Use of leptin receptor positive cells in the preparation of a product for preventing or treating intervertebral disc degeneration

Abstract

This invention relates to the field of biomedical technology, specifically to the use of leptin receptor-positive cells in the preparation of products for the prevention or treatment of intervertebral disc degeneration. This invention provides a novel treatment strategy for intervertebral disc degeneration, namely, repairing degenerated intervertebral discs by supplementing with LepR+ cells. This strategy has advantages such as minimal invasiveness, long-lasting repair effects, and fewer complications, overcoming the limitations of existing surgical and conservative treatments, and providing new ideas and technical support for the clinical treatment of intervertebral disc degenerative diseases. Furthermore, this invention constructs a mouse model susceptible to intervertebral disc degeneration, in which the rate of intervertebral disc degeneration after injury is much higher than in normal mice. This model provides an efficient experimental tool for studying the mechanisms of intervertebral disc degeneration and screening treatment strategies.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of leptin receptor-positive cells in the preparation of products for the prevention or treatment of intervertebral disc degeneration. Background Technology

[0002] Intervertebral disc degeneration is a common degenerative disease of the spine, mainly characterized by a reduction in the number of nucleus pulposus cells, degradation of the extracellular matrix, and destruction of the annulus fibrosus structure, leading to decreased disc height and weakened mechanical stability. This disease can cause symptoms such as chronic low back pain, neck and shoulder pain, and numbness and weakness in the limbs. In severe cases, it can also lead to secondary complications such as disc herniation, spinal stenosis, and segmental instability, significantly impacting the patient's quality of life. Epidemiological surveys show that intervertebral disc degeneration has a high incidence rate among middle-aged and elderly people, and this rate increases with age. Statistics show that over 80% of adults experience varying degrees of low back pain at some point in their lives, a significant portion of which is related to intervertebral disc degeneration. This disease not only causes physical suffering but also imposes a substantial socioeconomic burden, including medical expenses and loss of labor capacity.

[0003] Currently, clinical treatment for intervertebral disc degeneration is mainly divided into two categories: conservative treatment and surgical treatment. Conservative treatment includes drug therapy (such as nonsteroidal anti-inflammatory drugs and muscle relaxants), physical therapy, and behavioral intervention. Its purpose is to relieve pain and improve function, but it cannot reverse the degenerative process that has already occurred, and its long-term efficacy is limited. For patients with severe symptoms or those who do not respond to conservative treatment, surgical treatment is often used, such as discectomy, interbody fusion, and artificial disc replacement. Surgery can relieve nerve compression and restore spinal stability, but it has drawbacks such as significant trauma, long recovery periods, and high costs. Furthermore, interbody fusion sacrifices the mobility of the affected segment and may accelerate degeneration of adjacent segments; while artificial disc replacement preserves mobility to some extent, it carries risks such as prosthesis wear and loosening, and uncertain long-term survival rates. Overall, existing treatment methods mostly focus on symptom control or structural replacement, and are difficult to fundamentally restore the tissue structure and biological function of the intervertebral disc.

[0004] In recent years, cell-based regenerative medicine has shown promise in the treatment of various tissue injuries and degenerative diseases. For example, in disease models such as osteoarthritis, myocardial injury, and liver fibrosis, transplantation of specific cell types can promote tissue repair and slow disease progression. These studies suggest that cell therapy strategies may provide a new biological intervention for degenerative diseases.

[0005] However, research on cell therapy for intervertebral disc degeneration is still in the exploratory stage. It is necessary to gain a deeper understanding of the development and degeneration process of the intervertebral disc at the cellular level and to develop new treatment strategies based on this understanding. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, and in order to address the problems of insufficient understanding of intervertebral disc degeneration at the cellular level and the lack of fundamental and effective means to address intervertebral disc degeneration, the purpose of this application is to provide the use of leptin receptor-positive cells in the preparation of products for the prevention or treatment of intervertebral disc degeneration, and a method for constructing an animal model susceptible to intervertebral disc degeneration, so as to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, this application first provides the use of leptin receptor-positive cells (LepR+ cells) in the preparation of products for the prevention and / or treatment of intervertebral disc degeneration.

[0008] This application further provides a method for constructing an animal model susceptible to intervertebral disc degeneration, comprising the following steps: specifically knocking out leptin receptor-positive cells in an animal to obtain the animal model susceptible to intervertebral disc degeneration.

[0009] This application also provides the use of the intervertebral disc degeneration susceptible animal model obtained by the above construction method in at least one of the following:

[0010] A. Screening for drugs to prevent and / or treat intervertebral disc degeneration;

[0011] B. To study the etiology, course, or pathogenesis of intervertebral disc degeneration.

[0012] Finally, this application provides a method for screening drugs to prevent or treat intervertebral disc degeneration, comprising the following steps: applying candidate drugs to an animal model susceptible to intervertebral disc degeneration obtained by the above construction method.

[0013] Compared with the prior art, the beneficial effects of this application are as follows:

[0014] 1) This application clarifies for the first time the important role of LepR+ cells in intervertebral disc development and degeneration repair, fills the gap in the application of LepR+ cells in the field of intervertebral discs, provides new experimental evidence for the origin of intervertebral disc tissue development, and enriches the research on the molecular mechanisms of intervertebral disc development and degeneration.

