WO2021000640A1 - Dkk1抑制剂在预防和/或治疗肿瘤恶病质与糖尿病伴随疾病中的应用 - Google Patents

Dkk1抑制剂在预防和/或治疗肿瘤恶病质与糖尿病伴随疾病中的应用 Download PDF

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WO2021000640A1
WO2021000640A1 PCT/CN2020/086807 CN2020086807W WO2021000640A1 WO 2021000640 A1 WO2021000640 A1 WO 2021000640A1 CN 2020086807 W CN2020086807 W CN 2020086807W WO 2021000640 A1 WO2021000640 A1 WO 2021000640A1
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dkk1
lrp5
mice
protein
diabetes
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PCT/CN2020/086807
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English (en)
French (fr)
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朱伟东
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上海东慈生物科技有限公司
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Priority to US17/624,453 priority Critical patent/US20220349876A1/en
Priority to EP20834295.6A priority patent/EP4009051A4/en
Publication of WO2021000640A1 publication Critical patent/WO2021000640A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Definitions

  • the invention relates to the field of biomedicine. More specifically, the present invention relates to the use of DKK1 inhibitors in the prevention and/or treatment of tumor cachexia and diabetes-associated diseases.
  • Tumor cachexia is accompanied by weight loss, which severely impairs the quality of life, limits the treatment of cancer, and shortens the survival time.
  • diabetes is accompanied by damage to various organs in the body, which seriously endangers the life and health of patients, and restricts the treatment of many accompanying or inherent diseases.
  • various hypoglycemic therapies can effectively prevent the occurrence and development of the disease, insulin itself has strong toxic side effects on a large group of patients (such as patients with diabetes and heart failure), and how to judge that some hypoglycemic effects are unstable Whether the patient's organs continue to suffer damage, etc., there are still a large number of clinical problems that need to be resolved.
  • the purpose of the present invention is to provide a treatment plan that can effectively prevent and/or treat tumor cachexia and diabetes-associated diseases.
  • DKK1 gene or its encoded protein inhibitor to prepare a composition or preparation for preventing and/or treating tumor cachexia and diabetes-associated diseases.
  • the inhibitor includes an inhibitor that inhibits the binding of DKK1 to LRP5/LRP6.
  • the inhibitor inhibits the activity and/or expression of DKK1.
  • the inhibitor inhibits the formation of a complex of DKK1 and LRP5/LRP6.
  • the inhibitor inhibits the activity and/or expression of LRP5/LRP6.
  • the inhibitor inhibits the activity and/or expression of Kremen1/Kremen2.
  • the inhibitor is selected from the group consisting of antibodies, free DKK1 receptor binding domain or mutants thereof, other free ligands of DKK1 receptor or mutants thereof, small molecule compounds, microRNA, siRNA, shRNA, CRISPR reagent, or a combination thereof.
  • the inhibitor is selected from the group consisting of MDC, CDHC/STHC, IGFBP4 protein, IGFBP4 expression vector, IGFBP4 mutant, IGFBP4 mutant expression vector, LRP5/LRP6 neutralizing antibody, free LRP5 /LRP6 and DKK1 binding domain or its mutants, Kremen1/Kremen2 inhibitors (including neutralizing antibodies), free Kremen1/Kremen2 and DKK1 binding domain or its mutants, or combinations thereof
  • the vector expressing IGFBP4 or the vector expressing the IGFBP4 mutant includes a viral vector.
  • the viral vector is selected from the following group: adeno-associated virus vector, lentiviral vector, or a combination thereof.
  • the IGFBP4 protein includes a full-length protein or protein fragment.
  • the IGFBP4 protein also includes a derivative of IGFBP4 protein.
  • the derivative of the IGFBP4 protein includes a modified IGFBP4 protein, a protein molecule whose amino acid sequence is homologous to the natural IGFBP4 protein and has the activity of the natural IGFBP4 protein, a dimer or multimer of the IGFBP4 protein, A fusion protein containing the amino acid sequence of the IGFBP4 protein.
  • the "protein molecule whose amino acid sequence is homologous to the natural IGFBP4 protein and has natural IGFBP4 protein activity” means that its amino acid sequence has ⁇ 85% homology with the IGFBP4 protein, preferably ⁇ 90% homology, more preferably ⁇ 95% homology, and best ⁇ 98% homology; a protein molecule with natural IGFBP4 protein activity.
  • the IGFBP4 mutant is selected from the group consisting of IGFBP4/H95P, IGFBPDF/H95A, IGFBPDF/H95E, IGFBPDF/H95D, or a combination thereof.
  • the Kremen1/2 inhibitor refers to a substance that can antagonize the activity and/or content of the Kremen2 gene or its protein in vivo or in vitro; the substance can be a synthetic or natural compound or protein (Such as antibodies), nucleotides, etc.
  • the Kremen1/2 inhibitor includes a substance that antagonizes the expression of Kremen1/2.
  • the Kremen1/2 inhibitor includes a Kremen1/2 protein antagonist and/or a Kremen1/2 gene antagonist.
  • the inhibition of Kremen1/2 expression or activity refers to reducing the expression or activity of Kremen1/2 gene or protein by ⁇ 20%, preferably, ⁇ 50%, more preferably, ⁇ 70%.
  • the tumor cachexia includes tumor cachexia with normal or low Wnt expression.
  • the tumor cachexia includes tumor cachexia unrelated to the Wnt pathway.
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the tumor cells are derived from one or more tumors selected from the group consisting of colon cancer, lung cancer, gastric cancer, pancreatic cancer, head and neck malignant tumors, or a combination thereof.
  • the muscle protein marker includes myoglobin.
  • the cytokine in the muscle tissue is selected from the following group: TNF ⁇ , IL1 ⁇ , IL6, or a combination thereof.
  • the mammal includes a mammal suffering from tumor cachexia and diabetes-associated diseases.
  • the mammal includes a human or non-human mammal.
  • the non-human mammal includes rodents (such as mice, rats, or rabbits) and primates (such as monkeys).
  • the DKK1 is derived from mammals; preferably, it is derived from humans, mice, rats, or rabbits; more preferably, it is derived from humans.
  • the DKK1 gene includes wild-type DKK1 gene and mutant DKK1 gene.
  • the mutant type includes a mutant form in which the function of the encoded protein is not changed after mutation (that is, the function is the same or substantially the same as the wild-type encoded protein).
  • polypeptide encoded by the mutant DKK1 gene is the same or substantially the same as the polypeptide encoded by the wild DKK1 gene.
  • the mutant DKK1 gene includes homology of ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%, more preferably ⁇ 98% compared with wild DKK1 gene) Or 99%) polynucleotides.
  • mutant DKK1 gene is included in the 5'end and/or 3'end of the wild-type DKK1 gene, truncated or added 1-60 (preferably 1-30, more preferably 1 -10) nucleotide polynucleotides.
  • the DKK1 gene includes a cDNA sequence, a genomic sequence, or a combination thereof.
  • the DKK1 protein includes an active fragment of DKK1 or a derivative thereof.
  • the homology of the active fragment or its derivative with DKK1 is at least 90%, preferably 95%, more preferably 98%, 99%.
  • the active fragment or derivative thereof has at least 80%, 85%, 90%, 95%, 100% of DKK1 activity.
  • amino acid sequence of the DKK1 protein is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 1 or 3 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, which has the function of the protein, A polypeptide derived from (i); or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 1 or 3 is ⁇ 90% (preferably ⁇ 95%, more preferably ⁇ 98% or 99%), and has the protein function Peptides.
  • nucleotide sequence of the DKK1 gene is selected from the following group:
  • amino acid sequence of the DKK1 protein is shown in SEQ ID NO.: 1 or 3.
  • nucleotide sequence encoding the DKK1 protein is shown in SEQ ID NO.: 2 or 4.
  • the content of the inhibitor of the DKK1 gene or its encoded protein is 0.1 mg/kg-100 mg/kg, preferably, 1 mg/kg-50 mg/kg, more preferably, 2 mg/kg- 20mg/kg.
  • the composition includes a pharmaceutical composition.
  • the pharmaceutical composition contains (a) DKK1 gene or its encoded protein inhibitor; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is liquid, solid, or semi-solid.
  • the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral liquids, or injections.
  • the component (a) accounts for 1-99wt% of the total weight of the pharmaceutical composition, preferably 10-90wt%, more preferably 30-70wt% %.
  • the composition also includes other drugs for preventing and/or treating tumor cachexia and diabetes-associated diseases.
  • the other drugs for preventing and/or treating tumor cachexia and diabetes-associated diseases are selected from the following group: tumor drugs, diabetes treatment drugs, or a combination thereof.
  • the diabetes treatment drug is selected from the following group: insulin drugs, metformin hypoglycemic drugs, GLP drugs, ghrelin, or a combination thereof.
  • the tumor drug is selected from the group consisting of chemotherapy drugs, targeted therapy drugs, immunotherapy drugs, cell therapy drugs, or a combination thereof.
  • the chemotherapeutic drug is selected from the group consisting of gemcitabine, cisplatin, vincristine, paclitaxel, doxorubicin, 5-FU, or a combination thereof.
  • the targeted therapy drug is selected from the group consisting of lapatinib, erlotinib, apatinib, rituximab, bevacizumab, trastuzumab , Or a combination thereof.
  • the immunotherapy drug is selected from the group consisting of PD-1 monoclonal antibody, PDL-1 monoclonal antibody, CD47 monoclonal antibody, or a combination thereof.
  • the cell therapy drug is selected from the group consisting of NK cells, CART cells, TIL cells, or a combination thereof.
  • composition or preparation can be used alone or in combination in the prevention and/or treatment of tumor cachexia and diabetes-associated diseases.
  • the combined use includes: combined use with other drugs for preventing and/or treating tumor cachexia and diabetes-associated diseases.
  • the second aspect of the present invention provides a pharmaceutical composition comprising:
  • a first active ingredient for the prevention and/or treatment of tumor cachexia and diabetes-associated diseases includes: DKK1 gene or its encoded protein inhibitor;
  • a second active ingredient for preventing and/or treating tumor cachexia and diabetes-associated diseases includes: other drugs for preventing and/or treating tumor cachexia and diabetes-associated diseases;
  • the component (a1) accounts for 1-99wt% of the total weight of the pharmaceutical composition, preferably 10-90wt%, more preferably 30-70wt% %.
  • the component (a2) accounts for 1-99wt% of the total weight of the pharmaceutical composition, preferably 10-90wt%, more preferably 30-70wt% %.
  • the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.
  • all other drugs for preventing and/or treating tumor cachexia and diabetes-associated diseases are selected from the following group: tumor drugs, diabetes treatment drugs, or a combination thereof.
  • the diabetes treatment drug is selected from the following group: insulin drugs, metformin hypoglycemic drugs, GLP drugs, ghrelin, or a combination thereof.
  • the tumor drug is selected from the group consisting of chemotherapy drugs, targeted therapy drugs, immunotherapy drugs, cell therapy drugs, or a combination thereof.
  • the chemotherapeutic drug is selected from the group consisting of gemcitabine, cisplatin, vincristine, paclitaxel, doxorubicin, 5-FU, or a combination thereof.
  • the targeted therapy drug is selected from the group consisting of lapatinib, erlotinib, apatinib, rituximab, bevacizumab, trastuzumab , Or a combination thereof.
  • the immunotherapy drug is selected from the group consisting of PD-1 monoclonal antibody, PDL-1 monoclonal antibody, CD47 monoclonal antibody, or a combination thereof.
  • the cell therapy drug is selected from the group consisting of NK cells, CART cells, TIL cells, or a combination thereof.
  • the pharmaceutical composition can be a single compound or a mixture of multiple compounds.
  • the pharmaceutical composition is used to prepare drugs or preparations for treating or preventing tumor cachexia and diabetes-associated diseases.
  • the pharmaceutical dosage form is oral administration or non-oral administration dosage form.
  • the oral administration dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.
  • the non-oral administration dosage form is injection or injection.
  • the total content of the active ingredient (a1) and the active ingredient (a2) is 1 to 99 wt% of the total weight of the composition, more preferably 5 to 90 wt%.
  • the third aspect of the present invention provides a medicine kit including:
  • first container and the second container are the same or different containers.
  • the medicine in the first container is a unilateral preparation containing DKK1 gene or its encoded protein inhibitor.
  • the medicine in the second container is a single preparation containing other medicines for preventing and/or treating tumor cachexia and diabetes-associated diseases.
  • the dosage form of the drug is an oral dosage form or an injection dosage form.
  • the kit also contains instructions that describe the combined administration of the active ingredient (a1) and the active ingredient (a2) to prevent and/or treat tumor cachexia and diabetes-associated diseases.
  • the dosage forms of the preparation containing the active ingredient (a1) DKK1 gene or its encoded protein inhibitor or the preparation containing other drugs for preventing and/or treating tumor cachexia and diabetes-associated diseases respectively include Capsules, tablets, suppositories, or intravenous injections.
  • the concentration of the DKK1 gene or its encoded protein inhibitor is 0.1mg/kg-100mg/kg, which is more Preferably, 1mg/kg-50mg/kg, more preferably, 2mg/kg-20mg/kg.
  • the fourth aspect of the present invention provides a method for reducing the content of ⁇ -arrestin2 in tumor cells in vitro, which comprises the steps:
  • tumor cells are cultured to reduce the content of ⁇ -arrestin2 in tumor cells.
  • the tumor cells are derived from one or more tumors selected from the group consisting of colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, head and neck cancer, or a combination thereof.
  • the tumor cell is a cell cultured in vitro.
  • the method is non-diagnostic and non-therapeutic.
  • the concentration of the DKK1 gene or its encoded protein inhibitor is 0.1mg/kg-100mg/kg, preferably, 1mg/kg-50mg/kg, more preferably, 2mg/kg- 20mg/kg.
  • the fifth aspect of the present invention provides a pharmaceutical composition according to the second aspect of the present invention or the use of the kit according to the third aspect of the present invention for preparing a medicine for preventing and/or treating tumor cachexia and diabetes-associated diseases drug.
  • the concentration of DKK1 gene or its encoded protein inhibitor is 0.1 mg/kg-100 mg/kg, preferably, 1 mg/kg-50 mg/kg, more preferably , 2mg/kg-20mg/kg.
  • the sixth aspect of the present invention provides a method for screening potential therapeutic agents for tumor cachexia and diabetes-associated diseases, including:
  • test group in the culture system, culture the cells expressing the DKK1 gene or its protein for a period of T1 in the presence of the test compound, and detect the expression level of the DKk1 gene or its protein in the culture system of the test group E1 and/or active A1;
  • test compound is a potential therapeutic agent for tumor cachexia and diabetes-associated diseases. .
  • the "significantly higher" means E1/E2 ⁇ 1/2, preferably, ⁇ 1/3, more preferably, ⁇ 1/4.
  • the cells include tumor cells.
  • the tumor cells are derived from one or more tumors selected from the group consisting of colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, head and neck cancer, or a combination thereof.
  • the method is non-diagnostic and non-therapeutic.
  • the method includes step (c): administering the potential therapeutic agent determined in step (a) to the mammal, so as to determine its effect on tumor cachexia and diabetes-associated diseases in the mammal.
  • the mammal includes a human or non-human mammal.
  • the non-human mammals include rodents, primates, preferably, mice, rats, rabbits, and monkeys.
  • the seventh aspect of the present invention provides a method for screening potential therapeutic agents for tumor cachexia and diabetes-associated diseases, including:
  • test compound I If the number Q1 of the DKK1 and LRP6 complex formed in the test group is significantly lower than the number Q2 of the DKK1 and LRP6 complex formed in the control group, it means the test compound Is a candidate compound.
  • the method includes step (b): administering the candidate compound determined in step (a) to a mammal, thereby determining its effect on tumor cachexia and diabetes-associated diseases in the mammal.
  • the mammal includes a human or non-human mammal.
  • the non-human mammals include rodents, primates, preferably, mice, rats, rabbits, and monkeys.
  • the "significantly lower” means that Q1/Q2 ⁇ 1/2, preferably, ⁇ 1/3, more preferably ⁇ 1/4.
  • the method is non-diagnostic and non-therapeutic.
  • the tumor cells are derived from one or more tumors selected from the group consisting of colon cancer, lung cancer, gastric cancer, pancreatic cancer, and head and neck malignant tumors.
  • the eighth aspect of the present invention provides a method for preventing and/or treating tumor cachexia and diabetes-associated diseases, including the steps:
  • the DKK1 gene or its encoded protein inhibitor, the pharmaceutical composition according to claim 2 or the kit according to claim 3 are administered.
  • the administration includes oral administration.
  • the subject includes human or non-human mammals.
  • the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.
  • the dosage of the DKK1 gene or its encoded protein inhibitor is 0.1 mg/kg-100 mg/kg, preferably, 1 mg/kg-50 mg/kg, more preferably, 2 mg/kg- 20mg/kg.
  • the frequency of administration of the DKK1 gene or its encoded protein inhibitor is 1-7 times a week, preferably, 2-5 times a week, more preferably, 2-3 times a week.
  • the administration time of the DKK1 gene or its encoded protein inhibitor is 1-20 weeks, preferably, 2-12 weeks, more preferably, 4-8 weeks.
  • the ninth aspect of the present invention provides a method for determining a treatment plan for tumor cachexia and diabetes-associated diseases, including:
  • the subject is a human or non-human mammal.
  • the sample is derived from blood, tumor tissue, ascites or urine.
  • the sample is derived from peripheral blood.
  • the following methods are used for detection: immunological detection methods, enzymatic methods, or mass spectrometry methods.
  • the immunodetection method is selected from the group consisting of ELISA, Western blotting, immunofluorescence, chemiluminescence, or a combination thereof.
  • the treatment plan also includes other therapies for preventing and/or treating tumor cachexia and diabetes-associated diseases.
  • the other therapies for preventing and/or treating tumor cachexia and diabetes-associated diseases are selected from the following group:
  • the diabetes treatment drug is selected from the following group: insulin drugs, metformin hypoglycemic drugs, GLP drugs, ghrelin, or a combination thereof.
  • the tumor drug is selected from the group consisting of chemotherapy drugs, targeted therapy drugs, immunotherapy drugs, cell therapy drugs, or a combination thereof.
  • the chemotherapeutic drug is selected from the group consisting of gemcitabine, cisplatin, vincristine, paclitaxel, doxorubicin, 5-FU, or a combination thereof.
  • the targeted therapy drug is selected from the group consisting of lapatinib, erlotinib, apatinib, rituximab, bevacizumab, trastuzumab , Or a combination thereof.
  • the immunotherapy drug is selected from the group consisting of PD-1 monoclonal antibody, PDL-1 monoclonal antibody, CD47 monoclonal antibody, or a combination thereof.
  • the cell therapy drug is selected from the group consisting of NK cells, CART cells, TIL cells, or a combination thereof.
  • Figure 1 shows that the up-regulation of Dkk1 may be associated with cachexia-related tumor death.
  • Figure 1a shows the relationship between the TCGA database analysis of clinically different tumor types (including head and neck cancer, pancreatic cancer, gastric adenocarcinoma, and lung adenocarcinoma) in patients with Dkk1 expression and survival.
  • Figure 1b shows the ELISA method to detect the expression of Dkk1 in the blood of tumor-bearing mice (intestinal cancer cell implantation tumor and lung adenocarcinoma implantation tumor).
  • Figure 1c shows the RT-qPCR method to detect Dkk1 expression in different organs.
  • Figure 1d shows the effect of RT-qPCR detection of implanted tumor on the expression of Dkk1 in muscle and kidney.
  • Figure 1e shows the Kaplan-Meier analysis of the effect of Dkk1 on the survival of tumor-bearing mice.
  • Figure 1f shows the Kaplan-Meier analysis of the Dkk1 comprehensive antibody prolonging the survival time of tumor-bearing mice.
  • Figure 1g shows the changes in the tumor-free body weight and muscle weight of mice caused by the late tumor.
  • Figure 1h shows the HE staining to observe the muscle pathological state of cachexia mice.
  • Figure 1i shows the observation of the weight of the kidney in the late stage of tumor implantation, the observation of the pathological state of the kidney by HE staining, and the expression of renal function detected by the automatic biochemical instrument.
  • FIG. 2 shows that down-regulation of membrane proteins LRP6 and Kremen2 can cause tumor cachexia.
  • Figure 2a shows the Western blot method to detect the expression of LRP6, LRP5, Kremen1, and Kremen2 in advanced muscle media/total protein of cachexia.
  • Figure 2b shows the Western blot method to detect the expression of kidney media/total protein LRP6, LRP5, Kremen1, Kremen2 in the advanced stage of cachexia.
  • Figure 2c shows the Western blot method to detect the expression of total/nucleoprotein ⁇ -catenin in the advanced kidney of cachexia.
  • Figure 3 shows that reversing the downregulation of LRP6 and Kremen2 on the membrane can prevent tumor cachexia.
  • Figure 3a shows the Western blot method to detect the changes of membrane/plasma proteins LRP6 and Kremen2 caused by Dkk1 intramuscular injection, and the reversal effect of MDC on it.
  • Figure 3b shows the Kaplan-Meier analysis of the combined use of MDC and Dkk1 to prolong the survival shortening effect of Dkk1 in tumor-bearing mice.
  • Figure 3c shows that intraperitoneal injection of MDC and IGFBP4 can reverse tumor-induced weight loss and muscle atrophy.
  • Figures 3d and 3e show the Kaplan-Meier analysis of the effect of Clathin-TG2 inhibitors (MDC, Cystamine and Spermidine) on the survival of tumor-bearing mice.
  • Figure 3f shows the Western blot analysis of MDC to reverse the expression of membrane proteins LRP6 and Kremen2 caused by tumors, and the expression of total protein Myoglobin.
  • Figure 3g shows the Kaplan-Meier analysis of the effect of IGFBP4 on the survival of tumor-bearing mice.
  • Figure 3h shows the Western blot analysis of IGFBP4 affecting the expression of membrane/plasma proteins LRP6 and Kremen2 caused by Dkk1.
  • Figure 4 shows the RNA-Seq analysis: knocking down the LRP5/6 gene extensively alters the expression of GPCRs.
  • RNA-Seq analysis uses Venn diagrams to reveal the number distribution of GPCRs with expression fold change> 0.2.
  • B and C After LRP5/6 or CTNNB1 ( ⁇ -catenin) was knocked down by siRNA in HepG2 cells, RNA-Seq analysis used histogram to show the change of GPCR expression fold (B); heat map showed the expression up-regulation ( Red) or down-regulated (green) GPCR fold change (log 2 value) (C).
  • Figure 5 shows that exogenous LRP6N reverses cell DNA damage induced by knockdown of LRP5/6.
  • Figure 6 shows that knocking down MESD leads to downregulation of LRP5/6 on the membrane and attenuation of resistance to oxidative stress.
  • Figure 7 shows that Dkk1 induces LRP5/6 endocytosis and DNA damage on the cell membrane.
  • Figure 8 shows that Dkk1 activates ⁇ -arrestin1/2 signaling by down-regulating LRP5/6 on the membrane.
  • Figure 9 shows that ⁇ -arrestin1/2 mediates cellular DNA damage caused by downregulation of LRP5/6 on the membrane.
  • (A) The expression of ⁇ -arrestin1/2 in HUVEC cells 48h after ⁇ -arrestin1/2 knockdown. n 3.
  • (B) 48h after ⁇ -arrestin1/2 knockdown, Dkk1 protein and H 2 O 2 stimulate the ⁇ H2AX response induced by HUVEC cells alone or successively. n 3.
  • (C) The expression of ⁇ H2AX after BIM-46187 pretreatment and Dkk1 protein stimulation of HUVEC cells. n 3.
  • (D) 48h after ⁇ -arrestin1/2 combined with LRP5/6 or ⁇ -catenin knockdown, H 2 O 2 stimulation induced the ⁇ H2AX response of HUVEC cells. n 3.
  • Figure 10 shows that G protein agonists can induce DNA damage responses.
  • CTX A or PTX (B) stimulates the ⁇ H2AX response of HUVEC cells at the specified time and concentration.
  • n 3.
  • FIG 11 shows that LRP5/6 gene knockout causes heart damage in mice.
  • (A) Tamoxifen-induced control for 1 week, LRP5/6 -/- and ⁇ -catenin -/- mice, LRP5/6, ⁇ -catenin, ⁇ H2AX, p53, p21, Bcl-2, Bax in the heart And Cleaved Caspase-3 expression. n 9.
  • (B) Tamoxifen-induced 35-week control, LRP5/6 -/- and ⁇ -catenin -/- mouse body weight changes (left picture) and multiple organs (heart, lung, kidney, spleen, skeletal muscle and Fat) relative weight ratio (right). n 6.
  • Figure 12 shows that LRP5/6 gene knockout activates ⁇ -arrestin1/2 signal transduction in mouse hearts.
  • (A) Tamoxifen induced control for 1 week, LRP5/6 -/- and ⁇ -catenin -/- mice, cell membrane, cytoplasm and nucleus ⁇ -arrestin1/2(m/c/n ⁇ -arrestin1 /2) expression. n 6.
  • (B) Real-time quantitative PCR analysis. Tamoxifen induced 8-week control, LRP5/6 -/- and ⁇ -catenin -/- mice, and some GPCR expression in the heart. The data is standardized by GAPDH internal reference. n 6.
  • Figure 13 shows the up-regulation of blood Dkk1 in diabetic mice induced by hyperglycemia.
  • Figure 14 shows the close relationship between the down-regulation of LRP5/6 on the membrane of diabetic mice and heart damage.
  • FIG. 15 shows that Dkk1 induces diabetic heart damage by inducing LRP5/6 membrane endocytosis.
  • (B) Immunofluorescence staining of ⁇ H2AX (red) and DAPI (blue) in the heart of normal mice, STZ model and diabetic mice combined with insulin and MDC, the scale bar is 50 ⁇ m. n 5.
  • Figure 16 shows that the down-regulation of LRP5/6 on the membrane of Leptin -/- mice results in diabetic heart damage.
  • (D) Expression of m/tLRP5/6 and t ⁇ -catenin in the hearts of WT, Leptin +/- and Leptin -/- mice at 4 weeks of age. n 6.
  • (E) ELISA analysis of blood Dkk1 in 6-week-old WT, Leptin +/- and Leptin -/- mice. n 10.
  • (F) Glucose tolerance test (GTT) of 6-week-old WT and Leptin -/- mice. n 6.
  • Figure 17 shows that LRP5/6 gene knockout aggravated diabetic heart damage.
  • FIG. 18 shows that exogenous LRP6N prevents diabetic heart damage.
  • A A schematic diagram of the construction of the LRP6N/Tg mouse strain based on the Cre-loxP recombinase system (upper figure) and the genotype identification of UBC-Cre-positive LRP6N/Tg mice (lower figure). Tamoxifen induces the loxP sequence to be deleted by Cre recombinase, and the CAG promoter initiates the transcription of the myc-tagged LRP6N gene.
  • Figure 19 shows that the down-regulation of LRP5/6 on the membrane of diabetic mice specifically activates cardiac ⁇ -arrestin1/2 signaling.
  • Figure 20 shows a schematic diagram of the breeding of transgenic mice that can induce systemic LRP5 and LRP6 co-knockout or ⁇ -catenin single-knockout.
  • Mating LRP5/6floxp/floxp or ⁇ -cateninfloxp/floxp homozygous mice with UBC-Cre positive mice can produce inducible whole-body LRP5/6 co-knockout or ⁇ -catenin single-knockout transgenes Mouse (UBC-Cre-LRP5/6floxp/floxp or UBC-Cre- ⁇ -cateninfloxp/floxp).
  • mice induced by intraperitoneal injection of tamoxifen will achieve systemic LRP5/6 co-knockout or ⁇ -catenin single-knockout (Ctr is a negative control). Such mice can be expressed as LRP5/6- /- and ⁇ -catenin-/-.
  • FIG. 21 shows a schematic diagram of breeding transgenic mice that can induce systemic overexpression of LRP6N.
  • Mating mice carrying the LRP6N gene (STOPfloxp/floxpLRP6N) with UBC-Cre-positive mice can produce transgenic mice (UBC-Cre-STOPfloxp/floxpLRP6N) that overexpress LRP6N all over the body.
