CN110596406A - TMEM16A as osteoporosis marker, application thereof, osteoporosis diagnosis kit and medicament - Google Patents
TMEM16A as osteoporosis marker, application thereof, osteoporosis diagnosis kit and medicament Download PDFInfo
- Publication number
- CN110596406A CN110596406A CN201911041023.6A CN201911041023A CN110596406A CN 110596406 A CN110596406 A CN 110596406A CN 201911041023 A CN201911041023 A CN 201911041023A CN 110596406 A CN110596406 A CN 110596406A
- Authority
- CN
- China
- Prior art keywords
- osteoporosis
- tmem16a
- medicament
- content
- subject
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physical Education & Sports Medicine (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Urology & Nephrology (AREA)
- Animal Behavior & Ethology (AREA)
- Rheumatology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Epidemiology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a TMEM16A as an osteoporosis marker and application thereof, an osteoporosis diagnosis kit and a medicament, and relates to the technical field of disease markers. The osteoporosis diagnosis kit using TMEM16A as the osteoporosis marker judges whether the subject has osteoporosis or not by detecting the content of TMEM16A in peripheral blood mononuclear cells of the subject. The diagnosis is quick and accurate, the operation is simple, and special instruments and equipment are not needed. The medicine for treating osteoporosis takes TMEM16A as a target, and relieves the osteoporosis of patients by regulating the expression level of TMEM16A of the patients.
Description
Technical Field
The invention relates to the technical field of disease markers, in particular to a TMEM16A marker used as an osteoporosis marker, application thereof, an osteoporosis diagnosis kit and a medicament.
Background
Osteoporosis is a metabolic disease of the whole body bone, and is mainly characterized by a decrease in bone mass and a change in bone microstructure accompanied by a decrease in bone strength, an increase in bone fragility, an increase in fracture risk, and the like. According to epidemiological statistics, the prevalence rate of osteoporosis in people over 50 years old in China is 20.7% for women and 14.4% for men. And according to global epidemiological investigation, the prevalence of osteoporosis is high in all countries. The onset of osteoporosis is associated with a variety of factors, of which sex factors and age factors are the most relevant to the onset of osteoporosis. The pain and the fracture caused by the osteoporosis seriously affect the life quality of the patient and endanger the survival of the patient, and the existing treatment means has relatively limited effect on the osteoporosis. In the elderly women, after the pathological fracture is mostly followed by osteoporosis, the residual fatality rate caused by osteoporosis fracture is high, and the fatality rate of the osteoporosis fracture exceeds the sum of the fatality rates of the elderly women suffering from breast cancer, cervical cancer and uterine body cancer at present.
The current clinical methods for diagnosing osteoporosis mainly comprise: 1. the physician consults a past medical history which provides a high risk factor for osteoporosis, such as alcohol abuse, heavy smoking, pre-menopausal bilateral ovariectomy, etc., and then needs further examination. 2. Bone mass is measured based on the measurement of bone mineral content and bone density, but the occurrence of fractures and osteoporosis does not depend solely on bone mass. 3. Ultrasound bone determination measures the speed of ultrasound through bone tissue, amplitude attenuation and stiffness index reflecting bone structure and bone mass.
The existing methods for diagnosing osteoporosis mainly have the following defects: 1. the physician can only provide reference for inquiring the past medical history and can not accurately judge whether osteoporosis exists. 2. Bone mass measurement is a commonly used means at present, but whether fracture occurs or not is mainly determined by bone strength, and the bone strength is composed of various aspects such as bone mass, bone quality, bone composition and bone shape, so that the judgment by the bone mass alone is not accurate. 3. The result of ultrasonic measurement is not the bone density or the bone mineral content, and can not compare with the true value, needs more observation data, and the huge inconvenience of moving of ultrasonic equipment in addition is higher shortcoming such as the cost of each measurement. Therefore, an improved product for diagnosing osteoporosis is needed in the market.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide an osteoporosis diagnosis kit using TMEM16A as an osteoporosis marker, the diagnosis is quick and accurate, the operation is simple, and no special instrument or equipment is needed.
A second object of the present invention is to provide the use of TMEM16A in the manufacture of a medicament for the treatment of osteoporosis.
The third purpose of the invention is to provide a medicine for treating osteoporosis, which takes TMEM16A as a target point.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the present invention, there is provided an osteoporosis diagnosis kit using TMEM16A as a marker for osteoporosis, which judges whether a subject has osteoporosis by detecting the content of TMEM16A in peripheral blood mononuclear cells of the subject.
Preferably, the test sample of the osteoporosis diagnosis kit comprises peripheral blood mononuclear cells;
preferably, the test sample of the osteoporosis diagnosis kit includes osteoclast precursor cells.
