A bone-targeted trimeric protein and preparation method and application thereof

Abstract

The present application relates to a kind of bone-targeted trimeric protein and its preparation method and application, belong to biomedical technology field.The present application provides a kind of recombinant trimeric protein based on trimeric motif, including nuclear factor κB receptor activation factor extracellular segment, it is verified with in-vivo and in-vitro experiment with high bone targeting, possess inhibiting osteoclastogenesis and promoting osteoblast differentiation Dual control activity, it can also induce CD4 + Treg cell development, improve the bone marrow inflammatory microenvironment of osteoporosis model mice, can be used as the drug of osteoporosis, solves the problem that traditional single-function treatment drug in prior art will cause bone resorption rebound and bone loss after stopping drug, and further induces inflammation, provides new ideas and means for the research and treatment of osteoporosis, has good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to a bone-targeting trimeric protein and a preparation method and application thereof. BACKGROUND

[0002] Osteoporosis (OP) is a systemic degenerative disease characterized by decreased bone mass, microstructure damage, and easy fragility fracture. At present, the therapeutic drugs for osteoporosis mainly include two categories: bone formation promoters and bone resorption inhibitors. The bone formation promoters mainly include teriparatide, etc., and the bone resorption inhibitors mainly include bisphosphonate drugs, denosumab (a fully human monoclonal antibody against RANKL (Receptor Activator of Nuclear factor-kappa B (RANK) ligand), etc. A large number of clinical evidences show that denosumab can increase the bone mineral density of patients and reduce the risk of bone fracture in the vertebral body and other parts. However, after continuous medication for 2-3 years, the bone formation and resorption of patients are deeply inhibited (the median of bone formation rate is zero), and the therapeutic effect rapidly disappears after drug withdrawal: the bone resorption markers rapidly rise within 7-9 months after the last injection, and the peak value is 150% higher than the pre-treatment level after 1 year, and the bone mineral density also recovers to the baseline level within 18 months after drug withdrawal. In addition, patients may have jaw necrosis, atypical femoral fractures, and severe rebound of bone resorption after drug withdrawal, etc. There is a coupling effect between osteoblasts (OB) and osteoclasts (OC), and the enhancement of osteogenic differentiation will promote the secretion of coupling factors such as sRANKL, further promoting osteoclast differentiation, and osteoclasts will also secrete effectors that promote osteoblast differentiation. Therefore, drugs that only inhibit bone resorption or promote bone formation will destroy the coupling effect between OB-OC, and the ideal OP treatment drug should have the function of bidirectional regulation of promoting bone formation and inhibiting bone resorption.

[0003] RANKL-RANK / OPG (Osteoprotegerin) is a classic signal pathway of OB-OC coupling. RANKL is one of the members of the Tumor necrosis factor (TNF) superfamily, and exists in two forms of soluble (sRANKL) and membrane (mRANKL). Bone cells, stromal cells, etc. mainly promote osteoclast activation and bone resorption by producing sRANKL; and many members of the TNF superfamily have the characteristics of transmitting bidirectional signals, and RANK +Extracellular vesicles bind to mRANKL on the surface of osteoblasts, regulate the expression of runt-related transcription factor 2 (RUNX2) by activating mRANKL-mediated reverse signals, and promote osteoblast differentiation and bone formation. In addition, RANKL is first expressed in CD4 + It is found on T cells, and is closely related to the survival of dendritic cells (DC), the maintenance of immune tolerance, and the differentiation balance of CD4 + T cells. Studies have shown that the combination of denosumab and inhibitors of immune checkpoints such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) can enhance the body's anti-tumor immune response and improve the body's response to immune checkpoint inhibitors when treating melanoma. Therefore, while blocking RANKL-RANK, denosumab can inhibit the formation of regulatory T cells (Treg) and induce inflammation, which may be an important reason for the inflammatory side effects of denosumab. SUMMARY

[0004] To solve the above technical problems, the present application provides a recombinant trimeric protein based on a trimeric motif and containing an extracellular segment of nuclear factor kappa B receptor activator, which has been verified by in vivo and in vitro experiments to have high bone targeting, activate mRANKL-mediated reverse signals, and have bidirectional regulation activity of inhibiting osteoclastogenesis and promoting osteoblast differentiation. In addition, it can induce CDC4 + Treg cell development and improve the bone marrow inflammatory microenvironment of osteoporosis model mice, and can be applied to the preparation of osteoporosis treatment drugs.

[0005] The first object of the present application is to provide a bone-targeted trimeric protein, which comprises an extracellular segment of nuclear factor kappa B receptor activator, a trimeric motif and a bone-targeting peptide, and the amino acid sequence of the extracellular segment of the nuclear factor kappa B receptor activator is shown in SEQ ID NO. 1.

[0006] Further, the nucleotide sequence of the extracellular segment of the nuclear factor kappa B receptor activator is shown in SEQ ID NO. 2.

[0007] Further, the trimeric motif is an isoleucine zipper or a T4 phage fiber protein-derived domain sequence.

[0008] Further, the T4 phage fiber protein-derived domain sequence is a Foldon sequence.

[0009] Further, the amino acid sequence of the isoleucine zipper is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 4.

[0010] Further, the bone targeting peptide is (DSS)6 bone targeting peptide.

[0011] Further, the amino acid sequence of the bone targeting peptide is shown as SEQ ID NO. 5, and the nucleotide sequence is shown as SEQ ID NO. 6.

[0012] Further, the trimeric protein is connected by a linker.

[0013] Further, the trimeric protein further comprises a purification tag.

[0014] In an embodiment of the present application, the purification tag is a histidine tag.

[0015] A second object of the present application is to provide a preparation method of the above-mentioned trimeric protein, comprising the following steps:

[0016] Step S1, constructing a recombinant expression vector, wherein the recombinant expression vector comprises, in sequence, a coding gene sequence of a nuclear factor kappa B receptor activator extracellular fragment, a trimer motif and a bone targeting peptide;

[0017] Step S2, transferring the recombinant expression vector into an expression system for culture, and collecting the culture supernatant;

[0018] Step S3, purifying the obtained culture supernatant to obtain the trimeric protein.

[0019] Further, the recombinant expression vector takes pcDNA3.4 plasmid as a backbone.

[0020] In an embodiment of the present application, an IL-10 signal peptide is used to promote the secretion of the target protein.

[0021] Further, the expression system is a eukaryotic expression system.

[0022] In an embodiment of the present application, the eukaryotic expression system is human embryonic kidney cell Expi HEK293F.

[0023] A third object of the present application is to provide a sequence encoding the above-mentioned trimeric protein.

[0024] A fourth object of the present application is to provide the use of the above-mentioned trimeric protein in the preparation of a drug comprising at least one function of (a)-(e) as follows:

[0025] (a) inhibiting the activation of bone marrow-derived macrophages to osteoclasts;

[0026] (b) promoting mesenchymal stem cell differentiation into osteoblasts;

[0027] (c) activating membrane-type nuclear factor kappa B receptor activator ligand-mediated reverse signal;

[0028] (d) promoting CD4 + Treg cell development;

[0029] (e) improving bone marrow immune microenvironment.

[0030] Further, the amount of the trimeric protein is 0-0.2 μM.

[0031] A fifth object of the present application is to provide a medicament comprising at least one function of inhibiting bone marrow-derived macrophage activation into osteoclasts, promoting mesenchymal stem cell differentiation into osteoblasts, activating membrane-type nuclear factor kappa B receptor activator ligand-mediated reverse signal, promoting CD4 + Treg cell development and improving bone marrow immune microenvironment, the medicament comprising the above-mentioned trimeric protein.

[0032] A sixth object of the present application is to provide use of the above-mentioned trimeric protein in the preparation of an osteoporosis treatment medicament.

[0033] A seventh object of the present application is to provide an osteoporosis treatment medicament comprising the above-mentioned trimeric protein.

[0034] Further, the osteoporosis treatment medicament is in any pharmaceutically acceptable dosage form, including at least one of a tablet, a capsule, an injection, a granule, a suspension and a solution.

[0035] Advantages of the present application:

[0036] The trimeric protein provided by the present application has clear bone targeting, and can play a role in inhibiting osteoclast activation, promoting osteoblast differentiation and improving bone marrow immune microenvironment, etc. Compared with the traditional RANKL antibody single-target treatment, the unique bidirectional regulation mechanism of the trimeric protein avoids the risk of bone resorption rebound and bone mass decrease in a short period after drug withdrawal, overcomes the clinical bottleneck of compensatory rebound of bone metabolism in existing anti-bone resorption treatment, provides a new idea and tool for the research and treatment of osteoporosis, and at the same time verifies the universality of bidirectional regulation, which has obtained the same effect in humans, rats and mice. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which:

[0038] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which:Figure 1 Schematic diagram of the recombinant sequence of BTM-tri-RANK overexpression in Example 1 of the present application;

[0039] Figure 2 Coomasie brilliant blue staining diagram of the collection tubes in Example 1 of the present application, wherein A is the denatured reducing electrophoresis diagram of each collection tube after nickel column affinity purification, M lane is the protein molecular weight marker, and 1-14 lanes correspond to collection tubes 1-14 respectively; B is the denatured reducing electrophoresis diagram of each collection tube after molecular sieve purification, M lane is the protein molecular weight marker, and 1-4 lanes correspond to collection tubes 1-4 respectively; C is the denatured non-reducing electrophoresis diagram of each collection tube after molecular sieve purification, M lane is the protein molecular weight marker, and 1-4 lanes correspond to collection tubes 1-4 respectively;

[0040] Figure 3 Protein purity detection by high performance liquid chromatography in Example 1 of the present application, wherein A is the protein purity detection of BTM-tri-RANK, and B is the protein purity detection of non-bone-targeting RANK trimer NT-tri-RANK;

