Application of XCR1 in preparation of osteoporosis treatment and detection product

Abstract

The invention discloses application of XCR1 in preparation of osteoporosis treatment and detection products, and belongs to the technical field of disease markers. According to the present invention, the XCR1 is adopted as the osteoporosis detection marker, whether a subject suffers from osteoporosis is determined by detecting the expression content of the XCR1 in the bone tissue of the subject, the XCR1 is developed as the drug target so as to prevent and treat osteoporosis, and the expression quantity of the XCR1 in the bone tissue of the patient is regulated so as to relieve the osteoporosis and other bone related diseases of the patient.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of XCR1 in the preparation of osteoporosis treatment and detection products. Background Art

[0002] Osteoporosis is a common systemic bone disease regulated by multiple genes. Related genes affect bone density and bone quality by regulating key processes of bone metabolism, including bone resorption, bone formation and bone reconstruction. Osteoporosis is particularly prevalent in the elderly and postmenopausal women. The treatment of osteoporosis includes non-drug treatment and drug treatment. Non-drug treatment mainly focuses on nutritional intervention and physical exercise, while drug treatment mainly includes taking drugs that promote bone formation, inhibit bone resorption, and increase bone strength, such as bisphosphonates, recombinant human parathyroid hormone, bone morphogenetic protein, etc., which can improve bone mass and reduce the risk of fractures in the short term, but long-term use may bring side effects, such as mandibular osteonecrosis and atypical femoral fractures. Gene therapy, as an emerging treatment method, can achieve the regulation of genes related to bone formation and resorption through targeted gene repair or editing, thereby stimulating bone formation and slowing down bone resorption. In the long run, it can become a revolutionary method for the treatment of osteoporosis. Compared with traditional treatments, gene therapy has significant advantages in the treatment of osteoporosis, such as: it can effectively reduce the side effects of drugs, long-lasting treatment effects, can treat multiple targets at the same time, has strong targeting, can reduce the impact on other tissues, can achieve early intervention and prevention of the disease, and reduce patients' dependence on drugs. It is suitable for patients with refractory or specific types of osteoporosis. In view of the limitations of existing detection methods and treatment methods for osteoporosis, there is an urgent need to explore new therapeutic targets to quickly and effectively curb the continuous loss of bone mass and prevent the occurrence of pathological fractures in advance. Therefore, it is particularly important to find new biomarkers to improve diagnostic accuracy and develop more effective treatment strategies.

[0003] XCR1 (XC motif chemokine receptor 1) is part of the G protein-coupled superfamily and is mainly expressed by a specific subset of dendritic cells. As a receptor for chemokines XCL1 and XCL2, it plays an important role in immune response. Currently, the biological functions of XCR1 reported include antigen presentation, self-tolerance, and association with certain cancers (such as renal clear cell carcinoma, lung cancer bone metastasis, etc.), but there are no reports of XCR1 being associated with osteoporosis. Summary of the invention

[0004] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide the use of XCR1 in the preparation of osteoporosis treatment and detection products.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention discloses the use of an agent for promoting the expression of XCR1 in the preparation of a drug for treating osteoporosis.

[0006] Preferably, the drug is a drug that targets osteoblasts.

[0007] The second aspect of the present invention discloses the use of a recombinant adeno-associated virus expressing XCR1 in the preparation of a drug for treating osteoporosis.

[0008] The third aspect of the present invention discloses a drug for treating osteoporosis, comprising an agent for promoting the expression of XCR1 and a pharmaceutically acceptable excipient.

[0009] The fourth aspect of the present invention discloses the use of a reagent for detecting the expression level of XCR1 in the preparation of a product for detecting osteoporosis.

[0010] Preferably, when the XCR1 content in the subject's bone tissue is lower than a normal value, the subject is determined to have osteoporosis.

[0011] Preferably, the reagent for detecting the expression level of XCR1 is an upstream primer as shown in SEQ ID NO.5 and a downstream primer as shown in SEQ ID NO.6.

[0012] A fifth aspect of the present invention discloses a kit for detecting osteoporosis, comprising a reagent for detecting the expression level of XCR1.

[0013] The sixth aspect of the present invention discloses the application of XCR1 in constructing a computational model for osteoporosis detection.

[0014] A seventh aspect of the present invention discloses an osteoporosis detection system, comprising: Data storage device: used to store the expression information of XCR1; Data analysis device: Calculate the patient's osteoporosis risk based on the XCR1 expression information; Result output device: outputs the result of the data analysis device.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The invention discloses an application of an agent for promoting XCR1 expression in the preparation of a drug for treating osteoporosis. Experiments have shown that XCR1 can be used as a therapeutic target for osteoporosis, and that the XCR1 and COL1A1 contents in bone tissue and the bone formation rate can be increased by administering XCR1 drug therapy mediated by recombinant adeno-associated virus (rAAV), indicating that drug development targeting XCR1 can provide a new treatment option for osteoporosis patients and is expected to significantly improve the therapeutic effect of osteoporosis.

