WO2021082350A1 - Tmem16a作为骨质疏松的标志物及其应用、骨质疏松诊断试剂盒和药物 - Google Patents

Tmem16a作为骨质疏松的标志物及其应用、骨质疏松诊断试剂盒和药物 Download PDF

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WO2021082350A1
WO2021082350A1 PCT/CN2020/083131 CN2020083131W WO2021082350A1 WO 2021082350 A1 WO2021082350 A1 WO 2021082350A1 CN 2020083131 W CN2020083131 W CN 2020083131W WO 2021082350 A1 WO2021082350 A1 WO 2021082350A1
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osteoporosis
tmem16a
diagnostic kit
expression
content
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PCT/CN2020/083131
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French (fr)
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安海龙
李英贤
陈娅斐
凌树宽
郭帅
孙维佳
王徐朝
展永
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河北工业大学
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    • 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

Definitions

  • This application relates to the technical field of disease markers, in particular to a TMEM16A as a marker of osteoporosis and its application, an osteoporosis diagnostic kit and a medicine.
  • Osteoporosis is a systemic bone metabolism disease. Its main characteristics are decreased bone mass and changes in bone microstructure, accompanied by decreased bone strength, increased bone fragility, and increased fracture risk. According to epidemiological statistics, the prevalence of osteoporosis among people over 50 years old in my country is 20.7% for women and 14.4% for men. And according to global epidemiological surveys, the prevalence of osteoporosis is relatively high in all countries. The incidence of osteoporosis is related to many factors, among which gender factors and age factors are the most relevant to the occurrence of osteoporosis. The pain and fracture caused by osteoporosis seriously affect the quality of life of patients and endanger the survival of patients. However, the existing treatment methods have relatively limited therapeutic effects on osteoporosis. In elderly women, pathological fractures are mostly secondary to osteoporosis. Osteoporotic fractures have a high disability mortality rate. The current mortality rate has exceeded that of elderly women suffering from breast cancer, cervical cancer and uterine body cancer. sum.
  • the clinical methods for diagnosing osteoporosis mainly include: 1.
  • the doctor consults the past medical history and provides high-risk factors for osteoporosis through the medical history, such as alcoholism, heavy smoking, premenopausal bilateral oophorectomy, etc. Need further inspection.
  • the measurement of bone mass is based on the measurement of bone mineral content and bone density, but the occurrence of fractures and osteoporosis does not only depend on bone mass.
  • Ultrasonic bone determination measuring the speed of ultrasound passing through bone tissue, amplitude attenuation and hardness index reflect bone structure and bone mass.
  • the existing methods for diagnosing osteoporosis mainly have the following shortcomings: 1.
  • the doctor's inquiry of the past medical history can only provide reference, and cannot accurately determine whether there is osteoporosis.
  • Bone mass measurement is a commonly used method now, but whether a fracture occurs is mainly determined by bone strength, and bone strength is composed of bone mass, bone quality, bone composition and bone shape and other aspects, so only through bone The amount of judgment is not accurate enough.
  • the result of ultrasound measurement is not bone density or bone mineral content, and cannot be compared with the true value. It requires more observation data.
  • the ultrasound equipment is very inconvenient to move, and the cost of each measurement is high. Therefore, an improved product for diagnosing osteoporosis is currently needed in the market.
  • the first purpose of this application is to provide an osteoporosis diagnostic kit using TMEM16A as a marker of osteoporosis, which is fast and accurate in diagnosis, simple in operation, and does not require special instruments and equipment.
  • the second purpose of this application is to provide the application of TMEM16A in the preparation of medicines for treating osteoporosis.
  • the third objective of this application is to provide a medicine for treating osteoporosis, which uses TMEM16A as a target.
  • the present application provides an osteoporosis diagnostic kit using TMEM16A as a marker of osteoporosis.
  • the osteoporosis diagnostic kit detects TMEM16A in peripheral blood mononuclear cells of a subject. To determine whether the subject suffers from osteoporosis.
  • the test sample of the osteoporosis diagnosis kit includes peripheral blood mononuclear cells
  • the test sample of the osteoporosis diagnostic kit includes osteoclast precursor cells.
  • TMEM16A in the peripheral blood mononuclear cells of the subject is higher than the normal value, it is determined that the subject has osteoporosis.
  • the content of TMEM16A protein in the peripheral blood mononuclear cells of the subject is at least 1.5 times the normal value, it is determined that the subject has osteoporosis.
  • the present application also provides the application of TMEM16A as a marker of osteoporosis in the preparation of drugs for treating osteoporosis.
  • the present application also provides a drug for treating osteoporosis, the drug uses TMEM16A as a target.
  • the drug targets osteoclasts.
  • the medicine contains an active ingredient that inhibits the expression of TMEM16A.
  • the medicine uses small interfering RNA (siRNA) that inhibits the expression of TMEM16A gene as an active ingredient.
  • siRNA small interfering RNA
  • the siRNA that inhibits the expression of the TMEM16A gene includes siRNA containing the sequence shown in SEQ ID NO. 1, siRNA with the sequence shown in SEQ ID NO. 2 and siRNA with the sequence shown in SEQ ID NO. 3 At least one.
  • This application is based on comparing the content of TMEM16A in samples of osteoporosis patients and normal people, and finds the difference between the content of TMEM16A in samples of osteoporosis patients and the content of TMEM16A in the normal delivery population, and points out that TMEM16A can be used as a marker of osteoporosis
  • TMEM16A can be used as a marker of osteoporosis
  • TMEM16A is a marker of osteoporosis. Based on this, using TMEM16A as a marker of osteoporosis, this application provides applications in the fields of osteoporosis diagnostic kits, and evaluation of the efficacy of drugs for treating osteoporosis, by detecting the expression of TMEM16A in patients’ peripheral blood mononuclear cells The amount can quickly determine that the patient is suffering from osteoporosis, the diagnosis is fast and accurate, the operation is simple, and no special instruments and equipment are required.
