WO2018095264A1 - Notch信号通路调控剂调节骨生成 - Google Patents

Notch信号通路调控剂调节骨生成 Download PDF

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WO2018095264A1
WO2018095264A1 PCT/CN2017/111300 CN2017111300W WO2018095264A1 WO 2018095264 A1 WO2018095264 A1 WO 2018095264A1 CN 2017111300 W CN2017111300 W CN 2017111300W WO 2018095264 A1 WO2018095264 A1 WO 2018095264A1
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signaling pathway
notch signaling
compound
composition
notch
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French (fr)
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高嵩
柯永欣
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陈艳丽
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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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • 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

Definitions

  • the invention belongs to the technical field of biomedicine and relates to a method for treating osteoporosis by using a Notch signaling pathway regulator.
  • Osteoporosis is a metabolic disease characterized by low bone mass, abnormal bone structure, increased bone fragility, and prone to fracture. It is one of the most important public health problems. With the acceleration of population aging, lifestyle changes and the impact of metabolic diseases, osteoporosis and its fractures have placed an increasingly heavy burden on families and society. Bone is a dynamic connective tissue. During the normal bone metabolism cycle, bone formation of osteoblasts and bone resorption of osteoclasts maintain a dynamic balance. In patients with osteoporosis, bone resorption is greater than bone formation, which in turn causes a net loss of bone mass. A variety of literature reports that Notch signaling pathway plays an important regulatory role in both osteoblasts and osteoclasts.
  • the Notch signaling pathway is an evolutionarily highly conserved intracellular or intercellular signaling system that plays a crucial regulatory role in multiple life processes in multicellular organisms.
  • the Notch signaling pathway consists of a ligand, a receptor, and its downstream effector molecules.
  • Notch ligands In humans and rodents, Notch ligands have Dll1, Dll3, Dll4, Jag1 and Jag2, and Notch receptors have Notch1, Notch2, Notch3 and Notch4.
  • Notch receptors belong to the type I transmembrane protein and are composed of the extracellular region, the transmembrane region and the intracellular region. After the Notch ligand binds to the Notch receptor, the Notch receptor protein undergoes two proteolysis in succession.
  • the first proteolysis cleaves the extracellular portion of the Notch receptor by TNF- ⁇ converting enzyme (TACE), releasing the extracellular portion of the Notch receptor (NECD).
  • TACE TNF- ⁇ converting enzyme
  • NECD extracellular portion of the Notch receptor
  • the second proteolysis cleaves the intracellular portion of the Notch receptor by the ⁇ -secretase complex, releasing the Notch receptor intracellular domain (NICD). Subsequently, NICD is transferred to the nucleus to regulate the expression of downstream target genes.
  • mutations in some of the related factors in the Notch signaling pathway are closely related to multiple dysplasias in bone tissue.
  • mutations in the Notch receptor NOTCH2 or the ligand JAG1 cause Alagille syndrome, often accompanied by typical skeletal abnormalities such as anterior arch rupture and absence of scoliosis.
  • Mutations in Notch ligand DLL3, Notch downstream effector gene MESP2 or HES7, Notch receptor hydrolase LFNG, etc. result in hypoplasia of the spine ribs, accompanied by severe deformities of the spine and rib morphology.
  • mice model The type indicates that knockdown of Notch ligand Dll3, effector Rbpj, Mesp1, Mesp2, Mes7 and Notch receptor hydrolase Psen1, Psen2, Lfng and other genes all lead to severe skeletal dysplasia.
  • the Notch signaling pathway regulates bone metabolism in both in vivo and in vitro experiments, but its exact role in stem cell proliferation and differentiation into bone cells remains controversial.
  • osteoblasts differentiated by stem cells secrete extracellular matrix to the periphery and embed themselves in the extracellular matrix. With the mineralization of the extracellular matrix, osteoblasts mature into bone cells.
  • the Notch signaling pathway plays an important regulatory role in this process.
  • a primary cultured bone marrow mesenchymal stem cell (BMSC) for osteogenic differentiation an osteogenic differentiation using a bone marrow mesenchymal stem cell-like cell line
  • an osteogenic differentiation using an osteoblast cell line .
  • the differentiation of the cell population is consistent in the stem cell stage, so the activation of the Notch signaling pathway during osteogenic differentiation of these cells significantly inhibits the differentiation of these cells into osteoblasts.
  • the decrease in the number of osteoblasts leads to a decrease in the number of mineralized bone cells, which ultimately manifests in the inhibition of osteoblast differentiation and mineralization in these cells by the activation of Notch signaling pathways reported by different investigators.
  • activation of the Notch signaling pathway promotes osteogenic differentiation and mineralization of these cells.
  • the osteoblast cell Since the osteoblast cell is in the osteoblast stage, there are high expression of Alp, Ocn, etc., while the further differentiated mineralized bone cells have low expression of osteoblast markers such as Alp and Ocn, so the Notch signaling pathway is in progress.
  • the activation of the osteocyte line is manifested by a decrease in the expression of the osteoblast marker gene. This result is due to the further differentiation of osteoblasts into osteoblasts, and it cannot be simply assumed that activation of the Notch signaling pathway inhibits the differentiation of osteoblasts. . Further mineralization experiments indicate that activation of the Notch signaling pathway significantly promotes mineralization of osteoblasts.
  • Notch activity-reported transgenic mice the activated Notch signaling pathway is highly expressed in mature osteoblasts and in bone cells embedded in mineralized stroma.
  • Gain of function experiments in, Notch signaling pathway Osteoblasts previous period (Col3.6-NICD or Osx-Cre; Rosa NICD) activation, inhibits preosteoblast to osteoblast differentiation, resulting in adult mice Osteopenia and a decrease in the number of osteoblasts.
  • Activation of the Notch signaling pathway at the mature osteoblast or osteocyte stage (Col2.3-NICD, Col3.2CreERT2; Rosa NICD or Dmp1Cre; Rosa NICD ) significantly promotes osteoproliferative sclerosis.
  • the Notch signaling pathway is inactivated in the stem cell or mesenchymal stem cell stage (Prx1Cre; Notch1 -/f Notch2 f/f ), showing extensive chondrocytes and osteoblasts at early (8 weeks) Hyperplasia, and in the advanced stage (after 15 weeks) showed severe osteopenia.
  • Ostch signaling pathway cannot differentiate into bone cells due to the inactivation of the Notch signaling pathway, so we observed a large compensatory proliferation of osteoblasts at an early stage. Since osteoblasts cannot be further differentiated into bone cells, we have observed severe osteoporosis symptoms in the late stage.
  • Notch signaling pathway is not expressed during the differentiation of stem cells into osteoblasts, and the activation of Notch signaling pathway in this process inhibits the differentiation of stem cells into osteoblasts; At the same time, Notch signaling pathway is highly expressed in mature osteoblasts and bone cells, and promotes osteoblast differentiation and mineralization into bone cells, while the inactivation of Notch signaling pathway in this process inhibits the mineralization of osteoblasts.
  • Notch signaling pathway regulates bone development can almost explain the seemingly contradictory results of all current in vivo and in vitro experiments and is validated by these results.
  • Sodium valproate sodium 2-propylvalerate
  • valproic acid can activate Notch signaling pathway in various cell lines such as osteosarcoma and neuroblastoma, and its stimulation and differentiation of various cells can be specifically inhibited by inhibitors of Notch signaling pathway or Notch signaling pathway. The siRNA is blocked.
  • valproic acid promotes the mineralization of primary BMSC and BMSC-derived cell lines and promotes the expression of osteoblast marker genes during osteogenic differentiation of BMSCs. This phenomenon is related to the Notch signaling pathway. The activation effect in these cells is consistent, suggesting that valproic acid can regulate bone metabolism through the Notch signaling pathway.
  • Resveratrol (3,5,4'-trihydroxystilbene) is widely grown in grapes, knotweed, peanuts, etc.
  • a polyphenolic compound In vitro cell experiments have shown that resveratrol can activate Notch signaling pathway in various cells such as medulloblastoma, carcinoid cells, undifferentiated thyroid carcinoma, and osteosarcoma, and its stimulatory effect on various cells can be blocked by Notch signaling pathway. Specific siRNA blockade. In vitro cell culture experiments, resveratrol can promote the differentiation of osteoblasts, promote the mineralization of bone marrow mesenchymal stem cells, and promote the repair of skull defects in vivo.
  • resveratrol prevented bone loss in rats such as castrated osteoporosis model rats, osteoporosis model rats, and aging osteoporosis models.
  • rats such as castrated osteoporosis model rats, osteoporosis model rats, and aging osteoporosis models.
  • resveratrol promotes bone formation and increases bone density.
  • the stem cell development stage cannot differentiate into osteoblasts under the action of the activated Notch signaling pathway, leading to the proliferation of these stem cells.
  • Cells in the developmental stage of osteoblasts differentiate into mineralized bone cells under the action of the activated Notch signaling pathway.
  • the net result of transient activation of the Notch signaling pathway is the proliferation of cells in the early stem cell stage of differentiation, the proliferation of cells in the late stage of osteoblast differentiation, and the decrease in the number of osteoblasts in the mid-differentiation phase.
  • the activity of Notch signaling pathway returns to normal levels, the number of osteoblasts can be accelerated by the proliferation of cells in the early stem cell stage and the number of osteoblasts in the early stem cell stage. restore.
  • Notch signaling pathway promotes osteoblast differentiation and mineralization into osteoblasts and inhibits stem cell differentiation into osteoblasts, resulting in an increase in the number of cells and bone cells in the early stem cell stage and a decrease in the number of osteoblasts.
  • This process goes with N Intermittent activation of the otch signaling pathway occurs cyclically.
  • the net result of intermittent activation of the Notch signaling pathway is that stem cells accelerate differentiation into osteoblasts and bone cells, thereby promoting osteogenesis and increasing bone density.
  • the same inhibition of osteogenesis can be achieved by intermittently inhibiting Notch signaling activity.
  • the basic principle is that the inhibition of Notch signaling pathway blocks the differentiation and mineralization of osteoblasts into bone cells, resulting in an increase in the number of osteoblasts. As the inhibition of the Notch signaling pathway is lifted, the proliferating osteoblasts differentiate into more bone cells. This process occurs cyclically with the intermittent inhibition of the Notch signaling pathway, and the net result is that it promotes the differentiation of osteoblasts into bone cells, thereby promoting osteogenesis and increasing bone density. Similarly, alternately activating and inhibiting the Notch signaling pathway also promotes osteogenesis.
  • the basic principle is that during the activation phase of the Notch signaling pathway, the activated Notch signaling pathway promotes osteoblast differentiation and mineralization, and inhibits stem cell differentiation into osteoblasts, leading to proliferation and differentiation of late-stage stem cells.
  • the activated Notch signaling pathway inhibits osteoblast differentiation in the early stage of differentiation of stem cells, while inhibition of Notch signaling pathway promotes proliferation of cells in the early stage of differentiation.
  • the differentiation of bone cells therefore, in the inhibition phase of the Notch signaling pathway, the proliferating cells in the stem cell stage differentiate into more osteoblasts. This process occurs cyclically as the activation and inhibition of the Notch signaling pathway alternates. The net result is that it promotes the differentiation of stem cells into bone cells, thereby promoting osteogenesis and increasing bone density.
  • the invention encompasses promoting bone formation by modulating the Notch signaling pathway.
  • the present invention provides a novel strategy for treating bone loss caused by disease or disorder.
  • the method of the invention may be to treat a subject already exhibiting bone loss or to treat a subject at risk of bone loss.
  • the methods of the present invention include, but are not limited to, the treatment of diseases having bone loss characteristics such as osteoporosis, rickets, osteomalacia, McCune-Albright syndrome, Paget bone disease, bone loss due to treatment of other diseases, and others. Some similar diseases with symptoms of bone loss and so on.
  • the method of the invention can be applied to all mammals. These mammals include, but are not limited to, humans, companion animals, and the like.
  • Companion animals include domesticated animals (such as cats, dogs, horses, etc.), animals of important commercial value (such as cows, beef cattle, competitive animals, etc.), important scientific research animals (such as captive animals, endangered animals, etc.) or others. Mammals of great value.
  • Notch signaling pathway activity can be modulated by an effective dose of Notch signaling pathway modulator.
  • the Notch signaling pathway regulator may be a Notch signaling pathway activator or a Notch signaling pathway inhibitor,
  • the Notch signaling pathway activity can be altered directly or indirectly. Directly altering Notch signaling pathway activity as a Notch signaling regulator directly acts on Notch receptor molecules, altering the normal function of the Notch receptor.
  • Notch signaling pathway modulators may be gamma endocrine enzyme activators or inhibitors, Rbpj activators or inhibitors, Hes1 activators or inhibitors, activators or inhibitors of Notch receptor 1-4, Notch Ligand Jag1/2 or Dll1/3/4 and its activators or inhibitors.
  • Suitable Notch signaling pathway modulators include, but are not limited to, antibodies, polypeptides, proteins, small molecules, or combinations of the above, and the like.
  • One aspect of the present invention is to treat, prevent or delay bone loss in a subject by periodically intermittently administering a Notch signaling pathway activator, comprising the steps of: (1) applying a Notch signaling pathway activator to the subject (2) Waiting for the Notch signaling pathway activator to be degraded and metabolized below the effective concentration in the treated subject; (3) repeat steps (1)-(2).
