Self-absorption cell model after intervertebral disc protrusion as well as construction method and application of self-absorption cell model
By constructing a self-absorbing cell model after disc herniation, simulating the self-absorbing of lumbar disc herniation, the problem of lack of scientific basis in the existing technology was solved, and an in vitro drug screening and evaluation system was established to realize the self-absorbing study of lumbar disc herniation. An important breakthrough provides an efficient drug evaluation method.
Patent Information
- Application Number
- CN202411289142.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2024-12-17
AI Technical Summary
The existing technology cannot effectively simulate and study the self-absorbing mechanism and pathophysiological process after lumbar disc herniation, resulting in a lack of scientific basis for the treatment strategy and a lack of in vitro experimental models to evaluate the self-absorbing effect of drugs.
A model of self-absorbing cells after intervertebral disc herniation was constructed. By preparing a co-culture system for nucleus pulposterior cells and macrophage-osteoclasts, the self-absorbing of intervertebral disc herniation was simulated, and quantitative reverse transcription polymerase was used to quantify Western blotting and real-time fluorescence. Chain amplification technology detects the expression of related proteins and genes and establishes an in vitro experimental system for drug screening.
It provides a standardized, safe and efficient self-absorbing drug evaluation system for in vitro disc herniation, replacing traditional animal experiments, verifying the safety and efficacy of drugs, providing an important reference for the self-absorbing research of lumbar disc herniation, and promoting drug induction Prediction and evaluation of self-absorbing capacity.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cell biology, and in particular to a self-absorption cell model after intervertebral disc herniation, and a construction method and application thereof. Background Art
[0002] Lumbar disc herniation is a degenerative spinal disease and one of the main causes of adult labor force loss. It is characterized by high morbidity and high disability rates, and has placed a huge burden on patients and society. The outer layer of the intervertebral disc is an annulus fibrosus composed of many layers of collagen fibers arranged in concentric circles. The adjacent upper and lower vertebrae are connected by a layer of cartilage endplates. The annulus fibrosus is wrapped with a nucleus pulposus tissue with a high water content and rich in proteoglycans. This structure gives the intervertebral disc excellent mechanical properties to bear gravity loads. When degenerative changes occur in the intervertebral disc, the water content and proteoglycan components of the nucleus pulposus tissue decrease first, resulting in a decrease in mechanical properties, which in turn causes the annulus fibrosus tissue to bear excessive gravity loads. As a result, the outer annulus fibrosus is torn and ruptured, and the inner nucleus pulposus tissue protrudes outward, causing nerve compression.
[0003] At present, a large number of treatment technologies, including various minimally invasive technologies, fusion technologies, dynamic technologies, ablation technologies and other treatment strategies are used to treat lumbar disc herniation with severe symptoms, but they are all invasive operations and symptomatic treatment strategies. They will cause new trauma to patients and may affect their motor function and quality of life to a certain extent. Compared with patients treated conservatively, surgical treatment only quickly relieves patients' symptoms, but compared with conservative treatment and bed rest, surgery has no significant advantages in relieving pain. The large-scale development of surgery has led to a sharp increase in the corresponding treatment costs, and the related costs worldwide are as high as tens of billions of dollars. The currently commonly used conservative treatment strategies, including physical therapy such as traction and massage, and taking anti-inflammatory analgesics, muscle relaxants and other treatment measures, can relieve patients' symptoms to a certain extent, but these treatments cannot promote the absorption of protruding disc tissue, so the compression of nerves still persists. There is a phenomenon in clinical practice: in the process of conservative treatment of some patients with lumbar disc herniation, the protruding disc tissue will self-absorb, and the patient's symptoms will be partially or completely relieved due to the absorption of the disc tissue. This phenomenon is called disc herniation self-absorption. The specific mechanism leading to this phenomenon is still unclear, and there are three main hypotheses: first, the dehydration and contraction of the herniated disc itself reduces its volume; second, the tension of the posterior longitudinal ligament retracts the herniated disc into the intervertebral space; third, the inflammatory response and immune cell-mediated enzymatic reaction and phagocytosis gradually absorb the herniated disc. The third hypothesis is supported by the most clinical phenomena and basic research evidence, but there is currently no cell model that can be used for self-absorption of disc herniation. Therefore, more in vitro experiments need to be developed to study the mechanism and pathophysiological process of self-absorption of disc herniation. Summary of the invention
[0004] The purpose of the present invention is to construct a lumbar disc herniation self-absorption cell model with relatively simple operation, high stability and repeatability, in order to accurately simulate the pathological and physiological characteristics of lumbar disc herniation self-absorption and evaluate the effect of self-absorption after disc herniation.
