be used.

[0029] In this invention, PBS (phosphate buffer saline) is a commonly used buffer solution in biological experiments. It is prepared by mixing disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), sodium chloride (NaCl), and other components in a specific ratio. The pH value is usually maintained at 7.2-7.4, and the osmotic pressure is similar to that of human body fluids.

[0030] The CLU overexpression lentiviral vector used in this invention is a product of Hanheng Biotechnology (Shanghai) Co., Ltd.

[0031] In this invention, the CLU-CM medium is a CLU conditioned medium.

[0032] In this invention, the NC-CM culture medium is prepared by centrifuging the supernatant obtained from the empty lentivirus group at 500g for 10 minutes and at 2000g for 15 minutes to remove cells and cell debris, and then filtering it through a 0.22μm sterile filter membrane.

[0033] In this invention, HEK293 cells or HEK293T cells are products of ATCC (American Type Culture Collection).

[0034] The polybrene used in this invention is a product of Shanghai Beyotime Biotechnology Co., Ltd.

[0035] In this invention, the HBSS (Hanks' balanced salt solution) used is a product of Jing Sulebo Technology Co., Ltd.

[0036] In this invention, Collagenase IV and DNase I are products from Sigma.

[0037] In this invention, Dispase II is a product of Gibco.

[0038] In this invention, the fluorescently labeled antibodies for FN1, COL1A2, PDGFR and α-SMA can be products of Beijing TransGen Biotech Co., Ltd.

[0039] In this invention, the basal culture medium (DMEM or DMEM / F12) can be a product of Shanghai Beyotime Biotechnology Co., Ltd.

[0040] In this invention, fetal bovine serum and penicillin-streptomycin are products of Gibco.

[0041] In this invention, Mgp_Fibroblast is a fibroblast derived from the meningeal plexus.

[0042] A method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia includes the following steps: a. Cell resuscitation and passage: BV2 microglia in the logarithmic growth phase were digested, centrifuged and seeded, and cultured at 37-39°C with 5-7% CO2. b. Specific induction: When BV2 cells adhere to the well and reach the expected degree of confluence, discard the original culture medium and add negative control medium and / or cluster protein-rich specific conditioned medium to different wells, and incubate continuously for 24 to 72 hours.

[0043] In some embodiments, the method for preparing the cluster protein-rich specific conditioned medium includes the following steps: 1) Construct a lentiviral vector carrying a cluster protein overexpression sequence; 2) Inoculate host cells in good in vitro culture conditions into a culture device. After the cells adhere to the wall, transfect them with the constructed lentiviral vector carrying the cluster protein overexpression sequence. 3) Continue culturing the cells for 48–72 hours; 4) Collect the cell culture supernatant, remove cell debris by centrifugation and filter to obtain a specific conditioned medium rich in cluster protein.

[0044] In some embodiments, step 3) specifically involves: after transfecting a CLU-overexpressing lentiviral vector, when the lentiviral titer is 1×10⁻⁶... 8When the TU / mL concentration is reached, add the virus solution and polybrene at the same time, with a final concentration of 4-8 μg / mL. Replace with fresh culture medium 6-12 hours after infection and continue culturing for 24-48 hours.

[0045] In some embodiments, in step 3), the cell supernatant transfected with empty lentivirus is collected simultaneously as a negative control culture medium.

[0046] In some embodiments, the host cell is a HEK293 cell or a HEK293T cell.

[0047] In some embodiments, the method for preparing the cluster protein-rich specific conditioned medium includes the following steps: 1) A rat model of trigeminal neuralgia was established by infraorbital nerve ligation; 2) Dissect the arachnoid tissue or related skull base arachnoid tissue from TN model rats, prepare small tissue blocks in pre-cooled PBS or HBSS, add tissue digestion solution, digest, rinse with PBS, filter, and prepare a single-cell suspension; 3) Resuspend the cells in PBS and adjust the cell concentration to 1×10⁻⁶. 6 ~1×10 7 Cells / mL were incubated with fluorescently labeled antibodies against FN1, COL1A2, PDGFR, and α-SMA at 3–7°C in the dark for 30–45 minutes. After washing with PBS, FN1 cells were screened using flow cytometry. + COL1A2 + PDGFR + and α-SMA + Co-expressed Mgp_Fibroblast fibroblast subsets; 4) After sorting, the cells are seeded in a culture device. After the cells reach 70% to 90% confluence, they are washed several times with PBS and cultured for 24 to 48 hours with serum-free or fetal bovine serum at a concentration of 0.5% to 10%. 5) After collecting the culture supernatant, the cells and cell debris were removed by centrifugation multiple times, and the mixture was filtered to obtain Mgp_Fibroblast fibroblast conditioned medium. ELISA was used to confirm that CLU protein was continuously highly expressed in the fibroblast conditioned medium.

[0048] In some embodiments, the tissue digestion solution contains 1–2 mg / mL Collagenase IV, 1–2 mg / mL Dispase II, and 50–100 μg / mL DNase I.

