Application of Clmp in diagnosis and treatment of bone injury

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of Clmp in diagnosis and treatment of bone injury. Specifically, the specific high expression of the Clmp gene in bone regeneration promotion of beta-TCP (beta tricalcium phosphate) and the effect of the Clmp gene on ECM (extracellular matrix) formation are found for the first time through a multi-scale transcriptomics technology. Experiments prove that the Clmp gene is not only a key endogenous target spot for activating osteogenic differentiation by beta-TCP, but also plays an important role in promoting extracellular matrix formation and osteogenic differentiation. The invention lays a scientific foundation for the targeted design of bone repair materials and the application of the Clmp gene in bone injury diagnosis and treatment, so that the method has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and particularly relates to the application of Clmp in the diagnosis and treatment of bone injuries. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Globally, there are as many as 180 million fractures of various types each year, and the demand for bone injury repair is huge. However, many bone defects have not yet been able to achieve complete healing through self-repair. Since the 1970s, calcium phosphate ceramics have been widely used in clinical practice as a classic artificial bone repair material. Numerous studies have shown that they have good biocompatibility, osteoconductivity, and osteoinductivity. Among them, β-tricalcium phosphate seems to have better osteoinductivity, but its exact mechanism is still unclear. Unfortunately, bone repair biomaterials such as calcium phosphate ceramics generally face bottleneck problems such as insufficient bioactivity and are unable to meet the needs of clinical applications. As a result, autologous bone transplantation remains the irreplaceable "gold standard" to date, but it faces many limitations such as donor restrictions, complications, and patient complications.

[0004] The unsatisfactory performance of various bone repair biomaterials reflects the shortcomings of existing biomaterial design and optimization methods. The fundamental reason is believed to be the lack of effective methods to achieve a comprehensive and accurate understanding of the interaction between biomaterials and tissue cells. It is generally believed that the current classic biomaterial "trial and error" research methodology has three shortcomings. First, it relies on the "trial and error" method to conduct limited and biased evaluations of cell behavior, and is unable to comprehensively evaluate the complex biological effects and molecular mechanisms between biomaterials and cells. Second, it focuses too much on the study of biological effects of a single static node, and is unable to understand the interaction between biomaterials and cells and tissues in time and space. Third, screening and evaluation are inefficient, time-consuming and costly. Therefore, there is an urgent need to construct new evaluation methods to fully and accurately understand the effects and mechanisms of biomaterials and tissue cells, so as to promote breakthroughs in the research of the next generation of biomaterials.

[0005] Recent studies have shown that endogenous key genes during embryonic development, such as lineage-specific genes and regenerative genes, play a crucial role in tissue regeneration and repair. Combining single-cell transcriptomes with bulk transcriptomes can comprehensively and accurately reveal the regulatory roles of key endogenous genes, overcoming the limitations of single-cell methods, which often lack depth and accuracy. For example, studies combining single-cell and bulk transcriptomes have revealed the key role of hsd17b12a in embryonic digestive organ development, the regulatory function of Scx in tendon and fibrocartilage differentiation, and the role of Slco1c1 in endothelial cell differentiation in mouse embryos. However, for evaluating the relationship between biomaterials and cells, there is still a lack of reports on multi-scale omics evaluation methods that integrate single-cell and bulk transcriptomes.

[0006] Multi-omics evaluation methods are considered a key development direction for building next-generation biomaterials design and optimization methodologies, although systematic research in this area is currently lacking. Developing targeted multi-scale omics evaluation methods based on the specific characteristics of tissue development and growth, and from the perspective of the regulation of relevant endogenous key genes, is a valuable area of ​​exploration.

[0007] In recent years, the development of single-cell transcriptome technology has provided new ideas for analyzing the heterogeneity of cell populations and the functions of key genes, but multi-scale analysis methods that combine it with bulk transcriptomes have not been reported in the evaluation of bone repair materials. It is particularly noteworthy that although extracellular matrix (ECM) deposition is the core link of osteogenic differentiation, the core genes that regulate ECM formation and their mechanism of action in material-mediated bone regeneration are still unclear. Among them, the Clmp (CXADR-like membrane protein) gene is an important regulatory factor for cell adhesion and intercellular connection. Its encoded protein belongs to the tight junction protein family and is usually involved in maintaining cell polarity, regulating intercellular adhesion and transmembrane signaling, especially playing a key role in tissue development and homeostasis. Studies have shown that Clmp affects epithelial barrier function by mediating cell-cell interactions and has potential functions in inflammation, tumor microenvironment regulation and organ formation. However, the inventors found that there has been no relevant research on its role in osteogenic differentiation and bone regeneration. Summary of the Invention

[0008] In view of the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide the application of Clmp in the diagnosis and treatment of bone injuries. Specifically, the present invention discovered for the first time through multi-scale transcriptomics technology the specific high expression of the Clmp gene in β-TCP (β tricalcium phosphate)-promoted bone regeneration and its effect on ECM formation. Experiments have confirmed that the Clmp gene is not only a key endogenous target for β-TCP to activate osteogenic differentiation, but also plays an important role in promoting extracellular matrix formation and osteogenic differentiation. The present invention lays a scientific foundation for the targeted design of bone repair materials and the application of the Clmp gene in the diagnosis and treatment of bone injuries. Based on the above research results, the present invention is completed.

[0009] Specifically, the technical solution of the present invention is as follows:

[0010] The first aspect of the present invention provides a use of any one of the following (a1)-(a4) in preparing a bone injury detection product;

[0011] (a1) Clmp gene;

[0012] (a2) Clmp protein;

[0013] (a3) Reagents for detecting Clmp gene expression;

[0014] (a4) Reagent for detecting Clmp protein expression.

[0015] The bone injury detection includes screening, (auxiliary) diagnosis, monitoring or prognostic detection of bone injuries.

[0016] A second aspect of the present invention provides a system for bone injury detection, the system comprising:

[0017] An acquisition unit is configured to: acquire the expression level of Clmp in a sample to be tested by a subject;

[0018] The evaluation unit is configured to evaluate and judge the disease condition of the subject according to the subject Clmp obtained by the acquisition unit.

[0019] Wherein, the Clmp includes Clmp gene and / or Clmp protein.

[0020] The third aspect of the present invention provides the use of Clmp gene / protein as a target in the preparation and / or screening of bone injury drugs.

[0021] A fourth aspect of the present invention provides the use of a substance for promoting Clmp gene / protein expression or increasing its activity in any one or more of the following:

[0022] (c1) promoting osteogenic differentiation or preparing a product that promotes osteogenic differentiation;

[0023] (c2) promoting extracellular matrix formation or preparing a product that promotes extracellular matrix formation;

[0024] (c3) promoting bone tissue formation or preparing products that promote bone tissue formation;

[0025] (c4) preparing products that promote bone damage repair;

[0026] (c5) Products for the prevention and / or treatment of bone damage.

[0027] The product may be a drug, a medical device, or an experimental reagent for non-medical use.