  • Mice induced by intraperitoneal injection of tamoxifen will overexpress LRP6N (Ctr is a negative control).
  • Such mice can be expressed as LRP6N/Tg.
  • the inventors unexpectedly discovered for the first time that inhibiting the expression or activity of the DKK1 protein in the blood of tumor cachexia and diabetes model animals can effectively prevent and/or treat tumor cachexia and diabetes-associated diseases, and discovered that DKK1 is One activity has nothing to do with traditional Wnt signaling pathway inhibitors, but by inducing the endocytosis of its receptor LRP5/6, thereby inducing organ damage.
  • the applicant also unexpectedly discovered that inhibiting the combination of DKK1 and LRP5/6 (ie inhibiting the formation of the complex of DKK1 with LRP5 and LRP6) can also effectively prevent and/or treat tumor cachexia and diabetes-associated diseases.
  • DKK1 protein induces the endocytosis of LRP5 and LRP6 to cause organ damage, accelerate the death of animals caused by tumor cachexia and the death of animals with acute myocardial infarction due to diabetes, and does not affect the Wnt signaling pathway effector protein ⁇ -catenin.
  • Small molecule drugs or protein drugs prevent the down-regulation of membrane LRP5 and LRP6 caused by Dkk1, which can completely prevent tumor cachexia and diabetes-associated diseases, and prolong the survival time of mice with these diseases.
  • small-molecule drugs prevent the down-regulation of membrane LRP5 and LRP6 caused by Dkk1 and prevent changes in multiple GPCR signaling pathways and all major cachexia-related pathways.
  • Cachexia is also known as cachexia. It is manifested as extreme weight loss, skinny, skeleton, anemia, weakness, complete bed rest, inability to take care of oneself, extreme pain, and general exhaustion. Usually caused by cancer and other serious chronic diseases. It can be regarded as a state of poisoning caused by many organs in the body. Tumor cachexia is accompanied by weight loss, which severely impairs the quality of life, limits the treatment of cancer, and shortens the survival time. However, there is currently no effective treatment method. Muscle atrophy is a key feature of tumor cachexia and a multifactorial disease that has a negative impact on the prognosis and quality of life of patients. Regardless of body mass index (BMI), skeletal muscle loss is considered to be a meaningful prognostic factor in the development of cancer, and is associated with increased incidence of chemotherapy toxicity, shortened tumor progression time, poor surgical results, physical damage and shortened survival .
  • BMI body mass index
  • Diabetes diabetes is a group of systemic metabolic diseases characterized by chronic hyperglycemia and caused by multiple causes of insulin secretion or defect. Long-term disorder of sugar, fat and protein metabolism can cause multiple organ damage and homeostasis imbalance, which can lead to chronic disease, hypofunction, and even failure of heart, kidney, eye, nerve, blood vessel and other tissues and organs.
  • Type 1 diabetes diabetes (diabetes mellitus type 1, T1DM) and type 2 diabetes (diabetes mellitus type 2, T2DM) are two important types of diabetes, and their pathogenesis is different. Continuous increase in blood sugar is a key factor in the DNA damage of multiple organs caused by diabetes. Therefore, how to reduce the cell damage caused by blood sugar while using insulin to lower blood sugar is an important scientific issue.
  • the terms "protein of the present invention”, “DKK1 protein”, and “DKK1 polypeptide” are used interchangeably, and all refer to a protein or polypeptide having the amino acid sequence of DKK1. They include DKK1 protein with or without starting methionine. In addition, the term also includes the full-length DKK1 and its fragments.
  • the DKK1 protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutant and its functionally active fragments.
  • DKK1 secreted protein is a classic Wnt/ ⁇ -catenin inhibitor, which inhibits the activation of classic Wnt/ ⁇ -catenin by directly binding to LRP5/6 receptor and Kremen receptor to mediate LRP5/6 endocytosis.
  • Mouse Dkk1 (NP_034181.2), composed of 272 amino acids, and its sequence is shown in SEQ ID NO.: 3;
  • human Dkk1 (NP_036374.1), composed of 266 amino acids, and its sequence is shown in SEQ ID NO.:1 .
  • DKK1 gene and “DKK1 polynucleotide” are used interchangeably, and both refer to a nucleic acid sequence having a DKK1 nucleotide sequence.
  • the similarity of human and mouse DKK1 at the DNA level is 83%, and the similarity of protein sequence is 81%. It should be understood that when encoding the same amino acid, the substitution of nucleotides in the codon is acceptable. In addition, it should be understood that when conservative amino acid substitutions are generated by nucleotide substitutions, nucleotide changes are also acceptable.
  • a nucleic acid sequence encoding it can be constructed based on it, and a specific probe can be designed based on the nucleotide sequence.
  • the full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification, recombination, or artificial synthesis.
  • primers can be designed according to the DKK1 nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library serves as a template, and the relevant sequence is obtained by amplification. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This usually involves cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain a very long fragment.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant DKK1 polypeptide. Generally speaking, there are the following steps:
  • polynucleotide or variant encoding human DKK1 polypeptide of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • the DKK1 polynucleotide sequence can be inserted into a recombinant expression vector.
  • any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • an expression vector containing the DNA sequence encoding DKK1 and suitable transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic cleavage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • Adeno-associated virus is smaller than other viral vectors, is non-pathogenic, and can transfect dividing and undivided cells, gene therapy methods based on AAV vectors for genetic diseases have been affected. Widespread concern.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the Parvoviridae dependent virus genus. It is the simplest type of single-stranded DNA-deficient virus found so far and requires a helper virus (usually adenovirus). Viruses) participate in replication. It encodes the cap and rep genes in the inverted repeat (ITR) at both ends. ITRs play a decisive role in virus replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.
  • Recombinant adeno-associated virus vector is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), and low immunogenicity, it can express foreign genes in vivo. Long and other characteristics, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially in terms of its application effects in various cells, tissues and in vivo experiments.
  • rAAV is used in the research of gene therapy for various diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, disease model construction, and gene preparation. Knockout mice and other aspects.
  • the vector is a recombinant AAV vector.
  • AAVs are relatively small DNA viruses that can integrate into the genome of the cells they infect in a stable and site-specific manner. They can infect a large range of cells without any effect on cell growth, morphology or differentiation, and they do not seem to be involved in human pathology.
  • the AAV genome has been cloned, sequenced and characterized.
  • AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of replication of the virus. The rest of the genome is divided into two important regions with encapsidation functions: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.
  • ITR inverted terminal repeat
  • AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described in, for example, PCT Application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for in vitro and in vivo gene transfer has been described (see, for example, International Patent Application Publication Nos. WO91/18088 and WO93/09239; U.S. Patent Nos.
  • Replication-deficient recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (such as adenovirus): the nucleic acid sequence of interest is flanked by two AAV inverted terminal repeats (ITR) Region plasmids, and plasmids carrying AAV encapsidation genes (rep and cap genes).
  • a human helper virus such as adenovirus
  • the recombinant vector is capsidized to viral particles (e.g., including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 And AAV virus particles of AAV16). Therefore, the present disclosure includes recombinant virus particles (recombinant because they contain recombinant polynucleotides) containing any of the vectors described herein. Methods of producing such particles are known in the art and are described in US Patent No. 6,596,535.
  • various conventional screening methods can be used to screen out substances that interact with DKK1 gene or protein, especially inhibitors.
  • the DKK1 inhibitor (or antagonist) that can be used in the present invention includes any substance that can inhibit the expression and/or activity of the DKK1 gene or its encoded protein.
  • DKK1 inhibitors also include inhibitors that inhibit the binding of DKK1 to LRP5 and LRP6 (including inhibitors that inhibit the formation of complexes of DKK1 with LRP5 and LRP6).
  • DKK1 inhibitors also include substances that inhibit the activity and/or expression of LRP5 and LRP6.
  • the inhibitor of DKK1 includes a small molecule compound, an antibody of DKK1, antisense RNA of DKK1 nucleic acid, siRNA, shRNA, miRNA, or an inhibitor of DKK1 activity.
  • the methods and steps for inhibiting DKK1 include using an antibody of DKK1 to neutralize its protein, and using shRNA or siRNA or CRISPR reagent carried by a virus (such as adeno-associated virus) to silence the DKK1 gene.
  • a virus such as adeno-associated virus
  • the inhibition rate of DKK1 is generally at least 50% or more inhibition, preferably 60%, 70%, 80%, 90%, 95% inhibition, which can be based on conventional techniques, such as flow cytometry, fluorescent quantitative PCR or Western Methods such as blot control and detect the inhibition rate of DKK1.
  • the inhibitors of DKK1 protein of the present invention can inhibit the expression and/or activity of DKK1 protein when administered (administered) therapeutically. Or inhibit the expression and/or activity of LRP5 and LRP6, or inhibit the formation of the complex of DKK1 with LRP5 and LRP6, thereby preventing and/or treating tumor cachexia.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): local, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, autologous cell extraction and culture and reinfusion Wait.
  • the present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the inhibitor of the present invention (such as antibodies, compounds, CRISPR reagents, antisense sequences (such as siRNA), or inhibitors) and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injections, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules should be manufactured under sterile conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 1 ⁇ g-10 mg/kg body weight per day.
  • the present invention found for the first time that inhibiting the expression or activity of LRP5 and LRP6, or inhibiting the formation of DKK1, LRP5 and LRP6 complexes, can prevent and/or treat tumor cachexia and diabetes-associated diseases.
  • the present invention finds for the first time that the tumor cachexia and diabetes-associated diseases that are prevented and/or treated by the present invention are tumor cachexia and diabetes-associated diseases that are not related to the Wnt pathway, or tumor cachexia and diabetes-associated diseases with normal or low Wnt expression.
  • the present invention finds for the first time that Dkk1 protein accelerates the death of animals caused by tumor cachexia and diabetes-associated diseases by inducing the endocytosis of LRP5 and LRP6, without affecting ⁇ -catenin.
  • Small molecule drugs prevented the down-regulation of membrane LRP5 and LRP6 caused by Dkk1, which completely prevented tumor cachexia and diabetes-associated diseases, and prolonged the survival time of mice.
  • the present invention finds for the first time that small molecule drugs prevent the down-regulation of membrane LRP5 and LRP6 caused by Dkk1 and prevent changes in multiple GPCR signaling pathways and all major cachexia and diabetes-associated disease-related pathways.
  • the present invention found for the first time that the development of tumor cachexia and diabetes-associated diseases is through Dkk1-LRP5 and LRP6 as the main axis, which affects the GPCR signaling pathway and is accompanied by the occurrence of inflammatory reactions.
  • CT26 colon cancer cell line was cultured in RPMI-1640 medium, supplemented with L-glutamine (Biosera, USA), 1% penicillin/streptomycin (Corning, USA) and 10% fetal bovine serum (Biosera, USA) USA). Cultured in a 37°C incubator, recombinant mouse Dkk1 and IGFBP-4/H95P proteins were purchased from Genscript. Mouse monoclonal antibody (mAb) to human Dkk1 was produced by Shanghai Sanyou Biopharmaceutical Co., Ltd. MDC was purchased from Sigma, dissolved in DMSO, and diluted with PBS for use.
  • mAb monoclonal antibody
  • mice Male BALB/c six-week-old mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and raised in a special sterile environment. All animal studies were approved by the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine and conducted in accordance with the National Institutes of Health Ethics Guidelines. The mice were randomly divided into two groups. The tumor-bearing mice were injected subcutaneously with 1*106CT26 tumor cells on the right side, and the control group was injected with an equal volume of PBS at the same site. The body weight, food intake and tumor volume of the mice were recorded every 3 days. Eight mice in each group were sacrificed on the 15, 20, 25, 30, 40, and 50 days after tumor implantation to detect the early, middle and late stages of cachexia.
  • MDC 10 mg/kg containing 12% DMSO or PBS was subcutaneously injected every three days as a control.
  • the tumor weight was estimated by multiplying the tumor density by the tumor volume.
  • the tumor density was determined by comparing the tumor reagent weight and tumor volume when the mice were sacrificed. Then subtract the tumor weight from the mouse body weight to get the tumor-free body weight.
  • the mice were sacrificed, and the samples were taken: quadriceps, kidneys and other organs, and weighed at the same time.
  • the blood samples were placed in centrifuge tubes containing edta after cardiac puncture, and plasma samples were obtained at 2000 rpm and 4°C and stored at -80°C for testing.
  • An ELISA kit (R&D, USA) specifically designed for mouse Dkk1 was used to determine plasma Dkk1 levels according to the instructions.
  • Wild-type mice and ⁇ -arrestin2+/-KO mice were injected into the left quadriceps muscle (0.25 mg/kg) and an equal volume of PBS was injected into the right leg as controls.
  • Combined injection of MDC (10 mg/kg), IGFBP-4/H95P (1.7 mg/kg) or PBS with Dkk1 was used as a control to observe the effect of the drug on the signal transfer induced by Dkk1.
  • the mice were sacrificed, and the left and right quadriceps were taken to measure the plasma cytokine levels.
  • Membrane protein was extracted according to the instructions of the membrane protein extraction kit (Sangon Biotech, China). Western blot analysis is omitted.
  • the antibodies used for WB analysis were purchased from Cell Signaling Technology and Proteintech.
  • the total RNA was extracted and isolated according to the instructions (Takara, Japan).
  • the relative mRNA level was calculated by the comparative CT method and normalized to the control group.
  • the detection primers are as follows:
  • RNA sequencing library was constructed from the extracted RNA using IIIumina library preparation protocol, RNA-seq was performed on the lllumina HiSeq4000 platform, using both ends protocol, and the length of each region was 150bp. Sequencing reads used hisat2 (v2.1.0) to align with the annotated mouse transcript (mm10). Use GFOLD (v1.1.4) to calculate gene expression levels and find differentially expressed genes. The parameters are as follows: GFOLD value ⁇ 0, and the change value of the two samples ⁇ 1.35. RNA-seq data acquisition instrument GSE121873 uploaded to the gene expression database . KEGG pathway analysis of defined genes.
  • the up-regulated DEGs in the quadriceps femoris of the CT26 tumor mice were modeled for 50 days or the Dkk1 protein injected for 24 hours and the DEGs in the quadriceps femoris of the control mice were up-regulated and down-regulated respectively.
  • the muscles of the mice treated with MDC improved .
  • Graphpad Prism software is used for statistical analysis. The results are all expressed by ⁇ standard deviation.
  • the Kolmogorov-Smirnov test is used to analyze the normality of the converted data, and the T test is used to compare the differences between the two groups; the one-way analysis of variance is used to compare the differences between three and more than three components, and then Perform LSD-t post-mortem analysis. P ⁇ 0.05 is considered statistically different.
  • adherent cultured cells When the confluence of adherent cultured cells reaches 80%, they can be frozen, passaged or plated. Cells in a 10cm culture dish can be cryopreserved in 3 tubes or passaged into 3 10cm dishes. The cryopreservation and resuscitation of cells follow the principle of "slow freezing and quick dissolution". The temperature should be gradually lowered during freezing and thawed quickly during resuscitation.
  • cryopreservation solution (90% complete medium + 10% DMSO), mix well, and let stand and cool to room temperature.
  • Gene knockdown is a kind of RNA interference (RNA interference, RNAi) technology, which is different from gene knockout (gene knockout) which silences the expression of the target gene permanently. It uses double-stranded small interfering RNA (small interfering RNA). , SiRNA) efficiently and specifically degrade mRNA with homologous sequences in the cell, thereby blocking the expression of the target gene, and causing the cell to appear the phenotype of the target gene deletion.
  • RNA interference RNA interference
  • small interfering RNA small interfering RNA
  • the transfection system of a 3.5cm petri dish is prepared as follows: 1 ⁇ l siRNA Oligo/Red (stock solution concentration is 20 ⁇ M) is added to the EP tube containing 250 ⁇ l pure Opti-MEM and mixed, 1.5 ⁇ l RNAiMAX (transfection reagent) is added Put it in a flow tube containing 250 ⁇ l of pure Opti-MEM and mix it, then add all the liquid in the EP tube to the flow tube and mix it, and let it stand at room temperature for 15 minutes.
  • a standard Illumina library preparation protocol was used to construct a transcriptome sequencing library from the extracted RNA samples.
  • the library was analyzed by RNA-Seq using the Pair-End solution with a read length of 100bp on the Illumina HiSeq 2000 sequencing platform. Use two mismatched Tophat-2.0.9 to compare the read sequence with the existing human transcriptome data (hg19). The only read mapping will remain in the gene expression profile. Differentially expressed genes are determined by the tool Cuffdiff. For each transcript, the expression level of the gene is evaluated by counting the number of reads per kilo bases per million reads (Reads Per Kilo bases per Million reads, RPKM) per million reads.
  • Plasmid extraction was performed at room temperature in accordance with the instructions of the endotoxin-free plasmid extraction kit (Beijing TIANGEN).
  • the transfection system of a 3.5cm petri dish is prepared as follows: 3 ⁇ g plasmid and 9 ⁇ l L-PEI (transfection reagent, 1 ⁇ g/ ⁇ l) are added to 250 ⁇ l pure Opti-MEM and mixed, and then stand at room temperature for 15 minutes, then Add to 3.5cm petri dish.
  • the medium is changed (medium containing 10% FBS without double antibody), and the cells are cultured for 24 to 48 hours, and then the samples can be collected and the total cell protein can be extracted.
  • the culture supernatant can be collected, and the transfection effect of the plasmids can be identified by Western Blotting.
  • TOPFLASH Reporter Gene Assay is a common method to analyze whether the classic Wnt signaling pathway is activated and the intensity of activation.
  • the detection system uses Promega's dual luciferase reporter gene detection system ( Reporter (DLRTM) Assay System). Specific steps are as follows:
  • Reagent preparation 1 ⁇ PBS; 1 ⁇ PLB (use after diluting 5 ⁇ Passive Lysis Buffer (PLB) with ddH2O, store the mother liquor at -20°C, and store at 4°C after dilution); LAR II Reagent (defrost 10ml Luciferase at room temperature) Assay BufferII, then pour all into the lyophilized substrate (Luciferase Substrate), use as soon as possible after mixing, avoid repeated freezing and thawing, each well of 96-well plate needs 100 ⁇ l); 1 ⁇ Stop& Reagent (using Stop& Buffer will 50 ⁇ Stop& Substrate dissolves in brown Stop& In the Reagent bottle, use it as soon as possible after mixing, avoid repeated freezing and thawing, each well of 96-well plate needs 100 ⁇ l).
  • Plasmid transfection was carried out after 24 hours, 160ng plasmid was co-transfected per well, Fugene (transfection reagent) 0.48 ⁇ l/well, Opti-MEM 8 ⁇ l/well.
  • the 160ng plasmid contains 40ng SP-TOP (Tcf/Lef-luc) reporter gene expression vector and 1ng Renilla Renilla luciferase expression vector, as well as 1ng Wnt3a plasmid or GFP plasmid (to detect transfection efficiency), and finally complete with Vector To 160ng, add all to the culture medium.
  • cell lysis After the cells are cultured for 24 hours, perform cell lysis: first remove the medium in the 48-well plate; wash the AD293 cells with 1 ⁇ PBS to remove the washing solution; add 1 ⁇ PLB to the 48-well plate, 65 ⁇ l/well, fully lyse the cells and mix well.
  • Cre recombinase gene By fusing the Cre recombinase gene with the ligand-binding domain (LBD) of two estrogen receptors (ER) to produce a chimeric recombinase (mER-Cre-mER), The expression of the chimeric recombinase is controlled by a specific ubiquitin C (UBC) promoter.
  • Ubiquitin protein exists in all eukaryotic cells, so Cre recombinase can be expressed in tissues and organs throughout the body. Cre recombinase can bind to loxP sites and effectively remove the sequence between two loxP sites to achieve gene knockout.
  • the chimeric Cre cannot enter the nucleus spontaneously to exert its activity, and can enter the nucleus only after combining with estrogen.
  • the key amino acids of estrogen LBD were mutated so that it could not be combined with the physiological estrogen in the body, but only with exogenous estrogen analogues. Tamoxifen is combined to realize the dual expression of Cre recombinase in time and space. The above is the transgenic characteristics of UBC-Cre mice.
  • LRP5 floxp/floxp , LRP6 floxp/floxp and ⁇ -catenin floxp/floxp homozygous mice have similar construction methods, for example, LRP5 floxp/floxp or LRP6 floxp/floxp homozygous mice, firstly by gene Gene targeting and homologous recombination insert one loxP site on each side of the LRP5 or LRP6 gene region in the genome, and screen for transgenes containing floxp-LRP5-floxp or floxp-LRP6-floxp sites Mice were then bred with wild-type (WT) mice and backcrossed to breed, and finally LRP5 floxp/floxp or LRP6 floxp/floxp homozygous mice were obtained [131] .
  • WT wild-type
  • ⁇ -catenin floxp/floxp homozygous mice can be obtained [134] .
  • the LRP5 floxp/floxp LRP6 floxp/floxp transgenic mice used in this study were obtained by continuing to mate and reproduce LRP5 floxp/floxp and LRP6 floxp/floxp mice.
  • LRP5 floxp/floxp LRP6 floxp/floxp homozygous mice and the above UBC-Cre mice to breed and produce transgenic mice that can induce the whole body co-knockout of LRP5 and LRP6, using The characteristics of Cre recombinase, the mouse genotype after tamoxifen induction is expressed as LRP5/6 -/- (LRP5/6 double knockout). Similarly, a transgenic mouse with conditional ⁇ -catenin knockout whole body can also be obtained, and the genotype after knockout is expressed as ⁇ -catenin -/- ( ⁇ -catenin knockout).
  • the transposon system is a transgenic model animal production system with high integration efficiency, high target gene expression probability, and suitable for inserting larger target fragments.
  • the unique "cut and paste” mechanism of transposons enables the "free” transfer of DNA fragments between the vector and the genome, thereby effectively mediating the integration of foreign DNA fragments into the genome.
  • the transposase effectively recognizes the specific transposon sequences (ITRs) at the ends of the target fragments in the vector, combines with the ends of the transposon to form a short hairpin structure, "cuts" it out of the vector, and “pastes” it to Specific sites in the genome.
  • the constructed gene vector containing the transposon system and the CAG promoter-floxp-stop codon-floxp-LRP6N-myc cDNA fragment and the expression vector of the CMV promoter-transposase are used to carry out mouse fertilized eggs by microinjection technology.
  • Nuclear DNA microinjection the injected fertilized eggs are transplanted into the reproductive system of pseudopregnant mice to make them pregnant and obtain newborn mice.
  • the tail tips are collected, DNA is extracted, identified by PCR, and transposon-mediated Of transgenic mice carrying the LRP6N gene fragment (CAG-floxp-stop codon-floxp-LRP6N).
  • CAG is a highly efficient synthetic promoter, often used in mammalian expression vectors to drive high-level expression of genes.
  • CAG contains the early enhancer of CMV, the promoter of ⁇ -actin and the splice acceptor of ⁇ -globin [138].
  • the LRP6N of the above-mentioned transgenic mice does not express under normal conditions. Only after crossing with the corresponding tissue-specific Cre expressing positive mouse, the stop codon (stop codon) is deleted by Cre in the specific tissue, so that the CAG The ⁇ -actin promoter enables the expression of LRP6N gene fragments in specific tissues.
  • mice containing the LRP6N gene CAG-floxp-stop codon-floxp-LRP6N
  • UBC-Cre mice [133] to reproduce tamoxifen inducible whole body Transgenic mice overexpressing LRP6N
  • the induced mice are expressed as LRP6N/Tg.
  • LRP6N/Tg mice have overexpression of LRP6N protein in the heart (as shown in Figure 18 (A, B) and Figure 21), indicating that the tamoxifen induction is successful Yes, LRP6N/Tg mice can be used for further research, such as type 1 diabetes modeling.
  • Leptin knockout mice are homozygous mice that spontaneously develop an obesity phenotype. They are often used as animal models for the study of type 2 diabetes. They can be used at 4 weeks. Obesity phenotype was seen, marked hyperglycemia (concentration higher than 13.8mM) and impaired glucose tolerance symptoms appeared at 6 weeks. Leptin (leptin) is a protein hormone secreted by adipose tissue. It is involved in regulating the metabolism of sugar, fat and energy in the body, prompting the body to reduce food intake, increasing energy release, inhibiting the synthesis of fat cells, and reducing weight [139] .
  • Leptin -/- mice lack leptin ligands, have a strong appetite, and increase their body weight rapidly, leading to fat deposition and morbid obesity. The body weight can reach three times that of WT mice and is not affected by dietary restrictions. In addition to the above performance, Leptin -/- mice also have the characteristics of insulin resistance, decreased fertility, decreased metabolism, impaired wound healing ability, and elevated levels of pituitary and adrenal hormones. Since Leptin -/- mice are infertile, Leptin -/- mice are all produced by the mating of Leptin +/- mice.
  • the VisualSonics Vevo 2100 imaging system was used for two-dimensional echocardiography.
  • the left chest of the mouse was depilated in advance, and the mouse was anesthetized with 1% isoflurane through a vaporizer, and oxygen was supplied at the same time.
  • the physical indicators that need to be tested to reflect left ventricular function include left ventricular internal dimension in end-diastole (LVIDd), left ventricular posterior wall in end-diastole (LVPWd), diastolic Interventricular septum in end-diastole (IVSd) (measured during the maximum volume of the left ventricle), left ventricular internal dimension in end-systole (LVIDs) (that is, when the left ventricular wall contracts The maximum degree is measured during the period).
  • LVIDd left ventricular internal dimension in end-diastole
  • LVPWd left ventricular posterior wall in end-diastole
  • IVSd diastolic Interventricular septum in end-diastole
  • LVIDs left ventricular internal dimension in end-systole
  • LVEF left ventricular ejection fraction
  • LVFS left ventricular short axis shortening
  • STZ-induced diabetes is a method of chemical diabetes modeling. Its full name is Streptozotocin. It is a broad-spectrum antibiotic isolated from achromatic streptomycin. It can be selected in a short time Sexual damage to pancreatic ⁇ cells, causing ⁇ cell necrosis, leading to varying degrees of decline in blood insulin and increased blood sugar, forming insulin-dependent type 1 diabetes. Compared with other chemical diabetes modeling methods such as Alloxan (ALX) modeling, STZ-induced diabetic hyperglycemia and ketosis are milder, and the resulting diabetes model is also more stable. In this study, we injected STZ into the abdominal cavity of mice to induce the establishment of a type 1 diabetes model.
  • ALX Alloxan
  • mice were fasted for 12 hours, but water was not forbidden.
  • model mice were injected intraperitoneally with 2% STZ-citric acid solution (160mg/kg) at one time, and mice in the control group were injected intraperitoneally with corresponding volume of citrate buffer at one time, 2 hours after the injection was completed , Resuming food for mice.
  • Glucose tolerance test is a standard test that shows the body's removal efficiency of exogenous glucose from the blood, and is used to assess the maintenance of the balance of glucose metabolism in the body.
  • the absorption of glucose by cells from the blood is regulated by insulin. Insulin resistance can lead to impaired glucose tolerance, that is, it takes longer to clear the glucose content than normal organisms.
  • Leptin -/- and WT mice started from 4 weeks of age and measured their body weight and fasting 6-hour blood glucose at the same time every week. According to the body weight and blood glucose changes of Leptin -/- mice, we selected 6-week-old Leptin -/- mice (experimental group) and WT mice (control group) for glucose tolerance test. Specific steps are as follows:
  • mice were fasted for 12 hours (cannot help water), weighed, and blood was collected from the tail to determine the basic blood glucose level.
  • the insulin rescue experiment is to subcutaneously inject long-acting insulin glargine or a corresponding volume of normal saline (control group) to diabetic mice induced by STZ for 1 week. Specific steps are as follows:
  • mice modeled in STZ for 1 week were divided into groups according to the principle of randomization, and their body weight and random blood glucose were measured.
  • the insulin rescue group was injected subcutaneously with diluted insulin (1U/kg/day), once a day at the same time for 7 consecutive days, and the control group was given a corresponding volume of normal saline.