[0049] This invention discloses a drug screening method. The method uses an in vitro drug screening model for trigeminal neuralgia constructed based on the aforementioned method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia. The method includes the following steps: 1) The culture equipment inoculated with BV2 microglia should be divided into at least a normal control group, a model control group, and a drug screening group. The normal control group is cultured with negative control medium, the model control group is induced with cluster protein-rich specific conditioned medium for 24-72 hours, and the drug screening group is conditioned with cluster protein-rich specific conditioned medium and the candidate drug or inhibitor to be tested. 2) After drug treatment for 24–48 hours, extract cell proteins / nucleic acids or collect culture supernatant for quantitative detection and evaluation of molecular and phenotypic indicators (based on the literature: Bustin SA, et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4):611–622. DOI:10.1373 / clinchem.2008.11279). 7).

[0050] In some embodiments, the quantitative detection and assessment of molecular and phenotypic indicators include one or more of the following: transmembrane receptor and kinase pathway assessment, transcriptional repression and ubiquitination axis assessment, and endoplasmic reticulum stress and inflammatory endpoint assessment.

[0051] In some embodiments, the assessment of the transmembrane receptor and kinase pathway includes the following steps: extracting total cellular protein and detecting it by Western blot (based on the literature: Mahmood T, Yang PC. (2012) Western blot: technique, theory, and trouble shooting). North American Journal of Medical Sciences , 4(9):429–434.DOI:10.4103 / 1947-2714.100998PMID:23050259)TREM2 activates the phosphorylation level of downstream protein DAP12 and the activation status of the RAS / MEK / ERK pro-inflammatory signaling pathway.

[0052] In some embodiments, the assessment of the transcriptional repression and ubiquitination axis includes the following operations: by IP-WB (based on: Co-immunoprecipitation and protein blots. Methods in Molecular Biology, 655:377–387. DOI: 10.1007 / 978-1-60761-765-5_24. PMID: 20872854) or WB detection (based on: Mahmood T, Yang PC. (2012) Western blot: technique, theory, and troubleshooting). North American Journal of Medical Sciences , 4(9):429–434.DOI:10.4103 / 1947-2714.100998PMID: 23050259) Expression of intracellular deubiquitinase OTULIN, linear ubiquitination and phosphorylation levels of STAT1, and mRNA and protein expression levels of the α subunit of the nascent polypeptide-associated complex detected by RT-qPCR and WB.

[0053] In some embodiments, the assessment of endoplasmic reticulum stress and inflammation endpoints includes the following operations: detecting the phosphorylation level of endoplasmic reticulum stress sensor proteins by Western blotting and by immunofluorescence detection (based on the literature: Ramos-Vara JA. (2005) Immunofluorescence staining of formalin-fixed, paraffin-embedded tissues). Journal of Histotechnology , 28(3): 183–190.DOI: 10.1179 / 014768805X48586) The expression intensity of inducible nitric oxide synthase in cells, and its detection by the Griess method (based on the literature: Moorcroft MJ, Davis J, Compton RG. (2001) Detection and determination of nitrate and nitrite: A review. Talanta, 54(5):785–803.DOI: 10.1016 / S0039-9140(01)00323-X,PMID: 18968301) or ELISA kit (based on the literature: Crowther JR. (2009)). The ELISA Guidebook (2nd ed.). Humana Press (Springer) nitric oxide and tumor necrosis factor-α in cell culture supernatant.

[0054] Specific implementation examples are shown in Examples 1, 2 and 3.

[0055] Example 1: A method for preparing a specific conditioned medium rich in clustering proteins (CLU) includes the following steps: 1) Seed HEK293 cells or HEK293T cells in the logarithmic growth phase into 6-well plates or 10cm culture dishes at a seeding density of 4 × 10⁶ cells per well in a 6-well plate. 5 1 cell per 10cm culture dish, or 5 × 10 cells per dish in a 10cm culture dish. 6 Cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C and 5% CO2 for 12 hours to achieve 60% cell confluence.

[0056] 2) Subsequently, a lentiviral vector carrying the CLU overexpression sequence was added to the cells, with a multiplicity of infection (MOI) of 10; when the lentiviral titer was 1×10⁻⁶... 8 At TU / mL, add 10 μL of virus solution to each well of a 6-well plate and 100 μL of virus solution to each 10 cm culture dish. Simultaneously add polybrene to a final concentration of 4 μg / mL. Replace with fresh complete culture medium 6 hours after infection and continue culturing for 24 hours. The empty lentivirus group was treated using the same method, and the cell supernatant transfected with empty lentivirus was collected as a negative control medium.

[0057] 3) After the cells stabilized, wash once with PBS, add serum-free DMEM or DMEM containing 0.5% fetal bovine serum, and continue culturing for 48 hours. Collect the cell culture supernatant, centrifuge sequentially at 300g for 5 minutes and 1000g for 10 minutes to remove cells and cell debris, then filter through a 0.22μm sterile filter to obtain CLU conditioned medium, denoted as CLU-CM. The supernatant obtained from the empty lentivirus group was treated in the same manner and denoted as NC-CM. ELISA confirmed that the CLU protein concentration in CLU-CM was higher than that in NC-CM, with a final CLU concentration of 500 ng / mL, which was used for subsequent microglial cell induction experiments.