[0015] 2) This application confirms that LepR+ cells are stem and progenitor cells with multi-directional differentiation potential. They have stem cell characteristics and can directly participate in the repair and regeneration of intervertebral disc tissue. Compared with traditional treatment methods, they have stronger targeting and repair effects and can fundamentally delay or reverse the process of intervertebral disc degeneration.

[0016] 3) This application provides a new treatment strategy for intervertebral disc degeneration, namely, repairing degenerated intervertebral discs by supplementing LepR+ progenitor cells. This strategy has advantages such as minimal trauma, long-lasting repair effect, and fewer complications, overcoming the limitations of existing surgical and conservative treatments, and providing new ideas and technical support for the clinical treatment of intervertebral disc degenerative diseases.

[0017] 4) LepR+ cells can be directly sorted from intervertebral disc tissue, which is convenient and highly specific. After in vitro culture and expansion, they can be mass-produced, which is convenient for industrial application and has broad prospects for clinical translation.

[0018] 5) The intervertebral disc degeneration-susceptible mouse model constructed in this application exhibits a significantly faster rate of intervertebral disc degeneration after injury induction compared to normal mice. This characteristic allows for the observation of the continuous pathological process of the intervertebral disc from early degeneration to late-stage structural destruction within a shorter experimental period, thereby significantly shortening the model preparation and observation time and improving research efficiency. Simultaneously, the rapid degeneration rate means that a richer collection of cellular and molecular event samples can be accumulated within the same timeframe, facilitating the dynamic tracking of key aspects such as nucleus pulposus cell reduction, matrix degradation, and microenvironmental changes, which is beneficial for in-depth analysis of the cellular mechanisms of intervertebral disc degeneration. Regarding treatment strategy screening, this model can present a clear and quantifiable degeneration phenotype in a short period, making the evaluation of the effects of candidate cell therapies or drug interventions faster and more sensitive, reducing the lengthy experimental period and inter-group variability caused by the slow natural degeneration. Therefore, this model provides an efficient experimental tool for studying the mechanisms of intervertebral disc degeneration and screening treatment strategies. Attached Figure Description

[0019] Figure 1 LepR-cre;tdtomato fl / fl A schematic diagram of mouse construction. (The diagram shows...) Figure 1 A is a schematic diagram of the Cre-loxP system. When Cre-positive mice are crossed with TD-Tomato mice, Cre can recognize and bind to the LoxP sequence located at the upstream STOP site of TD-Tomato, thereby removing the STOP site and making Cre-positive cells labeled with red light. Figure 1 B demonstrates the creation of LepR-cre;tdtomato mice by crossing LepR-cre mice with R26R-Td-tomato mice. fl / fl A diagram of a mouse; Figure 1 C is for LepR-cre;tdtomato fl / fl Image showing the results of genotyping in mice.

[0020] Figure 2 LepR-cre;tdtomato fl / flAutofluorescence distribution in coronal sections of the L4-5 intervertebral disc of mice 7 days after birth.

[0021] Figure 3 LepR-cre;tdtomato fl / fl Autofluorescence distribution in coronal sections of the L4-5 intervertebral disc of mice 14 days after birth.

[0022] Figure 4 LepR-cre;tdtomato fl / fl Autofluorescence distribution in coronal sections of the L4-5 intervertebral disc of mice 4 weeks after birth.

[0023] Figure 5 LepR-cre;tdtomato fl / fl Autofluorescence distribution in coronal sections of the L4-5 intervertebral discs of mice 8 weeks after birth.

[0024] Figure 6 LepR-cre;tdtomato fl / fl Autofluorescence distribution in coronal sections of the L4-5 intervertebral discs of mice 16 weeks after birth.

[0025] Figure 7 LepR-cre;tdtomato fl / fl Autofluorescence distribution in coronal sections of the L4-5 intervertebral discs of mice at 52 weeks of age.

[0026] Figure 8 LepR-cre;tdtomato fl / fl Autofluorescence distribution in coronal sections of the L4-5 intervertebral discs of mice at 75 weeks of age.

[0027] Figure 9 The graph shows the change in the proportion of LepR+ cells in nucleus pulposus (NP) cells in mice of different ages. Scale bar = 50 micrometers.

[0028] Figure 10 A graph showing the change in the proportion of LepR+ cells in the annulus fibrosus (AF) in mice of different ages, scale bar = 50 micrometers.

[0029] Figure 11 A schematic diagram illustrating the construction of the LepR-cre;DTA mouse. (The diagram shows the mouse structure.) Figure 11A is a schematic diagram of the Cre-loxP system. When Cre-positive mice are crossed with DTA mice, Cre can recognize and bind to the LoxP sequence located at the STOP site upstream of DTA, thereby removing the STOP site and killing Cre-positive cells. Figure 11 B demonstrates the creation of LepR-cre;DTA mice by crossing LepR-cre mice with R26R-EGFP-DTA mice; Figure 11 Figure C shows the genotype identification results of LepR-cre;DTA mice.