[0028] A fifth aspect of the present invention provides a product for preventing and / or treating bone damage, the product comprising a substance that promotes the expression of Clmp gene / protein or increases its activity and a bone damage repair material.

[0029] A sixth aspect of the present invention provides the use of a substance that inhibits Clmp gene / protein expression or reduces its activity in constructing a bone injury repair model.

[0030] The present invention has demonstrated through experiments that knocking down the Clmp gene in osteoblasts can effectively inhibit the expression of Runx2 and Alp (key indicators of osteogenic differentiation), as well as the expression of Fn and Col1A (key indicators of extracellular matrix formation), and inhibit β-TCP-mediated bone tissue formation, thereby being used to construct bone damage repair cell and animal models for basic scientific research.

[0031] A seventh aspect of the present invention provides a method for preventing and / or treating bone damage, comprising: administering to a subject the substance that promotes the expression of the Clmp gene / protein or increases its activity.

[0032] Beneficial technical effects of one or more of the above technical solutions:

[0033] The above technical solution established a multi-scale transcriptomics method to discover the differences in the biological effects of different bone repair materials from the gene regulation level, revealing the key function of the Clmp gene in osteogenic differentiation and extracellular matrix formation. The Clmp gene is not only a key endogenous target for β-TCP to activate osteogenic differentiation, but also plays an important role in promoting extracellular matrix formation and osteogenic differentiation, thereby playing an important role in the diagnosis and treatment of bone injuries.

[0034] The above technical solution provides a new perspective for evaluating the relationship between biomaterials and tissue cells, and at the same time provides an important methodological basis for the design and optimization of the next generation of bone regeneration materials. Therefore, it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0036] Figure 1 Figure 1 shows the effects of the four calcium phosphate materials on the osteogenic differentiation capacity and volume of MC3T3 microspheres in Example 1. A. Bright field microspheres on day 7; B. Live / Dead staining of microspheres on day 7; C. ARS staining of microspheres on day 7; (D–F) Volume changes of microspheres treated with the four materials on days 1, 3, and 7; (G–I) ARS quantitative results of microspheres treated with the four materials on days 1, 3, and 7.

[0037] Figure 2 The transcriptome analysis of HA and β-TCP for 1-7 days in Example 2 of the present invention is as follows: A. Volcano plot of differentially expressed genes of HA and β-TCP on the first day, red: differential up-regulation, blue: differential down-regulation, gray: no statistical significance; B. Functional enrichment of differentially expressed genes on the first day, filtered by Pvalue<0.05; C. GSEA enrichment plot of Calcineurin-Nfat Signaling Cascade of differentially expressed genes on the third day; D. GSEA enrichment plot of Calcineurin-Mediated Signaling of differentially expressed genes on the third day; E. Volcano plot of differentially expressed genes of HA and β-TCP on the third day; F. Functional enrichment of differentially expressed genes on the third day, filtered by Pvalue<0.05; G. GSEA enrichment plot of Bone Mineralization of differentially expressed genes on the third day; H. GSEA enrichment plot of Collagen-Containing Extracellular Matrix of differentially expressed genes; I. Volcano plot of differentially expressed genes of HA and β-TCP on the seventh day; J. Functional enrichment of differentially expressed genes on the seventh day, filtered by Pvalue<0.05; K. Bone enrichment of differentially expressed genes on the seventh day. GSEA enrichment map of Mineralization; GSEA enrichment map of Collagen-Containing Extracellular Matrix of differentially expressed genes on day 7. Figure 3This is the process of establishing the embryonic bone development lineage in Example 3 of the present invention. A (left) Cell subpopulations during the E7.5-9.5 period, grouped as Seurat_clusters; A (right) Cell subpopulations during the E7.5-9.5 period, grouped as orig.ident; B (left) Cell subpopulations during the E9.5-13.5 period, grouped as Seurat_clusters; B (right) Cell subpopulations during the E9.5-13.5 period, grouped as orig.ident; C Cell subpopulations during the E7.5-9.5 period, transcriptional changes of the top 16 genes in the E7.5-E9.5 biological evolutionary trajectory, Moran's index <0.3; C Cell subpopulations during the E9.5-13.5 period, transcriptional changes of the top 16 genes in the E9.5-E13.5 biological evolutionary trajectory, Moran's index <0.3. Figure 4 This is the time series analysis of the AGSE186832 data set from day 0 to day 21 in Example 3 of the present invention; B GO function enrichment of Cluster 2 genes, filtered by p < 0.05; C Venn diagram of E7.5-9.5 biological evolution Track_gene, E9.5-13.5 biological evolution Track_gene and Cluster 2 genes; D GO function enrichment of 452 intersection genes, filtered by p < 0.05; E Intersection Venn diagram of day3H vs day3C and 452 intersection genes; F GO function enrichment of 6 HA-related endogenous genes, filtered by p < 0.05; G Intersection Venn diagram of day3β vs day3C and 452 intersection genes; GO function enrichment of 20 key genes related to Hβ-TCP, filtered by p < 0.05; I Violin plot of Clmp expression distribution; J UMAP plot of Clmp expression distribution; K Clmp expression levels on days 1-7.

[0038] Figure 5A is the expression change of Clmp protein under HA and β-TCP induction on the first day in Example 4 of the present invention; B is the expression change of Clmp protein under HA and β-TCP induction on the third day; C is the gray value analysis of Clmp expression on the first day, ***P<0.001; D is the gray value analysis of Clmp expression on the third day, **P<0.01; E is the expression change of Fn under HA and β-TCP induction on the first day (2D); F is the expression change of Fn under HA and β-TCP induction on the first day (3D); G is the expression change of Fn under HA and β-TCP induction on the third day (2D); H is the expression change of Fn under HA and β-TCP induction on the third day (3D); IL: the gray value analysis of Fn expression corresponding to EH, ***P<0.001, **P<0.01, *P<0.05; M, N, O: the expression changes of Clmp, Runx2 protein and Fura-2 AM in MSCs cells under HA and β-TCP induction on the third day; P UMAP plot of E9.5-E13.5 grouped by Clmp expression level; Q Clmp_high vs Clmp_low differential gene GO enrichment.

[0039] Figure 6 Comparison of knockdown efficiency of MC3T3-NC, sh1#, 2#, and 3# in Example 5 of the present invention; B Clmp expression in MC3T3-sh3# and shNC cells, ****P<0.0001; C (upper) Fn protein expression in MC3T3-sh3# and shNC cells after 3 days of β-TCP culture, ****P<0.0001; C (lower) Fn protein expression in MC3T3-sh3# and shNC cells after 7 days of β-TCP culture, ****P<0.0001; D-F Fura-2AM, Alp, and Runx2 protein expression levels in MC3T3-sh3# and shNC cell microspheres after 3 days of β-TCP culture, **P<0.01, ***P<0.001, ****P<0.0001; G-I Sp7, Spp1, and Alp gene expression levels in MC3T3-Widetype, shNC, and sh3# cell microspheres after 7 days of β-TCP culture, **P<0.01,

[0040] ***P<0.001,****P<0.0001.