[0005] The method for establishing a self-absorption model after intervertebral disc herniation of the present invention comprises the following steps:
[0006] 1) Prepare a self-absorption cell model of intervertebral disc herniation and observe the growth of nucleus pulposus cells and changes in extracellular matrix in the environment of intervertebral disc herniation;
[0007] 2) Prepare a self-absorption cell-tissue model after intervertebral disc herniation, observe the self-absorption by co-culturing macrophages-osteoclasts with nucleus pulposus tissue, and detect the expression of related proteins and genes in the culture system.
[0008] In step 1), the nucleus pulposus cells are prepared as follows: the nucleus pulposus tissue of the intervertebral disc is taken, trimmed into a size of about 1mm x 1mm, digested with a digestion solution for 4 hours, filtered and collected; the filtrate is centrifuged at 4°C, the supernatant is discarded to obtain a cell precipitate, and the cell suspension is inoculated into a DMEM culture solution containing 10% FBS after resuspending to obtain nucleus pulposus cells. The digestion solution is a type II collagenase solution, wherein the concentration of type II collagenase is 0.002g / ml.
[0009] In step 1), the inflammatory environment of the nucleus pulposus cells is induced by lipopolysaccharide (LPS) stimulation. The specific method is as follows: the nucleus pulposus cells are cultured in a 12-well plate. When the cells grow to 80% density, the original culture medium in each well is replaced with 2 ml of culture medium containing LPS. The culture time is 24 hours. The LPS concentration is 1 μg / ml.
[0010] The effect of inflammatory environment on the growth of nucleus pulposus cells in step 1) was observed by EdU cell proliferation experiment.
[0011] The toxicity of the inflammatory environment on nucleus pulposus cells in step 1) was observed by live / dead cell staining experiments.
[0012] In step 1), the extracellular matrix of nucleus pulposus cells was observed by Alcian blue staining and immunofluorescence staining.
[0013] The macrophage-osteoclast culture system in step 2) was prepared as follows: Raw 264.7 macrophages were inoculated in a 6-well plate at a density of 80%, and after the cells adhered to the wall, the original culture medium in each well was replaced with 2 ml of culture medium containing nuclear factor κB receptor activator ligand (RANKL), and the culture time was 5 days, during which the culture medium containing RANKL was replaced every other day. The concentration of RANKL was 50 ng / ml.
[0014] The nucleus pulposus tissue inoculated in step 2) is the nucleus pulposus tissue removed during surgical treatment of patients with lumbar disc herniation. The inoculation size is about 0.5 cm×0.5 cm.
[0015] After the nucleus pulposus tissue is inoculated in step 2), the culture system is placed in a living cell workstation for real-time observation for 48 hours.
[0016] In step 2), the related proteins and gene expression were detected by Western blotting (WB) and real-time fluorescence quantitative reverse transcription polymerase chain amplification (RT-qPCR) technology. The related proteins are tartrate acid phosphatase (TRAP), cathepsin K (CTSK), and metalloproteinase 9 (MMP9).
[0017] The present invention provides a self-absorption cell model after intervertebral disc herniation established by the above method.
[0018] The present invention also provides a method for evaluating the self-absorption effect after the above-mentioned intervertebral disc herniation.
[0019] The present invention also provides a method for screening a drug for promoting self-absorption after intervertebral disc herniation, comprising the following steps:
[0020] a. Establish a self-absorption cell model after intervertebral disc herniation according to the above method;
[0021] b. applying the candidate drug to the post-disc herniation self-absorption cell model described in step a;
[0022] c. Screen potential drugs by evaluating the self-absorption effect after disc herniation.