[0058] A method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia includes the following steps: a. Cell resuscitation and passage: BV2 microglia in the logarithmic growth phase were digested, centrifuged, and seeded at an appropriate cell density into 6-well or 24-well plates and cultured in a cell culture incubator containing 5% CO2 at 37°C.

[0059] b. Specific induction: When BV2 cells adhered and reached about 70% confluence, the original culture medium was discarded, and the negative control culture medium or specific conditioned medium (NC-CM / CLU-CM) prepared in Example 1 was added to different wells. The cells were incubated for 24 hours. During this process, CLU in the culture medium specifically bound to the microglial cell membrane receptor TREM2, inducing downstream cascade reactions.

[0060] like Figure 1 As shown, immunofluorescence results confirmed that CLU in the culture medium can bind significantly and specifically to the microglial cell membrane surface receptor TREM2.

[0061] Example 2: A method for preparing a specific conditioned medium rich in clustering proteins (CLU) includes the following steps: 1) Seed HEK293 cells or HEK293T cells in the logarithmic growth phase into 6-well plates or 10cm culture dishes at a seeding density of 4 × 10⁶ cells per well in a 6-well plate. 5 1 cell per 10cm culture dish, or 5 × 10 cells per dish in a 10cm culture dish. 6 Cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin for 24 hours at 37°C and 5% CO2 to achieve 80% cell confluence.

[0062] 2) Subsequently, a lentiviral vector carrying the CLU overexpression sequence was added to the cells, with a multiplicity of infection (MOI) of 50; when the lentiviral titer was 1×10⁻⁶... 8 At TU / mL, add 50 μL of virus solution to each well of a 6-well plate and 500 μL of virus solution to each 10 cm culture dish. Simultaneously add polybrene to a final concentration of 8 μg / mL. Replace with fresh complete culture medium 12 hours after infection and continue culturing for 48 hours. The empty lentivirus group was treated using the same method, and the cell supernatant transfected with empty lentivirus was collected as a negative control medium.

[0063] 3) After the cells stabilized, wash twice with PBS, add serum-free DMEM or DMEM containing 2% fetal bovine serum, and continue culturing for 72 hours. Collect the cell culture supernatant, centrifuge sequentially at 500g for 10 minutes and 2000g for 15 minutes to remove cells and cell debris, then filter through a 0.22μm sterile filter to obtain CLU conditioned medium, denoted as CLU-CM. The supernatant obtained from the empty lentivirus group was treated in the same manner and denoted as NC-CM. ELISA confirmed that the CLU protein concentration in CLU-CM was higher than that in NC-CM, with a final CLU concentration of 200 ng / mL, which was used for subsequent microglial cell induction experiments.

[0064] A method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia includes the following steps: a. Cell resuscitation and passage: BV2 microglia in the logarithmic growth phase were digested, centrifuged, and seeded into 24-well plates at an appropriate cell density. The plates were then cultured in a cell culture incubator containing 7% CO2 at 39°C.

[0065] b. Specific induction: When BV2 cells adhered and reached about 70% confluence, the original culture medium was discarded, and the negative control culture medium or specific conditioned medium (NC-CM / CLU-CM) prepared in Example 1 was added to different wells. The cells were incubated for 72 hours. During this process, CLU in the culture medium specifically bound to the microglial cell membrane receptor TREM2, inducing downstream cascade reactions.

[0066] Example 3: A method for preparing a specific conditioned medium rich in clustering proteins (CLU) includes the following steps: 1) Adult SD rats were selected, and a trigeminal neuralgia model was established by infraorbital nerve ligation. Anesthesia was performed by intraperitoneal injection of sodium pentobarbital 40 mg / kg or by inhalation anesthesia with isoflurane. On the 7th day after surgery, after behavioral verification of TN-like pain response, arachnoid tissue around the trigeminal nerve root or related skull base arachnoid tissue was harvested.

[0067] 2) Place the tissue in pre-cooled PBS and cut it into pieces approximately 1 mm in size. 3 Tissue blocks were added to a tissue digestion solution containing 1 mg / mL Collagenase IV, 1 mg / mL Dispase II, and 50 μg / mL DNase I, and digested at 37°C for 30 minutes. During digestion, the tissue was gently pipetted every 5 minutes. After digestion, culture medium containing 10% fetal bovine serum was added to terminate the process. The mixture was then filtered through a 400-mesh nylon mesh or a 70 μm cell sieve and centrifuged at 3000g for 5 minutes to obtain a single-cell suspension.