[0041] Figure 4 Protein molecular weight size detection results in Example 1 of the present application, wherein A is the molecular weight size of BTM-tri-RANK, and B is the molecular weight size of RANK monomer protein;

[0042] Figure 5 Protein identification results of BTM-tri-RANK in Example 1 of the present application, wherein A is the Coomasie brilliant blue staining results of the denatured non-reducing electrophoresis gel of BTM-tri-RANK, B is the Western blot detection results of Anti-Histag antibody, and C is the WB detection results of Anti-RANK antibody, M lane is the protein molecular weight marker, lane 1 is the BTM-tri-RANK protein, and lane 2 is the RANK monomer protein;

[0043] Figure 6 Bone targeting verification results of BTM-tri-RANK protein in Example 1 of the present application, wherein A is the bone targeting results of the trimer protein in vitro, and B is the bone targeting results of the trimer protein in vivo;

[0044] Figure 7Figure 2A shows representative images of TRAP staining to detect the inhibitory effect of different concentrations of BTM-tri-RANK on osteoclast activation, where A is a representative image of TRAP staining to detect the inhibitory effect of different concentrations of BTM-tri-RANK, Anti-RANKL and RANK monomer on osteoclastogenesis, the scale bar is 200 μm, B is a quantitative analysis of TRAP staining, *** represents a comparison between RANK monomer and BTM-tri-RANK, and ### represents a comparison between Anti-RANKL and BTM-tri-RANK;

[0045] Figure 8 Figure 2B shows the bone plate detection of the bone erosion ability of osteoclasts after BTM-tri-RANK intervention, where A is a representative image of the bone erosion ability of osteoclasts after BTM-tri-RANK intervention, the scale bar is 200 μm, and B is a quantitative result of the bone plate erosion area;

[0046] Figure 9 Figure 2C shows the real-time quantitative PCR detection of the expression of osteoclast-related genes inhibited by BTM-tri-RANK, where A is a quantitative chart of the relative expression of gene mRNA, B is a quantitative chart of the relative expression of gene mRNA, and C is a quantitative chart of the relative expression of gene mRNA; Ctsk Oscar Nfatc1

[0047] Figure 10 Figure 2D shows the inhibitory effect of BTM-tri-RANK on the osteoclast activation of human peripheral blood mononuclear cells, where A is a representative image of the osteoclastogenesis of human peripheral blood-derived mononuclear cells by RANK monomer and BTM-tri-RANK under a microscope, the scale bar is 100 μm, and B is a quantitative analysis result of the TRAP positive (TRAP + ) area;

[0048] Figure 11 Figure 3A shows the ALP staining results of BTM-tri-RANK promoting osteogenic differentiation of mouse BMSCs, where A is a general observation and a representative image under a microscope, the scale bar is 200 μm, B is a quantitative result of ALP, C is an alizarin red staining general observation and a representative image under a microscope, the scale bar is 200 μm, and D is a quantitative analysis result of alizarin red;

[0049] Figure 12 Figure 3B shows the real-time quantitative PCR detection of the expression of osteogenic-related genes promoted by BTM-tri-RANK in mouse BMSCs, where A is the expression of bone-related gene Opn , B is the expression of bone-related gene Col1a1 , C is the expression of bone-related gene Runx2 , and D is the expression of bone-related gene​​​Sp7 expression;

[0050] Figure 13 The WB assay for the effect of BTM-tri-RANK on mRNAKL reverse signaling and osteogenesis-related proteins in Example 3 of the present invention, wherein A is the WB assay for the expression of mRNAKL reverse signaling and osteogenesis-related proteins in mouse BMSCs after 3 days of intervention with 0.05 μM BTM-tri-RANK and Anti-RANKL in osteogenic induction medium (OB) or conventional medium (CTR), B is the quantitative analysis result of RUNX2 protein expression, and C is the quantitative analysis result of p-mTOR protein expression;

[0051] Figure 14 Figure 3 shows the promotion of osteogenic differentiation of human MSCs by BTM-tri-RANK in Example 3 of the present invention. The scale bar is 200 μm, wherein A is a representative image of ALP staining of osteogenic induction of human umbilical cord-derived MSCs, B is a representative image of alizarin red staining of human umbilical cord-derived MSCs, C is a representative image of ALP staining of human bone marrow-derived BMSCs under the microscope, D is a representative image of alizarin red staining of human bone marrow-derived BMSCs under the microscope, E is a quantitative analysis result of ALP staining of human bone marrow-derived BMSCs, and F is a quantitative analysis result of alizarin red staining of human bone marrow-derived BMSCs;

[0052] Figure 15 These are the Micro-CT results of the femurs of mice in each group after 4 weeks of treatment in Example 4 of the present invention, wherein A is a representative two-dimensional Micro-CT image of each group after 4 weeks of administration, B is a representative three-dimensional Micro-CT reconstruction image of each group, C is the quantitative analysis and statistical results of bone density of mice in each group, D is the quantitative analysis and statistical results of relative bone volume or bone volume fraction of mice in each group, E is the quantitative analysis and statistical results of trabecular thickness of mice in each group, and F is the quantitative analysis and statistical results of trabecular number of mice in each group;

[0053] Figure 16 These are the Micro-CT results of the femurs of mice in each group 3 months after drug withdrawal in Example 4 of the present invention, wherein A is a representative two-dimensional image of Micro-CT scans of mice in each group after 4 weeks of drug administration and 3 months after drug withdrawal, and B is a representative three-dimensional image reconstructed after scanning of mice in each group; C is the quantitative analysis and statistical results of bone density of mice in each group, D is the quantitative analysis and statistical results of relative bone volume or bone volume fraction of mice in each group, E is the quantitative analysis and statistical results of trabecular thickness of mice in each group, and F is the quantitative analysis and statistical results of trabecular number of mice in each group;

[0054] Figure 17Figure 6 is a H&E staining image of mouse femur in Example 4 of the present application, wherein the upper image is the result under 2.5 times objective, the scale is 500 μm, and the lower image is the result under 10 times objective, the scale is 125 μm;

[0055] Figure 18 Figure 7 is a Masson trichrome staining image of mouse femur in Example 4 of the present application, wherein the upper image is the result under 2.5 times objective, the scale is 500 μm, and the lower image is the result under 10 times objective, the scale is 125 μm;

[0056] Figure 19 Figure 8 is a TRAP staining detection of the number of mouse bone marrow osteoclasts in Example 4 of the present application, wherein A is a representative picture of TRAP staining of mouse femur after 4 weeks of treatment, the red area is TRAP + osteoclasts, the scale is 125 μm, and B is a quantitative analysis statistical result of the TRAP + area;

[0057] Figure 20 Figure 9 is an immunohistochemical staining analysis of mouse bone marrow osteogenic related molecules in Example 4 of the present application, the scale is 20 μm, wherein A is a representative picture of type I collagen immunohistochemical staining of mouse femur after 4 weeks of treatment, B is a representative picture of RUNX2 immunohistochemical staining of mouse femur after 4 weeks of treatment, C is a quantitative analysis statistical result of type I collagen, and D is a quantitative analysis statistical result of RUNX2;

[0058] Figure 21 Figure 10 is a calcein labeling detection of mouse dynamic bone formation ability in Example 4 of the present application, wherein A is a representative picture of dynamic bone formation ability detection of mouse cortical bone, the scale is 20 μm, and B is a quantitative analysis statistical chart of bone formation distance;

[0059] Figure 22 Figure 11 is an ELISA detection of bone metabolism markers in mouse serum in Example 4 of the present application, wherein A is an ELISA detection of bone formation marker P1NP level in mouse serum after 4 weeks of treatment, and B is an ELISA detection of bone resorption marker CTX-1 level in mouse serum after 4 weeks of treatment;

[0060] Figure 23 Figure 12 is a BTM-tri-RANK treatment to increase the number of osteogenic related cells in Example 4 of the present application, wherein A is a representative result of flow cytometry analysis of the proportion of mouse bone marrow osteogenic related cells (Sca-1 + CD51 - and early osteogenic related cells (Sca-1 - CD51 + , pOB) in the mouse bone marrow after 4 weeks of administration, and B is a representative result of flow cytometry analysis of the proportion of mouse bone marrow osteoclasts (CD115+ CD117 + A is a representative result of the proportion of pOB and OB cells in bone marrow cells, C is a quantitative statistical result of the proportion of OC cells in bone marrow cells, and D is a quantitative statistical result of the proportion of OC cells in bone marrow cells;

[0061] Figure 24 The BTM-tri-RANK in Example 5 of the present invention affects CD4 + A is flow cytometry analysis of CD4 T cell differentiation. + Representative images and quantitative analysis results of the proportion of Treg cells in T cells. B is a representative image and quantitative analysis results of the proportion of Th17 cells analyzed by flow cytometry;

[0062] Figure 25 These are the levels of inflammation-related factors in the serum of mice in each group after 4 weeks of drug administration in Example 5 of the present invention, wherein A is the level of inflammation-related factor IL-6 in the serum of mice after 4 weeks of treatment by multi-factor detection, B is the level of inflammation-related factor Kc / gro in the serum of mice after 4 weeks of treatment by multi-factor detection, C is the level of TNF-α in the serum of mice after 4 weeks of treatment by multi-factor detection, and D is the level of IL-10 in the serum of mice after 4 weeks of treatment by multi-factor detection;

[0063] Figure 26 The percentage of immune cells in the lymph nodes of each group after 4 weeks of administration in Example 5 of the present invention, wherein A is a representative result of flow cytometric analysis of the proportion of T cells and B cells in the total cells in the lymph nodes of mice after 4 weeks of treatment, and B is a representative result of flow cytometric analysis of CD4 T cells in the lymph nodes of mice after 4 weeks of treatment. + T cells, CD8 + Representative results of the proportion of T cells, C is the quantitative analysis of the proportion of B cells and T cells in the total cells of the lymph nodes, D is the CD4 + T cells, CD8 + Quantitative analysis and statistical results of the proportion of T cells in T cells;

[0064] Figure 27 The percentage of Treg and Th17 cells in the lymph nodes of each group of mice after 4 weeks of treatment in Example 5 of the present invention, where A is the percentage of Treg cells in the lymph nodes of each group of mice analyzed by flow cytometry after 4 weeks of treatment. + Representative results of the proportion of T cells. B is the flow cytometry analysis of the proportion of Th17 cells in CD4 + Representative results of the proportion of T cells, C is Treg cells in CD4 + Quantitative analysis of the proportion of T cells, D is the proportion of Th17 cells in CD4 + Quantitative analysis and statistical results of the proportion of T cells. DETAILED DESCRIPTION

[0065] The present application is further described below with reference to the accompanying drawings and specific examples, which are provided to give a better understanding of the application and enable its implementation by those skilled in the art, but the examples are not intended to limit the present application.