Preferably, a subject is determined to have osteoporosis when the level of TMEM16A in the subject's peripheral blood mononuclear cells is elevated compared to normal.
Preferably, the subject is determined to have osteoporosis when the content of TMEM16A protein in peripheral blood mononuclear cells of the subject is at least 1.5 times the normal value.
According to another aspect of the present invention, there is also provided the use of TMEM16A as a marker for osteoporosis in the manufacture of a medicament for the treatment of osteoporosis.
According to another aspect of the present invention, there is also provided a medicament for the treatment of osteoporosis, said medicament targeting TMEM 16A.
Preferably, the drug is targeted to osteoclasts.
Preferably, the medicament contains an active ingredient that inhibits TMEM16A expression.
Preferably, the medicament has, as an active ingredient, small interfering rna (sirna) that inhibits TMEM16A gene expression.
Preferably, the siRNA for inhibiting TMEM16A gene expression comprises at least one of siRNA comprising a sequence shown in SEQ ID NO.1, siRNA comprising a sequence shown in SEQ ID NO.2 and siRNA comprising a sequence shown in SEQ ID NO. 3.
Compared with the prior art, the invention has the following beneficial effects:
the invention is based on comparing the content of TMEM16A in samples of osteoporosis patients and normal people, finds that the content of TMEM16A in samples of osteoporosis patients is different from the content of TMEM16A in normal people, indicates that TMEM16A can be used as a marker of osteoporosis, determines the increase of TMEM16A and the correlation of osteoporosis by observing the mouse model of osteoporosis and the content change of TMEM16A in osteoclast differentiation, simultaneously reduces the expression of TMEM16A gene in osteoclasts, shows that all the related genes for characterizing the differentiation of osteoclasts into bones are reduced to different degrees, and indicates that TMEM16A is the marker of osteoporosis. Based on the fact that TMEM16A is used as the osteoporosis marker, the invention provides an osteoporosis diagnosis kit and application in the fields of curative effect evaluation of osteoporosis treatment drugs and the like, the osteoporosis of a patient can be judged rapidly through detecting the expression level of peripheral blood mononuclear cells TMEM16A of the patient, diagnosis is rapid and accurate, operation is simple, and special instruments and equipment are not needed.
Based on the inventive concept of the content correlation between the osteoporosis and the TMEM16A, the medicine for treating the osteoporosis provided by the invention takes the TMEM16A as a target point, and the osteoporosis condition of a patient is relieved by regulating and controlling the expression amount of the TMEM16A of the patient.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the comparison between the expression level of TMEM16A in bone tissues of a normal human and an osteoporosis patient in example 1 of the present invention;
FIG. 2 is a graph showing the comparison between the expression level of TMEM16A in bone tissues of a normal mouse and a tail-suspended osteoporosis-induced mouse in example 2 of the present invention;
FIG. 3 is a graph showing the expression level of TMEM16A protein in osteoclasts after 1, 3 and 5 days of induction in example 3 of the present invention;
FIG. 4 is a statistical graph showing the expression level of TMEM16A protein in osteoclasts after 1, 3 and 5 days of induction in example 3 of the present invention;
FIG. 5 shows the expression level of osteoclast differentiation related gene after the siRNA down-regulates the expression of TMEM16A in example 4 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that:
in the present invention, all the embodiments and preferred methods mentioned herein can be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
Osteoporosis is a metabolic and systemic disease of the skeletal system that can destroy the micro-architecture of a patient's bone, increase the patient's bone fragility, decrease its bone strength, and thereby increase the risk of fracture. The basic mechanism of osteoporosis is that dynamic balance of bone reconstruction is destroyed, osteoblasts and osteoclasts are out of balance in function, so that bone metabolism is unbalanced, bone resorption is accelerated, bone formation is reduced, and finally bone resorption is far greater than bone formation, and net bone loss is caused. The signal paths related to bone metabolism comprise a BMPs signal path, an OPG/RANKL/RANK signal path, a TGF-beta signal path, a MAPK signal path, a Wnt/beta-catenin signal path, a PPAR-gamma signal path and a Hedgehog signal path.
TMEM16A belongs to the family of proteins with multiple transmembrane structures-TMEM 16 family, TMEM16A is a calcium activated chloride channel (CaCCs). The TMEM16A protein structure has 10 transmembrane domains, with both the nitrogen and carbon termini located intracellularly. Cells or tissues distributed by TMEM16A are now found in vascular epithelial cells, pancreatic epithelial cells, salivary gland epithelial cells, and the submandibular acini, mammary glands and renal tubules of the bronchial mucosa. The function of TMEM16A was associated with diarrhea, hypertension and various cancers, and no prior studies revealed that TMEM16A expression levels were associated with osteoporosis.