  • the drug for the treatment of osteoporosis takes TMEM16A as a target, and regulates the expression of TMEM16A in patients to alleviate the patient's osteoporosis.
  • Figure 1 is a comparison of the expression levels of TMEM16A in bone tissues of normal people and osteoporotic patients in Example 1 of the application;
  • Figure 2 is a comparison of the expression levels of TMEM16A in bone tissues of normal mice and tail-sling-induced osteoporosis mice in Example 2 of the application;
  • Figure 3 shows the expression of TMEM16A protein in osteoclasts 1, 3, and 5 days after induction in Example 3 of the application;
  • Figure 4 is a statistical diagram of the expression of TMEM16A protein in osteoclasts 1, 3, and 5 days after induction in Example 3 of the application;
  • Figure 5 shows the expression of genes related to osteoclast differentiation after down-regulation of TMEM16A expression by siRNA in Example 4 of the application;
  • Figure 6 shows the relative expression levels of TMEM16A gene in peripheral blood mononuclear cells of 50 normal people and 50 osteoporosis patients
  • Figure 7 shows the relative expression levels of TMEM16A in osteoclast precursor cells of normal people and patients with osteoporosis.
  • Osteoporosis is a metabolic and systemic disease of the skeletal system, which can damage the bone microstructure of the patient, increase the fragility of the patient's bone, reduce its bone strength, and thereby increase the risk of fracture in the patient.
  • the basic mechanism of osteoporosis is that the dynamic balance of bone remodeling is disrupted, and the function of osteoblasts and osteoclasts is imbalanced, which leads to imbalance of bone metabolism, accelerated bone resorption, and decreased bone formation. Ultimately, bone resorption is much greater than bone formation and net bone mass. Lost.
  • the signaling pathways related to bone metabolism include BMPs signaling pathway, OPG/RANKL/RANK signaling pathway, TGF- ⁇ signaling pathway, MAPK signaling pathway, Wnt/ ⁇ -catenin signaling pathway, PPAR- ⁇ signaling pathway and Hedgehog signaling pathway.
  • TMEM16A belongs to the TMEM16 family of proteins with multiple transmembrane structures.
  • TMEM16A is a calcium-activated chloride channel (CaCCs).
  • the TMEM16A protein structure has 10 transmembrane domains, and both the nitrogen and carbon ends are located in the cell.
  • TMEM16A distributes in cells or tissues including vascular epithelial cells, pancreatic epithelial cells, salivary gland epithelial cells, bronchial mucosa, submandibular acinar cells, mammary glands and renal tubules.
  • the function of TMEM16A is related to diarrhea, hypertension and a variety of cancers.
  • Existing studies have not revealed that the expression level of TMEM16A is related to osteoporosis.
  • This application measures the expression level of TMEM16A in osteoclasts of osteoporosis patients and normal people. It is found that the expression level of TMEM16A in the bone tissue of osteoporosis patients is about 1.5 times higher than that of normal people. Therefore, TMEM16A is a sign of osteoporosis. Things. Furthermore, it was found from the mouse model of osteoporosis that the content of TMEM16A in bone tissue of mice with osteoporosis was significantly increased, which was about 4 times higher than that of normal mice, thus further confirming that TMEM16A is a marker of osteoporosis. .
  • TMEM16A in the cells of the induced osteoclasts increased drastically.
  • the content of TMEM16A in the osteoclasts increased on the fifth day of induction. Therefore, the increase of TMEM16A content is an important sign of osteoclast differentiation, and the higher the degree of osteoclast differentiation, the more likely the patient will have osteoporosis.
  • TMEM16A When using siRNA interference method to down-regulate TMEM16A, the genes Nfatc1, Acp5, Ctsk, Mmp9, and Clcn7, which are related to osteoclast differentiation into osteoclasts, all decreased to varying degrees, further indicating that TMEM16A is a marker of osteoporosis.
  • the present application provides an osteoporosis diagnostic kit using TMEM16A as a marker of osteoporosis.
  • the osteoporosis diagnostic kit judges the test subject by detecting the content of TMEM16A in the peripheral blood mononuclear cells of the subject Whether the person suffers from osteoporosis.
  • This application compares the content of TMEM16A in the samples of osteoporosis patients and normal people, and finds the difference between the content of TMEM16A in the samples of osteoporosis patients and the content of TMEM16A in the normal population, and points out that TMEM16A can be used as a marker of osteoporosis
  • TMEM16A can be used as a marker of osteoporosis
  • the osteoporosis diagnostic kit with TMEM16A as a marker can directly reflect whether the subject has osteoporosis and osteoporosis at the molecular level.
  • TMEM16A as a marker, whether it is the mRNA transcribed from all or part of the TMEM16A gene, or the TMEM16A protein as the target substance for detection, the detection method is more convenient than bone mass measurement and ultrasound measurement. There is no need for new equipment and instruments, just the corresponding primers or antibodies and other conventional reagents can complete the detection, and it is compatible with other molecular-level target substance methods and equipment and conventional reagents in the laboratory.
  • the test sample of the osteoporosis diagnostic kit includes peripheral blood mononuclear cells.
  • the content of TMEM16A in peripheral blood mononuclear cells can correctly reflect the condition of osteoporosis.
  • the test sample of the osteoporosis diagnostic kit includes osteoclast precursor cells.
