Research and development methods for histone methyltransferase Setdb1 in disuse osteoporosis

Abstract

The present invention discloses a research and development method for histone methyltransferase Setdb1 in disuse osteoporosis, specifically including the steps of studying the diagnostic application of Setdb1 in disuse osteoporosis; studying the effect of Setdb1 on osteoblast proliferation function; studying the effect of bone-targeted delivery of si-Setdb1 on mouse bone formation and studying the effect of overexpression of Setdb1 on cell proliferation function in a 2D rotational MC3T3-E1 cell unloading model. This scheme found that the expression of histone methyltransferase Sedtb1 was significantly reduced in the 2D rotational cell unloading model and the mouse hindlimb tail suspension (HLU) unloading model, and explored its effect on osteoblast proliferation function and bone formation, suggesting that it may serve as a potential biomarker for disuse osteoporosis and can be used as a new target to combat disuse osteoporosis, with good prospects for translational medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of application of histone methyltransferase Setdb1 in osteoporosis, and in particular to a research and development method of histone methyltransferase Setdb1 in diagnosing and treating disuse osteoporosis. Background Art

[0002] Osteoporosis is a common skeletal system disease, which is mainly manifested by decreased bone density and destruction of bone microstructure, thereby increasing the risk of fractures.

[0003] Bones are dynamically changing organs. Under normal mechanical stimulation, a dynamic balance exists between osteoblast-mediated bone formation and osteoclast-mediated bone resorption during bone remodeling. However, under long-term unloading conditions, this balance is disrupted, resulting in decreased bone formation and increased bone resorption. Osteoblasts play a crucial role in this process. Dysfunction of osteoblast differentiation, proliferation, and apoptosis is the primary cause of disuse osteoporosis, and this process is regulated by multiple factors.

[0004] Setdb1, also known as ESET or KMT1E, is a methyltransferase that belongs to the SET domain protein family. Setdb1 contains a Tudor domain at its N-terminus and a SET domain at its C-terminus. The C-terminal SET domain catalyzes the trimethylation of histone H3K9 (H3K9me3), which is associated with gene silencing or transcriptional repression.

[0005] Disuse osteoporosis (DOP) is a secondary osteoporosis caused by prolonged unloading of the skeleton. It primarily occurs in response to prolonged bed rest, immobilization with limited mobility, and weightlessness. Disuse osteoporosis, particularly during long-term spaceflight, poses a serious threat to astronauts' health. While existing anti-osteoporosis medications, such as calcium supplements, vitamins, bisphosphonates, estrogen, and parathyroid hormone, can reduce fracture rates in osteoporosis patients, their side effects, such as osteonecrosis, hypercalcemia, and thromboembolic disease, pose significant risks to human health. Therefore, understanding the molecular biological mechanisms of DOP and identifying new molecular therapeutic targets are crucial for its diagnosis and treatment. Summary of the Invention

[0006] The main purpose of the present invention is to provide a research and development method for the histone methyltransferase Setdb1 in disuse osteoporosis, so as to improve the shortcomings of existing anti-osteoporosis drugs in the relevant technology for the treatment of disuse osteoporosis and provide new molecular targets for the diagnosis and treatment of disuse osteoporosis.

[0007] To achieve the above objectives, the present invention provides a method for developing histone methyltransferase Setdb1 in disuse osteoporosis, which specifically comprises the following steps:

[0008] S1. Study the diagnostic application of Setdb1 in disuse osteoporosis: Determine the protein expression of Setdb1 by testing protein samples in MC3T3-E1 cells after unloading on a 2D rotator and in the femurs of tail-suspended mice.

[0009] S2. Study the effect of Setdb1 on osteoblast proliferation: Setdb1 overexpression vector pcDNA3.1-Setdb1 and siRNA-Setdb1 were transfected into MC3T3-E1 cells, and their osteoblast proliferation capacity was detected;

[0010] S3. Study the effect of bone-targeted delivery of si-Setdb1 on bone formation in mice;

[0011] S4. Study the effect of overexpression of Setdb1 on the proliferation function of MC3T3-E1 cells after unloading of the 2D rotator.