[0068] 3) Resuspend the cells in PBS containing 1% BSA to adjust the cell concentration to 1×10⁻⁶. 6 Cells / mL. Incubate with fluorescently labeled antibodies against FN1, COL1A2, PDGFR, and α-SMA at a concentration of 0.25 μg / mL for each antibody. 6 Cells, or diluted 1:50, were incubated at 4°C in the dark for 30 minutes, washed twice with PBS, and FN1 cells were screened by flow cytometry (based on Shapiro HM (2003) Practical flow cytometry (4th ed.). John Wiley & Sons). + COL1A2 +PDGFR + and α-SMA + Co-expressed Mgp_Fibroblast fibroblast subset.

[0069] 4) After sorting, the cells were seeded into collagen I or gelatin-coated culture plates at a density of 2 × 10⁶ cells per well in a 24-well plate. 4 10 cells, 1×10⁶ cells per well in a 6-well plate 5 10 cells, or 1×10 cells per 10cm culture dish 6 Cells were cultured in primary culture at 37°C in a 5% CO2 incubator using DMEM / F12 or DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Once the cells reached 70% confluence, they were washed once with PBS and cultured for another 24 hours in medium containing serum-free or 0.5%-10% fetal bovine serum.

[0070] 5) After collecting the culture supernatant, the culture was centrifuged at 300g for 5 minutes and then at 1000g for 10 minutes to remove cells and cell debris. The supernatant was then filtered through a 0.22μm sterile membrane to obtain Mgp_Fibroblast fibroblast conditioned medium, denoted as Mgp_Fibroblast+-CM. ELISA confirmed sustained high expression of CLU protein in this conditioned medium, with a final CLU concentration of 50 ng / mL, which was used for subsequent BV2 microglia induction and drug screening experiments.

[0071] A method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia includes the following steps: a. Cell resuscitation and passage: BV2 microglia in the logarithmic growth phase were digested, centrifuged, and seeded into 6-well plates at an appropriate cell density. The plates were then cultured in a cell culture incubator containing 5% CO2 at 37°C.

[0072] b. Specific induction: When BV2 cells adhered to the wall and reached about 70% confluence, the original culture medium was discarded, and the negative control culture medium or specific conditioned medium (NC-CM / CLU-CM) prepared in Example 3 was added to different wells. The cells were incubated for 24 hours. During this process, CLU in the culture medium specifically binds to the microglial cell membrane receptor TREM2, inducing downstream cascade reactions.

[0073] The flow cytometry cell sorting results of this embodiment are shown below. Figure 2 and Figure 3 .

[0074] like Figure 2As shown, in both the control group and this example (trigeminal neuralgia group), the proportion of PDGFR+ / α-SMA+ double-positive cells, representing activated myofibroblasts, was around 21%-23%, with little variation; indicating that the total proportion of fibroblasts in the trigeminal neuralgia group was not significantly different from that in the control group.

[0075] like Figure 3 As shown, the proportion of FN1 positive cells (V1R) in the control group was only 12.03%, and the vast majority of cells (87.97%) were in a negative / low expression state, indicating that the level of fibronectin secreted by the cells was very low at this time.

[0076] In this embodiment, the proportion of FN1 positive cells increased significantly to 46.09%, while the proportion of negative cells decreased to 53.91%, indicating that the treatment significantly induced FN1 expression.

[0077] In the control group, only 5.68% of cells were COL1A2 positive (V1R), and the vast majority of cells (94.32%) were negative or low in expression, indicating that the level of collagen synthesis in cells was very low at this time.

[0078] In this embodiment, the proportion of COL1A2 positive cells increased significantly to 41.92%, while the proportion of negative cells decreased to 58.08%, indicating that the treatment significantly induced the expression of COL1A2.

[0079] In PDGFR+ / α-SMA+ fibroblasts, treatment / modeling significantly upregulated the expression of fibrosis markers FN1 and COL1A2, with the proportion of positive cells increasing from single digits / 10% to over 40%.

[0080] The results showed that the proportion of Mgp_Fibroblast subtype fibroblasts was significantly increased in the total fibroblasts in the trigeminal neuralgia model, transitioning from a resting state to an activated state with high fibrin secretion.

[0081] Example 4: A method for preparing a specific conditioned medium rich in clustering proteins (CLU) includes the following steps: 1) Adult SD rats were selected, and a trigeminal neuralgia model was established by infraorbital nerve ligation. Anesthesia was performed by intraperitoneal injection of sodium pentobarbital 50 mg / kg or by inhalation anesthesia with isoflurane. On the 14th day after surgery, after behavioral verification of TN-like pain response, arachnoid tissue around the trigeminal nerve root or related skull base arachnoid tissue was harvested.

[0082] 2) Place the tissue in a pre-cooled HBSS and cut it into pieces of approximately 1 mm. 3Tissue blocks were added to a tissue digestion solution containing 2 mg / mL Collagenase IV, 2 mg / mL Dispase II, and 100 μg / mL DNase I, and digested at 37°C for 45 minutes. During digestion, the tissue was gently pipetted every 10 minutes. After digestion, culture medium containing 10% fetal bovine serum was added to terminate the process. The mixture was then filtered through a 400-mesh nylon mesh or a 70 μm cell sieve and centrifuged at 500g for 10 minutes to obtain a single-cell suspension.