[0066] Example 1: Preparation of bone-targeted RANK trimer

[0067] (1) Construction of expression plasmid and transfection

[0068] Using genetic engineering technology, a bone-targeted RANK trimer (BTM-tri-RANK) overexpression sequence was constructed, and the sequence structure is as shown in Figure 1 The recombinant expression sequence was synthesized into a pcDNA3.4 eukaryotic cell expression vector, and human IL-10 was used as a signal peptide to promote the secretion of the target protein, wherein the trimer motif is an isoleucine zipper (ILZ), and the histidine tag Histag is used for subsequent protein purification.

[0069] The amino acid sequence of the RANK extracellular segment is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2; the amino acid sequence of the isoleucine zipper is shown in SEQ ID NO. 3, and the nucleotide sequence is shown in SEQ ID NO. 4; the amino acid sequence of the bone-targeting sequence is shown in SEQ ID NO. 5, and the nucleotide sequence is shown in SEQ ID NO. 6; the nucleotide sequence of the signal peptide IL-10 is shown in SEQ ID NO. 7; and the nucleotide sequence of the histidine tag 6xHistag is shown in SEQ ID NO. 8.

[0070] The trimer protein is connected by a linker (Linker); the amino acid sequence of the linker is shown in SEQ ID NO. 9, and the amino acid sequence is shown in SEQ ID NO. 10.

[0071] Human embryonic kidney cells Expi HEK293F cells were used for transfection. After 5 days of transfection, the cell state was observed, and when the survival rate was less than 60%, the cells were collected at 4°C by centrifugation, and were stored at -80°C or directly subjected to protein purification.

[0072] A non-targeted RANK trimer NT-tri-RANK was also constructed as a control, and the construction method was the same as above, except that it did not contain the bone-targeting sequence.

[0073] (2) Purification of trimer protein

[0074] a. Affinity chromatography purification:

[0075] The cell supernatant was collected 5 days after cell transfection, and the supernatant was loaded onto a nickel column using an AKTA protein purification system, and the cell supernatant was purified by binding of the histone tag to the nickel column.

[0076] A preliminary test of the protein concentration of 1 mL / tube of the collected liquid was performed using Coomassie brilliant blue G250 staining solution, and the collection tubes with strong color development were sampled, and then denaturing and reducing SDS-PAGE electrophoresis was performed. The Coomassie brilliant blue staining results showed that almost all the collection tubes had obvious protein bands at about 35 kD (A in Figure 2 After dialysis, molecular sieving was performed.

[0077] b. Molecular sieving:

[0078] The product purified by nickel column affinity chromatography was further purified by molecular sieving, and PBS was used as the buffer system to remove other impurities that did not meet the molecular weight of the trimer.

[0079] SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) denaturing reduction and non-reduction electrophoresis was performed on the proteins in each collection tube of the molecular sieve, and the collection tubes with higher purity were selected for protein recovery (B and C in Figure 2 to obtain RANK trimer protein (BTM-tri-RANK or NT-tri-RANK) with higher purity and no RANK monomer contamination.

[0080] (3) Trimer protein purity identification and molecular weight size detection

[0081] a. SDS-PAGE electrophoresis

[0082] Denaturing non-reducing electrophoresis: part of the purified BTM-tri-RANK protein, NT-tri-RANK protein or commercial RANK monomer protein was taken out, the corresponding volume of denaturing non-reducing loading buffer was added according to the volume, and electrophoresis was directly performed.

[0083] b. Coomassie brilliant blue staining

[0084] After electrophoresis, Coomassie brilliant blue staining solution was used.

[0085] c. Western blot (WB) detection

[0086] After electrophoresis and membrane transfer, the nitrocellulose membrane was blocked at room temperature for 1 hour, Anti-6xHistag (horseradish peroxidase HRP labeled, 1:2000 dilution) and RANK antibody Anti-RANK (1:1000 dilution) were added, and incubated at 4°C overnight.

[0087] After overnight, the nitrocellulose membrane was placed in a shaker at room temperature for 5 minutes, repeated three times, and the Anti-6xHistag incubated band was directly developed, exposed; to the RANK antibody (Anti-RANK) incubated band, 1:1000 diluted horseradish peroxidase HRP labeled goat anti-rabbit secondary antibody was added, incubated at room temperature for 1 month, after incubation, the WB washing liquid was washed three times, 1:1 mixed color developing substrate was added, automatic exposure, and the image was saved.

[0088] d. High Performance Liquid Chromatography (HPLC) and Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) detection

[0089] HPLC detection: Biomix-SEC separation column was used to detect the purity of the sample;

[0090] SEC-MALS detection: microDAWN was connected to the HPLC-SEC system, and hydrophilic silica gel high efficiency molecular exclusion chromatography column TSK-G3000SWXL was used to directly determine the molecular weight of the obtained protein.

[0091] After further purification of the protein by molecular sieve chromatography, the high-purity component F3 was collected, concentrated, and the HPLC detection showed that the purity of the target protein was 98% (F3). Figure 3 ).

[0092] According to the principle of molecular exclusion, the molecular weight of BTM-tri-RANK protein was determined, and the SEC-MALS detection showed that the molecular weight of the target protein was 102.5 kD (A in Figure 4 ), and the corresponding RANK monomer was detected by MALS, with a molecular weight of 28.31 kD (B in Figure 4 ). Therefore, BTM-tri-RANK conforms to the theoretical molecular weight of the trimer protein.

[0093] SDS-PAGE electrophoresis was performed on the purified protein, and the results of Coomassie brilliant blue staining showed that the RANK monomer existed as a single band at about 25 kD under denaturing non-reducing conditions; and under denaturing non-reducing conditions, BTM-tri-RANK existed mainly as a band greater than 100 kD, and weak bands existed at 70 kD and 35 kD (A in Figure 5 ), which may be due to the dissociation of protein structure under different electrophoresis conditions.

[0094] Meanwhile, Western Blot (WB) qualitative detection was carried out, and the protein bands were incubated with Anti-Histag antibody and Anti-RANK antibody respectively, and the results showed that the purified protein was determined as RANK trimer—BTM-tri-RANK. Figure 5 B and C in the formula (I).

[0095] (4) Detection of bone targeting ability of the trimer protein

[0096] In vitro detection: the trimer BTM-tri-RANK protein was diluted with PBS buffer to 0.5, 1, 2, 4, 5, 8, 10, 15, 20 μM concentrations respectively, and non-targeted RANK trimer (NT-tri-RANK) groups were set up at 1, 4, 8, 10, 20 μM as controls, and PBS holes were blank controls, 3 repeated holes were set up in each group, 100 μL / hole was added to the bone plate (96-well plate), 4 ℃ incubation overnight, ELISA washing liquid was washed for three times, HRP labeled Anti-Histag antibody working solution (1:10000) was added, 37 ℃ incubation for 30 minutes, ELISA washing liquid was washed for three times, 90 μL of substrate working solution (TMB) was added, 37 ℃ incubation for 5-15 minutes, 50 μL of stop solution was added per hole after the color was obvious, and the value was read at an absorbance of 450 nm. With the increase of protein concentration, more Histag fusion protein can be detected on the bone plate, when the protein concentration is 1 μM, the OD value of the BTM-tri-RANK group is 2.96±0.178 times of the NT-tri-RANK group, and the BTM-tri-RANK is significantly attached to the non-targeted NT-tri-RANK at each concentration of ≥1 μM (P<0.05) Figure 6 A), which proves that the BTM-tri-RANK has good binding ability to the bone plate.