  • Osteoclast precursor cells are derived from monocyte/macrophage precursor cells in peripheral blood mononuclear cells, which form mature multinudeatedeells (MNCs) after fusion. Under normal physiological conditions, the bone resorption of osteoclasts in the bones and the bone production of osteoblasts maintain a dynamic balance, maintaining bone metabolism and bone hardness and elasticity.
  • the number of osteoclasts increases, the function is enhanced, the activity of bone resorption metabolism increases, and the dynamic balance of the body's bone tissue is disrupted, which leads to bone resorption diseases. Therefore, when the bones are diseased, the osteoclasts will also change compared with the osteoclasts in normal bones.
  • the content of TMEM16A in the peripheral blood mononuclear cells of the subject is higher than the normal value, it is determined that the subject has osteoporosis.
  • the content of TMEM16A can be the content of mRNA obtained by transcription of all or part of the gene of TMEM16A, and some of the genes can optionally be the more specific part of the gene sequence encoding TMEM16A; alternatively, the content of TMEM16A can also be the content of TMEM16A protein.
  • the normal value in this embodiment refers to the content of TMEM16A in osteoclasts in people who do not suffer from osteoporosis.
  • TMEM16A in osteoclasts in people who do not suffer from osteoporosis can be derived from Statistically significant measurement values in people with osteoporosis can also be derived from values that have been reasonably inferred and verified, and this application does not limit this.
  • the osteoporosis diagnostic kit is used to detect mRNA obtained by transcription of all or part of the gene of TMEM16A.
  • the osteoporosis diagnostic kit includes, but is not limited to, a set of reagents for extracting mRNA, primers for reverse transcription of mRNA, and a set of reagents for reverse transcription, which are used to detect cDNA obtained by reverse transcription. A set of reagents, or, related experimental consumables, etc.
  • the osteoporosis diagnostic kit is used to detect the protein expressed by the TMEM16A gene, and the osteoporosis diagnostic kit includes, but is not limited to, specific binding to the protein expressed by the TMEM16A gene. Antibodies, kits of reagents for ELISA experiments, kits of reagents for immunoblotting reactions, or consumables for related experiments, etc.
  • the application also provides the application of TMEM16A as a marker of osteoporosis in the preparation of medicines for the treatment of osteoporosis.
  • TMEM16A as a marker can be used to evaluate the efficacy of drugs for the treatment of osteoporosis, such as detecting the TMEM16A content level of subjects in clinical trials, testing the TMEM16A content level of test animals in animal experiments, or in cell experiments Detect the level of TMEM16A in the cells.
  • the application also provides a medicine for treating osteoporosis.
  • the medicine uses TMEM16A as a target and regulates the expression of TMEM16A in patients to alleviate the patient's osteoporosis.
  • the drug targets osteoclasts.
  • abnormal osteoclasts are the main cause of bone lesions.
  • TMEM16A is also distributed in vascular epithelial cells, pancreatic epithelial cells, salivary gland epithelial cells, bronchial mucosa, submandibular acinars, breast and renal tubules, etc.
  • targeted inhibition of TMEM16A expression in osteoclasts can avoid the interference of drugs on TMEM16A located in other cells and tissues, and reduce the side effects of drugs.
  • the drug contains an active ingredient that inhibits the expression of TMEM16A and down-regulates the expression of the patient's TMEM16A.
  • the application does not limit the active ingredient that inhibits the expression of TMEM16A, which is acceptable in the art to inhibit or interfere with gene expression. All the ingredients can be used as the active substance of the drug of the application.
  • the drug uses small interfering RNA (siRNA) that inhibits the expression of TMEM16A gene as an active ingredient.
  • siRNA is a double-stranded RNA with a length of 20 to 25 nucleotides that can interfere with the expression of target genes. So as to achieve the purpose of inhibiting the expression of the target gene.
  • the siRNA as the active ingredient of the siRNA can exist alone or integrated into the biological material.
  • the biological material that can express the siRNA that inhibits the expression of the TMEM16A gene is used as the active ingredient, the biological material as the active ingredient in the drug can be expressed SiRNA that inhibits the expression of TMEM16A gene to inhibit the expression of TMEM16A gene to achieve therapeutic effects.
  • the biological material includes, but is not limited to, one or more of gene cassettes, recombinant vectors, recombinant microorganisms, and recombinant cells capable of expressing siRNA that inhibits the expression of TMEM16A gene.
  • the dosage form of the above-mentioned drugs can be, but not limited to, injections, tablets, powders, granules, capsules or oral liquids, which are not limited in this application.
  • the drug can also select appropriate excipients acceptable in the field of pharmacy according to its dosage form.
  • the adjuvant of the drug can be, for example, but not limited to, a buffer, a lyophilized protective agent, and a preservative.
  • the adjuvant of the drug can be, for example, but not limited to at least one of a disintegrant, excipient, and filler; when the drug is a gel, the The medicine may contain hydrogel to directly fill the affected area.
  • TMEM16A amino acid sequence is shown in SEQ ID NO. 5; nucleotide sequence is shown in SEQ ID NO. 6
  • TMEM16A antibody to carry out cell immunoblotting experiment, compare and analyze the expression level of TMEM16A in osteoclasts of patients with osteoporosis and the expression level of normal people. The results are shown in Figure 1. It was found that the expression of TMEM16A in the bone tissue of patients with osteoporosis was about 1.5 times higher than that of normal people. Therefore, the increased expression of TMEM16A is a marker of osteoporosis.
  • a mouse osteoporosis model was first established, and then the TMEM16A content of the bone tissue of the osteoporotic mouse was measured.