[0083] 3) Resuspend the cells in PBS containing 2% BSA to adjust the cell concentration to 1×10⁻⁶. 7 Cells / mL. Incubate with fluorescently labeled antibodies against FN1, COL1A2, PDGFR, and α-SMA, at a concentration of 1 μg / mL for each antibody. 6 Cells, or diluted 1:200 for use. Incubate at 4°C in the dark for 45 minutes, wash twice with PBS, and sort by flow cytometry (based on: Melamed MR, Mullaney PF, Mendelsohn ML. (1979) Flow cytometry and sorting). John Wiley & Sons (Monograph) Screening for Mgp_Fibroblast fibroblast subsets co-expressing FN1⁺, COL1A2⁺, PDGFR⁺ and α-SMA⁺.

[0084] 4) After sorting, the cells were seeded into collagen I or gelatin-coated culture plates at a density of 5 × 10⁶ cells per well in a 24-well plate. 4 10 cells, 3 × 10⁶ cells per well in a 6-well plate 5 10 cells, or 3 × 10 cells per 10cm culture dish 6 Cells were cultured in primary culture at 37°C in a 5% CO2 incubator using DMEM / F12 or DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. After the cells reached 90% confluence, they were washed twice with PBS and cultured for another 48 hours in medium containing serum-free or 0.5%-10% fetal bovine serum.

[0085] 5) After collecting the culture supernatant, the culture was centrifuged sequentially at 500g for 0 minutes and then at 2000g for 15 minutes to remove cells and cell debris. The medium was then filtered through a 0.22μm sterile filter to obtain Mgp_Fibroblast fibroblast conditioned medium, denoted as Mgp_Fibroblast+-CM. ELISA confirmed sustained high expression of CLU protein in this conditioned medium, with a final CLU concentration of 200 ng / mL, which was used for subsequent BV2 microglia induction and drug screening experiments.

[0086] A method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia includes the following steps: a. Cell resuscitation and passage: BV2 microglia in the logarithmic growth phase were digested, centrifuged, and seeded into 24-well plates at an appropriate cell density. The plates were then cultured in a cell culture incubator containing 5% CO2 at 37°C.

[0087] b. Specific induction: When BV2 cells adhered to the wall and reached about 70% confluence, the original culture medium was discarded, and the negative control culture medium or specific conditioned medium (NC-CM / CLU-CM) prepared in Example 4 was added to different wells. The cells were incubated for 24 hours. During this process, CLU in the culture medium specifically binds to the microglial cell membrane receptor TREM2, inducing downstream cascade reactions.

[0088] Example 5: This invention discloses a drug screening method. The drug screening method is based on the in vitro drug screening model for trigeminal neuralgia constructed using the above-mentioned method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia. Example 5 uses the NC-CM culture medium and CLU-CM culture medium obtained in Examples 1 and 3, and includes the following steps: 1) Experimental grouping and drug administration: The culture plates inoculated with BV2 microglia were divided into the following three groups: Normal control group (marked as control group in the attached figure): cultured with NC-CM, without other treatment; Model control group (marked as negative control in the attached figure): induced with CLU-CM for 24 hours, without any candidate drugs; Drug screening group: The candidate drug or inhibitor to be tested is added along with CLU-CM.

[0089] like Figure 4 The image shows a comparison of surgical procedures in the rat trigeminal neuralgia animal model (infraorbital nerve CCI model) in Example 5 of this invention.

[0090] 2) Quantitative detection and evaluation of molecular and phenotypic indicators: Twenty-four hours after drug treatment, cell proteins / nucleic acids were extracted or culture supernatant was collected, and the results were analyzed and evaluated based on the following three levels of core molecular indicators: ① Assessment of transmembrane receptor and kinase pathways: Total cellular protein was extracted, and the phosphorylation level of DAP12, a downstream protein of TREM2 activation (pDAP12), and the activation status of the RAS / MEK / ERK pro-inflammatory signaling pathway (pMEK and pERK expression levels) were detected by Western Blot (WB).

[0091] In this embodiment, the specific procedure for detecting the phosphorylation level (pDAP12) of the TREM2-activated downstream protein DAP12 by Western Blot (WB) is as follows: BV2 microglia were seeded into 6-well plates, with 1 × 10⁶ cells per well. 5 Cells were collected and, after adhering to the culture vessel and reaching approximately 70% confluence, were treated with NC-CM and CLU-CM, respectively. In the drug screening group, the candidate drug was added simultaneously with CLU-CM. The final concentrations of small molecule candidate drugs were 0.01 μM, protein antibody candidate drugs were 0.10 μg / mL, and siRNA candidate drugs were 25 nM. Cells were collected after 24 hours of treatment.

[0092] like Figure 5 As shown, the CLU concentration in cells treated with NC-CM was significantly lower than that in cells treated with CLU-CM.