[0012] In one embodiment of the present invention, in S1 and S4, the mouse MC3T3-E1 cells are obtained by treating the mouse preosteoblasts MC3T3-E1 cells in a 2D rotator.

[0013] In one embodiment of the present invention, in S1, the femurs of the hind limbs of the tail-suspended mice need to be pre-treated, and the treatment method is as follows: the tails of C57BL / 6J male mice are suspended for 21 days, with the hind limbs suspended in the air so that the axis of the mouse body is at 30 degrees to the ground and the forelimbs of the mouse touch the ground, so that the mouse can move freely and obtain food and water, and the animal is subjected to horizontal unloading treatment;

[0014] Western blotting was used to detect protein samples extracted from mouse MC3T3-E1 cells and hind limb femurs of tail-suspended mice.

[0015] In one embodiment of the present invention, in said S1, the femur of the hind limb of the tail-suspended mouse is further processed by immunohistochemical staining to determine the expression of Setdb1 therein.

[0016] In one embodiment of the present invention, in said S2, the siRNA-Setdb1 sequence is: sense: 5'-CACUCAGUCAGAGCUUUAUTT-3'; antisense: 5'-AUAAAGCUCUGACUGAGUGTT-3'.

[0017] In one embodiment of the present invention, in S2, CCK-8, EdU and Western blotting are used to detect the expression of PCNA protein.

[0018] In one embodiment of the present invention, in S3, the effect of bone-targeted delivery of si-Setdb1 on bone formation in mice is explored, and the specific steps are:

[0019] S31. si-Setdb1 was delivered to the bone formation area of ​​mice via the bone-targeting material (DSS) 6-liposome, and the microstructure of the mouse femur was examined using micro-CT.

[0020] S32, Goldner staining was used to detect new bone formation;

[0021] S33, calcein double labeling experiment to detect the growth rate of cortical bone;

[0022] S34. Three-point bending test was used to detect the mechanical properties of the femur of the mouse hind limb.

[0023] In one embodiment of the present invention, the S4 is to study the effect of overexpression of Setdb1 on the proliferation function of MC3T3-E1 cells, which specifically includes the following steps:

[0024] The Setdb1 overexpression vector pcDNA3.1-Setdb1 was transfected into MC3T3-E1 cells, and the transfected cells were placed in a 2D rotator to remove the load for 48 h;

[0025] The expression of PCNA protein was detected by CCK-8, EdU and Western blotting to determine its osteogenic proliferation ability.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The changes of histone methyltransferase Sedtb1 in disuse osteoporosis were discovered, and its effects on osteoblast proliferation and bone formation were explored, suggesting that it may serve as a potential biomarker for disuse osteoporosis and a new target to combat disuse osteoporosis, with good prospects for translational medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A schematic diagram of a process according to an embodiment of the present invention;

[0029] Figure 2 The protein expression graph of Setdb1 in a 2D rotating MC3T3-E1 cell unloading model detected by western blotting technology according to an embodiment of the present invention is provided;

[0030] Figure 3 The protein expression graph of Setdb1 in MC3T3-E1 cells after 72 hours of unloading provided by an embodiment of the present invention;

[0031] Figure 4 The protein expression of Setdb1 in the mouse hindlimb tail suspension (HLU) unloading model was detected by western blotting technology according to an embodiment of the present invention;

[0032] Figure 5 This is an immunohistochemical staining image of Setdb1 in the hind limb bones of tail-suspended mice according to an embodiment of the present invention;

[0033] Figure 6 Schematic diagram of protein expression of Setdb1 and PCNA affected by osteoblast proliferation function according to an embodiment of the present invention;

[0034] Figure 7 This is a CCK-8 analysis diagram for detecting cell proliferation ability based on the osteoblast proliferation function provided by an embodiment of the present invention;

[0035] Figure 8 This is a diagram showing the EdU staining results of the osteoblast proliferation function provided by an embodiment of the present invention;

[0036] Figure 9 A micro-CT image of trabecular bone microstructure detected according to an embodiment of the present invention;

[0037] Figure 10 This is a micro-CT analysis diagram of trabecular bone microstructure according to an embodiment of the present invention;

[0038] Figure 11 A diagram of new bone formation detected by Goldner staining according to an embodiment of the present invention;