  • Mouse osteoporosis model establishment method select female C57 mice aged 3-6 months, the female mice are artificially removed from the female mouse ovaries through surgery, and cultured for four weeks to build a model to simulate female postmenopausal bone caused by lack of estrogen Poor texture; male mice are selected male C57 mice aged 2-4 months. The testes of female mice are artificially removed by surgery, and the model is cultured for four weeks to simulate the senile bone of elderly men due to the drastic reduction of androgen secretion. Porosity.
  • the main methods for measuring the expression of TMEM16A include: measuring the changes in the RNA content of TMEM16A by PCR, including: Trizol extraction of total RNA, reverse transcription reaction, PCR reaction, and electrophoresis experiment. Measure the changes of TMEM16A protein expression by western blotting, including: collecting protein samples, determining protein content, SDS-PAGE electrophoresis, transferring membranes, blocking, incubating antibodies, and exposure detection.
  • osteoclasts were induced to differentiate in vitro.
  • the method of osteoclast induced differentiation is as follows: bone marrow cells are extracted from 6-8 weeks old C57 mouse bone marrow, the red blood cell lysate is fully lysed and centrifuged to extract bone marrow stromal cells and add 10ng/ Cultivate with ml of M-CSF, draw the supernatant after 24 hours, centrifuge at 1500 rpm, add a medium containing 50ng/ml Rankl and 30ng/ml M-CSF and continue culturing for 5 days, change the medium once during this period, and use it after 5 days TRAP staining kit detects whether osteoclasts are successfully induced.
  • the expression level of TMEM16A was detected by PCR and immunoblotting experimental methods, and the experimental methods were the same as the above-mentioned examples.
  • the content of TMEM16A in the cells was detected 1, 3, and 5 days after the normal induction.
  • the results are shown in Figure 3 and Figure 4.
  • the TMEM16A in the cells will increase sharply.
  • the content of TMEM16A in osteoclasts increased by about 13 times on the fifth day. Therefore, the increased content of TMEM16A is an important sign of osteoclast differentiation, and the higher the degree of osteoclast differentiation, the patient has The greater the likelihood of osteoporosis.
  • the TMEM16A gene in osteoclasts was down-regulated by siRNA interference.
  • TMEM16A gene GenBank NM_178642
  • three sets of TMEM16A-shRNA lentiviral plasmids were designed, namely CCTGCTAAACAACATCATT(2,399-2,418nt, shRNA1, (As shown in SEQ ID NO.1), TCTCATGGTGGAGCTGTTT (2,924-2,943nt, shRNA2, as shown in SEQ ID NO.2), GAGTTATCATCTATAGAAT (1,747-1,766nt, shRNA3, as shown in SEQ ID NO.3), and control