[0016] The application of the reagent for detecting the expression level of XCR1 provided by the present invention in the preparation of a product for detecting osteoporosis, through experiments, it is found that XCR1 is significantly low-expressed in the osteoporosis environment, so XCR1 is used as a marker for detecting osteoporosis, and the content of XCR1 in the bone tissue of the subject can be detected to determine whether the subject suffers from osteoporosis, and when the content of XCR1 in the subject's tissue is reduced compared to the normal value, it is determined that the subject suffers from osteoporosis. The method is fast and simple to diagnose, can more sensitively and specifically reflect the state of osteoporosis, and improve the diagnostic accuracy of osteoporosis. In addition, based on the difference in the expression level of XCR1, individualized diagnosis and treatment of osteoporosis can be achieved, and the treatment strategy can be optimized. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the results of detecting the expression of XCR1 in the bone tissues of normal mice and osteoporotic mice of the present invention; Figure 2 This is a result diagram of the expression levels of XCR1 and COL1A1 in human osteosarcoma cells (U2OS cells) mediated by lentivirus overexpression of the human XCR1 gene in the present invention; wherein A is the expression level of XCR1, and B is the expression level of COL1A1; Figure 3 This is the immunohistochemical staining result diagram of the present invention; Figure 4 It is a statistical diagram of the immunohistochemical staining results of the present invention; wherein A is the average optical density level of XCR1, and B is the average optical density level of COL1A1; Figure 5 This is a result diagram showing the effect of XCR1 overexpression on bone formation rate of the present invention; Figure 6 It is a schematic diagram of the osteoporosis detection system of the present invention. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0019] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0020] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0021] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0022] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0023] The RT-qPCR technology (Real-Time Quantitative Polymerase Chain Reaction) used in this article is a mature technology that can be used for gene expression analysis. It is a molecular biology technology that combines reverse transcription (RT) and real-time quantitative PCR (qPCR), including RNA extraction, cDNA synthesis, qPCR amplification and data statistical analysis.

[0024] The immunohistochemistry (IHC) experimental method used in this article is a technology widely used in pathology and biological research. It introduces the immune reaction between antigens and antibodies into tissue (cell) specimens and displays the special markers carried by antibodies (or antigens) to complete the qualitative, localized and quantitative detection of a certain antigen (or antibody) in the tissue (cell).

[0025] After extensive research, the present invention found that XCR1 is significantly underexpressed in the osteoporosis environment, and speculated that it may be used as a marker for the diagnosis of osteoporosis. The reagent that promotes the high expression of XCR1 can be used as a therapeutic drug for osteoporosis. The drug enhances bone health by promoting the expression or function of XCR1. The specific drug form includes but is not limited to small molecule compounds, peptides, antibodies, gene therapy vectors, etc. The drug mechanism is to directly or indirectly regulate the expression of XCR1 gene, promote osteoblast activity, increase bone formation, and thus improve the osteoporosis condition.

[0026] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0027] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, are conventional commercial products, and their specifications are conventional specifications in the art.

[0028] 1. Application of XCR1 in osteoporosis detection 1. To explore the correlation between XCR1 content and osteoporosis in mice Female C57 mice aged 3 to 6 months were randomly divided into an osteoporosis group and a sham operation group.

[0029] Osteoporosis mouse group: Female C57 mice aged 3 to 6 months were selected, and their ovaries were artificially removed by surgery. The mice were cultured normally for 4 weeks to simulate osteoporosis caused by estrogen deficiency in postmenopausal women.

[0030] Sham operation group: The difference from the osteoporosis mouse group is that the ovaries were not removed. Instead, only slight operations were performed on the tissues around the ovaries of female C57 mice to simulate surgical trauma, but without causing a significant decrease in estrogen levels, thus forming a contrast with the real ovariectomized mice.

[0031] Measurement of mouse XCR1 expression: Total RNA was extracted from each group of mice and reverse transcribed into cDNA. The changes in the RNA content of mouse XCR1 were measured by RT-qPCR reaction. The sequences of the upstream primer and downstream primer of mouse XCR1 are shown in SEQ ID NO.2 and SEQ ID NO.3 in Table 1, respectively.

[0032] Table 1 Sequence Listing

[0033] Test results such as Figure 1 As shown, compared with normal mice (i.e., sham-operated mice), the XCR1 content in the bone tissue of mice with osteoporosis was significantly reduced, only 0.3 times that of normal mice, thus further confirming that the reduced XCR1 content is a sign of osteoporosis.