[0023] The beneficial effects of the present invention are:
[0024] The present invention provides an integrated detection strategy by establishing an integrated test system based on intervertebral disc herniation experiments, and develops a disc herniation self-absorption cell model for disc herniation self-absorption efficacy detection under an inflammatory environment. The constructed disc model is used to establish a standardized, safe, and efficient in vitro disc herniation self-absorption drug evaluation system, as one of the methods to replace traditional animal experiments, and the in vitro evaluation method for the safety and efficacy of a single drug verifies its experimental stability, repeatability, and safety as an alternative method, providing an important reference for the study of lumbar disc herniation self-absorption, and providing prediction and evaluation for the drug-induced lumbar disc self-absorption ability.
[0025] Lumbar disc herniation self-absorption is a relatively rare clinical phenomenon. Existing studies have shown that it is accompanied by inflammatory reactions and infiltration of new blood vessels. The rarity of self-absorption in clinical practice has also promoted the exploration of its mechanism. The in vitro constructed lumbar disc herniation self-absorption cell model has extremely high application value.
[0026] The invention can effectively establish a self-absorption cell model after intervertebral disc herniation, and the preparation method is simple and can be used for screening drugs that induce self-absorption of intervertebral disc herniation, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a diagram of different growth states of nucleus pulposus cells;
[0028] Figure 2 The state diagrams of normal nucleus pulposus cells and nucleus pulposus cells stimulated by LPS are shown respectively; A is a normal nucleus pulposus cell diagram; B is a nucleus pulposus cell diagram stimulated by LPS;
[0029] Figure 3The proliferation status of normal nucleus pulposus cells and nucleus pulposus cells stimulated by LPS is shown; A is the result of EdU cell proliferation staining of normal nucleus pulposus cells; B is the result of EdU cell proliferation staining of nucleus pulposus cells stimulated by LPS;
[0030] Figure 4 The live / dead staining results of normal nucleus pulposus cells and nucleus pulposus cells stimulated with LPS are shown; A is the live / dead staining results of normal nucleus pulposus cells; B is the live / dead staining results of nucleus pulposus cells stimulated with LPS;
[0031] Figure 5 The Alcian blue staining results of normal nucleus pulposus cells and nucleus pulposus cells stimulated by LPS are shown; A is the Alcian blue staining result of normal nucleus pulposus cells; B is the Alcian blue staining result of nucleus pulposus cells stimulated by LPS;
[0032] Figure 6 This is a picture showing the results of macrophage-osteoclast absorption of nucleus pulposus tissue observed by the living cell workstation;
[0033] Figure 7 This is the result of Trap staining of the macrophage-osteoclast culture system;
[0034] Figure 8 This is the result of Trap staining after the macrophage-osteoclast culture system was inoculated with nucleus pulposus tissue;
[0035] Fig. 9 The mRNA expression level of ACP5, CTSK, and MMP9 in the macrophage-osteoclast culture system inoculated with nucleus pulposus tissue;
[0036] Fig.10 This is a graph showing the expression levels of Trap, CTSK, and MMP9 proteins in the macrophage-osteoclast culture system inoculated with nucleus pulposus tissue. DETAILED DESCRIPTION
[0037] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.
[0038] The words "include" or "comprising" mentioned throughout the specification and claims are open-ended terms and should be interpreted as "including but not limited to". The following description of the specification is a preferred embodiment of the present application, but the description is for the purpose of the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.
[0039] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.
[0040] Example 1 Preparation of the self-absorption cell model after intervertebral disc herniation of the present invention
[0041] A cell model of self-absorption after intervertebral disc herniation was constructed in vitro, and the growth of nucleus pulposus cells and the expression of extracellular matrix were evaluated.
[0042] 1. Construction of a self-absorption cell model after intervertebral disc herniation
[0043] 1.1 Source and preparation of nucleus pulposus cells
[0044] The nucleus pulposus tissue of the intervertebral disc of C57 mice was taken, trimmed into a size of about 0.5 cm×0.5 cm, digested with digestive fluid for 4 hours, filtered and collected; the filtrate was centrifuged at 4°C, the supernatant was discarded to obtain a cell precipitate, and the cell suspension was inoculated into a DMEM culture fluid containing 10% FBS after resuspending to obtain nucleus pulposus cells. The digestive fluid was a type II collagenase solution, wherein the concentration of type II collagenase was 0.002 g / ml. The cell culture conditions were cultured in a 37°C, 5% CO2 incubator.