[0097] In vivo detection: Select 6-8 weeks of female C57BL / 6 mice of the same batch, randomly divided into 3 groups, 3 in each group, 150 μL of PBS buffer, BTM-tri-RANK (150 μg), non-bone targeted RANK trimer (NT-tri-RANK, 150 μg) were given by tail vein, 2 hours after injection, the mice were sacrificed, the femur and tibia of the mice were stripped, the cells in the bone marrow cavity were removed, the remaining femur and tibia were put into a mortar, the bone was ground into powder in liquid nitrogen, 100 μL of pre-cooled RIPA lysis buffer was added, 4 ℃ for 1 hour, 14800 rpm, 4 ℃ centrifugal 30 minutes, take supernatant. According to the BCA quantitative kit instructions, the protein concentration was detected, the concentration of all samples was adjusted to 5 μg / μL, denaturing reducing loading buffer was added, 100 ℃ for 5 minutes, the protein was divided, and frozen at -80 ℃. Each sample was loaded according to 15 μL, SDS-PAGE electrophoresis and membrane transfer were carried out, the nitrocellulose membrane after membrane transfer was incubated with HRP labeled Anti-Histag antibody (1:1000 dilution), after gel imaging, WB washing liquid was washed for 5 minutes, antibody stripping liquid was poured into room temperature for 30 minutes, after washing for 5 minutes, blocking solution was added, incubated at room temperature for 1 hour, after discarding the blocking solution, mouse Anti-β-actin antibody was added, after washing, the corresponding secondary antibody was incubated, and the gel imaging instrument was exposed. The results show that BTM-tri-RANK has good bone targeting effect in vivo (B). Figure 6

[0098] Example 2: Bone-targeted RANK trimer blocks RANKL positive signal to inhibit bone marrow-derived macrophage cell osteoclast activation

[0099] (1) Effect of different concentrations of trimer protein BTM-tri-RANK on osteoclast activation of mouse bone marrow-derived macrophage cells (BMMs)

[0100] After euthanizing 6-8 weeks of female C57BL / 6 mice, the femur and tibia of the mice were stripped, and the bone marrow cells were collected. 1 mL of red blood cell lysis solution was added to the cell precipitate to remove red blood cells.

[0101] The cells were resuspended with 10 mL of α-MEM complete medium containing 10% fetal bovine serum, cultured overnight (14-16 hours), and the suspended cells were taken, 30 ng / mL of macrophage colony-stimulating factor (M-CSF) was added, mixed, and then added to a 6-well plate for culture for 3 days. After 3 days, the supernatant was removed, PBS was added, and the adherent cells were scraped off with a cell scraper, and the cells were collected by centrifugation at 1200 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and the cells were counted to 2×10 4 ​Cells were seeded at 1 cell / well in 48-well plates (with M-CSF added during the period). After the cells adhered, the medium was changed and RANKL (50 ng / mL) and different concentrations of BTM-tri-RANK / RANK monomer / RANKL antibody Anti-RANKL were added to the medium according to different experimental groups. In order to eliminate the differences in the number of molecules and pharmacodynamics caused by the differences in molecular weight among the three drugs, BTM-tri-RANK, RANK monomer and RANKL antibody Anti-RANKL, the molar concentration of the drugs was used (the same number of effective molecules in the same volume). The medium was changed every 3 days, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed on the 3rd day, and tartrate-resistant acid phosphatase (TRAP) staining was performed after 5 days.

[0102] Osteoclast induction was performed for 5 days, and when obvious osteoclast morphology appeared under a microscope, tartrate-resistant acid phosphatase (TRAP) staining was performed. Before TRAP staining, the culture medium was discarded, 300 μL of 4% paraformaldehyde solution was added to each well, and the cells were fixed at room temperature for 10 minutes. After fixation, the paraformaldehyde was discarded, and the cells were washed with PBS buffer twice. Deionized water was preheated, and TRAP staining was performed according to the requirements of the kit instructions. Photographs were taken under a microscope, and the number of TRAP-positive cells was counted and statistically analyzed.

[0103] The results of TRAP staining showed that the number of osteoclasts in each field of view under RANKL induction was 37.33 ± 5.03, and 0.5 nM BTM-tri-RANK could effectively inhibit the generation of mature osteoclasts, but there were still some TRAP + Osteoclast precursor cells, with the increase of the concentration of BTM-tri-RANK, TRAP + cells also disappeared, and osteoclast differentiation was completely inhibited ( Figure 7 Therefore, the inhibitory effect of BTM-tri-RANK on RANKL-induced osteoclastogenesis has a concentration gradient effect. At the same time, we compared the ability of RANK monomer, Anti-RANKL and BTM-tri-RANK to inhibit osteoclastogenesis at different concentrations ( Figure 7The number of osteoclasts in each field was 34.33 ± 8.39, 36 ± 3.46, 34.33 ± 3.21, 25.33 ± 3.06, and 20.33 ± 1.53 under the intervention of 0.5 nM, 1 nM, 2 nM, 3 nM, and 4 nM RANK monomers, respectively; the number of osteoclasts in the 0.5 nM and 1 nM Anti-RANKL groups was 35.33 ± 0.58 and 4 ± 1.73 per field. The results showed that, at the same molar concentration, BTM-tri-RANK had better RANKL binding ability than RANK monomers and Anti-RANKL, and could better inhibit osteoclastogenesis.

[0104] (2) Detection of the effects of different concentrations of the trimeric protein BTM-tri-RANK on the activation of mouse BMMs and bone erosion

[0105] After culturing the mouse bone marrow cavity cells in α-MEM complete medium containing M-CSF for 3 days, the cells were scraped off using a cell scraper and inoculated in 96-well bone plates at 10 4 After the cells adhered, different concentrations of Anti-RANKL and BTM-tri-RANK were added to the α-MEM complete medium for intervention according to different groups. The medium was replaced every 3 days, and after the bone plate showed obvious osteoclast erosion boundary, the medium was removed, PBS was washed 3 times, 200 μL PBS was added to each well, and ultrasonic cleaning was performed for 30-60 minutes to remove residual cells. After the cells were completely removed, microscopic observation and photography were performed. Image J software was used to statistically analyze the bone plate erosion area in each field.

[0106] The bone plate absorption assay results showed that both BTM-tri-RANK and Anti-RANKL could significantly inhibit the formation of bone absorption pits mediated by osteoclasts. At a concentration of 0.5 nM, the bone absorption pit area of the BTM-tri-RANK intervention group was smaller than that of the Anti-RANKL group Figure 8 ), indicating that BTM-tri-RANK treatment could better inhibit the function of osteoclasts in vitro than Anti-RANKL treatment at the same concentration.

[0107] (3) Detection of BMMs osteoclast-related gene expression using quantitative real-time polymerase chain reaction (qRT-PCR)

[0108] Through qRT-PCR experiments, it was found that 3 days after RANKL induction, the expression of osteoclast-related genes in the RANKL-induced groupCtsk Gene expression was 18055.64 ± 2140.889 times (A) of the control group (M-CSF) Figure 9 Oscar Gene expression was 612.86 ± 20.12 times (B) Figure 9 Nfatc1 Gene expression was 6.48 ± 0.149 times (C), and after BTM-tri-RANK intervention, the gene expression was 192.56 ± 33.9, 37.64 ± 4.343, and 0.94 ± 0.134 times of the M-CSF group, respectively, significantly down-regulating the expression of osteoclast-related genes. Figure 9

[0109] (4) Detection of the inhibitory ability of BTM-tri-RANK on the differentiation of human peripheral blood mononuclear cells into osteoclasts

[0110] Ficoll density gradient centrifugation was used to isolate mononuclear cells (PBMCs) from the peripheral blood of healthy volunteers; 2 x 10 6 cells / 1 mL / well were inoculated in a 48-well plate, the control group was cultured with α-MEM complete culture medium containing 20 ng / mL human macrophage colony-stimulating factor (M-CSF), the RANKL induction group was added with 20 ng / mL human M-CSF and 50 ng / mL human RANKL recombinant protein in the culture medium, and the BTM-tri-RANK and RANK monomer intervention groups were added with different concentrations of BTM-tri-RANK and RANK monomers on the basis of the RANKL induction group for induction, and 3 replicate wells were set up for each group.

[0111] TRAP staining was performed according to the above method, and the results showed that BTM-tri-RANK had a concentration gradient effect on the inhibition of RANKL-mediated osteoclast formation, and its inhibitory effect was stronger than that of RANK monomer (0.94 ± 0.134 times) Figure 10 ), effectively inhibiting the osteoclast activation of human peripheral blood mononuclear cells.

[0112] Example 3: Bone-targeted RANK trimer activates RANKL reverse signaling on the cell membrane surface to promote osteogenic differentiation of mesenchymal stem cells

[0113] I. Detection of the osteogenic differentiation status of BMSCs

[0114] ​​​Osteogenic induction medium formula: α-MEM complete medium containing 10 mM β-glycerophosphate, 50 μg / mL vitamin c, 10 mM dexamethasone. 3 independent repeat experiments were performed, 3 replicate wells were set up in each group, and the data statistics and analysis of the independent experiment results of the same batch were shown in the results. The experiment was divided into the following groups:

[0115] (1) CTR: α-MEM complete medium treatment group;

[0116] (2) CTR-BTM-tri-RANK: α-MEM complete medium containing 0.05 μM BTM-tri-RANK treatment group;

[0117] (3) CTR-Anti-RANKL: α-MEM complete medium containing 0.05 μM Anti-RANKL treatment group;

[0118] (4) OB: Osteogenic induction medium group;

[0119] (5) OB-BTM-tri-RANK: Osteogenic induction medium containing 0.05 μM BTM-tri-RANK treatment group;

[0120] (6) OB-Anti-RANKL: Osteogenic induction medium containing 0.05 μM Anti-RANKL treatment group.

[0121] After 7 days of osteogenic induction culture of cells, ALP (alkaline phosphatase) staining can be performed. Before staining, remove the culture medium, wash with PBS at room temperature for 2-3 times, use pre-cooled 4% paraformaldehyde to fix at room temperature for 10 minutes, wash with PBS for 3 times, add BCIP / NBT (5-bromo-4-chloro-3-indole phosphate p-toluidine salt / tetrazolium chloride nitrogen blue) staining working solution (operate according to the instructions, add 300-fold diluted BCIP solution and 150-fold diluted NBT solution in the alkaline phosphatase color developing buffer), incubate at 37°C, observe the color depth every 5 minutes, and when the difference between groups is obvious, slowly immerse in deionized water to terminate the staining.