[0030] Figure 12 Imaging findings of spinal instability and puncture injury modeling. Among them, Figure 12 A shows a comparison of lumbar spine X-rays between the Lepr-cre and Lepr-cre;DTA groups 4 weeks after the establishment of the mouse spinal instability model. Figure 12 B shows a comparison of X-ray images of mouse tails in the Lepr-cre and Lepr-cre;DTA groups 4 weeks after the establishment of the mouse tail puncture injury model. Figure 12 C represents the intervertebral disc height index measurement method: 2*(D+E+F) / (A+B+C+G+H+I); Figure 12 D is a quantitative analysis chart of the intervertebral disc height index.

[0031] Figure 13 This image shows the pathological results after creating a model for spinal instability. Figure 13 Figures A and C show the staining results of HE, Safranin-Fix-Green, and Sirius Red, respectively, indicating that the L4-5 intervertebral discs of Lepr-cre;DTA mice showed more severe degenerative changes 4 weeks after spinal instability modeling. Scale bar = 200 micrometers.

[0032] Figure 14 This is a graph showing the histological scoring results.

[0033] Figure 15 This is a diagram illustrating the stemness analysis of leptin receptor-positive cells in the intervertebral disc. Among them, Figure 15 A is the result diagram of CFU-F in LepR+ cells; Figure 15 B shows the differentiation results of the three lineages of LepR+ cells: chondrogenesis (left), osteoogenesis (middle), and adipogenesis (right).

[0034] Figure 16 The image shows the effect of transplanting LepR+ positive cells on intervertebral disc degeneration caused by injury. Figure 16 A represents the cell transplantation process and key time points; Figure 16 BC shows the imaging changes of the intervertebral disc in mice 30 days after transplantation of LepR+ cells; Figure 16D shows the pathological changes (Safranin-Firmgreen) of the intervertebral disc in mice 30 days after transplantation of LepR+ cells. Detailed Implementation

[0035] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0036] The first aspect of this application provides the use of leptin receptor-positive cells in the preparation of products for the prevention and / or treatment of intervertebral disc degeneration.

[0037] In this invention, "leptin receptor positive cells" refers to cells that express leptin receptor (LepR).

[0038] In some specific embodiments, the leptin receptor-positive cells are intervertebral disc leptin receptor-positive cells.

[0039] In this invention, "intervertebral disc" refers to a fibrocartilaginous structure located between two adjacent vertebral bodies, including the central nucleus pulposus, the surrounding annulus fibrosus, and the cartilaginous endplates that connect to the vertebral bodies.

[0040] In this invention, "intervertebral disc degeneration" refers to the degenerative changes that occur in the structure and function of the intervertebral disc, including a reduction in nucleus pulposus cells, degradation of the extracellular matrix, a decrease in the water content of the nucleus pulposus, rupture or fissure formation of the annulus fibrosus, a decrease in the height of the intervertebral disc, and the resulting pathological process of decreased mechanical properties or decreased intervertebral stability of the intervertebral disc.

[0041] In some specific embodiments, the product is a drug or a medical device.

[0042] In this invention, "medications for preventing intervertebral disc degeneration" refers to drugs that can prevent or delay the occurrence of intervertebral disc degeneration, reduce the risk of intervertebral disc degeneration, or slow its progression, and are usually administered to individuals who do not show clinical manifestations or radiographic signs of intervertebral disc degeneration.

[0043] In this invention, "medications for treating intervertebral disc degeneration" refers to drugs that can reduce, improve or reverse the pathological changes of intervertebral disc degeneration, relieve related symptoms, restore or maintain the structure and function of the intervertebral disc, and prevent or alleviate further deterioration of intervertebral disc degeneration. They are usually administered to individuals who have clinical manifestations or imaging signs of intervertebral disc degeneration.

[0044] In some specific embodiments, the drug is an injectable drug.

[0045] Preferably, the drug is an injection drug for the intervertebral disc.

[0046] In this invention, "intervertebral disc injection drug" refers to a drug preparation that is delivered directly to the intervertebral disc or its adjacent area by local injection, thereby distinguishing it from systemic drug administration routes such as oral administration (absorption via the digestive tract) and intravenous administration (systemic distribution via blood circulation).

[0047] In some specific embodiments, the drug includes leptin receptor-positive cells and one or more pharmaceutically acceptable carriers or excipients.

[0048] In this invention, "pharmaceutically acceptable carriers or excipients" refers to carriers or excipients used to formulate leptin receptor-positive cell injection drugs that are compatible with leptin receptor-positive cells and have no toxic side effects on the receptors. These carriers or excipients can maintain the activity and function of leptin receptor-positive cells and facilitate their dispersion, delivery, or colonization in vivo. The carriers or excipients include, but are not limited to: physiological saline, phosphate-buffered saline (PBS), balanced salt solutions (such as Hank's balanced salt solution, Ringer's solution), glucose solution, human serum albumin, autologous serum, extracellular matrix components (such as hyaluronic acid, collagen, gelatin, fibrinogen / thrombin), trehalose, dimethyl sulfoxide (DMSO, for cryopreservation), hydroxyethyl starch, polyvinyl alcohol, polyethylene glycol (PEG), dextran, mannitol, sorbitol, amino acids, vitamins, inorganic salts, and gel-like or thermosensitive hydrogel materials suitable for leptin receptor-positive cell injection (such as Pluronic F127, chitosan-based gel).