  • the in vivo rescue experiment of MDC in STZ diabetic mice is to inject MDC (diluted in sterile PBS, 10mg/kg/day) into the abdominal cavity of diabetic mice induced by STZ for 3 days, and inject the corresponding volume of DMSO (diluted in sterile PBS) once a day ) Diabetic mice served as a control group. Body weight and random blood glucose were measured at different time points after the injection. After MDC intervened for 4 consecutive days, the mice were anesthetized and the samples were taken.
  • the in vivo rescue experiment of MDC in Leptin -/- mice is to pre-inject MDC (diluted in sterile PBS, 10mg/kg) or a corresponding volume of DMSO (diluted in sterile PBS) into 6-week-old Leptin -/- mice in advance .
  • MDC diluted in sterile PBS
  • DMSO diluted in sterile PBS
  • the injection dose of mouse MDC should be calculated based on the weight of 6-week-old WT mice born in the same litter. After MDC intervention for 1 hour, 50% D-glucose or corresponding volume of PBS was injected intraperitoneally.
  • the procedure for Leptin -/- mice to inject D-glucose is to inject once every 2 hours during 24 hours, a total of 12 times, and the dose of D-glucose for each injection is fixed as the first dose (2g/kg). After the injection, the mice were anesthetized and the materials were taken.
  • Dkk1 was injected locally (10 ⁇ g of Dkk1 per heart, dissolved in 30 ⁇ l PBS) or an equal volume of PBS at three locations in the heart. Among them, two sites are on the border around the left ventricle, and one site enters the myocardium of the left ventricle. After the injection, the wound was sutured, the anesthesia was removed, and the mice were awaited to wake up. After MDC intervenes continuously for 2 days, the mice are anesthetized and the materials are taken.
  • the LRP6N plasmid (with myc tag) used in this study needs to be identified in advance, that is, by transfecting AD293 cells in vitro, and then collecting the supernatant culture medium of the cells, using Western Blotting to prove that the LRP6N plasmid can express the secreted LRP6N protein, Then carry out the following operation of tail vein injection of plasmid.
  • Reagent preparation 1Hepes solution (50mM, pH 7.4, 0.22 ⁇ m filtration); 2D-glucose solution (50%, 0.22 ⁇ m filtration); 3L-PEI solution (1mg/ml, pH 5.0, 0.22 ⁇ m filtration).
  • LRP6N plasmid Western Blotting proved that the secreted LRP6N protein expressed by the LRP6N plasmid can be continuously expressed in vivo for at least 6 days.
  • the tail vein injection of LRP6N plasmid should be carried out 1 day after STZ induction, and 7 days after STZ modeling, the body weight and random blood glucose should be measured, the mice should be anesthetized and the materials should be taken.
  • mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (45 mg/kg).
  • the abdomen is upward and the limbs are fixed on the operating table.
  • the upper abdomen is cut laterally under the xiphoid process to expose the liver.
  • the xiphoid process is lifted with hemostatic forceps, and the diaphragm is cut into the chest cavity.
  • the ribs are cut along both sides of the xiphoid process and opened to the head. , Expose the heart.
  • Normal saline perfusion quickly put the catheter of the perfusion pump into the normal saline and start perfusion until the fluid flowing out from the right atrial appendage is colorless, and the mouse liver is pale in color, the limbs are white, and the intestines are swollen.
  • tissue samples extracted from protein or RNA after the normal saline perfusion is completed, they are quickly frozen in liquid nitrogen and stored at -80°C; for the tissue samples for frozen sections, after the normal saline perfusion is completed, blot the tissue samples attached to the sample. The liquid is directly embedded in OCT, then quickly frozen in liquid nitrogen, and stored at -80°C; for the tissue samples made for paraffin sections, after 4% paraformaldehyde is infused, remove the specimen and put it in 4% paraformaldehyde , And continue to fix on a shaker at 4°C for 24h for subsequent paraffin embedding and section staining.
  • step "7” The supernatant of step "7" was centrifuged, 4°C, 14000g, 30min, carefully collect the supernatant to a new EP tube, do not touch the precipitate, extract the pulp protein, and store it at -80°C for later use.
  • Each cell sample add 200 ⁇ l A solution.
  • Each tissue sample A solution and B solution are mixed according to a volume ratio of 20:1 (containing PMSF with a final concentration of 1 mM) to prepare 200 ⁇ l tissue homogenate and add to the tissue.
  • the BCA method is used to determine the protein concentration. The specific steps are as follows:
  • a solution and B solution are prepared with a volume ratio of 50:1, 200 ⁇ l/well, the 96-well plate is gently shaken to mix, and it is placed in a 37°C incubator for 30 minutes.
  • the sample volume with the smallest OD value (a ⁇ l) is used as the calibration volume, and the trim volume of other samples is [minimum OD value ⁇ a ⁇ l]/the OD value of other samples. Then use ddH 2 O to make up other sample volumes to the calibration volume (a ⁇ l ), so as to adjust the OD value of each sample to the level of the smallest OD value sample.
  • the protein electrophoresis gel is a double-layer gel, including a concentrated gel on the upper layer and a separating gel on the lower layer.
  • the concentration of the concentrated gel is 5%
  • the concentration of the separating gel is determined according to the molecular weight of the target protein.
  • large molecular weight proteins LRP5/6, ⁇ -catenin, ⁇ -arrestin1/2
  • small molecular weight proteins ⁇ H2AX, p53, p21, Bcl-2, Bax, Cleaved Caspase- 3
  • the number of holes (10 holes or 15 holes) and thickness (1mm or 1.5mm) of the gel prepared according to the number and volume of the protein sample loaded. See the experimental materials section for the gel formula, the preparation steps are as follows:
  • Sample loading arrange all protein samples according to the recorded loading sequence, pipette the same amount of sample into the gel hole.
  • the tip of the pipette tip is attached to the higher side of the glass, and the position along the channel is as low as possible. When resistance is encountered, it can be dripped. Since the sample contains loading buffer, the sample solution can sink into the bottom of the hole by itself; In order to avoid cross-contamination, the sample should be replaced with a new pipette tip for the next sample; the sample amount should be moderate, with a maximum of 40 ⁇ l per hole for 10-hole glue and 20 ⁇ l per hole for 15-hole glue.
  • pre-stained protein Marker 8 ⁇ l on the left and 4 ⁇ l on the right
  • Electrophoresis Cover the electrophoresis tank lid, turn on the power, start electrophoresis at room temperature, set the electrophoresis conditions: 80V, 30min, after the sample is run off the concentrated gel and pressed into a horizontal straight line (judged by bromophenol blue) , 120V, 90min, separate the dyes of different molecular weight in the Marker in the gel. According to the molecular weight of the Marker, judge whether the protein molecules of different sizes are sufficiently separated in the gel, stop the electrophoresis in time, and prepare for electrotransmission.
  • PVDF membrane For target protein transfer membrane greater than 20kDa, and 0.2 ⁇ m PVDF membrane for less than 20kDa. PVDF membrane needs to be treated with methanol to activate the positively charged groups on the membrane and make it easier to bind to negatively charged proteins. Specific steps are as follows:
  • the blocking solution (5% skim milk) can bind to the non-specific binding sites on the PVDF membrane, reducing the background of the PVDF membrane and ensuring the specific binding of the primary antibody to the target protein. Specific steps are as follows:
  • the protein or peptide on the PVDF membrane reacts specifically with the corresponding primary antibody, and then incubating the secondary antibody (horseradish peroxidase (HRP) labeled) to bind the primary antibody to the secondary antibody.
  • HRP horseradish peroxidase
  • the HRP connected to the substrate of the exposure solution and the secondary antibody undergoes a cascade amplification reaction, showing a chemiluminescent protein band that can be detected, so that the expression level of the specific target protein can be judged.
  • the primary antibody should be centrifuged briefly before use, and an antigen solution should be diluted with the primary antibody diluent according to the multiple recommended by the antibody manual.
  • Incubation of primary antibody According to the size of the Marker, cut out the corresponding part of the PVDF membrane containing the distribution of the target protein to be incubated, and different target proteins should be incubated with their corresponding specific primary antibodies. The incubation time depends on the affinity of the antibody and the protein and the abundance of the protein. Put the diluted primary antibody and PVDF membrane (with protein distribution side up) in the incubator together to cover the membrane evenly so that the primary antibody and membrane can be evenly combined. 4°C, slowly shake the shaker for overnight incubation. For protein samples with low affinity or concentration, it is recommended to use a higher antibody dilution concentration and a longer incubation time (generally no more than 18h) to ensure specific binding.
  • Membrane washing Recover the incubated primary antibody to a centrifuge tube (usually reused three times), and store at 4°C.
  • the PVDF membrane is placed on a shaker and washed three times with TBST for 5 minutes each time. After washing, prepare the secondary antibody for incubation.
  • the secondary antibody should be centrifuged briefly before use, and the secondary antibody should be diluted with TBST at the multiple recommended by the antibody instruction.
  • the development of protein bands adopts chemiluminescence method.
  • the HRP linked to the secondary antibody is a highly sensitive enzyme that can catalyze the production of a luminescent substance from the substrate of the ECL exposure solution.
  • the exposure function converts the luminescent signal into the band gray signal on the picture.
  • the band gray scale can reflect the target protein The expression level. Specific steps are as follows:
  • RNA preparation After sample collection, it is best to proceed to total RNA preparation immediately. If it cannot be extracted in time, it should be frozen with liquid nitrogen and stored at -80°C. When extracting tissue RNA, the sample should be quickly broken by grinding with liquid nitrogen. Do not thawed first to prevent endogenous RNase from causing RNA degradation. In addition, in order to prevent RNA from being degraded by exogenous RNase during the extraction process, pay attention to the use of RNase-removed pipette tips and EP tubes; grinding tools need to be used after high temperature and high pressure sterilization; wear disposable masks and gloves; avoid during operation Speech etc.
  • RNA samples Remove genomic DNA (gDNA) in RNA samples. Prepare the reaction solution on ice according to the table below, prepare the Master Mix according to the number of reactions + 2, and then divide it into each reaction tube, and finally add the RNA sample. The reaction solution was reacted at 42°C for 2 min. After completion, it was stored on ice, and the resulting RNA without gDNA was used for reverse transcription.
  • gDNA genomic DNA
  • Reverse transcription reaction Prepare the reaction solution on ice according to the table below, prepare Master Mix according to the number of reactions + 2, and then distribute them to each reaction tube, and mix gently. The reaction solution was reacted at 37°C for 15 minutes; at 85°C, the reaction was conducted for 5 seconds. After completion, store on ice. The obtained cDNA template was used in the SYBR Green qPCR reaction.
  • Step 1 pre-denaturation (95°C for 30s); Step 2: PCR reaction (95°C for 5s, 60°C for 34s) 40 ⁇ cycles.
  • Step 2 PCR reaction (95°C for 5s, 60°C for 34s) 40 ⁇ cycles.
  • 60°C set the fluorescence detection point and perform amplification. If necessary, a dissolution curve can be added after the reaction to detect the specificity of the amplified product.
  • the final CT value is calculated by the 2- ⁇ CT method to calculate the expression of a certain gene in different samples.
  • the laser scanning confocal microscope takes pictures and imaging (63 ⁇ oil lens), you can see Cy3 labeled red fluorescence (target protein) and DAPI labeled blue fluorescence (cell nucleus).
  • Soaking wax dipping the melted paraffin at 65°C for 3 to 4 hours.
  • Embedding Put the tissue in a self-made embedding box, add melted paraffin, adjust the position of the tissue with a pin, carefully place the embedding box on a cooling table at -20°C, and wait until most of the paraffin has solidified. Put the label on the paraffin block, mark the tissue specimen, and pry the wax after 10 minutes.
  • Sectioning Trim the paraffin block with a safety blade, fix it on a paraffin microtome, adjust the thickness of the section to 6 ⁇ m, flatten the tissue piece with a writing brush, transfer it to a 40°C water bath, and then use a slide glass to remove the tissue piece from the water bath Remove from the pot.
  • Sample preparation The freshly obtained mouse blood was stored in an EDTA anticoagulation tube, and after standing at room temperature for 10 minutes, centrifuged at 1000 g for 15 minutes, and the supernatant, namely plasma was collected. If it cannot be tested immediately, it should be stored at -80°C after aliquoting to avoid repeated freezing. If precipitation occurs during storage, it should be fully thawed at room temperature and centrifuged again.
  • Standard product Use reagent diluent to dilute the standard product to 1350, 675, 337.5, 168.8, 84.4, 42.2, 21.1pg/ml and other 7 concentration gradients.
  • Washing the plate carefully remove the sealing film, discard the liquid and spin dry, fill each well with the diluted washing liquid, let it stand for 30s and discard it, pat dry thoroughly with paper, and wash the sample plate 3 times.
  • Blocking Add 300 ⁇ l reagent diluent to each well and incubate for 1h at room temperature.
  • Sample loading Add 100 ⁇ l ddH 2 O (blank well), Dkk1 standard or plasma sample to each well, cover the sample plate, and incubate for 2 hours at room temperature.
  • Reading the plate zero adjustment with a blank hole, and read the OD value of each hole at a wavelength of 450nm.
  • sample preparation is the same as the measurement of plasma Dkk1 (ELISA method).
  • Standard product Use standard product diluent to dilute the standard product to 8 concentration gradients such as 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.157ng/ml.
  • Color development Add 200 ⁇ l of color development solution (reaction substrate containing AChE) to each well. Re-seal the ELISA plate with a sealing film, place it on a shaker, and react for 90-120 min at room temperature in the dark.
  • Reading the plate zero adjustment with a blank hole, and read the OD value of each hole at a wavelength of 412nm.
  • Example 1 The up-regulation of Dkk1 may be related to tumor death associated with cachexia.
  • CT26 tumor-bearing mice developed severe cachexia, accompanied by continuous weight loss and muscle weight loss (Figure 1g). Lean muscle fibers and blank areas were revealed by HE staining, suggesting muscle atrophy and some muscle hydrolysis (Figure 1h). It is very interesting that the kidney weight has always remained the same, and the two renal function indicators of HE staining and blood creatinine and urea nitrogen ( Figure 1i) have not changed, indicating that CT26 cells have no effect on the kidney. These results indicate that CT26 tumor implantation usually affects organs other than the kidney. Although an increase in Dkk1 was observed in mice bearing LLC cell tumors (Figure 1b), LLC cells have a strong ability to migrate. Therefore, in order to clarify the potential mechanism of the harmful effects of Dkk1 in tumor death unrelated to metastasis, we focused on CT26 tumor-bearing mice in the following experiments.
  • Muscle atrophy is a key feature of tumor cachexia and a multifactorial disease that has a negative impact on the prognosis and quality of life of patients. Regardless of body mass index (BMI), skeletal muscle loss is considered to be a meaningful prognostic factor in the development of cancer, and is associated with increased incidence of chemotherapy toxicity, shortened tumor progression time, poor surgical results, physical damage and shortened survival .
  • Kremen1/2 is a necessary receptor for Dkk1-mediated LRP5/6 submembrane. Therefore, in order to explore the potential mechanism of Dkk1's harmful effects in cancer death, we first detected the expression of Dkk1 binding proteins LRP5/6 and Kremen1/2 in CT26 tumor-bearing mice.
  • the Wnt co-receptor LRP5/6 is involved in activating the Wnt/ ⁇ -catenin pathway. Since LRP5/6 activates ⁇ -catenin into the nucleus, in order to explore whether the close influence of LRP5/6 itself on GPCRs is widespread, it is necessary to exclude the interference of ⁇ -catenin signals downstream of LRP5/6, that is, to have the corresponding ⁇ -catenin The experiment serves as the experimental control of LRP5/6.
  • RNAi technology to perform LRP5/6 and ⁇ -catenin gene knockdown experiments in several cells (HepG2, Hela, U2OS, HUVEC), and it was observed under the microscope that knocking down the LRP5/6 gene caused the above cell status to deteriorate.
  • Example 5 LRP6 extracellular segment (LRP6N) can reverse DNA damage induced by LRP5/6 deletion
  • the rescue effect of the LRP6N protein in cell DNA damage induced by knocking down LRP5/6 indicates that the lack of LRP5/6 on the cell membrane is the root cause of DNA damage in cells.
  • H 2 O 2 can simulate an abnormal culture environment in a disease state in vitro.
  • the peroxide group of H 2 O 2 can directly attack the DNA double strands of cells and simply cause oxidative DNA damage without causing changes in the expression of LRP5/6 and ⁇ -catenin, and the degree of DNA damage induced by H 2 O 2 Concentration and time dependence (Figure 5C).
  • an adverse stimulus H 2 O 2
  • HUVEC cells on the basis of the above experiment.
  • Example 6 Loss of MESD can enhance the oxidative stress effect
  • Example 7 Dkk1 can induce DNA damage effect
  • the secreted protein Dkk1 induces the endocytosis of LRP5/6 on the cell membrane and inhibits the Wnt/ ⁇ -catenin signaling pathway.
  • the mechanism is still unclear.
  • purified Dkk1 protein with different time and concentration gradients was added to HUVEC cells, and it was found that Dkk1 can rapidly induce the endocytosis of LRP5 (100ng/ml from 15min) and LRP6 (100ng/ml from 5min) on the cell membrane.
  • H 2 O 2 low concentration H 2 O 2 (50 ⁇ M) to further stimulate HUVEC cells on the basis of the above experiment.
  • H 2 O 2 itself can activate the ⁇ H2AX signal without affecting the expression of LRP5/6 on the cell membrane, and when Dkk1 induces the down-regulation of LRP5/6 on the membrane, H 2 O 2 can strongly up-regulate the ⁇ H2AX signal ( Figure 7B ).
  • MDC or LRP6N itself does not cause a ⁇ H2AX response and cannot prevent H 2 O 2 from ⁇ H2AX activation, but they significantly attenuate the ⁇ H2AX signal that is enhanced by H 2 O 2 in the presence of Dkk1 ( Figure 7B) .
  • Example 8 Dkk1 activates ⁇ -arrestin1/2 signal transduction through endocytosis of LRP5/6
  • RNA-Seq analysis proves that there is a close relationship between LRP5/6 and most GPCRs, so it is not difficult to speculate that the down-regulation of LRP5/6 on the membrane may lead to an imbalance in the homeostasis of the GPCR membrane and further complicate the downstream signal pathways. Change.
  • ⁇ -arrestin1/2 like various G proteins, are general direct downstream targets of GPCRs, but unlike GPCRs and G proteins, ⁇ -arrestin1/2 can quickly translocate to the cell membrane and directly It binds to the C-terminus of activated GPCRs and is specifically involved in the regulation of general GPCR signal transduction through its own cell membrane and cytoplasm/nucleus transport.
  • BIM-46187 (1 ⁇ M 30min) to pretreat HUVEC cells, which is a general inhibitor of G protein, which can directly bind to G protein ⁇ subunit, thereby preventing the formation of ligand-activated GPCR and G protein complex And the subsequent conformational changes of G protein, because this binding prevents the normal interaction between the receptor and the G protein heterotrimer, thereby inhibiting the GDP/GTP exchange of the G protein ⁇ subunit, resulting in the G protein and GPCR signal being affected. Extensive suppression.
  • Example 9 ⁇ -arrestin1/2 mediates cellular DNA damage caused by down-regulation of LRP5/6 on the membrane
  • ⁇ -arrestin1/2 the direct downstream target of GPCR, can not only participate in GPCR signal transmission through the transport between cell membrane and cytoplasm/nucleus, but also mediate DNA damage response. Therefore, on the one hand, LRP5/6 on HUVEC cell membrane While being endocytosed by Dkk1, it rapidly induces the transfer of ⁇ -arrestin1/2 from the cell membrane to the cytoplasm, which in turn leads to the activation of ⁇ H2AX; on the other hand, inhibiting Dkk1 from endocytosis of LRP5/6 by MDC can also prevent ⁇ - The translocation of arrestin1/2 in the cell and the activation of ⁇ H2AX.
  • HUVEC cells knocked down ⁇ -arrestin1/2 (Figure 9A), which not only blocked GPCR- ⁇ -arrestin1/2 signaling, but also inhibited the activation of ⁇ H2AX induced by Dkk1 stimulation ( Figure 9B) .
  • ⁇ -arrestin1/2 for Dkk1 and low-concentration H2O2 (50 ⁇ M) to stimulate the enhanced ⁇ H2AX signal, knocking down ⁇ -arrestin1/2 also has a significant rescue effect, but knocking down ⁇ -arrestin1/2 does not protect H2O2 itself.
  • DNA damage of cells ( Figure 9B), indicating the specific role of ⁇ -arrestin1/2 in promoting Dkk1-induced DNA damage.
  • knocking down ⁇ -arrestin1/2 in HUVEC cells can also inhibit the DNA damage and resistance caused by knocking down LRP5/6.
  • the ability of oxidative stress is weakened, and this protective effect does not depend on ⁇ -catenin ( Figure 9D).
  • ⁇ -arrestin1/2 the direct molecular target downstream of GPCR, is a direct regulator of cell DNA damage and homeostasis imbalance induced by LRP5/6 deletion.
  • Example 10 G protein agonist can induce DNA damage response
  • PTX pertussis toxin
  • long-term stimulation can activate Gs protein ⁇ subunit
  • ⁇ H2AX is activated by different doses of PTX in both short-term and long-term stimulation ( Figure 10B) .
  • Figure 10B The above results indicate that affecting the functions of different G proteins can induce DNA damage responses. Therefore, as a general downstream target of GPCRs, the destruction of G protein homeostasis will extensively change GPCR signals, which in turn leads to imbalances in cell homeostasis.
  • LRP5/6 -/- or ⁇ -catenin -/- mice induced by intraperitoneal injection of tamoxifen to LRP5/6 in adult tissues explain the role of They are obtained by mating LRP5/6 floxp/floxp or ⁇ -catenin floxp/floxp mice with Cre recombinase-containing mice (UBC-Cre/ESR1).
  • UBC-Cre/ESR1 mice Cre recombinase-containing mice
  • LRP5/6 short-term knockout of LRP5/6 can specifically induce adult mouse heart DNA damage response and cell cycle arrest, while long-term loss of LRP5/6 causes accumulation of DNA damage, which in turn leads to impaired cardiac function.
  • LRP5/6 can protect the normal function and homeostasis of the adult mouse heart.
  • Example 12 LRP5/6 gene knockout activates the signaling of ⁇ -arrestin1/2 in mouse heart
  • LRP5/6 -/- instead of ⁇ -catenin -/- mice can induce the translocation of ⁇ -arrestin1/2 in the heart (including the downregulation of ⁇ -arrestin1/2 on the cell membrane, ⁇ -arrestin1/2 and the up-regulation of ⁇ -arrestin1 in the nucleus), which indicates that ⁇ -arrestin1/2 can mediate the heart DNA damage caused by LRP5/6 knockout, and it also provides a direct relationship between LRP5/6 and GPCR. evidence.
  • Example 14 The close relationship between the down-regulation of LRP5/6 on the membrane of diabetic mice and heart damage
  • Dkk1 has the dual biological functions of inhibiting Wnt/ ⁇ -catenin signaling and LRP5/6 on the endocytic membrane.
  • Dkk1 has the dual biological functions of inhibiting Wnt/ ⁇ -catenin signaling and LRP5/6 on the endocytic membrane.
  • Blood 8-OHdG was also significantly up-regulated on the 7th day of STZ modeling ( Figure 14C), further confirming the occurrence of diabetes DNA damage.
  • the temporal consistency of these changes in STZ diabetic mice means that the down-regulation of LRP5/6 on the heart membrane and the occurrence of DNA damage are closely related to the up-regulation of Dkk1 in hyperglycemia.
  • the diabetic mice on the 7th day of STZ modeling also showed significant up-regulation of the levels of other injury markers (including heart p53 and p21, Bax/Bcl-2 and Cleaved Caspase-3) , which indicates that diabetes caused severe heart DNA damage and eventually apoptosis.
  • other injury markers including heart p53 and p21, Bax/Bcl-2 and Cleaved Caspase-3
  • Example 15 Dkk1 induces diabetic heart damage by inducing LRP5/6 membrane endocytosis
  • MDC also inhibited the up-regulation of p53, p21, Bax/Bcl-2 and Cleaved Caspase-3 in the diabetic heart, and the weight loss of diabetic mice.
  • HE staining of the heart showed that STZ modeling caused significant damage to the diabetic myocardial structure, including disordered and broken myocardial fibers, and expansion of interstitial spaces. These changes can be prevented by insulin (positive control) and MDC (Figure 15F) .
  • MDC did not change the blood glucose concentration of diabetic mice while preventing the aforementioned damage (Figure 15G), indicating that the diabetic damage caused by hyperglycemia is essentially due to the down-regulation of LRP5/6 on the membrane.
  • the above results preliminarily propose a molecular mechanism for LRP5/6 to regulate diabetic heart damage independently of ⁇ -catenin, that is, the hyperglycemic phenotype of diabetes induces the up-regulation of blood Dkk1, which leads to the down-regulation of LRP5/6 on the membrane, namely Dkk1 -The LRP5/6 axis has changed, which in turn affects the stability of GPCR signals, causing cardiac DNA damage, cell cycle arrest, and even apoptosis, and other steady-state imbalances, which eventually form the heart damage of diabetes.
  • Obesity is considered to be a high-risk factor leading to the onset of type 2 diabetes.
  • Leptin -/- mice show an obese phenotype and are accompanied by severe hyperglycemia. Therefore, we use it as a mouse model that mimics type 2 diabetes to investigate the relevant role of LRP5/6 in it.
  • Figures 16A and 16B compared with litter-born Leptin +/- or WT mice, 5-week-old Leptin -/- mice have significantly increased body weight and blood sugar.
  • the in vivo results of the above-mentioned type 1 and type 2 diabetic mouse models jointly indicate that hyperglycemia can induce the up-regulation of blood Dkk1 and the down-regulation of LRP5/6 on the cell membrane, which in turn leads to the heart DNA damage response and cell cycle arrest in diabetic mice. Even apoptosis. Therefore, the integrity of LRP5/6 on the cell membrane is essential for maintaining the homeostasis of tissues and organs.
  • MDC may be expected to become a clinical treatment drug for organ damage in patients with type 1 and type 2 diabetes.
  • Example 17 LRP5/6 gene knockout aggravated diabetic heart damage
  • LRP6N/Tg mice prevented the up-regulation of diabetic heart ⁇ H2AX, p53, p21, Bax/Bcl-2 and Cleaved Caspase-3 and blood 8-OHdG induced by STZ modeling ( Figure 18C And 18D).
  • LRP6N/Tg mice also inhibited the down-regulation of LRP5/6 on the endogenous membrane induced by STZ modeling ( Figure 18C).
  • LRP6N/Tg mice and mice injected with LRP6N plasmid tail vein overexpress LRP6N, which can effectively prevent STZ modeling-induced diabetic weight loss (Figure 18H).
  • LRP5/6 -/- but not ⁇ -catenin -/- mice can also induce cardiac ⁇ -arrestin1/2 shift changes (Figure 12A), which is LRP5/6 regulating ⁇ -arrestin1/2 Provided direct evidence.
  • the general inhibitor of G protein BIM-46187 can significantly prevent Dkk1's direct endocytosis of LRP5/6-induced cardiac ⁇ -arrestin1/2 translocation changes ( Figure 19F) and DNA damage ( Figure 15I) by inhibiting GPCR signal disturbances.
  • the loss of LRP5/6 can destroy the homeostasis of many GPCRs and cause downstream ⁇ -arrestin1/2 signal transduction, which promotes the DNA damage response, severe DNA damage and even apoptosis, and ultimately leads to impaired organ function.
  • LRP5/6 and insulin receptor (IR) are single-pass transmembrane proteins, and insulin receptor is considered to play a key role in the development of diabetes. It belongs to receptor tyrosine kinases.
  • the receptor tyrosine kinase (RTKs) family consists of two ⁇ subunits (IR- ⁇ ) and two ⁇ subunits (IR- ⁇ ) connected by disulfide bonds. The two ⁇ subunits are located on the outer side of the cell membrane, and there are insulin binding sites on them; the two ⁇ subunits are the transmembrane part of the receptor and play a signal transduction role.