[0045] The nucleus pulposus cell growth diagram obtained is as follows Figure 1 shown.
[0046] 1.2 Induction of inflammatory environment in nucleus pulposus cells after disc herniation
[0047] The inflammatory environment of nucleus pulposus cells was induced by lipopolysaccharide (LPS) stimulation. The specific method was as follows: the nucleus pulposus cells were cultured in a 12-well plate. When the cells grew to 80% density, the original culture medium in each well was replaced with 2 ml of culture medium containing LPS. The culture time was 24 hours. The LPS concentration was 1 μg / ml. The cell culture conditions were 37°C and 5% CO2 incubator. The results are shown in Figure 2. Figure 2 As shown in A and B, compared with the nucleus pulposus cells in normal culture, the nucleus pulposus cells in the inflammatory environment induced by LPS stimulation have shrinkage and deformation, and the number of cells is reduced.
[0048] 2 Model evaluation
[0049] 2.1 Evaluation of nucleus pulposus cell growth
[0050] EdU cell proliferation assay: After nucleus pulposus cells were stimulated with LPS (1 μg / mL) for 24 hours, the cells were TMAccording to the instruction manual of EdUCell Proliferation Kit with Alexa Fluor 555 (Biyuntian), normal nucleus pulposus cells and LPS-stimulated nucleus pulposus cells were labeled with EdU and stained with cell nuclei, and detected using laser confocal microscopy (CLSM, LSM880). Figure 3 As shown in A and B, compared with nucleus pulposus cells cultured normally, the proliferation ability of nucleus pulposus cells cultured in an inflammatory environment is weakened.
[0051] Cell live / dead staining: After the nucleus pulposus cells were stimulated with LPS (1 μg / mL) for 24 hours, the normal nucleus pulposus cells and the nucleus pulposus cells stimulated with LPS were stained with Calcein AM / PI in accordance with the instructions of the Calcein / PI Cell Viability / Cytotoxicity Assay Kit (Biyuntian), and detected using laser confocal microscopy (CLSM, LSM 880). Figure 4 As shown in A and B, compared with nucleus pulposus cells cultured normally, the number of dead nucleus pulposus cells cultured in an inflammatory environment increases.
[0052] 2.2 Evaluation of extracellular matrix expression by nucleus pulposus cells
[0053] 2.2.1 Preparation of cell slides: Nucleus pulposus cells were inoculated onto cell slides in 24-well plates, cultured for 24 hours until the cells adhered to the wall, and then stimulated with LPS for 24 hours. The LPS concentration was 1 μg / ml. The cell culture conditions were 37°C and 5% CO2 incubator.
[0054] 2.2.2 Alcian blue staining: Take out the cell slide prepared in 1.4.1 from the 24-well plate, immerse in Alcian blue staining solution (Biyuntian) for 3 minutes, wash with running water, seal the slide and take pictures under a microscope.
[0055] The results are as follows Figure 5 As shown in A and B, compared with nucleus pulposus cells cultured normally, the extracellular matrix expression of nucleus pulposus cells cultured in an inflammatory environment is weakened.
[0056] Example 2 Preparation of self-absorption cell-tissue model after intervertebral disc herniation
[0057] A self-absorption cell-tissue model after intervertebral disc herniation was constructed in vitro, and the absorption of the tissue was evaluated.
[0058] 1. Construction method of the self-absorption cell-tissue model after intervertebral disc herniation of the present invention
[0059] 1.1 Establishment of macrophage-osteoclast culture system
[0060] Raw 264.7 macrophages (Pnosai) were seeded in 6-well plates at a density of 80%. After the cells adhered to the wall, the original culture medium in each well was replaced with 2 ml of culture medium containing nuclear factor κB receptor activator ligand (RANKL). The culture time was 5 days, during which the culture medium containing RANKL was replaced every other day. The RANKL concentration was 50 ng / ml.