The invention measures the expression level of TMEM16A in osteoclast of osteoporosis patients and normal people, and finds that the expression level of TMEM16A in bone tissues of the osteoporosis patients is about 1.5 times higher than that of the normal people, so that TMEM16A is a marker of osteoporosis. Further, it was found from the osteoporosis mouse model that the content of TMEM16A in the bone tissue of the mice with osteoporosis was significantly increased, about 4 times higher than that of the normal mice, and thus it was further confirmed that TMEM16A is a marker of osteoporosis. When the osteoclast is induced and differentiated in vitro, the content of TMEM16A in the osteoclast after induction is found to be increased dramatically, and the content of TMEM16A in the osteoclast is increased by about 13 times in the fifth day of induction compared with the first day of induction, so that the content of TMEM16A is increased as an important mark for the differentiation of the osteoclast, and the higher the differentiation degree of the osteoclast is, the higher the possibility that the patient suffers from osteoporosis is increased. When TMEM16A is down-regulated by using a siRNA interference method, the related genes Nfatc1, Acp5, Ctsk, Mmp9 and Clcn7 which characterize osteoclast differentiation into bone are all reduced to different degrees, and further, the TMEM16A is a marker of osteoporosis.
Based on this, the present invention provides an osteoporosis diagnosis kit using TMEM16A as a marker of osteoporosis, which determines whether a subject has osteoporosis by detecting the content of TMEM16A in peripheral blood mononuclear cells of the subject. The invention finds the difference between the content of TMEM16A in the sample of the osteoporosis patient and the content of TMEM16A in the normal population by comparing the content of TMEM16A in the sample of the osteoporosis patient with the content of the normal population, indicates that the TMEM16A can be used as a marker of the osteoporosis, and determines that the increase of the TMEM16A is related to the osteoporosis by observing the content change of the TMEM16A in an osteoporosis mouse model and osteoclast differentiation. Therefore, an osteoporosis diagnosis kit using TMEM16A as an osteoporosis marker is proposed for the first time. Compared with the method of inquiring the past medical history, bone mass measurement, ultrasonic measurement and the like by a doctor, the osteoporosis diagnosis kit using the TMEM16A as the marker can directly reflect whether the subject suffers from osteoporosis and the degree of the osteoporosis on the molecular level; the TMEM16A is used as a marker, mRNA obtained by transcription of all or part of the gene of TMEM16A or TMEM16A protein is used as a target substance for detection, the detection method is more convenient compared with bone mass measurement and ultrasonic measurement, new equipment and instruments are not needed, the detection can be completed only by corresponding primers or antibodies and other conventional reagents, and the method, the equipment and the conventional reagents for detecting other target substances at a molecular level in a laboratory are compatible.
In some preferred embodiments, the test sample of the osteoporosis diagnostic kit comprises peripheral blood mononuclear cells. The content of TMEM16A in peripheral blood mononuclear cells correctly reflects the diseased status of osteoporosis. More preferably, the test sample of the osteoporosis diagnosis kit comprises osteoclast precursor cells. Osteoclast precursor cells are derived from monocyte/macrophage lineage precursor cells in peripheral blood mononuclear cells, and are fused to form mature multinucleated giant cells (MNCs). Under normal physiological conditions, bone resorption by osteoclasts in the bone and osteogenesis by osteoblasts maintain a dynamic balance, and bone metabolism and hardness and elasticity of the bone are maintained. Under pathological conditions, osteoclast is increased in number and function, bone resorption and metabolism activity is increased, and dynamic balance of bone tissues of the body is disturbed, so that bone resorption diseases are caused. Thus, when bone is diseased, osteoclasts are also altered compared to osteoclasts in normal bone.
In some preferred embodiments, a subject is determined to have osteoporosis when the level of TMEM16A in the subject's peripheral blood mononuclear cells is increased as compared to normal. Wherein the content of TMEM16A can be the content of mRNA obtained by transcription of all or part of the gene of TMEM16A, wherein part of the gene can be a part with better specificity in the gene sequence for coding TMEM 16A; optionally, TMEM16A content may also be the content of TMEM16A protein. The normal value in this embodiment refers to the content of TMEM16A in osteoclasts in people without osteoporosis, and the content of TMEM16A in osteoclasts in people without osteoporosis can be derived from a statistically significant measurement value in people without osteoporosis, or from a value that is reasonably inferred and verified, which is not limited by the present invention.
Since the present invention measures the expression level of TMEM16A in osteoclasts of patients with osteoporosis and normal persons, and finds that the expression level of TMEM16A in peripheral blood mononuclear cells of patients with osteoporosis is increased by about 1.5 times as much as that of normal persons, in some preferred embodiments, when the content of TMEM16A protein in peripheral blood mononuclear cells of a subject is at least 1.5 times of the normal value, the subject is judged to have osteoporosis.