[0041] Figure 7 a, immunofluorescence of mesenchymal stem cells MSC-sh3#; b, Runx2 expression of mesenchymal stem cells MSC-sh3#; c, Alizarin red staining results of mesenchymal stem cells MSC-sh3# in Example 5 of the present invention. Figure 8The following are the immunoblotting results of Clmp overexpression in Example 6 of the present invention (a); immunofluorescence results of Clmp overexpression; CCK8 assay (cell proliferation assay) of Clmp overexpression; and alkaline phosphatase level detection of Clmp overexpression. *P<0.1, **P<0.01.

[0042] Figure 9 A shows the volume change of subcutaneous bone tissue 7-14 days after NC, sh3# cells were premixed with β-TCP and injected into BALB / c nude mice in Example 7 of the present invention, *P<0.05; B shows HE staining of bone tissue in NC, sh3# groups at 14 days; C shows Fura-2 AM staining of bone tissue in NC, sh3# groups at 14 days; DE shows the expression of Clmp, Fn, Col1A, and Runx2 proteins in bone tissue in NC, sh3# groups at 14 days, *P<0.05, **P<0.01, ****P<0.0001; and Safranin Fast Green staining of bone tissue in NC, sh3# groups at 14 days. DETAILED DESCRIPTION

[0043] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0044] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0045] The present invention will now be further described with reference to specific examples. The following examples are intended only to illustrate the present invention and are not intended to limit its contents. Experimental conditions not specified in the examples are generally based on conventional conditions or those recommended by the reagent company. Reagents and consumables used in the following examples are commercially available unless otherwise specified.

[0046] In the present invention, the term "expression level" refers to the amount of a gene product present in a body or sample at a specific time point. Expression level can be measured / quantified / detected, for example, by protein or mRNA expressed by the gene. Expression level can be quantified, for example, as follows: the amount of the target gene product present in a normalized sample using the total amount (total protein or mRNA) of the same type of gene product in the same sample or reference sample (for example, a sample obtained from the same individual at the same time or a portion of the same size (weight, volume) of the same sample), or the sample size (weight, volume, etc.) for determining the amount / limitation of the target gene product. Expression level can be measured or detected by any method known in the art, such as a method (such as mass spectrometry) for direct detection and quantification of the target gene product, or a method for indirect detection and measurement of the target gene product, typically by combining the target gene product with one or more different molecules or detection devices (such as primers, probes, antibodies, protein scaffolds) specific for the target gene product. It is also known to the skilled person to determine the level of gene copies, which also includes determining the absence or presence of one or more fragments (e.g., by nucleic acid probes or primers, such as quantitative PCR, Multiplex ligation-dependent probe amplification (MLPA) PCR).

[0047] The terms "indicator" and "marker" are used interchangeably in the present invention and refer to a sign or signal of a condition or are used to monitor a condition. Such a "condition" refers to a biological state of a cell, tissue, or organ, or refers to the health and / or disease state of an individual. An indicator can be the presence or absence of a molecule including, but not limited to, peptides, proteins, and nucleic acids, or can be a change in the expression level or pattern of such a molecule in a cell, or tissue, organ, or individual. An indicator can be a sign of the onset, development, or presence of a disease in an individual or the further progression of such a disease. An indicator can also be a sign of the risk of developing a disease in an individual.

[0048] The terms "up-regulated", "increased" or "increased" in the level of an indicator refer to a decrease in the level of such indicator in a sample compared to a reference.

[0049] The terms "down-regulated", "decreased" or "decreased" in the level of an indicator refer to a decrease in the level of such indicator in a sample compared to a reference.

[0050] In principle, a reference amount can be calculated for a given group or cohort of subjects according to the present invention based on the mean or median of a given Clmp by applying standard statistical methods. In particular, the accuracy of a test, such as a method intended or not intended to detect an event, is best described by its receiver-operating characteristic (ROC) (see, inter alia, Zweig 1993, Clin. Chem. 39: 561-577). A ROC plot is a graph of all sensitivity versus specificity pairs obtained by varying the decision threshold over the entire observed data range. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to correctly assign a subject to a certain prognosis or diagnosis. A ROC plot represents the overlap between two distributions by plotting sensitivity against 1-specificity over the entire range of thresholds suitable for discrimination. On the y-axis is the sensitivity or true positive fraction, which is defined as the ratio of the number of true positive test results to the sum of the number of true positive and false negative test results. This is also referred to as positive in the presence of a disease or condition. It is calculated separately for the affected subgroup. On the x-axis is the false positive score or 1-specificity, which is defined as the ratio of the false positive result number to the true negative number and the sum of the false positive result number. It is a specific index, and is calculated completely by unaffected subgroups. Because true and false positive scores are calculated completely individually, therefore by using the test results from two different subgroups, ROC diagram is independent of the prevalence of events in the group. Each point on the ROC diagram represents the sensitivity / -specificity pair corresponding to a specific decision threshold. The test with perfect discrimination (no overlap in the two result distributions) has the ROC diagram through the upper left corner, wherein the true positive score is 1.0 or 100% (perfect sensitivity), and the false positive score is 0 (perfect specificity). The theoretical diagram of the test not distinguishing (the distribution of two groups of results is identical) is the 45 ° diagonal line from the lower left corner to the upper right corner. Most of the figures fall between these two extremes. If the ROC diagram falls below the 45 ° diagonal line completely, this is easily corrected by reversing the "positive" standard from "greater than" to "less than", and vice versa. In some embodiments, the present invention provides the method for the diagnosis of the present invention. The method for the diagnosis of the present invention is described in detail. Qualitatively, the closer the figure is to the upper left corner, the higher the overall accuracy of the test. According to the confidence interval of expectation, a threshold value can be derived from the ROC curve, allowing to diagnose or predict a given event with the appropriate balance of sensitivity and specificity respectively. Therefore, it is preferably possible to generate a reference for the inventive method by setting up the ROC for the group as described above and deriving a threshold value therefrom. According to the expected sensitivity and specificity of diagnostic method, the ROC figure allows to derive a suitable threshold value. Preferably, the reference amount is within such a value range, and it represents at least 75% sensitivity and at least 45% specificity or at least 80% sensitivity and at least 40% specificity or at least 85% sensitivity and at least 33% specificity or at least 90% sensitivity and at least 25% specificity.

[0051] The term "test kit" as used in the present invention refers to a collection of the above-mentioned components that are preferably provided separately or provided in a single container. The container also preferably includes instructions for implementing the method of the present invention. Examples of these components of the test kit and their use have been given in this specification. Preferably, the test kit includes the above-mentioned components in a ready-to-use formulation. Preferably, the test kit may additionally include instructions, such as a user's manual for adjusting components (e.g., the concentration of the detection agent) and for explaining the results of any determination of the diagnosis provided by the method of the present invention. In particular, such a manual may include information for determining that the amount of the gene product is assigned to the diagnosis type. Details are found elsewhere in this specification. In addition, such a user's manual may provide instructions for correctly using the test kit components for determining the amount of the corresponding biomarker. The user's manual may be provided in paper or electronic form (e.g., stored on a CD or CD ROM). The present invention also relates to the purposes of the test kit in any method according to the present invention.