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Abstract

一种DKK1基因或其编码蛋白抑制剂的用途，用于制备组合物或制剂，该组合物或制剂用于预防和/或治疗肿瘤恶病质与糖尿病伴随疾病。通过抑制肿瘤细胞中的DKK1基因或其编码蛋白的表达或活性，有效预防和/或治疗肿瘤恶病质与糖尿病伴随疾病，也提供了检测血中DKK1的蛋白表达水平方法，作为精准治疗与判断预后的指标。

Description

DKK1抑制剂在预防和/或治疗肿瘤恶病质与糖尿病伴随疾病中的应用 技术领域
本发明涉及生物医药领域。更具体地，本发明涉及DKK1抑制剂在预防和/或治疗肿瘤恶病质与糖尿病伴随疾病中的应用。
背景技术
肿瘤恶病质伴随体重减轻，严重损害生存质量，限制癌症的治疗，同时缩短生存期，然而，目前没有有效的治疗方法。同样，糖尿病伴随全身各个脏器的损伤，严重危害患者的生命健康，同时限制很多伴随疾病或固有疾病的治疗。虽然各类降糖疗法可以有效阻止疾病发生发展，然而，胰岛素本身对一大类病人(比如糖尿病伴有心衰的患者)有很强的毒副作用，还有如何判断部分降糖效果不稳定的病人其脏器是否仍在继续遭受损伤，等等，目前临床仍然存在大量亟待解决的问题。
因此，本领域迫切需要开发一种能够有效预防和/或治疗这些临床难以解决的疾病的治疗方案。
发明内容
本发明的目的就是提供一种能够有效预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的治疗方案。
在本发明第一方面，提供了一种DKK1基因或其编码蛋白抑制剂的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗肿瘤恶病质与糖尿病伴随疾病。
在另一优选例中，所述抑制剂包括抑制DKK1与LRP5/LRP6结合的抑制剂。
在另一优选例中，所述抑制剂抑制DKK1的活性和/或表达量。
在另一优选例中，所述抑制剂抑制DKK1与LRP5/LRP6的复合物的形成。
在另一优选例中，所述抑制剂抑制LRP5/LRP6的活性和/或表达量。
在另一优选例中，所述抑制剂抑制Kremen1/Kremen2的活性和/或表达量。
在另一优选例中，所述抑制剂选自下组：抗体、游离DKK1受体结合域或其突变体、DKK1受体的其它游离配体或其突变体、小分子化合物、microRNA、 siRNA、shRNA、CRISPR试剂、或其组合。
在另一优选例中，所述抑制剂选自下组：MDC、CDHC/STHC、IGFBP4蛋白、表达IGFBP4的载体、IGFBP4突变体、表达IGFBP4突变体的载体、LRP5/LRP6中和抗体、游离LRP5/LRP6与DKK1的结合域或其突变体、Kremen1/Kremen2抑制剂(包括中和抗体)、游离Kremen1/Kremen2与DKK1的结合域或其突变体、或其组合
在另一优选例中，所述表达IGFBP4的载体或表达IGFBP4突变体的载体包括病毒载体。
在另一优选例中，所述病毒载体选自下组：腺相关病毒载体、慢病毒载体、或其组合。
在另一优选例中，所述的IGFBP4蛋白包括全长蛋白或蛋白片段。
在另一优选例中，所述IGFBP4蛋白还包括IGFBP4蛋白的衍生物。
在另一优选例中，所述IGFBP4蛋白的衍生物包括经修饰的IGFBP4蛋白、氨基酸序列与天然IGFBP4蛋白同源且具有天然IGFBP4蛋白活性的蛋白分子、IGFBP4蛋白的二聚体或多聚体、含有IGFBP4蛋白氨基酸序列的融合蛋白。
在另一优选例中，所述“氨基酸序列与天然IGFBP4蛋白同源且具有天然IGFBP4蛋白活性的蛋白分子”是指其氨基酸序列与IGFBP4蛋白相比具有≥85％的同源性，较佳地≥90％的同源性，更佳地≥95％的同源性，最佳地≥98％同源性；并且具有天然IGFBP4蛋白活性的蛋白分子。
在另一优选例中，所述IGFBP4突变体选自下组：IGFBP4/H95P、IGFBPDF/H95A、IGFBPDF/H95E、IGFBPDF/H95D、或其组合。
在另一优选例中，所述Kremen1/2抑制剂是指能够在体内或体外拮抗Kremen2基因或其蛋白的活性和/或含量的物质；所述物质可以为人工合成的或天然的化合物、蛋白(如抗体)、核苷酸等。
在另一优选例中，所述Kremen1/2抑制剂包括拮抗Kremen1/2表达的物质。
在另一优选例中，所述Kremen1/2抑制剂包括Kremen1/2蛋白拮抗剂和/或Kremen1/2基因拮抗剂。
在另一优选例中，所述抑制Kremen1/2表达或活性指将Kremen1/2基因或蛋白的表达或活性降低≥20％，较佳地，≥50％，更佳地，≥70％。
在另一优选例中，所述肿瘤恶病质包括Wnt正常或低表达的肿瘤恶病质。
在另一优选例中，所述肿瘤恶病质包括与Wnt通路无关的肿瘤恶病质。
在另一优选例中，所述组合物或制剂还用于选自下组的一种或多种用途：
(a)降低肿瘤细胞内β-arrestin2的含量；
(b)抑制哺乳动物中的IκB和NFκB的激活；
(c)抑制p53和Bax/Bcl-2的上调；
(d)抑制肌肉蛋白标志物的下调；
(e)抑制哺乳动物肌肉组织中的细胞因子的上调。
在另一优选例中，所述肿瘤细胞来自选自下组的一种或多种肿瘤：肠癌、肺癌、胃癌、胰腺癌、头颈部恶性肿瘤、或其组合。
在另一优选例中，所述肌肉蛋白标志物包括肌红蛋白(myoglobin)。
在另一优选例中，所述肌肉组织中的细胞因子选自下组：TNFα、IL1β、IL6、、或其组合。
在另一优选例中，所述哺乳动物包括患有肿瘤恶病质与糖尿病伴随疾病的哺乳动物。
在另一优选例中，所述哺乳动物包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠、或兔)、灵长类动物(如猴)。
在另一优选例中，所述DKK1来源于哺乳动物；优选地，来源于人、小鼠、大鼠、或兔；更优选地，来源于人。
在另一优选例中，所述DKK1基因包括野生型DKK1基因和突变型DKK1基因。
在另一优选例中，所述的突变型包括突变后编码蛋白的功能未发生改变的突变形式(即功能与野生型编码蛋白相同或基本相同)。
在另一优选例中，所述的突变型DKK1基因编码的多肽与野生DKK1基因所编码的多肽相同或基本相同。
在另一优选例中，所述的突变型DKK1基因包括与野生DKK1基因相比，同源性≥80％(较佳地≥90％，更佳地≥95％，更佳地，≥98％或99％)的多核苷酸。
在另一优选例中，所述的突变型DKK1基因包括在野生型DKK1基因的5＇端和/或3＇端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸。
在另一优选例中，所述的DKK1基因包括cDNA序列、基因组序列、或其组合。
在另一优选例中，所述DKK1蛋白包括DKK1的活性片段或其衍生物。
在另一优选例中，所述活性片段或其衍生物与DKK1的同源性至少为90％，优 选为95％，更优选为98％、99％。
在另一优选例中，所述活性片段或其衍生物至少具有80％、85％、90％、95％、100％的DKK1活性。
在另一优选例中，所述DKK1蛋白的氨基酸序列选自下组：
(i)具有SEQ ID NO.:1或3所示氨基酸序列的多肽；
(ii)将如SEQ ID NO.:1或3所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，具有所述蛋白功能的、由(i)衍生的多肽；或
(iii)氨基酸序列与SEQ ID NO.:1或3所示氨基酸序列的同源性≥90％(较佳地≥95％，更佳地≥98％或99％)，具有所述蛋白功能的多肽。
在另一优选例中，所述DKK1基因的核苷酸序列选自下组：
(a)编码如SEQ ID NO.:1或3所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:2或4所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:2或4所示序列的同源性≥95％(较佳地≥98％，更佳地≥99％)的多核苷酸；
(d)在SEQ ID NO.:2或4所示多核苷酸的5’端和/或3’端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(e)与(a)-(d)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述DKK1蛋白的氨基酸序列如SEQ ID NO.:1或3所示。
在另一优选例中，所述编码DKK1蛋白的核苷酸序列如SEQ ID NO.:2或4所示。
在另一优选例中，所述DKK1基因或其编码蛋白的抑制剂的含量为0.1mg/kg-100mg/kg，较佳地，1mg/kg-50mg/kg，更佳地，2mg/kg-20mg/kg。
在另一优选例中，所述组合物包括药物组合物。
在另一优选例中，所述药物组合物含有(a)DKK1基因或其编码蛋白抑制剂；和(b)药学上可接受的载体。
在另一优选例中，所述药物组合物为液态、固体、或半固体。
在另一优选例中，所述药物组合物的剂型包括片剂、颗粒剂、胶囊、口服液、或注射剂。
在另一优选例中，所述的药物组合物中，所述组分(a)占所述药物组合物总 重量的1-99wt％，较佳地10-90wt％，更佳地30-70wt％。
在另一优选例中，所述组合物还包括其他的预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的药物。
在另一优选例中，所述其他的预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的药物选自下组：肿瘤药物、糖尿病治疗药物、或其组合。
在另一优选例中，所述糖尿病治疗药物选自下组：胰岛素类药物、二甲双胍类降糖药物、GLP类药物、胃饥饿素(Ghrelin)、或其组合。
在另一优选例中，所述肿瘤药物选自下组：化疗药物、靶向治疗药物、免疫治疗药物、细胞治疗药物、或其组合。
在另一优选例中，所述化疗药物选自下组：吉西他滨、顺铂、长春新碱、紫杉醇、多柔比星、5-FU、或其组合。
在另一优选例中，所述靶向治疗药物选自下组：拉帕替尼、厄洛替尼、阿帕替尼、利妥昔单抗、贝伐珠单抗、曲妥珠单抗、或其组合。
在另一优选例中，所述免疫治疗药物选自下组：PD-1单抗、PDL-1单抗、CD47单抗、或其组合。
在另一优选例中，所述细胞治疗药物选自下组：NK细胞、CART细胞、TIL细胞、或其组合。
在另一优选例中，所述组合物或制剂在预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的应用中，可单独使用，或联合使用。
在另一优选例中，所述的联合使用包括：与其它预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的药物联合使用。
本发明第二方面提供了一种药物组合物，包括：
(a1)用于预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的第一活性成分，所述第一活性成分包括：DKK1基因或其编码蛋白抑制剂；
(a2)预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的第二活性成分，所述第二活性成分包括：其他的用于预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的药物；和
(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物中，所述组分(a1)占所述药物组合物总重量的1-99wt％，较佳地10-90wt％，更佳地30-70wt％。
在另一优选例中，所述的药物组合物中，所述组分(a2)占所述药物组合物总 重量的1-99wt％，较佳地10-90wt％，更佳地30-70wt％。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为1:100至100：1，较佳地为1:10至10：1。
在另一优选例中，所其他的预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的药物选自下组：肿瘤药物、糖尿病治疗药物、或其组合。
在另一优选例中，所述糖尿病治疗药物选自下组：胰岛素类药物、二甲双胍类降糖药物、GLP类药物、胃饥饿素(Ghrelin)、或其组合。
在另一优选例中，所述肿瘤药物选自下组：化疗药物、靶向治疗药物、免疫治疗药物、细胞治疗药物、或其组合。
在另一优选例中，所述化疗药物选自下组：吉西他滨、顺铂、长春新碱、紫杉醇、多柔比星、5-FU、或其组合。
在另一优选例中，所述靶向治疗药物选自下组：拉帕替尼、厄洛替尼、阿帕替尼、利妥昔单抗、贝伐珠单抗、曲妥珠单抗、或其组合。
在另一优选例中，所述免疫治疗药物选自下组：PD-1单抗、PDL-1单抗、CD47单抗、或其组合。
在另一优选例中，所述细胞治疗药物选自下组：NK细胞、CART细胞、TIL细胞、或其组合。
在另一优选例中，所述药物组合物中可以是单一化合物，也可以是多个化合物的混合物。
在另一优选例中，所述的药物组合物用于制备治疗或预防肿瘤恶病质与糖尿病伴随疾病的药物或制剂。
在另一优选例中，所述的药物剂型为口服给药或非口服给药剂型。
在另一优选例中，所述的口服给药剂型是片剂、散剂、颗粒剂或胶囊剂，或乳剂或糖浆剂。
在另一优选例中，所述的非口服给药剂型是注射剂或针剂。
在另一优选例中，所述的活性成分(a1)和活性成分(a2)的总含量为组合物总重的1～99wt％，更佳地为5～90wt％。
本发明第三方面提供了一种药盒，包括：
(i)第一容器，以及位于该第一容器中的活性成分(a1)DKK1基因或其编码蛋白抑制剂，或含有活性成分(a)的药物；和
(ii)第二容器，以及位于该第二容器中的活性成分(a2)其他的用于预防和/ 或治疗肿瘤恶病质与糖尿病伴随疾病的药物,或含有活性成分(a2)的药物。
在另一优选例中，所述的第一容器和第二容器是相同或不同的容器。
在另一优选例中，所述的第一容器的药物是含DKK1基因或其编码蛋白抑制剂的单方制剂。
在另一优选例中，所述的第二容器的药物是含其他的用于预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的药物的单方制剂。
在另一优选例中，所述药物的剂型为口服剂型或注射剂型。
在另一优选例中，所述试剂盒还含有说明书，所述说明书中记载了联合给予活性成分(a1)和活性成分(a2)从而预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的说明。
在另一优选例中，所述含有活性成分(a1)DKK1基因或其编码蛋白抑制剂的制剂或含有其他的用于预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的药物的制剂的剂型分别包括胶囊、片剂、栓剂、或静脉注射剂。
在另一优选例中，所述含有活性成分(a1)DKK1基因或其编码蛋白抑制剂的制剂中，所述DKK1基因或其编码蛋白抑制剂的浓度为0.1mg/kg-100mg/kg，较佳地，1mg/kg-50mg/kg，更佳地，2mg/kg-20mg/kg。
本发明第四方面提供了一种体外降低肿瘤细胞内β-arrestin2的含量的方法，包括步骤：
在DKK1基因或其编码蛋白抑制剂存在的条件下，培养肿瘤细胞，从而降低肿瘤细胞内β-arrestin2的含量。
在另一优选例中，所述肿瘤细胞来自于选自下组的一种或多种肿瘤：肠癌、肺癌、胃癌、胰腺癌、头颈部恶性肿瘤、或其组合。
在另一优选例中，所述肿瘤细胞为体外培养的细胞。
在另一优选例中，所述方法为非诊断性和非治疗性的。
在另一优选例中，所述DKK1基因或其编码蛋白抑制剂的作用浓度为0.1mg/kg-100mg/kg，较佳地，1mg/kg-50mg/kg，更佳地，2mg/kg-20mg/kg。
本发明第五方面提供了一种本发明第二方面所述的药物组合物或本发明第三方面所述药盒的用途，用于制备用于预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的药物。
在另一优选例中，所述药物组合物中，DKK1基因或其编码蛋白抑制剂的作用浓度为0.1mg/kg-100mg/kg，较佳地，1mg/kg-50mg/kg，更佳地，2mg/kg-20mg/kg。
本发明第六方面提供了一种筛选肿瘤恶病质与糖尿病伴随疾病的潜在治疗剂的方法，包括：
(a)在测试组中，在培养体系中，在测试化合物的存在下，培养表达DKK1基因或其蛋白的细胞一段时间T1，检测测试组所述培养体系中的DKk1基因或其蛋白的表达量E1和/或活性A1；
并且在不存在所述测试化合物且其他条件相同的对照组中，检测对照组所述培养体系中DKK1基因或其蛋白的表达量E2和/或活性A2；和
(b)对E1和E2进行比较，如果E1显著低于E2，则表示所述测试化合物是肿瘤恶病质与糖尿病伴随疾病的潜在治疗剂；或
对A1和A2进行比较，如果A1显著低于A2，则表示所述测试化合物是肿瘤恶病质与糖尿病伴随疾病的潜在治疗剂。。
在另一优选例中，所述“显著高于”指E1/E2≤1/2，较佳地，≤1/3，更佳地，≤1/4。
在另一优选例中，所述细胞包括肿瘤细胞。
在另一优选例中，所述肿瘤细胞来自于选自下组的一种或多种肿瘤：肠癌、肺癌、胃癌、胰腺癌、头颈部恶性肿瘤、或其组合。
在另一优选例中，所述的方法是非诊断和非治疗性的。
在另一优选例中，所述的方法包括步骤(c)：将步骤(a)中所确定的潜在治疗剂施用于哺乳动物，从而测定其对哺乳动物的肿瘤恶病质与糖尿病伴随疾病的影响。
在另一优选例中，所述哺乳动物包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物、灵长目动物，较佳地，包括小鼠、大鼠、兔、猴。
本发明第七方面提供了一种筛选肿瘤恶病质与糖尿病伴随疾病的潜在治疗剂的方法，包括：
(a)在测试组中，在培养体系中，在测试化合物的存在下，培养肿瘤细胞一段时间T1，检测测试组所述培养体系中所述DKK1与LRP6复合物的形成情况；
并且在不存在所述测试化合物且其他条件相同的对照组中，检测对照组所述培养体系中所述DKK1与LRP6复合物的形成情况；
(b)如果所述测试组中的所述述DKK1与LRP6复合物的形成数量Q1显著低于所述对照组中的所述述DKK1与LRP6复合物的形成数量Q2，则表示所述测试化合 物是候选化合物。
在另一优选例中，所述的方法包括步骤(b)：将步骤(a)中所确定的候选化合物施用于哺乳动物，从而测定其对哺乳动物的肿瘤恶病质与糖尿病伴随疾病的影响。
在另一优选例中，所述哺乳动物包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物、灵长目动物，较佳地，包括小鼠、大鼠、兔、猴。
在另一优选例中，所述“显著低于”指Q1/Q2≤1/2,较佳地，≤1/3，更佳地≤1/4。
在另一优选例中，所述的方法是非诊断和非治疗性的。
在另一优选例中，所述肿瘤细胞来源于选自下组的一种或多种肿瘤：肠癌、肺癌、胃癌、胰腺癌、头颈部恶性肿瘤。
本发明第八方面提供了一种预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的方法，包括步骤：
给需要的对象，施DKK1基因或其编码蛋白抑制剂、权利要求2所述的药物组合物、或权利要求3所述的药盒。
在另一优选例中，所述的施用包括口服。
在另一优选例中，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物和灵长目动物，优选小鼠、大鼠、兔、猴。
在另一优选例中，所述DKK1基因或其编码蛋白抑制剂的施用剂量为0.1mg/kg-100mg/kg，较佳地，1mg/kg-50mg/kg，更佳地，2mg/kg-20mg/kg。
在另一优选例中，所述DKK1基因或其编码蛋白抑制剂的施用频率为每周1-7次，较佳地，每周2-5次，更佳地，每周2-3次。
在另一优选例中，所述DKK1基因或其编码蛋白抑制剂的施用时间为1-20周，较佳地，2-12周，更佳地，4-8周。
本发明第九方面提供了一种确定肿瘤恶病质与糖尿病伴随疾病的治疗方案的方法，包括：
a)提供来自受试者的测试样品；
b)检测测试样品中DKK1蛋白的水平；和
c)基于所述样品中的DKK1蛋白水平来确定肿瘤恶病质与糖尿病伴随疾病 的治疗方案。
在另一优选例中，所述的受试者为人或非人哺乳动物。
在另一优选例中，所述样品来源于血液、肿瘤组织、腹水或尿液。
在另一优选例中，所述样品来源于外周血。
在另一优选例中，用以下方法进行检测：免疫学检测方法、酶学方法、或质谱方法。
在另一优选例中，所述免疫检测方法选自下组：ELISA、Western blotting、免疫荧光、化学发光、或其组合。
在另一优选例中，所述治疗方案还包括其他的预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的疗法。
在另一优选例中，所述其他的预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的疗法选自下组：
(a)肿瘤药物疗法；和/或
(b)糖尿病治疗药物疗法。
在另一优选例中，所述糖尿病治疗药物选自下组：胰岛素类药物、二甲双胍类降糖药物、GLP类药物、胃饥饿素(Ghrelin)、或其组合。
在另一优选例中，所述肿瘤药物选自下组：化疗药物、靶向治疗药物、免疫治疗药物、细胞治疗药物、或其组合。
在另一优选例中，所述化疗药物选自下组：吉西他滨、顺铂、长春新碱、紫杉醇、多柔比星、5-FU、或其组合。
在另一优选例中，所述靶向治疗药物选自下组：拉帕替尼、厄洛替尼、阿帕替尼、利妥昔单抗、贝伐珠单抗、曲妥珠单抗、或其组合。
在另一优选例中，所述免疫治疗药物选自下组：PD-1单抗、PDL-1单抗、CD47单抗、或其组合。
在另一优选例中，所述细胞治疗药物选自下组：NK细胞、CART细胞、TIL细胞、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了Dkk1上调可能与恶病质相关的肿瘤死亡有关。
其中，图1a显示了TCGA数据库分析临床上不同的肿瘤类型(包括了头颈癌、胰腺癌、胃腺癌和肺腺癌)患者，血液中Dkk1的表达量域生存期的关系。
图1b显示ELISA法检测荷瘤小鼠(肠癌细胞种植瘤和肺腺癌细胞种植瘤)血液中Dkk1的表达。图1c显示了RT-qPCR法检测不同器官中Dkk1的表达量。
图1d显示了RT-qPCR法检测种植肿瘤对肌肉和肾脏中Dkk1表达量的影响。
图1e显示了Kaplan-Meier分析Dkk1对荷瘤小鼠生存期的影响。
图1f显示了Kaplan-Meier分析Dkk1综合抗体延长荷瘤小鼠生存期作用。
图1g显示了肿瘤晚期引起小鼠去瘤体重和肌肉重量的改变。
图1h显示了HE染色观察恶病质小鼠肌肉病理状态。
图1i显示了肿瘤种植晚期观察肾脏的重量、HE染色观察肾脏病理状态及自动生化仪检测肾功能的表达。
图2显示了下调膜蛋白LRP6和Kremen2能引起肿瘤恶病质。
图2a显示了Western blot法检测恶病质晚期肌肉中膜/总蛋白LRP6、LRP5、Kremen1、Kremen2的表达。
图2b显示了Western blot法检测恶病质晚期肾脏中膜/总蛋白LRP6、LRP5、Kremen1、Kremen2的表达。
图2c显示了Western blot法检测恶病质晚期肾脏中总/核蛋白β-catenin的表达。
图3显示了逆转膜上LRP6和Kremen2下调可以阻止肿瘤恶病质。
图3a显示了Western blot法检测Dkk1肌肉注射引起的膜/浆蛋白LRP6、Kremen2的改变，以及MDC对其逆转作用。
图3b显示了Kaplan-Meier分析MDC和Dkk1联用延长Dkk1引起的荷瘤小鼠生存期缩短作用。
图3c显示了腹腔注射MDC和IGFBP4可以逆转肿瘤引起的去瘤体重下降以及肌肉萎缩。
图3d和3e显示了Kaplan-Meier分析Clathin-TG2抑制剂(MDC、Cystamine和Spermidine)对荷瘤小鼠生存期的影响。
图3f显示了Western blot分析MDC逆转肿瘤引起的膜蛋白LRP6和Kremen2的表达，以及总蛋白Myoglobin的表达。
图3g显示了Kaplan-Meier分析IGFBP4对荷瘤小鼠生存期的影响。
图3h显示了Western blot分析IGFBP4影响Dkk1引起的膜/浆蛋白LRP6和Kremen2的表达。
图4显示了RNA-Seq分析：敲低LRP5/6基因广泛改变GPCR的表达。
(A)HepG2细胞中LRP5/6(红色)或CTNNB1(β-catenin，蓝色)被siRNA敲低后，RNA-Seq分析用文氏图揭示了表达倍数变化>0.2的GPCR的数量分布。(B和C)HepG2细胞中LRP5/6或CTNNB1(β-catenin)被siRNA敲低后，RNA-Seq分析用柱状图显示了GPCR表达倍数的变化(B)；用热图显示了表达上调(红色)或下调(绿色)的GPCR倍数变化(log 2值)(C)。
图5显示了外源性LRP6N逆转敲低LRP5/6诱导的细胞DNA损伤。
(A)LRP5，LRP6或β-catenin被梯度稀释的siRNA敲低48h后，HUVEC细胞中LRP5/6，β-catenin和γH2AX的表达。n＝3。(B)LRP5/6或β-catenin敲低48h后，LRP6N蛋白预处理和H 2O 2刺激诱导HUVEC细胞的γH2AX反应。n＝3。(C)H 2O 2按指定时间(左图)和浓度(右图)刺激HUVEC细胞后，γH2AX，细胞膜或总体LRP5/6(m/tLRP5/6)，细胞核或总体β-catenin(n/tβ-catenin)的表达。n＝3。
图6显示了敲低MESD导致膜上LRP5/6下调和氧化应激抵抗的减弱。
(A)MESD敲低48h后，HUVEC细胞中MESD，细胞膜或总体LRP5/6(m/tLRP5/6)和总体β-catenin(tβ-catenin)的表达。n＝3。(B)MESD被siRNA#1敲低48h后，LRP6N蛋白预处理和H 2O 2刺激诱导HUVEC细胞的γH2AX反应。n＝3。
图7显示了Dkk1诱导细胞膜上LRP5/6内吞和DNA损伤反应。
(A)Dkk1蛋白按指定时间(左图)和浓度(右图)刺激HUVEC细胞后，细胞膜或总体LRP5/6(m/tLRP5/6)，细胞核或总体β-catenin(n/tβ-catenin)和γH2AX的表达。n＝3。(B)MDC或LRP6N蛋白预处理合并Dkk1蛋白和H 2O 2单独或先后刺激HUVEC细胞诱导的γH2AX反应。n＝3。
图8显示了Dkk1通过下调膜上LRP5/6激活β-arrestin1/2信号的传导。
(A和B)MDC预处理合并指定时间(A)和浓度(B)的Dkk1蛋白刺激HUVEC细胞后，细胞膜或细胞浆β-arrestin1/2(m/cβ-arrestin1/2)和LRP5/6(m/cLRP5/6)的表达。n＝3。(C)BIM-46187预处理合并Dkk1蛋白刺激HUVEC细胞后，细胞膜或细胞浆β-arrestin1/2(m/cβ-arrestin1/2)的表达。n＝3。(D)LRP5/6或β-catenin敲低48h后，HUVEC细胞中β-arrestin1/2，LRP5/6和β-catenin的表达。n＝3。
图9显示了β-arrestin1/2介导了膜上LRP5/6下调引起的细胞DNA损伤。
(A)β-arrestin1/2敲低48h后，HUVEC细胞中β-arrestin1/2的表达。n＝3。(B)β-arrestin1/2敲低48h后，Dkk1蛋白和H 2O 2单独或先后刺激HUVEC细胞诱导的γH2AX反应。n＝3。(C)BIM-46187预处理合并Dkk1蛋白刺激HUVEC细胞后，γH2AX的表达。n＝3。(D)β-arrestin1/2合并LRP5/6或β-catenin敲低48h后，H 2O 2刺激诱导HUVEC细胞的γH2AX反应。n＝3。