In some alternative embodiments, the osteoporosis diagnostic kit is used to detect mRNA transcribed from all or part of the genes of TMEM 16A. The osteoporosis diagnosis kit comprises, but is not limited to, a kit for extracting mRNA, a primer for reverse transcription of mRNA, a kit for reverse transcription, a kit for detecting cDNA obtained by reverse transcription, or a consumable material for related experiments, etc. In some alternative embodiments, the osteoporosis diagnosis kit is used for detecting the protein expressed by the TMEM16A gene, and includes, but is not limited to, an antibody for specifically binding to the protein expressed by the TMEM16A gene, a kit for performing an ELISA experiment, a kit for performing an immunoblotting reaction, or a related experimental consumable, etc.
The invention also provides application of TMEM16A as an osteoporosis marker in preparing a medicament for treating osteoporosis. TMEM16A can be used as a marker for evaluating the therapeutic effect of drugs for treating osteoporosis, such as detecting the level of TMEM16A content in a subject in a clinical experiment, detecting the level of TMEM16A content in a subject animal in an animal experiment, or detecting the level of TMEM16A content in a cell experiment.
The invention also provides a medicine for treating osteoporosis, which takes the TMEM16A as a target point and relieves the osteoporosis of a patient by regulating the expression level of the TMEM16A of the patient.
In some preferred embodiments, the drug targets osteoclasts. On one hand, osteoclast abnormality is a main reason for causing bone pathological changes, and on the other hand, as TMEM16A is also distributed in a plurality of cells and tissues such as vascular epithelial cells, pancreatic epithelial cells, salivary gland epithelial cells, bronchial mucosa submandibular gland acinus, mammary gland, renal tubules and the like, the targeted inhibition of the expression of TMEM16A in osteoclasts can avoid the interference of a drug on TMEM16A located in other cells and tissues and reduce the side effects of the drug.
In some preferred embodiments, the medicament contains an active ingredient inhibiting TMEM16A expression to down-regulate the expression of TMEM16A in a patient, the invention is not limited to an active ingredient inhibiting TMEM16A expression, and active ingredients inhibiting or interfering with gene expression acceptable in the art may be used as the active substance of the medicament of the invention.
In some preferred embodiments, the drug comprises a small interfering RNA (siRNA) inhibiting the expression of TMEM16A gene as an active ingredient, and the siRNA is a double-stranded RNA with the length of 20 to 25 nucleotides, and can interfere the expression of a target gene, thereby achieving the purpose of inhibiting the expression of the target gene. The siRNA can exist independently as the siRNA in the active ingredients, and can also be integrated in the biological material, when the biological material containing the siRNA capable of expressing and inhibiting the TMEM16A gene expression is used as the active ingredients, the biological material used as the active ingredients in the medicine can express the siRNA capable of inhibiting the TMEM16A gene expression so as to inhibit the TMEM16A gene expression to achieve the therapeutic effect. The biological material includes, but is not limited to, one or more of a gene cassette, a recombinant vector, a recombinant microorganism and a recombinant cell capable of expressing an siRNA that inhibits TMEM16A gene expression.
It is understood that the dosage form of the above-mentioned drugs can be, but is not limited to, injections, tablets, powders, granules, capsules or oral liquids, and the present invention is not limited thereto. And the medicine can also select proper auxiliary materials acceptable in the pharmaceutical field according to the dosage form, optionally, when the medicine is an injection, the auxiliary materials of the medicine can be at least one of buffer solution, freeze-drying protective agent and preservative, for example and without limitation; when the medicament is an oral preparation, the auxiliary material of the medicament can be, but is not limited to, at least one of a disintegrating agent, an excipient and a filler; when the medicament is a gel, the medicament can contain hydrogel to be directly filled into an affected part.
To facilitate a clearer understanding of the contents of the present invention, reference will now be made in detail to the following specific embodiments. Unless otherwise specified, the sources of experimental animals, drugs and reagents used in the examples of the present invention are normal and readily available:
example 1
In this example, the expression level of TMEM16A in osteoclasts of patients with osteoporosis and normal persons was measured, and a trace amount of bone tissue was extracted from patients with osteoporosis by a minimally invasive surgery, from which primary osteoclasts were isolated, and cell culture was performed using a complete medium (DMEM- α medium: fetal bovine serum ═ 9: 1). The RNA content of TMEM16A in osteoclasts of patients with osteoporosis was analyzed by PCR experiments as compared with that of normal persons. The change of the expression level of TMEM16A in osteoclasts of osteoporosis patients and the expression level of normal people is compared and analyzed by using a cell immunoblotting experiment of the TMEM16A antibody. As shown in FIG. 1, it was found that the expression level of TMEM16A in the bone tissue of patients with osteoporosis was increased about 1.5 times as compared with that of normal persons, and thus the increased expression level of TMEM16A was a marker of osteoporosis.