[0034] 2. Exploring the correlation between human XCR1 content and osteoporosis The correlation between XCR1 content and osteoporosis was explored by lentivirus-mediated overexpression of human XCR1 gene. The specific steps are as follows: human XCR1 was used as the target gene, the CDS region of human XCR1 gene (nucleotide sequence as shown in SEQ ID NO.4 in Table 1) was constructed into a recombinant lentiviral expression vector backbone (pCDH-CMV-MCS-EF1-Puro (XhoI-EcoRI-XbaI-EcoRV-BamHI-NotI)), and a recombinant lentiviral expression vector plasmid expressing human XCR1 gene was obtained, and the recombinant lentiviral expression vector plasmid expressing human XCR1 gene was co-transfected with two lentiviral auxiliary packaging plasmids (i.e., pCMV-VSV-Gcat and psPAX2) into HEK 293T cell lines to prepare a lentivirus expressing human XCR1 gene. The lentivirus expressing the human XCR1 gene was used to infect U2OS cells with a confluence of 80% to 90% to overexpress the human XCR1 gene in the U2OS cells. Finally, RT-qPCR was used to detect the changes in the expression levels of XCR1 and COL1A1 in the U2OS cells. The sequences of the upstream primer and the downstream primer of the human XCR1 gene are shown in SEQ ID NO.5 and SEQ ID NO.6 in Table 1, respectively, and the sequences of the upstream primer and the downstream primer of the human COL1A1 gene are shown in SEQ ID NO.7 and SEQ ID NO.8 in Table 1, respectively.

[0035] At the same time, the empty lentiviral expression vector plasmid and two lentiviral auxiliary packaging plasmids were co-transfected into the HEK293T cell line to prepare the control group lentivirus, which was used to infect U2OS cells with a confluence of 80% to 90% for a negative control experiment.

[0036] Test results such as Figure 2 As shown, it was found that after overexpression of the XCR1 gene in U2OS cells, the expression levels of the XCR1 gene and the bone formation marker gene COL1A1 were significantly increased, indicating that XCR1 can be used as a potential target for the treatment of osteoporosis.

[0037] 2. Application of XCR1 in the treatment of osteoporosis 1. Improvement of osteoporosis-related indicators by rAAV-mediated XCR1 drug therapy 1) Preparation of adeno-associated virus overexpressing XCR1 (rAAV-XCR1) The mouse XCR1 gene CDS region (nucleotide sequence as shown in SEQ ID NO.1 in Table 1) replaced the GFP gene on the pAAV-GFP plasmid to construct a recombinant rAAV expression vector plasmid rAAV-XCR1 expressing mouse XCR1, which was co-transfected with AAV packaging plasmid and auxiliary packaging plasmid (pAAV2 / 9n and pHelper) into HEK 293T cell line to prepare adeno-associated virus (rAAV-XCR1) overexpressing XCR1. rAAV-XCR1 was purified by iodixanol density gradient ultracentrifugation and ultrafiltration tube filtration. The titer of rAAV-XCR1 was detected by RT-qPCR.

[0038] At the same time, the pAAV-GFP plasmid was co-transfected with the AAV packaging plasmid and the auxiliary packaging plasmid into the HEK 293T cell line to prepare rAAV for subsequent control experiments.

[0039] 2) Treatment of osteoporotic mice by rAAV overexpressing XCR1 Female C57 mice aged 3 to 6 months were randomly divided into sham operation group, osteoporosis group and osteoporosis + XCR1 overexpression group. The mouse models of sham operation group, osteoporosis group and osteoporosis + XCR1 overexpression group were constructed in the same way as described above. The knee joints of mice in sham operation group were injected with 20 μL phosphate buffered saline (PBS), and the knee joints of mice in osteoporosis group were injected with 20 μL of 1×10 13 rAAV with a titer of 1×10 13 rAAV-XCR1, each group of drugs were injected for 3 consecutive weeks, and the total amount of drug injected into each group of mice was 1.2×10 11 Three months after drug injection, the mice in each group were killed at the coccyx, and the bone tissues of the mice in the sham operation group, osteoporosis group, and osteoporosis + XCR1 overexpression group were isolated and extracted.

[0040] 3) Immunohistochemistry The expression of XCR1 and bone formation marker COL1A1 was detected by immunohistochemistry to verify its potential in the treatment of osteoporosis. The specific steps are as follows: the bone tissue samples of each group of mice extracted in step 2) were immersed in 4% paraformaldehyde fixative for 24 hours and decalcified. The decalcified samples were immersed in ethanol solutions of different concentrations for dehydration. The dehydrated tissue was immersed in xylene to make the tissue transparent and paraffin-embedded. Tissue sections were made by paraffin slicer, and then dewaxed and gradient hydrated. Antigen epitopes were repaired with antigen repair solution, peroxidase was inhibited with 3% hydrogen peroxide solution, and nonspecific binding sites were blocked with 5% bovine serum albumin. Primary antibody incubation, secondary antibody incubation, color development, counterstaining, dehydration, transparency and counterstaining were performed in sequence, and the sections were detected and photographed under a microscope.