图10显示了G蛋白激动剂能够诱导DNA损伤反应。
(A和B)CTX(A)或PTX(B)按指定时间和浓度刺激诱导HUVEC细胞的γH2AX反应。n＝3。
图11显示了LRP5/6基因敲除引起小鼠心脏损伤。
(A)他莫昔芬诱导1周的对照，LRP5/6 -/-和β-catenin -/-小鼠，心脏中LRP5/6，β-catenin，γH2AX，p53，p21，Bcl-2，Bax和Cleaved Caspase-3的表达。n＝9。(B)他莫昔芬诱导35周的对照，LRP5/6 -/-和β-catenin -/-小鼠的体重变化(左图)及多器官(心脏，肺脏，肾脏，脾脏，骨骼肌和脂肪)的相对重量比(右图)。n＝6。(C)对照，LRP5/6 -/-和β-catenin -/-小鼠左心室射血分数(EF％)和左心室短轴缩短率(FS％)在他莫昔芬诱导后指定时间的变化(左图)；他莫昔芬诱导35周的对照，LRP5/6 -/-和β-catenin -/-小鼠的代表性二维超声心动图，显示左心室收缩末期和舒张末期的波型模式。LRP5/6 -/-小鼠的平波模式表明左心室壁肌肉运动受损(右图)。n＝6。(D)实时定量PCR分析。他莫昔芬诱导35周的对照，LRP5/6 -/-和β-catenin -/-小鼠，心脏中ANP和BNP的表达。数据通过GAPDH内参标准化。n＝6。(E)对照，LRP5/6 -/-和β-catenin -/-小鼠在他莫昔芬诱导后指定时间测量的二维心脏超声各指标的数值。n＝6。
图12显示了LRP5/6基因敲除激活小鼠心脏β-arrestin1/2信号的传导。
(A)他莫昔芬诱导1周的对照，LRP5/6 -/-和β-catenin -/-小鼠，心脏中细胞膜，细胞浆和细胞核β-arrestin1/2(m/c/nβ-arrestin1/2)的表达。n＝6。(B)实时定量PCR分析。他莫昔芬诱导8周的对照，LRP5/6 -/-和β-catenin -/-小鼠，心脏中一些GPCR的表达。数据通过GAPDH内参标准化。n＝6。
图13显示了高血糖诱导糖尿病小鼠血液Dkk1的上调。
(A)正常小鼠，STZ造模7天或合并胰岛素治疗7天的糖尿病小鼠，血液Dkk1的ELISA分析。n＝8。(B)STZ造模(左图)或合并胰岛素治疗(右图)的糖尿病小鼠，指定时间的血糖浓度。n＝10。
图14显示了糖尿病小鼠膜上LRP5/6下调与心脏损伤的密切关系。
(A和B)STZ造模(A)或合并胰岛素治疗(B)的糖尿病小鼠，指定时间的心脏中细胞核或总体β-catenin(n/tβ-catenin)，细胞膜或总体LRP5/6(m/tLRP5/6)和γH2AX的表达。n＝9。(C)正常小鼠，STZ造模7天或合并胰岛素治疗7天的糖尿病小鼠，血液8-OHdG的ELISA分析。n＝5。(D和E)STZ造模7天(D)或合并胰岛素治疗7天(E)的糖尿病小鼠，心脏中p53，p21，Bcl-2，Bax和Cleaved Caspase-3的表达。n＝9。(F)STZ造模(左图)或合并胰岛素治疗(右图)的糖尿病小鼠，指定时间的体重变化。n＝10。
图15显示了Dkk1通过诱导LRP5/6膜内吞引起糖尿病心脏损伤。
(A)STZ造模7天或合并MDC干预4天的糖尿病小鼠，心脏中m/tLRP5/6和n/tβ-catenin的表达。n＝9。(B)正常小鼠，STZ造模以及分别合并胰岛素和MDC的糖尿病小鼠，心脏中γH2AX(红色)和DAPI(蓝色)的免疫荧光染色，比例尺为50μm。n＝5。(C)STZ造模7天或合并MDC干预4天的糖尿病小鼠，心脏中γH2AX，p53，p21，Bcl-2，Bax和Cleaved Caspase-3的表达。n＝9。(D)正常小鼠，STZ造模或合并MDC干预的糖尿病小鼠，血液8-OHdG的ELISA分析。n＝5。(E)STZ造模或合并MDC干预的糖尿病小鼠，指定时间的体重变化。n＝10。(F)正常小鼠，STZ造模以及分别合并胰岛素和MDC的糖尿病小鼠心脏HE染色，比例尺为70μm。n＝5。(G)STZ造模7天或合并MDC干预4天的糖尿病小鼠血糖浓度。n＝10。(H)野生型小鼠Dkk1蛋白心脏内注射或合并MDC干预2天，心脏中m/tLRP5/6，n/tβ-catenin，γH2AX，p53和p21的表达。n＝6。(I)野生型小鼠Dkk1蛋白合并BIM-46187心脏内注射2天，心脏中γH2AX，p53和p21的表达。n＝6。
图16显示了Leptin -/-小鼠膜上LRP5/6下调导致糖尿病心脏损伤。
(A和B)WT(雄性n＝17；雌性n＝9)和Leptin -/-(雄性n＝12；雌性n＝9)小鼠的6周龄外形图，基因型鉴定及不同周龄的体重和血糖浓度。(C)6周龄WT，Leptin +/-和Leptin -/-小鼠的心脏中m/tLRP5/6，n/tβ-catenin，γH2AX，p53，p21，Bcl-2，Bax和Cleaved Caspase-3的表达。n＝6。(D)4周龄WT，Leptin +/-和Leptin -/-小鼠的心脏中m/tLRP5/6和tβ-catenin的表达。n＝6。(E)6周龄WT，Leptin +/-和Leptin -/-小鼠血液Dkk1的ELISA分析。n＝10。(F)6周龄WT和Leptin -/-小鼠的糖耐量测试(GTT)。n＝6。(G和I)D-葡萄糖(每2h腹腔注射一次，持续24h)或合并MDC干预的6周龄Leptin -/-小鼠，血液Dkk1(G， n＝7)和8-OHdG(I，n＝5)的ELISA分析。(H)D-葡萄糖(每2h腹腔注射一次，持续24h)或合并MDC干预的6周龄Leptin -/-小鼠，心脏中m/tLRP5/6，n/tβ-catenin和γH2AX的表达。n＝10。(J和K)D-葡萄糖(每2h腹腔注射一次，持续24h)或合并MDC干预的6周龄Leptin -/-小鼠，心脏中γH2AX，p53，p21，Bcl-2，Bax和Cleaved Caspase-3的表达。n＝10。
图17显示了LRP5/6基因敲除加重了糖尿病心脏损伤。
(A)他莫昔芬诱导4周或合并STZ造模7天的对照，LRP6 -/-和β-catenin -/-小鼠，心脏中γH2AX，p53，p21，Bcl-2，Bax，Cleaved Caspase-3，LRP5/6和β-catenin的表达。n＝9。(B)他莫昔芬诱导4周或合并STZ造模14天的对照，LRP5/6 -/-和β-catenin -/-小鼠左心室射血分数(EF％)和左心室短轴缩短率(FS％)在STZ造模前后的变化(左图)；STZ造模14天的对照，LRP5/6 -/-和β-catenin -/-小鼠的代表性二维超声心动图，显示左心室收缩末期和舒张末期的波型模式。LRP5/6 -/-小鼠的平波模式表明左心室壁肌肉运动受损(右图)。n＝6。(C和D)他莫昔芬诱导4周或合并STZ造模7天的对照，LRP6 -/-和β-catenin -/-小鼠的体重变化(C)和血糖浓度(D)。n＝6。(E)他莫昔芬诱导4周或合并STZ造模14天的对照，LRP5/6 -/-和β-catenin -/-小鼠在STZ造模前后测量的二维心脏超声各指标的数值。n＝6。
图18显示了外源性LRP6N阻止了糖尿病心脏损伤。
(A)基于Cre-loxP重组酶系统的LRP6N/Tg小鼠品系构建的示意图(上图)和UBC-Cre阳性的LRP6N/Tg小鼠的基因型鉴定(下图)。他莫昔芬诱导使loxP序列被Cre重组酶删除，CAG启动子从而启动了带myc标签的LRP6N基因的转录。(B)他莫昔芬诱导4周的对照和LRP6N/Tg小鼠，心脏中带myc标签的LRP6N，m/tLRP5/6，tβ-catenin和γH2AX的表达。n＝6。(C)STZ造模7天的对照和LRP6N/Tg小鼠，心脏中γH2AX，p53，p21，Bcl-2，Bax，Cleaved Caspase-3，m/tLRP5/6和n/tβ-catenin的表达。n＝9。(D)正常小鼠，STZ造模或合并LRP6N/Tg过表达的糖尿病小鼠，血液8-OHdG的ELISA分析。n＝5。(E)尾静脉注射Vector和LRP6N质粒的野生型小鼠，指定时间的肺脏中带myc标签的LRP6N表达。n＝3。(F)STZ造模7天合并尾静脉注射Vector和LRP6N质粒的糖尿病小鼠，心脏中γH2AX，p53，p21，Bcl-2，Bax和Cleaved Caspase-3的表达。n＝8。(G)对照，LRP5/6 -/-和β-catenin -/-小鼠(左图，n＝12)在他莫昔芬诱导35周后，以及对照和LRP6N/Tg小鼠(中图，n＝16)，Vector和LRP6N质粒注射的小鼠(右图，n＝16)在STZ造模7天后的血糖浓度。(H)对照和 LRP6N/Tg小鼠(左图，n＝15)，Vector和LRP6N质粒注射的小鼠(右图，n＝15)在STZ造模7天后的体重变化。
图19显示了糖尿病小鼠膜上LRP5/6下调特异激活心脏β-arrestin1/2信号的传导。
(A)STZ造模7天的糖尿病小鼠，心脏中细胞膜，细胞浆和细胞核β-arrestin1/2及细胞膜和细胞浆insulin receptor-β(m/c/nβ-arrestin1/2和m/cIR-β)的表达。n＝6。(B)STZ造模14天合并胰岛素给药7天的糖尿病小鼠，心脏中m/c/nβ-arrestin1/2和m/cIR-β的表达。n＝6。(C)STZ造模7天合并MDC给药4天的糖尿病小鼠，心脏中m/c/nβ-arrestin1/2和m/cIR-β的表达。n＝6。(D)STZ造模7天的对照或LRP6N/Tg小鼠，心脏中m/c/nβ-arrestin1/2和m/cIR-β的表达。n＝6。(E)野生型小鼠Dkk1蛋白心脏内注射合并MDC给药2天，心脏中m/c/nβ-arrestin1/2和m/cIR-β的表达。n＝6。(F)野生型小鼠Dkk1蛋白合并BIM-46187心脏内注射2天，心脏中m/c/nβ-arrestin1/2的表达。n＝6。(G)对照，胰岛素单独给药7天，MDC单独给药4天，LRP6N/Tg小鼠的心脏中m/c/nβ-arrestin1/2和m/cIR-β的表达。n＝6。
图20显示了可诱导全身LRP5和LRP6共敲除或β-catenin单敲除的转基因小鼠配种示意图。将交配得到的LRP5/6floxp/floxp或β-cateninfloxp/floxp纯合型小鼠与UBC-Cre阳性小鼠交配，可繁殖出诱导性全身LRP5/6共敲除或β-catenin单敲除的转基因小鼠(UBC-Cre-LRP5/6floxp/floxp或UBC-Cre-β-cateninfloxp/floxp)。经他莫昔芬腹腔注射诱导后的小鼠体内会实现全身LRP5/6的共敲除或β-catenin的单敲除(Ctr为阴性对照)，这样的小鼠可分别表示为LRP5/6-/-和β-catenin-/-。
图21显示了可诱导全身过表达LRP6N的转基因小鼠配种示意图。将携带LRP6N基因的小鼠(STOPfloxp/floxpLRP6N)与UBC-Cre阳性小鼠交配，可繁殖出诱导性全身过表达LRP6N的转基因小鼠(UBC-Cre-STOPfloxp/floxpLRP6N)。经他莫昔芬腹腔注射诱导后的小鼠体内会有LRP6N的过表达(Ctr为阴性对照)，这样的小鼠可表示为LRP6N/Tg。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现，抑制肿瘤恶病质与糖 尿病模型动物血中的DKK1蛋白的表达或活性，可有效预防和/或治疗肿瘤恶病质与糖尿病伴随疾病，并且发现DKK1这一活性与传统的作为Wnt信号通路抑制剂无关，而是通过诱导其受体LRP5/6内吞，从而诱导器官损伤。并且，申请人还意外的发现，抑制DKK1和LRP5/6的结合(即抑制DKK1与LRP5和LRP6复合物的形成)，也可有效预防和/或治疗肿瘤恶病质与糖尿病伴随疾病。在此基础上，本发明人完成了本发明。
具体地，实验表明，DKK1蛋白通过诱导LRP5和LRP6的内吞作用，导致了器官损伤，加速了肿瘤恶病质导致的动物死亡和糖尿病伴随急性心肌梗死动物的死亡，同时不影响Wnt信号通路效应蛋白β-catenin。而小分子药物或者蛋白药物阻止了Dkk1引起的膜LRP5和LRP6下调可以完全阻止肿瘤恶病质与糖尿病伴随疾病，并且很好的延长了这些疾病小鼠生存期。通过全基因组的转录分析得到，在骨骼肌中，小分子药物阻止Dkk1引起的膜LRP5和LRP6下调阻止了多个GPCR信号通路的改变以及所有主要的恶病质相关的通路。而且，将Dkk1蛋白直接肌肉注射进小鼠下肢，马上就引起GPCR信号通路以及恶病质相关通路的激活。这些发现，建立了一个重要的途径，就是肿瘤恶病质与糖尿病伴随疾病的发展是通过不依赖于Wnt信号通路，而是以Dkk1-LRP5/LRP6为主轴，影响GPCR信号通路的(图1)。并为预防和/或治疗肿瘤恶病质与糖尿病伴随疾病提出了一个非常有前景的治疗方案。这样，虽然肿瘤恶病质与糖尿病有着本身特异的发病原因，DKK1-LRP5/6这条通路的改变在两种疾病上都扮演了同样的器官损伤作用。
肿瘤恶病质
恶病质亦称恶液质。表现为极度消瘦，皮包骨头，形如骷髅，贫血，无力，完全卧床，生活不能自理，极度痛苦，全身衰竭等综合征。多由癌症和其他严重慢性病引起。可看作是由于全身许多脏器发生障碍所致的一种中毒状态。肿瘤恶病质伴随体重减轻，严重损害生存质量，限制癌症的治疗，同时缩短生存期，然而，目前没有有效的治疗方法。肌肉萎缩是肿瘤恶病质的一个关键特征，是一个多因素疾病，对患者预后和生活质量有负面影响。无论体重指数(BMI)如何，骨骼肌损耗被认为是癌症发展过程中一个有意义的预后因素，并且与增加化疗毒性的发生率、缩短肿瘤进展时间、手术结果差、身体损害和缩短生存期有关。
糖尿病
糖尿病(diabetes mellitus，DM)是一组由多病因引起的胰岛素分泌或者作用缺陷，并以慢性高血糖为特征的全身代谢性疾病。长期的糖、脂肪及蛋白质代谢紊乱可引起多器官损害和稳态失衡，会导致心、肾、眼、神经、血管等组织器官慢性病变，功能减退，甚至衰竭等并发症。1型糖尿病(diabetes mellitus type 1，T1DM)和2型糖尿病(diabetes mellitus type 2，T2DM)是糖尿病的两个重要分型，两者的发病机制有所不同。血糖持续升高是糖尿病所致多器官DNA损伤的关键因素，因此利用胰岛素降低血糖进行治疗的同时如何降低血糖导致的细胞损伤作用是一个重要意义的科学问题。
DKK1蛋白和多核苷酸
在本发明中，术语“本发明蛋白”、“DKK1蛋白”、“DKK1多肽”可互换使用，都指具有DKK1氨基酸序列的蛋白或多肽。它们包括含有或不含起始甲硫氨酸的DKK1蛋白。此外，该术语还包括全长的DKK1及其片段。本发明所指的DKK1蛋白包括其完整的氨基酸序列、其分泌蛋白、其突变体以及其功能上活性的片段。
DKK1分泌蛋白是经典Wnt/β-catenin抑制剂，通过直接结合LRP5/6受体和Kremen受体介导LRP5/6内吞而抑制经典Wnt/β-catenin的激活状态。Mouse Dkk1(NP_034181.2)，由272个氨基酸组成，，序列如SEQ ID NO.:3所示；human Dkk1(NP_036374.1)，由266个氨基酸组成，序列如SEQ ID NO.:1所示。
在本发明中，术语“DKK1基因”、“DKK1多核苷酸”可互换使用，都指具有DKK1核苷酸序列的核酸序列。
人DKK1基因的基因组全长：
human DKK1 DNA序列(801 nt)，NCBI登录号：NM_012242.4
Figure PCTCN2020086807-appb-000001
Figure PCTCN2020086807-appb-000002
鼠DKK1基因的基因组全长：
Mouse Dkk1 DNA序列(819 nt)，NCBI登录号：NM_010051.3
Figure PCTCN2020086807-appb-000003
人和鼠DKK1，在DNA水平的相似性为83％，蛋白序列相似性为81％。需理解的是，当编码相同的氨基酸时，密码子中核苷酸的取代是可接受的。另外需理解的是，由核苷酸取代而产生保守的氨基酸取代时，核苷酸的变换也是可被接受的。
在得到了DKK1的氨基酸片段的情况下，可根据其构建出编码它的核酸序列，并且根据核苷酸序列来设计特异性探针。核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的DKK1核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板， 扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(如载体)和细胞中。
通过常规的重组DNA技术，可利用本发明的多核苷酸序列可用来表达或生产重组的DKK1多肽。一般来说有以下步骤：
(1).用本发明的编码人DKK1多肽的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，DKK1多核苷酸序列可插入到重组表达载体中。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含DKK1编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属的 细菌细胞；真菌细胞如酵母；植物细胞；昆虫细胞；动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
腺相关病毒
因腺相关病毒(Adeno-associated virus，AAV)较其他病毒载体小，无致病性，可转染正在分裂和未分裂的细胞等特性，基于AAV载体的针对遗传性疾病的基因治疗方法受到了广泛的关注。
腺相关病毒(adeno-associated virus，AAV),也称腺伴随病毒,属于微小病毒科依赖病毒属,是目前发现的一类结构最简单的单链DNA缺陷型病毒，需要辅助病毒(通常为腺病毒)参与复制。它编码两个末端的反向重复序列(ITR)中的cap和rep基因。ITRs对于病毒的复制和包装具有决定性作用。cap基因编码病毒衣壳蛋白，rep基因参与病毒的复制和整合。AAV能感染多种细胞。
重组腺相关病毒载体(rAAV)源于非致病的野生型腺相关病毒，由于其安全性好、宿主细胞范围广(分裂和非分裂细胞)、免疫源性低，在体内表达外源基因时间长等特点，被视为最有前途的基因转移载体之一，在世界范围内的基因治疗和疫苗研究中得到广泛应用。经过10余年的研究，重组腺相关病毒的生物学特性己被深 入了解，尤其是其在各种细胞、组织和体内实验中的应用效果方面已经积累了许多资料。在医学研究中，rAAV被用于多种疾病的基因治疗的研究(包括体内、体外实验)；同时作为一种有特点的基因转移载体，还广泛用于基因功能研究、构建疾病模型、制备基因敲除鼠等方面。
在本发明一个优选的实施例中，载体为重组AAV载体。AAV是相对较小的DNA病毒，其可以稳定和位点特异性方式整合到它们所感染的细胞的基因组中。它们能够感染一大系列的细胞而不对细胞生长、形态或分化产生任何影响，并且它们似乎并不涉及人体病理学。AAV基因组己被克隆、测序及表征。AAV在每个末端包含约145个碱基的反向末端重复序列(ITR)区域，其作为病毒的复制起点。该基因组的其余被分成两个带有衣壳化功能的重要区域：包含涉及病毒复制和病毒基因表达的rep基因的基因组左边部分；以及包含编码病毒衣壳蛋白的cap基因的基因组右边部分。
AAV载体可采用本领域的标准方法制备。任何血清型的腺相关病毒均是合适的。用于纯化载体的方法可见于例如美国专利No.6566118、6989264和6995006，它们的公开内容整体以引用方式并入本文。杂合载体的制备在例如PCT申请No.PCT/US2005/027091中有所描述，该申请的公开内容整体以引用方式并入本文。用于体外和体内转运基因的衍生自AAV的载体的使用己有描述(参见例如国际专利申请公布No.WO91/18088和WO93/09239；美国专利No.4,797,368、6,596,535和5,139,941，以及欧洲专利No.0488528，它们均整体以引用方式并入本文)。这些专利公布描述了其中rep和/或cap基因缺失并被所关注的基因替换的各种来源于AAV的构建体，以及这些构建体在体外(进入培养的细胞中)或体内(直接进入生物体)转运所关注的基因的用途。复制缺陷重组AAV可通过将以下质粒共转染进被人类辅助病毒(例如腺病毒)感染的细胞系而制备：所含的所关注核酸序列的侧翼为两个AAV反向末端重复序列(ITR)区域的质粒，和携带AAV衣壳化基因(rep和cap基因)的质粒。然后通过标准技术纯化所产生的AAV重组体。
在一些实施方案中，重组载体被衣壳化到病毒粒子(例如包括但不限于AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15和AAV16的AAV病毒粒子)中。因此，本公开包括含有本文所述的任何载体的重组病毒粒子(因其包含重组多核苷酸而为重组的)。产生这样的粒子的方法是本领域己知的，并在美国专利No.6,596,535中有所描述。
DKK1抑制剂和药物组合物
利用本发明蛋白，通过各种常规筛选方法，可筛选出与DKK1基因或蛋白发生相互作用的物质，尤其是抑制剂等。
可用于本发明的DKK1抑制剂(或拮抗剂)包括任何可以抑制DKK1基因或其编码蛋白的表达和/或活性的物质。
在本发明中，DKK1抑制剂还包括抑制DKK1与LRP5和LRP6结合的抑制剂(包括抑制DKK1与LRP5和LRP6复合物形成的抑制剂)。
在本发明中，DKK1抑制剂还包括抑制LRP5和LRP6的活性和/或表达量的物质。
例如，所述DKK1的抑制剂包括小分子化合物、DKK1的抗体、DKK1核酸的反义RNA、siRNA、shRNA、miRNA、或DKK1的活性抑制剂。
在一种优选的实施方式中，抑制DKK1的方法和步骤包括利用DKK1的抗体中和其蛋白，利用病毒(如腺相关病毒)携带的shRNA或siRNA或CRISPR试剂进行DKK1基因的沉默。
对DKK1的抑制率一般为达到至少50％以上的抑制，优选为60％、70％、80％、90％、95％的抑制，可以基于常规技术，例如流式细胞术、荧光定量PCR或Western blot等方法对DKK1的抑制率进行控制和检测。
本发明DKK1蛋白的抑制剂(包括抗体、小分子化合物、反义核酸、CRISPR试剂以及其他抑制剂)，当在治疗上进行施用(给药)时，可抑制DKK1蛋白的表达和/或活性、或抑制LRP5和LRP6的表达和/或活性、或抑制DKK1与LRP5和LRP6复合物的形成，从而预防和/或治疗肿瘤恶病质。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：局部、肌内、腹膜内、静脉内、皮下、皮内、局部给药、自体细胞提取培养后回输等。
本发明还提供了一种药物组合物，它含有安全有效量的本发明抑制剂(如抗体、化合物、CRISPR试剂、反义序列(如siRNA)、或抑制剂)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液 通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物，可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克-10毫克/千克体重。
本发明的主要优点包括：
(1)本发明首次发现，或抑制LRP5和LRP6的表达或活性、或抑制DKK1与LRP5和LRP6复合物的形成，可预防和/或治疗肿瘤恶病质与糖尿病伴随疾病。
(2)本发明首次发现，本发明所预防和/或治疗的肿瘤恶病质与糖尿病伴随疾病为与Wnt通路无关的肿瘤恶病质与糖尿病伴随疾病、或Wnt正常或低表达的肿瘤恶病质与糖尿病伴随疾病。
(3)本发明首次发现，Dkk1蛋白通过诱导LRP5和LRP6的内吞作用，加速了肿瘤恶病质与糖尿病伴随疾病导致的动物死亡，同时不影响β-catenin。而小分子药物阻止了Dkk1引起的膜LRP5和LRP6下调可以完全阻止肿瘤恶病质与糖尿病伴随疾病，并且很好的延长了小鼠生存期。
(4)本发明首次发现，小分子药物阻止Dkk1引起的膜LRP5和LRP6下调阻止了多个GPCR信号通路的改变以及所有主要的恶病质和糖尿病伴随疾病相关的通路。
(5)本发明首次发现，肿瘤恶病质与糖尿病伴随疾病的发展是通过Dkk1-LRP5和LRP6为主轴，影响GPCR信号通路并伴随炎症反应的发生。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非特别说明，否则本发明实施例中所用材料和试剂均为市售产品。
实验方法
恶病质研究相关实验方法:
1、细胞培养及试剂
CT26结肠癌细胞株在RPMI-1640培养基中培养，培养基中添加L-谷氨酰胺(Biosera,USA)和1％青霉素/链霉素(Corning,USA)和10％胎牛血清(Biosera,USA)。37摄氏度培养箱培养，重组小鼠Dkk1和IGFBP-4/H95P蛋白购自Genscript.小鼠对人Dkk1单克隆抗体(mAb)是由上海三友生物制药有限公司生产。从Sigma公司购买MDC，用DMSO溶解，PBS稀释后使用。
2、癌症患者生存期分析
根据指示基因的中位数表达，将某一肿瘤类型的患者分为Dkk1高表达和低表达组。基于TCGA数据库、Oncolinc软件分析在线生成总体生存曲线，采用Kaplan-Meier生存期分析结合Log-rank显著性检验进行生存期分析。
3、肿瘤种植造模
雄性BALB/c六周龄小鼠，购自上海斯莱克实验动物有限公司，在特殊无菌环境下饲养。所有动物研究均经过福建中医药大学动物伦理委员会批准并按照美国国立卫生研究院伦理学指南进行。小鼠随机分为两组，荷瘤小鼠右侧皮下注射1*106CT26肿瘤细胞，对照组同部位注等体积PBS。每3天记录小鼠体重、食物摄入量和肿瘤体积。分别于肿瘤植入后的第15、20、25、30、40、50天处死各组8只小鼠，检测恶病质病程的早期、中期和晚期。从CT26细胞植入后第20天气，每三天皮下注射含有12％DMSO的MDC(10mg/kg)或PBS作为对照。生存期实验从CT26细胞植入后第40天气，每三天腹腔注射PBS、Dkk1(0.25mg/kg)、Dkk1综合抗体(0.2gm/kg)、MDC(10mg/kg)或IGFBP-4/H95P(1.7mg/kg)，观察荷瘤小鼠生存期。在药物治疗前，随机对小鼠进行分析，以确保各组肿瘤平均体积相等。每4天用游标卡尺测量肿瘤大小、肿瘤体积的计算公式为V＝(L*W2)/2。用肿瘤密度乘以肿瘤体积估算肿瘤重量，肿瘤密度通过比较小鼠处死时肿瘤的试剂重量和肿瘤体积来确定。然后用小鼠体重减去肿瘤重量得到去瘤体重。实验终点牺牲小鼠，取材部位：股四头肌、肾脏和其他器官，同时称重。血液标本经过心脏穿刺放置于含edta的离心管中，2000rpm 4℃获得血浆样本，-80℃保存，待测。采用专为小鼠Dkk1涉及的ELISA试剂盒(R&D，USA)根据说明书测定血浆Dkk1水平。
4、肌肉注射Dkk1
野生型小鼠和β-arrestin2+/-KO小鼠左股四头肌注射(0.25mg/kg)、右腿注射等体积的PBS作为对照。与Dkk1联合注射MDC(10mg/kg)、IGFBP-4/H95P(1.7mg/kg)或PBS作为对照，观察药物对Dkk1诱导的信号转到 的影响。48小时后牺牲小鼠，取左右股四头肌，测定血浆细胞因子水平。
5、蛋白印记分析
膜蛋白是根据膜蛋白提取试剂盒(Sangon Biotech,China)说明书提取。Western blot分析略。用于WB分析的抗体购于Cell Signaling Technology和Proteintech.