Example 2
In the implementation, a mouse osteoporosis model is firstly established, and then the content of TMEM16A in bone tissues of an osteoporosis mouse is measured.
The method for establishing the mouse osteoporosis model comprises the following steps: selecting a female C57 mouse with the age of 3-6 months, artificially removing the ovary of the female mouse through an operation, normally culturing for four weeks to model, and simulating the osteoporosis of a female caused by the lack of estrogen after menopause; the male mice are male C57 mice selected from 2-4 months old, the testes of the female mice are artificially removed through an operation, and the male mice are cultured normally for four weeks to model, so that senile osteoporosis caused by great reduction of androgen secretion of the elderly males is simulated.
The method for measuring the expression level of TMEM16A mainly comprises the following steps: the measurement of the change in the RNA content of TMEM16A by PCR mainly comprised: extracting total RNA by a Trizol method, carrying out reverse transcription reaction, carrying out PCR reaction, and carrying out electrophoresis experiments. The change of the expression level of TMEM16A protein is measured by an immunoblotting experiment, and mainly comprises the following steps: collecting protein samples, determining protein content, performing SDS-PAGE electrophoresis, transferring membranes, sealing, incubating antibodies, and exposing and detecting.
As a result, as shown in fig. 2, it was found that the content of TMEM16A in the bone tissue of the mice with osteoporosis was significantly increased, about 4 times higher than that of the normal mice, and thus it was further confirmed that the increased content of TMEM16A was a marker of osteoporosis.
Example 3
In this example, osteoclast was induced to differentiate in vitro by the following method: bone marrow cells are extracted from bone marrow of a C57 mouse at 6-8 weeks, erythrocyte lysate is fully lysed and then centrifuged, bone marrow stromal cells are extracted, 10ng/ml M-CSF is added for culture, supernatant is extracted after 24 hours, centrifugation is carried out at 1500rpm, a culture medium containing 50ng/ml Rankl and 30ng/ml M-CSF is added for continuous culture for 5 days, the culture medium is replaced once, and a TRAP staining kit is used for detecting whether osteoclast induction is successful or not after 5 days. The expression level of TMEM16A was determined by PCR and immunoblotting as described in the above examples.
Then, the content of TMEM16A in the cells after 1, 3 and 5 days of normal induction was measured, and as a result, as shown in fig. 3 and 4, after 1, 3 and 5 days of normal induction, the content of TMEM16A in the cells was dramatically increased, and the content of TMEM16A in the osteoclasts was increased by about 13 times on the fifth day of induction compared with the first day of induction, so that the increase of TMEM16A content was an important marker of osteoclast differentiation, and the higher the degree of osteoclast differentiation, the higher the possibility that the patient has osteoporosis was.
Example 4
In this example, TMEM16A gene in osteoclast is down-regulated by siRNA interference, and three groups of TMEM16A-shRNA lentiviral plasmids, CCTGCTAAACAACATCATT (2, 399-. The plasmid is transferred into cells by adopting an instantaneous transfection method, the transfection quantity of each hole of a 24-hole plate is 0.5 mu g of lentiviral plasmid, 1.5 mu L of transfection reagent and 50 mu L of OptiMEM, the three are transferred and evenly mixed by shaking, and the mixture is added into the cells cultured normally after standing for 15 minutes. The expression level of TMEM16A was determined by PCR and immunoblotting as described above.
As shown in fig. 5, it was found that the genes Nfatc1, Acp5, Ctsk, Mmp9 and Clcn7, which characterize osteoclast differentiation into bone, are decreased to different extents after the expression of TMEM16A gene is down-regulated, so we finally determined that TMEM16A is a marker of osteoclast pathogenesis, and that the down-regulation of TMEM16A can inhibit osteoclast differentiation, thereby inhibiting the generation of osteoporosis.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Hebei university of industry
<120> TMEM16A as osteoporosis marker, application thereof, osteoporosis diagnosis kit and medicament
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence
<400> 1
cctgctaaac aacatcatt 19
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
tctcatggtg gagctgttt 19
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence
<400> 3
gagttatcat ctatagaat 19
<210> 4
<211> 19
<212> DNA
<213> Artificial sequence
<400> 4
ttctccgaac gtgtcacgt 19
Claims (10)
1. An osteoporosis diagnosis kit using TMEM16A as an osteoporosis marker, wherein the osteoporosis diagnosis kit judges whether a subject has osteoporosis or not by detecting the content of TMEM16A in peripheral blood mononuclear cells of the subject.