[0052] As used herein, the term "system" refers to a system of devices comprising at least the aforementioned devices that are operatively interconnected to allow for diagnosis. How the devices are operatively interconnected will depend on the type of device included in the device. For example, in the case of a device for automatically determining the status or amount of a gene product, the data obtained by the automated device can be processed, for example, by a computer program, to establish a diagnosis. Preferably, in this case, the device is contained in a single device. Thus, the device can include an analysis unit for determining the status or amount of a gene product in a sample and an evaluation unit for processing the resulting data for diagnosis. Preferred detection devices are disclosed above in conjunction with embodiments relating to the methods of the present invention. In this case, the devices are operatively interconnected so that the user of the system can combine the quantitative determination result and its diagnostic value, based on the instructions and explanations provided in the manual. In such embodiments, the devices can be presented as separate devices and preferably packaged together as a kit. A person skilled in the art will understand how to connect the devices without requiring further inventive skill. Preferred devices are those that can be used without the specific knowledge of a professional clinician, such as test strips or electronic devices that require only sample loading. The results can be output as raw data for parametric diagnosis, preferably given as absolute or relative quantities. It should be understood that these data will require interpretation by a clinician. However, expert system devices are also envisioned, wherein the output comprises processed diagnostic raw data, the interpretation of which does not require a professional clinician. Other preferred devices comprise an analysis unit / device (e.g., a biosensor, an array, a solid support coupled to a ligand that specifically recognizes a polypeptide, a plasma surface resonance device, an NMR spectrometer, a mass spectrometer, etc.) or an evaluation unit / device as mentioned above according to the method of the invention.

[0053] As previously mentioned, the Clmp (CXADR-like membrane protein) gene is a key regulator of cell adhesion and intercellular junctions. The protein it encodes belongs to the tight junction protein family and is commonly involved in maintaining cell polarity, regulating intercellular adhesion, and transmembrane signaling, playing a particularly critical role in tissue development and homeostasis. Studies have shown that Clmp influences epithelial barrier function by mediating cell-cell interactions and has potential roles in inflammation, tumor microenvironment regulation, and organogenesis. However, its role in osteogenic differentiation and bone regeneration has not been studied.

[0054] In view of this, in a typical embodiment of the present invention, there is provided a use of any one of the following (a1)-(a4) in preparing a bone injury detection product;

[0055] (a1) Clmp gene;

[0056] (a2) Clmp protein;

[0057] (a3) Reagents for detecting Clmp gene expression;

[0058] (a4) Reagent for detecting Clmp protein expression.

[0059] Wherein, the reagent for detecting Clmp gene expression may include reagents for detecting Clmp gene expression based on real-time fluorescence quantitative PCR, in situ hybridization, gene chip and gene sequencing;

[0060] The reagent for detecting Clmp protein expression can be a reagent for detecting Clmp protein expression level based on an immunoassay method.

[0061] The products include but are not limited to primers, probes, (gene or protein) chips, nucleic acid membrane strips, detection kits, detection devices and detection equipment for detecting Clmp gene and protein expression levels, which are not specifically limited here.

[0062] The bone injury detection includes screening, (auxiliary) diagnosis, monitoring or prognostic detection of bone injuries.

[0063] In the present invention, the bone injury may include abnormalities in bone structure or function, which may be caused by trauma, disease, metabolic abnormalities, etc., such as traumatic bone injury (such as fracture, bone crack, bone contusion, joint dislocation and subluxation, etc.), pathological bone injury (such as osteoporosis and osteoporotic fracture, bone tumor-related injury, infectious bone injury (such as osteomyelitis, bone tuberculosis), metabolic bone disease (such as rickets, osteomalacia, Paget's disease of bone), sports / strain bone injury (such as fatigue periostitis, bone / cartilage injury) and bone injury-related complications (such as nonunion / delayed union, infection, traumatic arthritis, neurovascular injury).

[0064] In another embodiment of the present invention, a system for detecting bone damage is provided, the system comprising:

[0065] An acquisition unit is configured to: acquire the expression level of Clmp in a sample to be tested by a subject;

[0066] The evaluation unit is configured to evaluate and judge the disease condition of the subject according to the subject Clmp obtained by the acquisition unit.

[0067] Wherein, the Clmp includes Clmp gene and / or Clmp protein.

[0068] The sample to be tested can be a blood sample (including peripheral blood) sample, a bone tissue sample, or a bone cell sample (such as osteoblast) of the subject.

[0069] The bone injury detection includes screening, (auxiliary) diagnosis, monitoring or prognostic detection of bone injuries. The bone injury may include abnormalities in bone structure or function, which may be caused by trauma, disease, metabolic abnormalities, etc., such as traumatic bone injuries (such as fractures, bone cracks, bone contusions, joint dislocations and subluxations, etc.), pathological bone injuries (such as osteoporotic fractures, bone tumor-related injuries, infectious bone injuries (such as osteomyelitis, bone tuberculosis), metabolic bone diseases (such as rickets, osteomalacia, Paget's disease of bone), sports / strain bone injuries (such as fatigue periostitis, bone / cartilage injuries) and bone injury-related complications (such as nonunion / delayed union, infection, traumatic arthritis, neurovascular injury).

[0070] In another embodiment of the present invention, the use of Clmp gene / protein as a target in the preparation and / or screening of bone injury drugs is provided.

[0071] Specifically, the effects of a candidate drug on the Clmp gene / protein before and after use can be used to determine whether the candidate drug can be used to prevent or treat bone damage.

[0072] Specifically, the method for screening drugs for preventing or treating bone damage includes:

[0073] (b1) treating a system expressing and / or containing the Clmp gene / protein with a candidate substance; setting up a parallel control without treatment with the candidate substance;

[0074] (b2) After completing step (b1), detecting the expression level of the Clmp gene / protein in the system; if the expression level of the Clmp gene / protein in the system treated with the candidate substance is significantly upregulated compared with the parallel control, the candidate substance can be used as a candidate drug for preventing or treating bone damage.

[0075] The system may be a cell (osteoblast) system, a solution system, a tissue (bone tissue) system, an organ system or an animal system, and is not specifically limited here.

[0076] In another embodiment of the present invention, there is provided the use of a substance for promoting the expression of Clmp gene / protein or increasing its activity in any one or more of the following:

[0077] (c1) promoting osteogenic differentiation or preparing a product that promotes osteogenic differentiation;

[0078] (c2) promoting extracellular matrix formation or preparing a product that promotes extracellular matrix formation;

[0079] (c3) promoting bone tissue formation or preparing products that promote bone tissue formation;

[0080] (c4) preparing products that promote bone damage repair;

[0081] (c5) Products for the prevention and / or treatment of bone damage.