[0093] After discarding the culture medium, the cells were washed twice with pre-cooled PBS. 100 μL of RIPA lysis buffer containing protease and phosphatase inhibitors was added to each well, and the cells were lysed on ice for 20 minutes. The lysis buffer was centrifuged at 12000 g and 4 °C for 10 minutes, and the supernatant was used as the total protein sample. The protein concentration was determined using the BCA method. 20 μg of total protein was loaded into each well, separated by SDS-PAGE, and then transferred to a PVDF membrane.

[0094] After transfer, the membrane was blocked with 5% BSA / TBST at room temperature for 1 hour. Then, pMEK1 / 2, MEK1 / 2, pERK1 / 2, ERK1 / 2, DAP12 / TYROBP, and β-actin were added, and the membrane was incubated overnight at 4°C. The phosphorylation level of DAP12 was determined using an immunoprecipitation-immunoblotting method. First, DAP12 protein was enriched with anti-DAP12 / TYROBP antibody, and then its phosphorylation level was detected with an anti-phosphotyrosine antibody (based on the literature: Morrison DK, et al. Detecting Tyrosine Phosphorylated Proteins by Western Blot Analysis. Cold Spring Harb Protoc. 2010;2010(4):pdb.prot5438). The next day, after washing with TBST, the membrane was incubated with HRP-labeled secondary antibody at room temperature for 1 hour, and then developed using ECL chemiluminescence.

[0095] like Figure 6As shown, in this embodiment, the levels of pDAP12, pMEK, or pERK in the drug screening group were significantly downregulated compared to the model control group, indicating that the drug has the activity of targeting and blocking CLU / TREM2 interaction or inhibiting its early pro-inflammatory signaling cascade.

[0096] ② Assessment of transcriptional repression and ubiquitination axis: The expression of the deubiquitinating enzyme OTULIN, the linear ubiquitination and phosphorylation levels of STAT1 were detected by IP-WB or WB (for specific procedures, please refer to the literature published above).

[0097] Specific test results are as follows: Figure 7 As shown, significant ubiquitination of STAT1 was observed in the negative control group (negative control +); after treatment with conditioned medium expressing CLU, the ubiquitination level of STAT1 decreased. The input control and internal control bands were normal, ruling out false positives and loading errors, and the results are reliable.

[0098] The expression level of the α subunit (NACA) of the neonatal peptide-associated complex was detected by Western blotting.

[0099] like Figure 8 As shown, the bands of pSTAT1, STAT1, OTULIN, and GAPDH in the negative control group and normal control group (the negative control is shown in the attached figure) are lighter than those in the conditioned medium of CLU-overexpressing cells in this embodiment. The conditioned medium of CLU-overexpressing cells in this embodiment induces OTULIN activation, STAT1 deubiquitination and phosphorylation into the nucleus, thereby inhibiting NACA transcription and significantly inhibiting the phosphorylation and activation of STAT1.

[0100] like Figure 9 As shown, this embodiment significantly rescued and upregulated the transcription and protein expression of NACA, thus determining that the candidate drug effectively blocked the key transcriptional abnormalities that lead to endoplasmic reticulum stress.

[0101] ③ Assessment of endoplasmic reticulum stress and inflammation endpoints: The phosphorylation levels of endoplasmic reticulum stress sensor proteins (IRE1α, PERK, ATF6) were detected by Western blotting.

[0102] In this embodiment, shNACA refers to short hairpin RNA (shRNA) designed targeting the NACA gene. After being introduced into cells via a lentiviral vector, it can specifically degrade NACA mRNA using the endogenous RNA interference (RNAi) mechanism, thereby stably inhibiting the expression of NACA protein at the posttranscriptional level.

[0103] The specific process is as follows: First, a shNACA lentiviral vector is constructed. Two to three specific shRNA sequences are designed targeting the coding region of the NACA gene. The corresponding sense and antisense oligonucleotides are synthesized and annealed to form double strands. These oligonucleotides are then ligated into a lentiviral transfer vector digested with restriction endonucleases. After transformation into E. coli, single clones are selected for PCR or enzyme digestion identification. Finally, the shNACA sequence is confirmed to be completely correct through sequencing.

[0104] Lentiviral packaging and preparation were then carried out. 293T cells were cultured to 70%-80% confluence. The shNACA transfer plasmid was mixed with three packaging plasmids in a certain proportion and co-transfected into 293T cells using transfection reagents. Supernatants containing viral particles were collected at 48 and 72 hours after transfection. Cell debris was removed by filtration through a 0.45 μm filter. If necessary, the virus was concentrated by ultracentrifugation. The viral titer was determined by serial dilution of 293T cells combined with puromycin screening.