2. The osteoporosis diagnostic kit according to claim 1, wherein the test sample of the osteoporosis diagnostic kit comprises peripheral blood mononuclear cells;
preferably, the test sample of the osteoporosis diagnosis kit includes osteoclast precursor cells.
3. The osteoporosis diagnostic kit of claim 1, wherein the subject is determined to have osteoporosis when the content of TMEM16A in peripheral blood mononuclear cells of the subject is increased compared to a normal value.
4. The osteoporosis diagnostic kit of any one of claims 1 to 3, wherein the subject is determined to have osteoporosis when the content of TMEM16A protein in peripheral blood mononuclear cells of the subject is at least 1.5 times the normal value.
Use of TMEM16A as a marker for osteoporosis in the manufacture of a medicament for the treatment of osteoporosis.
6. A medicament for the treatment of osteoporosis, said medicament targeting TMEM 16A.
7. The medicament of claim 6, wherein the medicament is targeted to osteoclasts.
8. The medicament according to claim 6 or 7, which comprises an active ingredient that inhibits the expression of TMEM 16A.
9. The drug of claim 8, which comprises as an active ingredient an siRNA inhibiting the expression of TMEM16A gene.
10. The medicament according to claim 8, wherein the siRNA for inhibiting TMEM16A gene expression comprises at least one of siRNA having the sequence shown in SEQ ID NO.1, siRNA having the sequence shown in SEQ ID NO.2 and siRNA having the sequence shown in SEQ ID NO. 3.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911041023.6A CN110596406B (en) | 2019-10-30 | 2019-10-30 | TMEM16A as osteoporosis marker, application thereof, osteoporosis diagnosis kit and medicament |
| PCT/CN2020/083131 WO2021082350A1 (en) | 2019-10-30 | 2020-04-03 | Tmem16a acting as osteoporosis marker and application thereof, osteoporosis diagnostic kit and drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911041023.6A CN110596406B (en) | 2019-10-30 | 2019-10-30 | TMEM16A as osteoporosis marker, application thereof, osteoporosis diagnosis kit and medicament |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110596406A true CN110596406A (en) | 2019-12-20 |
| CN110596406B CN110596406B (en) | 2021-05-07 |
Family
ID=68852030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911041023.6A Active CN110596406B (en) | 2019-10-30 | 2019-10-30 | TMEM16A as osteoporosis marker, application thereof, osteoporosis diagnosis kit and medicament |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN110596406B (en) |
| WO (1) | WO2021082350A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111450237A (en) * | 2020-03-17 | 2020-07-28 | 中国人民解放军63919部队 | Novel target for developing osteoporosis treatment drug |
| WO2021082350A1 (en) * | 2019-10-30 | 2021-05-06 | 河北工业大学 | Tmem16a acting as osteoporosis marker and application thereof, osteoporosis diagnostic kit and drug |
| CN113754764A (en) * | 2021-08-20 | 2021-12-07 | 四川大学华西医院 | anti-DOG-1 antibodies, conjugates, pharmaceutical compositions, pharmaceutical formulations and uses thereof |
| CN114107476A (en) * | 2021-04-12 | 2022-03-01 | 上海市同仁医院 | Application of miR-29 in osteoporosis |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154482A (en) * | 2011-02-16 | 2011-08-17 | 北京大学 | Application of calcium activated chloride channels ANO1/TMEM16A in diagnosis and treatment of prostate cancer |
| US20120308582A1 (en) * | 2011-06-02 | 2012-12-06 | Novartis Ag | Molecules and methods for modulating tmem16a activities |
| CN104422777A (en) * | 2013-09-10 | 2015-03-18 | 中国医学科学院肿瘤医院 | Application of ANO1 protein in prediction on prognosis of esophagus cancer and precancerous lesion risk |
| KR20150117504A (en) * | 2014-04-10 | 2015-10-20 | 연세대학교 산학협력단 | Pharmaceutical compositions for the treatment or prevention of cancers comprising idebenone |
| CN105106228A (en) * | 2015-08-12 | 2015-12-02 | 河北工业大学 | Medicine composition which is used as TMEM16A ion channel activator and contains ginsenoside Rb1 |
| KR20170019905A (en) * | 2015-08-13 | 2017-02-22 | 연세대학교 산학협력단 | Pharmacy composition for preventing or treating anti-cancer and expression depressant of ANO1 |