[0122] At the same time of ALP staining, RIPA lysis buffer was used to extract protein from the same batch of cells in the 6-well plate which were treated in the same way. The protein concentration was measured according to the operation of the BCA quantitative kit. At the same time, the standard of the ALP activity quantitative kit was diluted, and the blank control group was set up. The protein was diluted 10-50 times to a total volume of 50 μL using the detection buffer. 50 μL of color developing substrate was added to each well of the blank control group and the experimental group, and then mixed well and incubated at 37°C for 5-10 minutes. Then the OD value was measured at 405 nm. According to the standard concentration and OD value, a standard curve was drawn, and the product concentration of each well in the experimental group was calculated according to the formula. The amount of alkaline phosphatase required to hydrolyze the color developing substrate to produce 1 μmol of product per minute at 37°C was defined as one enzyme activity unit, i.e. the ALP activity detected in the diethanolamine buffer system. The enzyme activity between groups was calculated. In order to more accurately analyze the enzyme activity, the results were normalized in the form of enzyme activity units / protein concentration.

[0123] ALP is an important marker of osteoblast activity during early differentiation of osteoblasts. Through ALP staining and quantification, we found that the ALP activity of the OB-BTM-tri-RANK group was 2.68±0.022 times that of the OB group and 3.53±0.029 times that of the OB-Anti-RANKL group (P<0.05, P<0.01, respectively). Figure 11 We found that under the premise of osteogenic induction, OB-BTM-tri-RANK had stronger ALP activity than OB and OB-Anti-RANKL groups, which proved that it could promote the early development of osteoblasts.

[0124] After 21 days of osteogenic induction of the cells in the 24-well plate (3 replicate wells were set up for each group), the osteogenic induction group was observed under a microscope to see obvious calcium nodules. The culture medium was discarded, and the cells were washed with PBS for 3 times (note that the steps should be slow and gentle to prevent the calcium nodules from falling off). 4% paraformaldehyde was used for room temperature fixation for 10 minutes. The fixation solution was discarded, and the cells were washed with PBS for 2 times. Alizarin red staining solution was added, and the cells were incubated at room temperature for 10-20 minutes. When the color of the calcium nodules was obvious, the staining was discarded, and deionized water was added to remove the residual staining. Gross observation and microscope photography were performed.

[0125] After qualitative observation with Alizarin Red, 1 mL of 10% cetylpyridinium chloride was added to each well and the cells were incubated on a shaker at room temperature for 1 hour. After incubation, the same volume of calcium dissolution solution was transferred to a 96-well plate and the absorbance was measured at 540 nm using a microplate reader. The results of Alizarin Red staining also showed the same trend. Quantitative results of calcium nodule dissolution showed that the OB-BTM-tri-RANK group was 1.23 ± 0.028 times that of the OB group and 1.20 ± 0.027 times that of the OB-Anti-RANKL group ( Figure 11 (C and D) BTM-tri-RANK can promote the early differentiation and mineralization of mouse bone marrow-derived BMSCs compared with Anti-RANKL.

[0126] 2. Effect of qRT-PCR on the expression of osteogenesis-related genes

[0127] Mouse BMSCs were cultured at a rate of 2×10 5 Cells / 2 mL / well were seeded in 6-well plates. After 3 days of different interventions, RNA was extracted and qRT-PCR was used to detect genes related to osteogenic differentiation.

[0128] After BTM-tri-RANK and Anti-RANKL were used to treat mouse BMSCs for 3 days, qRT-PCR was performed to detect osteogenic differentiation-related genes in mouse BMSCs. Opn 、 Col1a1 、 Runx2 and Sp7 The expression levels were 1.13±0.020, 2.78±0.077, 1.14±0.029 and 1.58±0.036 times of those in the OB group ( Figure 12 Compared with Anti-RANKL, BTM-tri-RANK can significantly upregulate osteogenesis-related genes— Runx2 and Sp7 expression, promoting osteogenic differentiation.

[0129] 3. Western blot analysis of the expression of osteoblast-related transcription factors (RUNX2) and proteins related to the PI3K-Akt-mTORC signaling pathway associated with mRANKL reverse signaling

[0130] Mouse BMSCs were treated for 3 days under different conditions, the culture medium was discarded, and the cells were collected and lysed to extract proteins; after BCA protein quantification, SDS-PAGE electrophoresis was performed. After transfer, the corresponding primary antibody was added, and incubated at 4°C overnight. The next day, after washing, the corresponding secondary antibody was incubated, and after washing, the color developing substrate was added. The gel imaging system was exposed. After exposure, the nitrocellulose membrane can be washed, and after stripping with antibody stripping solution, it is resealed and other primary antibody working solution is added for detection of different target proteins. ImageJ software was used for quantitative analysis of protein bands.

[0131] We extracted the proteins from mouse BMSCs induced for 3 days, and after BCA quantification, we detected the expression of osteogenic-related transcription factor RUNX2. WB results showed that RUNX2 was significantly up-regulated after BTM-tri-RANK intervention compared with the OB group and the Anti-RANKL group (Fig. 4A and B). Figure 13

[0132] We performed WB detection of key proteins in the PI3K-Akt-mTORC1 signaling pathway. The results showed that BTM-tri-RANK promoted the phosphorylation of mTOR (Fig. 5A and C), while it had no significant effect on the overall expression and phosphorylation level of PI3K, indicating that it may exert a pro-osteogenic effect through the PI3K-Akt-mTORC1 signaling pathway. Therefore, compared with Anti-RANKL, BTM-tri-RANK can promote the expression of osteogenic-related transcription factor RUNX2, regulate the PI3K-Akt-mTORC1 signaling pathway, and promote osteogenic differentiation, while OPG, RANK-Fc, and Denosumab can block the positive signal, but they also block the bone formation mediated by the reverse signal of mRANKL. Figure 13 Four, detection of the effect of BTM-tri-RANK on the osteogenic differentiation of mesenchymal stem cells (MSCs) from healthy donors

[0133] MSCs from umbilical cord and BMSCs from bone marrow were inoculated in 24-well plates at 2×10 4 cells / 1 mL / well, and in 6-well plates at 10 5 cells / 2 mL / well. When the cells reached 80-90% confluence, the corresponding medium was replaced for intervention, and the cells were replaced every 3 days. After 7 days of culture, ALP staining and quantification were performed, and around 21 days, alizarin red staining and quantification were performed.

[0134]

[0135] ​​According to different groups, we added 0.05 μM of BTM-tri-RANK on the basis of osteogenic differentiation, and the results of ALP staining showed that BTM-tri-RANK could significantly enhance the early osteogenic differentiation ability of human umbilical cord-derived MSCs (A in Figure 14 ; the results of alizarin red staining showed that BTM-tri-RANK could promote the osteogenic mineralization of umbilical cord-derived MSCs (B in Figure 14 ).

[0136] We used pediatric bone marrow-derived BMSCs to induce osteogenesis and added BTM-tri-RANK intervention, and the results of ALP staining were consistent with those of umbilical cord-derived MSCs (C and E in Figure 14 ), and BTM-tri-RANK could also significantly enhance the early osteogenic ability of human BMSCs. Alizarin red staining and quantitative results showed that BTM-tri-RANK significantly increased the formation of calcium nodules (D and F in Figure 14 ). Therefore, BTM-tri-RANK can promote the early osteogenic differentiation and osteogenic mineralization of human MSCs.

[0137] Example 4: Therapeutic effect of bone-targeted RANK trimer on OVX mice in the treatment of bone metabolism

[0138] (1) Analysis of bone parameters related to mouse femur

[0139] 8-week-old female C57BL / 6 mice were obtained by removing both ovaries to obtain OVX (ovariectomy-induced osteoporosis) mice.

[0140] After 2 weeks, the sham operation group (Sham, using 3-0 suture to suture the inner and outer skin of the mouse, and then iodophor disinfection) mice were randomly divided into 2 groups; OVX group mice were randomly divided into 3 groups. The sham operation group and the OVX group were injected with 100 μL / each of PBS via the tail vein; the sham operation-BTM-tri-RANK group and the OVX-BTM-tri-RANK group were injected with 100 μL / each of BTM-tri-RANK protein (10 mg / kg) via the tail vein, once every two weeks; the OVX-Anti-RANKL group was given 10 mg / kg of Anti-RANKL antibody (100 μL / each) subcutaneously according to the previous study, once every two weeks. Each group had 17 mice, which were used for detection after 4 weeks of treatment, detection after 3 months of drug withdrawal, and mouse bone marrow monocyte sequencing, respectively.

[0141] After euthanizing the mice in each group, the left femur of the mouse was completely removed, ensuring the integrity of the bone marrow cavity, and was placed in 4% paraformaldehyde for 1 day of room temperature fixation, and 70% ethanol was used for long-term storage.

[0142] The distal femur of the mouse was placed in the capture interval of the Micro-CT scanning bin, and the distal femur of the mouse was scanned using Skyscan software according to a resolution of 4000, a voxel resolution of 9 μm, a rotation of 180°, and a 0.5 mm Al filter.

[0143] After the scanning was completed, the NRecon software was used to select the analysis target area, and the parameters were changed to 0-0.075. The distal femur of the mouse was reconstructed, and after the reconstruction was completed, the Dateview software was used to open the reconstructed data, select the 3D display form, adjust the femoral shaft direction and the transverse section direction to make the femoral longitudinal section direction consistent, record the layer number where the growth plate is located, set the BMD calculation formula in the analysis software CTan, select the area from the lower end of the growth plate to the lower layer 120 layer for analysis, adjust the gray threshold value to 65-255, manually circle the inside of the bone marrow cavity every few layers, set the Hu value to-1000-8521, and BMD=(AC-3.17) / 5676.23. The bone mineral density (BMD), bone volume per total volume (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and other related three-dimensional parameters of the mouse were exported, and a 3D file was formed.

