Application of polyinosinic-polycytidylic acid (Poly (I: C)) in preparation of medicine for treating and / or preventing bone defect and / or osteoporosis

Abstract

The invention relates to the technical field of development of medicines for bone defect and osteoporosis, in particular to application of poly (I: C) in preparation of medicines for treating and / or preventing bone defect and / or osteoporosis. The osteogenic differentiation function damage of the bone marrow mesenchymal stem cells is an important reason for slow bone defect repair and rapid osteoporosis development, and in-vivo and in-vitro experience proves that the bone repair is accelerated and the osteoporosis development is delayed by promoting the osteogenic function of the bone marrow mesenchymal stem cells. Therefore, the polyinosinic-polycytidylic acid has a good application prospect in clinical treatment of bone defect and osteoporosis.

Description

Technical Field

[0001] The present invention relates to the technical field of bone defect and osteoporosis drug development, and in particular to the use of poly (I:C) in the preparation of drugs for treating and / or preventing bone defects and / or osteoporosis. Background Art

[0002] Osteoporosis (OP) is a common orthopedic disease in the elderly and the main cause of chronic pain and fractures in the elderly. Its main pathological characteristics are low bone density and microstructural destruction of bone tissue caused by the disruption of the dynamic balance between bone formation and resorption. As a common aging bone disease, OP has a prevalence of up to 32.0% in the elderly over 65 years old, which brings a huge social burden and poses a major challenge to the health of the elderly. There are many drugs available for the treatment of osteoporosis in clinical practice, but serious side effects limit their further promotion and application. For example, bisphosphonates may cause excessive inhibition of bone turnover, leading to mild trauma, atypical fractures, esophagitis and atrial fibrillation. In addition, although estrogen replacement therapy has a good effect in preventing postmenopausal osteoporosis, it often increases the risk of endometrial cancer, ovarian cancer and breast cancer due to its strong estrogen activity. Therefore, it is of great significance to understand its exact pathogenesis and find drugs with sufficient efficacy and minimal adverse reactions.

[0003] Bone defect refers to a pathological state in which the integrity of the bone structure is destroyed and part of the bone is missing due to various reasons such as trauma, infection, tumor resection, and congenital malformation. It can occur in bones in various parts of the body, and the size, shape and complexity of the defect vary. Bone defects directly affect the normal support and movement functions of bones. For example, long bone defects can cause the limbs to be unable to bear weight normally and limited in activity, affecting the patient's ability to walk, hold objects and other daily activities; spinal bone defects may affect the stability of the spine and even compress the spinal cord, causing further damage to the limbs' sensory and motor functions. At present, clinical treatment is mainly based on autologous bone transplantation, allogeneic bone transplantation and artificial bone material implantation. At present, new research results are constantly emerging in the field of bone defect treatment, such as the development of new artificial bone materials with better bioactivity, and the exploration of tissue engineering bone (combining seed cells, biological scaffolds, etc. to construct repair materials closer to natural bones). However, clinically, there are still many challenges such as how to accurately select the best treatment plan for bone defects of different types, sizes and complexities, and how to further improve the repair effect, shorten the treatment cycle, and reduce the incidence of complications. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a new use of Poly(I:C), which has the function of promoting osteogenic differentiation of BMSCs. The present invention aims to effectively prevent and treat bone defects and osteoporosis by promoting osteogenic differentiation of BMSCs, and further provides the use of Poly(I:C) in the preparation of drugs for treating and / or preventing bone defects and / or osteoporosis.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of poly I:C in the preparation of a drug for treating and / or preventing bone defects and / or osteoporosis. The chemical formula of the poly I:C is 19 H 27 N7O 16 P2.

[0007] The structural formula of the poly I:C is:

[0008]

[0009] In the present invention, poly I:C (Poly (I:C)) accelerates bone formation and repair and delays the progression of osteoporosis by promoting the osteogenic differentiation function of bone marrow mesenchymal stem cells.

[0010] In the present invention, osteoporosis is postmenopausal osteoporosis.

[0011] The present invention also provides a medicine for treating and / or preventing bone defects and / or osteoporosis, wherein the effective ingredient comprises poly I:C.

[0012] In a preferred embodiment of the present invention, the dosage form is any one of tablets, capsules, oral liquids, lozenges, granules, granules, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, creams, sprays, drops or patches.

[0013] In a preferred embodiment of the present invention, the drug is administered orally, intraperitoneally, intravenously or subcutaneously.

[0014] Impaired osteogenic differentiation of bone marrow mesenchymal stem cells is an important reason for the slow repair of bone defects and the rapid progression of osteoporosis. In vivo and in vitro verification shows that poly I-cell accelerates bone repair and delays the progression of osteoporosis by promoting the osteogenic function of bone marrow mesenchymal stem cells. Therefore, poly I-cell has a good application prospect in the treatment of clinical bone defects and osteoporosis.

[0015] The present invention discloses the following technical effects:

[0016] Poly(I:C) can accelerate bone formation and repair and delay the progression of osteoporosis by promoting the osteogenic differentiation of BMSCs. Therefore, Poly(I:C) has a good application prospect in the treatment of clinical bone defects and osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 is the expression of TLR3 in aged mice; wherein, A is the HE staining picture of mouse femur; B is the statistics of mouse femur bone mass area, and the ordinate represents the positive area ratio of bone mass; C is the immunohistochemical staining picture of mouse femur TLR3; D is the statistics of mouse femur TLR3 immunohistochemical staining, and the ordinate represents the positive cell ratio of TLR3; E is the Western-Blot diagram of TLR3, and GAPDH represents the internal reference; F is the relative expression analysis result of TLR3 protein; G is the relative expression analysis result of TLR3 mRNA; (young group, Young; old group, Old; NS, no significance vs Old; *p<0.05vs Old, **p<0.01vs Old, ***p<0.001vs Old).

[0019] Figure 2 The expression of TLR3 in osteoporosis model (OVX) mice; A is the HE staining picture of mouse femur; B is the statistics of mouse femur bone mass area, and the ordinate represents the positive area ratio of bone mass; C is the immunohistochemical staining picture of mouse femur TLR3; D is the immunohistochemical statistics of mouse femur TLR3, and the ordinate represents the positive cell ratio of TLR3; E is the Western-Blot diagram of TLR3, and GAPDH represents the internal reference; F is the relative expression analysis result of TLR3 protein; G is the relative expression analysis result of TLR3 mRNA; (sham group, sham; model group, OVX; NS, no significance vs OVX; *p<0.05vs OVX, **p<0.01vs OVX, ***p<0.001vs OVX).