[0049] In this invention, a "medical device for preventing intervertebral disc degeneration" refers to a medical device containing an effective ingredient (leptin receptor-positive cells) that, when applied to an individual who has not shown clinical manifestations or imaging signs of intervertebral disc degeneration, can prevent or delay the occurrence of intervertebral disc degeneration, reduce the risk of intervertebral disc degeneration, or slow its progression.

[0050] In this invention, a "medical device for treating intervertebral disc degeneration" refers to a medical device containing an effective ingredient (leptin receptor-positive cells) that, when applied to an individual with clinical manifestations or imaging signs of intervertebral disc degeneration, can reduce, improve, or reverse the pathological changes of intervertebral disc degeneration, alleviate related symptoms, restore or maintain the structure and function of the intervertebral disc, and prevent or alleviate further deterioration of intervertebral disc degeneration.

[0051] In some specific embodiments, the medical device is a drug delivery device for the intervertebral disc.

[0052] In this invention, "intervertebral disc site drug delivery device" refers to a medical device that can deliver leptin receptor-positive cells to the intervertebral disc site or its adjacent area through local, direct or targeted means, thereby distinguishing it from systemic drug delivery routes such as oral delivery and intravenous delivery. It includes, but is not limited to, intervertebral disc puncture injection needles, microneedle arrays, pedicle catheters, intervertebral disc built-in sustained-release pumps, locally implantable sustained-release gel carriers, percutaneous endoscopic intervertebral disc drug delivery catheters, and intervertebral disc built-in stents or adhesive drug delivery patches loaded with leptin receptor-positive cells.

[0053] In some embodiments, the medical device includes leptin receptor-positive cells and one or more pharmaceutically acceptable carriers or excipients.

[0054] In some specific embodiments, the product has the function of improving the disordered state of the nucleus pulposus tissue structure.

[0055] In some specific embodiments, the product has the function of delaying or inhibiting the hardening of the extracellular matrix of nucleus pulposus cells.

[0056] In some specific embodiments, the product has the function of delaying or inhibiting the mineralization of the extracellular matrix of nucleus pulposus cells.

[0057] In some specific embodiments, the product has the function of reducing the decrease in intervertebral disc height.

[0058] In this invention, "improving the disordered state of the nucleus pulposus tissue structure" refers to the ability to correct or alleviate the disordered state caused by abnormal cell arrangement, fibrous structure or extracellular matrix distribution in the nucleus pulposus tissue, so as to restore it to a normal tissue structure or make it more orderly.

[0059] In this invention, "delaying or inhibiting the hardening of the extracellular matrix of nucleus pulposus cells" refers to the ability to slow down or prevent the process of hardening of the extracellular matrix of nucleus pulposus cells, such as increased stiffness and decreased elasticity, thereby maintaining or restoring its normal biomechanical properties.

[0060] In this invention, "delaying or inhibiting the mineralization of the extracellular matrix of nucleus pulposus cells" refers to the ability to slow down or prevent the abnormal calcium salt deposition, hydroxyapatite crystal formation, or calcified nodule formation in the extracellular matrix of nucleus pulposus cells, thereby inhibiting or delaying the pathological calcification of nucleus pulposus tissue.

[0061] In this invention, "reducing the decrease in intervertebral disc height" refers to the ability to slow down, stop or partially restore the process of decreasing intervertebral disc height, thereby maintaining or increasing the relative height of the intervertebral disc in imaging or morphology, and delaying or preventing the narrowing of the intervertebral space caused by intervertebral disc degeneration.

[0062] The second aspect of this application provides a method for constructing an animal model susceptible to intervertebral disc degeneration, comprising the following steps:

[0063] By specifically knocking out leptin receptor-positive cells in animals, the aforementioned animal model susceptible to intervertebral disc degeneration was obtained.

[0064] In this invention, an "animal model susceptible to intervertebral disc degeneration" refers to an animal model that, after specific treatment, exhibits a significantly higher rate of occurrence, progression, or severity of intervertebral disc degeneration than normal control mice when subjected to the same intervertebral disc injury induced by factors such as acupuncture, surgery, or load. This is manifested in pathological changes such as loss of nucleus pulposus cells, degradation of extracellular matrix, and decrease in intervertebral disc height occurring much faster than in normal mice.

[0065] In some specific embodiments, the specific knockout of leptin receptor-positive cells in the animal is achieved through genetic methods.

[0066] Preferably, the genetic method includes the following steps:

[0067] A first non-human animal that specifically expresses Cre recombinase in leptin receptor-positive cells is crossed with a second non-human animal carrying a conditionally expressed DTA element to obtain offspring animals. The conditionally expressed DTA element comprises a first loxP site, a transcription termination sequence, a second loxP site, and a DTA coding sequence connected in sequence. The offspring animals simultaneously inherit both the Cre recombinase and the conditionally expressed DTA element. In leptin receptor-positive cells, the Cre recombinase mediates the excision of the transcription termination sequence, thereby allowing the expression of the DTA coding sequence, and consequently, the leptin receptor-positive cells in the animal are cleared.

[0068] In some specific embodiments, the animal is selected from one or more of mice, rats, hamsters, pigs, rabbits, and monkeys.

[0069] Preferably, the animal is selected from mice.

[0070] The third aspect of this application provides for the use of the animal model susceptible to intervertebral disc degeneration obtained by the construction method described in the second aspect in at least one of the following:

[0071] A. Screening for drugs to prevent and / or treat intervertebral disc degeneration;

[0072] B. To study the etiology, course, or pathogenesis of intervertebral disc degeneration.