[0039] Figure 12 This is a first schematic diagram of detecting new bone formation using calcein according to an embodiment of the present invention;

[0040] Figure 13 A second schematic diagram of detecting new bone formation using calcein according to an embodiment of the present invention;

[0041] Figure 14 A graph showing the biomechanical properties of bones detected by a three-point bending test according to an embodiment of the present invention;

[0042] Figure 15 A diagram showing the results of a three-point bending test to detect the biomechanical properties of bones according to an embodiment of the present invention;

[0043] Figure 16A schematic diagram of PCNA protein expression in accordance with an embodiment of the present invention showing the effect on the proliferation function of unloaded MC3T3-E1 cells;

[0044] Figure 17 A CCK-8 analysis chart for detecting cell proliferation ability to show the effect of unloaded MC3T3-E1 cell proliferation function according to an embodiment of the present invention;

[0045] Figure 18 This is a diagram showing the EdU staining results of the effect on the proliferation function of unloaded MC3T3-E1 cells provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0046] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0047] Additionally, the term "plurality" shall mean two or more.

[0048] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0049] See also Figures 1-18 The present invention provides a method for developing histone methyltransferase Setdb1 in disuse osteoporosis, which specifically comprises the following steps:

[0050] S1. Investigate the diagnostic application of Setdb1 in disuse osteoporosis: Detect protein samples extracted from mouse MC3T3-E1 cells and the femurs of tail-suspended mice to determine the expression of Setdb1 in the protein samples; the femurs of tail-suspended mice must be pre-processed by tail suspension for 21 days and then subjected to animal-level unloading; Western blotting is used to detect protein samples extracted from mouse MC3T3-E1 cells and the femurs of tail-suspended mice.

[0051] The specific steps of Setdb1 in diagnosing disuse osteoporosis are:

[0052] Step 1: Preparation of experimental materials: prepare MC3T3-E1 cells and 12 6-month-old male C57BL / 6J mice as experimental subjects, and divide the mice into a control (Con) group and a tail-suspended (HLU) group in equal numbers.

[0053] Step 2: Prepare experimental reagents, including α-MEM culture medium, fetal bovine serum, double antibody, trypsin, RI PA cell lysis buffer, protease inhibitors, loading buffer, electrophoresis buffer, transfer buffer, primary antibody diluent, PVDF membrane, BCA protein quantification kit, Setdb1 antibody, secondary antibody, paraformaldehyde, and EDTA.

[0054] Step 3: Establish a 2D rotator unloaded cell model.

[0055] In a clean bench, cells reaching 90% confluence were passaged into spinner flasks and cultured in a 37°C incubator. After 6-8 hours of cell attachment, the flasks were filled with α-MEM medium containing 10% serum and 1% double-antibody and placed upright in a 37°C incubator overnight. Air bubbles were then completely expelled from the spinner flasks, which were then rotated on a rotator with a radius of 1.5 cm and a speed of 24 rpm. A control group was treated similarly and cultured in a 37°C incubator. Rotation times varied from 24 hours, 48 ​​hours, and 72 hours. After rotation, cell samples were extracted for testing as needed for subsequent experiments.

[0056] Step 4: Establish the hindlimb tail suspension (HLU) mouse unloading model.

[0057] Six-month-old male C57BL / 6J mice were housed under standard conditions (22°C, 12h light / 12h dark cycle). The mice were suspended by their tails, with their hind limbs suspended in the air, with their torso angled at 30° to the ground. Their forelimbs were placed on the ground, allowing them to move freely and access food and water. Mice were tail-suspended for 21 days, then anesthetized and euthanized. Bilateral femurs and tibias were harvested for experimental studies.

[0058] Step 5: Western blotting detection.

[0059] Total protein was extracted from MC3T3-E1 cells and hind tibiae using RIPA cell lysis buffer and protease inhibitors. Cells were ultrasonically disrupted, and protein concentration was determined by the BCA assay. Sample loading buffer was added and the cells were heated at 95°C for 10 minutes. Precast gels were used for electrophoresis, with 30 μl of sample loaded per well. Electrophoresis was performed at 90 V for 30 minutes, followed by 120 V for 90 minutes. The membranes were transferred to the membranes as usual and blocked with 5% skim milk for 4 hours. Antibodies to Setdb1 and GAPDH (1:1000) were added, respectively, and the cells were incubated overnight at 4°C. The next day, the membranes were washed three times with TBST (10 minutes each) on a rocking platform. The membranes were then incubated with a goat anti-rabbit secondary antibody (1:5000) for 60 minutes and washed three times with TBST (10 minutes each) on a rocking platform. ECL luminescence was used, and images were captured. Image J software was used to analyze the grayscale values ​​of the captured bands and calculate the relative protein content.

[0060] Step 6: Immunohistochemical staining (IHC) of bone tissue.

[0061] Mouse femurs were fixed in 4% paraformaldehyde for 2 days and then decalcified in 10% EDTA for 3 weeks. The decalcified specimens were dehydrated in graded ethanol and embedded in paraffin. Paraffin-embedded bone tissue samples were then sliced ​​along the long axis of the tibia with a thickness of 5 μm and placed on slides in a 37°C incubator to dry for 72 hours. Antigen retrieval was performed using 0.01 M citrate buffer (pH 6.0). After blocking with goat serum, the prepared Setdb1 primary antibody reagent was added dropwise and incubated at 4°C overnight. After incubation with a radish peroxidase-conjugated secondary antibody (HRP), DAB staining and hematoxylin staining were performed and observed under a microscope.

[0062] It has been verified by experiments that Figure 2-5 It was found that the expression of Setdb1 was reduced, and the decrease was most obvious 48 hours after cell rotation.

[0063] The femur of the hind limb of the tail-suspended mouse can also be processed by immunohistochemical staining to determine the expression of Setdb1. Immunohistochemical staining technology found that Setdb1 expression was reduced in the hind limb bones of the tail-suspended mouse. Figure 5 .

[0064] S2. Study the effect of Setdb1 on osteoblast proliferation function: The Setdb1 overexpression vector pcDNA3.1-Setdb1 and siRNA-Setdb1 were transfected into MC3T3-E1 cells, respectively. The siRNA-Setdb1 sequence was: sense: 5'-CACUCAGUCAGAGCUUUAUTT-3'; antisense: 5'-AUAAAGCUCUGACUGAGUGTT-3'. The specific serial numbers are shown in Table 1. CCK-8, EdU and Western blotting were used to detect PCNA protein expression to determine its osteoblast proliferation ability.

[0065] The specific steps to study the effect of Setdb1 on osteoblast proliferation are as follows:

[0066] Step 1: Prepare experimental materials and MC3T3-E1 cells.

[0067] Step 2: Prepare the experimental reagents: α-MEM medium, fetal bovine serum, double antibody, trypsin, RIPA cell lysis buffer, protease inhibitors, loading buffer, electrophoresis buffer, transfer buffer, primary antibody diluent, PVDF membrane, BCA protein quantification kit, Setdb1 antibody, secondary antibody, overexpression vector pcDNA3.1-Setdb1, si-Setdb1, Lipo2000 transfection reagent, CCK-8 kit, EdU cell proliferation detection kit.

[0068] Step 3: Cell transfection.

[0069] Lyophilized powders of pcDNA3.1-Setdb1 and si-Setdb1 were diluted with double-distilled water in a clean hood and aliquoted for use. siRNA sequences targeting Setdb1 are listed in Table 1. Cells were passaged into six-well plates at a confluence of 90% in a clean hood and transfected when the confluence reached 60%-70%. 5 μl of Lipofectamine 2000 was added to 250 μl of Opti-MEM medium. Another 250 μl of Opti-MEM medium was then added to 80 mM of si-Setdb1 or 2.5 μg of the pcDNA3.1-Setdb1 diluted stock solution. The cells were allowed to stand for 5 minutes, mixed thoroughly, and allowed to stand for 20 minutes. The mixed solution was then randomly distributed into six-well plates, and 2 ml of serum-free, antibody-free α-MEM medium was added to each well. The cells were incubated at 37°C. After 6 hours, the medium was replaced with α-MEM medium containing 10% serum and no double antibody, and cell proteins were extracted after 24-48 hours.

[0070]

[0071] Table 1 Nucleotide sequence correspondence table

[0072] Step 4: Western blotting was used to detect the protein expression level of PCNA in MC3T3-E1 cells using the same method as in S1.

[0073] Step 5: CCK-8 was used to detect the proliferation ability of osteoblasts.

[0074] In a clean bench, cells with a confluence of 90% were passaged into 96-well plates at a density of 2000 cells / well, with 100 μl of culture medium per well. The plates were pre-cultured in an incubator (37°C, 5% CO2). CCK-8 enhancement solution (10 μl / well) was added to the 96-well plates and incubated in a cell culture incubator for 1 hour. The absorbance of the reaction solution at 450 nm was then measured using a microplate reader.

[0075] Step 6: EdU staining to detect osteoblast proliferation ability.

[0076] MC3T3-E1 cells were seeded on cover slips and then transfected. TM EdU labeling was performed using an EdU cell proliferation kit. Half of the culture medium was replaced with fresh medium containing 20 μM EdU and incubated in an incubator for 2 hours. The coverslips seeded with cells were then fixed in 4% paraformaldehyde for 15 minutes at room temperature. The cells were then soaked in 0.3% Triton X-100 in PBS for 15 minutes at room temperature and incubated with Alexa Fluor 594 for 30 minutes at room temperature. Finally, osteoblasts were incubated in PBS containing Hoechst 33342 for 10 minutes at room temperature. Stained cells were observed using confocal microscopy, and quantification was performed by assessing the proportion of EdU-positive cells.

[0077] like Figure 6-Figure 8 Experimental results show that overexpression of Setdb1 can promote osteoblast proliferation, while knockdown of Setdb1 can inhibit osteoblast proliferation.

[0078] S3. Study the effect of bone-targeted si-Setdb1 on bone formation in mice. The specific steps are as follows:

[0079] Step 1: Preparation of experimental materials. Prepare 15 experimental mice as experimental subjects and divide them into Con group, (DSS)6-liposome-si-NC group, and (DSS)6-liposome-si-Setdb1 group.

[0080] Step 2: Prepare the experimental reagents: bone-targeted delivery material (DSS), 6-liposome, si-RNA, paraformaldehyde, EDTA, ethanol, iron hematoxylin staining solution, polipochrome acid fuchsin stain, orange G solution, brilliant green stain, neutral resin, polymethyl acrylate, calcein, and saline.

[0081] Step 3: Use bone-targeting material (DSS) 6-liposome to deliver si-Setdb1 to the bone formation area of ​​mice.

[0082] The specific dosage of si-Setdb1 was 10 mg / kg body weight. For a single injection, the targeted drug was prepared in 200 μl of saline, mixed with the si-RNA, and shaken on a shaker for 30 minutes. The mixture was then allowed to rest at room temperature for 1 hour. The drug was drawn into a 1 ml syringe. The mouse's tail was exposed using a venous visualization mouse tail injection fixture, and the tail vein was located. The needle was inserted parallel to the tail vein. A successful injection was considered successful if there was no resistance. Injections were repeated twice every two weeks for a total of four weeks.

[0083] Step 4: If Figure 9-10 , micro-CT was used to examine the microstructure of mouse femur.

[0084] For micro-CT, the left femur and tibia of each mouse were fixed in 4% paraformaldehyde for 24 hours and scanned 360°. A 2.5 × 2.5 × 3 mm³ cube was selected approximately 1.5 mm from the proximal epiphyseal plate as the region of interest (ROI), representing the microarchitecture of the femur and tibia. Structural parameters such as BMD, Tb.Th, BV / TV, and Tb.N were analyzed within the ROI.

[0085] Step 5: If Figure 11 Goldner staining was used to detect new bone formation.