[0061] 1.2 Co-culture of macrophage-osteoclast system and nucleus pulposus tissue
[0062] 1.2.1 Obtaining nucleus pulposus tissue
[0063] The nucleus pulposus tissue removed during surgery for lumbar disc herniation was disinfected, repeatedly rinsed with saline to remove blood and other components, and then trimmed to a size of 0.5 cm × 0.5 cm.
[0064] 1.2.2 Cell-tissue co-culture
[0065] Raw 264.7 macrophages (Pnosai) were seeded in a 6-well plate at a density of 80%. After the cells adhered to the wall, the nucleus pulposus tissue obtained in 1.2.1 was added to each well of the 6-well plate, and the original culture medium in each well was replaced with 2 ml of culture medium containing nuclear factor κB receptor activator ligand (RANKL). The culture time was 5 days, during which the culture medium containing RANKL was replaced every other day. The concentration of RANKL was 50 ng / ml.
[0066] 2. Model Evaluation
[0067] 2.1 Live cell workstation detection
[0068] After the macrophage-osteoclast system and nucleus pulposus tissue were prepared, they were placed in a living cell workstation to observe the phagocytosis of nucleus pulposus tissue by macrophages and osteoclasts. Figure 6 As shown, macrophages and osteoclasts phagocytized the nucleus pulposus tissue.
[0069] 2.2 Osteoclast Trap staining
[0070] Discard the culture medium and nucleus pulposus tissue in the 6-well plate in 1.2, fix with 4% tissue cell fixative (Solabo) for 15 minutes, cover the cells with 0.2% Triton X-100 solution (Solabo) for 20-30 minutes, and wash with distilled water 3 times. Then add TRAP working solution (Sevier) to the cell well plate to cover the cells, incubate at 37℃ in the dark for 15-20 minutes, and wash with distilled water 3 times. Observe and take pictures under a microscope.
[0071] The results are as follows Figure 7 and Figure 8As shown, nucleus pulposus tissue can induce more osteoclast formation, further promoting the digestion and absorption of nucleus pulposus tissue.
[0072] 2.3 RT-qPCR assay to detect the expression of self-absorption-related genes
[0073] Macrophages and nucleus pulposus cells were collected after 5 days of co-culture with nucleus pulposus tissue in 1.2, and total RNA was extracted using Small Volume Total RNA Extraction Kit (Jianshi), and complementary DNA was prepared from 10 μg of total RNA using reverse transcriptase and oligodeoxyribonucleic acid primers. qRT-PCR was performed using the CFX96 Touch qRT-PCR system (Bio-Rad). All reactions were repeated three times and normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
[0074] The results are as follows Fig. 9 As shown, the self-absorption cell-tissue model after intervertebral disc herniation can promote the mRNA expression of enzymes related to intervertebral disc tissue self-absorption, thereby promoting intervertebral disc self-absorption.
[0075] 2.4WB experiment to detect the expression of self-absorption related proteins
[0076] Macrophages and nucleus pulposus cells co-cultured with nucleus pulposus tissue for 5 days in 1.2 were collected and lysed using RIPA lysis buffer (Biyuntian). Protease phosphatase inhibitors (Biyuntian) were added during lysis. After lysis, the protein concentration was determined using a NanoVue spectrophotometer. After SDS-PAGE (80V, 120 minutes), the membranes were transferred to polyvinylidene difluoride membranes. After blocking with 5% milk, they were incubated with the following primary antibodies (anti-Trap, anti-CTSK, anti-MMP9) (1:1000 dilution) at 4°C overnight, and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution) at room temperature for 1 hour. Enhanced chemiluminescence was used to detect signals (KPL, MD, USA). β-actin was used as a control. Western blot was repeated 3 times for each batch of cells.
[0077] The results are as follows Fig.10 As shown, the self-absorption cell-tissue model after intervertebral disc herniation can promote the high expression of enzymes related to intervertebral disc tissue self-absorption, thereby promoting intervertebral disc self-absorption.