[0073] In this invention, "the cause of intervertebral disc degeneration" refers to various initial factors or inducing factors that lead to or promote the occurrence and progression of intervertebral disc degeneration.

[0074] In this invention, "the course of intervertebral disc degeneration" refers to the entire time process of intervertebral disc degeneration from its occurrence and development to its outcome, as well as the various stages it goes through.

[0075] In this invention, "pathogenesis of intervertebral disc degeneration" refers to the inherent laws and mechanisms of biology, pathology, and biomechanics involved in the occurrence and development of intervertebral disc degeneration.

[0076] The fourth aspect of this application provides a method for screening drugs to prevent or treat intervertebral disc degeneration, comprising the following steps: applying a candidate drug to an animal model susceptible to intervertebral disc degeneration obtained by the construction method described in the second aspect.

[0077] Specifically, the candidate drugs to be screened are administered to animal models susceptible to intervertebral disc degeneration. The changes in intervertebral disc degeneration-related indicators (such as intervertebral disc height, nucleus pulposus structure, extracellular matrix status, etc.) in the animal models are then observed to assess whether the candidate drugs have the effect of preventing or treating intervertebral disc degeneration.

[0078] The present invention will be further illustrated by the following examples, but these examples do not limit the scope of the invention.

[0079] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are conventional products that can be purchased commercially. In addition to the specific methods, equipment, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described, used, and materials in the embodiments of this invention may be used to implement this invention.

[0080] Example 1: Constructing LepR-cre;tdtomato fl / fl Mouse, tracing LepR+ cells

[0081] Lineage tracing technology has become a popular technique in recent years. It uses site-specific recombinase systems in gene targeting technology to permanently label specific cells at a genetic level at a specific time, recording the activity of all labeled cells and their progeny cells, thereby enabling the tracking and observation of cell fate. Therefore, lineage tracing technology can better reveal the clonal dynamics and proliferation characteristics of proliferating cells, as well as their contributions to tissue and organ growth, development, degeneration, and repair processes.

[0082] The Cre / loxP recombinase system is the most commonly used site-specific recombinase system for lineage tracing. Specifically, when Cre-positive mice are crossed with TD-Tomato mice, Cre can recognize and bind to the LoxP sequence located upstream of the STOP site in TD-Tomato mice, thereby removing the STOP site and allowing Cre-positive cells to be labeled with red light (…). Figure 1 A).

[0083] To investigate the expression and distribution characteristics of LepR+ cells in mouse intervertebral discs and their role in disc development and repair, this application crossbred LepR-cre mice with R26R-Td-tomato mice to construct LepR-cre;tdtomato fl / fl Mice (Fig. 1B). Both LepR-cre and R26R-Td-tomato mice were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd.

[0084] LepR-cre;tdtomato fl / fl Genomic DNA was extracted from mouse tail tip tissue, and the target transgene / knock-in fragment was specifically amplified by polymerase chain reaction (PCR) to identify LepR-cre;tdtomato fl / fl Mouse genotype (e.g.) Figure 1 (As shown in C). The PCR system used in this application was a mutant (MT) specific amplification system. The wild-type (WT) genome had no amplification products (labeled as NA, i.e., no amplification). After separation by agarose gel electrophoresis, the size and position of the bands were observed using a UV imaging system. The results confirmed that the above mice successfully knocked into the tdTomato fluorescent reporter gene at the ROSA26 site, with a mutant positive genotype, and simultaneously carried the ObRb-Cre recombinase transgene.

[0085] Example 2: Quantity and distribution characteristics of LepR+ cells in various tissue components of the intervertebral disc in mice of different ages.

[0086] To further determine the distribution characteristics of LepR+ cells, this application conducted an in-depth study of LepR-cre;tdtomato fl / fl The autofluorescence distribution of coronal sections of the L4-5 intervertebral discs in mice at different ages (7d, 14d, 4w, 8w, 16w, 52w and 75w) from early birth to late adulthood was shown, with R26R-Td-tomato mice used as negative controls (Figures 2-8).

[0087] The results showed that: LepR-cre;tdtomato fl / flSeven days after birth, the proportion of LepR+ cells in the nucleus pulposus (NP) of mice was 89.3%. This proportion gradually decreased, reaching 70.6% by 52 weeks of age. However, no further decrease was observed in aged mice at 75 weeks of age. Figure 9 The proportion of LepR+NP cells gradually decreases after birth, revealing the potential role of LepR+NP cells in intervertebral disc homeostasis, since the loss of notochord (NC) cells in the postnatal period is considered a marker of the onset of intervertebral disc degeneration.

[0088] Furthermore, this application discovered LepR-cre;tdtomato fl / fl Seven days after birth, LepR+ cells constituted only a small proportion of the annulus fibrosus (AF) cells. This proportion increased significantly from 14 days to 4 weeks of development, reaching 44.4% by 52 weeks. Figure 10 It is noteworthy that all LepR+ AF cells were outer annulus fibrosus (OAF) cells, rather than inner annulus fibrosus (IAF) cells. This suggests that LepR+ cells residing in the OAF after birth are unlikely to have migrated from NP cells, but may have migrated from the IVD niche (between the OAF and the cartilaginous endplate) or the bloodstream, which could promote calcification of the annulus fibrosus during intervertebral disc degeneration.