[0082] Substances that promote Clmp gene / protein expression or enhance its activity include substances that upregulate Clmp expression and / or promote its activity using gene-specific Mimics technology; such as promoters or lentiviruses that upregulate Clmp gene expression; and also include compound promoters.

[0083] As mentioned above, the bone injury may include abnormalities in bone structure or function, which may be caused by trauma, disease, metabolic abnormalities, etc., such as traumatic bone injury (such as fracture, bone crack, bone contusion, joint dislocation and subluxation, etc.), pathological bone injury (such as osteoporotic fracture, bone tumor-related injury, infectious bone injury (such as osteomyelitis, bone tuberculosis), metabolic bone disease (such as rickets, osteomalacia, Paget's disease of bone), sports / strain bone injury (such as fatigue periostitis, bone / cartilage injury) and bone injury-related complications (such as nonunion / delayed union, infection, traumatic arthritis, neurovascular injury).

[0084] The product may be a drug, a medical device, or an experimental reagent for non-medical use.

[0085] According to the present invention, when the product is a medicine, the medicine further comprises at least one inactive pharmaceutical ingredient.

[0086] The inactive pharmaceutical ingredients may be carriers, excipients, diluents, etc. commonly used in pharmacy. Furthermore, the inactive pharmaceutical ingredients such as carriers, excipients, and diluents that may be included are well known in the art, and a person of ordinary skill in the art can determine whether they meet clinical standards.

[0087] The carrier can be a microcapsule, liposome, nanoparticle or polymer and any combination thereof. The delivery vehicle of the pharmaceutically acceptable carrier can be a liposome, a biocompatible polymer (including natural polymers and synthetic polymers), a lipoprotein, a lipopolysaccharide, an artificial viral envelope, an inorganic (including metal) particle, and a bacterium or virus (such as baculovirus, adenovirus and retrovirus, etc.), a phage, a cosmid or a plasmid vector.

[0088] The medicine can also be administered separately to other preventive and / or therapeutic compounds in a separate composition or in a dosage form different from the main active ingredient. Partial doses of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered separately. During the course of treatment, the dosage of the medicine of the present invention can be adjusted according to the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

[0089] The medicine of the present invention can be administered to the body in a known manner. For example, it can be delivered to the tissue of interest by systemic intravenous delivery or local injection. Alternatively, it can be administered intravenously, percutaneously, intranasally, through the mucosa, or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered in the present invention can vary depending on various factors to a great extent, such as the target cell, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0090] In the present invention, promoting bone damage repair specifically refers to promoting the repair of bone damage by bone damage repair materials.

[0091] In the present invention, the bone damage repair material may be a β-TCP biomaterial.

[0092] The subjects of drug administration can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys and gorillas, and are not specifically limited here.

[0093] When the product is a medical device, it may be an implantable medical device, specifically a drug-device combination product, which refers to a product consisting of a drug and a medical device, manufactured as a single entity. A specific embodiment of the drug-device combination product may include the aforementioned bone damage repair material (β-TCP biomaterial), and / or the β-TCP biomaterial may also be loaded with a drug, wherein the drug may be a substance that promotes expression or enhances the activity of the Clmp gene / protein.

[0094] The non-medical experimental reagents can be used for basic research. For example, the product can be used to regulate osteoblast differentiation and extracellular matrix formation in vitro, thereby creating osteoblast and animal-related biological models, thereby facilitating research on the mechanisms of occurrence and development of diseases such as bone injury.

[0095] In another embodiment of the present invention, a product for preventing and / or treating bone damage is provided, comprising:

[0096] (d1) Substances that increase the expression or activity of Clmp gene / protein;

[0097] (d2) Substances that enhance Clmp gene / protein expression or activity and bone damage repair materials.

[0098] As mentioned above, the bone injury may include abnormalities in bone structure or function, which may be caused by trauma, disease, metabolic abnormalities, etc., such as traumatic bone injury (such as fracture, bone crack, bone contusion, joint dislocation and subluxation, etc.), pathological bone injury (such as osteoporotic fracture, bone tumor-related injury, infectious bone injury (such as osteomyelitis, bone tuberculosis), metabolic bone disease (such as rickets, osteomalacia, Paget's disease of bone), sports / strain bone injury (such as fatigue periostitis, bone / cartilage injury) and bone injury-related complications (such as nonunion / delayed union, infection, traumatic arthritis, neurovascular injury).

[0099] It is particularly important to note that the failure of a fracture to heal is called nonunion. Bone tissue has a strong ability to repair itself. When a fracture is given appropriate treatment, most fractures will heal well. However, some fractures are difficult to heal. When a fracture heals slowly, it is called delayed healing. When a fracture cannot heal, it is called nonunion. Among patients with fractures, about 5% of patients have difficulty healing. Due to the continuous movement of the fracture site, nonunion is usually accompanied by pain, which greatly reduces the patient's quality of life. The present invention has demonstrated through experiments that the expression level of Clmp in patients with nonunion is significantly reduced, and the expression of Clmp is related to intercellular adhesion and extracellular matrix production. Therefore, by overexpressing Clmp and combining it with bone injury repair materials, the recovery of nonunion / delayed healing is promoted, thereby achieving the purpose of bone injury treatment.

[0100] The substances that promote Clmp gene / protein expression or enhance its activity include substances that upregulate Clmp expression and / or promote its activity based on gene-specific Mimics technology; such as promoters or lentiviruses that upregulate Clmp gene expression; and also include compound promoters.

[0101] The product may be a medicine or a medical device.

[0102] As previously mentioned, when the product is a medical device, it may be an implantable medical device, specifically a drug-device combination product. A drug-device combination product refers to a product consisting of a drug and a medical device, manufactured as a single entity. Specifically, it may include a device coated with, impregnated with, or combined with a drug, a pre-filled drug delivery device / system, a pre-filled biologic delivery device / system, a device coated with or combined with a biologic, or a topical liquid or gel product, etc., without further limitation.

[0103] Among them, a specific embodiment of the drug-device combination product can be a bone damage repair material (such as β-TCP biomaterial), and / or the β-TCP biomaterial can also be loaded with drugs, and the drugs can be substances that promote Clmp gene / protein expression or increase its activity.

[0104] In another embodiment of the present invention, there is provided the use of a substance that inhibits the expression of Clmp gene / protein or reduces its activity in constructing a bone injury repair model.

[0105] The substance that inhibits the expression of Clmp gene / protein or reduces its activity may include but is not limited to RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA targeting Clmp gene, substances for implementing lentiviral infection or gene knockout, and antibodies or protein inhibitors against Clmp protein, etc., and is not specifically limited here.

[0106] The present invention has demonstrated through experiments that knocking down the Clmp gene in osteoblasts can effectively inhibit the expression of Runx2 and Alp (key indicators of osteogenic differentiation), as well as the expression of Fn and Col1A (key indicators of extracellular matrix formation), and inhibit β-TCP-mediated bone tissue formation, thereby being used to construct bone damage repair cell and animal models for basic scientific research.