| CN108315414A (en) * | 2018-02-06 | 2018-07-24 | 江苏省人民医院（南京医科大学第附属医院） | Biomarker for predicting esophageal squamous cell carcinoma prognosis |
| CN108938625A (en) * | 2017-05-27 | 2018-12-07 | 北京大学 | Purposes and preparation method of the 2- virtue formamido thiophene -3- carboxylic acid derivative as ANO1 protein inhibitor |
| CN109806249A (en) * | 2019-03-05 | 2019-05-28 | 河北工业大学 | Application, activator, kit and drug of the carotenoid in the product of preparation activation TMEM16A ion channel |
| CN109833320A (en) * | 2019-03-19 | 2019-06-04 | 河北工业大学 | Application, activator, kit and drug of the ginsenoside in the product of preparation activation TMEM16A ion channel |
| CN110167588A (en) * | 2016-12-22 | 2019-08-23 | 南京大学 | Reduce the smooth muscle contraction response induced by inflammatory contracting agent |
Family Cites Families (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2560643A1 (en) * | 2010-04-20 | 2013-02-27 | Institute for OneWorld Health | Compounds, compositions and methods comprising 1,3,4-oxadiazole derivatives |
| KR101463505B1 (en) * | 2012-02-06 | 2014-11-19 | 아주대학교산학협력단 | Biomarker composition comprising UBAP2 for diagnosis and evaluation of clinical therapeutic effect for osteoporosis |
| KR101486368B1 (en) * | 2012-06-08 | 2015-01-26 | 재단법인 아산사회복지재단 | Marker composition comprising S1P protein for prediction of risk in osteoporotic fracture and osteoporosis |
| KR20160048442A (en) * | 2014-10-24 | 2016-05-04 | 가톨릭대학교 산학협력단 | Biomarkers related to rheumatoid arthritis |
| WO2018108165A1 (en) * | 2016-12-15 | 2018-06-21 | 深圳瑞健生命科学研究院有限公司 | Drug for preventing and treating osteoporosis and uses thereof |
| US20210156844A1 (en) * | 2017-05-02 | 2021-05-27 | Bayer Aktiengesellschaft | TMEM16A Modulation for Diagnostic or Therapeutic Use in Pulmonary Hypertension (PH) |
| CN107090513A (en) * | 2017-06-27 | 2017-08-25 | 首都医科大学附属北京友谊医院 | GM2A genes diagnose the mark with prognosis as Male Osteoporosis |
| CN109722474A (en) * | 2017-10-31 | 2019-05-07 | 中国科学院深圳先进技术研究院 | MiRNA marker miR-19b relevant to bone metabolic disease and its application |
| CN107904305B (en) * | 2017-12-29 | 2019-01-04 | 北京泱深生物信息技术有限公司 | Biomarker of the HEATR4 gene as Diagnosis of osteoporosis |
| CN109557198B (en) * | 2018-11-20 | 2021-08-31 | 苏州大学 | Protein biomarker for osteoporosis diagnosis and application thereof |
| CN109468375B (en) * | 2018-12-28 | 2021-05-14 | 固安博健生物技术有限公司 | Application of molecular marker in osteoporosis |
| CN110596406B (en) * | 2019-10-30 | 2021-05-07 | 河北工业大学 | TMEM16A as osteoporosis marker, application thereof, osteoporosis diagnosis kit and medicament |
-
2019
- 2019-10-30 CN CN201911041023.6A patent/CN110596406B/en active Active
-
2020
- 2020-04-03 WO PCT/CN2020/083131 patent/WO2021082350A1/en active Application Filing
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154482A (en) * | 2011-02-16 | 2011-08-17 | 北京大学 | Application of calcium activated chloride channels ANO1/TMEM16A in diagnosis and treatment of prostate cancer |
| US20120308582A1 (en) * | 2011-06-02 | 2012-12-06 | Novartis Ag | Molecules and methods for modulating tmem16a activities |
| CN104422777A (en) * | 2013-09-10 | 2015-03-18 | 中国医学科学院肿瘤医院 | Application of ANO1 protein in prediction on prognosis of esophagus cancer and precancerous lesion risk |
| KR20150117504A (en) * | 2014-04-10 | 2015-10-20 | 연세대학교 산학협력단 | Pharmaceutical compositions for the treatment or prevention of cancers comprising idebenone |
| CN105106228A (en) * | 2015-08-12 | 2015-12-02 | 河北工业大学 | Medicine composition which is used as TMEM16A ion channel activator and contains ginsenoside Rb1 |
| KR20170019905A (en) * | 2015-08-13 | 2017-02-22 | 연세대학교 산학협력단 | Pharmacy composition for preventing or treating anti-cancer and expression depressant of ANO1 |
| CN110167588A (en) * | 2016-12-22 | 2019-08-23 | 南京大学 | Reduce the smooth muscle contraction response induced by inflammatory contracting agent |
| CN108938625A (en) * | 2017-05-27 | 2018-12-07 | 北京大学 | Purposes and preparation method of the 2- virtue formamido thiophene -3- carboxylic acid derivative as ANO1 protein inhibitor |
| CN108315414A (en) * | 2018-02-06 | 2018-07-24 | 江苏省人民医院（南京医科大学第附属医院） | Biomarker for predicting esophageal squamous cell carcinoma prognosis |