[0105] Next, target cell transduction and stable cell line screening were performed. Target cells were cultured to the logarithmic growth phase, and lentivirus was added according to the optimal multiplicity of infection determined in the preliminary experiment. At the same time, polybrene was added to improve transduction efficiency. After culturing at 37°C for 24 hours, the culture medium was replaced with fresh medium to remove the virus and polybrene. 48-72 hours after transduction, puromycin at the screening concentration was added. The drug-containing culture medium was replaced every 2-3 days. Screening was carried out for 7-10 days until all uninfected cells died. The surviving cells are the cell population that stably expresses shNACA. Single clones can be selected for further amplification as needed for experiments.

[0106] Finally, to verify the knockdown efficiency, total RNA and total protein were extracted from stable cell lines. The relative expression level of NACA mRNA was detected by real-time quantitative PCR, and the expression level of NACA protein was detected by Western blot. Cells transduced with disordered shRNA were used as negative controls to confirm whether the knockdown effect of the NACA gene met the experimental requirements.

[0107] Test results as follows Figure 10 As shown in the example, the treatment of CLU-overexpressing cells with conditioned medium resulted in a significant reduction in the phosphorylation levels of NACA-1 and NACA-2 compared to the negative control group (the negative control group served as the control).

[0108] The expression intensity of inducible nitric oxide synthase (iNOS) in cells was detected by immunofluorescence. like Figure 11 As shown in the figure, the treatment of CLU cells with conditioned medium to knock down NACA-1 and NACA-2 expression levels was significantly higher than that in the normal control group (negative control).

[0109] Nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in cell culture supernatant were detected by the Griess method or ELISA kit.

[0110] The specific procedure for detecting NO release levels using the Griess method is as follows: Since NO is unstable in aqueous solution, it is usually detected by measuring its stable metabolite, nitrite (NO2). - The level can be used to indirectly reflect the amount of NO released.

[0111] In this embodiment, the Griess reagent kit was used to detect NO2 in the cell culture supernatant. - concentration.

[0112] Before testing, bring Griess reagent, sodium nitrite standard, and the supernatant to room temperature. Prepare a standard curve using sodium nitrite standard; the concentration range can be set to 0, 1, 2, 5, 10, 20, 40, or 80 μM, or according to the kit instructions. Add 100 μL of the standard and the test sample to each well of a 96-well plate. Then add an equal volume of Griess reagent to each well. The Griess reagent can consist of reagent A and reagent B; for example, add 50 μL of Griess reagent A first, mix gently, then add 50 μL of Griess reagent B, and react at room temperature in the dark for 15 minutes.

[0113] After the reaction, the absorbance was measured at 540 nm or 550 nm using a microplate reader. The NO2 concentration in each sample was calculated based on the sodium nitrite standard curve. - NO2 concentration was calculated and expressed as the NO release level. At least three replicates were performed for each sample. Results can be expressed as μM NO2. - It can also be further normalized based on total cellular protein content or cell number, and expressed as μM NO2. - / mg protein or μM NO2 - / 10 5 cells.

[0114] like Figure 12 As shown, NO2 in the drug screening group (NACA knockdown) - The concentration was significantly higher than that of the model control group, suggesting that the candidate drug can promote CLU-CM-induced NO release and pro-inflammatory activation in microglia.

[0115] The specific procedure for ELISA detection of TNF-α secretion concentration is as follows: In one embodiment, a sandwich ELISA kit was used to detect the concentration of TNF-α in the cell culture supernatant. Since BV2 cells are derived from mouse microglia, a mouse TNF-α ELISA kit was used; if rat primary microglia were used, a rat TNF-α ELISA kit was used.

[0116] Before testing, allow the ELISA plate, standards, sample diluent, washing buffer, detection antibody, enzyme conjugate, and chromogenic substrate to reach room temperature. Prepare the TNF-α standard according to the kit instructions, setting the standard curve range to 0–1000 pg / mL or as recommended by the kit. Add 500 μL of the standard, blank wells, and test supernatant to each antibody-coated ELISA plate. If the sample concentration is high, it can be diluted twofold using sample diluent.

[0117] After adding the samples, seal the plate and incubate at 37°C for 1 hour, or incubate at room temperature according to the kit instructions. After incubation, discard the liquid in the wells, wash the plate three times with washing buffer, allowing it to stand for about 30 seconds after each addition of washing buffer, then pat dry thoroughly. Next, add 50 μL of biotinylated detection antibody or enzyme-labeled detection antibody to each well and continue incubation for 30 minutes. After washing, add 50 μL of HRP-labeled streptavidin or HRP-labeled secondary antibody to each well and incubate for 20 minutes. After washing again, add 50 μL of TMB chromogenic buffer to each well and react in the dark for 10 minutes. Once a clear blue gradient appears in the standard curve wells, add 50 μL of stop solution to each well to stop the reaction.

[0118] Use a microplate reader to read the absorbance at 450 nm; if the instrument supports this, 570 nm or 630 nm can be read simultaneously as calibration wavelengths. Calculate the TNF-α concentration of each sample based on the standard curve. Each sample should have at least three replicates. Results are expressed in pg / mL, or normalized based on total cellular protein content or cell number.