  • the plasmid NC shRNA, TTCTCCGAACGTGTCACGT is shown in SEQ ID NO.4.
  • Transfect the plasmid into the cells by transient transfection method The transfection amount per well of the 24-well plate is 0.5 ⁇ g lentiviral plasmid, 1.5 ⁇ L transfection reagent, 50 ⁇ L OptiMEM, transfer the three by shaking and mixing, and let stand for 15 minutes Then added to the normal cultured cells.
  • the expression level of TMEM16A was detected by PCR and western blotting, and the experimental method was the same as above.
  • Example 5 Using peripheral blood as a sample
  • peripheral blood of 50 normal people and 50 osteoporotic patients were extracted, and the relative expression of TMEM16A gene in peripheral blood mononuclear cells was detected by RT-PCR method.
  • the results are shown in Figure 6, 48 out of 50 patients had relatively high expression of TMEM16A gene, while only 1 out of 50 normal persons in the control group had high expression of TMEM16A protein.
  • the test method is exactly the same as the osteoclast described above, and the test results are shown in Figure 7. Compared with normal people, the expression of TMEM16A in osteoclast precursor cells of patients with osteoporosis is significantly increased.

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Abstract

TMEM16A作为骨质疏松的标志物及其应用、骨质疏松诊断试剂盒和用于治疗骨质疏松的药物，涉及疾病标志物技术领域。以TMEM16A作为骨质疏松的标志物的骨质疏松诊断试剂盒通过检测受试者外周血单核细胞中TMEM16A的含量来判断受试者是否患有骨质疏松。诊断快速准确、操作简单、无需特殊的仪器和设备。该用于治疗骨质疏松的药物以TMEM16A作为靶点，通过调控患者TMEM16A的表达量以缓解患者的骨质疏松病症。

Description

TMEM16A作为骨质疏松的标志物及其应用、骨质疏松诊断试剂盒和药物 技术领域
本申请涉及疾病标志物技术领域，尤其是涉及一种TMEM16A作为骨质疏松的标志物及其应用、骨质疏松诊断试剂盒和药物。
背景技术
骨质疏松是一种全身骨代谢性疾病，其主要特征是骨量减少及骨微结构改变，伴随骨强度下降、骨脆性增加及骨折危险性增加等。根据流行病学统计，我国50岁以上人群中骨质疏松的患病率女性为20.7％，男性为14.4％。并且根据全球的流行病学调查，各国的骨质疏松的患病率均较高。骨质疏松的发病与多种因素相关，其中性别因素和年龄因素对骨质疏松发生的相关性最大。骨质疏松导致的疼痛和骨折严重的影响了患者的生活质量，危及患者生存，而现有的治疗手段对骨质疏松的治疗效果相对有限。在老年女性中，病理性骨折多继发于骨质疏松症之后，骨质疏松性骨折致残致死率高，目前其病死率已超过老年女性罹患乳腺癌、宫颈癌及子宫体癌病死率的总和。
目前临床上诊断骨质疏松的方法主要有：1.医生询查既往病史，通过病史提供可能患有骨质疏松的高危因素，比如酗酒，大量吸烟，绝经前双侧卵巢切除术等，然后还需要进一步的检查。2.测量骨量，以测量骨矿含量和骨密度为依据，但是骨折及骨质疏松的发生不仅仅依赖于骨量。3.超声骨测定，测量超声通过骨组织的速度，振幅衰减和硬度指数反映骨结构和骨量。
现有诊断骨质疏松的方法主要有如下缺点：1.医生询查既往病史只能提供参考，并不能准确判断是否患有骨质疏松。2.骨量测量是现在常用的手段，但是是否发生骨折主要是有骨强度决定的，而骨强度由骨量、骨质量、骨骼的构成成分及骨的外形等多方面组成，因此仅仅通过骨量进行判断也不够准确。3.超声测量的结果不是骨密度或骨矿含量，不能与真值相比，需要更多的观察资料，此外超声设备巨大不便移动，每次测量成本较高等缺点。因此一种改进的诊断骨质疏松的产品是目前市场需要的。
有鉴于此，特提出本申请。
发明内容
本申请的第一目的在于提供一种以TMEM16A作为骨质疏松的标志物的骨质疏松诊断试剂盒，诊断快速准确、操作简单、无需特殊的仪器和设备。
本申请的第二目的在于提供TMEM16A在制备用于治疗骨质疏松的药物中的应用。
本申请的第三目的在于提供一种用于治疗骨质疏松的药物，该药物以TMEM16A作为靶点。
为解决上述技术问题，本申请特采用如下技术方案：
根据本申请的一个方面，本申请提供了一种以TMEM16A作为骨质疏松的标志物的骨质疏松诊断试剂盒，所述骨质疏松诊断试剂盒通过检测受试者外周血单核细胞中TMEM16A的含量来判断受试者是否患有骨质疏松。