[0144] The Minics software was used to visually display the data after three-dimensional reconstruction.

[0145] The two-dimensional image showed that the administration of BTM-tri-RANK significantly increased the number of trabeculae, and the Anti-RANKL group showed obvious thickening of the growth plate; the 3D image showed that the bone in the distal femur region of the mouse in the BTM-tri-RANK group was more compact (Figs. Figure 15 The related parameter results showed that the BMD value of the OVX group after ovariectomy was 0.58 ± 0.042 times that of the Sham group, proving that the mouse model of osteoporosis was successfully established; after 4 weeks of administration, the BMD value of the OVX-BTM-tri-RANK group was 2.08 ± 0.360 times that of the OVX group, and the BMD value of the OVX-Anti-RANKL group increased to 1.93 ± 0.442 times that of the OVX group (Figs. Figure 15OVX-BTM-tri-RANK group was not significantly different from OVX-Anti-RANKL group. Meanwhile, the parameters such as BV / TV, Tb.Th, Tb.N, etc. showed a significant upward trend, and the BV / TV, Tb.Th and Tb.N of OVX-Anti-RANKL group were 2.41±0.056 times, 1.29±0.196 times and 1.83±0.595 times of that of OVX group. The BV / TV of OVX-BTM-tri-RANK group was 2.67±0.639 times of that of OVX group (Fig. 1D), the Tb.Th was 1.31±0.081 times of that of OVX group (Fig. 1E), and the Tb.N was 2.04±0.433 times of that of OVX group (Fig. 1F). Figure 15 Figure 15 Figure 15

[0146] Micro-CT scanning was performed on mice 3 months after drug withdrawal, and two-dimensional images and 3D simulation images showed that the trabecular bone number of OVX-BTM-tri-RANK group was still more than that of OVX group, and the bone was more compact (Fig. 2A and B); the trabecular bone number of OVX-Anti-RANKL group decreased significantly. The BMD value of OVX-BTM-tri-RANK group was 1.40±0.022 times of that of OVX group (Fig. 2C), the BV / TV was 2.32±0.141 times of that of OVX group (Fig. 2D), the Tb.N was 1.98±0.104 times of that of OVX group (Fig. 2E), and the Tb.Th of OVX-BTM-tri-RANK group was 1.32±0.020 times of that of OVX group (Fig. 2F). In contrast, there was no significant difference between the values of femur of OVX-Anti-RANKL group and OVX group. Figure 16 Figure 16 Figure 16 Figure 16 Figure 16

[0147] (2) Hematoxylin & Eosin (H&E) and Masson's trichrome histological staining of femur of mice after 4 weeks of administration

[0148] After Micro-CT scanning of the femur of mice, the decalcification was performed by replacing the decalcification solution containing 14% EDTA (ethylene diamine tetraacetic acid), and the fresh decalcification solution was replaced every day. After 2 weeks of decalcification, the degree of decalcification of the femur of mice was checked, and after complete decalcification, dehydration was performed using gradient ethanol.

[0149] After fixation and decalcification of the femur of mice, paraffin embedding and sectioning were performed, and H&E staining was performed on the paraffin sections. The results showed that the trabecular bone of BTM-tri-RANK group increased (Fig. 4A and B). Figure 17 ​​​​​​​​), which is consistent with the two-dimensional images of Micro-CT. Masson trichrome staining was performed on paraffin sections of mouse femurs, and the results showed that BTM-tri-RANK increased the newly formed bone (blue area) of the OVX group (0.48 ± 0.160) and the OVX-Anti-RANKL group (0.07 ± 0.072) (p < 0.05) (Fig. 4). Figure 18 ).

[0150] (3) TRAP, RUNX2, and type I collagen immunohistochemical staining of mouse femur sections after 4 weeks of administration

[0151] After the paraffin specimen was sectioned, deparaffinated, antigen repaired, and blocked, the diluted anti-mouse RUNX2 primary antibody working solution (1:1000 dilution) was added, and incubated overnight at 4°C. The next day, PBS was washed 3 times for 5 minutes each time, and the 1:50 diluted HRP-labeled goat anti-rabbit secondary antibody working solution was added and incubated at 37°C for 1 hour. After incubation, DAB developing solution was added dropwise, and incubated at 37°C for several minutes until obvious brown positive appeared, and the reaction was terminated. After antibody staining, hematoxylin was used for re-staining for 1 minute, gradient alcohol was used for dehydration, xylene was used for transparency, and neutral resin was used for mounting, and the bright field of the microscope was observed.

[0152] The type I collagen staining procedure was the same as above, with a 1:200 dilution ratio of the primary antibody, and an HRP-labeled goat anti-mouse secondary antibody was used.

[0153] The results showed that the OVX-BTM-tri-RANK group had the largest area of TRAP-positive (TRAP + ) regions in the distal femur of the mice, which was 0.48 ± 0.160 times that of the OVX group and 0.07 ± 0.072 times that of the OVX-Anti-RANKL group (p < 0.05) (Fig. 4). Figure 19 ), and had the least area of TRAP-positive (TRAP + ) regions. Therefore, BTM-tri-RANK can rescue bone loss in OVX mice by inhibiting osteoclast activation.

[0154] At the same time, in order to further analyze the effect of BTM-tri-RANK treatment on the expression of type I collagen and RUNX2 in mice, we performed immunohistochemical staining on the mouse femurs, and the results showed that the OVX-BTM-tri-RANK group had stronger expression of type I collagen and RUNX2 than the OVX group and the OVX-Anti-RANKL group (p < 0.05) (Fig. 4). Figure 20 ).

[0155] (4) Detection of dynamic bone formation in mice after 4 weeks of administration using calcein labeling and hard tissue sections

[0156] After 4 weeks of treatment, the mice in each group were injected intraperitoneally with 20 mg / kg of calcein 13 days and 2 days before sampling.

[0157] After the mouse femur is isolated, it is fixed in 4% paraformaldehyde at room temperature for 24 hours; washed with PBS for 3 times, 5 minutes each time; dehydrated with 70% ethanol for 24 hours; dehydrated with 75% ethanol for 24 hours; dehydrated with 80% ethanol for 24 hours; dehydrated with 85% ethanol for 24 hours; dehydrated with 90% ethanol for 24 hours; dehydrated with 95% ethanol for 24 hours; dehydrated with 100% ethanol for 24 hours; dehydrated with 100% ethanol for 24 hours; transparentized with xylene for 3-4 hours;

[0158] A 50 mL syringe is plugged with loose cotton, and a layer of 200 mesh column chromatography silica gel powder is laid on the piston to make a simple filter device. The polymer of methyl methacrylate and butyl methacrylate is filtered using the simple filter device (filtering as needed), and the filtered 30 mL methyl methacrylate and 17.5 mL butyl methacrylate, 2.5 mL methyl benzoate and 600 μL polyethylene glycol PEG400 are mixed. The transparent femur is placed in the mixture and left to stand for 2 days. After 2 days, the mixture is prepared again, the solution is replaced, and the sample is soaked for another day. The embedding agent is prepared before embedding, mixed and divided into 20 mL glass bottles. The soaked femur sample is placed in each glass bottle and labeled, and stored at -20 ℃ for 2 days. After 2 days, the embedding agent is observed for solidification. When it is slightly solidified, the sample is transferred to room temperature and left to stand.

[0159] When the sample is solidified to the state that it cannot be poked by tweezers, knock off the outer glass bottle, fix the sample on a hard tissue slicer, adjust the knife holder to the appropriate height, and adjust the back-and-forth speed of the blade to trim the tissue at a thickness of 20 μm. When the desired tissue sample is reached, adjust the section thickness to 10 μm, and use tweezers to pick up the section (to make the section as flat as possible, a cotton ball dipped in water can be used to moisten the tissue surface). Place the section in a 70 ℃ section lifter, and use the prepared adhesive glass slide to lifter the section. After liftering, cover the glass slide containing the section with a layer of plastic film, and then cover it with a clean glass slide. Place the glass slide in a 70 ℃ section press, and evenly press a weight on it. After 2 hours of pressing, accumulate the glass slides together, use a dovetail clamp to fix both ends, and place the glass slide in a 60 ℃ oven. After 1 day, remove the plastic film on the top of the glass slide, place it in ethylene glycol ethyl ether acetate for plastic removal, and observe the section state. After plastic removal, place the section in 100% ethanol, and use neutral resin for mounting.

[0160] The results show that the distance between the two fluorescent lines labeled by calcein in the OVX-BTM-tri-RANK group is larger than that in the OVX group and the OVX-Anti-RANKL group (A and B in FIG. 6), indicating that the bone formation ability is stronger. Figure 21

[0161] ​(5) Detection of the levels of bone formation markers and bone resorption markers in serum of mice after 4 weeks of administration

[0162] After 4 weeks of treatment of mice in each group, the mice were anesthetized and blood was taken from the orbit, and after standing at 4°C for 1 hour, centrifugation was performed at 1500 g and 4°C for 10 minutes, and the upper serum was taken into a new EP tube.

[0163] After the mouse serum was diluted 50 times, the bone metabolism factor content was detected according to the steps of the CTX-I (type I collagen cross-linked carboxy-terminal peptide) and P1NP (type I procollagen amino-terminal propeptide) factor ELISA detection kit instructions.

[0164] The P1NP and CTX-I factor contents in serum are important indicators of bone metabolism status, so we performed ELISA detection of P1NP and CTX-I in serum of mice after 4 weeks of treatment. The results showed that the P1NP of the OVX-BTM-tri-RANK group was 1.48±0.205 times that of the OVX group, while the OVX-Anti-RANKL group had no statistical difference with the OVX group (A in Figure 22 ; the CTX-I factor contents of the OVX-BTM-tri-RANK group and the OVX-Anti-RANKL group decreased to 0.77±0.046 times and 0.78±0.050 times of the OVX group (B in Figure 22 ).