[0107] In another embodiment of the present invention, a method for preventing and / or treating bone damage is provided, comprising: administering to a subject the above-mentioned substance that promotes the expression of Clmp gene / protein or increases its activity.

[0108] Substances that promote Clmp gene / protein expression or increase its activity include substances that upregulate Clmp expression and / or promote its activity based on gene-specific Mimics technology; such as promoters or lentiviruses that upregulate Clmp gene expression; and also include compound promoters. Furthermore, the compound promoters include the bone repair material β-TCP.

[0109] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods in the following examples are generally carried out under conventional conditions.

[0110] Example 1 Effect of calcium phosphate-based materials on the osteogenic differentiation of MC3T3 cells

[0111] 1.1 Materials and Methods

[0112] Cell culture: Pre-treat Aggrewell culture plates with 1 mL / well of PBS and sterilize with UV for 30 min; rinse with 2 mL of warm basal medium and add 1 mL of warm complete medium. MC3T3 cells were cultured at 2.4 × 10 5 The cells were seeded in culture plates at a final concentration of / mL and co-cultured with calcium phosphate materials (HA, α-TCP, β-TCP, TTCP, particle size 10-20 μm) at the same cell number and concentration. Osteogenic differentiation medium (Precellia) was used to induce differentiation, and the medium was changed every 3 days.

[0113] Detection indicators:

[0114] Microsphere volume: The changes in microsphere volume were recorded from culture day 0 to 7.

[0115] Cell activity: On the 7th day, cells were stained with a live / dead cell double staining kit (KTA1001) and observed under a confocal microscope.

[0116] Mineralization capacity: Alizarin Red S staining was performed on days 3, 5, and 7, and the product was dissolved in 10% hexadecylpyridine and the absorbance at 450 nm was measured using a microplate reader.

[0117] 1.2 Experimental Results

[0118] β-TCP can significantly promote the increase of cell microsphere volume on days 3, 5 and 7 ( Figure 1 A, DF). Subsequently, the Live / Dead staining results on the 7th day showed that there was no significant difference in the activity of the cell microspheres among the four materials ( Figure 1 B) Alizarin red staining was used to observe microsphere mineralization on days 3, 5, and 7, and quantified using cetylpyridine. The results showed that β-TCP significantly promoted microsphere mineralization at each time point. These results indicate that β-TCP has the best ability to promote osteogenic differentiation and mineralization of MC3T3 cells among the four calcium phosphate materials.

[0119] Example 2: Transcriptomic analysis of the mechanism of HA and β-TCP mediating osteogenic differentiation

[0120] 1.1 Materials and Methods

[0121] Transcriptome sequencing: Samples of MC3T3 cells from the HA group and β-TCP group were collected on days 1, 3, and 7, and total RNA was extracted and subjected to bulk transcriptome sequencing.

[0122] Data analysis: The Limma package was used to identify differentially expressed genes, and GSEA and GO enrichment analysis were used to screen key signaling pathways, focusing on calcium signaling pathways, bone mineralization, and extracellular matrix-related pathways.

[0123] 1.2 Experimental Results

[0124] On day 1, there were 603 differentially expressed genes in the β-TCP group compared with the HA group (194 up-regulated and 409 down-regulated) ( Figure 2 A), Enrichment analysis and GSEA results showed that the calcium signaling pathway was significantly activated ( Figure 2 B-2D, P<0.05). On day 3, there were 123 differentially expressed genes between the two groups (72 up-regulated and 51 down-regulated) ( Figure 2 E), further analysis revealed that biological signals such as bone mineralization (GO:0031214) and Collagen-Containing Extracellular Matrix (GO:0062023) were upregulated ( Figure 2 On day 7, there were 133 differentially expressed genes between the two groups ( Figure 2 I), and its enrichment signals were also concentrated in functions such as bone trabecula formation (GO:0031214) and extracellular matrix organization (GO:0031214). Figure 2 JK), indicating that β-TCP has a significant promoting effect on extracellular matrix formation during the entire osteogenesis process. This indicates that β-TCP significantly enhances osteogenic differentiation ability by activating calcium signaling pathways and promoting extracellular matrix formation.

[0125] Example 3: Construction and application of a multi-scale transcriptomics evaluation system

[0126] 1.1 Materials and Methods

[0127] Single-cell RNA sequencing analysis: Single-cell transcriptome data from mouse embryos (E7.5–E13.5) were obtained (TOME database). Analysis objects were constructed using Seurat V4, data were integrated using the Harmony package, and visualized using UMAP. Pseudo-time series analysis was performed using Monocle 3.0 to identify key genes involved in paraxial mesoderm formation and osteogenic lineage differentiation.

[0128] Batch transcriptome association analysis: Combined with the GSE186832 dataset (MC3T3-E1 osteogenic differentiation data for 0–21 days), the osteogenesis-related gene clusters were screened using the fuzzy c-means algorithm, and 452 endogenous key genes were obtained by intersection with embryonic development genes.

[0129] 1.2 Experimental Results

[0130] The results are as follows Figure 3 As shown in the figure, in terms of establishing the embryonic bone development lineage, two pseudo-time-sequential trajectories were constructed: paraxial mesoderm formation (E7.5–E9.5) and osteoblast differentiation (E9.5–E13.5). A total of 13 cell subpopulations were annotated in the E7.5–9.5 stage ( Figure 3 A (left side), Mesenchymal stromal cells, Chondrocyte and osteoblast progenitors, Paraxial mesoderm, Skeletal muscle progenitors, osteoblast progenitors osteogenic related subpopulations were also obtained at E9.5–13.5 stage ( Figure 3 B (right)). The results of differential gene and function enrichment analysis showed that the Regulation of actin cytoskeleton and Nucleocytoplasmic transpor signals were activated at E9.5 compared with E7.5. Wnt signaling and Adherensjunction were upregulated at E13.5 compared with E9.5. Furthermore, based on the emergence of the paraxial mesoderm as the central node in this study, two biological evolutionary trajectories, the paraxial mesoderm formation (E7.5-E9.5) and the paraxial mesoderm-osteoblast (E9.5-E13.5), were established, and the important node genes on the trajectory were displayed through heat maps ( Figure 3 C, Figure 3 D). The results showed that during paraxial mesoderm formation (embryonic bone development), gene expression trends along the pseudo-chronological trajectory can be divided into six clusters, including dynamic trends in genes such as Run1t1, Rgs6, Dpf3, Plagl1, and Pde4d. In the pseudo-chronological trajectory of paraxial mesoderm to osteoblasts (osteoblast differentiation), we also observed six clusters distributed along the evolutionary trajectory. Among them, Cadm1, Alx1, Actg2, Alcam, and Meis2 play a decisive role in skeletal development and early osteoblast differentiation.

[0131] In terms of material regulation differences, the results are as follows Figure 4As shown in the results, HA regulates 6 endogenous genes (tended to bone development and Wnt signaling pathway), and β-TCP regulates 20 endogenous genes (enriched in the extracellular matrix, such as collagen-containing extracellular matrix, extracellular matrix structural constituent, collagen trimer), among which the Clmp gene is specifically expressed in osteoblast-related cell subpopulations.