优选地，所述骨质疏松诊断试剂盒的检测样本包括外周血单核细胞；
优选地，所述骨质疏松诊断试剂盒的检测样本包括破骨细胞前体细胞。
优选地，当受试者外周血单核细胞中的TMEM16A含量相比于正常值升高时，判定受试者患有骨质疏松。
优选地，当受试者外周血单核细胞中的TMEM16A蛋白的含量至少为正常值的1.5倍时，判定受试者患有骨质疏松。
根据本申请的另一个方面，本申请还提供了TMEM16A作为骨质疏松的标志物在制备用于治疗骨质疏松的药物中的应用。
根据本申请的另一个方面，本申请还提供了一种用于治疗骨质疏松的药物，所述药物以TMEM16A作为靶点。
优选地，所述药物靶向作用于破骨细胞。
优选地，所述药物含有抑制TMEM16A表达的活性成分。
优选地，所述药物以抑制TMEM16A基因表达的小干扰RNA(siRNA)作为活性成分。
优选地，所述抑制TMEM16A基因表达的siRNA包括含有如SEQ ID NO.1所示序列的siRNA、如SEQ ID NO.2所示序列的siRNA和如SEQ ID NO.3所示序列的siRNA中的至少一种。
与现有技术相比，本申请具有如下有益效果：
本申请基于比较骨质疏松患者和正常人的样本中TMEM16A的含量，发现骨质疏松患者的样本中的TMEM16A的含量和正产人群中TMEM16A的含量的差异，指出TMEM16A可作为骨质疏松的标志物，又通过观察骨质疏松小鼠模型和破骨细胞分化中TMEM16A的含量变化确定了TMEM16A的升高和骨质疏松相关，同时下调破骨细胞中的TMEM16A基因表达，表征破骨细胞分化成骨的相关基因均有不同程度的下降，说明了TMEM16A是骨质疏松的标志物。基于此，以TMEM16A作为骨质疏松的标志物，本申请提供了骨质疏松诊断试剂盒，以及治疗骨质疏松的药物的疗效评估等领域中的应用，通过检测病人外周血单核细胞TMEM16A表达量可以快速判断病人患有骨质疏松，诊断快速准确、操作简单、无需特殊的仪器和设备。
基于骨质疏松和TMEM16A的含量相关性的申请构思，本申请提供的用于治疗骨质疏松的药物以TMEM16A作为靶点，通过调控患者TMEM16A的表达量以缓解患者的骨质疏松病症。
附图说明
为了更清楚地说明本申请具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见 地，下面描述中的附图是本申请的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本申请实施例1中正常人与骨质疏松患者骨组织中TMEM16A表达量比较；
图2为本申请实施例2中正常小鼠与尾吊诱发骨质疏松小鼠骨组织中TMEM16A表达量比较；
图3为本申请实施例3中诱导1、3和5天后破骨细胞中TMEM16A蛋白表达量；
图4为本申请实施例3中诱导1、3和5天后破骨细胞中TMEM16A蛋白表达量统计图；
图5为本申请实施例4中siRNA下调TMEM16A表达后破骨细胞分化相关基因表达量；
图6示出了50名正常人和50名骨质疏松患者的外周血单核细胞中TMEM16A基因的相对表达量；
图7示出了正常人与骨质疏松患者破骨前体细胞中TMEM16A相对表达量。
具体实施方式
下面将结合实施例对本申请的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
需要说明的是：
本申请中，如果没有特别的说明，本文所提到的所有实施方式以及优选实施方法可以相互组合形成新的技术方案。
本申请中，如果没有特别的说明，本文所提到的所有技术特征以及优选特征可以相互组合形成新的技术方案。
除非另有说明，本文中所用的专业与科学术语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法或材料也可应用于本申请中。
骨质疏松症属于一种代谢性和全身性的骨骼系统疾病，能够对患者的骨微结构造成破坏，增加患者的骨脆性、降低其骨强度，从而增加患者骨折的风险。骨质疏松的基本发生机制为骨重建动态平衡被破坏，成骨细胞与破骨细胞功能失衡，导致骨代谢失衡，骨吸收加快，骨形成减少，最终造成骨吸收远大于骨形成，骨净量丢失。和骨代谢相关的信号通路有BMPs信号通路、OPG/RANKL/RANK信号通路、TGF-β信号通路、MAPK信号通路、Wnt/β-catenin信号通路、PPAR-γ信号通路和Hedgehog信号通路。
TMEM16A属于具有多次跨膜结构的蛋白质家族-TMEM16家族，TMEM16A是一种钙激活氯离子通道(CaCCs)。TMEM16A蛋白质结构具有10个跨膜域，氮端和碳端均位于细胞内。目前发现TMEM16A分布的细胞或组织 有血管上皮细胞、胰腺上皮细胞、唾液腺上皮细胞和支气管粘膜下颌下腺腺泡、乳腺和肾小管。TMEM16A的功能与腹泻、高血压和多种癌症相关，现有的研究并没有揭示TMEM16A表达水平和骨质疏松相关。
本申请测量了骨质疏松病人和正常人的破骨细胞中TMEM16A的表达量，发现骨质疏松病人骨组织中TMEM16A表达量比正常人升高约1.5倍，因此TMEM16A是骨质疏松的一个标志物。进一步的，从骨质疏松小鼠模型中发现，患有骨质疏松小鼠骨组织的TMEM16A含量显著升高，比正常小鼠高约4倍，因此进一步确认了TMEM16A是骨质疏松的标志物。随后在体外对破骨细胞进行诱导分化，发现诱导后的破骨细胞内细胞内的TMEM16A会发生剧烈的升高，诱导第五天与诱导第一天相比，破骨细胞中TMEM16A含量升高了约13倍，因此TMEM16A含量升高是破骨细胞分化的重要标志，而破骨细胞分化程度越高则患者患有骨质疏松可能性越大。当使用siRNA干扰的方法对TMEM16A进行下调，表征破骨细胞分化成骨的相关基因Nfatc1，Acp5，Ctsk，Mmp9，Clcn7都有不同程度的下降，进一步说明了TMEM16A是骨质疏松的标志物。