[0020] Figure 3Figure 2 is the effect of different concentrations of Poly (I: C) on the osteogenic differentiation of BMSCs; A is the effect of different concentrations of Poly (I: C) on the cell viability of BMSCs; B is the Western-Blot diagram of TLR3, GAPDH represents the internal reference; C is the relative expression analysis result of TLR3 protein; D is the alkaline phosphatase staining diagram; E is the relative quantitative analysis of alkaline phosphatase staining results; F is the Alizarin Red staining diagram; G is the relative quantitative analysis of Alizarin Red staining results; H is the Western-Blot diagram of COL1A1, ALP, Runx2, and OCN, GAPDH represents the internal reference; I is the relative expression analysis result of ALP protein; J is the relative expression analysis result of OCN protein; K is the relative expression analysis result of COL1A1 protein; L is the relative expression analysis result of Runx2 protein; M is the relative expression analysis result of TLR3 mRNA; N is the relative expression analysis result of ALP The relative expression analysis results of mRNA were as follows: ((Poly(I:C) low-dose group, 5μg / mL; (Poly(I:C) medium-dose group, 10μg / mL; (Poly(I:C) medium-dose group, 20μg / mL; Ctrl group, 0μg / mL; *p<0.05vs Ctrl group, **p<0.01vs Ctrl group, ***p<0.001vs Ctrl group).

[0021] Figure 4 The figure is an analysis of bone mass in mice with femoral defect model after intervention with different concentrations of Poly(I:C); A is the Micro-CT scanning result (2D represents two-dimensional and 3D represents three-dimensional); B is ABH / OG staining; C is HE staining; D is the bone density (BMD) analysis result; E is the trabecular relative volume (BV / TV) analysis result; F is the trabecular thickness (Tb.Th) analysis result; G is the trabecular number (Tb.N) analysis result; H is the trabecular separation (Tb.Sp) analysis result; (Vehicle group, 0 mg / kg; Poly(I:C) low-dose group, 5 mg / kg; Poly(I:C) high-dose group, 10 mg / kg; NS, no significance vs Vehicle; *p<0.05vs Vehicle, **p<0.01vs Vehicle, ***p<0.001vs Vehicle).

[0022] Figure 5Analysis of the osteogenic effect of different concentrations of Poly (I:C) on femoral defect model mice; wherein, A is the result of ALP immunohistochemical staining; B is the result of OCN immunohistochemical staining; C is the result of Runx2 immunohistochemical staining; D is the result of Trap staining; E is the statistical analysis of ALP immunohistochemical staining results; F is the statistical analysis of OCN immunohistochemical staining results; G is the statistical analysis of Runx2 immunohistochemical staining results; H is the statistical analysis of Trap staining results; (Vehicle group, 0 mg / kg; Poly (I:C) low-dose group, 5 mg / kg; Poly (I:C) high-dose group, 10 mg / kg; NS, no significance vs Vehicle; *p<0.05vsVehicle, **p<0.01vsVehicle, ***p<0.001vsVehicle).

[0023] Figure 6 Figure 2 is the bone mass analysis of OVX model mice after intervention with different concentrations of Poly(I:C); A is the Micro-CT scan result (2D represents two-dimensional and 3D represents three-dimensional); B is ABH / OG staining; C is HE staining; D is the bone mineral density (BMD) analysis result; E is the trabecular relative volume (BV / TV) analysis result; F is the trabecular thickness (Tb.Th) analysis result; G is the trabecular number (Tb.N) analysis result; H is the trabecular separation (Tb.Sp) analysis result; I is the structural model index (SMI) analysis result; J is the connection density (Conn.Dn) analysis result; K is the trabecular area analysis result; (sham group, Vehicle group, 0 mg / kg; Poly(I:C) low-dose group, 5 mg / kg; Poly(I:C) high-dose group, 10 mg / kg; NS, no significance vs Vehicle; *p<0.05 vs Vehicle, **p<0.01 vs Vehicle, ***p<0.001vs Vehicle).

[0024] Figure 7Analysis of the osteogenic effect of different concentrations of Poly (I: C) on OVX model mice after intervention; A is the result of ALP immunohistochemical staining; B is the result of OCN immunohistochemical staining; C is the result of Runx2 immunohistochemical staining; D is the result of Trap staining; E is the statistical analysis of ALP immunohistochemical staining; F is the statistical analysis of OCN immunohistochemical staining; G is the statistical analysis of Runx2 immunohistochemical staining; H is the statistical analysis of Trap staining results; (Vehicle group, 0 mg / kg; Poly (I: C) low-dose group, 5 mg / kg; Poly (I: C) high-dose group, 10 mg / kg; NS, no significance vs Vehicle; *p<0.05vsVehicle, **p<0.01vs Vehicle, ***p<0.001vs Vehicle). DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Bone marrow mesenchymal stem cells (BMSCs) were first discovered in adult bone marrow and can self-renew and differentiate into multiple cell lineages, including osteoblasts, chondrocytes, and adipocytes. In osteoporosis and bone defect conditions, changes in the pathological microenvironment can trigger abnormal changes in BMSCs, such as oxidative stress, apoptosis, and abnormal autophagy, which seriously damage the osteogenic ability of BMSCs and cause the body's bone formation ability to decline. Therefore, restoring the normal function of BMSCs promotes their differentiation into osteoblasts, participates in the formation of new bone, increases bone mass, helps improve bone structure changes such as sparse trabeculae caused by osteoporosis, and accelerates bone defect repair.

[0031] Poly(I:C), also known as polyinosinic acid-cytidylic acid, is a synthetic double-stranded RNA (dsRNA) that is mainly used as a Toll-like receptor 3 (TLR3) agonist. It is a double-stranded RNA structure formed by polyinosinic acid (poly I) and polycytidylic acid (poly C) connected by phosphodiester bonds. This structure simulates the double-stranded RNA genome component of the virus and is an effective viral double-stranded RNA mimic. It is currently mainly used in the field of immunity to activate immune cells and regulate interferon response. The present invention found that TLR3 is downregulated in osteoporosis, and Poly(I:C) is used as a TLR3 activator to intervene in BMSCs, and bone defect and osteoporosis model mice are injected for intervention treatment. The results showed that Poly(I:C) has the effect of promoting osteogenic differentiation of BMSCs and effectively treats bone defect and osteoporosis model mice. The present invention uses Poly(I:C) as the research object and clarifies its possible intervention mechanism from the perspective of osteogenic differentiation of BMSCs: TLR3 agonist Poly(I:C) promotes osteogenic differentiation of BMSCs, promotes bone repair of bone defects and delays the occurrence and development of osteoporosis. Therefore, the present invention not only has new insights and breakthroughs in the regulation of TLR3 on stem cells, but also provides new opportunities and ideas for exploring the treatment of bone defects and osteoporosis.

[0032] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Poly I:C (P1530, Sigma-Aldrich, USA) used in the examples of the present invention.

[0035] Example 1 TLR3 expression in aged mice

[0036] Objective: To observe the expression of TLR3 in aged mice

[0037] method

[0038] 1.1 Experimental Grouping

[0039] (A) Control group (Young group); (B) Elderly group (Old group). There were 6 mice in each group.

[0040] 1.2 Obtaining femoral tissue and preparing slices

[0041] After washing with PBS for several times, the samples of each group were placed in 4% paraformaldehyde fixative and immersed for 2 days to achieve full fixation. After fixation, they were rinsed with deionized water for 6 times (15 minutes / time), and then decalcified with 14% EDTA aqueous solution for 10 days, with the solution changed every day. After decalcification was completed (no obvious blockage when punctured with a needle), they were rinsed with 1×PBS 3 times (15 minutes / time), rinsed with deionized water 3 times (15 minutes / time), and dehydrated with alcohol (30%, 50%, 70%) for 15 minutes each stepwise, and then processed by a fully automatic tissue dehydrator and embedded in paraffin to prepare donor wax blocks. When slicing, slice with a thickness of 4μm.

[0042] 1.3 Histological and bone morphometric analysis

[0043] 1.3.1 HE staining

[0044] 1) Take slices of femoral tissue from each group and bake at 60°C overnight;

[0045] 2) The next day, remove the sections, wait for them to return to room temperature, and then dewax and rehydrate them step by step (dewaxing with xylene 3 times, 5 minutes each time; 100% and 95% alcohol 2 times, 5 minutes each time; 70% alcohol 1 time, 5 minutes each time; deionized water 2 times, 1 minute each time);

[0046] 3) Place the slices in hydrochloric acid alcohol for 30 seconds and drain the excess water on paper briefly;

[0047] 4) Place the sections in the hematoxylin solution for 30 seconds, then rinse with deionized water several times until the water is clear (approximately 3 times);

[0048] 5) Place the slices in hydrochloric acid alcohol for 3 seconds, then wash with deionized water 3 times, 1 minute each time;

[0049] 6) Place the slices in 0.5% ammonia water for 15 seconds, then wash twice with deionized water, 1 minute each time;

[0050] 7) Place the sections in 95% alcohol for 1 minute, then transfer to Eosin working solution for staining for 1 minute;

[0051] 8) Dehydrate and make transparent step by step (95% alcohol 3 times, 1 minute / time; 100% alcohol 2 times, 1 minute / time; xylene dewaxing 3 times, 1 minute / time), and seal with sealing medium. Observe and complete the slide collection under a Zeiss upright microscope.

[0052] 1.3.2 Bone morphometry

[0053] The morphological changes of femoral tissue in each group were observed and compared, and bone tissue morphometric analysis was performed. Slice images of femoral tissue in each group were taken, and the trabecular area of ​​each femoral sample was statistically analyzed using Image J 1.46r software.

[0054] 1.4 Immunohistochemistry

[0055] Bake the slides overnight; dewax and rehydrate (three cylinders of xylene, gradient alcohol (100%×2, 95%×2, 75%)); pay attention to whether the wax is removed completely; wash 3 times with 1×PBS 3min / cylinder; dissolve 0.2g pepsin in 50ml of 0.01M HCl solution, preheat HCl to 37℃, and soak the slides for 60min (sodium citrate 60℃ soak for 4 hours); wash 3 times with 1×PBS 3min / cylinder; permeabilize 0.3% Triton 100×10min (1×PBS1000mL+3mLTriton stock solution); wash 3 times with 1×PBS 3min / cylinder; block with peroxidase blocker for 15min; (if the primary antibody is mouse antibody, use steps 9 and 11 and peroxidase blocking for 20min; omit step 10); wash 3 times with 1×PBS 3min / cylinder; serum blocking for 15min; pour out the serum without washing with water, add primary antibody, and incubate at 4℃ overnight. The next day, take out the tube and place it at room temperature for half an hour, then wash it 3 times with 1×PBS for 3 minutes / tank; reagent 3: block with biotin-labeled goat anti-rabbit IgG polymer for 15 minutes; wash it 3 times with 1×PBS for 3 minutes / tank; reagent 4: block with horseradish enzyme-labeled streptavidin working solution for 15 minutes; wash it 3 times with 1×PBS for 3 minutes / tank; develop color with DAB (if DAB is prepared with Intrivogen kit, add 1 mL of DAB substrate solution + 1 drop of DAB concentrate to the Ep tube); wash it 3 times with 1×PBS for 1 minute / tank; add CAT Hemato×ylin (hematoxyl) for 30-45 seconds;

[0056] Wash 3 times with 1×PBS for 1 min / tank; 5-10s with 1% hydrochloric acid alcohol; 3 times with 1×PBS for 1 min / tank; 20s with 0.5% ammonia water;

[0057] Wash 3 times with 1×PBS 1min / tank; dry the slides in a 37℃ oven for 3-4 hours, and use xylene 1min / tank 3 times to make them transparent; seal the slides with neutral resin. Observe and complete the slide collection under a Zeiss microscope, and perform statistical analysis on each femur sample using Image J 1.46r software.

[0058] 1.5 Western-Blot detection of TLR3 expression in femoral tissue

[0059] 1.5.1 Total protein extraction

[0060] 1) Wash the femoral tissue once with pre-cooled 1× PBS, break the sample with a steel ball, add pre-cooled cell lysis buffer (containing 1% protease phosphatase inhibitor), let stand on ice for 30 minutes, and vortex every 10 minutes.

[0061] 2) Centrifuge at 4°C and 13,000 rpm for 10 minutes, carefully aspirate the supernatant into a new 1.5 mL centrifuge tube, and place on ice to obtain the total protein extract.

[0062] 1.5.2 Total protein quantification

[0063] 1) Add 1 mL of the protein sample to be tested, cell lysis buffer (containing 1% protease phosphatase inhibitors), and 1×PBS into labeled 1.5 mL centrifuge tubes and place them together in crushed ice for later use.

[0064] 2) Preparation of standard test solutions: 2 mg / mL BSA standard (10×1 mL ampules, containing 2 mg / mL bovine serum albumin (BSA), 0.9% salt and 0.05% sodium azide) was diluted with cell lysis buffer (containing 1% protease phosphatase inhibitors) to obtain protein standard test solutions with BSA concentrations of 2000 μg / mL, 1500 μg / mL, 1000 μg / mL, 750 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, and 0 μg / mL.

[0065] 3) Preparation of BCA working solution: According to the number of standard substances and samples to be tested, add BCA reagent A:BCA reagent B in the BCA protein concentration kit (#23225, Thermo Fisher Scientific, USA) at a ratio of 50:1 (v / v) into the sample adding tank and mix evenly. The solution is stable at room temperature for 24 hours.

[0066] Total volume of BCA working solution = (number of standards + number of samples) × 2 replicate wells × 200uL;

[0067] 4) Take 10uL of protein standard test solution and prepared sample test solution and add them to the standard well and sample well of 96-well ELISA plate respectively, and prepare 2 wells in parallel for each concentration.

[0068] 5) Add 200uLBCA working solution to each well, cover, and incubate at 37°C for 30 minutes.

[0069] 6) After the incubation, the cells were allowed to return to room temperature naturally and placed in a cell imaging microplate detection system. The OD value was measured at λ = 562 nm to prepare a standard curve. Figure 1 As shown, the regression equation is: y(OD)=0.0005×(C)+0.0059, R2=0.9983, that is, the concentration of the protein sample to be loaded=(OD-0.0059) / 0.0005.

[0070] 1.5.3 Polyacrylamide gel electrophoresis

[0071] 1) According to the above measured protein concentrations of each group, prepare equal volumes and masses of protein samples to be loaded. Take 20ug of protein from each group of samples, calculate its volume, fill it up to 40uL with cell lysis buffer (containing 1% protease phosphatase inhibitors), and finally add 10uL 5×loading buffer, the final volume is 50uL. At the same time, prepare equal volumes of protein marker (8uL Maker + 32uLRIPA lysis buffer + 10uL 5×loading buffer).

[0072] 2) Incubate each group of protein samples in a metal bath at 100°C for 10 minutes, return to room temperature, centrifuge briefly, and place on ice for later use.

[0073] 3) Take the precast protein gel, carefully pull out the "comb" of the gel plate, and tear off the green seal at the bottom; insert the gel plate into the vertical electrophoresis tank containing 1× electrophoresis buffer.

[0074] 4) Add 5uL of protein marker to well 1 of the gel, and add 10uL of the protein sample to be loaded to the remaining wells.

[0075] 5) After the sample is loaded, insert the electrode into the appropriate position of the vertical electrophoresis tank; turn on the power of the electrophoresis instrument, adjust the voltage to 80V, wait for 30 minutes, adjust the voltage to 110V, wait for 1.5-2.0 hours, until 10kDa reaches the bottom of the gel, and stop the electrophoresis instrument.

[0076] 1.5.4 Transfer

[0077] 1) Take out the gel sheet from the vertical electrophoresis tank, rinse the gel sheet with tap water, carefully remove the short gel sheet, cut off the excess gel with a gel knife, carefully transfer the gel from the long gel sheet to deionized water, and soak for later use.

[0078] 2) Take a small PVDF transfer package, place the transfer layer containing PVDF membrane on the electrode plate, and be careful to remove bubbles; carefully transfer the glue from deionized water to the PVDF membrane, and be careful to remove bubbles; then place the transfer layer without PVDF membrane on the PVDF membrane, and be careful to remove bubbles. After completion, cover the electrode cover, insert the transfer plate into the transfer instrument, and transfer for 7 minutes at 2.5A and 25V.

[0079] 1.5.5 Western Blot

[0080] 1) Blocking: After the transfer is completed, the protein-loaded PVDF membrane is quickly placed in 3-5 mL of blocking solution (1×TBST containing 5% BSA) and shaken for 1 hour.

[0081] 2) Primary antibody incubation: After blocking, add an appropriate amount of TLR3 primary antibody according to the dilution factor (1:1000), mix thoroughly, and place on a shaker at 4°C overnight.

[0082] 3) Secondary antibody incubation: The next day, remove the primary antibody blocking solution, add an appropriate amount of 1×TBST and wash three times for 10 minutes each; add an appropriate amount of blocking solution, dilute it according to the specific dilution factor (1:4000), add an appropriate amount of corresponding secondary antibody, mix well, and shake at room temperature for 1 hour.

[0083] 4) Development: After the secondary antibody incubation is completed, remove the secondary antibody blocking solution, add an appropriate amount of 1×TBST and wash 3 times, 10 minutes each time; carefully absorb excess water with absorbent paper, quickly place it in a transparent paper protective cover, add pre-mixed ECL developer solution, make it evenly distributed on the test membrane, and remove excess bubbles; slightly absorb excess ECL developer solution with absorbent paper; place the test membrane on the ChemiDocTM MP imaging system for imaging, and use Image J for semi-quantitative analysis.

[0084] 1.6 Real-time PCR detection of TLR3 mRNA expression level in femoral tissue

[0085] 1.6.1 Extraction of mRNA from femur tissue

[0086] 1) The sample was quickly frozen with liquid nitrogen, then crushed and collected into a 1.5 mL centrifuge tube. 0.5 mL of Trizol was directly added to the centrifuge tube. After repeated pipetting and mixing, the mixture was allowed to stand at room temperature for 5 minutes.

[0087] 2) Add 0.1 mL of chloroform (1 / 5 of the total volume of Trizol) to the centrifuge tube in step 1), mix by inverting for 15-30 seconds, and let stand at room temperature for 2-3 minutes; after completion, centrifuge at 13,000 rpm and 4°C for 15 minutes, carefully aspirate the supernatant and transfer it to another 1.5 mL centrifuge tube.

[0088] 3) Add 1 volume of 70% alcohol to the supernatant in step 2), shake and mix for 30 seconds, then add it to the 2 mL collection tube provided by the RNA extraction kit, and further purify the mRNA according to the operating instructions in the kit.

[0089] 1.6.2 mRNA concentration determination and reverse transcription

[0090] 1.5 μL of mRNA was taken from each group of samples, and the RNA concentration was measured using an ultra-micro UV spectrophotometer; 500 ng of mRNA was taken from each group of samples to prepare the reaction system (20 μL) according to the operating instructions of the reverse transcription kit to synthesize cDNA, and the remaining mRNA was stored in a -80°C refrigerator.

[0091] The reverse transcription reaction system was as follows: 500 ng of mRNA, 16 μL of RNase-free water, and 4 μL of 5×iscript.

[0092] The reverse transcription program was as follows: primer extension at 25°C for 5 min, reverse transcription reaction at 46°C for 20 min, and reverse transcriptase inactivation (termination reaction) at 95°C for 1 min.

[0093] 1.6.3 Real-time polymerase chain reaction (RT-PCR) quantitative analysis of genes

[0094] The complementary deoxyribonucleic acid (cDNA) obtained by reverse transcription in each group was diluted 5 times with 80 μL of RNase-free water and used as a PCR reaction template. 500 ng of mRNA was taken from each group of samples to prepare the reaction system (20 μL) according to the instructions in the SYBR kit to amplify the target gene, and the remaining mRNA was stored in a -80°C refrigerator. In this experiment, TLR3 was selected as the target gene, GAPDH was used as the internal reference gene (primer sequences are shown in Table 1), the target gene copy number / GAPDH ratio was used as the statistical value, the 2-ΔΔCT method was used to analyze the real-time fluorescence PCR data, and the expression level of TLR3 in femoral tissue was analyzed by Real-time PCR.

[0095] The real-time PCR reaction system is as follows:

[0096] 2 μL of cDNA template, 0.5 μL of upstream primer (10 μL M), 0.5 μL of downstream primer (10 μL M), 10 μL of Sybr green mi×, and 7 μL of RNase-free water.

[0097] Real-time PCR reaction procedure is as follows:

[0098] Initial denaturation at 95°C for 3 minutes, denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 60 seconds (40 cycles of denaturation, annealing and extension), and melting curve at 60-95°C for 5 seconds hold / 1°C.

[0099] The primer sequences are shown in Table 1:

[0100] Table 1

[0101]

[0102] result

[0103] (I) Histological and bone morphometric analysis results

[0104] HE staining results and bone tissue morphometric analysis showed that ( Figure 1 AB), the trabecular volume of the old group of mice was lower than that of the young group, and the difference was statistically significant (p<0.05).

[0105] (II) Immunohistochemical staining test results

[0106] TLR3 immunohistochemical staining results showed that ( Figure 1 In CD), the number of TLR3-positive cells in the old group was lower than that in the young group, and the difference was statistically significant (p<0.05).

[0107] (III) Western-Blot detection of TLR3 protein expression in femoral tissue

[0108] Western-Blot results showed that ( Figure 1 The expression level of TLR3 protein in the femoral tissue of the old group mice was lower than that in the young group, and the difference was statistically significant (p<0.05).

[0109] (IV) Real-time PCR detection of TLR3 mRNA expression level in femoral tissue

[0110] The results of real-time PCR showed that ( Figure 1 Middle G), the mRNA expression level of TLR3 in the femoral tissue of the elderly group of mice was lower than that in the young group, and the difference was statistically significant (p<0.05).

[0111] in conclusion

[0112] TLR3 expression was significantly downregulated in aged mice.

[0113] Example 2 Expression of TLR3 in OVX model mice

[0114] Objective: To observe the expression of TLR3 in OVX model mice

[0115] method

[0116] 2.1 Experimental Grouping

[0117] Control group (sham group); (B) model group (OVX group). There were 6 mice in each group.

[0118] 2.2 OVX model construction

[0119] Ovariectomy was used (10-week-old mice were intraperitoneally injected with Shutai anesthetic, and the mice were anesthetized 3min to 5min after the injection. The mice were fixed in the supine position, depilated, and then disinfected with iodine and alcohol respectively in the surgical area, covered with a sterile towel, cut the skin, cut the peritoneum in the folds, opened the abdominal cavity, and white fat was visible in the incision field. The fat layer was pushed aside to find the uterus, and one side of the uterine horn was gently pulled out. The fat-wrapped ovary was visible at the end. The ovary was pink and mulberry-shaped. The fallopian tube under the ovary was clamped with a tissue clamp, and the fallopian tube and fat were tied with silk thread. The ovary was cut off and the uterine horn was returned to the abdomen. The ovary on the other side is removed by the same method. The abdominal cavity and muscle layer are sutured continuously (the muscle layer is sutured together with the peritoneum), the skin is sutured intermittently, the skin suture is disinfected again with iodine, and penicillin is injected into the abdominal cavity.) OVX modeling success criteria: ① OVX model surgery: The successful removal of the bilateral ovaries of the mouse during the operation indicates that the ovariectomy surgery is successful; ② Bone phenotype: Since OVX mice usually have a more obvious bone phenotype 8 weeks after ovariectomy, the measure of successful modeling is usually to perform CT detection of the femoral bone mass, bone density and pathological staining of the mice 8 weeks after modeling to observe the changes in bone mass. If the differences in the femoral bone mass, bone density and pathological staining of the mice detected by CT are statistically significant, it means that the modeling is successful. The differences in the femoral bone mass, bone density and pathological staining of the modeling mice of the present invention are statistically significant, indicating that the modeling of the present invention is successful.

[0120] 2.3 Obtaining femoral tissue and preparing slices

[0121] Samples were collected 8 weeks after modeling, and the subsequent experimental steps refer to the experimental section 1.2.

[0122] 2.4 Histological and bone morphometric analysis

[0123] For specific experimental steps, please refer to 1.3 Experimental Section.

[0124] 2.5 Immunohistochemistry

[0125] For specific experimental steps, please refer to 1.4 Experimental Section.

[0126] 2.6 Western-Blot detection of TLR3 expression in femoral tissue

[0127] For specific experimental steps, please refer to 1.5 Experimental Section.

[0128] 2.7 Real-time PCR detection of TLR3 mRNA expression level in femoral tissue

[0129] For specific experimental steps, please refer to 1.6 Experimental Section.

[0130] result

[0131] (I) Histological and bone morphometric analysis results

[0132] HE staining results and bone tissue morphometric analysis showed that ( Figure 2 AB), the trabecular bone volume of OVX group mice was lower than that of sham group, and the difference was statistically significant (p<0.05).

[0133] (II) Immunohistochemical staining test results

[0134] TLR3 immunohistochemical staining results showed that ( Figure 2 In the middle CD), the number of TLR3-positive cells in the OVX group was lower than that in the sham group, and the difference was statistically significant (p<0.05).

[0135] (III) Western-Blot detection of TLR3 protein expression in femoral tissue

[0136] Western-Blot results showed that ( Figure 2 In the EF, the expression level of TLR3 protein in the femoral tissue of mice in the OVX group was lower than that in the sham group, and the difference was statistically significant (p<0.05).

[0137] (IV) Real-time PCR detection of TLR3 mRNA expression level in femoral tissue

[0138] The results of real-time PCR showed that ( Figure 2 Middle G), the mRNA expression level of TLR3 in the femoral tissue of OVX mice was lower than that in the sham group, and the difference was statistically significant (p<0.05).

[0139] in conclusion

[0140] The expression of TLR3 was significantly downregulated in OVX model mice.

[0141] Example 3 Effect of Poly(I:C) on the differentiation of BMSCs

[0142] Objective: To observe the effect of Poly(I:C) on the osteogenic differentiation of BMSCs

[0143] method

[0144] 3.1 Extraction of primary BMSCs

[0145] Items to prepare: Sterilized scissors, forceps, and vascular clamps, at least two sets. Drapes, 1mL syringes, small beakers, 75% alcohol, culture dishes, 15mL centrifuge tubes, and MEMα culture medium.

[0146] Animal preparation: 3-4 4-week-old C57 mice.

[0147] Steps:

[0148] 1) Place the items in the clean bench for UV sterilization in advance.

[0149] 2) The animals were killed by dislocating the neck, and the skin of both lower limbs was removed. The hair of the mice was washed off with PBS, and the animals were placed in a small beaker containing 75% alcohol and soaked for 10-15 minutes. The animals were then moved to a clean bench for subsequent operations.

[0150] 3) Move the lower limbs of the mouse into a culture dish filled with PBS, and use the first set of scissors and forceps on the towel to remove the lower limb muscles. Separate the femur and tibia, and soak them in another dish and wait for the next step. After all the treatments are completed, proceed to the next step.

[0151] 4) Pour 8-10ml of serum-free MEMα medium into a clean culture dish, and use a 1mL syringe to extract 1ml of medium for later use. Use the second set of instruments, clamp the femur or tibia in one hand with a vascular clamp, and use scissors in the other hand to cut off the ends of the femur or tibia. Use a 1mL syringe to flush the bone marrow in the femoral or tibia medullary cavity into the culture dish containing the culture medium, and repeat the flushing 2-3 times until there is no red bone marrow in the femoral medullary cavity. After all the processing is completed, proceed to the next step.

[0152] 5) Use a 1 mL gun to blow and disperse the bone marrow clumps, collect them in a centrifuge tube, centrifuge them normally, add serum-containing culture medium to resuspend them, and add them to a culture dish to culture for 48 hours.

[0153] 6) After 48 hours, discard the culture medium of the culture dish, wash it twice with PBS, and replace it with new culture medium. (If the medium is not changed after 48 hours, it will still be turbid, because blood cells and immune cells basically do not adhere to the wall. After changing the medium, you can see the adherent cells, and the cells are still round.)

[0154] Change the medium every 2 days. On the 7th day, you can see the cells growing in colonies. If the number of cells reaches more than 80%, you can subculture. Subculture once or twice. The number of cells must be guaranteed. If there are too few, they will not grow.

[0155] 3.2 Drug toxicity testing

[0156] The cells were seeded into 96-well plates at 2000 cells / well. After 12 hours, the medium was changed and different concentrations of Poly(I:C) (0, 5, 10, 20, 40, 80, 100 μg / mL) were added. After 24 hours of intervention, the medium was changed and CCK8 reagent was added. After incubation for 2 hours, the OD value was detected using an enzyme reader for statistical analysis.

[0157] 3.3 Experimental Grouping

[0158] Control group (0 μg / mL); (B) Poly(I:C) low-dose group (5 μg / mL); (C) Poly(I:C) medium-dose group (10 μg / mL); (D) Poly(I:C) high-dose group (20 μg / mL). The concentration of Poly(I:C) was set according to the results of drug toxicity tests.

[0159] 3.4 Osteogenesis induction and related staining

[0160] The cells were seeded into 24-well plates at 50,000 cells / well. After 12 hours, the medium was changed and osteogenic induction solution containing different concentrations of Poly(I:C) was added. The formula of osteogenic induction solution is: 50μg / mL vitamin C, 10mM sodium β-glycerophosphate and 0.1μM dexamethasone. The medium was changed every two days. After one week, alkaline phosphatase staining was performed, photos were taken and statistical analysis was performed using Image J 1.46r software. After 3 weeks of induction, alizarin red staining was performed, photos were taken and statistical analysis was performed using Image J 1.46r software.

[0161] 3.5 Western-Blot detection of protein expression of TLR3, COL1A1, ALP, Run×2, and OCN in BMSCs

[0162] The cells were seeded into 6-well plates at 100,000 cells / well. After 12 hours, the medium was changed and osteogenic induction solution containing different concentrations of Poly (I: C) was added. Total protein was extracted after one week of induction. The culture medium of each group in the culture plate was aspirated, washed once with pre-cooled 1× PBS, and pre-cooled cell lysis solution (containing 1% protease phosphatase inhibitor) was added. After standing for 5 minutes, the cells were carefully scraped from the culture plate with a cell scraper, blown into a cell suspension and transferred to a 1.5 mL centrifuge tube, and stood on ice for 30 minutes, vortexing every 10 minutes. For subsequent experimental steps, refer to the experimental section 1.5.

[0163] 3.6 Real-time PCR detection of mRNA expression levels of TLR3, COL1A1, ALP, SP7, Runx2, and OPN in femoral tissue

[0164] The cells were seeded into 6-well plates at 100,000 cells / well. After 12 hours, the medium was changed and osteogenic induction solution containing different concentrations of Poly (I: C) was added. mRNA was extracted after one week of induction. The original culture medium was aspirated, washed once with 1× PBS, and 0.5 mL of Trizol was directly added to the culture plate. After repeated pipetting and even mixing, it was transferred to a separation gel tube (centrifuged at 13,000 rpm for 1 minute before use) and allowed to stand at room temperature for 5 minutes. For subsequent experimental steps, refer to the experimental section 1.6. The primer sequences are shown in Table 2:

[0165] Table 2

[0166]

[0167] result

[0168] (I) Drug toxicity test results

[0169] The results of CCK-8 test showed that ( Figure 3 In the figure (A), cell viability decreased when the Poly(I:C) concentration was ≥40 μg / mL, and the difference was statistically significant (p<0.05), so the subsequent experimental studies used concentrations of 5, 10, and 20 μg / mL.

[0170] (II) Western-Blot analysis of TLR3 protein expression after Poly(I:C) administration

[0171] Western-Blot results showed that ( Figure 3 After the administration of Poly(I:C), the expression level of TLR3 protein was significantly increased compared with the control group, and the difference was statistically significant (p<0.05).

[0172] (III) Alkaline phosphatase staining to detect the osteogenic effect of Poly(I:C) after administration

[0173] The results of alkaline phosphatase staining showed that ( Figure 3 After the administration of Poly(I:C), the expression of alkaline phosphatase was significantly increased compared with the control group, and the difference was statistically significant (p<0.05).

[0174] (IV) Alizarin red staining to detect the osteogenic effect of Poly(I:C) after administration

[0175] The results of Alizarin red staining showed that ( Figure 3 FG), after the administration of Poly(I:C), the expression of mineralized nodules was significantly increased compared with the control group, and the difference was statistically significant (p<0.05).

[0176] (V) Western-Blot detection of protein expression of COL1A1, ALP, Runx2, and OCN after administration of Poly(I:C)

[0177] Western-Blot results showed that ( Figure 3 In the HL group, after the administration of Poly(I:C), the expression levels of COL1A1, ALP, Runx2, and OCN proteins were significantly increased compared with those in the control group, and the differences were statistically significant (p<0.05).

[0178] (VI) Real-time PCR detection of mRNA expression levels of TLR3, COL1A1, ALP, SP7, Runx2, and OPN after administration of Poly(I:C)

[0179] The results of real-time PCR showed that ( Figure 3 After the administration of Poly(I:C), the mRNA expression levels of TLR3, COL1A1, ALP, SP7, Runx2, and OPN were significantly increased compared with the control group, and the differences were statistically significant (p<0.05).

[0180] in conclusion

[0181] Poly(I:C) promotes the osteogenic differentiation of BMSCs.

[0182] Example 4 Intervention effects of different concentrations of Poly(I:C) on femoral defect model mice

[0183] Objective: To observe the therapeutic and osteogenesis effects of Poly(I:C) on femoral defect model mice

[0184] method

[0185] 4.1 Experimental Grouping

[0186] (A) Model group (Vehicle group); (B) Poly(I:C) low-dose group (5 mg / kg-PIC group); (C) Poly(I:C) high-dose group (10 mg / kg-PIC group). 8 mice in each group.

[0187] 4.2 Establishment of femoral defect model

[0188] The femoral defect model was constructed as follows: 10-week-old C57 male mice were selected, and the mice were intraperitoneally injected with sutane anesthetic, and were anesthetized 3-5 minutes after the injection. The mice were fixed in the supine position, depilated, and then disinfected with iodine and alcohol respectively in the surgical area, covered with sterile drapes, cut the skin, pushed the patella to one side, exposed the distal femur, and used a 0.6mm thick turner to punch holes in the trochlea part between the medial and lateral femoral condyles until it entered the medullary cavity. After using cotton balls to stop bleeding, the patella was reset and the skin was sutured. The femoral defect modeling success criteria: ① Defect model surgery: During the operation, the middle of the medial and lateral femoral condyles of the mouse were punched until the bone marrow flowed out of the gap, indicating that the femoral defect surgery was successful; ② Bone phenotype: After modeling, the femoral defect mouse will have a defect on the distal femoral surface and a turner tunnel in the medullary cavity, so the success of the modeling can usually be measured by CT and pathological staining to observe whether there is a defect on the surface of the mouse femur and whether there is a tunnel in the femoral medullary cavity. CT and pathological staining showed that the surface of the femur of the mouse was defective and a tunnel was left in the femoral medullary cavity, indicating that the modeling was successful. CT and pathological staining showed that the surface of the femur of the mouse modeling of the present invention was defective and a tunnel was left in the femoral medullary cavity, indicating that the modeling of the present invention was successful.

[0189] 4.3 Drug intervention

[0190] One day after the model was established, the mice were divided into groups for intraperitoneal drug intervention. The Vehicle group received 0.5 mL of normal saline per mouse per day, and the Poly(I:C) group received 0.5 mL of 5 mg / kg and 10 mg / kg Poly(I:C) per mouse three times a week. The intervention lasted for 1 week.

[0191] 4.4 Micro-CT detection

[0192] Mouse femur samples were fixed in 4% paraformaldehyde for 48 hours and then scanned by Micro-CT (Skyscan1176, Bruker, Belgium). Subsequently, images were corrected, analyzed, and reconstructed using DataViewer, CTAn, and CTvox software. The parameters evaluated and recorded included trabecular bone density (Tra.BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.Sp), structural model index (SMI), and connection density (Conn.Dn).

[0193] 4.5 Histological and bone morphometric analysis

[0194] The experimental steps for pathological sample processing and HE staining refer to Experiment 1.3. The ABH / OG staining steps are as follows: slices of femoral tissue from each group were taken and baked at 60°C overnight; the next day, the slices were taken out and dewaxed and rehydrated step by step after returning to room temperature (xylene dewaxing 3 times, 5 minutes / time; 100% and 95% alcohol 2 times, 5 minutes / time; 70% alcohol 1 time, 5 minutes / time; deionized water 2 times, 1 minute / time); the slices were placed in hydrochloric acid alcohol for differentiation for 30 seconds, and the excess water was briefly drained on paper; the slices were placed in alicin blue hematoxylin solution for staining for 45 minutes, and then rinsed with deionized water several times until the water was clear (about 3 times); the slices were placed in hydrochloric acid alcohol for differentiation for 3 seconds, and then washed with deionized water 3 times, 1 minute / time; the slices were placed in 0.5% ammonia water for 15 seconds, and then washed with deionized water twice, 1 minute / time; the slices were placed in 95% alcohol for 1 minute, and then transferred to Eosin / Orange Stain in G working solution for 1 minute and 30 seconds; dehydrate and make transparent step by step (95% alcohol 3 times, 1 minute / time; 100% alcohol 2 times, 1 minute / time; xylene dewaxing 3 times, 1 minute / time), and seal with sealing medium. Observe and complete the collection under Zeiss upright microscope.

[0195] 4.6 Immunohistochemistry

[0196] For specific experimental steps, please refer to 1.4 Experimental Section.

[0197] 4.7 Trap staining

[0198] Dewax and rehydrate the paraffin sections; prepare the hexazo para-carmine solution; prepare the incubation solution: add 1 mL of hexazo para-carmine to 18 ml of acetate solution, then add 1 mL of AS-BI phosphate solution, then add 282 mg of potassium sodium tartrate, adjust the pH to 5.0, filter and set aside; negative control incubation solution: add 1 mL of hexazo para-carmine to 18 ml of acetate solution, then add 282 mg of potassium sodium tartrate. Adjust the pH to 50 and filter and set aside; wash the sections with 37°C distilled water for 30 seconds; incubate in the incubation solution at 37°C for 50-60 minutes; wash with deionized water for 3 minutes; counterstain with hematoxylin for 40 seconds; wash with tap water for 10 minutes to turn blue; seal the sections with glycerol gelatin.

[0199] result

[0200] (I) Micro-CT test results

[0201] The 2D and 3D reconstruction results of Micro-CT showed that the bone mass at the femoral defect site in the Poly(I:C) group was significantly higher than that in the Vehicle group ( Figure 43D morphometric analysis showed that the BMD, BV / TV and Tb.N of the femoral defect site in the Poly(I:C) group were significantly higher than those in the Vehicle group, while Tb.Sp was lower than those in the Vehicle group, and the differences were statistically significant (p<0.05), with the 10 mg / kg Poly(I:C) treatment group being the most significant ( Figure 4 (in DH).

[0202] (II) Histological and bone morphometric analysis results

[0203] ABH / OG and HE staining results and bone tissue morphometric analysis showed that the bone mass at the femoral defect site in the Poly(I:C) group was significantly higher than that in the Vehicle group ( Figure 4 Chinese BC).

[0204] (III) Immunohistochemical staining test results

[0205] The results of ALP immunohistochemical staining showed that ( Figure 5 In Figures 5A and 5E), the ALP-positive area of ​​the femoral defect in the Poly(I:C) group was significantly higher than that in the Vehicle group (p<0.05). Figure 5 In Figure 5B and Figure 5F), the number of OCN-positive cells in the femoral defect of the Poly(I:C) group was significantly higher than that of the Vehicle group (p<0.05). Figure 5 (C) and (G) in Figure 5, the number of Runx2-positive cells in the femoral defect site of the Poly(I:C) group was significantly higher than that of the Vehicle group (p<0.05).

[0206] (IV) Trap staining test results

[0207] Trap staining results showed ( Figure 5 (D) and (H) in Figure 5 ), there was no significant difference in the number of Trap-positive cells in the femoral defect site between the Poly(I:C)-treated group and the Vehicle group (p>0.05).

[0208] in conclusion

[0209] Poly(I:C) promoted bone repair in a femoral defect model mouse model by promoting osteogenesis.

[0210] Example 5 Intervention effects of different concentrations of Poly(I:C) on OVX model mice

[0211] Objective: To observe the therapeutic and osteogenesis effects of Poly(I:C) on OVX model mice

[0212] method

[0213] 5.1 Experimental Grouping

[0214] Sham operation group (sham group); (B) model group (OVX group); (C) Poly(I:C) low-dose group (5mg / kg-PIC group); (D) Poly(I:C) high-dose group (10mg / kg-PIC group). 8 mice in each group.

[0215] 5.2 Building the OVX Model

[0216] The experimental steps refer to 2.2 Experimental Section.

[0217] 5.3 Pharmacological intervention

[0218] Seven days after the model was established, the mice were divided into different groups for intraperitoneal drug intervention. The Vehicle group received 0.5 mL of normal saline per mouse per day, and the Poly(I:C) group received 0.5 mL of 5 mg / kg and 10 mg / kg Poly(I:C) per mouse three times a week. The intervention lasted for 7 weeks.

[0219] 5.4 Micro-CT detection

[0220] The experimental steps refer to Section 4.4 Experimental.

[0221] 5.5 Histological and bone morphometric analysis

[0222] The experimental steps refer to Section 4.5 Experimental.

[0223] 5.6 Immunohistochemistry

[0224] For specific experimental steps, please refer to 1.4 Experimental Section.

[0225] 5.7 Trap staining

[0226] The experimental steps refer to Section 4.7 Experimental Section.

[0227] result

[0228] (I) Micro-CT test results

[0229] The 2D and 3D reconstruction results of Micro-CT showed that the bone mass in the femur of the Poly(I:C) group was significantly higher than that of the OVX group ( Figure 6 3D morphometric analysis showed that the BMD, BV / TV, Tb.N and Conn.Dn of the femur in the Poly(I:C) group were significantly higher than those in the OVX group, while Tb.Sp and SMI were lower than those in the OVX group, and the differences were statistically significant (p<0.05), with the 10 mg / kg Poly(I:C) treatment group being the most significant ( Figure 6 Chinese DJ).

[0230] (II) Histological and bone morphometric analysis results

[0231] ABH / OG and HE staining results and bone tissue morphometric analysis showed that ( Figure 6 In BC and K in 6), the bone mass in the femur of the Poly(I:C) group was significantly higher than that of the Vehicle group, and the difference was statistically significant (p<0.05).

[0232] (III) Immunohistochemical staining test results

[0233] The results of ALP immunohistochemical staining showed that ( Figure 7 In Figures A and E), the ALP-positive area in the femur of the Poly(I:C) group was significantly higher than that of the OVX group (p<0.05). Figure 7 In Figure 7B and Figure 7F), the number of OCN-positive cells in the femur of the Poly(I:C) group was significantly higher than that of the OVX group (p<0.05). Figure 7 (C) and (G) in Figure 7, the number of Runx2-positive cells in the femur of the Poly(I:C)-treated group was significantly higher than that of the OVX group (p<0.05).

[0234] Trap staining results

[0235] Trap staining results showed ( Figure 7 (D) and (H) in Figure 7 ), there was no significant difference in the number of Trap-positive cells in the femur of the Poly(I:C)-treated group compared with the Vehicle group, and the difference was not statistically significant (p>0.05).

[0236] in conclusion

[0237] Poly(I:C) delays bone loss in OVX model mice by promoting osteogenesis.

[0238] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the contents disclosed herein. The present application is intended to cover any variations, uses or adaptations of the present application, which follow the general principles of the present application and include common knowledge or customary technical means in the art that are not disclosed in the present application.

Claims

1. Use of poly (I:C) in the preparation of a drug for treating and / or preventing bone defects and / or osteoporosis, characterized in that: The chemical formula of the poly (I:C) is C 19 H 27 N7O 16 P2.

2. A drug for treating and / or preventing bone defects and / or osteoporosis, characterized in that: The active ingredient includes poly I:C.

3. The drug according to claim 2, characterized in that The dosage form is any one of tablets, capsules, oral liquids, lozenges, granules, granules, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, creams, sprays, drops or patches.

4. The drug according to claim 2, characterized in that The administration route of the drug is oral, intraperitoneal, intravenous or subcutaneous injection.

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