[0132] By establishing a multi-scale transcriptomics system, we revealed the unique mechanism by which β-TCP promotes extracellular matrix formation by regulating the Clmp gene. We preliminarily judged that Clmp may be an endogenous key gene that regulates osteogenesis through β-TCP.

[0133] Example 4: β-TCP promotes osteogenic differentiation and extracellular matrix production by upregulating Clmp

[0134] 1.1 Materials and Methods

[0135] Protein and gene expression detection:

[0136] Western blot: The expression of Clmp protein in MC3T3 cells of β-TCP group and HA group on the 1st and 3rd day was detected.

[0137] Immunofluorescence: Detect the expression of fibronectin (FN) and Runx2 in MC3T3 cells and primary mouse MSCs under 2D / 3D culture, and Fura-2 AM probe to detect Ca 2+ level.

[0138] Single-cell sequencing validation: Based on E9.5–E13.5 data, the differential gene functions of Clmp_high and Clmp_low cell subpopulations were compared.

[0139] 1.2 Experimental Results

[0140] The Clmp protein level of MC3T3 cells in the β-TCP group was significantly higher than that in the HA group on days 1 and 3 ( Figure 5 A-5D). At the same time, the expression of extracellular matrix-related fibronectin (FN) in MC3T3 cells in the β-TCP group increased significantly on day 1 and day 3 ( Figure 5 E-5L). To eliminate the error introduced by a single cell line, primary mouse bone marrow mesenchymal stem cells (MSCs) were extracted and cultured in 3D. MSCs were stimulated with HA and β-TCP for 3 days, and immunofluorescence experiments showed that the expression of Clmp, Runx2, and Fura-2 AM probes in MSCs increased significantly under β-TCP stimulation, with significant differences ( Figure 5Single-cell analysis revealed that differentially expressed genes in the Clmp_high subpopulation were enriched for extracellular matrix formation. Therefore, β-TCP promotes the expression of osteogenic differentiation-related proteins and extracellular matrix production by upregulating Clmp gene expression.

[0141] Example 5: Effect of Clmp gene knockdown on β-TCP-induced osteogenic differentiation

[0142] 1.1 Materials and Methods

[0143] shRNA knockdown: Clmp shRNA was designed (sequences shown in Table 1), ligated into the PLKO.1 vector, and packaged into MC3T3-E1 cells. Stable knockdown strains were selected using puromycin.

[0144] Table 1

[0145]

[0146]

[0147] At the same time, knockdown MSC stem cells were cultured based on Sh3#.

[0148] Functional Verification:

[0149] Immunofluorescence: Detection of Ca in knockdown cells 2+ , Alp, and Runx2 expression.

[0150] RT-PCR: Detect the mRNA levels of osteogenic marker genes (Spp1, Sp7, Alp).

[0151] Western blot: Detect FN protein expression.

[0152] CCK8 experiment: detect the proliferation of cells after knockdown.

[0153] Alkaline phosphatase assay: Detect the alkaline phosphatase content in knockdown cells.

[0154] 1.2 Experimental Results

[0155] The Sh3# group had the highest Clmp gene knockdown efficiency ( Figure 6 A-6B), the ability of MC3T3 cells to produce extracellular matrix after β-TCP knockdown was significantly restricted at 3 days and 7 days, and the expression of FN protein was significantly reduced after knockdown, with significant differences ( Figure 6 C). Further studies were conducted by collecting NC and sh3# cell spheres stimulated with β-TCP for 3 days in 3D culture and performing immunofluorescence staining. The results showed that after Clmp knockdown, intracellular Ca 2+ production, and the expression of Alp and Runx2 proteins was inhibited ( Figure 6 D-6F). Furthermore, Realtime-PCR was used to detect osteogenic differentiation markers: Spp1, Sp7, and Alp in the cell spheres of the NC group and the sh3# group stimulated by β-TCP for 7 days. The results showed that after Clmp knockdown, the expression of Spp1, Sp7, and Alp genes was significantly inhibited ( Figure 6 G-6I). This indicates that Clmp gene deletion significantly weakens the osteogenic differentiation and extracellular matrix production induced by β-TCP. Figure 7 As shown in Figure 3, compared with the NC group, the proliferation ability and alkaline phosphatase secretion ability of MC3T3 cells in the Sh3# group were significantly reduced. This was also confirmed in the stem cell experiment. Compared with the NC group, the expression of Clmp and Runx2 in MSC cells in the Sh3 group was significantly reduced, and the cell mineralization ability was also significantly reduced.

[0156] Example 6: Effect of overexpression of Clmp gene on MC3T3 cells

[0157] 1.1 Materials and Methods

[0158] Clmp overexpression: The Clmp DNA fragment was ligated into the PLVG vector and co-packaged with psPAX2 and pMD2.G to form a viral vector. The plasmid was transfected into 293T cells using Lipofectamine 2000. After 48 hours, the viral supernatant was collected and used to infect MC3T3-E1 cells for 24 hours. Finally, Clmp-overexpressing cell lines were selected using puromycin at a final concentration of 2.5 μg / ml.

[0159] Functional Verification:

[0160] Immunoblotting and immunofluorescence: Detect Clmp expression in overexpressed cells;

[0161] CCK8 assay: Detect cell proliferation after Clmp overexpression.

[0162] Alkaline phosphatase assay: Detect the alkaline phosphatase content in cells after overexpressing Clmp.

[0163] 1.2 Experimental Results

[0164] like Figure 8 As shown in the results, compared with the NC group, the expression level of Clmp in the OE group was significantly increased, and the cell proliferation ability and alkaline phosphatase secretion ability of the OE group were significantly enhanced, indicating that Clmp promoted the proliferation and osteogenic differentiation of osteoblasts.

[0165] Example 7: Verification of the regulatory effect of Clmp on β-TCP-mediated osteogenesis in nude mice

[0166] 1.1 Materials and Methods

[0167] Animal experiment: MC3T3 cell spheres (2×10 6 Cells / 200 μL) were transplanted subcutaneously into the backs of BALB / c nude mice. The longest diameter (L), perpendicular diameter (W), and height (H) of the transplanted tissue were measured at 1 and 2 weeks, and the volume was calculated using the formula V = π / 6 × L × W × H. After sampling, the tissues were fixed, dehydrated, embedded in paraffin, and sectioned. Animal experiments were approved by the Medical Ethics Committee of the Affiliated Hospital of Qingdao University (Approval No. 2YFYWZLL2963) and conformed to relevant ethical standards.

[0168] After collecting the materials:

[0169] HE staining: observe the formation of ossification centers.

[0170] Immunofluorescence: Detect the expression of Clmp, FN, Runx2, and Col1A.

[0171] Safranin O-Fast Green staining: Assessment of collagen matrix formation.