[0165] (6) Analysis of OB-related cells and OC-related cells in bone marrow of mice after 4 weeks of administration

[0166] Flow staining group 1: CD45 (fluorescein isothiocyanate FITC dye, eBioscience, 11-0451-85, 0.5 μg / sample), CD3 (FITC dye, BioLegend, 100204, 1 μg / sample), B220 (FITC dye, BioLegend, 103206, 1 μg / sample), Ter (FITC dye, eBioscience, 11-5921-82, 0.25 μg / sample), Gr-1 (FITC dye, BioLegend, 108406, 0.25 μg / sample), CD31 (PE-Vvio770 TM dye, Meitenyi, 130-102-902, 0.6 μg / sample), Sca-1 (SB436 dye, eBioscience, 62-5981-82, 0.25 μg / sample), CD51 (phycoerythrin PE dye, BioLegend, 104106, 1 μg / sample)

[0167] Flow staining group 2: CD115 (APC / cy7 dye, BioLegend, 135536, 0.5 μg / sample), CD117 (PE / cy7 dye, BioLegend, 105814, 0.25 μg / sample)

[0168] After the right femur of mice was taken out, bone marrow cells were separated, and 100 μL of cell suspension per sample was taken for flow staining group 1 and group 2 staining after lysing red blood cells. After adding antibodies of each group, the cells were incubated at 4 °C for 30 minutes, then centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. The cell precipitate was resuspended with 400 μL / tube of F-PBS (PBS buffer with fetal bovine serum), and the cells were detected by machine.

[0169] Each flow staining group and different tissues need to take part of the cells for single staining tube of each antibody for compensation adjustment, and a homologous control tube is set up as a control.

[0170] Mesenchymal stem cells (MSCs) play a key role in tissue regeneration, and these MSCs have the ability of self-renewal and differentiation into osteogenic, chondrogenic and adipogenic lineages. In order to isolate MSCs from mouse bone marrow, various combinations of phenotypic markers have been proposed, in particular PDGFRa (platelet-derived growth factor receptor), Sca-1 (stem cell antigen 1) and CD51. PDGFRa + Sca-1 + (PaS) cells are an adult stromal population near the artery, rich in MSC activity, while the osteoblast lineage cell (OBC) subpopulation is defined as Lin-CD45-CD31-Sca1-CD51 + . We used a combination of FITC-labeled anti-mouse CD45, CD3, B220, Ter, Gr-1 antibodies to remove other cells such as hematopoietic cells, B cells, T cells, etc. in bone marrow, and selected the FITC-negative cell population for Sca-1 and CD51 expression analysis. Sca-1 + CD51 - cells were defined as osteogenic-related cells, and Sca-1-CD51 + cells were defined as early osteogenic-related cells. The results showed that the OB-related cells in the OVX-BTM-tri-RANK group were 1.11 ± 0.095 times that of the OVX group, while the OVX-Anti-RANKL group had no significant difference from the OVX group (A and C in FIG. 6). There was no significant difference between the early osteogenic-related cells in each group. Figure 23

[0171] ​M-CSF receptor CD115 can be used as a marker for osteoclast progenitor cells, and CD117, also known as c-kit, is a receptor for stem cell factor and a consistent marker for early hematopoietic lineage including early osteoclast progenitor cells. Therefore, in flow cytometry, we used CD115 + and CD117 + to label osteoclast-related cells. The results showed that in the mouse bone marrow, the CD115 + CD117 + cell amount of the OVX-Anti-RANKL group was 1.49 ± 0.309 times that of the OVX group and 1.23 ± 0.256 times that of the OVX-BTM-tri-RANK group (P < 0.05 in B and D of FIG. 6). The OVX-Anti-RANKL group significantly increased the number of osteoclast-related cells in the bone marrow compared to the OVX-BTM-tri-RANK group. Figure 23

[0172] Example 5: Bone-targeted RANK trimer improves the inflammatory microenvironment of OVX mice bone marrow

[0173] (1) In vitro induction of Treg and T helper 17 cells (Th17) cell differentiation medium, the CD4 + T cell subpopulation changes

[0174] Select 6-8 week old female C57BL / 6 mice, after euthanasia, sterilely separate the mouse spleen in a clean bench, grind the spleen to a single cell suspension in a 40 μm cell screen using the tail end of a 5 mL syringe, transfer to a 15 mL centrifuge tube, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, resuspend the cells with 6 mL of PBS, slowly add to a 15 mL centrifuge tube that has been pre-dispensed with 3 mL of mouse lymphocyte separation medium, centrifuge at 1900 rpm, room temperature, acceleration 3, deceleration 0 for 8 minutes; add 10 mL of PBS to the 15 mL centrifuge tube, pipette the white membrane layer into it, mix well and centrifuge at 1200 rpm for 5 minutes, discard the supernatant, and the cell pellet is the peripheral blood mononuclear cells (PBMC).

[0175] Treg induction and differentiation: PBMC was inoculated in a 24-well plate coated with Anti-CD3 and CD28 at 2 × 10 6 cells / 2 mL / well, the Treg induction medium was replaced, and BTM-tri-RANK (0.05 μM) and Anti-RANKL (0.05 μM) were added according to different groups for induction and differentiation.

[0176] Th17 induction and differentiation: 10 8 ​PBMC were resuspended in volume of 1 x 106cells / mL, 10 μL / 10 7 CD4 direct labeling magnetic beads were added, and the mixture was incubated at 4 °C for 15-20 minutes. The Miltenyi magnetic column was placed in a magnetic stand, and 500 μL of PBS was used to rinse the column. The incubated cell suspension was added, and after the liquid was dropped, 500 μL of PBS was used to rinse the column. After the liquid was dropped, the column was moved away from the magnetic field, 2-3 mL of PBS was added, and the piston was quickly pressed to collect the cells. After centrifugation, the cells were counted, and 2 x 10 6 The cells were inoculated in a 24-well plate coated with Anti-CD3 and CD28 at a concentration of 2 x 10

[0177] CTR: Treg or Th17 induction medium culture group; BTM-tri-RANK: Treg or Th17 induction medium culture group containing 0.05 μM BTM-tri-RANK; Anti-RANKL: Treg or Th17 induction medium culture group containing 0.05 μM Anti-RANKL.

[0178] After 48 hours of different induction treatment of Treg and Th17 cells, the cells were collected and centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. (Note: All staining operations need to be carried out in the dark)

[0179] Treg group: add 100 μL of F-PBS (PBS containing 2% fetal bovine serum) to resuspend the cells; add 0.25 μg / sample of Anti-CD4 (eFluor™ 450, eBioscience, 48-0041-82), 1 μg / sample of Anti-CD8 (APC / cy7, BioLegend, 100714) and 0.25 μg / sample of APC-Anti-CD25 (APC, BioLegend, 101910) antibodies, incubate at 4 ℃ for 30 minutes; centrifuge at 2000 rpm for 5 minutes, add 1 mL / sample of the broken nucleus working solution (broken nucleus solution:diluent = 3:1), incubate at room temperature for 30 minutes, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, add 1 mL / sample of the broken nucleus termination buffer, resuspend, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, add 0.25 μg / sample of anti-mouse Foxp3 (PE, eBioscience, 12-4771-82) antibody, incubate at 4 ℃ for 30 minutes, add 1 mL of F-PBS, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, add 1 mL of F-PBS for washing once, and finally resuspend in 400 μL of F-PBS. Set up the isotype control tube and each marker single-staining tube for adjusting the compensation between each channel, and the staining is the same as that of the experimental group and all the broken nucleus operations are the same.

[0180] Th17 group: add 1 mL of RPMI complete medium containing 2 μL of stimulation blocking agent to resuspend the cell pellet, open the cover, incubate at 37 ℃, 5% CO2 for 5 hours, tap the cells every 1 hour, centrifuge at 2000 rpm for 5 minutes after stimulation, discard the supernatant, add 100 μL of F-PBS containing 0.25 μg of anti-mouse CD4 (eFluor™ 450 dye, eBioscience, 48-0041-82) and 1 μg of anti-mouse CD8 (APC / cy7 dye, BioLegend, 100714) antibody to each sample, incubate at 4 ℃ for 30 minutes, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, add 300 μL of membrane breaking working solution per sample, incubate at 4 ℃ for 20 minutes, then add 1 mL of membrane breaking termination working solution (5 mL of 10x membrane breaking termination solution added to 45 mL of deionized water), centrifuge at 2000 rpm for 5 minutes, add 100 μL of F-PBS containing 0.5 μg of anti-mouse IL-17a antibody (PE / cy7 dye, eBioscience, 25-7177-82), incubate at 4 ℃ for 30 minutes, add 1 mL of F-PBS per sample after incubation, centrifuge to remove the supernatant, resuspend in 1 mL of F-PBS per sample, centrifuge, discard the supernatant, and add 400 μL of F-PBS for detection. Set up isotype control tubes and single marker staining tubes for each channel to adjust the compensation between channels. All stimulation blocking and membrane breaking operations must be consistent between samples.