[0089] Example 3: LepR+ cells play an important role in the development and degenerative repair of mouse intervertebral discs.

[0090] 3.1 Construct Lepr-cre;DTA mice and specifically knock out LepR+ cells.

[0091] The Cre / loxP recombinase system was used again. Specifically, when Cre-positive mice were crossed with DTA mice, Cre could recognize and bind to the LoxP sequence located at the upstream STOP site of DTA, thereby removing the STOP site and killing Cre-positive cells (Figure 11A).

[0092] To investigate the specific role of LepR+ cells in intervertebral disc development, this application crossbred LepR-cre mice with R26R-EGFP-DTA mice to construct LepR-cre;DTA mice (… Figure 11 B). Both LepR-cre mice and R26R-EGFP-DTA mice were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd.

[0093] Genomic DNA was extracted from the tail tip tissue of experimental mice, and the target transgene / knock-in fragment was specifically amplified by polymerase chain reaction (PCR) to identify the genotype of the experimental mice. The PCR system used in this study was a mutant (MT) specific amplification system; wild-type (WT) genomes showed no amplification products (labeled NA, i.e., no amplification). After separation by agarose gel electrophoresis, the size and position of the bands were observed using a UV imaging system. The results confirmed the PCR agarose gel electrophoresis identification of the experimental mouse genotypes, detecting three elements: Rosa26-LSL-tdTomato (expected 196 bp), ObRb(LepR)-Cre (expected 225 bp), and Rosa26-LSL-EGFP-DTA (expected 357 bp). The results showed that specific bands of the expected size appeared in tdTomato positive samples (lanes 2, 4, 5, 6), Cre positive samples (lanes 1, 2, 4, 5, 6), and DTA positive samples (lanes 18, 19, 24). The results for the positive control, wild-type, and blank control were all normal, indicating that the PCR system was reliable and that LepR-cre;tdTomato and LepR-Cre;DTA double transgenic mice suitable for subsequent experiments were successfully screened.

[0094] 3.2 Effects of LepR+ cell knockout on intervertebral disc degeneration repair in mice

[0095] To further explore the role of LepR+ cells in the repair of intervertebral disc degeneration, this application constructed a mouse spinal instability model and a mouse tail acupuncture degeneration model, respectively.

[0096] Construction of a mouse model of spinal instability:

[0097] 1) Preoperative preparation

[0098] The equipment and supplies required for the surgery include surgical microscopes, gas anesthesia machines, surgical instruments, sterile suture needles, iodine solution, saline solution, sterile gauze, gloves, and disinfectant alcohol.

[0099] 2) Animal preparation

[0100] C57BL / 6J mice, LepR-cre mice, and LepR-cre;DTA mice were used, and all mice were housed in an SPF-grade animal facility. Five mice were housed in each cage, with ample food and water provided. The animal facility was set to a 12-hour diurnal lighting system.

[0101] 3) Preoperative anesthesia

[0102] Mice were placed in an anesthesia induction chamber for induction of anesthesia, with the oxygen flow rate set at 0.4 liters per minute and the isoflurane concentration at 4%. Once the mice reached a comatose state, they were switched to endotracheal anesthesia to maintain the anesthetic effect. At this point, the isoflurane concentration was adjusted to 1.5%, while the oxygen flow rate remained at 0.4 liters per minute. Surgical procedures were only performed after confirming that the mice's heart rate and respiration were stable.

[0103] 4) Modeling process

[0104] a) Skin preparation: Use a small animal trimmer to remove hair from the surgical area on the mouse's back and clean up any remaining hair.

[0105] b) Expose the facet joints.

[0106] The lumbar region was identified, and a posterior midline incision was made. The L4-5 facet joints were exposed by blunt dissection layer by layer. Under an operating microscope, the spinous process, bilateral inferior facet joints, and adjacent interspinous ligaments were removed using microsurgical needle forceps.

[0107] Incision suturing: The subcutaneous fascia and skin were sutured layer by layer using 5-0 sutures. The surgical area was disinfected to prevent infection. Postoperatively, the animal was placed on a temperature-controlled plate to await recovery from anesthesia.

[0108] Construction of a mouse tail acupuncture degeneration model:

[0109] 1) Preparation

[0110] First, the mice were anesthetized by intraperitoneal injection of avodin, and then the mice were fixed supine on the modeling table. Next, the tails of the mice were disinfected three times in sequence using physiological saline, povidone-iodine solution, and 75% alcohol.

[0111] 2) Positioning process

[0112] The caudal intervertebral space was precisely located using small animal X-ray imaging equipment, with the target being Co6-8.

[0113] 3) Setting up experimental and control groups

[0114] For the Co6-7 intervertebral disc as the experimental group, the needle was inserted vertically into the Co6-7 intervertebral disc 1.5 mm, ensuring the needle remained parallel to the lamina. Then, the needle was rotated 180° axially and held in this position for 10 seconds. The Co7-8 intervertebral disc remained intact as the control segment. Finally, the needle was removed, and the mouse tail was disinfected three times with iodine solution and 75% alcohol to complete the modeling process.

[0115] Intervertebral disc condition was continuously observed in LepR-cre mice and LepR-cre;DTA mice for 4 weeks after modeling.