[0078] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for constructing a self-absorption cell model after intervertebral disc herniation, comprising: 1) Take intervertebral tissue from the intervertebral disc with intervertebral disc herniation, obtain the nucleus pulposus cells therein, and observe the growth status of the nucleus pulposus cells and the state of the extracellular matrix; The nucleus pulposus cells are prepared according to the following method: the nucleus pulposus tissue of the intervertebral disc is taken, trimmed into a size of about 1mm x 1mm, digested with a digestion solution for 4 hours, filtered and collected the digestion solution; the filtrate is centrifuged at 4°C, the supernatant is discarded to obtain a cell precipitate, and the cell suspension is inoculated into a DMEM culture solution containing 10% FBS after resuspending to obtain nucleus pulposus cells, wherein the digestion solution is a type II collagenase solution, wherein the concentration of the type II collagenase is 0.002g / ml; and / or, 2) By co-culturing macrophages-osteoclasts with nucleus pulposus tissue, the self-absorption was observed and the expression of related proteins and genes was detected.
2. The construction method according to claim 1, characterized in that: Step 1) also includes using lipopolysaccharide (LPS) to stimulate and induce an inflammatory environment in the nucleus pulposus cells. Preferably, the inflammatory environment in the nucleus pulposus cells is achieved by a method comprising the following steps: the nucleus pulposus cells are cultured in a 12-well plate, and when the cells grow to 80% density, the original culture medium in each well is replaced with 2 ml of culture medium containing LPS, and the culture time is 24 hours; the LPS concentration is 1 μg / ml.
3. The construction method according to claim 2, characterized in that: Step 1) also includes detecting cell proliferation, toxicity of inflammatory environment to nucleus pulposus cells, and / or the state of extracellular matrix of nucleus pulposus cells; Preferably, the effect of the inflammatory environment on the growth of nucleus pulposus cells is detected by EdU cell proliferation assay; Preferably, the toxicity of the inflammatory environment to the nucleus pulposus cells is detected by live / dead cell staining experiments; Preferably, the detection of the state of the extracellular matrix of the nucleus pulposus cells is performed by Alcian blue staining and immunofluorescence staining.
4. The construction method according to claim 1, characterized in that: The macrophage-osteoclast culture system described in step 2) is prepared according to the following method: Raw 264.7 macrophages are inoculated in a 6-well plate at a density of 80%. After the cells adhere to the wall, the original culture medium in each well is replaced with 2 ml of culture medium containing nuclear factor κB receptor activator ligand (RANKL). The culture time is 5 days, during which the culture medium containing RANKL is replaced every other day, and the RANKL concentration is 50 ng / ml.
5. The construction method according to claim 4, characterized in that: The nucleus pulposus tissue inoculated in step 2) is the nucleus pulposus tissue removed during surgical treatment of patients with lumbar disc herniation, and the inoculation size is about 0.5 cm×0.5 cm.
6. The construction method according to claim 4, characterized in that: After the nucleus pulposus tissue is inoculated in step 2), the culture system is placed in a living cell workstation for real-time observation for 48 hours.
7. The construction method according to claim 4, characterized in that: In step 2), the detection of related protein and gene expression is achieved by Western blotting (WB) and / or real-time fluorescence quantitative reverse transcription polymerase chain amplification (RT-qPCR); preferably, the related proteins are tartrate acid phosphatase (TRAP), cathepsin K (CTSK), and metalloproteinase 9 (MMP9).
8. The self-absorbing cell model of intervertebral disc herniation constructed by the construction method according to any one of claims 1 to 7.
9. Use of the intervertebral disc herniation self-absorption cell model as claimed in claim 8 in screening drugs for promoting intervertebral disc herniation self-absorption.
10. A method for screening a drug for promoting self-absorption after intervertebral disc herniation, comprising: a. Constructing a self-absorption cell model after intervertebral disc herniation according to the method of any one of claims 1 to 7; b. applying the candidate drug to the post-disc herniation self-absorption model described in step a; c. Screen and obtain drugs that promote self-absorption after intervertebral disc herniation based on the evaluation of the self-absorption effect after intervertebral disc herniation.