6、Real-time PCR分析
总RNA按说明书提取分离(Takara,Japan).比较CT法计算相对mRNA水平，归一化至对照组。
检测引物如下:
Figure PCTCN2020086807-appb-000004
Figure PCTCN2020086807-appb-000005
7、RNA-seq分析
总RNA采用Trizol试剂盒(Lige technology)提取，RNA测序文库采用IIIumina文库准备协议从提取的RNA中构建，RNA-seq在lllumina HiSeq4000平台上进行，采用两端协议，都区长度为150bp。测序读取使用hisat2(v2.1.0)与带注释的小鼠转录本(mm10)对齐。使用GFOLD(v1.1.4)计算基因表达水平，发现差异表达基因，参数如下：GFOLD值≠0，并且两个样本的变化值≥1.35.RNA-seq数据采集仪GSE121873号上传到基因表达综合数据库中。对定义基因进行KEGG通路分析。将CT26肿瘤小鼠造模50天的股四头肌中的上调的DEGs或注射Dkk1蛋白24小时与对照组小鼠股四头肌中DEGs分别上调和下调，经过MDC治疗的小鼠肌肉有改善。
8、统计分析
采用Graphpad Prism软件进行统计分析。结果均已±标准差表示，使用Kolmogorov-Smirnov检验分析为转换数据的正态性，采用T检验比较两组之间 的差异；采用单因素方差分析比较三个火三个以上组件的差异，然后进行LSD-t事后分析。P＜0.05为有统计学差异。
糖尿病研究相关实验方法：
1、细胞的冻存，复苏，传代及分盘
当贴壁培养的细胞汇合度达到80％，即可进行冻存、传代或者分盘。一个10cm培养皿细胞可以冻存3管或传代3个10cm盘。细胞的冻存和复苏遵循“缓冻速溶”的原则，冻存时要梯度降温，复苏时要快速解冻。
A、细胞冻存：
(1)吸去10cm培养皿中的培养液，加入3ml PBS，轻轻摇晃培养皿。
(2)吸去PBS，加入1ml胰酶，轻轻摇晃培养皿，使胰酶完全覆盖贴壁细胞，将培养皿置于细胞培养箱中5min。
(3)取出培养皿，观察细胞是否完全变成球形或从壁上脱离，若已变圆，加入1ml完全培养基，终止胰酶消化，轻轻吹打细胞使其悬浮，收集细胞悬液到15ml离心管。
(4)室温，1000rpm，离心5min。
(5)配制冻存液(90％完全培养基+10％DMSO)，混匀后，静置冷却至室温。
(6)吸去离心后的细胞上清，先用1ml冻存液打匀细胞沉淀，再加入剩余冻存液，继续吹打混匀。
(7)将细胞悬液分装到冻存管中，1ml/管，迅速将其放入冻存盒，再放入-80℃冰箱，48h后冻存管转移到液氮罐中永久保存。
B、细胞复苏：
(1)从液氮罐中取出细胞，在37℃水浴锅中迅速解冻。
(2)室温，1000rpm，离心5min。
(3)吸去上清，加入1ml完全培养基，打匀细胞沉淀，再转移到10cm培养皿(含有7ml完全培养基)，在培养箱中前后左右摇晃混匀，静置培养。
C、细胞传代：
(1)先按照冻存的前4步进行操作。
(2)吸去离心后的细胞上清，先用1ml完全培养基打匀细胞沉淀，再加入适量培养基，继续吹打混匀。
(3)每个10cm培养皿(含有7ml完全培养基)加入1ml细胞悬液，在培 养箱中前后左右摇晃混匀，静置培养。
D、细胞分盘：
(1)先按照冻存的前4步进行操作。
(2)吸去上清，加入5ml完全培养基，吹打混匀。
(3)取100μl细胞悬液稀释10倍，再取20μl稀释液到血球计数板进行计数，数出四个大方格中的细胞数，计算平均值乘以105，得出原细胞悬液中每毫升的细胞数。
(4)每个3.5cm培养皿(含有2ml完全培养基)加入含有一定数量细胞的细胞悬液，在培养箱中混匀后，静置培养。
2、细胞的基因敲低
基因敲低(gene knockdown)是一种RNA干扰(RNA interference,RNAi)技术，不同于基因敲除(gene knockout)使目标基因永久性的表达沉默，它是通过双链小干扰RNA(small interfering RNA，siRNA)高效特异地降解细胞内具有同源序列的mRNA，从而阻断靶基因的表达，使细胞出现靶基因缺失的表型。
(1)先对3.5cm培养皿中已培养24h的HUVEC细胞换液，将完全培养基换成不含双抗、含10％FBS的培养基。
(2)一个3.5cm培养皿的转染体系配制如下：1μl siRNA Oligo/Red(原液浓度为20μM)加入含250μl纯Opti-MEM的EP管中并混匀，1.5μl RNAiMAX(转染试剂)加入含250μl纯Opti-MEM的流式管中并混匀，再将EP管中的液体全部加入流式管中并混匀，室温静置15min。
(3)将配好的转染体系加入3.5cm培养皿中，细胞继续培养24～48h，即可收样并抽提细胞总蛋白，通过蛋白免疫印迹(Western Blotting)鉴定基因的敲低效果。
3、RNA-Seq分析
使用标准Illumina文库制备方案从提取的RNA样本构建得到转录组测序文库。在Illumina HiSeq 2000测序平台上使用阅读长度为100bp的Pair-End方案对文库进行RNA-Seq分析。使用两个不匹配的Tophat-2.0.9将读取序列与已有的人转录组数据(hg19)进行比对。唯一的读取映射会保留在基因表达谱中。差异表达的基因通过工具Cuffdiff确定。对于每个转录本，通过计算每百万读取中来自于某基因每千个碱基长度的读取数(Reads Per Kilo bases per Million reads，RPKM)来评估基因的表达水平。
4、质粒的转化、扩增大抽和细胞转染
A、质粒的转化：
(1)从-80℃冰箱中取出感受态细菌在冰上融化。
(2)打开细菌专用超净操作台的紫外灯灭菌10min，打开水浴锅加热至42℃。
(3)标记若干灭菌EP管，每个EP管依次加入50μl感受态菌液和2.5μl质粒后轻轻打匀。
(4)将EP管放在冰上孵育25min，然后放入42℃水浴锅热激60s，再将其插入冰块90s。
(5)每个EP管加入1ml细菌培养基后放入37℃摇床，225rpm，摇45min。
(6)将制备好的细菌培养板(已灭菌，含Amp 1:1000)放入37℃培养箱预热。
(7)将灭菌枪头折弯，吸入菌液后均匀涂在细菌培养板上(每盘100μl)。
(8)待液体稍干后将细菌培养板倒置放入37℃培养箱中培养12～16h。
B、质粒的扩增与大抽：
(1)从细菌培养板中挑取中等大小，饱满，且没有和其他克隆接触的单克隆，接种到含3ml 2.5％LB细菌培养基(已灭菌，含Amp 1:1000)的15ml离心管中，盖子轻旋，放入37℃摇床，225rpm，摇14h。
(2)从小摇LB培养液中取1ml用作质粒小量抽提，并对小量抽提产物测序鉴定，选取阳性克隆的培养液，1:1000接种到含100～200ml 2.5％LB培养基(已灭菌，含Amp 1:1000)的大摇瓶中，放入37℃摇床，225rpm，摇14h。
(3)从大摇LB培养液中取500μl菌液和500μl 50％灭菌甘油混合，加入冻存管，记录好菌种信息，放入-20℃冰箱长期保存。
(4)将其余大摇LB培养液(菌液OD600一般在2.0～3.0之间)室温5000g，离心10min，收集菌体沉淀至灭菌50ml离心管中，并尽可能吸去上清。
(5)质粒大抽按照无内毒素质粒大提试剂盒(北京TIANGEN)的说明书在室温下进行操作。
(6)使用NanoDrop测定质粒DNA的浓度(μg/ml)和纯度(OD260/OD280)，纯度通常在1.7～1.9为佳。
C、质粒的细胞转染：
(1)先对3.5cm培养皿中已培养24h的AD293细胞换液，将完全培养基换成不含双抗、含10％FBS的培养基。
(2)一个3.5cm培养皿的转染体系配制如下：3μg质粒和9μl L-PEI(转染试剂，1μg/μl)依次加入到250μl纯Opti-MEM中并混匀，室温静置15min，然后加入到3.5cm培养皿中。
(3)转染4h后，进行换液(不含双抗、含10％FBS的培养基)，细胞继续培养24～48h，即可收样并抽提细胞总蛋白。对于表达分泌型蛋白的质粒，可以收取培养液上清，通过Western Blotting鉴定质粒的转染效果。
5、报告基因检测
报告基因检测(TOPFLASH Reporter Gene Assay)，是分析经典Wnt信号通路是否被激活及激活强度的常用方法，检测系统采用Promega公司的双萤光素酶报告基因检测系统(
Figure PCTCN2020086807-appb-000006
Reporter(DLRTM)Assay System)。具体步骤如下：
(1)试剂准备：1×PBS；1×PLB(用ddH2O将5×Passive Lysis Buffer(PLB)稀释后使用，母液-20℃储存，稀释后4℃储存)；LAR II Reagent(室温解冻10ml Luciferase Assay BufferⅡ，随后全部倒入底物冻干粉(Luciferase Substrate)中，混匀后尽快使用，避免反复冻融，96孔板每孔需100μl)；1×Stop&
Figure PCTCN2020086807-appb-000007
Reagent(用Stop&
Figure PCTCN2020086807-appb-000008
Buffer将50×Stop&
Figure PCTCN2020086807-appb-000009
Substrate溶解于棕色Stop&
Figure PCTCN2020086807-appb-000010
Reagent瓶中，混匀后尽快使用，避免反复冻融，96孔板每孔需100μl)。
(2)样品准备：使用两代以内的AD293细胞在48孔板中铺盘，做3个复孔，细胞数量为30000个/孔，培养基为300μl/孔，过夜贴壁后，我们以考察LRP6或β-catenin敲低是否会抑制Wnt3a激活的经典Wnt通路为例，进行如下操作。
先进行基因敲低，每孔的转染体系配制如下：0.4μl LRP6或β-catenin的siRNA Oligo(使用Annealing Buffer将0.4μl Oligo按1:1至1:32倍比稀释为6个浓度梯度)/Red(Red为对照)加入20μl纯Opti-MEM中并混匀，0.6μl RNAiMAX(转染试剂)加入20μl纯Opti-MEM中并混匀，将两者进一步混匀，室温静置15min，全部加入培养基中。
24h后进行质粒转染，每孔共转染质粒160ng，Fugene(转染试剂)0.48μl/孔，Opti-MEM 8μl/孔。160ng的质粒包含40ng SP-TOP(Tcf/Lef-luc)报告基因表达载体和1ng Renilla海肾萤光素酶表达载体，以及1ng Wnt3a质粒或GFP质粒(检测转染效率)，最后用Vector补齐至160ng，全部加入培 养基中，细胞继续培养24h后，进行细胞裂解：先去除48孔板中的培养基；用1×PBS清洗AD293细胞，去除清洗液；将1×PLB加入48孔板，65μl/孔，将细胞充分裂解混匀。
(3)上机操作和数据处理：检测用96孔板每孔内加入上述细胞裂解液20μl。通过Promega公司的
Figure PCTCN2020086807-appb-000011
96Microplate Luminometer和
Figure PCTCN2020086807-appb-000012
Luciferase Assay System依次得到萤火虫荧光素酶(Photinus Pyralis Luciferase)和海肾萤光素酶(Renilla Reniformis Luciferase)的表达情况。先将萤光素酶检测试剂II(Luciferase Assay Reagent II，LAR II)加入样品中，100μl/孔，萤火虫萤光素酶产生的光信号持续至少1min。定量萤火虫萤光强度后，再加入Stop&
Figure PCTCN2020086807-appb-000013
Reagent，100μl/孔，终止萤火虫萤光的同时启动海肾萤光素酶反应，在完成两种萤光素酶的检测后，根据海肾萤光素酶数据对萤火虫荧光素酶数据做归一化处理。
6、特定基因型的转基因小鼠
A、含UBC-Cre的转基因小鼠
通过将Cre重组酶基因与两个雌激素受体(estrogen receptor，ER)的配体结合区(ligand-binding domain，LBD)进行融合，产生一种嵌合重组酶(mER-Cre-mER)，该嵌合重组酶的表达受特异的泛素C(ubiquitin C，UBC)启动子的控制。泛素蛋白存在于所有真核细胞之中，因此，Cre重组酶能在全身的组织和器官表达。Cre重组酶能与loxP位点结合，有效切除两个loxP位点间的序列，实现基因敲除。但是嵌合的Cre不能自发地进入细胞核内发挥其活性，只有结合雌激素后才能入核。为避免内源性雌激素引起的非特异性基因敲除，对雌激素LBD的关键氨基酸进行突变，使其不能与体内的生理性雌激素结合，而只能与外源性的雌激素类似物他莫昔芬(Tamoxifen)结合，从而实现了Cre重组酶时间和空间的双重表达。以上就是UBC-Cre小鼠的转基因特点。
B、可诱导LRP5/6或β-catenin全身敲除的转基因小鼠
LRP5 floxp/floxp，LRP6 floxp/floxp和β-catenin floxp/floxp这三种纯合型小鼠的构建方法类似，例如，LRP5 floxp/floxp或LRP6 floxp/floxp纯合型小鼠，首先是通过基因打靶(gene targeting)和同源重组(homologous recombination)在基因组中LRP5或LRP6基因区域的两侧各插入1个loxP位点，筛选得到含有floxp-LRP5-floxp或floxp-LRP6-floxp位点的转基因小鼠，然后将其分别与野生型(wild-type，WT)小鼠交配及回交繁殖，最终得到LRP5 floxp/floxp或LRP6 floxp/floxp纯合型小鼠 [131]。同理，可 以获得β-catenin floxp/floxp纯合型小鼠 [134]。本研究中用到的LRP5 floxp/floxpLRP6 floxp/floxp转基因小鼠是将LRP5 floxp/floxp和LRP6 floxp/floxp小鼠继续进行交配繁殖而得。
接下来，如图20所示，我们通过LRP5 floxp/floxpLRP6 floxp/floxp纯合型小鼠与上述UBC-Cre小鼠进行配种繁殖产生可诱导LRP5和LRP6全身共敲除的转基因小鼠，利用Cre重组酶的特性，经他莫昔芬诱导后的小鼠基因型表示为LRP5/6 -/-(LRP5/6double knockout)。同理，也可以获得条件性β-catenin全身敲除的转基因小鼠，其敲除后的基因型表示为β-catenin -/-(β-catenin knockout)。本研究中，我们分别对UBC-Cre-LRP5/6 floxp/floxp纯合型小鼠、UBC-Cre-β-catenin floxp/floxp纯合型小鼠和Ubc-Cre阳性小鼠(对照组)腹腔注射1％他莫昔芬(30mg/kg/day)，连续注射5天，继续等待1周或4周后，Western Blotting检测小鼠心脏的LRP5/6或β-catenin的敲除效果，发现相较于对照组小鼠，LRP5/6 -/-小鼠的心脏LRP5/6和β-catenin -/-小鼠的心脏β-catenin的蛋白表达水平均显著降低(如图17(A)和图20所示)，表明他莫昔芬诱导作用是成功的，可以对LRP5/6 -/-和β-catenin -/-小鼠用做进一步的研究，如1型糖尿病造模。
C、可诱导LRP6N全身过表达的转基因小鼠
转座子系统是一种高整合效率、高目的基因表达概率、适合较大目的片段插入的转基因模式动物制作系统。转座子特有的“剪切和粘贴”机制，使DNA片段在载体和基因组之间能“自由”的转移，从而有效介导外源DNA片段对基因组的整合。转座时，转座酶有效地识别载体中目的片段两端的特异转座子序列(ITRs)，与转座子末端结合形成短暂的发夹结构，“剪切”后脱离载体，“粘贴”至基因组的特异性位点。具体地，将构建好的含有转座子系统和CAG promoter-floxp-stop codon-floxp-LRP6N-myc cDNA片段的基因载体和CMV promoter-transposase的表达载体通过显微注射技术，进行小鼠受精卵细胞核DNA显微注射，注射后的受精卵移植到假孕小鼠生殖系统中，使其怀孕并获得新生小鼠，采集尾尖，提取DNA，利用PCR进行鉴定，筛选制备出转座子介导的携带LRP6N基因片段的转基因小鼠(CAG-floxp-stop codon-floxp-LRP6N)。
CAG是一种高效的合成启动子，常用于哺乳动物的表达载体中驱动基因的高水平表达。CAG中含有CMV的早期增强子，β-actin的启动子及β-珠蛋白的剪接受体[138]。上述转基因小鼠的LRP6N在正常情况下并不表达，只有与相应的组织特 异性Cre表达阳性的小鼠杂交后，因Stop终止密码子(stop codon)在特定组织中被Cre删除，从而CAG的β-actin启动子能够让LRP6N基因片段在特定组织中实现表达。因此，如图21所示，我们将含有LRP6N基因的小鼠(CAG-floxp-stop codon-floxp-LRP6N)与上述UBC-Cre小鼠[133]交配，繁殖出他莫昔芬可诱导的全身过表达LRP6N的转基因小鼠，诱导后的小鼠表示为LRP6N/Tg。本研究中，我们对UBC-Cre-STOPfloxp/floxpLRP6N小鼠腹腔注射1％他莫昔芬(30mg/kg/day)，连续注射5天，继续等待4周后，Western Blotting检测发现，相较于UBC-Cre阳性小鼠(对照组)，LRP6N/Tg小鼠在心脏中有LRP6N蛋白的过表达(如图18(A，B)和图21所示)，表明他莫昔芬诱导作用是成功的，可以对LRP6N/Tg小鼠用做进一步的研究，如1型糖尿病造模。
D、Leptin基因敲除小鼠
Leptin基因敲除小鼠(Leptin knockout mice，Leptin -/-mice，ob/ob mice)是一种自发产生肥胖表型的纯合子小鼠，常作为研究2型糖尿病的动物模型，4周时可以看到肥胖表型，6周时出现显著高血糖(浓度高于13.8mM)和糖耐量受损症状。Leptin(瘦素)是由脂肪组织分泌的蛋白质类激素，参与调节体内的糖、脂肪及能量代谢，促使机体减少摄食，增加能量释放，抑制脂肪细胞的合成，进而使体重减轻 [139]。Leptin -/-小鼠由于缺乏瘦素配体，食欲旺盛，体重会迅速增加，导致脂肪沉积和病态肥胖，体重可达WT小鼠的三倍，且不受饮食限制的影响。除上述表现外，Leptin -/-小鼠还具有胰岛素抵抗，生殖力降低，代谢减退，创伤愈合能力受损及垂体和肾上腺激素水平升高等特点。由于Leptin -/-小鼠不育，故Leptin -/-小鼠均由Leptin +/-小鼠交配产生。
7、小鼠基因型鉴定
A、组织基因组DNA抽提：
(1)剪脚趾给小鼠标记序号，并将剪下的脚趾放入EP管，用于基因组DNA提取，每次剪完脚趾的剪刀要用酒精棉球擦拭干净，避免基因组的交叉污染。
(2)加入裂解液，50mM NaOH 200μl/管，完全覆盖样品，在金属浴锅100℃煮1h，30min时拿出来小心弹打，将组织和裂解液充分混合，使组织尽可能被煮烂并释放出基因组DNA，注意盖紧EP管盖子，防止裂解液蒸发。最后，检查脚趾是否被NaOH完全裂解，对于未充分裂解的组织，需适当延长裂解时间。
(3)溶解完全后，1000g，离心1min，加入Tris-HCl(pH 8.0)20μl/管以中和裂解液，Vortex震荡混匀后，进行基因型鉴定。
B、普通PCR反应：
(1)普通PCR反应体系配制如下：
Figure PCTCN2020086807-appb-000014
(2)将加好反应体系的8联管瞬时离心，放入PCR仪，按如下所示的不同转基因小鼠的PCR反应条件设置好反应程序，进行PCR扩增。
(2.1)UBC-Cre小鼠：
Figure PCTCN2020086807-appb-000015
(2.2)LRP5 floxp/floxp、LRP6 floxp/floxp或β-catenin floxp/floxp小鼠：
Figure PCTCN2020086807-appb-000016
Figure PCTCN2020086807-appb-000017
(2.3)STOP floxp/floxpLRP6N小鼠：
Figure PCTCN2020086807-appb-000018
(2.4)Leptin -/-小鼠：
Figure PCTCN2020086807-appb-000019
C、PCR反应产物琼脂糖核酸电泳，鉴定小鼠基因型：
(1)根据欲分离DNA片段大小用1×TAE缓冲液配制适宜浓度的琼脂糖溶液。在微波炉内加热熔化，冷却片刻，加入一滴荧光染料(Gel-Red)，旋转混匀后，倒入制胶平板中，室温下，待溶液凝固。
(2)完全凝固，小心拔出梳子，上样孔一侧靠近负极，放入电泳槽内。使电泳槽中的1×TAE缓冲液完全没过胶面，避免孔内产生气泡。向孔内点样，10μl/孔，根据被鉴定的DNA条带大小选择对应分子量范围的DNA Marker。
(3)打开电源，开始电泳。一般电压为60～100V，电泳20～40min即可。
(4)电泳完毕，关闭电源。在核酸成像仪上使电泳条带成像，根据DNA Marker判断条带大小，确定小鼠基因型。特定基因型小鼠的条带大小参见下表：
小鼠基因型 DNA条带大小
UBC-Cre 400bp
LRP5 floxp/floxp 288bp
LRP6 floxp/floxp 411bp
β-catenin floxp/floxp 400bp
STOP floxp/floxpLRP6N 835bp
Leptin -/- 184bp
8、二维超声心动图
本研究使用VisualSonics Vevo 2100成像系统进行二维超声心动图检测。提前对小鼠的左胸部进行脱毛，用1％异氟烷通过汽化器将小鼠麻醉，同时补给氧气。需检测的反映左心室功能的物理指标包括舒张末期左心室内径(left ventricular internal dimension in end-diastole，LVIDd)、舒张末期左心室后壁厚度(left ventricular posterior wall in end-diastole，LVPWd)、舒张末期室间隔厚度(interventricular septum in end-diastole，IVSd)(即在左心室体积最大期间测量)，收缩末期左心室内径(left ventricular internal dimension in end-systole，LVIDs)(即在左心室后壁收缩程度最大期间测量)。左心室容量的计算方法为Teichholtz校正公式：V＝[7.0/(2.4+D)]×D 3，计算舒张末期左心室容积(left ventricular end-diastolic volume，LVEDV)的D代表LVIDd，计算收缩末期左心室容积(left ventricular end-systolic volume，LVESV)的D代表LVIDs。左心室射血分数(left ventricular ejection fraction，LVEF)的计算公式为：EF＝(LVEDV-LVESV)/LVEDV×100％，左心室短轴缩短率(left ventricular fractional shortening，LVFS)的计算公式为：FS＝(LVIDd-LVIDs)/LVIDd×100％。
9、1型糖尿病小鼠造模
STZ诱导糖尿病发病是一种化学性糖尿病造模的方法，其全称为链脲佐菌素(Streptozotocin)，是一种从无色链霉素分离出来的广谱抗生素，它能够在短时间内选择性损伤胰岛β细胞，引起β细胞坏死，导致血胰岛素不同程度下降伴血糖升高，形成胰岛素依赖型的1型糖尿病。和其他化学性糖尿病造模方法如四氧嘧啶(Alloxan，ALX)造模相比，STZ引起的糖尿病高血糖反应及酮症均较缓和，所致糖尿病模型也较稳定。本研究中，我们对小鼠腹腔注射STZ诱导建立1型糖尿病模型，使用电子秤测量小鼠体重，使用便携式血糖仪从小鼠尾端采血，测量其血糖浓度。成模标准是非空腹血糖值高于13.8mM(250mg/dl)。造模步骤如下：
(1)将8周龄野生型雄性小鼠按照随机化原则分组，进行脚趾标记，测量体重和随机血糖。
(2)造模前，对小鼠禁食12h，但不禁水。
(3)配制2％STZ-柠檬酸溶液，即2g STZ粉末溶解于100ml柠檬酸缓冲液(0.05M，pH 4.5)，混匀成为淡黄色透明液体。STZ在室温下极不稳定，易升华，操作应在冰上进行，所配溶液需注意避光，并及时使用。
(4)按照所称体重，造模组小鼠一次性腹腔注射2％STZ-柠檬酸溶液(160mg/kg)，对照组小鼠一次性腹腔注射相应体积的柠檬酸缓冲液，注射完成2h后，恢复小鼠进食。
(5)在注射后的不同时间点测量体重和随机血糖，并将小鼠麻醉，取材。
10、葡萄糖耐量测试
葡萄糖耐量测试(glucose tolerance test，GTT)是显示机体对外源葡萄糖从血液中清除效率的一个标准测试，用以评估体内糖代谢平衡的维持情况。细胞从血液中吸收葡萄糖受到胰岛素的调节，胰岛素抵抗会导致糖耐量异常，即相比正常机体，需要更长时间来清除葡萄糖含量。本研究中，Leptin -/-和WT小鼠从4周龄开始，连续每周同一时间测量其体重和空腹6h血糖。根据Leptin -/-小鼠的体重和血糖变化，我们选取6周龄Leptin -/-小鼠(实验组)和WT小鼠(对照组)进行葡萄糖耐量测试。具体步骤如下：
(1)对小鼠禁食12h(不禁水)，称量体重，尾端采血测定基础血糖水平。
(2)用灭菌PBS配成20％和30％D-葡萄糖溶液，并用0.45μm滤器过滤。
(3)按照所称体重，用20％D-葡萄糖溶液对WT小鼠进行腹腔注射，用30％D-葡萄糖溶液对Leptin -/-小鼠进行腹腔注射，剂量均为2g/kg。
(4)在注射后的不同时间点(15、30、60、90、120、240min)，尾端采血测量小鼠血糖浓度。
11、胰岛素体内拯救
胰岛素体内拯救实验是对STZ诱导1周的糖尿病小鼠皮下注射长效甘精胰岛素(Insulin Glargine)或相应体积的生理盐水(对照组)。具体步骤如下：
