WO2022227449A1 - 骨硬化蛋白及其在制备治疗或预防阿尔兹海默症的相关产品上应用 - Google Patents

骨硬化蛋白及其在制备治疗或预防阿尔兹海默症的相关产品上应用 Download PDF

Info

Publication number
WO2022227449A1
WO2022227449A1 PCT/CN2021/126531 CN2021126531W WO2022227449A1 WO 2022227449 A1 WO2022227449 A1 WO 2022227449A1 CN 2021126531 W CN2021126531 W CN 2021126531W WO 2022227449 A1 WO2022227449 A1 WO 2022227449A1
Authority
WO
WIPO (PCT)
Prior art keywords
sclerostin
protein
disease
alzheimer
treating
Prior art date
Application number
PCT/CN2021/126531
Other languages
English (en)
French (fr)
Inventor
蒋青
郭保生
石天舒
Original Assignee
蒋青
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 蒋青 filed Critical 蒋青
Publication of WO2022227449A1 publication Critical patent/WO2022227449A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a new application in the field of biomedicine, belongs to the field of related drug research of Alzheimer's disease, and specifically relates to sclerostin and its application in preparing related products for treating or preventing Alzheimer's disease.
  • AD Alzheimer's disease
  • cerebral cortex and hippocampus of patients leading to progressive memory loss in patients and seriously affecting the cognitive function of normal elderly people. It brings a serious life burden to patients and a heavy economic pressure to the society.
  • AD Alzheimer's disease
  • the specific mechanism is not very clear, and there is no significantly improved drug treatment.
  • the current pathogenic cause of Alzheimer's disease may be related to the accumulation of plaque-like insoluble amyloid beta (A ⁇ ) in the extracellular space and blood vessel walls, as well as increased neural tangles resulting from the accumulation of microtubule tau protein in neurons .
  • a ⁇ protein is cleaved from APP protein and released into the extracellular environment in the form of diffusible oligomers (A ⁇ O).
  • a ⁇ O can be cleared from the brain mediated by APOE, and it can also be taken up by astrocytes through low-density lipoprotein receptor-related protein 1 (LRP1) to reduce the concentration of A ⁇ O in brain tissue.
  • LRP1 low-density lipoprotein receptor-related protein 1
  • a ⁇ O can also aggregate in the intercellular space to form fibrillar structures, which are then assembled into plaques.
  • a ⁇ plaques can be cleared by macrophages and microglia by phagocytosis, and can also be degraded by endoproteases (IDE, NEP, MMPs) in astrocytes.
  • IDE endoproteases
  • MMPs endoproteases
  • Sclerostin is a glycoprotein encoded, expressed and translated by the sost gene. Before adulthood, it was found that bone cells, liver cells, heart, and vascular endothelial cells can secrete it. It is expressed and secreted in the blood to regulate peripheral bone mass. Previous studies have found that sclerostin is a specific inhibitor of Wnt signaling, which can bind to cell surface LRP5/6 and activate the downstream GSK-3 ⁇ activity, thereby increasing the phosphorylation level of ⁇ -catenin and promoting its protein degradation. to inhibit Wnt signaling. At the same time, the Wnt signaling pathway also plays an important role in regulating neuronal synaptic plasticity and memory function in the adult brain.
  • the present invention provides sclerostin and its application in the preparation of related products for the treatment or prevention of Alzheimer's disease.
  • the amino acid sequence of the sclerostin is shown in SEQ NO.1, It is applied to the preparation of a drug for the preparation of neutralizing antibodies for the treatment and/or prevention of Alzheimer's disease, and it shows a good effect of reducing the expression of BACE1 in neuron cells and inhibiting the production of A ⁇ protein, which is a good method for the treatment and/or prevention of Alzheimer's disease.
  • Alzheimer's disease provides a new option and thinking, broadening the field of options for treating Alzheimer's disease.
  • Sclerostin sclerostin
  • amino acid sequence of said sclerostin is shown in SEQ NO.1.
  • nucleotide sequence encoding the above-mentioned sclerostin is shown in SEQ NO.2.
  • a recombinant plasmid comprising the above-mentioned nucleotide sequence of sclerostin.
  • a recombinant strain is characterized in that the strain expressing the above-mentioned recombinant plasmid.
  • the above-mentioned sclerostin, recombinant plasmids or recombinant strains are used in the preparation of neutralizing antibodies for treating or preventing Alzheimer's disease.
  • a neutralizing antibody characterized in that its active ingredient is the above-mentioned sclerostin, recombinant plasmid or recombinant strain.
  • a product for treating or preventing Alzheimer's disease comprising the above-mentioned sclerostin, recombinant plasmid, recombinant strain or neutralizing antibody as active ingredients; the product is a pharmaceutical composition or a health care product.
  • the dosage form of the pharmaceutical composition is powder for injection, injection for injection or powder for powder.
  • sclerostin of the present invention is a protein that regulates bone mass secreted by osteocytes.
  • the inventor synthesized recombinant protein by copying the nucleotide sequence of sclerostin, and then verified through cell and animal experiments, and found that sclerostin protein can accelerate the production of A ⁇ protein outside the brain tissue and accelerate cognitive function decline. After the neutralizing antibody, it has the effect of relieving Alzheimer's disease by significantly inhibiting the expression of BACE1 protein, thereby inhibiting the production of A ⁇ protein.
  • the neutralizing antibody prepared based on sclerostin has the effect of treatment and/or prevention, and can be used to prepare medicines or health products for the treatment and/or Alzheimer's disease, which is the treatment and/or prevention of Alzheimer's disease. It provides a brand new option and thinking, broadening the field of choice for the treatment of Alzheimer's disease.
  • Figure 1 shows that increasing bone-derived sclerostin protein significantly increases amyloid deposition and accelerates cognitive decline in mice with Alzheimer's disease.
  • A After injection of bone-targeted overexpression of sost virus, the level of sclerostin protein in mouse serum Significantly increased;
  • B-C In Alzheimer's disease mouse model APP/PS1 mice, increasing the concentration of sclerostin protein in serum significantly increased amyloid deposition pattern (B) and statistical graph (C);
  • D-G In APP/PS1 mice, a mouse model of Alzheimer's disease, increasing the concentration of sclerostin protein in serum significantly increased the production of A ⁇ 1-40 and 1-42 proteins in the cerebral cortex and hippocampus; In the APP/PS1 mouse model of Alzheimer's disease, increasing the concentration of sclerostin protein in serum significantly damaged the spatial memory ability of Alzheimer's disease mice; In the mouse model APP/PS1 mice, increasing the concentration of sclerostin protein in serum significantly impaired the ability of Alzheimer's mice to
  • Figure 2(A) shows the changes of intracellular BACE1 mRNA levels after 24 hours and 48 hours after adding sclerostin recombinant protein to neuronal cell line N2a;
  • Figure 2(B) shows the expression of BACE1 protein in neuronal N2a cells after adding 200ng/ml of recombinant sclerostin to N2a cells;
  • Figure 2(C) shows that in N2a cells, adding 200ng/ml of recombinant sclerostin can significantly promote the luciferase activity of BACE1;
  • Figure 2(D) shows that the addition of 200ng/ml sclerostin recombinant protein to the Alzheimer's neuron cell line N2a-APP cells significantly promoted the protein levels of the protein fragments sAPP ⁇ and ⁇ -CTF during the production of A ⁇ protein;
  • Figure 2(E) shows the production of A ⁇ 1-40 protein by adding 200ng/ml of sclerostin recombinant protein to Alzheimer's neuron cell line N2a-APP cells;
  • Figure 2(F) shows the production of A ⁇ 1-42 protein by adding 200ng/ml sclerostin recombinant protein to the Alzheimer's neuron cell line N2a-APP cells;
  • Figure 3(A) shows the production of BACE1 protein in neuronal cell N2a by adding sclerostin neutralizing antibody in the co-culture of neuronal cell line N2a cells and osteocyte MLO-Y4;
  • Figure 3(B) shows the co-culture of neuronal cell line N2a-APP cells and osteocytes MLO-Y4 in Alzheimer's disease cell model, adding sclerostin neutralizing antibody, and the A ⁇ protein production process in N2a-APP cells. Protein fragment sAPP ⁇ and ⁇ -CTF protein levels;
  • Figure 3(C) shows the changes of A ⁇ 1-40 protein level in the culture medium by adding sclerostin neutralizing antibody in the co-culture of neuronal cell line N2a-APP cells and bone cells MLO-Y4 in Alzheimer's disease cell model;
  • Figure 3(D) shows the changes of A ⁇ 1-42 protein level in the culture medium by adding sclerostin neutralizing antibody in the co-culture of neuronal cell line N2a-APP cells and bone cells MLO-Y4 in Alzheimer's disease cell model .
  • amino acid sequence of the chemically synthesized mouse sclerostin with a purity greater than 98% is shown in SEQ NO.1:
  • the synthesized sclerostin powder was dissolved in double-distilled water to prepare a solution with a concentration of 1 ⁇ g/ ⁇ l, which was then stored at -80° C. for later use. When in use, dilute it with double distilled water to a corresponding concentration and save it for later use. In this example, it was diluted to 200ng/ml.
  • mice According to the amino acid sequence of mouse sclerostin, a neutralizing antibody that can be injected into mice is constructed.
  • the specific construction method is as follows:
  • Quality control using polyacrylamide gel electrophoresis (SDS-PAGE) method, purity >75%.
  • a conventional immunization protocol was used, and the target protein-Immunoplus was used to immunize 3 BALB/C mice + 3 C57 mice;
  • the routine immunization schedule is shown in the table below.
  • Cell fusion and plating 2 rounds of cell fusion were performed using the electrofusion method.
  • the average fusion efficiency is that about 2500 spleen cells can be fused to produce 1 hybridoma cell, so it is expected that the number of hybridoma cells can reach about 2 ⁇ 10 4 .
  • All cells from each round of fusion will be plated into 96-well plates, up to a maximum of 15 96-well plates.
  • Primary screening Screen the supernatant of the fusion cells by indirect ELISA, and select the supernatant that is positive for the target protein.
  • the specific screening plan is as follows:
  • the positive parent clones were transferred to 24-well plates for expansion, up to 10 strains. 2 ml of supernatant was collected from each expanded clone for indirect ELISA detection. All specific positive clones were cryopreserved to avoid loss of clones.
  • Subcloning Subcloning the positive parent clones (up to 5 strains) by limiting dilution method to ensure that these positive parent clones are derived from a single parent clone cell. A maximum of 3 rounds of subcloning were performed. The general success rate of subcloning is 80%. Positive losses may occur at the subcloning stage. (If the obtained positive parent clones cannot meet the specificity requirements, other parent clones will be additionally selected for re-subcloning).
  • Subclonal Screening was performed using indirect ELISA and blocking ELISA methods.
  • Cryopreservation of monoclonal cells 2 stable daughter clones were selected from each parent clone for expansion and cryopreservation. 5 ml of supernatant was collected from each subclone before cryopreservation. Each subclone provides 2 tubes of cells.
  • Antibody production Small-scale antibody production using established cells and in vitro culture.
  • Antibody purification The antibody was purified by Protein A/G affinity chromatography, and the purified antibody was stored in phosphate buffered saline (PBS) by dialysis.
  • PBS phosphate buffered saline
  • QC detection was performed by polyacrylamide gel electrophoresis (SDS-PAGE), indirect ELISA and blocking ELISA, and antibody concentration was determined on NanoDrop 2000.
  • a sost overexpression plasmid was constructed and combined with the relatively mature bone-targeting virus AAV9-DSS-Nlter (standard reference: Yang, Y.S., et al. (2019)."Bone -targeting AAV-mediated silencing of Schnurri-3prevents bone loss in osteoporosis.”Nat Commun 10(1):2958.), construct a sost virus called bone-targeting overexpression, and use it to enter mice for relevant experimental verification.
  • the target gene is obtained by PCR or enzyme digestion, constructed into the target vector by ligation or homologous recombination/assembly, and transformed into E.coli DH5 ⁇ competent cells. Transformants were screened by colony PCR, and positive clones were sent for sequencing. Validated clones were subjected to plasmid extraction. In this example, PCR was used for construction.
  • the forward primer is shown in SEQ NO.2: CGCAAATGGGGCGGTAGGCGTG;
  • the reverse primer is shown in SEQ NO.3: CATAGCGTAAAAGGAGCAACA;
  • the PCR product was subjected to agarose gel electrophoresis to detect the amplification effect, and the target gene band was cut from the gel after agarose gel electrophoresis.
  • TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0 was used as a gel for recovery.
  • the expression plasmid sequence is shown in SEQ NO.4:
  • the optimal amount of insert used [0.02x base number of insert]ng (0.03pmol),
  • Optimum amount of linearized carrier used [0.02 ⁇ number of linearized carrier bases]ng(0.03pmol),
  • mice In order to verify whether sclerostin is involved in the pathogenesis of Alzheimer's disease, we constructed a bone-targeted overexpression sost plasmid virus and injected it into 5-month-old APP/PS1 mice at a concentration of 4*10 11 through the tail vein. The mice were kept in single cages for 3 months, and the relevant experimental analysis was carried out. It mainly includes sclerostin concentration in serum, ⁇ protein concentration in mouse brain tissue, mouse behavioral testing (including water maze, cognition of new things), synaptic plasticity and integrity testing, etc. Affecting ⁇ protein production alters cognitive function in mice.
  • sclerostin recombinant protein 200ng/ml sclerostin recombinant protein was added to the neuron cell line N2a cell culture medium for detection (the detection method was referred to Liu, C.C., et al. (2014). "Deficiency in LRP6-mediated Wnt signaling contributes to synaptic abnormalities and amyloid pathology in Alzheimer's disease.”Neuron 84(1):63-77.Parr,C.,et al.(2015).”Activation of the Wnt/beta-catenin pathway represses the transcription of the beta-amyloid precursor protein cleaving enzyme(BACE1 ) via binding of T-cell factor-4 to BACE1 promoter.”FASEB J 29(2):623-635.) Intracellular content of ⁇ -catenin protein and BACE1 protein.
  • the Luciferase activity after the addition of sclerostin was detected to determine the transcription of BACE1 mRNA by sclerostin.
  • sclerostin could significantly reduce the intracellular ⁇ -catenin protein level and increase the BACE1 protein expression (Fig. 2(B)).
  • sclerostin increased BACE1 transcript level by Luciferase (Fig. 2(C)).
  • Fig. 2(D) we constructed the Alzheimer's disease cell line N2a-APP cells, and found that the addition of sclerostin recombinant protein could significantly promote the protein levels of protein fragments sAPP ⁇ and ⁇ -CTF during the production of A ⁇ protein.
  • the culture medium was tested and found that after adding sclerostin recombinant protein, the concentration of A ⁇ 1-40 and A ⁇ 1-42 proteins in the medium increased significantly.
  • MEM Hyclone, SH30023.01B
  • fetal bovine serum Gibco: 10099-141
  • Polybrene Sigma: H9268
  • Puromycin Sigma: P8833
  • 24-well plate Corning: 3524
  • 6-well plate Corning: 3516
  • Neuro-2a cells were seeded into 6 cm dishes at 30% confluency.
  • Neuro-2a cells were prepared into a cell suspension of 0.33 ⁇ 10 5 cells/ml and were to be plated.
  • the amount of virus is shown in the table below.
  • Construct Alzheimer's disease cell model and N2a-APP cell line add 200ng/ml sclerostin recombinant protein to neuronal cell line N2a cell culture medium, extract N2a-APP cell protein, and detect protein fragments in the process of A ⁇ protein production
  • ⁇ -CTF, sAPP ⁇ protein, and the concentration of A ⁇ 1-40 and A ⁇ 1-42 proteins in the medium are measured by ELISA of R&D Company, catalog number: DAB142, DAB140B).
  • the bone cell line MLO-Y4 was co-cultured with the neuron cell line N2a cells, and 200ng/ml of The sclerostin neutralizing antibody was placed in an incubator for 24 hours, and the N2a cell protein was extracted. Western Blot technology was used to detect the expression of BACE1 protein. The results are shown in Figure 3(A). The addition of the antibody significantly reduced the expression of BACE1 protein in N2a cells.
  • the bone cell line MLO-Y4 was co-cultured with the constructed Alzheimer's disease cell model and N2a-APP cell line. 200ng/ml sclerostin neutralizing antibody was added to + streptomycin), and after 24 hours of incubation, N2a-APP cell protein was extracted to detect the expression of BACE1 protein and protein fragments such as ⁇ -CTF, sAPP ⁇ protein in the process of A ⁇ protein production (detection Using Western Blot technology), and A ⁇ 1-40 and A ⁇ 1-42 protein concentrations in the culture medium (detected using Elisa technology, product company R&D), so as to clarify the effect of sclerostin neutralizing antibody on the production process of A ⁇ protein.
  • BACE1 protein and protein fragments such as ⁇ -CTF, sAPP ⁇ protein in the process of A ⁇ protein production (detection Using Western Blot technology), and A ⁇ 1-40 and A ⁇ 1-42 protein concentrations in the culture medium (detected using Elisa technology, product company R&D), so
  • Sclerostin can significantly promote the production of A ⁇ protein and accelerate the decline of cognitive function both at the animal and cellular levels.
  • the neutralizing antibody constructed based on sclerostin recombinant protein can reduce the level of BACE1 protein in neurons at the cellular level, thereby inhibiting the production of A ⁇ protein, and has the effect of alleviating and/or treating Alzheimer's disease.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Food Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Psychiatry (AREA)
  • Hospice & Palliative Care (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Polymers & Plastics (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

属于阿尔兹海默症的相关药物研究领域，具体公开了骨硬化蛋白及其在制备治疗或预防阿尔兹海默症的相关产品上应用。骨硬化蛋白作为一种骨细胞分泌的调控骨量的蛋白，其氨基酸序列如SEQ NO.1所示。通过合成骨硬化蛋白重蛋白以及相应的中和抗体，再通过细胞、动物学实验验证，sclerostin重组蛋白可以加速脑组织外Aβ蛋白的产生，加速认知功能衰退；在加入中和抗体之后，通过显著抑制BACE1蛋白表达，抑制Aβ蛋白的产生，具有缓解阿尔滋海默症的作用。基于sclerostin制备的中和抗体具有治疗和/或预防的作用，可被用于制备治疗和/或阿尔兹海默症的药物或保健品，这为治疗和/或预防阿尔兹海默症提供了一种全新的选择和思路，拓宽了治疗阿尔兹海默症的选择领域。

Description

骨硬化蛋白及其在制备治疗或预防阿尔兹海默症的相关产品上应用 技术领域
本发明涉及生物医药领域的新用途，属于阿尔兹海默症的相关药物研究领域，具体涉及骨硬化蛋白(sclerostin)及其在制备治疗或预防阿尔兹海默症的相关产品上应用。
背景技术
阿尔兹海默症(Alzheimer's Disease，AD)是一种逐渐加重的神经退行性疾病，广泛影响到患者的大脑皮质和海马区域，导致患者进行性记忆减退，严重影响正常老年人的认知功能，给患者带来严重的生活负担，也给社会带来沉重的经济压力。到2018年，全球大约有0.44亿人患有阿尔兹海默症，而这个数字将会在2050年达到1.5亿人，然而其中具体机制不是很清楚，并且尚无显著改善的药物治疗。
目前对于阿尔兹海默症的致病原因可能与细胞外空间和血管壁上的斑块状不溶淀粉状蛋白β(Aβ)的积累以及神经元中微管Tau蛋白聚集导致的神经缠结增加有关。Aβ蛋白是由APP蛋白上裂解下来，并以可扩散的寡聚物(AβΟ)的方式释放到细胞外环境中。AβO可以通过APOE介导从大脑中清除，也可以通过低密度脂蛋白受体相关蛋白1(LRP1)被星形胶质细胞摄取从而降低脑组织中AβO的浓度。此外，AβO还可以在细胞间隙中聚集形成原纤维结构，然后将其组装成斑块。Aβ斑块可通过吞噬作用被巨噬细胞和小胶质细胞清除，也可以在星形胶质细胞的内切蛋白酶(IDE、NEP、MMPs)作用下降解。然而目前研究认为，在阿尔兹海默症临床前期，就可以出现脑脊液中AβO清除效率降低，增加脑脊液AβO浓度，这可能与这些降解蛋白酶功能失调有关。此外，并不是所有AβO都可以被清除，一些从Aβ原纤维解离的低聚物可能未被清除，并且对相邻的突触有一定毒性，促使微管相关Tau蛋白的过度磷酸化和聚集，使其失去了与微管结合和稳定微管的作用，聚集形成成对螺旋丝，加重认知功能的退变。
骨硬化蛋白(sclerostin)是由sost基因编码表达翻译的糖蛋白，在成年以前，发现骨细胞、肝脏细胞、心脏、血管内皮细胞都可以分泌，在成年以后，sost基因主要由骨细胞、软骨细胞表达，分泌于血液中调控外周骨量。之前的研究发现，sclerostin是Wnt信号的特异性抑制因子，可以与细胞表面LRP5/6结合，激活了下游的GSK-3β活性，从而增加β-catenin的磷酸化水平，促进其蛋白降解，从而起到抑制Wnt信号作用。同时Wnt信号通路在成人大脑中调控神经元突触可塑性和记忆功能中也起到重要作用，之前的研究发现在家族性PS1突变的家族性AD患者中，β-catenin蛋白表达显著下降，且在前脑神经元细胞中特异性敲除LRP6受体，可以显著损伤突触可塑性，增加Aβ蛋白的表达，这可能与Wnt信号受到抑制时，激活BACE1蛋白的表达，促进Aβ蛋白剪切体的形成。因此，提示sclerostin可能在阿尔兹海默症发病过程中以及Aβ蛋白产生过程中起到重要作用。
发明内容
针对现有技术的不足，本发明提供了骨硬化蛋白(sclerostin)及其在制备治疗或 预防阿尔兹海默症的相关产品上应用，该骨硬化蛋白的氨基酸序列如SEQ NO.1所示，将其应用到制备治疗和/或预防阿尔兹海默症的中和抗体的药物中，表现出较好的减少神经元细胞BACE1表达，以及抑制Αβ蛋白产生的作用，为治疗和/或预防阿尔兹海默症提供了一种全新的选择和思路，拓宽了治疗阿尔兹海默症的选择领域。
为解决现有技术问题，本发明采取的技术方案为：
骨硬化蛋白(sclerostin)，所述的骨硬化蛋白的氨基酸序列如SEQ NO.1所示。
作为改进的是，编码上述骨硬化蛋白的核苷酸序列如SEQ NO.2示。
一种重组质粒，含有上述的骨硬化蛋白的核苷酸序列的质粒。
一种重组菌株，其特征在于，表达上述的重组质粒的菌株。
上述骨硬化蛋白、重组质粒或重组菌株在制备治疗或预防阿尔兹海默症的中和抗体上应用。
一种中和抗体，其特征在于，其有效成分为上述的骨硬化蛋白、重组质粒或重组菌株。
一种治疗或预防阿尔兹海默症的产品，包含上述骨硬化蛋白、重组质粒、重组菌株或中和抗体作为活性成分；所述产品为药物组合物或保健品。
作为改进的是，所述药物组合物的剂型为注射粉针、注射针剂或散剂粉末。
有益效果：
与现有技术相比，本发明骨硬化蛋白(sclerostin)及其在制备治疗或预防阿尔兹海默症的相关产品上应用，骨硬化(sclerostin)作为一种骨细胞分泌的调控骨量的蛋白，发明人通过复制骨硬化蛋白的核苷酸序列合成重组蛋白，再通过细胞、动物学实验验证，发现sclerostin蛋白可以加速脑组织外Aβ蛋白的产生，加速认知功能衰退，同时，在加入合成的中和抗体之后，通过显著抑制BACE1蛋白表达，从而抑制Aβ蛋白的产生，具有缓解阿尔滋海默症的作用。可见，基于sclerostin制备的中和抗体具有治疗和/或预防的作用，可被用于制备治疗和/或阿尔兹海默症的药物或保健品，这为治疗和/或预防阿尔兹海默症提供了一种全新的选择和思路，拓宽了治疗阿尔兹海默症的选择领域。
附图说明
图1为增加骨源性sclerostin蛋白显著增加淀粉样蛋白沉积，加速阿尔兹海默症小鼠认知功能衰退，(A)在打入骨靶向过表达sost病毒后，小鼠血清中sclerostin蛋白水平显著增加；(B-C)在阿尔兹海默症小鼠模型APP/PS1小鼠中，增加血清中sclerostin蛋白浓度，显著增加淀粉样蛋白沉积模式图(B)以及统计图(C)；(D-G)在阿尔兹海默症小鼠模型APP/PS1小鼠中，增加血清中sclerostin蛋白浓度，显著增加大脑皮质和海马区Aβ1-40、1-42蛋白产生；(H)水迷宫检测发现，在阿尔兹海默症小鼠模型APP/PS1小鼠中，增加血清中sclerostin蛋白浓度,显著破坏阿尔兹海默症小鼠空间记忆能力；(I)新事物认知检测发现，在阿尔兹海默症小鼠模型APP/PS1小鼠中，增加血清中sclerostin蛋白浓度，显著破坏阿尔兹海默症小鼠对新事物探索的能力；
图2(A)为在神经元细胞系N2a中，加入sclerostin重组蛋白，在24小时和48小时后，细胞内BACE1 mRNA水平改变情况；
图2(B)为在N2a细胞中，加入200ng/ml的重组sclerostin后，神经元细胞N2a中的 BACE1蛋白表达情况；
图2(C)为在N2a细胞中，加入200ng/ml的重组sclerostin可以显著促进BACE1的luciferase活性变化情况；
图2(D)为在阿尔兹海默症神经元细胞系N2a-APP细胞中，加入200ng/ml的sclerostin重组蛋白，显著促进了Aβ蛋白产生过程中的蛋白片段sAPPβ和β-CTF蛋白水平；
图2(E)为在阿尔兹海默症神经元细胞系N2a-APP细胞中，加入200ng/ml的sclerostin重组蛋白，Aβ1-40蛋白的产生情况；
图2(F)为在阿尔兹海默症神经元细胞系N2a-APP细胞中，加入200ng/ml的sclerostin重组蛋白，Aβ1-42蛋白产生情况；
图3(A)为在神经元细胞系N2a细胞和骨细胞MLO-Y4共培养中，加入sclerostin中和抗体，神经元细胞N2a中的BACE1蛋白产生情况；
图3(B)为在阿尔兹海默症细胞模型的神经元细胞系N2a-APP细胞和骨细胞MLO-Y4共培养中，加入sclerostin中和抗体，N2a-APP细胞中Aβ蛋白产生过程中的蛋白片段sAPPβ和β-CTF蛋白水平；
图3(C)为在阿尔兹海默症细胞模型的神经元细胞系N2a-APP细胞和骨细胞MLO-Y4共培养中，加入sclerostin中和抗体，培养基中Aβ1-40蛋白水平的变化；
图3(D)为在阿尔兹海默症细胞模型的神经元细胞系N2a-APP细胞和骨细胞MLO-Y4共培养中，加入sclerostin中和抗体，培养基中Aβ1-42蛋白水平的含量变化。
具体实施方式
下面结合附图和具体实施例对本发明作进一步描述。
实施例1Sclerostin准备
化学合成纯度大于98％的小鼠骨硬化蛋白(sclerostin)的氨基酸序列如SEQ NO.1所示：
Figure PCTCN2021126531-appb-000001
将合成的骨硬化蛋白(sclerostin)粉末溶于双蒸水中，配制浓度为1μg/μl溶液，分装后-80℃的低温保存备用。使用时用双蒸水稀释成相应浓度保存备用，本实施例中稀释成200ng/ml。
实施例2基于sclerostin重组蛋白构建sclerostin中和抗体
根据小鼠骨硬化蛋白(sclerostin)氨基酸序列，构建可以打入小鼠体内的中和抗体，具体构建方法如下：
材料准备阶段：免疫原验证
免疫原制备：根据实施例1构建的小鼠骨硬化蛋白作为免疫原，采用SDS-PAGE方法检验蛋白质抗原。负责将其偶联Immunoplus作为免疫原，用于打破小鼠的免疫耐受。
质量控制(QC)：采用聚丙烯酰胺凝胶电泳(SDS-PAGE)方法，纯度>75％。
第一阶段：动物免疫SC2033-PF
选用常规免疫方案，采用靶蛋白-Immunoplus免疫3只BALB/C小鼠+3只C57小鼠；
常规方案免疫计划表具体下表所示。
Figure PCTCN2021126531-appb-000002
Figure PCTCN2021126531-appb-000003
采血检测：每次免疫7天后，用间接ELISA方法检测免疫动物血清，以此确定免疫应答的水平。
饲养：小鼠饲养
常规免疫第一阶段结束后，如果免疫动物能够达到针对免疫原的免疫应答水平(OD值>1.0，效价达到1:8,000)，则进入第二阶段。
第二阶段：细胞融合和筛选
细胞融合和铺板：采用电融合方法进行2轮细胞融合。平均融合效率是用大约2500个脾细胞就能融合产生1个杂交瘤细胞，因此预计杂交瘤细胞数目可以达到约2×10 4个。每轮融合的所有细胞都将被铺到96孔板中，至多铺15块96孔板。
初筛：用间接ELISA方法筛选融合细胞的上清液，挑选出针对靶蛋白呈阳性的上清。
具体筛选方案如下：
Figure PCTCN2021126531-appb-000004
确认筛选：对初筛阶段得到的所有阳性克隆，采用间接ELISA方法筛选所有的阳性母克隆细胞上清，针对阻断物受体有较好的阻断效果。
克隆扩大培养和冻存：
将阳性母克隆细胞转到24孔板扩大培养，最多10株。每个扩大培养的克隆收集2ml上清，用于间接ELISA检测。冻存所有特异性阳性克隆细胞，以避免克隆丢失。
第三阶段：亚克隆，扩大培养和低温保存
亚克隆：采用有限稀释法对阳性母克隆进行亚克隆(最多选取5株)，以确保这些阳性母克隆分别来自于单个母克隆细胞。最多进行3轮亚克隆。一般亚克隆的成功率在80％。亚克隆阶段可能出现阳性丢失。(如果得到的阳性母克隆不能够满足特异性要求，将额外选择其它母克隆重新进行亚克隆)。
亚克隆筛选：用间接ELISA和阻断ELISA方法进行亚克隆筛选。
单克隆细胞低温保存：每个母克隆选择2个稳定的子克隆进行扩增和低温保存。低温冻存前每个子克隆收集5ml上清。每个子克隆提供2管细胞。
第四阶段：小规模抗体生产
抗体生产：采用定株细胞进行小规模抗体生产，体外培养。
抗体纯化：采用Protein A/G亲和层析方法纯化抗体，用透析方法将纯化抗体保存在磷酸盐缓冲液(PBS)中。
质量控制(QC)：用聚丙烯酰胺凝胶电泳(SDS-PAGE)，间接ELISA和阻断ELISA进行QC检测，在NanoDrop2000测定抗体浓度。
实施例3
根据骨硬化蛋白(sclerostin)的氨基酸序列，构建sost过表达质粒，并与目前较为成熟的骨靶向病毒AAV9-DSS-Nlter结合(标准参考：Yang,Y.S.,et al.(2019)."Bone-targeting AAV-mediated silencing of Schnurri-3prevents bone loss in osteoporosis."Nat Commun 10(1):2958.)，构建称为骨靶向过表达sost病毒，用于打入小鼠体内进行相关实验验证。
通过PCR或者酶切获得目的基因，通过连接或者同源重组/组装构建入目的载体，转化E.coli DH5α感受态细胞。用菌落PCR对转化子进行筛选，阳性克隆送测序。经验证无误的克隆进行质粒抽提。本实施例中采用PCR的方式进行构建。
从GenBank中查询目的基因以及上、下游的序列，用VectorNTI软件进行引物设计，引物的序列如下所示：
正向引物如SEQ NO.2所示：CGCAAATGGGCGGTAGGCGTG；
反向引物如SEQ NO.3所示：CATAGCGTAAAAGGAGCAACA；
使用高保真的PrimeSTAR酶扩增目的基因，反应体系和条件如下。
①.按照下列组分配置PCR反应液：
Figure PCTCN2021126531-appb-000005
Figure PCTCN2021126531-appb-000006
PCR产物进行琼脂糖凝胶电泳检测扩增效果，并把目的基因条带从琼脂糖凝胶电泳后的胶中割下来，用TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0做胶回收，所得sost过表达质粒序列如SEQ NO.4所示所示：
Figure PCTCN2021126531-appb-000007
③、使用无缝组装试剂盒(多个目的基因插入片段适用)，将目的基因构建入线性化表达载体中，即将目的基因片段和线性化载体以摩尔比1:1加到离心管中进行重组反应：
Figure PCTCN2021126531-appb-000008
Figure PCTCN2021126531-appb-000009
最适插入片段使用量＝[0.02x插入片段碱基数]ng(0.03pmol)，
最适线性化载体使用量＝[0.02x线性化载体碱基数]ng(0.03pmol)，
混匀后在37℃孵育30分种，然后转移到冰上放置5分钟。直接转化或者置于-20℃保存等需要转化时解冻转化。
实施例4
为了验证sclerostin是否参与阿尔滋海默症发病过程，我们将构建骨靶向过表达sost质粒病毒通过尾静脉以4*10 11浓度打入5月龄APP/PS1小鼠体内，将APP/PS1小鼠以单笼饲养3个月，进行相关实验分析。主要包括血清中sclerostin浓度，小鼠脑组织Αβ蛋白浓度，小鼠行为学检测(包括水迷宫、新事物认知)、突触可塑性和完整性检测等，以明确增加血清中sclerostin之后，是否可以影响Αβ蛋白产生改变小鼠认知功能。
我们对通过尾静脉注射骨靶向sost过表达病毒的APP/PS1阿尔兹海默症小鼠血清检测发现，打入sost过表达病毒后，小鼠血清中骨硬化蛋白(sclerostin浓度)显著增加(如图1(A))。之后检测小鼠皮质、海马区中Αβ蛋白沉积进行检测，发现在APP/PS1小鼠中，增加血清中sclerostin蛋白浓度可以显著增加皮质和海马区淀粉样蛋白沉积现象(图1(B-G))。我们对不同组小鼠进行行为学检测发现(检测方法和标准参照Vorhees,C.V.and M.T.Williams(2006)."Morris water maze:procedures for assessing spatialand related forms of learning and memory."Nat Protoc 1(2):848-858.Leger,M.,et al.(2013)."Object recognition test in mice."Nat Protoc 8(12):2531-2537.)，增加血清中sclerostin可以显著破坏小鼠的空间记忆(图1(H))、新事物认知(图1(I))。这表明增加血清中的sclerostin蛋白通过促进淀粉样蛋白形成和沉积，进而加速阿尔兹海默症小鼠认知功能衰退。
实施例5
将200ng/ml sclerostin重组蛋白加入神经元细胞系N2a细胞培养基中，检测(检测方法参照Liu,C.C.,et al.(2014)."Deficiency in LRP6-mediated Wnt signaling contributes to synaptic abnormalities and amyloid pathology in Alzheimer's disease."Neuron 84(1):63-77.Parr,C.,et al.(2015)."Activation of the Wnt/beta-catenin pathway represses the transcription of the beta-amyloid precursor protein cleaving enzyme(BACE1)via binding of T-cell factor-4to BACE1 promoter."FASEB J 29(2):623-635.)β-catenin蛋白、BACE1蛋白的细胞内含量。之后通过对N2a细胞内转入β-catenin过表达质粒和BACE1 promoter质粒，进行在sclerostin加入后的Luciferase活性检测，明确sclerostin对BACE1 mRNA转录情况。
我们采用神经元细胞系N2a细胞进行细胞检测，在细胞中加入重组的骨硬化蛋白(sclerostin)，分别在24小时、48小时后检测细胞内BACE1 mRNA水平改变情况，发现加入不同浓度sclerostin蛋白可以显著增加BACE1 mRNA转录水平如图2(A)所示，从图中可以看出，发现加入100ng/ml和200ng/ml的sclerostin重组蛋白可以显著促进BACE1 mRNA水平Sclerostin重组蛋白可以显著促进BACE1蛋白表达，促进Aβ蛋白产生。之后对N2a细胞内蛋白进行检测，进一步明确sclerostin可以显著降低细胞内β-catenin蛋白水平，增加BACE1 蛋白表达(图2(B))。之后我们通过Luciferase明确sclerostin增加BACE1转录水平(图2(C))。之后我们构建阿尔兹海默症细胞系N2a-APP细胞，发现加入sclerostin重组蛋白后，可以显著促进Αβ蛋白产生过程中的蛋白片段sAPPβ和β-CTF蛋白水平(图2(D))。对培养基进行检测发现，加入sclerostin重组蛋白后，培养基中Αβ1-40、Αβ1-42蛋白浓度显著增加。
上述使用所用N2a-APP细胞系的构建方法如下：
一、实验材料：
1.细胞株和慢病毒：
病毒(pRLenti-CMV-MCS-3FLAG-PGK-Puro，和元构建：GL102)；
病毒(pRLenti-CMV-APP695swe(K595N/M596L)-3FLAG-PGK-Puro，和元构建：H13319)；Neuro-2a细胞来源于库存，
培养基：MEM+10％FBS，37℃，5％CO 2培养。
2.主要试剂耗材：
MEM(Hyclone,SH30023.01B)；胎牛血清(Gibco：10099-141)；Polybrene(Sigma:H9268)；Puromycin(Sigma:P8833)；24孔板(Corning:3524)；6孔板(Corning:3516)；60mm dish(Corning:430166)；100mm dishCorning:430167)
3.主要仪器设备：
荧光显微镜(DMI8，LEICA)；CO 2培养箱(311，Thermo)；生物安全柜(BSC-Ⅱ-A2，上海净化)。
二、实验步骤：
1、细胞铺板：
将Neuro-2a细胞按30％汇合度接种到6cm培养皿。
1)Neuro-2a细胞配成0.33×10 5cells/ml细胞悬液，待铺板。
2)每皿铺3ml，即1×10 6cells/well，铺6个6cm培养皿。
2、感染慢病毒：
12～20小时后感染病毒
加病毒量计算方法：(细胞数×MOI值/病毒原液滴度)×10 3＝病毒加药量(μl)
病毒量，见下表所示。
Figure PCTCN2021126531-appb-000010
1)加polybrene：每皿加15μl 1mg/ml polybrene，最终在细胞样品中polybrene终浓度为5μg/ml；
2)感染12-20小时后换培养基：弃去培养基，每皿加入3ml新鲜的培养基。
3、稳定株筛选
1)72h以后，加入终浓度1μg/ml puromycin；每隔2-3天换一次终浓度1μg/ml  puromycin新鲜培养基；
2)药物筛选约两周后，拍荧光照片；
3)稳定细胞株冻存。
实施例6
构建阿尔兹海默症细胞模型及N2a-APP细胞系，将200ng/ml sclerostin重组蛋白加入神经元细胞系N2a细胞培养基中培养，提取N2a-APP细胞蛋白，检测Αβ蛋白产生过程中的蛋白片段如β-CTF,sAPPβ蛋白，以及培养基中Aβ1-40和Aβ1-42蛋白浓度(测定采用R&D公司的ELISA，货号：DAB142，DAB140B)。
实施例7
将骨细胞系MLO-Y4与神经元细胞系N2a细胞共培养，在培养基(培养基具体为：αMEM培养基+10％胎牛血清+2％青霉素+链霉素)中加入200ng/ml的sclerostin中和抗体，放入培养箱中孵育24小时后，提取N2a细胞蛋白，利用Western Blot技术检测BACE1蛋白表达情况，结果如图3(A)所示，从图中可以看出，sclerostin中和抗体的加入，显著降低N2a细胞内BACE1蛋白表达。
之后再将骨细胞系MLO-Y4与构建的阿尔兹海默症细胞模型及N2a-APP细胞系共培养，在培养基(培养基具体为：αMEM培养基+10％胎牛血清+2％青霉素+链霉素)中加入200ng/ml的sclerostin中和抗体，孵育24小时后，提取N2a-APP细胞蛋白检测BACE1蛋白表达情况以及Αβ蛋白产生过程中的蛋白片段如β-CTF,sAPPβ蛋白(检测使用Western Blot技术)，以及培养基中Aβ1-40和Aβ1-42蛋白浓度(检测采用Elisa技术，产品公司R&D)，从而明确sclerostin中和抗体对Αβ蛋白产生过程的影响。加入sclerostin中和抗体之后可以显著抑制Αβ蛋白产生过程中的蛋白片段sAPPβ和β-CTF蛋白水平，结果如图3(B)所示，同时对培养基进行检测发现，加入sclerostin中和抗体后，培养基中Αβ1-40(图3(C))、Αβ1-42(图3D)蛋白的浓度显著降低。
综上所述，骨硬化蛋白(Sclerostin)在动物和细胞水平中都可以显著促进Aβ蛋白的产生，加速认知功能的衰退。同时根据sclerostin重组蛋白构建的中和抗体，可以在细胞层面中减少神经元细胞内BACE1蛋白水平，进而抑制Aβ蛋白的产生，具有缓解和/或治疗阿尔兹海默症的作用。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Figure PCTCN2021126531-appb-000011
Figure PCTCN2021126531-appb-000012

Claims (8)

  1. 骨硬化蛋白，其特征在于，所述骨硬化蛋白的氨基酸序列如SEQ NO.1所示。
  2. 根据权利要求1所述的骨硬化蛋白，其特征在于，编码所述骨硬化蛋白的核苷酸序列如SEQ NO.2示。
  3. 一种重组质粒，其特征在于，含有编码权利要求1所述的骨硬化蛋白的核苷酸序列的质粒。
  4. 一种重组菌株，其特征在于，表达权利要求3所述的重组质粒的菌株。
  5. 基于权利要求1所述的骨硬化蛋白、权利要求3所述的重组质粒或权利要求4所述的重组菌株在制备治疗或预防阿尔兹海默症的中和抗体上应用。
  6. 一种中和抗体，其特征在于，其有效成分为权利要求1所述的骨硬化蛋白、权利要求3所述的重组质粒或权利要求4所述的重组菌株。
  7. 一种治疗或预防阿尔兹海默症的产品，其特征在于，包含权利要求1所述骨硬化蛋白、权利要求3所述的重组质粒、权利要求4所述的重组菌株或权利要求6所述的一种中和抗体作为活性成分；所述产品为药物组合物或保健品。
  8. 根据权利要求7所述的治疗或预防阿尔兹海默症的产品，其特征在于，所述药物组合物的剂型为注射粉针、注射针剂或散剂粉末。
PCT/CN2021/126531 2021-04-28 2021-10-27 骨硬化蛋白及其在制备治疗或预防阿尔兹海默症的相关产品上应用 WO2022227449A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110467570.1 2021-04-28
CN202110467570.1A CN113354724A (zh) 2021-04-28 2021-04-28 骨硬化蛋白及其在制备治疗或预防阿尔兹海默症的相关产品上应用

Publications (1)

Publication Number Publication Date
WO2022227449A1 true WO2022227449A1 (zh) 2022-11-03

Family

ID=77525673

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/126531 WO2022227449A1 (zh) 2021-04-28 2021-10-27 骨硬化蛋白及其在制备治疗或预防阿尔兹海默症的相关产品上应用

Country Status (2)

Country Link
CN (1) CN113354724A (zh)
WO (1) WO2022227449A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113354724A (zh) * 2021-04-28 2021-09-07 蒋青 骨硬化蛋白及其在制备治疗或预防阿尔兹海默症的相关产品上应用
CN113913463B (zh) * 2021-09-19 2023-08-18 郭保生 抑制sost基因表达的重组质粒及其骨靶向重组腺相关病毒与应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101821291A (zh) * 2007-10-12 2010-09-01 诺瓦提斯公司 抗硬骨素抗体的组合物和应用方法
US20130251735A1 (en) * 2003-09-22 2013-09-26 Enzo Biochem, Inc. Sclerostin and the inhibition of wnt signaling and bone formation
CN108101984A (zh) * 2017-12-18 2018-06-01 中国人民解放军总医院 骨硬化蛋白单链抗体的制备方法和用途
CN113354724A (zh) * 2021-04-28 2021-09-07 蒋青 骨硬化蛋白及其在制备治疗或预防阿尔兹海默症的相关产品上应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT2261335T (pt) * 1998-11-27 2017-09-08 Ucb Pharma Sa Composições e métodos para aumentar a mineralização óssea
US9133272B2 (en) * 2011-03-01 2015-09-15 Amgen Inc. Bispecific binding agents

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130251735A1 (en) * 2003-09-22 2013-09-26 Enzo Biochem, Inc. Sclerostin and the inhibition of wnt signaling and bone formation
CN101821291A (zh) * 2007-10-12 2010-09-01 诺瓦提斯公司 抗硬骨素抗体的组合物和应用方法
CN108101984A (zh) * 2017-12-18 2018-06-01 中国人民解放军总医院 骨硬化蛋白单链抗体的制备方法和用途
CN113354724A (zh) * 2021-04-28 2021-09-07 蒋青 骨硬化蛋白及其在制备治疗或预防阿尔兹海默症的相关产品上应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE 12 May 2008 (2008-05-12), ANONYMOUS : "Mus musculus sclerostin, mRNA (cDNA clone MGC:169457 IMAGE:8860852), complete cds", XP055981114, retrieved from NCBI Database accession no. BC137833.1 *
WANG ZIWEI, JING TIAN: "Sclerostin monoclonal antibody in the treatment of osteoporosis", CHINESE JOURNAL OF TISSUE ENGINEERING RESEARCH JUNE, vol. 17, no. 33, 13 August 2013 (2013-08-13), pages 6021 - 6026, XP055981072, ISSN: 2095-4344, DOI: 10.3969/j.issn.2095-4344.2013.33.019 *
YUAN JUN, MELONI BRUNO P., SHI TIANXING, BONSER ANNE, PAPADIMITRIOU JOHN M., MASTAGLIA FRANK L., ZHANG CHANGQING, ZHENG MINGHAO, G: "The Potential Influence of Bone-Derived Modulators on the Progression of Alzheimer’s Disease", JOURNAL OF ALZHEIMER`S DISEASE, IOS PRESS, NL, vol. 69, no. 1, 7 May 2019 (2019-05-07), NL , pages 59 - 70, XP055981111, ISSN: 1387-2877, DOI: 10.3233/JAD-181249 *

Also Published As

Publication number Publication date
CN113354724A (zh) 2021-09-07

Similar Documents

Publication Publication Date Title
WO2022227449A1 (zh) 骨硬化蛋白及其在制备治疗或预防阿尔兹海默症的相关产品上应用
Croce et al. Neuroprotective effect of neuropeptide Y against beta-amyloid 25–35 toxicity in SH-SY5Y neuroblastoma cells is associated with increased neurotrophin production
JP5231214B2 (ja) エリスロポエチン変異体
Tortarolo et al. Amyotrophic lateral sclerosis, a multisystem pathology: insights into the role of TNFα
JP4351043B2 (ja) アミロイド毒性の阻害方法
Tang et al. Expression of the group I metabotropic glutamate receptor in the hippocampus of patients with mesial temporal lobe epilepsy
JP2024012437A (ja) ラミノパチーを処置するためのｌｉｎｃ複合体の破壊
US20160129073A1 (en) Method for Inhibiting HIV Replication in Mammal and Human Cells
Meng et al. Rosiglitazone ameliorates spinal cord injury via inhibiting mitophagy and inflammation of neural stem cells
JPWO2008038599A1 (ja) 軸索再生促進剤
JP7037486B2 (ja) 医薬としてのヒト由来免疫抑制タンパク質およびペプチドの使用
US9023798B2 (en) Cystinosin replacement factor
US10036016B2 (en) Methods for inducing glucose uptake
US20110287014A1 (en) Antibodies directed against calcium channel subunit alpha2/delta and methods using same
KR20160116848A (ko) Tspan7 억제제를 포함하는 파골세포 형성 억제 또는 골 질환의 치료용 조성물
WO2021228052A1 (zh) 生物大分子靶向特异性补体抑制剂及其制备方法与应用
KR20080025173A (ko) 양막 세포 및 그의 사용방법
KR100885545B1 (ko) Ｒａｎｋ 저해 활성을 갖는 폴리펩티드, 그를 포함하는약학적 조성물, 그를 이용한 개체의 파골세포 분화를억제하는 방법 및 파골세포의 분화 및 활성화로 인하여야기되는 질병을 치료 또는 예방하는 방법, 그를 코딩하는핵산, 상기 핵산을 포함하는 벡터 및 숙주세포
WO2014060580A1 (en) Lxvp-mediated calcineurin inhibition in macrophages
KR102580041B1 (ko) 오스테오폰틴 억제제를 유효성분으로 포함하는 퇴행성 신경계질환의 예방, 개선 또는 치료용 조성물
US20220380454A1 (en) Methods and compositions for treating alzheimer's disease
US20230390359A1 (en) Use of reelin for treating cardiac diseases
US20240024418A1 (en) Method for treating cancer and inflammatory diseases using stem cell- derived extracellular vesicles comprising sirp
WO2016129627A1 (ja) 疼痛抑制物質のスクリーニング方法および疼痛の予防または治療用医薬組成物
KR20180120341A (ko) 폐 섬유증의 예방 또는 치료용 약학적 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21938912

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21938912

Country of ref document: EP

Kind code of ref document: A1