[0119] like Figure 13 As shown, the TNF-α concentration in the drug screening group was significantly higher than that in the model control group, indicating that the candidate drug can promote the release of pro-inflammatory cytokines induced by CLU in microglia.

[0120] Combination Figure 12 and Figure 13 It can be seen that the levels of pPERK and pIRE1α in the drug screening group were significantly increased, and the expression of iNOS and the release of NO and TNF-α were significantly higher than those in the model control group. Therefore, it was finally determined that the candidate drug can effectively promote endoplasmic reticulum stress and pathological inflammatory activation in microglia.

[0121] The above description is merely a preferred embodiment of the present invention and does not limit the scope of patent protection of the present invention. Any equivalent structural transformations made based on the description and drawings of the present invention, whether directly or indirectly applied to other related technical fields, are similarly included within the scope of protection of the present invention.

Claims

1. A method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia, characterized in that, Includes the following steps: a. Cell resuscitation and passage: BV2 microglia in the logarithmic growth phase were digested, centrifuged and seeded, and cultured at 37-39°C with 5-7% CO2. b. Specific induction: When BV2 cells adhere to the wall and reach the expected degree of confluence, discard the original culture medium and add negative control medium and cluster protein-rich specific conditioned medium to different wells, and incubate continuously for 24-72 hours; The method for preparing the cluster protein-rich specific conditioned medium includes the following steps: 1) A rat model of trigeminal neuralgia was established by infraorbital nerve ligation; 2) Dissect the arachnoid tissue or related skull base arachnoid tissue from TN model rats, prepare small tissue blocks in pre-cooled PBS or HBSS, add tissue digestion solution, digest, rinse with PBS, filter, and prepare a single-cell suspension; 3) Resuspend the cells in PBS and adjust the cell concentration to 1×10⁻⁶. 6 ~1×10 7 Cells / mL were incubated with fluorescently labeled antibodies against FN1, COL1A2, PDGFR, and α-SMA at 3–7°C in the dark for 30–45 minutes. After washing with PBS, FN1 cells were screened using flow cytometry. + COL1A2 + PDGFR + and α-SMA + Co-expressed fibroblast subsets; 4) After sorting, the cells are seeded in a culture device. After the cells reach 70% to 90% confluence, they are washed several times with PBS and cultured for 24 to 48 hours with serum-free or fetal bovine serum at a concentration of 0.5% to 10%. 5) After collecting the culture supernatant, the cells and cell debris were removed by centrifugation multiple times, filtered, and fibroblast conditioned medium was obtained. The continuous expression of CLU protein in the fibroblast conditioned medium was confirmed by ELISA.

2. The method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia as described in claim 1, characterized in that, The tissue digestion fluid contains 1–2 mg / mL Collagenase IV, 1–2 mg / mL Dispase II, and 50–100 μg / mL DNase I.

3. A drug screening method, wherein the drug screening method is based on an in vitro drug screening model for trigeminal neuralgia constructed using the method for constructing an in vitro drug screening model for trigeminal neuralgia targeting the interaction between fibroblasts and microglia as described in any one of claims 1 to 2, characterized in that, Includes the following steps: 1) The culture equipment inoculated with BV2 microglia should be divided into at least a normal control group, a model control group, and a drug screening group. The normal control group is cultured with negative control medium, the model control group is induced with cluster protein-specific conditioned medium for 24-72 hours, and the drug screening group is conditioned with cluster protein-specific conditioned medium and the candidate drug or inhibitor to be tested. 2) After 24–48 hours of drug treatment, extract cell proteins / nucleic acids or collect culture supernatant for quantitative detection and evaluation of molecular and phenotypic indicators.

4. The drug screening method as described in claim 3, characterized in that, Quantitative detection and assessment of molecular and phenotypic indicators include one or more of the following: assessment of transmembrane receptor and kinase pathways, assessment of transcriptional repression and ubiquitination axis, and assessment of endoplasmic reticulum stress and inflammatory endpoints.

5. The drug screening method as described in claim 4, characterized in that, The assessment of the transmembrane receptor and kinase pathway includes the following procedures: extracting total cellular protein, detecting the phosphorylation level of DAP12, a downstream protein activated by TREM2, and the activation status of the RAS / MEK / ERK pro-inflammatory signaling pathway by Western blotting; or The assessment of the transcriptional repression and ubiquitination axis includes the following procedures: detecting the expression of the deubiquitinating enzyme OTULIN, the linear ubiquitination and phosphorylation levels of STAT1 by IP-WB or WB, and detecting the mRNA and protein expression levels of the α subunit of the nascent polypeptide-associated complex by RT-qPCR and WB; or The assessment of endoplasmic reticulum stress and inflammation endpoints includes the following procedures: detecting the phosphorylation level of endoplasmic reticulum stress sensor proteins by Western blotting, detecting the expression intensity of intracellular inducible nitric oxide synthase by immunofluorescence, and detecting nitric oxide and tumor necrosis factor-α in cell culture supernatant by Griess assay or ELISA kit.

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