(1)取出胰岛素注射笔，室温放置3h，恢复胰岛素生物活性。连接好注射笔专用针头，小心挤出10μl胰岛素液体(最小单位剂量体积)。用移液枪小心吸出，分装在EP管，2μl/管。使用前，每个EP管加入998μl灭菌生理盐水，将胰岛素浓度稀释为0.0002U/μl(原液浓度为0.1U/μl)，配好后应及时使用。
(2)将STZ造模1周的小鼠按照随机化原则分组，测量体重和随机血糖。
(3)按照所称体重，胰岛素拯救组皮下注射稀释好的胰岛素(1U/kg/day)，每天同一时间注射一次，连续注射7天，对照组为相应体积的生理盐水。
(4)在胰岛素干预的不同时间点测量体重和随机血糖，并将小鼠麻醉，取材。
12、MDC体内拯救
STZ糖尿病小鼠的MDC体内拯救实验是对STZ诱导3天的糖尿病小鼠腹腔注射MDC(灭菌PBS稀释，10mg/kg/day)，每天同一时间注射一次，注射相应体积DMSO(灭菌PBS稀释)的糖尿病小鼠作为对照组。在注射后的不同时间点测量体重和随机血糖，待MDC连续干预4天后，将小鼠麻醉，取材。
Leptin -/-小鼠的MDC体内拯救实验是预先对6周龄Leptin -/-小鼠腹腔注射MDC(灭菌PBS稀释，10mg/kg)或者相应体积的DMSO(灭菌PBS稀释)，Leptin -/-小鼠MDC的注射剂量应根据6周龄同窝出生的WT小鼠的体重计算而得。MDC干预1h后，腹腔注射50％D-葡萄糖或者相应体积的PBS。Leptin -/-小鼠注射D-葡萄糖的程序是，24h期间每隔2h注射一次，共计12次，每次D-葡萄糖的注射剂量固定为首次使用剂量(2g/kg)。注射结束，将小鼠麻醉，取材。
Dkk1纯化蛋白局部注射心脏合并MDC干预的实验，选取10周龄野生型雄性小鼠，对其左胸部进行脱毛，并腹腔注射MDC(灭菌PBS稀释，10mg/kg)或者相应体积的DMSO(灭菌PBS稀释)，以后每天同一时间注射一次。MDC干预1h后，进行心脏Dkk1蛋白注射。先使用1％异氟烷给小鼠持续吸入麻醉，并使用呼吸机供氧，然后打开小鼠近胸骨端第四肋间隙，暴露出心脏。使用200μl微量注射器，在心脏的三个位点局部注射Dkk1(每颗心脏Dkk1用量为10μg，溶解于30μl PBS)或者等体积PBS。其中，两个位点在左心室周围的边界，一个位点进入左心室的心肌。注射结束，缝合伤口，撤去麻醉，等待小鼠苏醒。待MDC连续干预2天后，将小鼠麻醉，取材。
除了给心脏单独注射Dkk1蛋白外，我们还需要进行Dkk1联合BIM-46187局部注射小鼠心脏的实验。根据实验分组，分为四种不同溶液的心脏注射，包括PBS，Dkk1，BIM-46187以及Dkk1与BIM-46187的混合液。每颗心脏Dkk1用量为10μg，BIM-46187用量为2.4μg，使用30μl PBS稀释后进行心脏注射，具体操作步骤同上。注射结束，缝合伤口，撤去麻醉，等待小鼠苏醒。待注射2天后，将小鼠麻醉，取材。
13、LRP6N质粒尾静脉注射
本研究中使用的LRP6N质粒(带myc标签)需要事先进行鉴定，即通过体外转染AD293细胞，随后收取细胞的上清培养液，使用Western Blotting检测证明LRP6N质粒能够表达出分泌型LRP6N蛋白之后，再进行以下质粒尾静脉注射的操作。试剂准备：①Hepes溶液(50mM，pH 7.4，0.22μm过滤)；②D-葡萄糖溶液(50％，0.22μm过滤)；③L-PEI溶液(1mg/ml，pH 5.0，0.22μm过滤)。具体方法：先将50μg LRP6N或Vector质粒溶于100μl含有5％D-葡萄糖的Hepes溶液中，室温静置10min。再缓慢加入65μl L-PEI到上述溶液中，迅速混匀。室温孵育10min，将混合物通过尾静脉注射到小鼠体内。前期我们发现，小鼠尾静脉注射荧光素酶(luciferase)质粒1天后，再经尾静脉给予荧光素底物(luciferin)，通过小动物活体成像仪观察全身的荧光素酶活性，其在肺脏、肝脏及脾脏处均有表达，且肺部表达水平最高。因此，我们选取肺脏来观察LRP6N质粒在尾静脉注射后不同时间的表达情况。Western Blotting证明LRP6N质粒表达的分泌型LRP6N蛋白能在体内持续表达至少6天。LRP6N质粒尾静脉注射应在STZ诱导1天后进行，在STZ造模7天后，测量体重和随机血糖，并将小鼠麻醉，取材。
14、小鼠取材
(1)2％戊巴比妥钠腹腔注射将小鼠麻醉(45mg/kg)。
(2)腹部朝上四肢固定在操作台，剑突下横向剪开上腹部，暴露肝脏，用止血钳提起剑突，剪开膈肌进入胸腔，沿剑突两侧剪断肋骨，向头侧掀开，暴露心脏。
(3)使用1ml注射器快速穿刺右心耳抽取适量血液放入EDTA抗凝管中。
(4)找到心尖部并用镊子提起，将灌流针刺入心尖部，用止血钳固定针尖，剪开右心耳，开始心脏灌流。
(5)生理盐水灌注：迅速将灌注泵的导管放入生理盐水中，开始灌流，直到从右心耳流出的液体为无色，同时小鼠肝脏色淡，四肢发白，肠管肿胀为止。
(6)多聚甲醛灌注：接着将灌注泵的导管小心移至4％多聚甲醛中，开始灌流。如看到小鼠四肢突然紧张，尾部卷曲，组织器官逐渐变硬，即达到固定效果。
(7)取材并进行器官称重。进一步处理：对于蛋白或RNA抽提的组织样本，生理盐水灌注完毕后，液氮迅速冷冻，-80℃保存；对于制作冰冻切片的组织样本，生理盐水灌注完毕后，吸干附着在样本上的液体，直接用OCT包埋好，再用液氮迅 速冷冻，-80℃保存；对于制作石蜡切片的组织样本，4％多聚甲醛灌注完毕后，再取下标本放入4％多聚甲醛中，4℃摇床上继续固定24h，用于后续的石蜡包埋和切片染色。
15、细胞和组织的蛋白抽提
细胞和组织的总蛋白抽提
(1)按下表配制总蛋白抽提的裂解液，配好混匀后放在冰上：
成分 用量(1000μl体系)
RIPA(2×) 500μl
蛋白酶抑制剂(100×) 10μl
NaF(0.5M) 100μl
β-Glycerophosphate(0.5M) 40μl
Na 3VO 4(0.2M) 5μl
ddH 2O 345μl
(2)3.5cm培养皿中的贴壁细胞汇合度达到90％可用于总蛋白抽提。先用吸管吸掉培养液，再用PBS洗两遍，吸干PBS残液后，置于冰上。
(3)每个细胞样品加入150μl总蛋白裂解液，使裂解液和细胞充分接触，用刮子刮下细胞裂解液收集到预冷EP管中，冰浴15min。
(4)每100mg组织加入500μl预冷灭菌PBS(含终浓度为1mM的蛋白酶和磷酸化酶抑制剂)，在PBS中尽可能将组织块剪碎。4℃，12000rpm，5min，弃PBS上清，保留沉淀。再向每个样品加入150μl总蛋白裂解液，使用匀浆器在冰上将组织沉淀充分匀浆，冰浴15min。
(5)4℃，14000g，10min，小心收集总蛋白上清液到新的EP管，-80℃保存备用。
细胞和组织的膜蛋白抽提
(1)分别向膜蛋白抽提A液和B液加入PMSF(终浓度为1mM)。
(2)3.5cm培养皿中的贴壁细胞汇合度达到90％可用于膜蛋白抽提。先用吸管吸掉培养液，再用PBS洗两遍，吸干PBS残液后，置于冰上。
(3)每个样品加入500μl预冷灭菌PBS(含终浓度为1mM的PMSF)，用刮子刮下细胞收集到预冷EP管中。在PBS中尽可能将组织块剪碎。
(4)4℃，12000rpm，5min，弃PBS上清，保留沉淀。
(5)每个样品加入100μl(细胞)或200μl(组织)A液，用移液枪将细胞沉淀重悬，使沉淀完全散开。组织沉淀需用匀浆器在冰上将其充分匀浆，冰浴15min。
(6)先用液氮将样品迅速冻结，然后放入37℃水浴锅，当样品融化到几乎没有冰晶时取出，反复冻融3～5次，勿使样品融化过度，结束后放回冰上。
(7)最高速Vortex 5s，4℃，700g，10min，小心收集上清液到新的EP管，勿触及沉淀。
(8)步骤“7”的上清液继续离心，4℃，14000g，30min，小心收集上清液到新的EP管，勿触及沉淀，抽提得到浆蛋白，-80℃保存备用。
(9)4℃，14000g，1min，尽可能吸尽上清，保证膜蛋白不被混入浆蛋白成分。
(10)每个样品加入50μl(细胞)或100μl(组织)B液，最高速Vortex 10s重悬沉淀，冰浴10min，重复2次，以充分抽提膜蛋白。
(11)4℃，14000g，5min，小心收集上清液到新的EP管，抽提得到膜蛋白，-80℃保存备用。
细胞和组织的核蛋白抽提
(1)分别向浆蛋白抽提A液和核蛋白抽提液加入PMSF(终浓度为1mM)。
(2)3.5cm培养皿中的贴壁细胞汇合度达到90％可用于核蛋白抽提。先用吸管吸掉培养液，再用PBS洗两遍，吸干PBS残液后，置于冰上。
(3)每个样品加入500μl预冷灭菌PBS(含终浓度为1mM的PMSF)，用刮子刮下细胞收集到预冷EP管中。在PBS中尽可能将组织块剪碎。
(4)4℃，12000rpm，5min，弃PBS上清，保留沉淀。
(5)每个细胞样品：加入200μl A液。每个组织样品：A液和B液按体积比20:1混匀(含终浓度为1mM的PMSF)配成200μl组织匀浆液加入组织中。
(6)最高速Vortex 5s，使沉淀完全散开。组织沉淀需用匀浆器在冰上将其充分匀浆，冰浴15min。
(7)组织：4℃，1500g，5min。吸上清到预冷EP管，抽提得到部分浆蛋白。组织沉淀接着从步骤“5”开始按照细胞的处理方法进行浆、核蛋白的抽提。
(8)每个样品加入10μl B液。最高速Vortex 5s，冰浴1min。
(9)最高速Vortex 5s，4℃，14000g，5min。
(10)吸上清到预冷EP管，勿触及沉淀，抽提得到浆蛋白，-80℃保存备用。组织抽提得到的浆蛋白可以和步骤“7”中的浆蛋白合并。
(11)完全吸尽残余上清，每个样品加入50μl核蛋白抽提液。
(12)最高速Vortex 30s，使沉淀完全散开，放回冰上，每隔2min再高速Vortex 30s，共持续30min。
(13)4℃，14000g，10min。
(14)吸上清到预冷EP管，抽提得到核蛋白，-80℃保存备用。
蛋白浓度的测定与校准
A、蛋白浓度的测定采用BCA法，具体步骤如下：
(1)冰上溶解混匀待测蛋白样品，96孔板每孔先加入18μl ddH 2O，再加入2μl样品，空白孔只加入20μl ddH 2O。每个样品做2个复孔。
(2)蛋白浓度测定A液和B液按体积比50:1配制蛋白浓度测定液，200μl/孔，将96孔板轻轻摇动混匀，放入37℃恒温箱孵育30min。
(3)取出96孔板，以空白孔调零，在562nm波长读取各孔的OD值。
B、蛋白浓度的校准采用相对定量法，具体步骤如下：
(1)OD值最小的样品体积(aμl)作为校准体积，其他样品的配平体积为[最小OD值×aμl]/其他样品OD值，再用ddH 2O补齐其他样品体积至校准体积(aμl)，从而将每个样品的OD值都调至最小OD值样品的水平。
(2)每个样品中加入0.25倍蛋白液体积的5×上样缓冲液并混匀，100℃金属浴锅中煮5min，短暂离心后，-80℃保存备用。
16、蛋白免疫印迹
A、配制SDS-PAGE凝胶：
蛋白电泳凝胶为双层胶，包括上层的浓缩胶和下层的分离胶。浓缩胶浓度为5％，分离胶浓度根据目的蛋白分子量的大小确定。本研究中大分子量蛋白(LRP5/6，β-catenin，β-arrestin1/2)使用8％-5％双层胶，小分子量蛋白(γH2AX，p53，p21，Bcl-2，Bax，Cleaved Caspase-3)使用12％-5％双层胶。另外，根据蛋白样品的上样数量和体积选择配制凝胶的孔数(10孔或15孔)和厚度(1mm或1.5mm)。凝胶配方见实验材料部分，配制步骤如下：
(1)用自来水将玻璃板冲洗干净，烘箱烘干后，放入配胶架，防止底部漏液。
(2)先配制分离胶，依次加入ddH 2O、30％Acrylamide、Tris-HCl、10％SDS、10％APS、TEMED，混匀后进行注胶，每块胶用量7.5ml，避免产生气泡，最后再加正丁醇压平液面，让其凝固30min。
(3)再配制浓缩胶(方法同分离胶)，每块胶用量2ml，避免产生气泡，迅速插 入干净的成孔梳子，让其凝固30min。
B、蛋白电泳：
通过蛋白分子量及电荷量的差异，导致不同的蛋白分子在电泳凝胶内的移动速度不同，从而使各种蛋白分子在凝胶中区分开来。具体步骤如下：
(1)将配制好的凝胶夹入夹子，放入电泳槽中，倒入1×电泳缓冲液，完全浸没凝胶的上样孔，轻轻取下成孔梳子，避免胶孔变形。
(2)冰上溶解混匀蛋白样品，100℃金属浴锅中煮5min，短暂离心后放回冰上，准备上样。
(3)上样：将所有蛋白样品按记录好的上样顺序排好，移液枪吸取等量样品加入胶孔中。上样时，枪头贴着较高侧玻璃，沿孔道所在位置尽量放低，遇到阻力时即可滴加，由于样品含上样缓冲液，故样品液可以自行沉入孔底；每完成一次上样，为避免交叉污染，需更换新枪头进行下一次上样；上样量要适中，10孔胶每孔最多40μl，15孔胶每孔最多20μl。上样结束后，在上样两侧的孔内各加入适量预染蛋白Marker(左侧8μl，右侧4μl)，通过Marker颜色的深浅方便分辨上样的左右顺序。
(4)电泳：盖上电泳槽盖子，接通电源，室温下开始电泳，设置电泳条件：先80V，30min，待样品跑下浓缩胶并压成一条水平直线后(通过溴酚蓝来判断)，再120V，90min，使Marker中不同分子量的染料在凝胶中分离开，根据Marker的分子量大小，判断不同大小的蛋白分子在凝胶中是否充分分离开，适时终止电泳，准备电转。
C、凝胶电转：
大于20kDa的目的蛋白转膜选用0.45μm的PVDF膜，小于20kDa则选用0.2μm的PVDF膜。PVDF膜需用甲醇处理，以活化膜上的正电基团，使其更容易与带负电的蛋白结合。具体步骤如下：
(1)预冷的1×电转缓冲液倒入电转槽中，转膜夹板及其内容物浸泡在1×电转缓冲液中备用。
(2)按照凝胶大小(5.5×8.5cm 2)准备好PVDF膜，剪角做好标记，以区分膜的正反面，在甲醇中活化1min后，放入1×电转缓冲液中备用。
(3)电泳结束后，胶板用水冲洗干净，并用刮板撬开，取下凝胶，打开转膜夹板，按照海绵衬垫→滤纸→凝胶→PVDF膜→滤纸→海绵衬垫的排列顺序，将内容物放入转膜夹板黑面的正中位置上，并轻轻压平，合上转膜夹板的白面。插入电转 槽(黑面对黑面，白面对红面)，补满电转缓冲液。转膜过程中，应避免PVDF膜和凝胶之间产生气泡，可用刮板轻轻赶走间隙中的微小气泡。
(4)电转：电转槽放入冰块中，盖上电转槽盖子，接通电源开始电转，设置电转条件：100V，120min。电转过程中，如发现电流过大或电转缓冲液过热，应及时更换新预冷的电转缓冲液，防止因过热导致转膜失败。
D、封闭：
封闭液(5％脱脂牛奶)能够和PVDF膜上的非特异性结合位点结合，降低PVDF膜的背景，确保一抗与目的蛋白的特异性结合。具体步骤如下：
(1)电转结束后，打开转膜夹板，用镊子取出PVDF膜，迅速放入封闭液中，室温下，摇床缓慢摇动1h。
(2)洗膜。倒掉封闭液，PVDF膜放在摇床上，用TBST洗三次，每次5min，洗好后准备一抗孵育。
E、抗体(一抗和二抗)孵育：
通过孵育一抗，PVDF膜上的蛋白质或多肽与对应的一抗起特异性免疫反应，然后通过孵育二抗(辣根过氧化物酶(HRP)标记)，使一抗与二抗相结合，经过曝光液底物与二抗连接的HRP发生级联放大反应，显示出能被检测到的化学发光的蛋白条带，从而能够判断特异性目的蛋白的表达水平。具体步骤如下：
(1)一抗的稀释：一抗使用前先要短暂离心，用一抗稀释液按抗体说明书推荐的倍数稀释一抗原液。
(2)一抗的孵育：根据Marker的大小，将PVDF膜剪出包含所孵育目的蛋白分布的相应部位，不同的目的蛋白要使用其对应的特异性一抗进行孵育。孵育时间取决于抗体与蛋白的亲和性及蛋白的含量丰度。将稀释好的一抗和PVDF膜(有蛋白分布的一面朝上)一起放在孵育盒内，使膜均匀覆没，让一抗与膜能够均匀结合。4℃，缓慢摇动摇床，进行过夜孵育。亲和性或者浓度较低的蛋白样品，建议使用较高的抗体稀释浓度和较长的孵育时间(一般不超过18h)来保证其特异性结合。
(3)洗膜：回收孵育后的一抗到离心管(一般重复利用三次)，4℃保存。PVDF膜放在摇床上，用TBST洗三次，每次5min，洗好后准备二抗孵育。
(4)二抗的稀释：二抗使用前先要短暂离心，用TBST按抗体说明书推荐的倍数稀释二抗原液。
(5)二抗的孵育：将稀释好的二抗和PVDF膜(有蛋白分布的一面朝上)一起放在孵育盒内，室温下，缓慢摇动摇床，孵育1～2h。
(6)洗膜。倒掉二抗(不回收)，PVDF膜放在摇床上，用TBST洗三次，每次5min，洗好后准备曝光显影。
F、曝光显影：
蛋白条带的显影采用化学发光法。二抗连接的HRP是一种高灵敏度酶，可以催化ECL曝光液底物产生一种发光物质，曝光机能将发光信号转化为图片上的条带灰度信号，条带灰度能够反映目的蛋白的表达水平。具体步骤如下：
(1)配制曝光液：将ECL曝光A液和B液等体积混匀，并用锡箔纸避光。
(2)将PVDF膜上的TBST用纸吸干，平放在一层透明薄膜上(有蛋白分布的一面朝上)，将曝光液均匀敷在膜上，反应1min左右，吸走曝光液，将薄膜放置在曝光机的镜头正下方，调整好视野，使用Quantity One 4.4软件对PVDF膜进行曝光，目的蛋白的曝光条件会因抗体效价不同而各异，需要自行摸索最佳的参数。最后将曝光数据导出TIFF图片格式保存，必要时可使用Image-Pro Plus 6.0软件进行蛋白条带的灰度值定量分析。
17、总RNA抽提和实时定量PCR
A、总RNA抽提：
样品收集之后，最好立刻进行总RNA制备。若不能及时抽提，应使用液氮将其冷冻后，-80℃保存。在组织RNA抽提时，应以液氮研磨的方式迅速破碎样品，勿先行解冻，以免内源性RNA酶引起RNA降解。此外，为防止RNA在提取过程中被外源性RNA酶降解，注意使用去RNA酶的枪头和EP管；研磨工具需要高温高压灭菌后使用；戴好一次性口罩和手套；操作时避免讲话等。
(1)每50～100mg组织加入1ml Trizol对液氮研磨后的样品进行裂解，用移液枪反复吹打以裂解组织样品。
(2)将Trizol裂解液转入EP管，室温放置5min。
(3)每1ml Trizol加入0.2ml三氯甲烷，盖上EP管盖子，在手中用力震荡15s，室温放置3min后，12000g，4℃，15min。
(4)取上层水相置于新的EP管中，每1ml Trizol加入0.5ml异丙醇，上下颠倒混匀，室温放置10min，12000g，4℃，10min，弃上清，保留沉淀。
(5)每1ml Trizol加入1ml用DEPC水配制的75％乙醇进行洗涤，涡旋混合，7500g，4℃，5min，弃上清，保留沉淀。同样操作再洗一次，共两次。
(6)让沉淀的RNA在室温下自然干燥。
(7)加入50μl DEPC水，冰上放置5～10min，使沉淀溶解，抽提得到RNA。
(8)使用NanoDrop测定RNA样品的浓度(μg/ml)和纯度(OD260/OD280)，纯度通常在1.8～2.0为佳。
B、实时定量PCR：
(1)去除RNA样品中的基因组DNA(gDNA)。按下表冰上配制反应液，按反应数+2的量配制Master Mix，再分装到每个反应管中，最后加入RNA样品。反应液在42℃，反应2min，结束后，冰上保存，得到的去gDNA的RNA用于反转录。
Figure PCTCN2020086807-appb-000020
(2)反转录反应。按下表冰上配制反应液，按反应数+2的量配制Master Mix，再分装到每个反应管中，轻柔混匀。反应液在37℃，反应15min；85℃，反应5s，结束后，冰上保存，得到的cDNA模板用于SYBR Green qPCR反应。
Figure PCTCN2020086807-appb-000021
(3)制备PCR反应体系。按下表冰上配制反应液，建议20μl反应液中cDNA模板用量所相当于的RNA样品不要超过100ng。
Figure PCTCN2020086807-appb-000022
(4)上机检测和结果分析
检测某一基因的样品需要做3个复孔，以排除加样误差。加好的96/384孔板用透明贴膜密封，并简短离心，离下管壁的残液。放入PCR仪，设置反应条件：Step1：预变性(95℃30s)；Step 2：PCR反应(95℃5s，60℃34s)40×循环。在60℃时，设定荧光检测点，进行扩增。必要时，反应结束后可加做溶解曲线，以检测扩增产物的特异性。最终得到的CT值，通过2 -ΔΔCT法计算出某一基因在不同样本中的表达情况。
18、组织冰冻切片和免疫荧光染色
(1)从-80℃冰箱取出OCT包埋块，用安全刀片将标本底部修平后迅速粘附于圆形支撑器上，置入-24℃冰冻切片机的冷冻台。待包埋块完全粘附于支撑器后，固定在标本台上，先快速切掉不要的OCT，直到暴露出待切组织块部位。
(2)调整切片厚度6μm，切好后室温放置30min以防脱片，-20℃保存备用。
(3)从-20℃冰箱取出冰冻切片，先室温放置30min，再用预冷的4％多聚甲醛室温固定20min，PBS洗3×10min，用纸吸干样本周围液体。
(4)0.2％Triton X-100(PBS稀释)通透细胞膜10min，PBS洗3×10min。
(5)免疫组化笔画圈，5％BSA室温封闭1h。
(6)滴加一抗(1％BSA稀释)，封口膜均匀覆盖，放在湿盒中防止表面干燥，4℃孵育过夜。
(7)PBST洗3×10min，用纸吸干样本周围液体。
(8)滴加Cy3荧光二抗(1％BSA稀释)，封口膜均匀覆盖，室温避光孵育1h。
(9)PBST洗3×10min，注意避光，用纸吸干样本周围液体。
(10)DAPI染色(2μg/ml，PBS稀释)10min，注意避光。
(11)PBST洗3×10min，注意避光，用纸吸干样本周围液体。
(12)滴加防荧光淬灭剂，封片。
(13)激光扫描共聚焦显微镜拍照成像(63×油镜)，能够看到Cy3标记的红色荧光(目的蛋白)和DAPI标记的蓝色荧光(细胞核)。
19、组织石蜡切片和HE染色
A、组织石蜡切片的制作：
(1)取出4％多聚甲醛固定24h后的组织标本，用大头针将标签和组织连接起 来，流水过夜冲洗去除固定液后，用ddH 2O配制的梯度乙醇进行脱水处理。
(2)脱水：70％乙醇1h→80％乙醇1h→90％乙醇1h→95％乙醇1h(×2)→100％乙醇1h(×2)。
(3)二甲苯透明30min(×2)。
(4)浸蜡：65℃融化好的石蜡浸3～4h。
(5)包埋：将组织放在自制包埋盒中，加入融化的石蜡，用大头针调整好组织的位置，小心将包埋盒放在-20℃冷却台上，待大部分石蜡凝固后，将标签贴在石蜡块上面，标记好组织标本，10min后撬蜡。
(6)切片：用安全刀片修剪石蜡块，固定在石蜡切片机上，调整切片厚度6μm，用毛笔将组织片摊平，转移到40℃水浴锅，再用防脱载玻片将组织片从水浴锅中捞出。
(7)将载玻片放到37℃烤片机上烤过夜，室温保存。
B、HE染色：
(1)将上述载玻片65℃烤片5min，开始脱蜡：二甲苯10min→二甲苯5min。
(2)水化：100％乙醇5min(×2)→95％乙醇5min(×2)→85％乙醇3min→75％乙醇2min→ddH 2O 1min。
(3)苏木素染5min。水洗15min。
(4)1％盐酸酒精分化30s。水洗15min。
(5)ddH 2O 2min→75％乙醇2min→85％乙醇2min。
(6)伊红染15s。
(7)脱水：95％乙醇5min(×2)→100％乙醇5min(×2)。
(8)二甲苯透明5min(×2)。
(9)封片：在载玻片中间滴一滴中性树胶，将盖玻片放在中性树胶上，缓慢按压使中性树胶充满盖玻片覆盖的整个空间，避免产生气泡。24h后用二甲苯擦去表面的中性树胶。通风橱中放置24h，使二甲苯充分挥发。
(10)光学显微镜下分别使用低倍(12.5×)和高倍(400×)观察拍照。
20、血浆Dkk1的测定(ELISA法)
(1)样品准备：将新鲜取得的小鼠血液存放在EDTA抗凝管中，室温静置10min后，1000g，离心15min，收集上清液，即血浆。如果不能立即检测，应分装后，-80℃保存，避免反复冷冻。保存过程中如出现沉淀，使用时应在室温下充分解冻， 并再次离心。
(2)所有试剂室温恢复30min并充分混匀。链霉亲和素-HRP(1:200)，生物素标记的山羊抗小鼠Dkk1检测抗体(1:180)，重组小鼠Dkk1标准品(1:100)，血浆样品(1:4)先使用试剂稀释液(reagent diluent)稀释到指定浓度；山羊抗小鼠Dkk1包被抗体(1:180)先使用包被缓冲液(coating buffer)稀释到指定浓度。稀释好的试剂用于以下各步骤。
(3)标准品：使用试剂稀释液将标准品倍比稀释为1350、675、337.5、168.8、84.4、42.2、21.1pg/ml等7个浓度梯度。
(4)上样板包被：立即将Dkk1包被抗体按每孔100μl加入96孔上样板中进行抗体包被，用封板膜密封好，室温下孵育过夜。
(5)洗板：小心揭掉封板膜，弃去液体并甩干，用稀释后的洗涤液注满每孔，静置30s弃去，用纸彻底拍干，充分清洗上样板3次。
(6)封闭：每孔加入300μl试剂稀释液(reagent diluent),室温下孵育1h。
(7)重复步骤“4”。
(8)上样：每孔加入100μl ddH 2O(空白孔)、Dkk1标准品或血浆样品，盖好上样板，室温下孵育2h。
(9)重复步骤“4”。
(10)检测：每孔加入100μl Dkk1检测抗体，盖好上样板，室温下孵育2h。
(11)重复步骤“4”。
(12)每孔加入100μl链霉亲和素-HRP，盖好上样板，室温下避光孵育20min。
(13)重复步骤“4”。
(14)每孔加入100μl显色液(底物A和底物B 1:1混匀，15min内使用)，盖好上样板，室温下避光孵育20min。
(15)每孔加入50μl终止液，轻轻摇晃上样板使液体混匀。
(16)读板：以空白孔调零，在450nm波长读取各孔的OD值。
(17)统计：根据标准品的浓度(横坐标)及其OD平均值(纵坐标)，生成标准曲线和线性回归方程，由公式计算出上样微孔中的Dkk1浓度(pg/ml)，再乘以稀释倍数得到Dkk1在血浆中的实际浓度(pg/ml)。
21、血浆8-OHdG的测定(ELISA法)
(1)样品准备同血浆Dkk1的测定(ELISA法)。
(2)分组：试剂盒室温放置30min。取出酶标板，根据标准品和血浆样品的数量确定所需板条数。分别设置空白孔(Blank)、非特异结合检测孔(Non-Specific Binding，NSB)、标准品/血浆样品组。为减少实验误差，设置2个复孔。