基于此，本申请提供了以TMEM16A作为骨质疏松的标志物的骨质疏松诊断试剂盒，所述骨质疏松诊断试剂盒通过检测受试者外周血单核细胞中TMEM16A的含量来判断受试者是否患有骨质疏松。本申请通过比较骨质疏松患者和正常人的样本中TMEM16A的含量，发现骨质疏松患者的样本中的TMEM16A的含量和正常人群中TMEM16A的含量的差异，指出TMEM16A可作为骨质疏松的标志物，又通过观察骨质疏松小鼠模型和破骨细胞分化中TMEM16A的含量变化确定了TMEM16A的升高和骨质疏松相关。因此首次提出以TMEM16A作为骨质疏松的标志物的骨质疏松诊断试剂盒。相比于医生询查既往病史、骨量测量和超声测量等方法，以TMEM16A作为标志物的骨质疏松诊断试剂盒可以直接从分子水平上反应受试者是否患有骨质疏松以及骨质疏松的患病程度；以TMEM16A作为标志物，无论是以TMEM16A的全部或者部分基因转录得到的mRNA，或TMEM16A蛋白作为检测的靶物质，其检测方法相比于骨量测量和超声测量均更便捷，也无需新的设备和仪器，只需相应的引物或者抗体等常规试剂就可以完成检测，和实验室中检测其他的分子水平上的靶物质方法和设备以及常规试剂兼容。
在一些优选的实施方式中，所述骨质疏松诊断试剂盒的检测样本包括外周血单核细胞。外周血单核细胞中TMEM16A的含量能正确的反应骨质疏松的患病情况。更优选地，所述骨质疏松诊断试剂盒的检测样本包括破骨细胞前体细胞。破骨细胞前体细胞来源于外周血单核细胞中的单核/巨噬细胞系前体细胞，经融合后形成成熟的多核巨细胞(Multinudeatedeells，MNCs)。正常生理条件下，骨骼中的破骨细胞的骨吸收与成骨细胞的骨生成维持动态平衡，维持骨骼新陈代谢和骨骼的硬度与弹性。病理条件下破骨细胞的数量增多，功能增强，骨吸收代谢的活性增加，机体骨组织的动态平衡被打乱，从而引发了骨吸收性疾病。因此当骨骼发生病变时，破骨细胞相较于正常骨骼中的破骨细胞也会发生改变。
在一些优选的实施方式中，当受试者外周血单核细胞中的TMEM16A含量相比于正常值升高时，判定受试者患有骨质疏松。其中TMEM16A含量可以为TMEM16A的全部或者部分基因转录得到的mRNA的含量，其中部分基因可选的为编码TMEM16A的基因序列中特异性较佳的部分；可选地，TMEM16A含量也可以为TMEM16A蛋白的含量。该实施方式中的正常值指的是未患有骨质疏松的人群中破骨细胞中的TMEM16A含量，未患有骨质疏松的人群中破骨细胞中的TMEM16A含量可以来源于对未患有骨质疏松的人群中具有统计学意义的测量值，也可以来源于经过合理推断和验证的值，本申请对此不做限制。
由于本申请测量了骨质疏松病人和正常人的破骨细胞中TMEM16A的表达量，发现骨质疏松病人外周血单核细胞中TMEM16A表达量比正常人升高约1.5倍，因此在一些优选的实施方式中，当受试者外周血单核细胞中的TMEM16A蛋白的含量至少为正常值的至少1.5倍时，判定受试者患有骨质疏松。
在一些可选的实施方式中，所述骨质疏松诊断试剂盒用于检测TMEM16A的全部或者部分基因转录得到的mRNA。所述骨质疏松诊断试剂盒中包括但不限于用于提取mRNA的成套试剂，用于反转录mRNA的引物及用于反转录的成套试剂，用于检测经反转录得到的cDNA的成套试剂，或，相关实验用耗材等。在一些可选的实施方式中，所述骨质疏松诊断试剂盒用于检测TMEM16A基因表达的蛋白，所述骨质疏松诊断试剂盒中包括但不限于用于和TMEM16A基因表达的蛋白特异性结合的抗体，用于进行ELISA实验的成套试剂，用于进行免疫印迹反应的成套试剂，或相关实验用耗材等。
本申请还提供了TMEM16A作为骨质疏松的标志物在制备用于治疗骨质疏松的药物中的应用。以TMEM16A作为标志物可以用于评估治疗骨质疏松的药物的疗效，例如在临床实验中检测受试者的TMEM16A含量水平，在动物实验中检测受试动物的TMEM16A含量水平，或在细胞实验中检测细胞中TMEM16A含量水平。
本申请还提供了一种用于治疗骨质疏松的药物，该药物以TMEM16A作为靶点，通过调控患者TMEM16A的表达量以缓解患者的骨质疏松病症。
在一些优选的实施方式中，所述药物靶向作用于破骨细胞。一方面，破骨细胞异常是导致骨骼发生病变的主要原因，另一方面，由于TMEM16A还分布于血管上皮细胞、胰腺上皮细胞、唾液腺上皮细胞和支气管粘膜下颌下腺腺泡、乳腺和肾小管等多处细胞和组织，针对性的抑制破骨细胞中的TMEM16A表达可以避免药物对位于其他细胞和组织的TMEM16A的干扰，降低药物的副作用。
在一些优选的实施方式中，所述药物含有抑制TMEM16A表达的活性成分，以下调患者TMEM16A的表达，本申请不限制抑制TMEM16A表达的活性成分，本领域中可接受的抑制或干扰基因表达的活性成分都可以作为本申请药物的活性物质。
在一些优选的实施方式中，所述药物以抑制TMEM16A基因表达的小干扰RNA(siRNA)作为活性成分，siRNA是一个长20到25个核苷酸的双股RNA，能够干扰靶基因的表达，从而达到抑制靶基因表达的目的。所述siRNA作为活 性成分中的siRNA可以单独存在，也可以整合于生物材料中，当以含有能够表达抑制TMEM16A基因表达的siRNA的生物材料作为活性成分时，药物中作为活性成分的生物材料能够表达抑制TMEM16A基因表达的siRNA，以抑制TMEM16A基因表达以达到治疗作用。所述生物材料包括但不限于为能够表达抑制TMEM16A基因表达的siRNA的基因盒、重组载体、重组微生物和重组细胞中的一种或者多种。
可以理解的是，上述药物的剂型例如可以为但不限于为注射剂、片剂、粉剂、粒剂、胶囊或口服液，本申请对此不做限制。并且所述药物还可以根据其剂型选择合适的药学领域可接受的辅料，可选地，当所述药物为注射剂，药物的辅料例如可以为但不限于为缓冲液、冻干保护剂和防腐剂中的至少一种；当所述药物为口服剂，药物的辅料例如可以为但不限于为崩释剂、赋形剂和填充剂中的至少一种；当所述药物为凝胶剂，所述药物中可以含有水凝胶，以直接填充至患处。
为了有助于更清楚的理解本申请的内容，现结合具体的实施例详细介绍如下。如无特别说明，本申请实施例中使用的实验动物、药品及试剂均来源为正规而易购渠道：
实施例1
本实施例测量了骨质疏松病人和正常人的破骨细胞中TMEM16A(氨基酸序列如SEQ ID NO.5所示；核苷酸序列如SEQ ID NO.6所示)的表达量，通过微创手术从骨质疏松病人中提取微量骨组织，从中分离出原代破骨细胞，使用完全培养基(DMEM-α培养基：胎牛血清＝9：1)进行细胞培养。通过PCR实验，分析骨质疏松病人破骨细胞中TMEM16A的RNA含量与正常人相比的变化。使用TMEM16A抗体进行细胞免疫印迹实验，比较分析骨质疏松病人破骨细胞中TMEM16A表达量与正常人表达量的变化。结果如图1所示，发现骨质疏松病人骨组织中TMEM16A表达量比正常人升高约1.5倍，因此TMEM16A表达量升高是骨质疏松的一个标志物。