[0116] like Figure 12 As shown in AB, compared with the LepR-cre group, the intervertebral discs of the LepR-cre group mice showed a trend of intervertebral disc degeneration, such as decreased height and subchondral bone sclerosis.

[0117] To investigate the progression of intervertebral disc degeneration, this application measured the disc-height index (DHI) of LepR-cre and LepR-cre;DTA mice after tail needle injury (Figure 12C) and compared it with the intervertebral disc of the LepR-cre intact control group.

[0118] Radiographic results showed that, compared with the intact control group (LepR-cre), the disc degeneration index (DHI) of LepR-cre and LepR-cre;DTA mice gradually decreased starting in the first week after injury-induced disc degeneration. Quantitative analysis showed that the mean DHI of the LepR-cre group mice was 100% before injury, and was 82.1%, 64.0%, and 50.8% at weeks 1, 2, and 4 after injury, respectively. In contrast, the DHI of the LepR-cre;DTA group mice decreased to 69.3%, 39.5%, and 19.8% at weeks 1, 2, and 4, respectively (Figure 12D).

[0119] Meanwhile, the pathological staining results, consisting of HE staining, safranin-fast green staining, and Sirius red staining, also indicated that the intervertebral discs of LepR-cre;DTA mice showed more severe degenerative changes 4 weeks after spinal instability modeling. Figure 13 AC, specifically manifested as a decrease in the number of nucleus pulposus cells, disordered collagen fiber arrangement, and disruption of the NP / AF boundary. Correspondingly, the pathological score of Lepr-cre;DTA mice was significantly higher than that of LepR-cre mice 4 weeks after modeling ( Figure 14 ).

[0120] The pathological scoring adopted the classic histological scoring standard for intervertebral disc degeneration (modified Thompson score), as follows:

[0121] The total score is 0-12 points, with higher scores indicating more severe degeneration.

[0122] 1) Nucleus pulposus morphology (0-3 points)

[0123] 0 points: The nucleus pulposus is full, with abundant cells and an intact structure;

[0124] 1 point: Mild shrinkage of the nucleus pulposus, with a small reduction in cells;

[0125] 2 points: The nucleus pulposus is significantly atrophied, with sparse cells and increased cavities;

[0126] 3 points: The nucleus pulposus is severely collapsed, with a large number of cells missing, almost to the point of fibrosis.

[0127] 2) Disordered fibrous ring structure (0-3 points)

[0128] 0 points: The fiber rings are neatly arranged and clearly layered;

[0129] 1 point: Localized fiber misalignment and a few cracks;

[0130] 2 points: The annulus fibrosus is obviously torn and delaminated, with increased gaps;

[0131] 3 points: The fiber rings are extensively broken, and the structure is loose and disordered.

[0132] 3) Changes in cell number and morphology (0-3 points)

[0133] 0 points: Cells are normal in morphology and evenly distributed;

[0134] 1 point: A small number of cells show vacuolation and mild hypertrophy;

[0135] 2 points: Significant apoptosis and vacuolar degeneration of cells;

[0136] 3 points: Extremely reduced cells, with extensive necrosis and calcification.

[0137] 4) Degree of extracellular matrix loss / calcification (0-3 points)

[0138] 0 points: Rich matrix, deep Safranin O staining, no calcification;

[0139] 1 point: Slight reduction in matrix, lighter staining, no calcification;

[0140] 2 points: Significant loss of matrix, with localized calcifications;

[0141] 3 points: Severe depletion of the matrix, extensive calcification and ossification.

[0142] Level Classification:

[0143] 0-3 points: Normal intervertebral disc;

[0144] 4-6 points: Mild degeneration;

[0145] 7-9 points: Moderate degeneration;

[0146] 10-12 points: Severe degeneration.

[0147] Example 4: LepR+ cells in the intervertebral disc are a type of stem cell.

[0148] In this application, flow cytometry was used to sort LepR+ cells from the damaged intervertebral discs of 4-week-old LepR-cre;tdtomato mice. The specific process is as follows:

[0149] 1) Prepare the flow cytometer and ensure that the equipment has been calibrated and is in normal working order.

[0150] 2) The cell suspension obtained after digesting the damaged intervertebral disc tissue of LepR-cre;tdtomato mice was transferred to the sample tube of the flow cytometer, and a suitable laser and filter were set to detect the tdTomato fluorescence signal.

[0151] 3) Start the flow cytometer, select the cell sorting mode, set the gate based on the tdTomato positive signal, and start sorting LepR+ cells.

[0152] 4) Collect the sorted LepR+ cells.

[0153] Then, its function was verified using the CFU-F assay. The results showed that approximately 89.1% of LepR+ cells isolated from the injured intervertebral disc were able to form CFU-F colonies in vitro. Figure 15 A).

[0154] To assess whether LepR+ cells from injured intervertebral discs exhibit stem cell-like characteristics in cell culture, osteogenic, chondrogenic, and adipogenic differentiation experiments were performed on collected LepR+ cells. The results showed that LepR+ cells possess the potential to differentiate into three lineages: chondrocytes (Alcian blue positive chondrocytes), osteoblasts (Alizarin Red positive mineralized matrix), and adipocytes (Oil Red positive). Figure 15 B).

[0155] All these findings suggest that LepR+ cells are important progenitor cells for intervertebral disc repair.

[0156] Example 5: In vivo study of the role of LepR+ cells in intervertebral disc degeneration repair

[0157] To verify whether supplementing LepR+ cells is a potential therapeutic strategy, this study conducted efficacy experiments in mice.

[0158] First, a rat tail acupuncture degeneration model was established according to the aforementioned method to ensure that the injury site was clearly visible. The puncture injury was located in the Co4-5, Co5-6, and Co6-7 intervertebral discs of the coccyx, with Co7-8 used as a non-surgical control.

[0159] Then, the LepR+ cells sorted in Example 4 were transferred to Matrigel-containing medium and gently mixed to ensure uniform cell distribution. The Matrigel-containing medium was prepared as follows: First, a normal medium (containing 10% fetal bovine serum, 1% penicillin-dextrose antibody, and 89% DMEM) was prepared. Then, Matrigel (purchased from Corning) was diluted with this normal medium to a final concentration of 3–5 mg / mL. The diluted mixture was then dispensed into each well at a concentration of 50–100 μL (210 μL / cm²). 2 Add to the culture plate and incubate at 37°C for 30 minutes to form a gel.

[0160] Using a microsyringe, administer at a rate of 1×10 5 A dose of 1 × 10⁺ cells was administered via slow injection of Matrigel suspension containing LepR+ cells into the site of the intervertebral disc injury (Co6-7 intervertebral disc), ensuring uniform cell distribution within the injury area. Similarly, at the same dose (1 × 10⁺ cells), 5 A Matrigel suspension containing LepR-cells was slowly injected into the Co5-6 intervertebral disc injury site, while a Matrigel suspension without cells was slowly injected into the Co4-5 intervertebral disc injury site. After injection, the reaction at the injection site was observed to ensure there was no significant bleeding or other adverse reactions.

[0161] Experimental results showed that puncture led to a decrease in the height index of the Co4-5 to Co6-7 intervertebral discs, while the administration of LepR+ cells significantly alleviated the degenerative process compared with LepR- cells and the control group (as shown in Figures 16B and 16C).

[0162] Based on the results of safranin-fast green staining ( Figure 16 As shown in D), this application found that one month after the acupuncture injury, all model groups showed different degrees of degeneration. Among them, the intervertebral discs of the control group and the LepR- group showed severe degeneration, obvious disorder of the nucleus pulposus tissue structure, and pathological remodeling of hardening and mineralization of the extracellular matrix, while the degree of degeneration in the LepR+ group was relatively mild.

[0163] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. Use of leptin receptor-positive cells in the preparation of products for the prevention and / or treatment of intervertebral disc degeneration.

2. Use according to claim 1, characterized in that, The leptin receptor-positive cells are intervertebral disc leptin receptor-positive cells.

3. Use according to claim 1, characterized in that, The product is a drug or a medical device.

4. Use according to claim 3, characterized in that, The drug is an intervertebral disc injection drug; and / or, the drug includes leptin receptor-positive cells, and one or more pharmaceutically acceptable carriers or excipients. And / or, the medical device is an intervertebral disc site delivery device; and / or, the medical device includes leptin receptor-positive cells and one or more pharmaceutically acceptable carriers or excipients.

5. The use according to claim 1, characterized in that, The product has at least one of the following functions: a) Improve the disordered state of the nucleus pulposus tissue structure; b) Delay or inhibit the hardening of the extracellular matrix of the nucleus pulposus; c) Delay or inhibit the mineralization of the extracellular matrix of nucleus pulposus cells; d) Reduce the decrease in intervertebral disc height.

6. A method for constructing an animal model susceptible to intervertebral disc degeneration, comprising the following steps: By specifically knocking out leptin receptor-positive cells in animals, the aforementioned animal model susceptible to intervertebral disc degeneration was obtained.

7. The construction method of claim 6, wherein, The specific knockout of leptin receptor-positive cells in the animal was achieved through genetic methods, which included the following steps: The first non-human animal that specifically expresses Cre recombinase in leptin receptor-positive cells was crossed with the second non-human animal that conditionally expresses DTA elements to obtain offspring animals; The conditionally expressed DTA element comprises a first loxP site, a transcription termination sequence, a second loxP site, and a DTA coding sequence connected in sequence. The offspring animals simultaneously inherit the Cre recombinase and the conditionally expressed DTA element. In leptin receptor-positive cells, the Cre recombinase mediates the excision of the transcription termination sequence, thereby allowing the expression of the DTA coding sequence, and subsequently clearing leptin receptor-positive cells from the animal body.

8. The construction method of claim 6, wherein, The animal is selected from one or more of mice, rats, hamsters, pigs, rabbits, and monkeys; preferably, the animal is selected from mice.

9. The use of the intervertebral disc degeneration susceptible animal model obtained by the construction method according to any one of claims 6 to 8 in at least one of the following: A. Screening for drugs to prevent and / or treat intervertebral disc degeneration; B. To study the etiology, course, or pathogenesis of intervertebral disc degeneration.

10. A method for screening drugs for the prevention or treatment of intervertebral disc degeneration, comprising the following steps: administering a candidate drug to an animal model susceptible to intervertebral disc degeneration obtained by the construction method as described in any one of claims 6 to 8.

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