[0041] Test results such as Figure 3 and Figure 4 As shown in the results, it was found that the levels of XCR1 and COL1A1 in the bone tissue of osteoporotic mice were significantly lower than those in the sham operation group, and the levels of XCR1 and COL1A1 in the bone tissue of osteoporotic + XCR1 overexpression group mice were significantly higher than those in the osteoporosis group. This indicates that XCR1 can be used as a therapeutic target for osteoporosis, and after rAAV-mediated XCR1 drug treatment, osteoporosis-related indicators were significantly improved.

[0042] 2. Improvement of bone formation in osteoporotic mice by overexpression of mouse XCR1 Female C57 mice aged 3 to 6 months were randomly divided into sham operation group, osteoporosis group and osteoporosis + XCR1 overexpression group. The treatment of mice in each group was the same as step 1. At 3 months after drug injection, each group of mice was intraperitoneally injected with calcein for the first time. On the 7th day after the injection of calcein, the mice were intraperitoneally injected with calcein for the second time. Calcein was used to mark the surface of bone formation to mark the front of bone mineralization. Three days after the second injection of calcein, the mice in each group were killed, and the bone tissues of the mice in each group were isolated and extracted. They were decalcified and paraffin-embedded according to the above method, and then sliced ​​and observed under a fluorescence microscope. The bone formation rate was calculated by measuring the distance between the two calcein-labeled lines.

[0043] The results are as follows Figure 5 As shown in the figure, the osteoporosis model group had a significantly lower bone formation rate than the sham operation group due to severe bone loss. In contrast, the bone formation rate of mice in the osteoporosis + XCR1 overexpression group was significantly higher than that in the osteoporosis group, close to the level of the sham operation group, indicating that XCR1 overexpression can significantly increase the bone formation rate of osteoporotic mice, that is, XCR1 as a therapeutic target can restore the bone formation ability of osteoporotic mice, and rAAV-mediated XCR1 gene drugs have a significant therapeutic effect on osteoporosis.

[0044] 3. Osteoporosis test kit The osteoporosis detection kit includes reagents for detecting the expression level of XCR1. The specific components can be selected according to the specific experimental method. Molecular biological techniques (such as RT-qPCR, Western blot, flow cytometry, etc.) are used to detect the mRNA or protein level of XCR1 in the subject's trace bone tissue to determine whether the subject suffers from osteoporosis. In addition, the kit can also include standards and controls (such as quantitatively standardized normal human bone tissue samples, etc.) and normal reference values.

[0045] 4. Osteoporosis Detection System like Figure 6 As shown, the osteoporosis detection system includes: Data storage module: used to store the expression information of XCR1; Data analysis module: Calculate the patient's osteoporosis risk based on the expression information of XCR1; Result output module: outputs the results of the data analysis module.

[0046] It is understandable that the osteoporosis detection system provided by the present invention corresponds to the aforementioned down-regulated expression level of XCR1 in the bone tissue of osteoporosis patients and can be used for the detection of osteoporosis.

[0047] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Use of an agent that promotes XCR1 expression in the preparation of a drug for treating osteoporosis.

2. The use according to claim 1, characterized in that: The drug is a drug that targets osteoblasts.

3. Application of recombinant adeno-associated virus expressing XCR1 in the preparation of drugs for the treatment of osteoporosis.

4. A drug for treating osteoporosis, characterized in that: The invention comprises an agent for promoting the expression of XCR1 and a pharmaceutically acceptable auxiliary material.

5. Use of a reagent for detecting XCR1 expression level in the preparation of a product for detecting osteoporosis.

6. The use according to claim 5, characterized in that: When the XCR1 content in the bone tissue of the subject is lower than the normal value, the subject is determined to have osteoporosis.

7. The use according to claim 5, characterized in that The reagents for detecting the expression level of XCR1 are an upstream primer as shown in SEQ ID NO.5 and a downstream primer as shown in SEQ ID NO.

6.

8. A kit for detecting osteoporosis, characterized in that: Reagents for detecting the expression level of XCR1 are included.

9. Application of XCR1 in constructing a computational model for osteoporosis detection.

10. An osteoporosis detection system, characterized in that: include: Data storage device: used to store the expression information of XCR1; Data analysis device: Calculate the patient's osteoporosis risk based on the XCR1 expression information; Result output device: outputs the result of the data analysis device.

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