实施例2
本实施例首先建立了小鼠骨质疏松模型，然后测量了骨质疏松小鼠骨组织的TMEM16A含量。
小鼠骨质疏松模型建立方法：选择3-6月龄的雌性C57小鼠，雌性小鼠通过手术人为摘除雌性小鼠卵巢，正常培养四周造模，模拟女性绝经后由于雌性激素缺乏导致的骨质疏松；雄性小鼠为选择2-4个月月龄的雄性C57小鼠，通过手术人为摘除雌性小鼠睾丸，正常培养四周造模，模拟老年男性由于雄性激素分泌大幅减少导致的老年性骨质疏松症。
TMEM16A表达量的测量方法主要有：通过PCR测量TMEM16A的RNA含量变化，主要包括：Trizol法提取总RNA，逆转录反应，PCR反应，电泳实验。通过免疫印迹实验测量TMEM16A蛋白表达量的变化，主要包括：收集蛋白样品，蛋白含量测定，SDS-PAGE电泳，转膜，封闭，孵育抗体，曝光检测。
结果如图2所示，发现患有骨质疏松小鼠骨组织的TMEM16A含量显著升高，比正常小鼠高约4倍，因此进一步确认了TMEM16A含量升高是骨质疏松的标志物。
实施例3
本实施例在体外对破骨细胞进行诱导分化，破骨细胞诱导分化方法如下：从6-8周C57小鼠骨髓提取骨髓细胞，红细胞裂解液充分裂解以后离心，提取骨髓基质细胞，加入10ng/ml的M-CSF进行培养，24小时后吸取上清，1500rpm离心，加入含有50ng/ml的Rankl和30ng/ml的M-CSF的培养基继续培养5天，期间更换培养基一次，5天后使用TRAP染色试剂盒检测破骨细胞是否诱导成功。TMEM16A表达量通过PCR和免疫印迹实验方法检测，实验方法同上述实施例。
然后分别检测正常诱导1、3和5天后细胞内的TMEM16A的含量，结果如图3和图4所示，在正常诱导1、3和5天后，细胞内的TMEM16A会发生剧烈的升高，诱导第五天与诱导第一天相比，破骨细胞中TMEM16A含量升高了约13倍，因此TMEM16A含量升高是破骨细胞分化的重要标志，而破骨细胞分化程度越高则患者患有骨质疏松可能性越大。
实施例4
本实施例中，将破骨细胞中TMEM16A基因通过siRNA干扰的方法进行下调，针对TMEM16A基因(GenBank NM_178642)，设计了三组TMEM16A-shRNA慢病毒质粒，分别为CCTGCTAAACAACATCATT(2,399–2,418nt，shRNA1，如SEQ ID NO.1所示)，TCTCATGGTGGAGCTGTTT(2,924–2,943nt，shRNA2，如SEQ ID NO.2所示)，GAGTTATCATCTATAGAAT(1,747–1,766nt，shRNA3，如SEQ ID NO.3所示)，以及对照质粒NC shRNA，TTCTCCGAACGTGTCACGT，如SEQ ID NO.4所示。采用瞬时转染的方法将质粒转入细胞，24孔板每孔转染量为0.5μg慢病毒质粒，1.5μL的转染试剂，50μL的OptiMEM，转让将三者震荡混匀，静置15分钟后加入到正常培养的细胞中。TMEM16A表达量通过PCR和免疫印迹实验方法检测，实验方法同上。
结果如图5所示，发现通过下调TMEM16A基因的表达后，表征破骨细胞分化成骨的相关基因Nfatc1，Acp5，Ctsk，Mmp9，Clcn7都有不同程度的下降，因此我们最终确定了TMEM16A是破骨细胞发病的标志物，通过下调TMEM16A可以抑制破骨细胞的分化，从而抑制骨质疏松的产生。
实施例5使用外周血作为样本
提取50名正常人和50名骨质疏松患者的外周血，RT-PCR方法检测外周血单核细胞中TMEM16A基因的相对表达量。结果如图6所示，50名患者中48人的TMEM16A基因相对表达量较高，而对照组的50名正常人中只有1人的TMEM16A蛋白高表达。
实施例6使用破骨细胞前体细胞作为样本
以破骨细胞前体细胞作为检测样本，检测方法与前文所述破骨细胞完全一致，检测结果如图7所示。与正常人相比，骨质疏松病人的破骨细胞前体细胞中TMEM16A表达显著升高。
最后应说明的是：以上各实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述各实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的范围。

Claims (11)

  1. 一种以TMEM16A作为骨质疏松的标志物的骨质疏松诊断试剂盒，所述骨质疏松诊断试剂盒通过检测受试者外周血单核细胞中TMEM16A的含量来判断受试者是否患有骨质疏松。
  2. 根据权利要求1所述的骨质疏松诊断试剂盒，其中，所述骨质疏松诊断试剂盒的检测样本包括外周血单核细胞。
  3. 根据权利要求1所述的骨质疏松诊断试剂盒，其中，所述骨质疏松诊断试剂盒的检测样本包括破骨细胞前体细胞。
  4. 根据权利要求1-3中任一项所述的骨质疏松诊断试剂盒，其中，当受试者外周血单核细胞中的TMEM16A含量相比于正常值升高时，判定受试者患有骨质疏松。
  5. 根据权利要求1-4任一项所述的骨质疏松诊断试剂盒，其中，当受试者外周血单核细胞中的TMEM16A蛋白的含量至少为正常值的1.5倍时，判定受试者患有骨质疏松。
  6. TMEM16A作为骨质疏松的标志物在制备用于治疗骨质疏松的药物中的应用。
  7. 一种用于治疗骨质疏松的药物，其中，所述药物以TMEM16A作为靶点。
  8. 根据权利要求7所述的药物，其中，所述药物靶向作用于破骨细胞。
  9. 根据权利要求7或8所述的药物，其中，所述药物含有抑制TMEM16A表达的活性成分。
  10. 根据权利要求9所述的药物，其中，所述药物包含抑制TMEM16A基因表达的siRNA作为活性成分。
  11. 根据权利要求10所述的药物，其中，所述抑制TMEM16A基因表达的siRNA包括含有如SEQ ID NO.1所示序列的siRNA、如SEQ ID NO.2所示序列的siRNA和如SEQ ID NO.3所示序列的siRNA中的至少一种。
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