| CN109806249A (en) * | 2019-03-05 | 2019-05-28 | 河北工业大学 | Application, activator, kit and drug of the carotenoid in the product of preparation activation TMEM16A ion channel |
| CN109833320A (en) * | 2019-03-19 | 2019-06-04 | 河北工业大学 | Application, activator, kit and drug of the ginsenoside in the product of preparation activation TMEM16A ion channel |
Non-Patent Citations (3)
| Title |
|---|
| LIANG DENG等: "Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma", 《ONCO TARGETS AND THERAPY》 * |
| UMAMAHESWAR DUVVURI等: "TMEM16A Induces MAPK and Contributes Directly to Tumorigenesis and Cancer Progression", 《CANCER RESEARCH》 * |
| ZONGTAO LIU等: "Inhibition of Ca2+-activated chloride channel ANO1 suppresses ovarian cancer through inactivating PI3K/Akt signaling", 《INTERNATIONAL JOURNAL OF CANCER》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021082350A1 (en) * | 2019-10-30 | 2021-05-06 | 河北工业大学 | Tmem16a acting as osteoporosis marker and application thereof, osteoporosis diagnostic kit and drug |
| CN111450237A (en) * | 2020-03-17 | 2020-07-28 | 中国人民解放军63919部队 | Novel target for developing osteoporosis treatment drug |
| CN114107476A (en) * | 2021-04-12 | 2022-03-01 | 上海市同仁医院 | Application of miR-29 in osteoporosis |
| CN113754764A (en) * | 2021-08-20 | 2021-12-07 | 四川大学华西医院 | anti-DOG-1 antibodies, conjugates, pharmaceutical compositions, pharmaceutical formulations and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110596406B (en) | 2021-05-07 |
| WO2021082350A1 (en) | 2021-05-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110596406B (en) | TMEM16A as osteoporosis marker, application thereof, osteoporosis diagnosis kit and medicament | |
| Ho et al. | Egr-1 deficiency protects from renal inflammation and fibrosis | |
| EP2215482A2 (en) | Biomarker for assessing response to fms treatment | |
| Hedhli et al. | Multimodal assessment of mesenchymal stem cell therapy for diabetic vascular complications | |
| JP5680744B2 (en) | Combination therapy and evaluation method | |
| WO1997028179A1 (en) | Method for inhibiting tumor cell growth | |
| Hao | Monitoring of renal ischemia reperfusion injury in rabbits by ultrasonic contrast and its relationship with expression of VEGF in renal tissue | |
| CN105400894A (en) | Intervertebral disc degenerative change diagnosis and treatment marker | |
| CN115429879B (en) | Application of targeted inhibition GATA3 in promoting liver regeneration and improving liver injury | |
| Zhang et al. | Bioinformatics analysis of breast cancer bone metastasis related gene-CXCR4 | |
| WO2019053295A1 (en) | Methods for improving the cell therapy efficacy with mesenchymal stem cell populations | |
| CN112280742A (en) | Anti-aging pharmaceutical composition or health product prepared from stem cells | |
| JP2008540361A (en) | GPR18 as a biomarker of Th1-mediated immune response | |
| CN115120723B (en) | Application of macrophage Piezo1 knockout in promoting ischemic tissue angiogenesis | |
| TWI333978B (en) | Granulysin and uses thereof | |
| CN110988346A (en) | Marker for auxiliary diagnosis of lung cancer and detection method | |
| CN111793686A (en) | Diagnostic and prognostic marker for luminal and HER2 breast cancers, and therapeutic PPAR γ inhibitor | |
| Oda et al. | Ovariectomy fails to augment bone resorption and marrow B lymphopoiesis in granulocyte colony-stimulating factor transgenic mice | |
| Peng et al. | NEK2 overexpression aggravates IL-22-induced keratinocyte proliferation and cytokine level increases and IMQ-induced psoriasis-like dermatitis | |
| JP7190567B2 (en) | Biomarkers for proliferating and migrating ability prediction of mesenchymal stem cells and their uses | |
| JP4044842B2 (en) | Screening method for drugs that can treat obesity | |
| CN115772557A (en) | Application of miR-148a in preparation of postmenopausal osteoporosis detection product | |
| Xu et al. | SOD3 regulates FLT1 to affect bone metabolism by promoting osteogenesis and inhibiting adipogenesis through PI3K/AKT and MAPK pathways | |
| CN116298301A (en) | Myocardial ischemia injury marker CAPG and application thereof | |
| HE et al. | Analysis of the pedigreed population genetics of SPF Yorkshire and Landrace pigs imported from Canada |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |