CN109554455B - Application of CMKLR1 gene in diagnosing or treating bone growth or bone abnormal increasing diseases - Google Patents
Application of CMKLR1 gene in diagnosing or treating bone growth or bone abnormal increasing diseases Download PDFInfo
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- CN109554455B CN109554455B CN201710865652.5A CN201710865652A CN109554455B CN 109554455 B CN109554455 B CN 109554455B CN 201710865652 A CN201710865652 A CN 201710865652A CN 109554455 B CN109554455 B CN 109554455B
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Abstract
The invention relates to an application of CMKLR1 gene in diagnosing or treating bone metabolism related diseases, and particularly discloses an application of CMKLR1 gene or CMKLR1 protein as a drug target in screening and/or preparing a drug for preventing and/or treating bone metabolism diseases, and an application of an agonist of CMKLR1 in preparing a drug for preventing and/or treating bone metabolism diseases.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of a CMKLR1 gene in diagnosis or treatment of diseases with bone growth or abnormal increase of bone mass.
Background
Osteoporosis is a congenital disease characterized by high bone density, and it has long been thought to be caused by bone resorption disorders due to defective osteoclast numbers or functions. However, it has recently been found that osteoblast function is abnormally enhanced in patients with osteoporosis, resulting in increased bone mass and increased bone fragility in patients. According to the difference of clinical manifestations and pathogenic genes, the bone disease can be divided into autosomal dominant hereditary scleroderma (ADO), autosomal recessive hereditary scleroderma (ARO) and rare X-chromosome linked recessive hereditary scleroderma (XLO). Based on the treatment difficulty and the unclear pathogenesis, a treatment scheme of symptomatic treatment by combined administration is usually adopted, such as treating anemia by prednisone and treating bone injury by channels-relaxing and blood-activating tablets, but the treatment effect is poor, the toxic and side effects are large, and an accurate treatment means aiming at a targeted gene is almost absent. CMKLR1(chemokine receptor-1), one of the receptors for the adipokine Chemerin, belongs to the family of G protein-coupled receptors. The invention utilizes the gene knockout technology or the specific antagonist to interfere the action of CMKLR1 receptor, can find that the bone density of mice is reduced, and can inhibit the differentiation of mesenchymal stem cells to osteoblasts and promote the generation of osteoclasts in bone marrow. Aiming at the new discovery that the CMKLR1 signal channel regulates bone marrow osteoblasts, osteoclasts and bone mass, the novel mechanism of the novel target gene CMKLR1 in regulating the bone mass of mice is verified, and a novel target is provided for developing and treating the osteoporosis. Although osteoporosis is a rare type of hereditary bone disease, the average incidence of ADO is about 5/100000, the average incidence of ARO is about 1/250000, and XLO is even more rare, based on its epidemiological data. The skeletal deformity and the possible multi-system involvement that result from it all cause a reduction in quality of life and potential psychological problems for the patient, and even the heavier may be life threatening. The clinical characteristics of the osteoporosis are that the clinical manifestations of the osteoporosis are widely heterogeneous, and some patients show fatal clinical features, such as anemia, pancytopenia, sepsis, secondary hepatosplenomegaly and the like. However, some patients may be asymptomatic or milder, and may only be detected by imaging examination of the bone.
The bone maintains its integrity throughout its life through bone remodeling, which is divided into four phases of resorption, reversal, formation and quiescence, which are mainly dependent on the action of osteoclasts and osteoblasts. The osteoclast is derived from mononuclear macrophage, and is formed into osteoclast with absorption activity through a series of proliferation and fusion, and the osteoclast is adhered to the surface of bone to perform bone absorption. After the bone absorption of osteoclast is completed, the osteoclast is dropped from the bone surface to die, and the bone is rebuilt into the reverse phase. When osteoclasts are exfoliated, mesenchymal stem cells in bone marrow differentiate into osteoblasts, which mainly function to synthesize osteoid and promote bone mineralization by expressing some special proteins such as alkaline phosphatase, thereby exerting bone formation effect. Abnormal bone disease can be caused once the balance of osteoclasts and osteoblasts is disrupted. Bone resorption dysfunction of osteoclasts, for example, causes osteoporosis. The causative genes associated with osteoporosis that have been discovered to date are a3V-ATPase (classical ARO), CLCN7 (ADO-II, IRO, neurogenic ARO), OSTM1 (neurogenic ARO), PLEKHM1(IRO), CA II (RTA), RANKL (ARO), RANK (ARO), NEMO (XLO), CTSK (Pycnodysostosis). The bone mass of the Pten knockout mouse is progressively increased, the detection of 4-month-old mice shows that the bone density (BMD) of the mice is obviously increased no matter in males or in females, and X-ray films and micro CT three-dimensional reconstruction show that the Pten knockout mouse has the occurrence of the osteopetrosis. At present, gene therapy means for the osteoporosis are insufficient, and a method for treating the osteoporosis is urgently needed to be developed.
The maintenance of bone mass in healthy humans requires a well-coordinated balance between bone formation, osteoblasts derived from Mesenchymal Stem Cells (MSCs) of bone marrow, and bone resorption, osteoclasts differentiated from bone marrow mononuclear cells. These mesenchymal stem cells belong to the multipotent stem cells, and can also differentiate into adipocytes, which do not directly act on bone formation, but can affect bone remodeling by releasing signal molecules. Chemokine (Chemerin) is a novel adipokine that promotes adipocyte differentiation, is mainly secreted from white adipose tissue, and is also abundantly expressed in bone marrow. The research finds that Chemerin and a receptor CMKLR1 thereof play a determinant role in the differentiation process of the preosteoblast 7F2 and the mesenchymal stem cells into osteoblasts or adipocytes, and the expression and secretion of Chemerin promote the differentiation of the cells into the adipocytes. Blockade of the Chemerin signaling pathway will inhibit differentiation of hematopoietic stem cells into osteoclasts, thus revealing a novel function of Chemerin. Both MSCs and MSC-differentiated cell types (e.g. osteoblasts and adipocytes) can express and secrete Chemerin at higher levels than hematopoietic stem cells, whereas the CMKLR1 receptor is expressed at higher levels in hematopoietic stem cells. Therefore, it is not clear that Chemerin may help to mediate the interaction between osteoblasts and osteoclasts, promote the self-balance between bone formation/bone resorption, and that Chemerin high-level secretion is related to the number of mesenchymal cells (e.g., adipocytes) in bone marrow, thereby causing bone metabolism-related diseases.
It is worth mentioning that silencing the gene CMKLR1 by RNAi technology has been found in recent years to inhibit adipocyte differentiation, clonal expansion and proliferation of mesenchymal stem cells. In contrast, the gene silencing of CMKLR1 reduces the expression of osteoblast bone-related active genes and inhibits mineralization, and the new discovery that CMKLR1, as a Wnt-targeted gene, regulates the differentiation of mesenchymal stem cells suggests that the Chemerin/CMKLR1 signaling pathway may be related to the function of mesenchymal stem cells, and also suggests that if the function of CMKLR1 is affected, the occurrence or development of osteoporosis is also affected, so far, there is no direct evidence that CMKLR1 is related to osteoporosis.
Disclosure of Invention
One aspect of the invention provides an application of CMKLR1 gene or CMKLR1 protein as a drug target in screening drugs for treating or preventing bone metabolic diseases.
In another aspect, the invention provides a CMKLR1 gene or CMKLR1 protein for use as a therapeutic target for preventing and/or treating bone metabolic diseases, or as a diagnostic target for bone metabolic diseases.
In the technical scheme of the invention, the bone metabolism disease is related diseases of abnormal increase of bone growth or bone quality, abnormal increase of osteoblast number or activity and/or abnormal decrease of osteoclast number or activity, and related diseases caused by enhancement of bone differentiation activity of bone marrow mesenchymal stem cells and/or reduction of bone marrow mononuclear cell osteoclast differentiation activity.
Wherein the bone metabolic disease is osteopetrosis.
Another aspect of the invention provides an animal model for mimicking a disease associated with abnormal reduction of bone growth or bone mass, wherein the CMKLR1 gene of the animal model is knocked out without other treatment (e.g., testicular resection).
In another aspect of the invention, there is provided a method for preparing an animal model for simulating a disease associated with abnormal reduction of bone growth or bone mass, wherein only the CMKLR1 gene of the animal is knocked out without other treatment (e.g., testicular resection treatment).
Another aspect of the invention provides an animal model in which the CMKLR1 gene has been knocked out as an animal model for studying diseases associated with abnormal reduction in bone growth or bone mass. The disease associated with abnormal reduction of growth or bone mass is selected from the group consisting of osteoporosis, Paget's disease (Paget), osteogenesis imperfecta, dysplasia of bone fibers, hypophosphatasia, and humoral hypercalcemia of malignancy, and multiple myeloma-induced osteolysis.
In another aspect of the invention, the invention provides an application of an inhibitor of CMKLR1 gene or CMKLR1 protein in preparing a medicament for preventing or treating bone metabolic diseases.
Another aspect of the invention provides a use of an inhibitor of CMKLR1 gene or CMKLR1 protein for preventing or treating bone metabolic diseases.
Wherein the inhibitor of CMKLR1 gene or CMKLR1 protein is selected from alpha-NETA, CMKLR1 antibody, CMKLR1 antagonistic polypeptide, CMKLR1shRNA, CMKLR1siRNA and CMKLR1 miRNA.
The invention provides a use of CMKLR1 gene or CMKLR1 protein in regulating differentiation of osteoblasts and osteoclasts in bone marrow cells, wherein the defect of the CMKLR1 gene causes reduction of differentiation of osteoblasts and increase of differentiation of osteoclasts in the bone marrow cells, and the excitation of the CMKLR1 gene causes reduction of differentiation of osteoblasts and osteoclasts in the bone marrow cells.
In the invention, CMKLR1(chemokine receptor-1) is a chemokine-like receptor-1, which is one of the receptors of the adipokine Chemerin and belongs to the G protein coupled receptor family.
In the present invention, a bone metabolic disease refers to abnormal growth, development, repair, absorption, degradation or homeostasis-related disorder of bone tissue; further, the present invention relates to a disease associated with an abnormal increase in bone growth or bone mass, an abnormal increase in the number or activity of osteoblasts and/or an abnormal decrease in the number or activity of osteoclasts, and a disease associated with a decreased osteogenic differentiation activity of mesenchymal stem cells and/or an increased osteoclastic differentiation activity of bone marrow mononuclear cells.
In the present invention, antibodies are in their broadest sense and include, for example, but are not limited to, whole antibodies as well as single chain antibodies, antibody fragments and chimeric antibodies.
The gene of the invention is a known gene in the prior art and is a unique sequence, GeneID:14747 at NCBI. The specific sequence is shown as SEQ ID No. 1.
ATGGAGTACGACGCTTACAACGACTCCGGCATCTATGATGATGAGTACTCTGATGGCTTTGGCTACTTTGTGGACTTGGAGGAGGCGAGTCCGTGGGAGGCCAAGGTGGCCCCGGTCTTCCTGGTGGTGATCTACAGCTTGGTGTGCTTCCTCGGTCTCCTAGGCAACGGCCTGGTGATTGTCATCGCCACCTTCAAGATGAAGAAGACCGTGAACACTGTGTGGTTTGTCAACCTGGCTGTGGCCGACTTCCTGTTCAACATCTTTTTGCCGATGCACATCACCTACGCGGCCATGGACTACCACTGGGTGTTCGGGAAGGCCATGTGCAAGATCAGCAACTTCTTGCTCAGCCACAACATGTACACCAGCGTCTTCCTGCTGACTGTCATCAGCTTTGACCGCTGCATCTCCGTGCTGCTCCCCGTCTGGTCCCAGAACCACCGCAGCATCCGCCTGGCCTACATGACCTGCTCGGCCGTCTGGGTCCTGGCTTTCTTCTTGAGCTCCCCGTCCCTTGTCTTCCGGGACACCGCCAACATTCATGGGAAGATAACCTGCTTCAACAACTTCAGCTTGGCCGCGCCTGAGTCCTCCCCACATCCCGCCCACTCGCAAGTAGTTTCCACAGGGTACAGCAGACACGTGGCGGTCACTGTCACCCGCTTCCTTTGCGGCTTCCTGATCCCCGTCTTCATCATCACGGCCTGCTACCTTACCATCGTCTTCAAGCTGCAGCGCAACCGCCTGGCCAAGAACAAGAAGCCCTTCAAGATCATTATCACCATCATCATCACCTTCTTCCTCTGCTGGTGCCCCTACCACACCCTCTACCTGCTGGAGCTCCACCACACAGCTGTGCCAAGCTCTGTCTTCAGCCTGGGGCTACCCCTGGCCACGGCCGTCGCCATCGCCAACAGCTGCATGAACCCCATTCTGTACGTCTTCATGGGCCACGACTTCAGAAAATTCAAGGTGGCCCTCTTCTCCCGCCTGGCCAACGCCCTGAGTGAGGACACAGGCCCCTCCTCCTACCCCAGTCACAGGAGCTTCACCAAGATGTCGTCTTTGAATGAGAAGGCTTCGGTGAATGAGAAGGAGACCAGTACCCTCTGA SEQ ID No.1
Advantageous effects
The invention proves the connection between the CMKLR1 gene and metabolic bone disease for the first time, and provides basis for the prevention, diagnosis and treatment of the disease.
Drawings
FIG. 1 is a graph of the results of micro CT scan analysis of femur and tibia of wild type (CMKLR1+/+) and CMKLR1 knockout mice (CMKLR 1-/-). Wherein A is a micro CT scanning data 3D reconstruction diagram for showing the distribution condition of cancellous bone trabeculae under the distal femur and proximal tibia growth plate areas; b is a bone parameter index comparing femurs of wild type and CMKLR1 knockout mice: bone density (BMD), bone volume score (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th); c is a bone parameter index comparing tibia of wild-type and CMKLR1 knockout mice: bone density (BMD), bone mass fraction (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th). Suitable for bone marrow, bone.
FIG. 2 is a graph showing the distribution of trabecular and osteoclast cells in wild type (CMKLR1+/+) and CMKLR1 knock-out mice (CMKLR 1-/-) femoral paraffin sections. Wherein A is a staining graph of HE and TRAP of the bone slice, B is a statistical condition of the number of osteoclasts in the bone slice, B is a statistical condition of the number of osteoblasts in the bone slice, C is a statistical condition of the number of osteoblasts in the bone slice, and P is less than 0.05.
FIG. 3 is a graph showing the results of osteogenic-adipogenic differentiation of bone marrow mesenchymal stem cells and osteoclastic differentiation of monocytes in wild type (CMKLR1+/+) and CMKLR1 knock-out mice (CMKLR 1-/-). Wherein A is a calcium nodule map of differentiation of alizarin red staining mesenchymal stem cells into osteoblasts, B is a comparison osteoblast mineralization condition, and C fluorescence real-time quantitative detection is performed on expression changes of osteogenic differentiation marker genes ALP, Runx2 and Osterix of the mesenchymal stem cells; d is an oil red O staining mesenchymal stem cell differentiation into adipocyte lipid drop diagram, E is a comparison adipocyte number condition, F is the change of fluorescence real-time quantitative detection mesenchymal stem cell adipogenic differentiation marker gene Fabp4, PPAR gamma and Adiponectin expression; g is TRAP staining osteoclast map, H is comparing osteoclast number, I is fluorescence real-time quantitative determination monocyte osteoclast differentiation marker gene TRAP, Cathepsin K and NFATc1 expression change, P < 0.05.
FIG. 4 shows the CMKLR1 antagonist α -NETA acting on bone formation in wild type mice. Wherein A is a micro CT scanning data 3D reconstruction diagram showing the distribution of cancellous bone trabeculae under the distal growth plate area of mouse thighbone of a control group and a CMKLR1 antagonist group, and B is a bone parameter index for comparing mouse thighbone: bone density (BMD), number of bone body integrals (BV/TV) and number of trabeculae (Tb.N). P is less than 0.05.
Detailed Description
The invention will be described in more detail below with reference to examples and the accompanying drawings, but the scope of the invention is not limited thereto.
Example 1 Collection of bone samples from mice
CMKLR1 knockout mice (CMKLR 1-/-) on C57BL/6J background were purchased from Deltagen, Inc., USA and supplied by the doctor Zable laboratory, Stanford university. C57BL/6J wild-type mice (CMKLR1+/+) from the Experimental animals center of Shenzhen advanced technology research institute, academy of sciences, China. These mice were housed in a constant temperature, humidity pathogen free (SPF) environment with a 12 hour light and dark cycle, and were allowed to eat and drink autonomously at all times. The welfare and the experimental procedures of the mice strictly follow the regulations of the animal operating rules of the experimental animals and the animal committee of Shenzhen advanced technology research institute of Chinese academy of sciences on the animal ethics. After the mice are sacrificed at the neck, the right femur and tibia are fixed by 70% ethanol for CT analysis, and the left femur is fixed by 4% PFA and then is decalcified and embedded by paraffin.
Example 2 mouse bone CT scan analysis
The bone of the mice was scanned using SkyScan1176MicroCT from BRUKER and the bone position was fixed using a foam plate. CT scans were performed using a voltage of 60kV, a current of 425 μ A, and a resolution of 9 μm. And after the scanning reconstruction is completed, performing structural analysis on the metaphyseal trabecula. And continuously discarding 50 layers from the disappearance position of the distal femur or proximal tibia metaphysis growth plate, selecting 200 layers as a trabecular bone reconstruction region for analysis, and reconstructing by using micro CT (micro computed tomography) self-contained software, wherein the threshold value is 80. The main analysis indexes comprise four indexes of bone density (BMD), bone body integral number (BV/TV), trabecular bone number (Tb.N) and trabecular bone thickness (Tb.Th).
Example 3 staining of bone sections H & E (hepatoxin-eosin) and TRAP (tartrate-resistant acid phosphatase)
Bone sections were H & E stained using the BOSTER biosystems BOSTER H & E staining kit (cat # AR1180) according to the kit instructions:
paraffin is sliced and dewaxed to water conventionally.
② hematoxylin is dripped to cover the specimen and is stained for 3 min.
Thirdly, washing the hematoxylin clean by tap water.
And fourthly, immersing the slices into a bluing solution for 60 seconds, and washing the slices with tap water.
Fifthly, immersing the slices in 95% alcohol for 30s, and directly dropwise adding eosin for dyeing for 1 min.
Sixthly, washing with tap water.
And seventhly, dehydrating, transparent, sealing and photographing.
Bone sections were TRAP stained using Takara TRAP staining kit (cat No. MK300) according to the kit instructions:
paraffin is sliced and dewaxed to water conventionally.
② dripping substrate reaction liquid to cover the specimen, and incubating for 30min at 37 ℃.
And thirdly, washing the reaction solution by distilled water to be clean, and terminating the reaction.
And fourthly, washing the mixture for 3 times with distilled water, and each time lasts for 5 min.
And fifthly, directly dripping glycerin sealing sheet and taking a picture.
Example 4 isolation and Induction of Mesenchymal Stem Cells (MSCs)
Soaking the mouse in 70% alcohol for 5min, aseptically dissecting in a super clean bench, taking out the long bone of the mouse, carefully shaving off the muscle on the long bone with forceps and sharp-pointed scissors to expose the growth plate of the long bone.
Cutting off the connection part of the growth plate and the backbone part to expose the marrow cavity at the two ends of the backbone.
③ the backbone of the long bone was placed in a10 cm dish containing 1.5mL of α -MEM medium, and the α -MEM medium was aspirated by a 1mL syringe to flush the bone marrow from the backbone.
Fourthly, the flushed cells are cultured for 3 days by using a complete culture medium (alpha-MEM, 10 percent FBS and 1 percent penicillin-streptomycin double antibody), and the liquid is changed once after the cells are completely attached to the wall.
Fifthly, continuing to culture for 7 days, after the clone is completely formed, washing with PBS for 3 times, digesting with 0.25% pancreatin for 2min, collecting cells and continuing to culture to obtain MSCs.
Sixthly, replacing the complete culture medium every 3 days, digesting with 0.25 percent of pancreatic enzyme when the cells are approximately 70 to 80 percent confluent, carrying out passage according to the proportion of 1: 3, and taking the 4 th to 6 th generation cells for experiment.
⑦ at 5 × 10 per hole4Cell graftingWhen MSCs grow to about 80% of the culture dish area, the complete culture medium is replaced by osteogenic induction medium (Gibco, Cat. No. A10072-01) or adipogenic induction medium (Gibco, Cat. No. A10070-01), and the induction medium is replaced every 3 days. Extracting cell RNA to identify the gene expression condition of osteogenic or adipogenic marker, staining alizarin red cells to calcify nodules, and staining oil red O to identify the formation of fat cells and the distribution of fat droplets.
Example 5 isolation of bone marrow mononuclear cells and Induction of osteoclast differentiation
Soaking the mouse in 70% alcohol for 5min, aseptically dissecting in a super clean bench, taking out the long bone of the mouse, carefully shaving off the muscle on the long bone with forceps and sharp-pointed scissors to expose the growth plate of the long bone.
Cutting off the connection part of the growth plate and the backbone part to expose the marrow cavity at the two ends of the backbone.
③ the backbone of the long bone was placed in a10 cm dish containing 1.5mL of α -MEM medium, and the α -MEM medium was aspirated by a 1mL syringe to flush the bone marrow from the backbone.
Fourthly, the flushed bone marrow cells are centrifuged at 1200rpm for 5min and collected, 0.5mL of erythrocyte lysate ACKLysing Buffer (Invitrogen, cat # A1049201) is added, and the reaction is carried out for 2min at room temperature.
Fifthly, adding a complete culture medium (alpha-MEM + 10% FBS + 1% penicillin-streptomycin double antibody) to terminate the lysis reaction, centrifuging at 1200rpm for 5min, and collecting cells.
Sixthly, inoculating the cells into a10 cm culture dish, adding a complete culture medium containing 10ng/mL M-CSF, and continuously culturing the cells.
Seventhly, after 24 hours of culture, removing suspended cells, washing with PBS 3 times, then blowing and beating the cells with 1mL of complete culture medium, collecting adherent cells to obtain bone marrow mononuclear cells, inoculating 1 × 104 cells per hole into a 24-hole plate, adding complete culture medium containing 10ng/mL M-CSF and 50ng/mL RANKL, changing the culture medium once every day, and inducing osteoclast differentiation for 9 days.
EXAMPLE 6 test of the Effect of CMKLR1 antagonists on the treatment of osteoporosis
Control group (control group): c57BL/6J wild type mice 8 weeks old are implanted with a slow release pump air pump (ALZET, MODEL 1004) subcutaneously for a total of 28 days;
② CMKLR1 antagonist group (α -NETA group): an 8-week-old C57BL/6J wild-type mouse was subcutaneously implanted with a 1mg CMKLR1 antagonist α -NETA (abcam, cat # ab144314) sustained release pump for 28 days with a daily release of 33 micrograms.
Mice were sacrificed by cervical dislocation at the end of the 28 day experiment and bone from the mice was fixed in 70% ethanol for CT analysis. The bone of the mice was scanned using SkyScan1176MicroCT from BRUKER and the bone position was fixed using a foam plate. CT scans were performed using a voltage of 60kV, a current of 425 μ A, and a resolution of 9 μm. And after the scanning reconstruction is completed, performing structural analysis on the metaphyseal trabecula. And continuously discarding 50 layers from the disappearance position of the distal femur or proximal tibia metaphysis growth plate, selecting 200 layers as a trabecular bone reconstruction region for analysis, and reconstructing by using micro CT (micro computed tomography) self-contained software, wherein the threshold value is 80. The main analysis indexes comprise three indexes of bone density (BMD), bone body integral number (BV/TV) and trabecular bone number (Tb.N).
Results
[ problem ] deletion of the CMKLR1 Gene results in decreased bone density in mice
The Micro CT3D reconstruction plot showed significant trabecular reduction of the CMKLR1 knockout mice in the cancellous bone region distal to the femur and proximal to the tibia, as compared to wild type mice (fig. 1A). Subsequently, the inventor analyzes the bone parameter data by Micro CT self-contained software CT analyzer & CT-volume, and the bone density BMD, bone trabecular bone volume fraction (BV/TV), trabecular bone thickness (tb.th) and trabecular bone number (tb.n) of the CMKLR1 knockout mouse are all significantly reduced in the distal femur (fig. 1B) and proximal tibia (fig. 1C) of the mouse, which is consistent with the 3D reconstruction map, and further proves that the CMKLR1 knockout mouse has a bone density reduction phenomenon, which is the first in vivo number reported in the world for CMKLR1 mediated mouse bone formation. To study the structural changes occurring in the bones of mice, the inventors decalcified the femurs of the mice, then sectioned in paraffin-embedded, HE-stained to clearly observe the structural changes of the trabecular bone of the mice, and fig. 2A shows that the number of trabecular bone was significantly reduced in the far cancellous bone region of the femurs of CMKLR1 knockout mice. Osteogenesis was also associated with osteoclasts, and then distribution of osteoclasts in trabeculae was analyzed by TRAP staining, and FIGS. 2B and 2C showed that osteoclasts were significantly increased in femurs of CMKLR1 knock-out mice, interestingly, there was no change in the number of osteoblasts, and thus, it was confirmed that osteoblast activity was decreased.
② the deletion of CMKLR1 gene breaks the differentiation balance between osteoblast and osteoclast in bone marrow
The present inventors flushed bone marrow from the bone marrow cavity of wild-type (CMKLR1+/+) and CMKLR1 knock-out (CMKLR 1-/-) mice to obtain Mesenchymal Stem Cells (MSCs) and monocytes, respectively, wherein the MSCs induced adipogenic osteogenic differentiation and the myelomonocytes induced osteoclastic differentiation. FIG. 3 shows that MSCs derived from CMKLR1 knockout mice have increased adipogenic differentiation and decreased osteogenic differentiation, and osteoclast differentiation induced by bone marrow mononuclear cells has also increased significantly, which indicates that CMKLR1 gene deletion causes bone marrow mesenchymal stem cells to have decreased osteogenic differentiation activity and bone marrow mononuclear cells to have increased osteoclast differentiation activity, and that in vitro experiments and in vivo experiments are consistent. Further intensive research shows that the total RNA of the cells is extracted by the inventor, and the fluorescent real-time quantitative detection result shows that the expressions of osteogenic marker genes ALP, Runx2 and Osterix are obviously reduced, which indicates that the osteogenic activity is reduced, and the expressions of lipid marker genes Fabp4, PPAR gamma and adiponectin are enhanced. The significantly increased expression of the osteoclastarker genes Trap, Cathepsin K and NFATc1 indicates increased osteoclast activity. In conclusion, the CMKLR1 gene deletion causes the reduction of the bone density of the mice and mainly aims at the bone marrow mesenchymal stem cells and the mononuclear cells to exert the bone reduction function.
③ antagonizing CMKLR1 to cause bone loss of experimental mice
The inventors implanted a slow-release pump containing 1mg of the CMKLR1 antagonist α -NETA subcutaneously into 8-week-old wild-type mice and investigated whether antagonism of the effect of CMKLR1 resulted in decreased bone density in the mice. FIG. 4 shows that the Micro CT3D reconstructed image shows that the CMKLR1 antagonist group (alpha-NETA group) mice have a significant trabecular reduction in the distal cancellous bone region of the femur compared to the control group. Meanwhile, the bone density BMD, the bone volume fraction (BV/TV) of the trabecular bone and the number (Tb.N) of the trabecular bone of the alpha-NETA group of mice are all remarkably reduced, and the further demonstration that the antagonism of the CMKLR1 gene can inhibit the bone formation of the mice, and finally the phenomenon of bone density reduction of the mice is caused, namely the demonstration that the CMKLR1 gene can be used for treating or preventing the osteopetrosis.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various conditions and applications.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute
Application of <120> CMKLR1 gene in diagnosis or treatment of diseases with increased bone growth or bone abnormality
<160>1
<170>PatentIn version 3.3
<210>1
<211>1116
<212>DNA
<213> CMKLR1 Gene
<400>1
atggagtacg acgcttacaa cgactccggc atctatgatg atgagtactc tgatggcttt 60
ggctactttg tggacttgga ggaggcgagt ccgtgggagg ccaaggtggc cccggtcttc 120
ctggtggtga tctacagctt ggtgtgcttc ctcggtctcc taggcaacgg cctggtgatt 180
gtcatcgcca ccttcaagat gaagaagacc gtgaacactg tgtggtttgt caacctggct 240
gtggccgact tcctgttcaa catctttttg ccgatgcaca tcacctacgc ggccatggac 300
taccactggg tgttcgggaa ggccatgtgc aagatcagca acttcttgct cagccacaac 360
atgtacacca gcgtcttcct gctgactgtc atcagctttg accgctgcat ctccgtgctg 420
ctccccgtct ggtcccagaa ccaccgcagc atccgcctgg cctacatgac ctgctcggcc 480
gtctgggtcc tggctttctt cttgagctcc ccgtcccttg tcttccggga caccgccaac 540
attcatggga agataacctg cttcaacaac ttcagcttgg ccgcgcctga gtcctcccca 600
catcccgccc actcgcaagt agtttccaca gggtacagca gacacgtggc ggtcactgtc 660
acccgcttcc tttgcggctt cctgatcccc gtcttcatca tcacggcctg ctaccttacc 720
atcgtcttca agctgcagcg caaccgcctg gccaagaaca agaagccctt caagatcatt 780
atcaccatca tcatcacctt cttcctctgc tggtgcccct accacaccct ctacctgctg 840
gagctccacc acacagctgt gccaagctct gtcttcagcc tggggctacc cctggccacg 900
gccgtcgcca tcgccaacag ctgcatgaac cccattctgt acgtcttcat gggccacgac 960
ttcagaaaat tcaaggtggc cctcttctcc cgcctggcca acgccctgag tgaggacaca 1020
ggcccctcct cctaccccag tcacaggagc ttcaccaaga tgtcgtcttt gaatgagaag 1080
gcttcggtga atgagaagga gaccagtacc ctctga 1116
Claims (5)
1. Use of a CMKLR1 gene or CMKLR1 protein in screening for a drug target for treating or preventing bone metabolic diseases;
the bone metabolism disease is related diseases caused by abnormal reduction of osteoclast number or activity, or related diseases caused by weakening of bone marrow monocyte osteoclast differentiation activity.
2. The use according to claim 1, wherein the bone metabolic disease is osteopetrosis.
3. Use of an inhibitor of the CMKLR1 gene or CMKLR1 protein for the preparation of a medicament for the prevention or treatment of a bone metabolism disorder; the bone metabolic disease is abnormal increase of bone growth or bone quality;
and the bone metabolism disease is related diseases caused by abnormal reduction of osteoclast number or activity or related diseases caused by reduction of bone marrow monocyte osteoclast differentiation activity.
4. The use according to claim 3, wherein the inhibitor of the CMKLR1 gene or CMKLR1 protein is selected from the group consisting of alpha-NETA, CMKLR1 antibody, CMKLR1 antagonist polypeptide, CMKLR1shRNA, CMKLR1 siRNA.
5. The use according to any one of claims 3 to 4, wherein the bone metabolic disease is osteopetrosis.
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| CN201710865652.5A CN109554455B (en) | 2017-09-22 | 2017-09-22 | Application of CMKLR1 gene in diagnosing or treating bone growth or bone abnormal increasing diseases |
| PCT/CN2017/116598 WO2019056609A1 (en) | 2017-09-22 | 2017-12-15 | Application of cmklr1 gene in diagnosis or treatment of bone growth or hyperostosis diseases |
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| CN201710865652.5A CN109554455B (en) | 2017-09-22 | 2017-09-22 | Application of CMKLR1 gene in diagnosing or treating bone growth or bone abnormal increasing diseases |
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| CN109554455B true CN109554455B (en) | 2020-09-25 |
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| Chemokine-Like Receptor 1 Is a Novel Wnt Target Gene that Regulates Mesenchymal Stem Cell Differentiation;SHANMUGAM MURUGANANDAN;《STEM CELLS》;20161012;第35卷(第3期);第711页摘要,第718页右栏倒数第1-3行，719页左栏第1行 * |
| Chemokine-Like Receptor 1 信号通路对双氢睾酮诱导小鼠骨密度增加的影响;姜晓彤等;《集成技术》;20170331;第6卷(第2期);第37页左栏最后1行、右栏第1行、图3 * |
| 姜晓彤等.Chemokine-Like Receptor 1 信号通路对双氢睾酮诱导小鼠骨密度增加的影响.《集成技术》.2017,第6卷(第2期),第32-40页. * |
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