[0086] Goldner staining procedures involved fixing the left tibia in 4% paraformaldehyde for 24 hours and decalcifying it in 10% EDTA for 3 weeks. The decalcified specimens were then dehydrated with graded ethanol and embedded in paraffin. After paraffin embedding, 5-μm sections were cut along the long axis of the tibia and placed on slides. The sections were then dried in a 37°C incubator for 72 hours before Goldner staining. (1) The sections were stained with iron hematoxylin solution for 15 minutes, rinsed with tap water, differentiated with hydrochloric acid alcohol solution for 4 seconds, rinsed with tap water, and rinsed with distilled water; (2) The sections were stained with fuchsin acid fuchsin for 15 minutes and rinsed twice with 1% acetic acid; (3) The sections were stained with orange G solution for 5 minutes and rinsed twice with 1% acetic acid; (4) The sections were stained with brilliant green stain for 10 minutes and rinsed three times with 1% acetic acid; (5) The sections were rinsed once with 70% ethanol, dehydrated twice with 100% ethanol, transparentized with xylene for 5 minutes, sealed with neutral resin, and observed under a microscope.

[0087] Step 6: If Figure 12 and Figure 13 , Calcein double labeling experiment was used to detect the cortical bone growth rate.

[0088] To measure the rate of bone formation, mice were intraperitoneally injected with calcein (8 mg / kg body weight) 10 and 3 days before euthanasia. The right tibia of the mice was fixed with 4% paraformaldehyde, embedded in polymethyl acrylate, and then cut into 50 μm sections using a hard tissue slicer. The two calcein-labeled lines were observed using a confocal microscope. The mineral attachment rate (MAR) of the trabecular bone was measured by the distance between the two fluorescent markers.

[0089] Step 7: If Figure 14 and Figure 15 ,The three-point bending test was used to detect the mechanical properties of the mouse hind limb femur.

[0090] Mouse right femurs were wrapped in saline-soaked gauze and stored at -80°C. After thawing, the femoral specimens were placed in an 8 mm diameter bending fixture. Load was applied perpendicular to the femoral midshaft at a rate of 0.02 mm / s until fracture occurred. The length, width, and cortical thickness of the fracture site were measured using a vernier caliper. The maximum load (N), stiffness (N / mm), and elastic modulus (GPa) were calculated from the load-deflection curves.

[0091] Experimental verification shows that targeted delivery of si-Setdb1 can significantly promote osteoporosis in tail-suspended mice, which is manifested by bone microstructure destruction, new bone formation disorders and impaired biomechanical properties.

[0092] S4. Study the effect of overexpression of Setdb1 on the proliferation function of MC3T3-E1 cells after unloading of the 2D rotator.

[0093] like Figure 16-18 In this example, the effect of overexpression of Setdb1 on the proliferation function of MC3T3-E1 cells was studied, which specifically included the following steps:

[0094] The Setdb1 overexpression vector pcDNA3.1-Setdb1 was transfected into MC3T3-E1 cells. 12 hours later, the cells were placed in a 2D rotator and rotated for 48 hours. CCK-8, EdU and PCNA protein detection were then performed using the same detection methods as above.

[0095] The expression of PCNA protein was detected by CCK-8, EdU and Western blotting to determine its osteogenic proliferation ability. Figure 16-18 Experimental results show that overexpression of Setdb1 can alleviate the inhibition of osteoblast proliferation caused by 2D rotation simulated weightlessness.

[0096] It should be noted that in S1 and S4, mouse MC3T3-E1 cells were obtained by treating mouse preosteoblasts with a 2D rotator; in S2, mouse MC3T3-E1 cells were obtained by transfecting mouse preosteoblasts with pcDNA3.1-Setdb1 or si-Setdb1.

[0097] After experimental verification, the conclusions are as follows:

[0098] Setdb1 was found to be significantly down-expressed in both osteoblast unloading models (2D-rotated MC3T3-E1 cells) and hindlimb unloading models (mouse hindlimb tail suspension), suggesting that it may serve as a potential biomarker for disuse osteoporosis.

[0099] It was found that knocking down Setdb1 significantly inhibited the proliferation of MC3T3-E1 cells, and overexpressing Setdb1 partially alleviated the osteoblast proliferation disorder caused by the 2D rotator, suggesting its importance to osteoblast proliferation and providing a reference for the treatment of disuse osteoporosis.

[0100] The researchers used the bone-targeting material (DSS) 6-liposome to deliver si-Setdb1 to the bone formation area of ​​mice and found that it led to impaired new bone formation, bone microarchitecture destruction, and impaired biomechanical properties in mice, further establishing the diagnostic role of Sedtb1 in disuse osteoporosis.