  • Periodically intermittent administration of the Notch signaling pathway activator in the present invention means that the Notch signaling pathway is continuously activated in vivo for a certain period of time after administration of an effective dose of the Notch signaling pathway activator, and then the administration of the Notch signaling pathway activator is stopped for a certain period of time.
  • the treatment method of the present invention can be carried out repeatedly and continuously for a long period of time.
  • the time during which the Notch signaling pathway is continuously activated in vivo may be several hours, several days or several weeks, and the continuous period of time after the Notch signal activator is reduced to below the effective dose may be several hours or several days. It is a few weeks, which may or may not be equal to the activation time of the Notch signaling pathway in vivo.
  • the subject is continuously administered the Notch signaling pathway activator and continues to activate the Notch signaling pathway for 1-7 consecutive days, stopping the application of the Notch signaling pathway activator to the subject and awaiting degradation of the Notch signaling pathway activator to The duration below the effective concentration is 1-7 consecutive days.
  • the Notch signaling pathway activator is continuously applied to the treated subject.
  • the duration of the Notch signaling pathway was continuously activated for 2-3 consecutive days, and the duration of the application of the Notch signaling pathway activator to the treatment subject and waiting for the degradation of the Notch signaling pathway activator to below the effective concentration was for 4-5 consecutive days.
  • the subject is continuously administered the Notch signaling pathway activator and continues to activate the Notch signaling pathway for 3-4 consecutive days, stopping the application of the Notch signaling pathway activator to the subject and awaiting degradation of the Notch signaling pathway activator to The duration below the effective concentration is 3-4 consecutive days.
  • the subject is continuously administered with a Notch signaling pathway activator and continues to activate the Notch signaling pathway for 2-5 consecutive days, stopping the application of the Notch signaling pathway activator to the subject and awaiting degradation of the Notch signaling pathway activator to The duration below the effective concentration is 2-5 consecutive days.
  • a sodium valproate to intermittently activate the Notch signaling pathway in large mice.
  • BMD bone mineral density
  • BV/TV bone volume fraction
  • One aspect of the invention is to treat, prevent or delay bone loss in a subject by periodically administering a Notch signaling pathway inhibitor intermittently, comprising the steps of: (1) applying a Notch signaling pathway inhibitor to the subject (2) Waiting for the Notch signaling pathway inhibitor to fall in the body of the treated subject Decompose to below the effective concentration; (3) Repeat steps (1)-(2).
  • Periodically intermittent administration of a Notch signaling pathway inhibitor in the present invention means that the Notch signaling pathway is continuously inhibited in vivo for a certain period of time after administration of an effective dose of a Notch signaling pathway inhibitor, and then the administration of the Notch signaling pathway inhibitor is stopped for a certain period of time.
  • the treatment method of the present invention can be carried out repeatedly and continuously for a long period of time.
  • the time during which the Notch signaling pathway is continuously inhibited in vivo may be several hours, several days, or several weeks, and the continuous period of time after the Notch signaling inhibitor is reduced to below the effective dose in the body may be several hours or several days. It may be several weeks, and this time may or may not be equal to the time in which the Notch signaling pathway is inhibited in vivo.
  • the treatment of the subject continues to use Notch signaling pathway inhibitors and continues to inhibit the Notch signaling pathway for 1-7 consecutive days, stop the application of Notch signaling pathway inhibitors in the treatment subject and wait for the degradation of the Notch signaling pathway inhibitor to The duration below the effective concentration is 1-7 consecutive days.
  • the treatment of the subject continues to use Notch signaling pathway inhibitors and continues to inhibit the Notch signaling pathway for 2-3 consecutive days, stop the application of Notch signaling pathway inhibitors in the treatment subject and wait for the degradation of Notch signaling pathway inhibitors to The duration below the effective concentration is 4-5 consecutive days.
  • the treatment of the subject continues to use the Notch signaling pathway inhibitor and continues to inhibit the Notch signaling pathway for 3-4 consecutive days, stop the application of Notch signaling pathway inhibitors in the treatment subject and wait for the degradation of the Notch signaling pathway inhibitor to The duration below the effective concentration is 3-4 consecutive days.
  • the treatment of the subject continues to use the Notch signaling pathway inhibitor and continues to inhibit the Notch signaling pathway for 2-5 consecutive days, stopping the application of the Notch signaling pathway inhibitor to the subject and awaiting degradation of the Notch signaling pathway inhibitor to The duration below the effective concentration is 2-5 consecutive days.
  • Notch signaling pathway inhibitors significantly promoted the proliferation and differentiation of bone marrow mesenchymal stem cells cultured in vitro into osteoblasts, the knockdown of Notch receptors in mesenchymal stem cells leads to an increase in osteoblasts in mice, so Notch signals Pathway inhibitors can result in an increase in osteoblasts in a subject.
  • the concentration of the Notch signaling pathway inhibitor When the concentration of the Notch signaling pathway inhibitor is degraded in vivo to below the effective concentration, the inhibition of the Notch signaling pathway is abolished, leading to activation of the Notch signaling pathway, and the activated Notch signaling pathway promotes osteoblast differentiation.
  • Mineralization is a bone cell.
  • the net result of intermittent administration of a Notch signaling pathway inhibitor is to promote the differentiation and mineralization of stem cells into bone cells, thereby promoting osteogenesis.
  • the induction knockdown of Notch ligand Jag1 in the Notch signaling pathway in bone marrow mesenchymal stem cells significantly promotes the differentiation of bone marrow mesenchymal stem cells into osteoblasts, which is expressed as alkaline phosphatase.
  • the number of positive cells increased significantly.
  • the mineralized nodules induced by the culture were significantly lower than the control group, indicating that the inhibition of Notch signaling pathway can promote the differentiation of stem cells into osteoblasts, but inhibit the further differentiation and mineralization of osteoblasts. Therefore, intermittent inhibition of the Notch signaling pathway can intermittently promote the proliferation of osteoblasts, with the net result of promoting osteogenesis.
  • One aspect of the invention is to treat, prevent or delay bone loss in a subject by altering the administration of a Notch signaling pathway activator and a Notch signaling pathway inhibitor, characterized in that it comprises the steps of: (1) applying a Notch signal to the subject Pathway activator; (2) Waiting or not waiting for Notch signaling pathway activator to degrade and metabolize below the effective concentration in the subject; (3) Apply Notch signaling pathway inhibitor to the subject; (4) Wait or not wait for Notch signaling pathway The inhibitor is degraded and metabolized below the effective concentration in the subject; (5) steps (1)-(4) are repeated.
  • Alternate administration of a Notch signaling pathway activator and a Notch signaling pathway inhibitor in the present invention means that the Notch signaling pathway is activated in vivo for a certain period of time after administration of an effective dose of a Notch signaling pathway activator, and then a Notch signaling pathway inhibitor is administered. Time, then again, give an effective dose of the Notch signaling pathway activator for a certain period of time, and so on.
  • administration of a Notch signaling pathway activator and maintenance of a sustained activation of the Notch signaling pathway can be performed in vivo for hours, days, or weeks, administration of a Notch signaling pathway inhibitor and maintenance of sustained inhibition of Notch signaling pathway activity in vivo
  • the time may be hours, days, or weeks, and this time interval may or may not be equal to the time that the Notch signaling pathway is continuously activated in the body.
  • Waiting for the Notch signaling pathway activator after waiting for the Notch signaling pathway activator to degrade to a concentration below the effective concentration can be short or no, or it can be hours or days, waiting for the Notch signaling pathway inhibitor after administration of the Notch signaling pathway inhibitor
  • the period of degradation to below the effective concentration can be short or no, and can be hours or days.
  • the time to continue to use the Notch signaling pathway activator and to continue to activate the Notch signaling pathway is 1-7 consecutive days in the subject, and the time to continue to apply the Notch signaling pathway inhibitor to the subject and continuously inhibit the activity of the Notch signaling pathway is 1-7 consecutive days.
  • the Notch signaling pathway activator is continuously applied to the treated subject and continues to be stimulated.
  • the time to live the Notch signaling pathway was 2-3 consecutive days, and the time to continue to use the Notch signaling pathway inhibitor in the treated subject and to continuously inhibit the activity of the Notch signaling pathway was 4-5 consecutive days.
  • the time to continue to use the Notch signaling pathway activator and to continue to activate the Notch signaling pathway in the subject is 3-4 consecutive days, and the time to continue to apply the Notch signaling pathway inhibitor to the subject and continuously inhibit the activity of the Notch signaling pathway is For 3-4 consecutive days.
  • the time to continue to use the Notch signaling pathway activator and to continue to activate the Notch signaling pathway is 2-5 consecutive days in the subject, and the time to continue to apply the Notch signaling pathway inhibitor to the subject and continuously inhibit the activity of the Notch signaling pathway is 2-5 days in a row.
  • MCL/lpr systemic lupus erythematosus model mice
  • BMSCs bone marrow mesenchymal stem cells
  • valproic acid (2-propylpentanoic acid) and resveratrol (3,4',5-trihydroxy-1,2-stilbene) promote bone formation via the Notch signaling pathway.
  • the Notch signaling pathway has different activities in many different cells. During the differentiation of stem cells into terminal cells, some cells require activation of the Notch signaling pathway in the early stage of differentiation, and some cells require activation of the Notch signaling pathway in the late stage of differentiation. During the development of endocrine cells, activation of the Notch signaling pathway inhibits the development of endocrine precursor stem cells to endocrine cells and promotes the differentiation of mature endocrine cells.
  • the activation of Notch signaling pathway in neuroblasts inhibits the differentiation of neuroblasts into neuronal cells, while activation in neuronal cells promotes neuronal maturation and function.
  • the activation of Notch signaling pathway promotes the proliferation and metastasis of some tumor cells, inhibits the proliferation and metastasis of some tumor cells and promotes the differentiation of these tumor cells.
  • Notch signaling pathway inhibits the proliferation and metastasis of specific tumor cells and promotes the differentiation of these tumor cells and the expression of Notch signaling pathway in the differentiation of stem cells into bone cells Similarly, activation of the Notch signaling pathway promotes differentiation of osteoblasts into osteoblasts and inhibition of differentiation of stem cells into osteoblasts to maintain the undifferentiated state of stem cells. Because the Notch signaling pathway plays a role in endocrine cell differentiation, neuronal cell differentiation, and certain tumor cells, and its role in stem cell differentiation into bone cells, we believe that it can be in endocrine cells, neuronal cells, or some Notch.
  • sodium valproate significantly promotes the formation of mineralized nodules during osteogenic differentiation of mouse bone marrow mesenchymal stem cells (BMSCs), while Notch signaling pathway inhibitors are strongly The ability of sodium valproate to promote the formation of mineralized nodules was inhibited, indicating that sodium valproate regulates the osteogenic differentiation of mouse bone marrow mesenchymal stem cells (BMSC) through the Notch signaling pathway.
  • BMSCs mouse bone marrow mesenchymal stem cells
  • resveratrol significantly promotes the formation of mineralized nodules during osteogenic differentiation of mouse bone marrow mesenchymal stem cells (BMSCs), whereas Notch signaling pathway inhibitors are strongly Inhibition of the ability of resveratrol to promote the formation of mineralized nodules, indicating that resveratrol regulates the osteogenic differentiation of mouse bone marrow mesenchymal stem cells (BMSC) through the Notch signaling pathway.
  • BMSC mouse bone marrow mesenchymal stem cells
  • the activator of the Notch signaling pathway is valproic acid.
  • valproic acid means valproic acid and pharmaceutically acceptable salts, esters, amides, valproic acid phospholipid derivatives, valproic acid amide derivatives, 2-propylpentenoic acid amide derivatives, 2- One or more of the n-propyl-4-hexynoic acid derivatives.
  • the Notch signaling pathway activator is valproic acid, sodium valproate, sodium divalproate, calcium valproate, magnesium valproate, valproic acid amide, 2-ethyl-3-methylpentanamide
  • SPD sec-butyl-propylacetamide
  • 2-propylpentenoic acid amide 2-n-propyl-4-hexynoic acid.
  • Sodium valproate is a widely used broad-spectrum antiepileptic drug.
  • valproic acid can activate Notch signaling pathway in various cell lines such as osteosarcoma and neuroblastoma, and its inhibitory growth and stimulation of various cells can be inhibited by Notch signaling pathway or Notch. Signal pathway-specific siRNA blockade.
  • Notch signaling pathway or Notch.
  • Signal pathway-specific siRNA blockade In in vitro cell culture experiments, several research groups reported that valproic acid inhibited the proliferation of primary BMSC and BMSC-derived cell lines and promoted mineralization of these cells.
  • mice, rats and humans several experiments have shown that long-term continuous ingestion of valproic acid results in bone loss and a decrease in bone density. And in a child and adolescent spinal muscular atrophy patient II In clinical trials, valproic acid significantly increased bone mineral density in patients.
  • valproic acid The half-life of valproic acid in adults is 10 to 20 hours, while in children it is 6 to 9 hours. Because valproic acid has a long half-life in adults, long-term continuous intake of valproic acid in adults leads to the continuous activation of Notch signaling pathway in mesenchymal stem cells, osteoblasts, and bone cells, thereby inhibiting mesenchymal stem cell orientation. Osteoblast differentiation promotes the differentiation and differentiation of osteoblasts into bone cells, which leads to a decrease in the number of osteoblasts. The decrease in the number of osteoblasts will further lead to a decrease in the number of bone cells.