[0181] Flow staining group 1: CD3 (FITC dye, BioLegend, 100204, 1 μg / sample), CD4 (eFluor™ 450 dye, eBioscience, 48-0041-82, 0.25 μg / sample), CD8 (APC / cy7 dye, BioLegend, 100714, 1 μg / sample), CD25 (APC dye, BioLegend, 101910, 0.25 μg / sample), Foxp3 (PE dye, eBioscience, 12-4771-82, 0.25 μg / sample);

[0182] Flow staining group 2: CD8 (APC / cy7 dye, BioLegend, 100714, 1 μg / sample), CD4 (eflour450 dye, eBioscience, 48-0041-82, 0.25 μg / sample), IL-17a (PE / cy7 dye, eBioscience, 25-7177-82, 0.5 μg / sample);

[0183] Flow staining group 3: CD3 (FITC dye, BioLegend, 100204, 1 μg / sample), CD4 (eFluor™ 450 dye, eBioscience, 48-0041-82, 0.25 μg / sample), CD8 (APC / cy7 dye, BioLegend, 100714, 1 μg / sample), B220 (APC dye, BioLegend, 103212, 0.25 μg / sample).

[0184] The mice were modeled and administered according to the method of Example 4.

[0185] Sham group: the mice in the sham group were administered PBS via the tail vein; Sham-BTM-tri-RANK group: the mice in the sham group were administered BTM-tri-RANK (10 mg / kg) once every two weeks via the tail vein; OVX group: the mice in the OVX group were administered PBS via the tail vein; OVX-Anti-RANKL group: the mice in the OVX group were administered Anti-RANKL (10 mg / kg) once every two weeks subcutaneously; OVX-BTM-tri-RANK group: the mice in the OVX group were administered BTM-tri-RANK (10 mg / kg) once every two weeks via the tail vein.

[0186] After the mice were administered for 4 weeks, the mice were taken blood from the orbit, and the inguinal lymph nodes, axillary lymph nodes and facial lymph nodes of the mice were taken out into PBS, and stored on ice. The lymph nodes were ground into a single cell suspension using a 1 mL syringe at the end of a 40 μm cell screen, and then centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and the cell pellet was resuspended in 1 mL of F-PBS. 100 μL of the cell suspension was taken for flow staining, and the cells were divided into three groups for flow staining. In group 1, CD3, CD4, CD8 and CD25 membrane surface antibodies were added, and the mixture was incubated at 4°C for 30 minutes. After incubation, the mixture was centrifuged at 2000 rpm for 5 minutes. The supernatant was removed, and 1 mL of nucleic acid breaking solution was added. The subsequent nucleic acid breaking and Foxp3 staining were performed as described above. In group 2, after the cells were centrifuged, the cell pellet was resuspended in 1 mL of RPMI complete medium containing 2 μL of stimulation blocking agent. The mixture was placed in a 37°C incubator for 5 hours. The cell pellet was tapped every hour. After the incubation was completed, the mixture was centrifuged at 2000 rpm for 5 minutes. The supernatant was removed, and the cell pellet was resuspended in 100 μL of F-PBS. CD8 and CD4 membrane surface antibodies were added, and the mixture was incubated at 4°C for 30 minutes. After incubation, the mixture was centrifuged. The cell pellet was resuspended in 300 μL of membrane breaking reagent. The subsequent membrane breaking and IL-17a antibody staining were performed as described above. In group 3, CD3, CD4, CD8 and B220 membrane surface antibodies were added, and the mixture was incubated at 4°C for 30 minutes. After incubation, the mixture was centrifuged at 2000 rpm for 5 minutes. The supernatant was removed, and 400 μL of F-PBS was added for detection.

[0187] Each flow staining group needs to take part of the cells to make single staining tube for each antibody to compensate for adjustment when preparing samples, and set up sample tube for isotype control antibody staining as control. All operations of membrane breaking, nucleus breaking and stimulation blocking should be consistent among groups.

[0188] mRANKL reverse signal may be involved in the differentiation of CD4 + T cells, affecting the development of Treg cells. Therefore, we used 0.05 μM of BTM-tri-RANK and Anti-RANKL to intervene with mouse spleen-derived CD4 + T cells in vitro, respectively. 48 hours after intervention, flow cytometry was used to analyze the proportion of Treg and Th17 cells. The results showed that compared with the OB and Anti-RANKL groups, the BTM-tri-RANK group had more Treg proportion and lower Th17 cell proportion ( Figure 24 ). Therefore, BTM-tri-RANK promotes the differentiation of CD4 + T cells to Treg cells and inhibits the differentiation of Th17 cells.

[0189] (2) Detection of immune-related factor levels in serum of OVX mice treated for 4 weeks using a multi-factor detection kit

[0190] After 4 weeks of treatment, the mice were taken blood from the orbit, and the mouse serum was collected. The MSD U-PLEX platform was used to detect multiple factors (TNF-a, IFN-g, IL-10, IL-12p70, IL-1b, IL-2, IL-4, IL-5, IL-6, KC / GRO) in mouse serum, and the standard curve was used for quantification. The quantitative range of each factor is: TNF-a (572-0.140 pg / mL); IFN-g (764-0.187 pg / mL); IL-10 (2630-0.642 pg / mL); IL-12p70 (29800-7.275 pg / mL); IL-1b (1510-0.369 pg / mL); IL-2 (2430-0.593 pg / mL); IL-4 (1660-0.405 pg / mL); IL-5 (945-0.231 pg / mL); IL-6 (5120-1.25 pg / mL); KC / GRO (2150-0.525 pg / mL). Some data is not shown due to being below the detection range or having large differences within the group.

[0191] Keratinocyte chemoattractant / growth-related oncogene homolog (Kc / gro) is a CXC chemokine, also known as chemokine (C-X-C motif) ligand 1 (CXCL1). It is involved in the activation and recruitment of neutrophils in the inflammatory environment; IL-6 is a typical cytokine associated with inflammatory diseases. After 4 weeks of administration, we detected the levels of immune-related factors in the serum of mice, including TNF-a, IFN-g, IL-10, IL-12p70, IL-1b, IL-2, IL-4, IL-5, IL-6, Kc / gro. In this study, we observed that some factors (IFN-g, IL-1b, IL-2, IL-5) had high individual variation coefficients in the comparison between groups, and did not pass the significance test; IL-12p70 and IL-4 had serum concentrations lower than the minimum limit of detection sensitivity in most experimental samples, so the above indicators were not shown in the results. The results showed that after ovariectomy, the contents of IL-6 and Kc / gro in the serum of the OVX group were 1.94 ± 0.415 times and 1.51 ± 0.099 times that of the sham operation group, respectively, and there was no significant difference between the OVX-Anti-RANKL group and the OVX group, the IL-6 of the OVX-BTM-tri-RANK group was 0.503 ± 0.101 times that of the OVX group (A in Figure 25 ), and the Kc / gro was 0.65 ± 0.080 times that of the OVX group (B in Figure 25 ); there was no significant difference in the levels of TNF-a and IL-10 factors in the OVX mice of each group (C and D in Figure 25 ), and the treatment of BTM-tri-RANK significantly down-regulated the secretion of inflammatory factors in the serum.

[0192] (3) FCM analysis of changes in related immune cell subsets in the lymph nodes of OVX mice after treatment;

[0193] After 4 weeks of treatment, the lymph node cells of each group of mice were analyzed by flow cytometry, and the flow cytometry results showed that the proportion of T cells in the lymph nodes of the OVX-BTM-tri-RANK group was significantly lower than that of the other groups (A and C in Figure 26 ), while there was no significant effect on the proportions of B cells and CD4 + T and CD8 + T subsets (B and D in Figure 26 ).

[0194] To further monitor the changes of Treg cells and Th17 cells in the peripheral organs of mice after administration, we ground the lymph nodes of each group of mice and analyzed the single cell suspension by flow cytometry, defining CD25 + Foxp3 + cells in CD4 + T cells as Treg cells, and IL-17A +The cells were Th17 cells. The results showed that the proportion of Treg cells in the OVX-BTM-tri-RANK group was 1.28±0.15, 1.11±0.13 and 1.37±0.16 times that of the sham group, OVX group and OVX-Anti-RANKL group, respectively ( Figure 27 In A and B, BTM-tri-RANK administration significantly increased the proportion of Treg cells in lymph nodes. At the same time, the proportion of Th17 cells in the OVX-BTM-tri-RANK group and the OVX-Anti-RANKL group were 0.74±0.132 times and 0.623±0.084 times that of the OVX group, respectively ( Figure 27 (C and D) BTM-tri-RANK treatment increased the proportion of Treg cells and decreased the proportion of Th17 cells in the lymph nodes of OVX mice.

[0195] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A bone-targeted trimeric protein, characterized in that: The trimeric protein includes an extracellular segment of a nuclear factor κB receptor activator, a trimeric motif and a bone-targeting peptide. The amino acid sequence of the extracellular segment of the nuclear factor κB receptor activator is shown in SEQ ID NO.1, the trimeric motif is an isoleucine zipper, the amino acid sequence of the isoleucine zipper is shown in SEQ ID NO.3, and the bone-targeting peptide is a (DSS) 6 bone-targeting peptide, the amino acid sequence of the bone-targeting peptide is shown in SEQ ID NO.

5.

2. The method for preparing the trimeric protein according to claim 1, characterized in that: The following steps are involved: Step S1, constructing a recombinant expression vector, wherein the recombinant expression vector sequentially comprises the coding gene sequences of the extracellular fragment of the receptor activator of nuclear factor κB, the trimer motif, and the bone-targeting peptide; Step S2: transferring the recombinant expression vector into the expression system for cultivation, and collecting the culture supernatant; Step S3: purifying the obtained culture supernatant to obtain the trimeric protein.

3. A polynucleotide encoding the trimeric protein according to claim 1.

4. Use of the trimeric protein according to claim 1 in the preparation of a drug for treating osteoporosis.

5. A drug for treating osteoporosis, characterized in that: The osteoporosis therapeutic drug comprises the trimeric protein according to claim 1.

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