[0172] 1.2 Experimental Results

[0173] The results showed that after Clmp knockdown, the growth of transplanted tissues in mice was significantly restricted ( Figure 9 A). Histological observations showed that on day 14, tissues similar to the ossification center, the Haversian canal, appeared in the tissues of the NC group. Microscopically, osteoblasts self-assembled into ring-like structures with a large amount of scattered fibrous connective tissue and extracellular matrix in the middle. In contrast, the tissues of the sh3# group only had a large number of scattered fibrous structures ( Figure 9 B). It is worth mentioning that the results of intracellular calcium ion detection showed that after Clmp knockdown, less calcium ions were produced in the tissue ( Figure 9 C). Furthermore, immunofluorescence assay results showed that the expression level of Clmp was reduced in the knockdown tissues, and the expression of Fn, Runx2, and Col1A was restricted ( Figure 9 Safranin fast green staining results showed that a large number of green-gray loose collagen-covered areas appeared in the NC group, which confirmed that Clmp plays an important role in maintaining the production of extracellular matrix during osteogenic differentiation ( Figure 9 H). These results indicate that Clmp regulates β-TCP-induced osteogenic differentiation and extracellular matrix formation in vivo, providing a new target for the design of bone regeneration materials.

[0174] Matters not covered by the present invention are known technologies.

[0175] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. Use of any one of the following (a1)-(a4) in the preparation of a bone injury detection product; (a1) Clmp gene; (a2) Clmp protein; (a3) Reagents for detecting Clmp gene expression; (a4) Reagent for detecting Clmp protein expression.

2. The use according to claim 1, characterized in that The reagents for detecting Clmp gene expression include reagents for detecting Clmp gene expression based on real-time fluorescence quantitative PCR, in situ hybridization, gene chip and gene sequencing; The reagent for detecting Clmp protein expression is a reagent for detecting Clmp protein expression level based on an immunoassay method; The products include primers, probes, (gene or protein) chips, nucleic acid membrane strips, detection kits, detection devices and detection equipment for detecting Clmp gene and protein expression levels.

3. The use according to claim 1, characterized in that The bone injury detection includes screening, (auxiliary) diagnosis, monitoring or prognostic detection of bone injuries; The bone injury includes abnormalities in bone structure or function, including traumatic bone injury (further including fractures, bone cracks, bone contusions, joint dislocations and subluxations), pathological bone injury (further including osteoporosis and osteoporotic fractures, bone tumor-related injuries, infectious bone injury (further including osteomyelitis, bone tuberculosis), metabolic bone disease (further including rickets, osteomalacia, Paget's disease of bone), sports / strain bone injury (further including fatigue periostitis, bone / cartilage injury) and bone injury-related complications (further including nonunion / delayed union, infection, traumatic arthritis and neurovascular injury).

4. A system for bone injury detection, characterized in that: The system comprises: An acquisition unit is configured to: acquire the expression level of Clmp in a sample to be tested by a subject; The evaluation unit is configured to evaluate and judge the disease condition of the subject according to the subject Clmp obtained by the acquisition unit. Wherein, the Clmp includes Clmp gene and / or Clmp protein.

5. The system according to claim 4, wherein: The sample to be tested is a blood sample (including peripheral blood), a bone tissue sample, and a bone cell sample (including osteoblasts) of the subject; Furthermore, the bone injury detection includes screening, (auxiliary) diagnosis, monitoring or prognostic detection of bone injuries.

6. Application of Clmp gene / protein as a target in the preparation and / or screening of bone injury drugs.

7. Use of a substance that promotes Clmp gene / protein expression or enhances its activity in any one or more of the following: (c1) promoting osteogenic differentiation or preparing a product that promotes osteogenic differentiation; (c2) promoting extracellular matrix formation or preparing a product that promotes extracellular matrix formation; (c3) promoting bone tissue formation or preparing products that promote bone tissue formation; (c4) preparing products that promote bone damage repair; (c5) Products for the prevention and / or treatment of bone damage; Furthermore, substances that promote Clmp gene / protein expression or enhance its activity include substances that upregulate Clmp expression and / or promote its activity based on gene-specific Mimics technology; further include promoters or lentiviruses that upregulate Clmp gene expression; and also include compound promoters. Furthermore, the bone injury includes abnormalities in bone structure or function, including traumatic bone injury (further including fractures, bone cracks, bone contusions, joint dislocations and subluxations), pathological bone injury (further including osteoporosis and osteoporotic fractures, bone tumor-related injuries, infectious bone injury (further including osteomyelitis and bone tuberculosis), metabolic bone disease (further including rickets, osteomalacia, Paget's disease of bone), sports / strain bone injury (further including fatigue periostitis, bone / cartilage injury) and bone injury-related complications (further including nonunion / delayed union, infection, traumatic arthritis and neurovascular injury); Furthermore, promoting bone damage repair specifically includes promoting the repair of bone damage by bone damage repair materials; Furthermore, the product is a drug, medical device, or experimental reagent for non-medical use; Furthermore, when the product is a medical device, the medical device is an implantable medical device, specifically a drug-device combination product; Furthermore, the drug-device combination product contains a bone damage repair material, and the bone damage repair material is loaded with a drug, and the drug is a substance that promotes the expression of Clmp gene / protein or increases its activity; Furthermore, the bone damage repair material is β-TCP biomaterial.

8. A product for preventing and / or treating bone damage, characterized in that: The products include: (d1) Substances that increase the expression or activity of Clmp gene / protein; (d2) Substances that increase Clmp gene / protein expression or activity and bone damage repair materials; Furthermore, the substance that promotes Clmp gene / protein expression or enhances its activity includes substances that upregulate Clmp expression and / or promote its activity based on gene-specific Mimics technology; such as promoters or lentiviruses that upregulate Clmp gene expression; and also includes compound promoters; The product is a pharmaceutical or medical device; Furthermore, when the product is a medical device, the medical device is an implantable medical device, specifically a drug-device combination product; Furthermore, the drug-device combination product contains a bone damage repair material, and the bone damage repair material is loaded with a drug, and the drug is a substance that promotes the expression of Clmp gene / protein or increases its activity; Furthermore, the bone damage repair material is β-TCP biomaterial; The bone injury includes abnormalities in bone structure or function, including traumatic bone injury (further including fractures, bone cracks, bone contusions, joint dislocations and subluxations), pathological bone injury (further including osteoporosis and osteoporotic fractures, bone tumor-related injuries, infectious bone injury (further including osteomyelitis, bone tuberculosis), metabolic bone disease (further including rickets, osteomalacia, Paget's disease of bone), sports / strain bone injury (further including fatigue periostitis, bone / cartilage injury) and bone injury-related complications (further including nonunion / delayed union, infection, traumatic arthritis and neurovascular injury).

9. Application of substances that inhibit Clmp gene / protein expression or reduce its activity in constructing bone injury repair models.

10. The use according to claim 9, characterized in that The substances that inhibit Clmp gene / protein expression or reduce its activity include RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA targeting Clmp gene, substances for implementing lentiviral infection or gene knockout, and antibodies or protein inhibitors against Clmp protein.

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