  • the net result of long-term continuous ingestion of valproic acid is that the process of differentiation of stem cells into bone cells is inhibited, resulting in a decrease in net bone mass and a decrease in bone density.
  • valproic acid has a relatively short half-life in children, valproic acid appears to be intermittently activated in the Notch signaling pathway in children. Intermittent activation of the Notch signaling pathway has a positive osteogenesis, so this property can explain the osteogenesis of valproic acid in phase II clinical trials in children and adolescents with spinal muscular atrophy.
  • the role of valproic acid in the body is not only related to the half-life, but also closely related to the therapeutic dose.
  • the activator of the Notch signaling pathway is resveratrol.
  • resveratrol refers to resveratrol (3,5,4'-trihydroxystilbene), resveratrol derivatives, resveratrol analogs, resveratrol oligomers.
  • the resveratrol derivative refers to a compound obtained by changing the substituent on the two benzene rings of resveratrol or changing the cis-trans structure by using stilbene in the molecule of resveratrol as a skeleton.
  • the derivatives of white reed alcohol include hydroxylated derivatives of resveratrol, methylated derivatives, nitroated derivatives, sulfated derivatives, aminated derivatives, acetamido derivatives or Their combination and the compound obtained by introducing a new element to each of the two benzene rings of the molecular skeleton.
  • the resveratrol analog refers to a compound having a molecular skeleton of diphenylethane, a molecular skeleton in which a carbon in stilbene is replaced by an isosteric nitrogen, or a B ring in a molecular skeleton substituted with a thienyl group.
  • Resveratrol oligomers are compounds in which resveratrol and its derivatives are dehydrogenated to form different degrees of polymerization, including but not limited to glucosin, alpha-glucoside, ⁇ . - Grapevine, ⁇ -glucoside.
  • the hydroxylated derivatives of resveratrol include, but are not limited to, compounds obtained by increasing or decreasing the number of hydroxyl groups and changing the position of the hydroxyl groups on the two benzene rings of the molecular skeleton of resveratrol.
  • the methylated derivative of resveratrol includes, but is not limited to, a compound obtained by partially or fully methylating a hydroxyl group on two benzene rings of a molecular skeleton in resveratrol or a hydroxylated derivative thereof.
  • the nitroated derivative, sulfoated derivative, aminated derivative, acetamide derivative of resveratrol includes, but is not limited to, resveratrol, resveratrol hydroxylated derivative or white Molecular skeleton in resveratrol methylated derivatives A compound obtained by partially or completely replacing a hydroxyl group on two benzene rings with a nitro group, a sulfo group, an amino group or an acetamide group.
  • the compound obtained by introducing a new element to the two benzene rings of the molecular skeleton of the resveratrol includes, but is not limited to, introducing a trifluoromethyl group, a chlorine element, respectively, on the two benzene rings of the molecular skeleton thereof.
  • resveratrol refers to resveratrol, 3'-hydroxyresveratrol (baclitaxel), 4,3', 5'-trihydroxy resveratrol, 4-hydroxy white cucurbit Alcohol, trans 3,5-dihydroxystilbene, 3,5,4'-trimethoxystilbene (TMB), 3,4,4'-trimethoxystilbene, 3,4,5 , 4'-tetrahydroxystilbene (4-HR), 3,4,5,4'-tetramethoxystilbene (MR-4), 4-hydroxytrans stilbene (4-HS) , 4'-hydroxystilbene (4'-HS), 3,4'-hydroxytrans stilbene (3,4'-DHS), 3,4',5-trihydroxyl -3- ⁇ -mono-D-glucoside, red sandalwood (3,5-dimethoxy-4'-hydroxystilbene), 3'-hydroxy rosewood, 3,5,2',4'- Tetramethoxystilbene, 5,4'-dihydroxy-3-methoxystilbene, 3,5-dihydroxy-4'-
  • resveratrol like valproic acid, activates Notch signaling pathways in a variety of cells, including medulloblastoma, carcinoid cells, undifferentiated thyroid carcinoma, and osteosarcoma, and inhibits multiple cells. Proliferation and differentiation-promoting effects are blocked by siRNA specific for the Notch signaling pathway.
  • resveratrol can promote the differentiation of osteoblasts, promote the mineralization of bone marrow mesenchymal stem cells, and promote the repair of skull defects in vivo, showing that resveratrol is regulated by activation of Notch signaling pathway. Osteoblast differentiation.
  • resveratrol prevents bone loss in normal or osteoporotic model animals such as rats, mice, and rabbits.
  • resveratrol promotes bone formation and increases bone density.
  • resveratrol did not significantly increase bone density. It has been reported that the total resveratrol content in plasma after oral administration is less than 2% of the oral dose.
  • the concentration of free resveratrol in plasma reached the highest in 30 minutes after oral resveratrol, followed by a rapid decrease and accelerated metabolism in an exponential manner after six hours.
  • resveratrol and its metabolites in plasma reached baseline levels within 4 hours.
  • resveratrol Since resveratrol has low bioavailability and rapid metabolism in vivo, the daily intake of administration is similar to intermittent stimulation of activation of the Notch signaling pathway, and intermittent activation of the Notch signaling pathway is beneficial for osteogenesis, so we In some animal and human experiments, it was observed that daily intake of resveratrol has a statistically significant osteogenesis phenomenon. In different rat experiments, we observed that only a very high oral dose of resveratrol could detect significant osteogenesis. We believe that because resveratrol has low bioavailability and rapid metabolism, only a high dose of resveratrol can maintain an effective activation of the Notch signaling pathway in animals, thereby promoting osteogenesis.
  • resveratrol significantly promotes the formation of late mineralized nodules in osteogenic differentiation of mouse mesenchymal stem cells, whereas Notch signaling pathway inhibitors significantly inhibit reductive mineralization of resveratrol
  • Notch signaling pathway inhibitors significantly inhibit reductive mineralization of resveratrol
  • Figure 1 Schematic diagram of the role of the Notch signaling pathway in the osteogenic differentiation of stem cells into bone cells.
  • FIG. 1 Effect of Notch signaling pathway activator valproic acid on trabecular bone in femur of 12-week female C57BL/6 mice.
  • Female C57BL/6 mice were randomly divided into 5 groups, 8 rats in each group. The treatment was continued for 12 weeks according to the following protocol: PBS, intraperitoneal injection of PBS every day from Monday to Sunday; VPA2, intraperitoneal injection of C by weight per day from Monday to Tuesday Sodium valerate (dosage 100mg/kg body weight), intraperitoneal injection of PBS every day from Wednesday to Sunday; VPA3, intraperitoneal injection of sodium valproate (100mg/kg body weight) per day by weight from Monday to Wednesday, every day from Thursday to Sunday Intraperitoneal injection of PBS; VPA4, intraperitoneal injection of sodium valproate (100 mg/kg body weight) per day by weight from Monday to Thursday, intraperitoneal injection of PBS every day from Friday to Sunday; VPA7, daily intraperitoneal injection of C by weight from Monday to Sunday
  • A is the bone volume fraction (BV/TV) changes; B is the change in bone density (BMD).
  • BMD bone density
  • FIG. 3 Effect of Notch signaling pathway activator valproic acid on trabecular bone in the femur of 20-week female C57BL/6 mice.
  • Female C57BL/6 mice were randomly divided into 5 groups, 12 rats in each group. The treatment was continued for 12 weeks according to the following protocol: PBS, intraperitoneal injection of PBS every day from Monday to Friday; VPA2, intraperitoneal injection of C by weight per day from Monday to Tuesday Sodium valerate (dosage 100 mg/kg body weight), intraperitoneal injection of PBS every day from Wednesday to Friday; VPA3, intraperitoneal injection of sodium valproate (100 mg/kg body weight) per day by weight from Monday to Wednesday, every Thursday to Friday Intraperitoneal injection of PBS; VPA4, intraperitoneal injection of sodium valproate (100 mg/kg body weight) per day by weight from Monday to Thursday, and intraperitoneal injection of PBS on Friday; VPA5, intraperitoneal injection of sodium valproate per day by weight from Monday to Friday ( The
  • A is the change in bone volume fraction (BV/TV); B is the change in bone density (BMD).
  • BMD bone density
  • FIG. 4 Effect of Notch signaling pathway activator valproic acid on trabecular bone in the femur of 24-week female SD rats.
  • Female Sprague-Dawley rats were randomly divided into 5 groups, 12 rats in each group, and treated continuously for 12 weeks according to the following protocol: PBS, intraperitoneal injection of PBS every day from Monday to Friday; VPA2, intraperitoneal injection of valproic acid per day by weight from Monday to Tuesday Sodium (dosage 100mg/kg body weight), intraperitoneal injection of PBS every day from Wednesday to Friday; VPA3, intraperitoneal injection of sodium valproate (100mg/kg body weight) per day by weight from Monday to Wednesday, intraperitoneal injection every Thursday to Friday PBS; VPA4, intraperitoneal injection of sodium valproate (100 mg/kg body weight) per day by weight from Monday to Thursday, intraperitoneal injection of PBS on Friday; VPA5, intraperitoneal injection of sodium valproate per day by weight from Monday to Friday (dose
  • A is the change in bone volume fraction (BV/TV); B is the change in bone density (BMD).
  • FIG. 5 Effect of Notch signaling pathway activator valproic acid on trabecular bone in femur of 24 week castrated female SD rats.
  • Sixteen-week-old female SD rats were selected for bilateral ovariectomy. After 8 weeks of surgery, they were randomly divided into 5 groups, 12 in each group, and treated continuously for 12 weeks according to the following protocol: PBS, intraperitoneal injection of PBS every day from Monday to Friday; VPA2, intraperitoneal injection of sodium valproate per day by weight from Monday to Tuesday (dose 100mg/kg body weight), intraperitoneal injection of PBS every day from Wednesday to Friday; VPA3, daily intraperitoneal injection of sodium valproate (100mg/kg body weight) per day from Monday to Wednesday, intraperitoneal injection of PBS every Thursday to Friday; VPA4 , Monday through Thursday Body weight was intraperitoneally injected with sodium valproate (100 mg/kg body weight) per day, and PBS was intraperitoneally injected on Friday; VPA5, sodium valproate (100
  • A is the change in bone volume fraction (BV/TV); B is the change in bone density (BMD).
  • BMD bone density
  • the femur and tibia of C57BL/6 mice were taken, the bone marrow cavity was exposed, and bone marrow mesenchymal stem cells were washed out with ⁇ MEM. Subsequently, 10,000,000 cells per well were suspended in 2 ml of basal medium ( ⁇ MEM containing 10% fetal bovine serum and 1% penicillin) in a six-well plate at 37 ° C, 5% CO 2 , saturated humidity ⁇ 95% Cultivate in an incubator. The culture medium was changed every three days from the fourth day of inoculation. On days 1 to 7, culture in basal medium.
  • basal medium ⁇ MEM containing 10% fetal bovine serum and 1% penicillin
  • the culture was continued by transferring to an osteogenic induction medium (basal medium component was added with 20 mM ⁇ -glycerophosphate, 10 nM dexamethasone, and 50 ⁇ g/ml ascorbic acid).
  • basic medium component was added with 20 mM ⁇ -glycerophosphate, 10 nM dexamethasone, and 50 ⁇ g/ml ascorbic acid.
  • von Kossa mineralized nodule staining was performed as follows.
  • Notch signaling pathway is activated in the late stage of osteogenic differentiation of bone marrow mesenchymal stem cells
  • TNR transgenic Notch reporter mice
  • Example 3 Notch signaling pathway inhibitors inhibit mineralization of bone marrow mesenchymal stem cells in the late stage of osteogenic differentiation
  • Example 2 12 weeks old MX1Cre; Jag1 f/f mice, MX1Cre mice and Jag1 f/f mice were injected intraperitoneally with pIpC every day for 7 days. The mice were sacrificed and the mouse bone marrow mesenchymal stem cells were taken. The method described in Example 1 was carried out for osteogenic differentiation for 21 days. On the 8th day of culture, a portion of the cells were taken from each group, and alkaline phosphatase staining was performed using a white blood cell alkaline phosphatase kit (Sigma, Cat. No. 85L2) according to the standard procedure outlined in the manufacturer's product specification. On the 21st day of culture, von Kossa staining was performed to detect the formation of mineralized nodules.
  • a white blood cell alkaline phosphatase kit Sigma, Cat. No. 85L2
  • Jag1 is an important Notch ligand, and MX1Cre can be highly expressed in bone marrow mesenchymal stem cells under pIpC-induced conditions. Therefore, in MX1Cre; Jag1 f/f mice, pIpC induction can selectively be used in bone marrow mesenchymal stem cells. The Jag1 gene was knocked out to inhibit the activity of the Notch signaling pathway. Cell culture experiments showed that after induction with pIpC, MSC1Cre; Jag1 f/f mouse bone marrow mesenchymal stem cells (BMSC) were compared with control mice (MX1Cre and Jag1 f/f ) in osteogenic differentiation.
  • BMSC bone marrow mesenchymal stem cells
  • the alkaline phosphatase-positive cells were significantly increased in 8 days, while the mineralized nodules were significantly reduced on the 21st day of osteogenic differentiation culture.
  • the cell culture experiment showed that knocking out the Jag1 gene in the bone marrow mesenchymal stem cell stage, thereby inhibiting the activity of the Notch signaling pathway at this stage, significantly promoted the differentiation and proliferation of BMSC into osteoblasts, whereas the proliferating osteoblasts due to The knockdown of the Jag1 gene failed to effectively activate the Notch signaling pathway, which resulted in the inhibition of osteoblast differentiation and mineralization into bone cells, and thus the mineralized nodules were significantly reduced compared with the control group.
  • 12-week-old female C57BL/6 mice were randomly divided into five groups of 8 animals each.
  • the first group was intraperitoneally injected with PBS every day from Monday to Sunday (control PBS group).
  • the second group was intraperitoneally injected with 100 mg/kg sodium valproate daily from Monday to Tuesday, and intraperitoneal injection of PBS (VPA2 group) every day from Wednesday to Sunday.
  • the third group received intraperitoneal injection of 100 mg/kg sodium valproate daily from Monday to Wednesday, and intraperitoneal injection of PBS (VPA3 group) every day from Thursday to Sunday.
  • the fourth group was intraperitoneally injected with 100 mg/kg sodium valproate daily from Monday to Thursday, and PBS (VPA4 group) was intraperitoneally injected every Friday through Sunday.
  • the fifth group was intraperitoneally injected with 100 mg/kg sodium valproate (VPA7 group) every day from Monday to Sunday. After 12 weeks of continuous injection, the animals were sacrificed and the femurs of the mice were taken. Microscopic CT scans were performed to analyze the bone mineral density and bone volume percentage of the femoral trabeculae according to the micro-CT standard analysis procedure.
  • the bone mineral density and bone volume percentage of the valproic acid injection group were significantly higher than those of the control group.
  • the VPA2 group had the greatest stimulating effect on osteogenesis compared with the control group (Fig. 2), suggesting that valproic acid intermittently activates Notch.
  • the signaling pathway can significantly promote osteogenesis in mice.
  • mice 20-week-old female C57BL/6 mice were randomly divided into five groups of 12 animals each.
  • the first group was intraperitoneally injected with PBS every day from Monday to Friday (control PBS group).
  • the second group was intraperitoneally injected with 100 mg/kg sodium valproate daily from Monday to Tuesday, and intraperitoneally injected with PBS (VPA2 group) every Wednesday to Friday.
  • the third group was intraperitoneally injected with 100 mg/kg sodium valproate daily from Monday to Wednesday, and intraperitoneally injected with PBS (VPA3 group) every Thursday to Friday.
  • the fourth group was intraperitoneally injected with 100 mg/kg sodium valproate daily from Monday to Thursday, and PBS (VPA4 group) was injected intraperitoneally on Friday.
  • the fifth group was intraperitoneally injected with 100 mg/kg sodium valproate (VPA5 group) every week from Monday to Friday. After 12 weeks of continuous injection, the animals were sacrificed and the femurs of the mice were taken. Microscopic CT scans were performed to analyze the bone mineral density and bone volume percentage of the femoral trabeculae according to the micro-CT standard analysis procedure.
  • Female SD rats of 24 weeks old were randomly divided into five groups, 12 in each group.
  • the first group was intraperitoneally injected with PBS every day from Monday to Friday (control PBS group).
  • the second group was intraperitoneally injected with 100 mg/kg sodium valproate daily from Monday to Tuesday, and intraperitoneally injected with PBS (VPA2 group) every Wednesday to Friday.
  • the third group was intraperitoneally injected with 100 mg/kg sodium valproate daily from Monday to Wednesday, and intraperitoneally injected with PBS (VPA3 group) every Thursday to Friday.
  • the fourth group was intraperitoneally injected with 100 mg/kg sodium valproate daily from Monday to Thursday, and PBS (VPA4 group) was injected intraperitoneally on Friday.
  • the fifth group was intraperitoneally injected with 100 mg/kg sodium valproate (VPA5 group) every week from Monday to Friday. After 12 weeks of continuous injection, the animals were sacrificed and the femur of the rats was taken. The microscopic CT scan was performed to analyze the bone mineral density and bone volume percentage of the femoral trabeculae according to the micro-CT standard analysis procedure.
  • the first group was intraperitoneally injected with PBS every day from Monday to Friday (control PBS group).
  • the second group was intraperitoneally injected with 100 mg/kg sodium valproate daily from Monday to Tuesday, and intraperitoneally injected with PBS (VPA2 group) every Wednesday to Friday.
  • the third group was intraperitoneally injected with 100 mg/kg sodium valproate daily from Monday to Wednesday, and intraperitoneally injected with PBS (VPA3 group) every Thursday to Friday.
  • the fourth group was intraperitoneally injected with 100 mg/kg sodium valproate daily from Monday to Thursday, and PBS (VPA4 group) was injected intraperitoneally on Friday.
  • the fifth group was intraperitoneally injected with 100 mg/kg sodium valproate (VPA5 group) every week from Monday to Friday.
  • the microscopic CT scan was performed to analyze the bone mineral density and bone volume percentage of the femoral trabeculae according to the micro-CT standard analysis procedure.
  • RESULTS Castrated female rats were a classic model of postmenopausal osteoporosis in women. The bilateral ovariectomized female rats showed severe osteoporosis.
  • valproic acid injection group VPA2 group The bone mineral density and bone volume percentage were significantly higher than those of the control PBS group, while the VPA3, VPA4, and VPA5 groups had no significant difference in bone mineral density and bone volume percentage compared with the control group (Fig. 5), suggesting valproic acid.
  • Intermittent activation of the Notch signaling pathway significantly promoted osteogenesis in ovariectomized rats, whereas valproic acid-induced sustained activation of the Notch signaling pathway had no significant effect on osteogenesis in ovariectomized rats.

Abstract

含Notch信号通路激活剂和/或抑制剂的化合物或组合物在制备用于预防或治疗骨质疏松症的药物中的应用，Notch信号通路的激活剂是丙戊酸或白藜芦醇。

Description

Notch信号通路调控剂调节骨生成 技术领域
本发明属于生物医药技术领域，涉及一种利用Notch信号通路调节剂治疗骨质疏松症的方法。
背景技术
骨质疏松症是以低骨量、骨结构失常、骨脆性增加、易发生骨折为特征的代谢性疾病，是最重要的公共健康问题之一。随着人口老龄化的加速，生活方式的改变及代谢性疾病的影响，骨质疏松症及其引起的骨折给家庭和社会带来越来越沉重的负担。骨骼是一种动态的结缔组织。在正常骨代谢周期，成骨细胞的骨形成和破骨细胞的骨吸收维持着动态平衡。在骨质疏松症患者中，骨吸收大于骨形成，进而造成了骨量净丢失。多种文献报道Notch信号通路对成骨细胞和破骨细胞均起到重要的调控作用。
Notch信号通路是进化高度保守的细胞内或细胞间信息传递系统，在多细胞生物体的多种生命过程中起至关重要的调控作用。Notch信号通路由配体、受体及其下游效应分子组成。在人类和啮齿类动物，Notch配体有Dll1、Dll3、Dll4、Jag1和Jag2，Notch受体有Notch1、Notch2、Notch3和Notch4。Notch受体都属于I型跨膜蛋白，由胞外区、跨膜区和胞内区组成。Notch配体与Notch受体结合后，Notch受体蛋白相继发生两次蛋白水解。第一次蛋白水解由TNF-α转化酶(TACE)对Notch受体的胞外部分进行切割，释放出Notch受体胞外段(NECD)。第二次蛋白水解由γ-分泌酶复合体对Notch受体胞内部分进行酶切，释放出Notch受体胞内段(NICD)。随后，NICD转移至细胞核内，调控下游靶基因的表达。
人类遗传学研究表明，Notch信号通路中一些相关因子的突变与骨组织的多种发育异常密切相关。比如，Notch受体NOTCH2或配体JAG1的突变导致Alagille综合征，常伴有脊椎前弓裂开、无脊柱侧突等典型的骨骼异常症状。而Notch配体DLL3、Notch下游效应基因MESP2或HES7、Notch受体水解酶LFNG等的突变，导致脊椎肋骨发育不全，伴随脊柱和肋骨形态的严重畸形。同时，实验小鼠模 型表明，Notch配体Dll3、效应分子Rbpj、Mesp1、Mesp2、Mes7和Notch受体水解酶Psen1、Psen2、Lfng等基因的敲除，均导致严重的骨骼发育异常。
Notch信号通路能在体内和体外实验中调节骨代谢，然而其在干细胞增生分化为骨细胞的过程中所起的确切作用仍然有很大的争议。在干细胞向骨细胞分化过程中，由干细胞分化的成骨细胞向周围分泌细胞外基质，并将自身包埋在细胞外基质中。随着细胞外基质的矿化，成骨细胞成熟为骨细胞。Notch信号通路在这一过程中起重要的调控作用。在体外细胞培养实验中，存在三种细胞成骨性分化体系。一种利用原代培养的骨髓间充质干细胞(BMSC)进行成骨性分化，一种利用骨髓间充质干细胞样细胞系进行成骨性分化，一种利用成骨细胞系进行成骨性分化。在原代培养的骨髓间充质干细胞，由于细胞属于混合群体，因此有处于不同分化阶段的细胞。Notch信号通路在这些细胞的激活，不仅促进了处于成骨细胞阶段的细胞的进一步分化和矿化，而且促进了处于骨髓间充质干细胞阶段的细胞的增生，其净结果表现为Notch的激活促进了原代BMSC的成骨性分化和矿化。在原代培养的骨髓间充质干细胞成骨性分化过程中，抑制Notch信号通路活性则显著抑制了矿化结节的形成，提示Notch信号通路在成骨细胞分化的矿化阶段发挥关键作用。在骨髓间充质干细胞来源的细胞系，由于细胞群体分化状态比较一致，均处于干细胞阶段，因此Notch信号通路在这些细胞成骨性分化过程中的激活，显著抑制了这些细胞分化为成骨细胞，而成骨细胞数量的减少则导致矿化的骨细胞数量的减少，最终表现为不同研究人员所报道的Notch信号通路的激活在这些细胞抑制成骨细胞分化和矿化。在成骨细胞系，Notch信号通路的激活促进了这些细胞的成骨性分化和矿化。由于成骨细胞系细胞处于成骨细胞阶段，有高表达的Alp，Ocn等，而进一步分化矿化的骨细胞则有低表达的Alp、Ocn等成骨细胞标志基因，因此Notch信号通路在成骨细胞系的激活表现为成骨细胞标志基因表达的下降，这一结果是由于成骨细胞进一步分化为骨细胞的结果，而不能简单认为是Notch信号通路的激活抑制了成骨细胞系的分化。进一步的矿化实验表明Notch信号通路的激活显著促进了成骨细胞的矿化。
在Notch活性报道转基因小鼠中，活化的Notch信号通路在成熟的成骨细胞和包埋在矿化基质的骨细胞中高表达。在功能获得实验中，Notch信号通路在前成骨 细胞时期(Col3.6-NICD或Osx-Cre；RosaNICD)的激活，抑制了前成骨细胞向成骨细胞的分化，从而导致成年小鼠的骨质减少和成骨细胞数量的降低。而Notch信号通路在成熟的成骨细胞或骨细胞阶段(Col2.3-NICD、Col3.2CreERT2；RosaNICD或Dmp1Cre；RosaNICD)的激活，则显著促进骨质增生硬化。在功能缺失实验中，Notch信号通路在干细胞或间充质干细胞阶段(Prx1Cre；Notch1-/fNotch2f/f)的失活，在早期(8周)表现为广泛的软骨细胞和成骨细胞的增生，而在晚期(15周以后)表现为严重的骨质减少。我们认为，由于Notch信号通路的失活，成骨细胞不能分化为骨细胞，因此我们在早期观察到了成骨细胞的代偿性大量增生。由于成骨细胞不能进一步分化矿化为骨细胞，因此我们在晚期观察到了严重的骨质疏松症状。而Notch信号通路在成骨细胞阶段(Col2.3Cre；Psen1f/fPsen2-/-)的失活，则导致严重的年龄相关的骨质疏松症状，这是由于Notch信号通路的失活抑制了成骨细胞分化矿化为骨细胞。
基于现有的体内体外实验结果，我们认为，Notch信号通路在干细胞分化为成骨细胞的过程中不表达，而Notch信号通路在这一过程中的激活则抑制了干细胞向成骨细胞的分化；同时Notch信号通路在成熟的成骨细胞和骨细胞高表达，并促进成骨细胞分化矿化为骨细胞，而Notch信号通路在这一过程中的失活则抑制了成骨细胞的矿化。这一新的Notch信号通路调控骨发育的机理几乎能解释目前所有的体内和体外实验看似矛盾的结果并被这些结果所验证。
丙戊酸钠(2-丙基戊酸钠)是一种目前常用的广谱抗癫痫药，其对癫痫患者的长期持续治疗，常常导致骨密度的降低。体外细胞培养实验表明，丙戊酸能在骨肉瘤、神经母细胞瘤等多种细胞系激活Notch信号通路，其对多种细胞的刺激分化作用能被Notch信号通路的抑制剂或Notch信号通路特异的siRNA阻断。而在骨髓间充质干细胞，丙戊酸能促进原代BMSC及BMSC来源的细胞系的矿化并促进BMSC成骨性分化过程中的成骨细胞标志基因的表达，这一现象与Notch信号通路在这些细胞内的激活效应一致，提示丙戊酸能通过Notch信号通路调控骨代谢。
白藜芦醇(3，5，4’-三羟基二苯乙烯)是广泛存在于葡萄、虎杖、花生等多种植 物中的一种多酚化合物。体外细胞实验表明，白藜芦醇能在髓母细胞瘤、类癌细胞、未分化甲状腺癌、骨肉瘤等多种细胞激活Notch信号通路，而其对多种细胞的刺激效应能被Notch信号通路特异的siRNA阻断。在体外细胞培养实验中，白藜芦醇能促进成骨细胞的分化、促进骨髓间充质干细胞的矿化，并在体内促进颅骨缺损的修复。在体内实验中，白藜芦醇能防止去势骨质疏松模型大鼠、废用骨质疏松模型大鼠、老龄化骨质疏松模型等大鼠中的骨丢失。在一项肥胖男性的临床实验中，白藜芦醇能促进骨形成，提高骨密度。
技术问题
问题的解决方案
技术解决方案
本发明的目的在于提供一种利用Notch信号通路调控剂治疗骨质疏松症的方法。
体外细胞培养实验和体内转基因动物实验均表明，Notch信号通路的无选择性和持续性地失活或激活，均导致严重的骨质疏松症状，而这些看似矛盾的实验结果均可以被我们的理论合理解释，那就是Notch信号通路在间充质干细胞阶段的激活抑制这些细胞向成骨细胞分化，而Notch信号通路在成骨细胞阶段的激活则促进成骨细胞分化和矿化(图1)。基于这一理论，我们认为间歇性地激活或抑制Notch信号通路，能促进成骨，提高骨密度。其基本作用原理为：当Notch信号通路激活时，产生两个方面的效应。首先在干细胞发育阶段的细胞在激活的Notch信号通路作用下，不能向成骨细胞分化，导致了这些干细胞的增生。而在成骨细胞发育阶段的细胞则在激活的Notch信号通路的作用下，分化矿化为骨细胞。因此，短暂性激活Notch信号通路的净结果为分化早期干细胞阶段的细胞的增生、分化晚期骨细胞阶段的细胞的增生以及分化中期的成骨细胞数量的下降。当Notch信号通路活性恢复到正常水平，由于处于早期干细胞阶段的细胞的增生和成骨细胞数量的下降，促使增生的处于早期干细胞阶段的细胞加速分化为成骨细胞，因此成骨细胞的数量可以恢复。随后Notch信号通路的再次激活又促进成骨细胞分化矿化为骨细胞并抑制干细胞分化为成骨细胞，从而导致处于早期干细胞阶段的细胞和骨细胞数量增加，而成骨细胞的数量减少。这一过程随着N otch信号通路的间歇性激活而循环发生。因此间歇性激活Notch信号通路的净结果为干细胞加速向成骨细胞和骨细胞分化，从而促进成骨，提高骨密度。理论上，间歇性地抑制Notch信号活性也可以达到同样的促进成骨的作用。其基本原理为：Notch信号通路的抑制阻断了成骨细胞向骨细胞的分化和矿化，导致成骨细胞数量增多。随着对Notch信号通路抑制的解除，增生的成骨细胞分化矿化为更多的骨细胞。这一过程随着Notch信号通路的间歇性抑制而循环发生，其净结果为促进了成骨细胞向骨细胞的分化，从而促进成骨，提高骨密度。同理，交替性地激活和抑制Notch信号通路也同样具有促进成骨作用。其基本原理为：在Notch信号通路的激活阶段，激活的Notch信号通路促进成骨细胞分化和矿化，并抑制干细胞分化为成骨细胞，从而导致分化早期干细胞阶段的细胞的增生、分化晚期骨细胞阶段的细胞的增生以及分化中期的成骨细胞数量的下降。在Notch信号通路的抑制阶段，由于激活的Notch信号通路对处于分化早期干细胞阶段的细胞向成骨细胞分化有抑制作用，而抑制Notch信号通路则促进了增生的处于分化早期干细胞阶段的细胞向成骨细胞的分化，因此在Notch信号通路的抑制阶段，增生的处于干细胞阶段的细胞分化为更多的成骨细胞。这一过程随着Notch信号通路的激活和抑制交替进行而循环发生，其净结果为促进了干细胞向骨细胞的分化，从而促进成骨，提高骨密度。
本发明包含通过调节Notch信号通路来促进骨形成。本发明提供了治疗疾病或失调引起的骨丢失的新策略。本发明的方法可以是对已经表现为骨丢失的治疗对象进行治疗或对有骨丢失风险的对象进行治疗。本发明的方法包括但不限于治疗以下具有骨丢失特性的疾病，比如骨质疏松症、软骨病、骨软化症、McCune-Albright综合征、Paget骨病、由于治疗其它疾病导致的骨丢失和其它一些相似的具有骨丢失症状的疾病等等。本发明方法可以被应用到所有的哺乳动物。这些哺乳动物包括但不仅限于人类、伴侣动物等。伴侣动物包括驯养的动物(例如猫、狗、马等)、重要商业价值的动物(例如奶牛、肉牛、竞技动物等)、重要的科研价值动物(例如圈养动物、濒危动物等)或其它一些有重要价值的哺乳动物。Notch信号通路活性可以通过有效剂量的Notch信号通路调控剂来调节。Notch信号通路调控剂可以是Notch信号通路激活剂或Notch信号通路抑制剂， 可以直接或者间接改变Notch信号通路活性。直接改变Notch信号通路活性为Notch信号调控剂直接作用于Notch受体分子，改变了Notch受体的正常功能。间接改变Notch信号通路活性可以发生在Notch信号通路的各个步骤，包括但不仅限于Notch受体的表达、Notch受体胞内段NICD的释放及其对Notch靶基因的调控等的各个步骤，这些改变可以发生在基因组水平、转录水平、翻译水平、蛋白折叠水平、酶切割水平等。在具体实施中，这些Notch信号通路调控剂可以是γ内分泌酶激活剂或抑制剂、Rbpj激活剂或抑制剂、Hes1激活剂或抑制剂、Notch受体1-4的激活剂或抑制剂、Notch配体Jag1/2或Dll1/3/4及其激活剂或抑制剂等。合适的Notch信号通路调控剂包括但不仅限于抗体、多肽、蛋白质、小分子或以上成分的组合等。
本发明的一个方面是通过周期性地间歇性给予Notch信号通路激活剂来治疗、预防或延迟治疗对象的骨丢失，其特征在于，包括如下步骤：(1)对治疗对象应用Notch信号通路激活剂；(2)等待Notch信号通路激活剂在治疗对象体内降解代谢到有效浓度以下；(3)重复步骤(1)-(2)。本发明中周期性地间歇性给予Notch信号通路激活剂是指给予有效剂量的Notch信号通路激活剂之后，在体内持续激活Notch信号通路一定的时间，然后停止给予Notch信号通路激活剂一定的时间。随着Notch信号通路激活剂在体内的代谢，直到Notch信号通路激活剂在体内的含量降低到有效剂量以下并维持一段时间后，然后再给予有效剂量的Notch信号通路激活剂，再等待其体内浓度降低到有效剂量以下并维持一段时间后，然后再次给予有效剂量的Notch信号通路激活剂，如此反复进行。由于骨质疏松症是一种长期的慢性代谢性疾病，因此本发明的治疗方法可以长期反复持续地进行。其中，Notch信号通路在体内连续激活的时间可以是几小时、几天也可以是几周，Notch信号激活剂体内含量降低到有效剂量以下后维持的一段连续时间可以是几小时、几天也可以是几周，这一时间可以和Notch信号通路在体内激活的时间相等，也可以不相等。作为一种优选，对治疗对象持续应用Notch信号通路激活剂并持续激活Notch信号通路的时间为连续1-7天，对治疗对象停止应用Notch信号通路激活剂并等待Notch信号通路激活剂降解代谢到有效浓度以下的持续时间为连续1-7天。作为一种优选，对治疗对象持续应用Notch信号通路激活剂 并持续激活Notch信号通路的时间为连续2-3天，对治疗对象停止应用Notch信号通路激活剂并等待Notch信号通路激活剂降解代谢到有效浓度以下的持续时间为连续4-5天。作为一种优选，对治疗对象持续应用Notch信号通路激活剂并持续激活Notch信号通路的时间为连续3-4天，对治疗对象停止应用Notch信号通路激活剂并等待Notch信号通路激活剂降解代谢到有效浓度以下的持续时间为连续3-4天。作为一种优选，对治疗对象持续应用Notch信号通路激活剂并持续激活Notch信号通路的时间为连续2-5天，对治疗对象停止应用Notch信号通路激活剂并等待Notch信号通路激活剂降解代谢到有效浓度以下的持续时间为连续2-5天。
在本发明的具体实施例中(图2、图3、图5)，我们用丙戊酸钠在大小鼠体内间歇性地激活Notch信号通路。在12周的实验周期内，我们通过每周连续2天、3天、4天、5天或7天腹腔注射丙戊酸钠来每周间歇性地激活Notch信号通路。结果表明，每周连续2天间歇性激活Notch信号通路引起12周龄和20周龄雌性小鼠、24周龄双侧卵巢切除雌性大鼠的骨密度(BMD)及骨体积分数(BV/TV)与对照组相比显著提高，而每周连续3天及以上时间激活Notch信号通路则不能引起相应大小鼠骨密度与骨体积分数的显著提高。由于丙戊酸钠在大鼠和小鼠体内半衰期都很长，因此注射丙戊酸钠的当天相当于Notch信号通路在当天的持续激活，而不注射的当天则相当于等待Notch信号通路的活性恢复到正常水平。因此这一结果表明，在大小鼠，间歇性地激活Notch信号通路显著促进了骨形成。在本发明的具体实施例中(图4)，我们用丙戊酸钠在24周龄雌性大鼠体内间歇性地激活Notch信号通路，在12周的实验周期内，我们通过每周连续2天、3天、4天、5天腹腔注射丙戊酸钠来每周间歇性地激活Notch信号通路，结果表明，每周连续3天间歇性激活Notch信号通路引起24周龄雌性大鼠的骨密度(BMD)及骨体积分数(BV/TV)与对照组相比显著提高，而每周连续2天、4天或5天激活Notch信号通路则与对照组相比不能引起骨密度与骨体积分数的显著改变。这一结果表明，间歇性地激活Notch信号通路在雌性大鼠显著促进了骨形成。
本发明的一个方面是通过周期性地间歇性给予Notch信号通路抑制剂来治疗、预防或延迟治疗对象的骨丢失，其特征在于，包括如下步骤：(1)对治疗对象应用Notch信号通路抑制剂；(2)等待Notch信号通路抑制剂在治疗对象体内降 解代谢到有效浓度以下；(3)重复步骤(1)-(2)。本发明中周期性地间歇性给予Notch信号通路抑制剂是指给予有效剂量的Notch信号通路抑制剂之后，在体内持续抑制Notch信号通路一定的时间，然后停止给予Notch信号通路抑制剂一定的时间。随着Notch信号通路抑制剂在体内的代谢，直到Notch信号通路抑制剂在体内的含量降低到有效剂量以下并维持一段时间后，然后再给予有效剂量的Notch信号通路抑制剂，再等待其体内浓度降低到有效剂量以下并维持一段时间后，然后再次给予有效剂量的Notch信号通路抑制剂，如此反复进行。由于骨质疏松症是一种长期的慢性代谢性疾病，因此本发明的治疗方法可以长期反复持续地进行。其中，Notch信号通路在体内连续抑制的时间可以是几小时、几天也可以是几周，Notch信号抑制剂在体内含量降低到有效剂量以下后维持的一段连续时间可以是几小时、几天也可以是几周，这一时间可以和Notch信号通路在体内抑制的时间相等，也可以不相等。作为一种优选，对治疗对象持续应用Notch信号通路抑制剂并持续抑制Notch信号通路的时间为连续1-7天，对治疗对象停止应用Notch信号通路抑制剂并等待Notch信号通路抑制剂降解代谢到有效浓度以下的持续时间为连续1-7天。作为一种优选，对治疗对象持续应用Notch信号通路抑制剂并持续抑制Notch信号通路的时间为连续2-3天，对治疗对象停止应用Notch信号通路抑制剂并等待Notch信号通路抑制剂降解代谢到有效浓度以下的持续时间为连续4-5天。作为一种优选，对治疗对象持续应用Notch信号通路抑制剂并持续抑制Notch信号通路的时间为连续3-4天，对治疗对象停止应用Notch信号通路抑制剂并等待Notch信号通路抑制剂降解代谢到有效浓度以下的持续时间为连续3-4天。作为一种优选，对治疗对象持续应用Notch信号通路抑制剂并持续抑制Notch信号通路的时间为连续2-5天，对治疗对象停止应用Notch信号通路抑制剂并等待Notch信号通路抑制剂降解代谢到有效浓度以下的持续时间为连续2-5天。由于Notch信号通路抑制剂显著促进了体外培养的骨髓间充质干细胞向成骨细胞的增生分化，而Notch受体在间充质干细胞的敲除导致小鼠体内成骨细胞的增多，因此Notch信号通路抑制剂能导致治疗对象体内成骨细胞的增加。当Notch信号通路抑制剂在体内的浓度降解代谢到有效浓度以下，则解除了对Notch信号通路的抑制，从而导致Notch信号通路的激活，而激活的Notch信号通路则促进了成骨细胞分化 矿化为骨细胞。因此间歇性地给予Notch信号通路抑制剂的净结果是促进了干细胞向骨细胞的分化矿化，进而促进了成骨。在本发明的具体实施例中，Notch信号通路中Notch配体Jag1在骨髓间充质干细胞的诱导敲除，显著促进了骨髓间充质干细胞向成骨细胞的分化，表现为呈碱性磷酸酶阳性的细胞数量显著增加，然而其诱导培养的矿化结节显著低于对照组，说明Notch信号通路的抑制能促进干细胞分化为成骨细胞，但是却抑制了成骨细胞的进一步分化和矿化，因此间歇性地抑制Notch信号通路能间歇性地促进成骨细胞的增生，其净结果为促进了成骨。
本发明的一个方面是通过交替性给予Notch信号通路激活剂和Notch信号通路抑制剂来治疗、预防或延迟治疗对象的骨丢失，其特征在于，包括如下步骤：(1)对治疗对象应用Notch信号通路激活剂；(2)等待或不等待Notch信号通路激活剂在治疗对象体内降解代谢到有效浓度以下；(3)对治疗对象应用Notch信号通路抑制剂；(4)等待或不等待Notch信号通路抑制剂在治疗对象体内降解代谢到有效浓度以下；(5)重复步骤(1)-(4)。本发明中交替性给予Notch信号通路激活剂和Notch信号通路抑制剂的意思是给予有效剂量的Notch信号通路激活剂之后，在体内激活Notch信号通路一定的时间，然后给予Notch信号通路抑制剂一定的时间，然后再次给予有效剂量的Notch信号通路激活剂一定的时间，如此反复进行。其中，给予Notch信号通路激活剂并维持持续激活Notch信号通路在体内活性的时间可以是几小时、几天也可以是几周，给予Notch信号通路抑制剂并维持持续抑制Notch信号通路在体内活性的时间可以是几小时、几天也可以是几周，而且这一时间间隔可以和Notch信号通路在体内持续激活的时间相等，也可以不相等。给予Notch信号通路激活剂之后等待Notch信号通路激活剂降解代谢到有效浓度以下的一段时间可以很短或没有，也可以是几小时或几天，给予Notch信号通路抑制剂之后等待Notch信号通路抑制剂降解代谢到有效浓度以下的一段时间可以很短或没有，也可以是几小时或几天。作为一种优选，对治疗对象持续应用Notch信号通路激活剂并持续激活Notch信号通路的时间为连续1-7天，对治疗对象持续应用Notch信号通路抑制剂并持续抑制Notch信号通路活性的时间为连续1-7天。作为一种优选，对治疗对象持续应用Notch信号通路激活剂并持续激 活Notch信号通路的时间为连续2-3天，对治疗对象持续应用Notch信号通路抑制剂并持续抑制Notch信号通路活性的时间为连续4-5天。作为一种优选，对治疗对象持续应用Notch信号通路激活剂并持续激活Notch信号通路的时间为连续3-4天，对治疗对象持续应用Notch信号通路抑制剂并持续抑制Notch信号通路活性的时间为连续3-4天。作为一种优选，对治疗对象持续应用Notch信号通路激活剂并持续激活Notch信号通路的时间为连续2-5天，对治疗对象持续应用Notch信号通路抑制剂并持续抑制Notch信号通路活性的时间为连续2-5天。在系统性红斑狼疮模型小鼠中(MRL/lpr)，骨髓间充质干细胞有高表达的Notch信号活性，伴随严重的骨丢失。由于在该模型小鼠的骨髓间充质干细胞(BMSC)中有高表达的Notch1、Notch2、NICD和Jag1信号，该模型小鼠为一个典型的在干细胞阶段激活Notch信号通路的小鼠。在系统性红斑狼疮模型小鼠(MRL/lpr)中间歇性地抑制Notch活性(在28天实验中，每周前三天每天通过DAPT抑制Notch活性，随后四天不处理)，该小鼠骨密度显著增加，提示在该小鼠有显著的成骨作用。在这一实验中，Notch信号通路在每周前三天通过每天的DAPT处理而受到强烈抑制，而在每周后四天无DAPT处理的情况下，该模型小鼠表现为Notch信号通路活性激活的状态。这样Notch信号通路活性抑制与激活交替作用四个循环后，小鼠表现为显著的骨密度增加。
本发明的一个方面是丙戊酸(2-丙基戊酸)和白藜芦醇(3，4’，5-三羟基-1，2-二苯乙烯)通过Notch信号通路促进骨形成。Notch信号通路在多种不同的细胞有不同的活性。在干细胞分化为终末细胞的过程中，有些细胞分化早期需要Notch信号通路的激活，有些细胞分化晚期需要Notch信号通路的激活。在内分泌细胞发育过程中，Notch信号通路的激活抑制内分泌前体干细胞向内分泌细胞的发育，而促进成熟的内分泌细胞的分化。而在神经元发育过程中，Notch信号通路在神经母细胞的激活抑制了神经母细胞向神经元细胞分化，而在神经元细胞内的激活则促进了神经元的成熟和功能。在肿瘤发生过程中，Notch信号通路的激活既促进一些肿瘤细胞的增生和转移，又抑制一些肿瘤细胞的增生和转移并促进这些肿瘤细胞的分化。Notch信号通路的激活抑制特定肿瘤细胞的增生转移并促进这些肿瘤细胞分化的作用与Notch信号通路在干细胞向骨细胞分化过程中的表现 类似，即Notch信号通路的激活既促进成骨细胞分化为骨细胞又抑制干细胞向成骨细胞分化从而维持干细胞的未分化状态。由于Notch信号通路在内分泌细胞分化、神经元细胞分化和某些特定肿瘤细胞中的作用与其在干细胞向骨细胞分化过程中的作用类似，因此我们认为能在内分泌细胞、神经元细胞或某些Notch信号通路分子低表达或无表达的如上所述的肿瘤细胞激活Notch信号通路的因子也能在成骨细胞促进Notch信号通路的激活并促进成骨细胞向骨细胞的分化矿化。白藜芦醇和丙戊酸在类癌细胞、骨肉瘤细胞等多种细胞系诱导Notch的高表达，并抑制这些细胞的生长，而白藜芦醇和丙戊酸也能显著促进成骨细胞的矿化。在本发明的一个具体实施例中，在小鼠骨髓间充质干细胞(BMSC)成骨性分化过程中，丙戊酸钠显著促进了矿化结节的形成，而Notch信号通路抑制剂则强烈抑制了丙戊酸钠促进矿化结节形成的能力，说明丙戊酸钠通过Notch信号通路来调控小鼠骨髓间充质干细胞(BMSC)的成骨性分化。在本发明的一个具体实施例中，在小鼠骨髓间充质干细胞(BMSC)成骨性分化过程中，白藜芦醇显著促进了矿化结节的形成，而Notch信号通路抑制剂则强烈抑制了白藜芦醇促进矿化结节形成的能力，说明白藜芦醇通过Notch信号通路来调控小鼠骨髓间充质干细胞(BMSC)的成骨性分化。
本发明中，Notch信号通路的激活剂是丙戊酸。这里，丙戊酸是指丙戊酸及其药学上可接受的盐、酯、酰胺、丙戊酸磷脂衍生物、丙戊酸酰胺衍生物、2-丙基戊烯酸酰胺衍生物、2-正-丙基-4-己炔酸衍生物中的一种或几种。优选的，Notch信号通路激活剂是丙戊酸、丙戊酸钠、双丙戊酸钠、丙戊酸钙、丙戊酸镁、丙戊酸酰胺、2-乙基-3-甲基戊酰胺、仲丁基-丙基乙酰胺(SPD)、2-丙基戊烯酸酰胺、2-正-丙基-4-己炔酸中的一种或几种。丙戊酸钠是一种目前常用的广谱抗癫痫药。体外细胞培养实验表明，丙戊酸能在骨肉瘤、神经母细胞瘤等多种细胞系激活Notch信号通路，其对多种细胞的抑制增长和刺激分化作用能被Notch信号通路的抑制剂或Notch信号通路特异的siRNA阻断。在体外细胞培养实验中，多个研究组报道丙戊酸能抑制原代BMSC及BMSC来源的细胞系的增生并促进这些细胞的矿化。在小鼠、大鼠和人体试验中，多个实验表明长期持续地摄入丙戊酸导致骨丢失和骨密度的降低。而在一项儿童和青少年的脊髓性肌肉萎缩症病人II 期临床实验中，丙戊酸显著提高了病人的骨密度。丙戊酸在成人中的半衰期为10到20小时，而在儿童的半衰期为6到9小时。由于丙戊酸在成人有很长的半衰期，因此成人长期持续地摄入丙戊酸导致Notch信号通路在间充质干细胞、成骨细胞以及骨细胞均持续活化，从而抑制了间充质干细胞向成骨细胞分化并促进了成骨细胞向骨细胞分化矿化，进而导致成骨细胞数量下降，而成骨细胞数量的下降将进一步导致骨细胞数量的下降。因此长期持续地摄入丙戊酸的净结果为干细胞分化为骨细胞的过程受到抑制，最后导致净骨量减少，骨密度降低。由于丙戊酸在儿童有相对短的半衰期，因此丙戊酸在儿童患者表现为对Notch信号通路的间歇性激活。间歇性激活Notch信号通路有正面的成骨作用，因此这一特性可以解释在儿童和青少年脊髓性肌肉萎缩症病人II期临床实验中丙戊酸的促进成骨作用。然而丙戊酸在体内的作用不仅与半衰期有关，而且也与治疗剂量密切相关，因此无论在儿童还是在成人，我们观察到在一些实验中长期持续摄入丙戊酸能降低骨密度，而在另一些实验中长期持续摄入丙戊酸不降低骨密度。在具体实施例中，我们持续或间歇性地在小鼠、大鼠或双侧卵巢切除的大鼠注射丙戊酸。结果表明，与对照组相比，间歇性注射丙戊酸显著促进了小鼠、大鼠和双侧卵巢切除大鼠的成骨，提高了骨体积百分数及骨密度，而持续性注射丙戊酸则对小鼠或大鼠的骨体积百分数和骨密度没有显著影响，表明间歇性注射的丙戊酸通过Notch信号通路调控成骨。
本发明中，Notch信号通路的激活剂是白藜芦醇。这里，白藜芦醇是指白藜芦醇(3，5，4’-三羟基二苯乙烯)、白藜芦醇衍生物、白藜芦醇类似物、白藜芦醇低聚体其中的一种或几种。白藜芦醇衍生物是指以白藜芦醇分子内的二苯乙烯为骨架，通过改变白藜芦醇两个苯环上的取代基或改变顺反式结构得到化合物。优选的，白芦藜醇的衍生物包括白藜芦醇的羟基化衍生物、甲基化衍生物、硝基化衍生物、磺基化衍生物、氨基化衍生物、乙酰胺基衍生物或它们的组合以及在其分子骨架的两个苯环上分别引入新的元素得到的化合物。白藜芦醇类似物是指分子骨架为二苯乙烷的化合物、分子骨架为二苯乙烯中的碳被电子等排体氮替换得到的化合物或分子骨架中B环被噻吩基取代所得到的化合物。白藜芦醇低聚体是指白藜芦醇及其衍生物以同种或异种的单体经脱氢后形成聚合度不 等的化合物，包括但不仅限于葡萄素、α-葡萄素、ε-葡萄素、δ-葡萄素。优选的，白藜芦醇的羟基化衍生物包括但不限于在白藜芦醇分子骨架的两个苯环上增加或减少羟基数量以及改变羟基的位置得到的化合物。优选的，白藜芦醇的甲基化衍生物包括但不限于在白藜芦醇或其羟基化衍生物中的分子骨架两个苯环上的羟基部分或全部甲基化得到的化合物。优选的，白藜芦醇的硝基化衍生物、磺基化衍生物、氨基化衍生物、乙酰胺基衍生物包括但不限于在白藜芦醇、白藜芦醇羟基化衍生物或白藜芦醇甲基化衍生物中的分子骨架两个苯环上的羟基部分或全部分别被硝基、磺基、氨基、乙酰胺基取代得到的化合物。优选的，白藜芦醇的在其分子骨架的两个苯环上分别引入新的元素得到的化合物包括但不限于在其分子骨架的两个苯环上分别引入三氟甲基、氯元素、碘元素、安替比林等得到的化合物。优选的，白藜芦醇是指白藜芦醇、3’-羟基白藜芦醇(白皮杉醇)、4，3’，5’-三羟基白藜芦醇、4-羟基白藜芦醇、反式3，5-二羟基二苯乙烯、3，5，4’-三甲氧基二苯乙烯(TMB)、3，4，4’-三甲氧基二苯乙烯、3，4，5，4’-四羟基二苯乙烯(4-HR)、3，4，5，4’-四甲氧基二苯乙烯(MR-4)、4-羟基反式二苯乙烯(4-HS)、4’-羟基二苯乙烯(4’-HS)、3，4’-羟基反式二苯乙烯(3，4’-DHS)、3，4’，5-三羟基
Figure PCTCN2017111300-appb-000001
-3-β-单-D-葡萄糖苷、紫檀芪(3，5-二甲氧基-4’-羟基二苯乙烯)、3’-羟基紫檀芪、3，5，2’，4’-四甲氧基二苯乙烯、5，4’-二羟基-3-甲氧基二苯乙烯、3，5-二羟基-4’-甲氧基二苯乙烯、顺式-3，4，5-三甲氧基-4’-溴二苯乙烯、葡萄素、α-葡萄素、ε-葡萄素、δ-葡萄素、SRT501或其组合物。体外细胞实验表明，白藜芦醇与丙戊酸一样，能在髓母细胞瘤、类癌细胞、未分化甲状腺癌、骨肉瘤等多种细胞激活Notch信号通路，并且其对多种细胞的抑制增生和促进分化效应能被Notch信号通路特异的siRNA阻断。在体外细胞培养实验中，白藜芦醇能促进成骨细胞的分化、促进骨髓间充质干细胞的矿化，并在体内促进颅骨缺损的修复，显示白藜芦醇通过激活Notch信号通路调控成骨细胞分化。在动物体内实验中，白藜芦醇能防止大鼠、小鼠、兔子等正常或骨质疏松模型动物中的骨丢失。在一项肥胖男性的临床实验中，白藜芦醇能促进骨形成，提高骨密度。然而在其它一些动物和人体实验中，白藜芦醇并没有显著提高骨密度。有报道表明口服后血浆中总的白藜芦醇含量低于口服剂量的2％。在一些健康志愿者实验中，口服 白藜芦醇后血浆中游离白藜芦醇浓度在30分钟内达到最高，随后快速降低，并在六小时后以指数形式加速代谢。在另一些健康志愿者实验中，血浆中的白藜芦醇及其代谢物在4小时内达到基线水平。由于白藜芦醇在生物体内生物利用度低，代谢迅速，因此每天摄入给药的方式类似于间歇性地刺激Notch信号通路的激活，而间歇性激活Notch信号通路有利于成骨，因此我们在部分动物及人体实验中观察到每天摄入白藜芦醇有统计意义上显著的成骨现象。在不同的大鼠实验中，我们观察到只有非常高的白芦藜醇口服剂量才能检测到显著的促进成骨现象。我们认为，由于白藜芦醇生物利用度很低并代谢迅速，因此只有很高的白芦藜醇摄入剂量才能在动物体内维持有效的激活Notch信号通路的作用，进而促进成骨。不同的人群对白藜芦醇的代谢速率不同，而且不同人体试验采用的口服剂量不同，因此在不同的人体实验中，我们观察到白芦藜醇对成骨作用的不同结果。在具体实施例中，白藜芦醇显著促进了小鼠间充质干细胞成骨性分化晚期矿化结节的形成，而Notch信号通路抑制剂则显著抑制了白藜芦醇的促进矿化结节生成的作用，说明白藜芦醇通过Notch信号通路促进成骨细胞的分化和矿化。
发明的有益效果
对附图的简要说明
附图说明
图1 Notch信号通路在干细胞成骨性分化为骨细胞过程中的作用示意图。
图2 Notch信号通路激活剂丙戊酸对12周雌性C57BL/6小鼠股骨中的小梁骨的影响。选取12周雌性C57BL/6小鼠，随机分成5组，每组8只，按以下方案连续处理12周：PBS，周一至周日每天腹腔注射PBS；VPA2，周一至周二按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周三至周日每天腹腔注射PBS；VPA3，周一至周三按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周四至周日每天腹腔注射PBS；VPA4，周一至周四按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周五至周日每天腹腔注射PBS；VPA7，周一至周日按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)。12周后，处死动物，取股骨，经显微CT扫描，对股骨远端小梁骨进行形态计量学分析。A为骨体积分数 (BV/TV)的变化；B为骨密度(BMD)的变化。其中**，p＜0.01；***，p＜0.001。
图3 Notch信号通路激活剂丙戊酸对20周雌性C57BL/6小鼠股骨中的小梁骨的影响。选取20周雌性C57BL/6小鼠，随机分成5组，每组12只，按以下方案连续处理12周：PBS，周一至周五每天腹腔注射PBS；VPA2，周一至周二按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周三至周五每天腹腔注射PBS；VPA3，周一至周三按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周四至周五每天腹腔注射PBS；VPA4，周一至周四按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周五腹腔注射PBS；VPA5，周一至周五按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)。12周后，处死动物，取股骨，经显微CT扫描，对股骨远端小梁骨进行形态计量学分析。A为骨体积分数(BV/TV)的变化；B为骨密度(BMD)的变化。其中*，p＜0.05；ns，p＞0.05。
图4 Notch信号通路激活剂丙戊酸对24周雌性SD大鼠股骨中的小梁骨的影响。选取24周雌性SD大鼠，随机分成5组，每组12只，按以下方案连续处理12周：PBS，周一至周五每天腹腔注射PBS；VPA2，周一至周二按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周三至周五每天腹腔注射PBS；VPA3，周一至周三按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周四至周五每天腹腔注射PBS；VPA4，周一至周四按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周五腹腔注射PBS；VPA5，周一至周五按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)。12周后，处死动物，取股骨，经显微CT扫描，对股骨远端小梁骨进行形态计量学分析。A为骨体积分数(BV/TV)的变化；B为骨密度(BMD)的变化。其中**，p＜0.01；***，p＜0.01；ns，p＞0.05。
图5 Notch信号通路激活剂丙戊酸对24周去势雌性SD大鼠股骨中的小梁骨的影响。选取16周龄雌性SD大鼠，进行双侧卵巢切除。手术8周后，随机分成5组，每组12只，按以下方案连续处理12周：PBS，周一至周五每天腹腔注射PBS；VPA2，周一至周二按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周三至周五每天腹腔注射PBS；VPA3，周一至周三按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周四至周五每天腹腔注射PBS；VPA4，周一至周四按 体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)，周五腹腔注射PBS；VPA5，周一至周五按体重每天腹腔注射丙戊酸钠(剂量为100mg/kg体重)。注射12周后，处死动物，取股骨，经显微CT扫描，对股骨远端小梁骨进行形态计量学分析。A为骨体积分数(BV/TV)的变化；B为骨密度(BMD)的变化。其中*，p＜0.05；ns，p＞0.05。
发明实施例
本发明的实施方式
下面结合具体实施例，进一步阐述本发明，应当理解，这些实施例仅用于说明本发明而不用于限制本发明要求保护的范围。
实施例1小鼠骨髓间充质干细胞的培养和成骨性分化
取C57BL/6小鼠股骨和胫骨，暴露骨髓腔，用αMEM冲洗出骨髓间充质干细胞。随后以每孔10,000,000个细胞悬浮于2毫升基础培养基(含有10％胎牛血清和1％青链霉素的αMEM)接种于六孔板，在37℃、5％CO2、饱和湿度≥95％的培养箱中培养。接种第四天开始，每三天更换一次培养液。第1至第7天，在基础培养基培养。第8天至第21天，转到成骨诱导培养基(基础培养基成分加入20mMβ-甘油磷酸钠、10nM地塞米松和50μg/ml抗坏血酸)继续培养。在第21天，按如下所述进行von Kossa法矿化结节染色。
矿化结节染色(von Kossa法)：(1)培养皿用PBS冲洗2次，4％多聚甲醛固定5分钟，双蒸水冲洗3次。(2)培养皿中加入5％硝酸银溶液1ml，将培养板开盖在紫外灯下照射1小时。(3)倒出残留的硝酸银溶液，蒸馏水冲洗3遍。(4)培养皿中加入5％硫代硫酸钠溶液1ml，中和残留的硝酸银溶液，吸出残液。(5)室温下晾干，封固。
结果：在小鼠骨髓间充质干细胞成骨性分化培养的第21天，von Kossa染色发现培养皿内有明显的散在的染成黑色的矿化结节。
相同的小鼠骨髓间充质干细胞成骨性分化培养和von Kossa染色方法同样应用于实施例2到实施例6中的骨髓间充质干细胞成骨性分化培养实验。
实施例2 Notch信号通路在骨髓间充质干细胞成骨性分化晚期激活
在转基因Notch报道小鼠(TNR)，Notch信号通路的激活诱导绿色荧光蛋白的 表达，因此有Notch活性的细胞在荧光显微镜下呈现绿色而无Notch活性的细胞没有绿色荧光。取12周龄TNR小鼠骨髓间充质干细胞，按照实施例1所述方法进行成骨性分化培养21天。
结果：在TNR小鼠骨髓间充质干细胞成骨性分化培养的第1天至第12天，没有检测到显示绿色荧光的细胞，而从第13天开始逐渐有显示绿色荧光的细胞出现，并在第16天左右显示绿色荧光的细胞数量最多，随后越来越多的显示绿色荧光的细胞被包裹在纤维化的细胞外基质内，导致绿色荧光强度的降低。这一结果说明Notch信号通路只在小鼠骨髓间充质干细胞成骨性分化培养的晚期激活。
实施例3 Notch信号通路抑制剂抑制骨髓间充质干细胞成骨性分化晚期的矿化
取12周龄C57BL/6小鼠骨髓间充质干细胞，按照实施例1所述方法进行成骨性分化培养21天。从培养的第4天起，在每3天换培养液的时候，一组培养基中加入10μM的Notch信号通路抑制剂DAPT，一组加入等量的DMSO(对照组)。在培养的第21天进行von Kossa染色检测矿化结节的形成。
结果：在小鼠骨髓间充质干细胞成骨性分化培养过程中，DAPT组的矿化结节显著少于对照组，提示Notch信号通路抑制剂DAPT显著抑制矿化结节的形成，说明Notch信号通路在骨髓间充质干细胞成骨性分化及矿化过程中发挥重要的作用。
实施例4 Notch 信号通路激活剂白藜芦醇促讲骨髓间充质干细胞成骨件分化晚 期的矿化
取12周龄C57BL/6小鼠骨髓间充质干细胞，按照实施例1所述方法进行成骨性分化培养21天。从培养的第8天起，在每3天换培养液的时候，一组培养基中加入20μM的Notch信号通路激活剂白藜芦醇(白藜芦醇组)，一组培养基中加入20μM的Notch信号通路激活剂白藜芦醇和10μM的Notch信号抑制剂DAPT(白藜芦醇+DAPT组)，一组加入等量的DMSO(对照组)。在培养的第21天进行von Kossa染色检测矿化结节的形成。
结果：在小鼠骨髓间充质干细胞成骨性分化培养过程中，白藜芦醇组的矿化结节显著多于对照组，而白芦藜醇+DAPT组的矿化结节显著低于白藜芦醇组，提示Notch信号通路激活剂白藜芦醇显著促进矿化结节的形成，而Notch信号通路抑 制剂抑制了白藜芦醇的促进矿化作用，说明Notch信号通路在骨髓间充质干细胞成骨性分化及矿化过程中发挥重要的作用。
实施例5 Notch 信号通路激活剂丙戊酸促讲骨髓间充质干细胞成骨性分化晚期 的矿化
取12周龄C57BL/6小鼠骨髓间充质干细胞，按照实施例1所述方法进行成骨性分化培养21天。从培养的第8天起，在每3天换培养液的时候，一组培养基中加入2mM的Notch信号通路激活剂丙戊酸钠(丙戊酸钠组)，一组培养基中加入2mM的Notch信号通路激活剂丙戊酸钠和10μM的Notch信号抑制剂DAPT(丙戊酸钠+DAPT组)，一组加入等量的DMSO(对照组)。在培养的第21天进行von Kossa染色检测矿化结节的形成。
结果：在小鼠骨髓间充质干细胞成骨性分化培养过程中，丙戊酸钠组的矿化结节显著多于对照组，而丙戊酸钠+DAPT组的矿化结节显著低于丙戊酸钠组，提示Notch信号通路激活剂丙戊酸钠显著促进矿化结节的形成，而Notch信号抑制剂抑制了丙戊酸钠的促进矿化作用，说明Notch信号通路在骨髓间充质干细胞成骨性分化及矿化过程中发挥重要的作用。
实施例6 Notch受体Jag1的缺失影响了骨髓间充质干细胞的成骨件分化
选取12周龄MX1Cre；Jag1f/f小鼠、MX1Cre小鼠和Jag1f/f小鼠，每天腹腔注射pIpC，连续注射7天后，处死小鼠，取小鼠骨髓间充质干细胞，分别按照实施例1所述方法进行成骨性分化培养21天。在培养的第8天，各组均取一部分细胞，按照生产厂家产品说明书所列的标准操作步骤，利用白细胞碱性磷酸酶试剂盒(Sigma公司，货号85L2)进行碱性磷酸酶染色。在培养的第21天，进行von Kossa染色检测矿化结节的形成。
结果：Jag1是重要的Notch配体，而MX1Cre可以在pIpC诱导条件下在骨髓间充质干细胞高表达，因此在MX1Cre；Jag1f/f小鼠，pIpC诱导可以选择性地在骨髓间充质干细胞敲除Jag1基因，进而抑制Notch信号通路的活性。细胞培养实验表明，经pIpC诱导后，MX1Cre；Jag1f/f小鼠的骨髓间充质干细胞(BMSC)与对照小鼠(MX1Cre和Jag1f/f)相比，在成骨性分化培养的第8天表现为碱性磷酸酶阳性的细胞显著增多，而在成骨性分化培养 的第21天则表现为矿化结节显著减少。该细胞培养实验表明，在骨髓间充质干细胞阶段敲除Jag1基因，进而抑制Notch信号通路在这一阶段细胞的活性，显著促进了BMSC向成骨细胞的分化增生，然而增生的成骨细胞由于Jag1基因的敲除而不能有效激活Notch信号通路，导致成骨细胞分化矿化为骨细胞这一阶段受到抑制，因此表现为矿化结节比对照组显著减少。
实施例7间歇性地激活Notch信号通路促讲小鼠的骨形成
选取12周龄雌性C57BL/6小鼠，随机分为五组，每组8只。第一组周一至周日每天腹腔注射PBS(对照PBS组)。第二组周一至周二每天腹腔注射100mg/kg的丙戊酸钠，周三至周日每天腹腔注射PBS(VPA2组)。第三组周一至周三每天腹腔注射100mg/kg的丙戊酸钠，周四至周日每天腹腔注射PBS(VPA3组)。第四组周一至周四每天腹腔注射100mg/kg的丙戊酸钠，周五至周日每天腹腔注射PBS(VPA4组)。第五组周一至周日每天腹腔注射100mg/kg的丙戊酸钠(VPA7组)。连续注射12周后，处死动物，取小鼠股骨，经显微CT扫描，按显微CT标准分析步骤分析股骨骨小梁的骨密度和骨体积百分比。
结果：丙戊酸注射组的骨密度与骨体积百分比均比对照组显著提高，其中VPA2组与对照组相比对成骨的刺激作用最大(图2)，提示丙戊酸间歇性地激活Notch信号通路能在小鼠显著促进骨生成。
实施例8间歇性地激活Notch信号通路促进小鼠的骨形成
选取20周龄雌性C57BL/6小鼠，随机分为五组，每组12只。第一组每周的周一至周五每天腹腔注射PBS(对照PBS组)。第二组每周的周一至周二每天腹腔注射100mg/kg的丙戊酸钠，周三至周五每天腹腔注射PBS(VPA2组)。第三组每周的周一至周三每天腹腔注射100mg/kg的丙戊酸钠，周四至周五每天腹腔注射PBS(VPA3组)。第四组每周的周一至周四每天腹腔注射100mg/kg的丙戊酸钠，周五腹腔注射PBS(VPA4组)。第五组每周的周一至周五每天腹腔注射100mg/kg的丙戊酸钠(VPA5组)。连续注射12周后，处死动物，取小鼠股骨，经显微CT扫描，按显微CT标准分析步骤分析股骨骨小梁的骨密度和骨体积百分比。
结果：和对照PBS组相比(图3)，丙戊酸注射组VPA2组的骨密度与骨体积百分比显著提高，而VPA3组、VPA4组和VPA5组与对照组相比对成骨的刺激作用 不明显，提示丙戊酸间歇性地激活Notch信号通路能在小鼠显著促进骨生成，而丙戊酸诱导Notch信号通路的持续性激活则对成骨的作用不明显。
实施例9间歇性地激活Notch信号通路促讲大鼠的骨形成
选取24周龄雌性SD大鼠，随机分为五组，每组12只。第一组每周的周一至周五每天腹腔注射PBS(对照PBS组)。第二组每周的周一至周二每天腹腔注射100mg/kg的丙戊酸钠，周三至周五每天腹腔注射PBS(VPA2组)。第三组每周的周一至周三每天腹腔注射100mg/kg的丙戊酸钠，周四至周五每天腹腔注射PBS(VPA3组)。第四组每周的周一至周四每天腹腔注射100mg/kg的丙戊酸钠，周五腹腔注射PBS(VPA4组)。第五组每周的周一至周五每天腹腔注射100mg/kg的丙戊酸钠(VPA5组)。连续注射12周后，处死动物，取大鼠股骨，经显微CT扫描，按显微CT标准分析步骤分析股骨骨小梁的骨密度和骨体积百分比。
结果：丙戊酸注射组VPA3组的骨密度与骨体积百分比和对照PBS组相比显著提高，而VPA2组、VPA4组和VPA5组的骨密度与骨体积百分比与对照组相比没有显著性差异(图4)，提示丙戊酸间歇性地激活Notch信号通路能在大鼠显著促进骨生成，而丙戊酸诱导Notch信号通路的持续性激活则对大鼠的骨生成作用不明显。
实施例10间歇性地激活Notch信号通路促进大鼠的骨形成
选取16周龄雌性SD大鼠60只，进行双侧卵巢切除。在第24周，随机分为五组，每组12只。第一组每周的周一至周五每天腹腔注射PBS(对照PBS组)。第二组每周的周一至周二每天腹腔注射100mg/kg的丙戊酸钠，周三至周五每天腹腔注射PBS(VPA2组)。第三组每周的周一至周三每天腹腔注射100mg/kg的丙戊酸钠，周四至周五每天腹腔注射PBS(VPA3组)。第四组每周的周一至周四每天腹腔注射100mg/kg的丙戊酸钠，周五腹腔注射PBS(VPA4组)。第五组每周的周一至周五每天腹腔注射100mg/kg的丙戊酸钠(VPA5组)。连续注射12周后，处死动物，取大鼠股骨，经显微CT扫描，按显微CT标准分析步骤分析股骨骨小梁的骨密度和骨体积百分比。
结果：去势雌性大鼠是经典的女性绝经后骨质疏松模型，双侧卵巢切除的去势雌性大鼠表现为严重的骨质疏松症状。在雌性去势大鼠，丙戊酸注射组VPA2组 的骨密度与骨体积百分比和对照PBS组相比显著提高，而VPA3组、VPA4组和VPA5组的骨密度与骨体积百分比与对照组相比没有显著性差异(图5)，提示丙戊酸间歇性地激活Notch信号通路能在去势大鼠显著促进骨生成，而丙戊酸诱导Notch信号通路的持续性激活则对去势大鼠的骨生成作用不明显。

Claims (14)

  1. 含Notch信号通路激活剂的化合物或组合物在制备用于预防或治疗骨质疏松症的药物中的应用，其特征在于，化合物或组合物的活性成分为Notch信号通路激活剂。
  2. 含Notch信号通路抑制剂的化合物或组合物在制备用于预防或治疗骨质疏松症的药物中的应用，其特征在于，化合物或组合物的活性成分为Notch信号通路抑制剂。
  3. 含Notch信号通路激活剂和Notch信号通路抑制剂的化合物或组合物在制备用于预防或治疗骨质疏松症的药物中的应用，其特征在于，化合物或组合物的活性成分为Notch信号通路激活剂和Notch信号通路抑制剂。
  4. 如权利要求1所述的化合物或组合物，其特征在于，该化合物或组合物间歇性地激活Notch信号通路，其作用方式依次包括如下步骤：步骤1：对治疗对象应用该化合物或组合物；步骤2：等待该化合物或组合物在治疗对象体内降解代谢到有效浓度以下；步骤3：重复步骤1至步骤2，直到检测到显著的骨形成效应为止。
  5. 如权利要求2所述的化合物或组合物，其特征在于，该化合物或组合物间歇性地抑制Notch信号通路，其作用方式依次包括如下步骤：步骤1：对治疗对象应用该化合物或组合物；步骤2：等待该化合物或组合物在治疗对象体内降解代谢到有效浓度以下；步骤3：重复步骤1至步骤2，直到检测到显著的骨形成效应为止。
  6. 如权利要求3所述的化合物或组合物，其特征在于，该化合物或组合物交替性地激活和抑制Notch信号通路，其作用方式依次包括如下步骤：步骤1：对治疗对象应用含Notch信号通路激活剂的化合物或组合物；步骤2：等待或不等待含Notch信号通路激活剂的化合物或组合物在治疗对象体内降解代谢到有效浓度以下；步骤3：对治疗对象应用含Notch信号通路抑制剂的化合物或组合物；步骤4：等待或不等待含Notch信号通路抑制剂的化合物或组合物在治 疗对象体内降解代谢到有效浓度以下；步骤5：重复步骤1至步骤4，直到检测到显著的骨形成效应为止。
  7. 如权利要求4所述的化合物或组合物，其特征在于，对治疗对象应用含Notch信号激活剂的化合物或组合物的时间为连续1-7天，对治疗对象不应用含Notch信号通路激活剂的化合物或组合物的时间为连续1-7天。
  8. 如权利要求5所述的化合物或组合物，其特征在于，对治疗对象应用含Notch信号通路抑制剂的化合物或组合物的时间为连续1-7天，对治疗对象不应用含Notch信号通路抑制剂的化合物或组合物的时间为连续1-7天。
  9. 如权利要求6所述的化合物或组合物，其特征在于，对治疗对象应用含Notch信号通路激活剂的化合物或组合物的时间为连续1-7天，对治疗对象不应用含Notch信号通路抑制剂的化合物或组合物的时间为连续1-7天。
  10. 如权利要求1或3所述的化合物或组合物，其特征在于，所述的Notch信号通路激活剂为丙戊酸及其药学上可接受的盐、酯、酰胺、丙戊酸磷脂衍生物、丙戊酸酰胺衍生物、2-丙基戊烯酸酰胺衍生物、2-正-丙基-4-己炔酸衍生物其中的一种或几种。
  11. 如权利要求1或3所述的化合物或组合物，其特征在于，所述的Notch信号通路激活剂为丙戊酸、丙戊酸钠、双丙戊酸钠、丙戊酸钙、丙戊酸镁、丙戊酸酰胺、2-乙基-3-甲基戊酰胺、仲丁基-丙基乙酰胺(SPD)、2-丙基戊烯酸酰胺、2-正-丙基-4-己炔酸其中的一种或几种。
  12. 如权利要求1或3所述的化合物或组合物，其特征在于，所述的Notch信号通路激活剂为白藜芦醇(3，5，4’-三羟基二苯乙烯)、白藜芦醇衍生物、白藜芦醇类似物、白藜芦醇低聚体其中的一种或几种。
  13. 如权利要求1或3所述的化合物或组合物，其特征在于，所述的No tch信号通路激活剂为白藜芦醇的羟基化衍生物、甲基化衍生物、硝基化衍生物、磺基化衍生物、氨基化衍生物、乙酰胺基衍生物、在白藜芦醇分子骨架的两个苯环上分别引入新的元素得到的化合物其中的一种或几种。
  14. 如权利要求1或3所述的化合物或组合物，其特征在于，所述的Notch信号通路激活剂为白藜芦醇、3’-羟基白藜芦醇(白皮杉醇)、4，3’，5’-三羟基白藜芦醇、4-羟基白藜芦醇、反式3，5-二羟基二苯乙烯、3，5，4’-三甲氧基二苯乙烯(TMB)、3，4，4’-三甲氧基二苯乙烯、3，4，5，4’-四羟基二苯乙烯(4-HR)、3，4，5，4’-四甲氧基二苯乙烯(MR-4)、4-羟基反式二苯乙烯(4-HS)、4’-羟基二苯乙烯(4’-HS)、3，4’-羟基反式二苯乙烯(3，4’-DHS)、3，4’，5-三羟基
    Figure PCTCN2017111300-appb-100001
    -3-β-单-D-葡萄糖苷、紫檀芪(3，5-二甲氧基-4’-羟基二苯乙烯)、3’-羟基紫檀芪、3，5，2’，4’-四甲氧基二苯乙烯、5，4’-二羟基-3-甲氧基二苯乙烯、3，5-二羟基-4’-甲氧基二苯乙烯、顺式-3，4，5-三甲氧基-4’-溴二苯乙烯、葡萄素、α-葡萄素、ε-葡萄素、δ-葡萄素、SRT501其中的一种或几种。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113481153A (zh) * 2021-07-25 2021-10-08 南通美韦德生命科学有限公司 一种脂肪间充质干细胞的分离培养方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090156510A1 (en) * 2007-11-09 2009-06-18 Washington University In St. Louis Use of notch signaling regulators for modulating osteogenesis
CN101481300A (zh) * 2009-02-20 2009-07-15 中国科学院广州化学研究所 一种反式多羟基二苯乙烯的制备方法
WO2013102193A1 (en) * 2011-12-29 2013-07-04 Trustees Of Tufts College Functionalization of biomaterials to control regeneration and inflammation responses

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090156510A1 (en) * 2007-11-09 2009-06-18 Washington University In St. Louis Use of notch signaling regulators for modulating osteogenesis
CN101481300A (zh) * 2009-02-20 2009-07-15 中国科学院广州化学研究所 一种反式多羟基二苯乙烯的制备方法
WO2013102193A1 (en) * 2011-12-29 2013-07-04 Trustees Of Tufts College Functionalization of biomaterials to control regeneration and inflammation responses

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIU, PENG ET AL.: "Anabolic Actions of Notch on Mature Bone", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 113, no. 15, 28 March 2016 (2016-03-28), pages E2152, XP055487412, ISSN: 0027-8424 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113481153A (zh) * 2021-07-25 2021-10-08 南通美韦德生命科学有限公司 一种脂肪间充质干细胞的分离培养方法

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