[0101] The relationship between Sedtb1 and disuse osteoporosis has been studied in depth and systematically. Based on the above findings, Sedtb1 expression level can be used as a new biomarker to assist in the diagnosis of disuse osteoporosis and as a new target to combat disuse osteoporosis, with good prospects for translational medicine.

[0102] In summary, the research and development method of histone methyltransferase Setdb1 in osteoporosis discovered the changes of histone methyltransferase Sedtb1 in disuse osteoporosis, explored its effects on osteoblast proliferation function and bone formation, suggesting that it may serve as a potential biomarker for disuse osteoporosis and can be used as a new target to combat disuse osteoporosis, with good prospects for translational medicine.

[0103] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of histone methyltransferase Setdb1 in preparing a preparation for diagnosing or treating disuse osteoporosis, characterized in that: The specific steps include: S1. Study the diagnostic application of Setdb1 in disuse osteoporosis: Determine the protein expression of Setdb1 by testing protein samples in MC3T3-E1 cells after unloading on a 2D rotator and in the femurs of tail-suspended mice. S2. Study the effect of Setdb1 on osteoblast proliferation: Setdb1 overexpression vector pcDNA3.1-Setdb1 and siRNA-Setdb1 were transfected into MC3T3-E1 cells, and their osteoblast proliferation capacity was detected; S3. Study the effect of bone-targeted delivery of si-Setdb1 on bone formation in mice; S4. Study the effect of Setdb1 overexpression on the proliferation of MC3T3-E1 cells after unloading of the 2D gyrator; In the S2, the siRNA-Setdb1 sequence is: sense: 5'- CACUCAGUCAGAGCUUUAUTT -3'; antisense: 5'- AUAAAGCUCUGACUGAGUGTT -3'; In S3, the effect of bone-targeted si-Setdb1 on bone formation in mice was investigated. The specific steps were as follows: S31. si-Setdb1 was delivered to the bone formation area of ​​mice via the bone-targeting material (DSS) 6-liposome, and the microstructure of the mouse femur was examined using micro-CT staining. S32, Goldner staining was used to detect new bone formation; S33, calcein double labeling experiment to detect the growth rate of cortical bone; S34, three-point bending test to detect the mechanical properties of mouse hind limb femur; In S4, the effect of overexpression of Setdb1 on the proliferation function of MC3T3-E1 cells was studied, which specifically included the following steps: The Setdb1 overexpression vector pcDNA3.1-Setdb1 was transfected into MC3T3-E1 cells, and the transfected cells were placed in a 2D rotator to remove the load for 48 h; The expression of PCNA protein was detected by CCK-8, EdU and Western blotting to determine its osteogenic proliferation ability.

2. Use of the histone methyltransferase Setdb1 according to claim 1 in preparing a preparation for diagnosing or treating disuse osteoporosis, characterized in that: In S1 and S4, mouse MC3T3-E1 cells were obtained by treating mouse preosteoblasts MC3T3-E1 cells in a 2D rotator.

3. Use of the histone methyltransferase Setdb1 according to claim 1 in preparing a preparation for diagnosing or treating disuse osteoporosis, characterized in that: In S1, the hind limb femurs of tail-suspended mice need to be pre-treated. The treatment method is as follows: the tails of C57BL / 6J male mice are suspended for 21 days, with the hind limbs suspended in the air so that the axis of the mouse body is at 30 degrees to the ground and the forelimbs of the mouse touch the ground, so that the mouse can move freely and has access to food and water, and the animal is subjected to horizontal unloading treatment; Western blotting was used to detect protein samples extracted from mouse MC3T3-E1 cells and hind limb femurs of tail-suspended mice.

4. Use of the histone methyltransferase Setdb1 according to claim 1 in preparing a preparation for diagnosing or treating disuse osteoporosis, characterized in that: In S1, the femurs of the hind limbs of the tail-suspended mice were also processed by immunohistochemical staining to determine the expression of Setdb1 therein.

5. Use of the histone methyltransferase Setdb1 according to claim 1 in preparing a preparation for diagnosing or treating disuse osteoporosis, characterized in that: In S2, CCK-8, EdU and Western blotting were used to detect PCNA protein expression and osteoblast proliferation ability.

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