(3)标准品：使用标准品稀释液将标准品倍比稀释为20、10、5、2.5、1.25、0.625、0.313、0.157ng/ml等8个浓度梯度。
(4)加样：按下表在不同设置的微孔中加入所需试剂：
Figure PCTCN2020086807-appb-000023
(5)孵育：酶标板用封板膜密封后，4℃孵育18h。
(6)洗板：小心揭掉封板膜，弃去液体并甩干，用稀释后的洗涤液注满每孔，静置30s弃去，用纸彻底拍干，充分清洗酶标板5次。
(7)显色：向每孔加入显色液(含有AChE的反应底物)200μl。将酶标板重新密封一张封板膜，放在摇床上，室温避光反应90～120min。
(8)终止：显色反应结束后，轻轻揭掉封板膜，防止液体溅出孔外，立即读板。
(9)读板：以空白孔调零，在412nm波长读取各孔的OD值。
(10)统计：标准品和血浆样品的OD值须先减去NSB孔OD平均值进行校正。根据标准品的浓度(横坐标)及其校正的OD平均值(纵坐标)，生成标准曲线和线性回归方程，由公式计算出上样微孔中的8-OHdG浓度(ng/ml)，再乘以稀释倍数得到8-OHdG在血浆中的实际浓度(ng/ml)。
22、统计学分析
本研究中，数据的统计学分析由GraphPad Prism 5软件完成，所有数据表示为平均值±标准差。两组样本间的不配对双尾t检验(Student’s t-test)和多组样本间的单因素方差分析(one-way analysis of variance with Bonferroni’s post hoc test，one-way ANOVA含Bonferroni法验后比较)用于评估两组数据的统计学差异。对于所有实验，当P值小于0.05时，组间差异具有显著统计学意义。图例中所示*号含义为P值的显著程度：*P<0.05， **P<0.01，***P<0.001。
实施例1 Dkk1上调可能与恶病质相关的肿瘤死亡有关。
通过TCGA分析，Dkk1在多种肿瘤的临床患者中表达量升高，同时高表达的Dkk1会显著减少患者生存期(图1a)通过ELISA分析得到，在小鼠皮下种植CT26和LLC肿瘤细胞后，也能观察到血液中的Dkk1上调(图1b)。Real-time PCR分析显示，相比肌肉、肾脏、心脏和肝脏等器官相比，CT26肿瘤细胞中的Dkk1表达量最高(图1c)，说明植入肿瘤可能会释放Dkk1和提高局部升值循环Dkk1.甚至，CT26肿瘤的种植引起了肌肉和肾脏中Dkk1表达量的升高(图1d)。值得注意的是，即使在CT26肿瘤种植之后，肾脏表达的Dkk1几乎可以忽略不计(图1c,d)。所有的CT26荷瘤小鼠在种植肿瘤后的大约80天全部死亡，而注射Dkk1可以明显缩短荷瘤小鼠生存期，(从40天开始间隔2天一次注射Dkk1)(图1e)，表明DKK1可以直接影响癌症死亡。然而Dkk1的中和抗体可以延长CT26荷瘤小鼠寿命(图1f)。由于CT26肿瘤细胞不转移，所以这个模型的癌症死亡不是转移引起的。值得注意的是，CT26荷瘤小鼠出现了严重的恶病质，伴随着持续的体重下降和肌肉重量下降(图1g)。显瘦的肌肉纤维和空白区域被HE染色显示出来，提示肌肉萎缩和一些肌肉水解(图1h)。非常有趣的是，肾脏重量始终保持不变，且HE染色和血液中的肌酐、尿素氮(图1i)这两个肾功能指标也没有改变，表明种植CT26细胞对肾脏没有影响。这些结果表明，CT26肿瘤种植通常会影响除了肾脏以外的器官。虽然，在LLC细胞荷瘤小鼠中也观察到了Dkk1的升高(图1b)，但是LLC细胞有较强的迁移能力。所以，为了阐明Dkk1在与转移无关肿瘤死亡中有害作用的潜在机制，我们在接下来的试验中重点研究了CT26荷瘤小鼠。
实施例2 下调膜蛋白LRP6和Kremen2能引起肿瘤恶病质
肌肉萎缩是肿瘤恶病质的一个关键特征，是一个多因素疾病，对患者预后和生活质量有负面影响。无论体重指数(BMI)如何，骨骼肌损耗被认为是癌症发展过程中一个有意义的预后因素，并且与增加化疗毒性的发生率、缩短肿瘤进展时间、手术结果差、身体损害和缩短生存期有关。此外，Kremen1/2是Dkk1介导的LRP5/6下膜所必须的受体。因此，为了探究Dkk1在癌症死亡中有害作用的潜在机制，我们首先检测了CT26荷瘤小鼠中Dkk1结合蛋白LRP5/6和Kremen1/2的表达。在种植肿瘤第50天的时候，体循环的Dkk1显著升高(图1b)，同时肌肉萎缩也明显发 生(图1g和图1h)，肌肉上的膜蛋白和总蛋白的LRP6以及Kremen2均显著下调，而LRP5和Kremen1下调不明显(图2a)。相比较起来，即使到了恶病质的最终阶段，肾中的LRP6表达没有改变，而Kremen2的表达反而显著的升高了(图2b)。基于肿瘤植入对肾脏无影响这些结果(实施例1i)，提示膜上的LRP6和Kremen2的调节可能是Dkk1诱导器官损伤的原因。此外，活化的β-catenin和核内的β-catenin的表达没有改变(图2c)，提示，恶病质发展引起LRP6改变机制与β-catenin无关。
实施例3 逆转膜上LRP6和Kremen2下调可以阻止肿瘤恶病质
为了验证膜上LRP6的下调参与肿瘤恶病质发生发展，我们用了一个小分子化学药物MDC，来阻止Dkk1引起的膜LRP5/6的下调。往正常小鼠下肢肌肉注射Dkk1，引起膜上LRP6和Kremen2的内吞(图3a)，而同时注射MDC可以阻止这一现象(图3a)。更重要的是，MDC可以抑制Dkk1注射引起的荷瘤小鼠快速死亡(图3b)。令人惊讶的是，在种植肿瘤的同时注射MDC，可以逆转膜上的LRP6和Kremen2下调从而完全的阻止体重和肌肉重量的下降(图3c)。即使是种植肿瘤40天以后，MDC干预也可以延长CT26小鼠的生存期(图3d)。进一步用clathrin-TG2相关的内吞抑制剂Cystamine和Spermindine看到了与MDC相似的延长肿瘤小鼠生存期的结果(图3e)。结合肌肉蛋白水平检测结果(图3f)表明，阻止Dkk1引起的膜上LRP6和Kremen2的下调可以防止肿瘤恶病质，并且延长生存期。有趣的是，失去IGF结合能力的重组突变体IGFBP4/H95P蛋白，可以结合膜上的LRP6而阻止Dkk1信号传导，同样也能延长荷瘤小鼠的生存期(图3g)。与MDC一样，同时注射IGFBP-4/H95P可以完全阻止体重和肌肉重量的下降(图3c)，其机制也是通过抑制Dkk1引起的膜LRP6表达的下调(图3h)。
实施例4 LRP5/6缺失后GPCR表达谱改变(转录组测序)
Wnt共受体LRP5/6参与激活Wnt/β-catenin通路。鉴于LRP5/6会激活β-catenin入核，所以为了探索LRP5/6自身对GPCR的密切影响是否普遍存在，则需要排除LRP5/6下游β-catenin信号的干扰，即要有相应β-catenin的实验作为LRP5/6的实验对照。首先，我们使用RNAi技术在若干细胞(HepG2、Hela、U2OS、HUVEC)中分别进行LRP5/6和β-catenin的基因敲低实验，显微镜下观察到敲低LRP5/6基因引起以上细胞状态变差和生长停滞，尤其在HepG2细胞中表现最为明显，接着通过WB检测发现LRP5/6敲低后γH2AX信号被上调，暗示敲低LRP5/6 基因的细胞均发生了不同程度的DNA损伤，而对照组和敲低β-catenin基因都没有发现这一现象，这说明LRP5/6能够不依赖β-catenin特异保护细胞稳态的平衡。考虑到在单个细胞中，存在大量GPCR可以与LRP5/6相互作用的可能性，所以我们猜测，敲低LRP5/6基因会同时影响到这些和LRP5/6相互作用的GPCR，进而会通过正负反馈回路的调控导致这些GPCR在转录水平的表达发生变化，最终改变了GPCR信号，打破了细胞稳态，引起诸如细胞生长停滞和DNA损伤等改变。为了验证这一假设，又考虑到HepG2细胞受LRP5/6基因敲低的影响较为显著，因此，我们利用高通量RNA-Seq，分析了HepG2细胞在敲低LRP5/6或者β-catenin之后全基因组转录水平的改变，尝试考察敲低LRP5/6基因本身对GPCR表达水平造成的影响。
HepG2细胞的RNA-Seq分析结果显示，相较于敲低β-catenin基因，敲低LRP5/6基因后导致表达水平发生改变的GPCR在数量和程度上均占有显著优势(图4，A-C)，这暗示敲低LRP5/6基因能够经由反馈回路特异地引起与之相互作用的GPCR在转录水平上的变化。这些结果表明了LRP5/6和GPCR之间具有广泛的密切关系，这种关系与Wnt/β-catenin通路无关，同时也为我们研究LRP5/6调控细胞及糖尿病心脏损伤的分子机制提供了依据。
实施例5 LRP6胞外段(LRP6N)可逆转LRP5/6缺失诱导的DNA损伤
最初我们发现，通过在HUVEC细胞中敲低LRP5/6或者β-catenin，观察到敲低LRP5/6使细胞出现了DNA损伤(γH2AX信号上调)，而对照组和敲低β-catenin都没有发生这一现象(图5B)。除了同时敲低LRP5/6以外，WB结果显示，与敲低β-catenin相比，使用不同浓度梯度的siRNA对HUVEC细胞单独敲低LRP5或LRP6也能导致DNA损伤发生(γH2AX信号上调)(图5A)。这些结果表明敲低LRP5/6基因能引起HUVEC细胞的稳态紊乱，并且LRP5/6的这种功能不依赖于β-catenin。有趣的是，通过向HUVEC细胞培养基中预先加入LRP6胞外段的可溶性蛋白质(LRP6ectodomain，LRP6N)，发现敲低LRP5/6诱导的γH2AX信号上调能够被LRP6N显著减弱(图5B)。由于分泌型LRP6N蛋白是在细胞膜表面起作用，因而LRP6N蛋白在敲低LRP5/6诱导的细胞DNA损伤中的拯救作用表明了LRP5/6在细胞膜上的缺失是导致细胞发生DNA损伤的根本原因。
H 2O 2可以在体外模拟一种疾病状态下的异常培养环境。H 2O 2的过氧基团可以直接攻击细胞的DNA双链单纯导致氧化性DNA损伤而不会引起LRP5/6和β-catenin 的表达变化，并且，H 2O 2诱导的DNA损伤程度呈现浓度和时间的依赖性(图5C)。为了进一步考察LRP5/6维持细胞稳态的生物学作用，我们使用模拟疾病状态的一种不良刺激(H 2O 2)在上述实验基础上对HUVEC细胞做进一步处理。当我们使用低浓度H 2O 2(50μM)刺激HUVEC细胞后发现，与对照组和敲低β-catenin相比，敲低LRP5/6上调的γH2AX信号被H 2O 2进一步激活，并且也能够通过外源性LRP6N蛋白的预处理让这种改变显著减弱(图5B)。相比之下，LRP6N蛋白对于对照组和敲低β-catenin的HUVEC细胞中H 2O 2激活的γH2AX信号则没有影响(图5B)。这些结果表明细胞膜上LRP5/6的缺失会减弱HUVEC细胞对氧化应激的抵抗能力，并且这一现象不依赖于β-catenin。
实施例6 MESD缺失可增强氧化应激效应
MESD蛋白作为LRP5/6的伴侣蛋白，能够帮助其从细胞浆转移到细胞膜上，成为成熟的LRP5/6发挥生物学功能。WB结果显示HUVEC细胞中敲低MESD基因，能够促使细胞膜上的LRP5/6降低(图6A)，但没有发生明显的细胞损伤(图6B)。进一步研究表明，经过低浓度H2O2(50μM)处理极大地增强了敲低MESD细胞中的γH2AX信号，而且外源性LRP6N蛋白的预处理能够显著减弱这种改变(图6B)。这些结果充分表明完整的膜上LRP5/6对于增强细胞抵抗氧化应激的能力是至关重要的。
实施例7 Dkk1可诱导DNA损伤效应
分泌蛋白Dkk1诱导细胞膜上LRP5/6内吞而抑制Wnt/β-catenin信号通路。由于成年时期许多慢性疾病(如糖尿病、慢性心肌缺血、恶性肿瘤等)患者的血液Dkk1水平均明显上调且伴有不良预后，但其中机制还不清楚。为了深入探讨该机制，在HUVEC细胞中加入了不同时间和浓度梯度的纯化Dkk1蛋白，发现Dkk1能够快速诱导细胞膜上LRP5(100ng/ml从15min)和LRP6(100ng/ml从5min)的内吞(总蛋白表达水平不变，而膜蛋白表达水平下调)(图7A)。值得注意的是，Dkk1(100ng/ml)在5min内显著激活γH2AX，并在15min时达到峰值(图7A)。这表明Dkk1能够在内吞LRP5/6的同时导致细胞DNA损伤发生的可能性。其次，我们还发现Dkk1(100ng/ml)是在60min后下调核内β-catenin的表达水平(总蛋白表达水平不变，而核蛋白表达水平下调)，但是较高剂量的Dkk1在30min内并没有下调核内β-catenin的表达，这提示Dkk1不是通过抑制β-catenin信号来诱导细胞DNA 损伤反应(图7A)。另外，像在图6B显示的,通过单纯使用siRNA来更大程度引起β-catenin减低同样没有改变γH2AX信号，也更加支持这一观点。为了进一步证明LRP5/6膜内吞与γH2AX激活之间的内在关系，我们使用Dkk1内吞LRP5/6的抑制剂MDC预处理HUVEC细胞，结果如图7B，发现Dkk1诱导的γH2AX激活被MDC减弱。而且与直接敲低LRP5/6类似的是，通过外源性LRP6N蛋白的预处理也可以抑制Dkk1诱导的γH2AX激活(图7B)。这些结果暗示分泌型LRP6N蛋白可以替代细胞膜上的内源性LRP5/6发挥保护细胞稳态的作用。综合以上数据可知，由Dkk1诱导的膜上LRP5/6的下调是细胞DNA损伤发生的起始原因，也即Dkk1导致的细胞损伤是通过下调膜上LRP5/6而不是通过阻止β-catenin的核移位来实现的。
此外，为了考察膜上LRP5/6对处于不良环境中的细胞是否也具有上述保护作用，我们使用低浓度H 2O 2(50μM)在上述实验基础上对HUVEC细胞做进一步刺激。实验表明，H 2O 2本身能够在不影响细胞膜上LRP5/6表达的同时，激活γH2AX信号，而且当Dkk1诱导膜上LRP5/6下调以后，H 2O 2能够强烈地上调γH2AX信号(图7B)。值得注意的是，MDC或LRP6N本身不引起γH2AX反应，并且也不能阻止H 2O 2诱导的γH2AX激活，但是它们显著减弱了在Dkk1存在下被H 2O 2诱导增强的γH2AX信号(图7B)。这些结果进一步表明完整的膜上LRP5/6对于抵抗氧化应激和维持细胞稳态是至关重要的。
实施例8 Dkk1通过内吞LRP5/6激活β-arrestin1/2信号的传导
上述RNA-Seq分析证明了LRP5/6和大多数GPCR之间存在密切关系，因而不难推测，膜上LRP5/6的下调可能会导致GPCR膜上稳态的失衡，进一步引起下游信号通路发生复杂的改变。如前所述，β-arrestin1/2和各种G蛋白一样，都是GPCR一般的直接下游靶标，但不同于GPCR和G蛋白的是，β-arrestin1/2可以快速地移位到细胞膜并直接结合到活化GPCR的C端，并通过自身的细胞膜和细胞浆/细胞核之间的转运，特异地参与调节一般GPCR信号的传导，因此，GPCR信号改变的一个普遍结果是细胞内β-arrestin1/2发生了移位改变。为了验证广泛的GPCR信号紊乱与LRP5/6下调是否存在相关性，我们考察了Dkk1诱导膜上LRP5/6内吞对β-arrestin1/2信号改变的具体影响，即用胞内β-arrestin1/2的信号传导进一步阐述膜上LRP5/6与GPCR之间的关系。
我们首先分析了Dkk1刺激HUVEC细胞后β-arrestin1/2的移位改变。结果显示，Dkk1可以在内吞LRP5/6的同时快速上调HUVEC细胞浆中的 β-arrestin1/2，而下调细胞膜上的β-arrestin1/2(图8A)，并且经过Dkk1内吞LRP5/6的抑制剂MDC的预处理可以阻止以上β-arrestin1/2的移位变化(图8B)。在短时间(15min)内Dkk1刺激诱导LRP5/6和β-arrestin1/2发生相似的快速变化，提示Dkk1首先结合并快速诱导细胞膜上LRP5/6发生内吞，然后导致β-arrestin1/2的移位改变，这表明膜上LRP5/6和GPCR之间具有直接而密切的关系(图8A)。一般而言，当GPCR激活时，首先发生的是质膜内侧G蛋白相对于这些GPCR的移位，然后才出现胞内β-arrestin1/2相对于这些GPCR的移位。为此，我们使用小分子BIM-46187(1μM 30min)预处理HUVEC细胞，它是G蛋白一般抑制剂，能够直接结合G蛋白α亚基，进而阻止配体活化的GPCR和G蛋白复合物的形成及后续G蛋白的构象改变，由于这样的结合阻止了受体与G蛋白异三聚体间正常的相互作用，从而抑制了G蛋白α亚基的GDP/GTP交换，导致G蛋白及GPCR信号受到了广泛抑制。事实上，我们发现BIM-46187能够阻止HUVEC细胞中Dkk1诱导的β-arrestin1/2移位(图8C)，表明Dkk1诱导的膜上LRP5/6下调影响了GPCR信号，进而导致GPCR一般下游靶标β-arrestin1/2的移位改变。值得注意的是，通过RNAi技术引起的LRP5/6下调是一个长期过程，可能会继发难以预料的二次效应。因此，在HUVEC细胞中敲低LRP5/6而不是β-catenin会上调β-arrestin1/2的表达水平(图8D)，可能是由于LRP5/6和GPCR之间的密切关系导致反馈系统在转录水平间接启动了以上变化。总之，这些结果暗示Dkk1可以通过诱导LRP5/6内吞影响GPCR信号，并且进一步证明LRP5/6确实与GPCR密切相关。
实施例9 β-arrestin1/2介导了膜上LRP5/6下调引起的细胞DNA损伤
目前已知，GPCR直接下游靶标β-arrestin1/2通过细胞膜和细胞浆/细胞核之间的转运不仅可以参与GPCR信号传递，还能介导DNA损伤应答，所以，一方面，HUVEC细胞膜上LRP5/6被Dkk1内吞的同时，快速诱导了β-arrestin1/2从细胞膜到细胞浆的转移，继而导致细胞γH2AX激活；另一方面，通过MDC抑制Dkk1内吞LRP5/6的同时，也能阻止β-arrestin1/2在细胞内的移位和γH2AX的激活。与之类似的还有，HUVEC细胞敲低β-arrestin1/2后(图9A)，不仅阻断了GPCR-β-arrestin1/2信号传导，还抑制了由Dkk1刺激诱导的γH2AX激活(图9B)。值得注意的是，对于Dkk1和低浓度H2O2(50μM)先后刺激诱导增强的γH2AX信号，敲低β-arrestin1/2 同样具有明显的拯救作用，但敲低β-arrestin1/2并没有保护H2O2本身引起的细胞DNA损伤(图9B)，表明β-arrestin1/2在促进Dkk1诱导的DNA损伤中的特异作用。此外，Dkk1刺激诱导的γH2AX激活也被G蛋白一般抑制剂BIM-46187(1μM，30min)预处理所抑制(图9C)。综合以上实验结果可知，Dkk1-LRP5/6轴是通过激活GPCR-β-arrestin1/2信号传导(β-arrestin1/2移位改变)介导了LRP5/6内吞引起的细胞DNA损伤和稳态失衡。
值得一提的是，与上述敲低β-arrestin1/2抑制Dkk1对细胞损害的结果相似，在HUVEC细胞中敲低β-arrestin1/2也能够抑制敲低LRP5/6所引起的DNA损伤及抗氧化应激能力的削弱，并且这种保护作用不依赖于β-catenin(图9D)。以上结果均证明，GPCR下游的直接分子靶标β-arrestin1/2是导致LRP5/6缺失诱导的细胞DNA损伤和稳态失衡的直接调控因子。
实施例10 G蛋白激动剂能够诱导DNA损伤反应
上述实验结果揭示了Dkk1-LRP5/6-GPCR-β-arrestin1/2通路在Dkk1诱导的细胞DNA损伤中的重要功能。接下来，为了进一步验证GPCR信号紊乱与细胞DNA损伤的密切关系，我们通过使用G蛋白激动剂干扰GPCR下游的直接靶标G蛋白，观察是否同样可以诱导细胞DNA损伤。实验表明，CTX(霍乱毒素)能持续激活Gs蛋白α亚基，其刺激HUVEC细胞能以剂量和时间依赖性方式显著上调γH2AX(图10A)。值得注意的是，PTX(百日咳毒素)可以快速抑制Gi蛋白α亚基，而长期刺激可以激活Gs蛋白α亚基，并且γH2AX在短期和长期刺激中都被不同剂量的PTX所激活(图10B)。以上结果表明，影响不同G蛋白的功能均能够诱导DNA损伤反应。因此，作为GPCR的一般下游靶标，G蛋白稳态的破坏会广泛改变GPCR信号，进而导致细胞稳态失衡。
实施例11 LRP5/6基因敲除引起小鼠心脏损伤
我们使用经腹腔注射他莫昔芬诱导后的8周龄LRP5/6或β-catenin全身敲除(LRP5/6 -/-或β-catenin -/-)小鼠对LRP5/6在成体组织中的作用加以阐述。它们是通过LRP5/6 floxp/floxp或β-catenin floxp/floxp小鼠分别与含Cre重组酶的小鼠(UBC-Cre/ESR1)交配获得，该重组酶表达受全身表达的UBC启动子的控制，能够实现基因的全身敲除。以心脏为例，Western Blotting从蛋白水平证实，他莫昔芬诱导1周的LRP5/6 -/-或β-catenin -/-小鼠心脏中的特定基因被成功敲除(图11A)。
在实验过程中发现，与对照组小鼠相比，经他莫昔芬诱导35周的LRP5/6 -/-小鼠显示出体重及多种器官(包括心脏，肺脏，肾脏，脾脏，骨骼肌和脂肪)重量的减轻(图11B)，而β-catenin -/-小鼠却没有出现这些变化，提示LRP5/6缺失特异地诱导了机体和多器官的严重损害。随后，我们选取感兴趣的心脏作为器官损伤的研究对象，进一步考察了LRP5/6敲除对心脏造成的影响。我们发现他莫昔芬诱导1周的LRP5/6 -/-而不是β-catenin -/-小鼠心脏中的γH2AX信号明显增加，p53和p21表达水平也显著上调，但LRP5/6 -/-小鼠心脏的DNA损伤并没有引起凋亡发生(图11A)。此外，二维超声心动图显示经他莫昔芬诱导35周的LRP5/6 -/-小鼠的心脏物理功能受损，包括左心室射血分数(EF％)和左心室短轴缩短率(FS％)的减少(图11C和11E)；实时定量PCR分析也显示LRP5/6 -/-小鼠心衰标志物ANP和BNP显著上调(图11D)，这些结果进一步表明LRP5/6敲除可以导致心脏损伤。相比之下，β-catenin -/-小鼠和同窝出生且只表达Cre重组酶的对照组小鼠直到敲除35周仍无明显差异。综上可知，短期敲除LRP5/6能特异诱导成年小鼠心脏DNA损伤反应和细胞周期停滞，而长期LRP5/6的缺失引起DNA损伤的积累，进而造成心脏功能受损。总之，上述实验初步证明了LRP5/6能够保护成年小鼠心脏的正常功能和稳态平衡。
实施例12 LRP5/6基因敲除激活小鼠心脏β-arrestin1/2信号的传导
细胞实验表明，GPCR的一般下游靶标β-arrestin1/2通过细胞膜和细胞浆/细胞核之间的转运不仅可以参与GPCR信号传递，还能介导DNA损伤应答。类似地，为了论证LRP5/6 -/-小鼠心脏的上述损伤变化是否与GPCR-β-arrestin1/2信号改变有关，我们考察了LRP5/6 -/-小鼠心脏的β-arrestin1/2。如图12A所示，LRP5/6 -/-而不是β-catenin -/-小鼠能够诱导心脏中β-arrestin1/2的移位改变(包括细胞膜上β-arrestin1/2的下调，细胞浆中β-arrestin1/2及细胞核中β-arrestin1的上调)，这说明β-arrestin1/2能够介导LRP5/6敲除引起的心脏DNA损伤，同时也为LRP5/6与GPCR的密切关系提供了直接证据。此外，实时定量PCR也显示(图12B)，LRP5/6 -/-而不是β-catenin -/-小鼠诱导了心脏中多个重要GPCR基因水平的改变，进一步支持了LRP5/6与多种GPCR密切相关的观点。总之，上述结果初步表明了LRP5/6具有不依赖β-catenin维持成年小鼠GPCR-β-arrestin1/2信号稳定，进而阻止心脏DNA损伤和功能紊乱的生物学功能。
实施例13 高血糖诱导糖尿病小鼠血液Dkk1的上调
我们的ELISA测定显示，STZ诱导的1型糖尿病小鼠血液Dkk1水平大幅增加(图13A)。有趣的是，通过每日给予胰岛素对糖尿病小鼠进行治疗，不仅降低了STZ诱导的血糖升高(图13B),也使其高浓度的血液Dkk1降回到了基础水平(图13A)。这些结果表明，STZ诱导的糖尿病高血糖能够特异地上调血液Dkk1，而胰岛素可以借助其降血糖作用使升高的血液Dkk1水平恢复正常。由于STZ诱导的糖尿病小鼠类似于血液中含有高表达Dkk1的糖尿病患者，因而，我们使用该小鼠模型来考察Dkk1对糖尿病有害作用的形成机制。
实施例14 糖尿病小鼠膜上LRP5/6下调与心脏损伤的密切关系
如前所述，Dkk1具有抑制Wnt/β-catenin信号和内吞膜上LRP5/6的双重生物学功能，为了确定高水平的血液Dkk1在糖尿病中发挥何种作用，我们考察了STZ诱导的1型糖尿病小鼠心脏β-catenin和LRP5/6的表达。如图14A所示，在STZ造模的不同时间点，我们发现心脏细胞核和总体β-catenin均没有发生改变，提示Wnt/β-catenin信号通路不参与糖尿病的发病过程。值得注意的是，伴随着血糖浓度(STZ造模第5天显著上升)及血液Dkk1的上调，心脏膜上LRP5/6的表达水平在造模第5天显著下调，并在造模第7天变得更加明显，但总体LRP5/6表达水平始终不受影响，提示在STZ诱导的1型糖尿病小鼠中，心脏膜上LRP5/6的内吞可能与高血糖诱导的血液Dkk1上调有关(图14A)。此外，表明心脏DNA损伤的γH2AX信号也从STZ造模第5天显著增加，并在第7天达到峰值(图14A)。血液8-OHdG在STZ造模第7天也明显上调(图14C)，进一步确认了糖尿病DNA损伤的发生。STZ糖尿病小鼠的这些改变在时间上的一致性意味着心脏膜上LRP5/6的下调及DNA损伤的发生与高血糖上调的Dkk1密切相关。同时，如图14D所示，在STZ造模第7天的糖尿病小鼠中，也显示出其他损伤标志物水平的明显上调(包括心脏p53和p21，Bax/Bcl-2和Cleaved Caspase-3)，这表明糖尿病导致了严重的心脏DNA损伤，最终引起了凋亡。
值得注意的是，如图14B所示，每日给予STZ诱导的糖尿病小鼠胰岛素治疗后，血糖浓度从治疗第1天开始降低并在随后的时间始终保持在低水平，同时血液Dkk1由于血糖浓度下降也处于基础水平。当胰岛素治疗第3天时，心脏的膜上LRP5/6开始上调，伴随着γH2AX信号减弱，这些改变在治疗第7天变得更加明显。并且如图14C和14E所示，在胰岛素治疗第7天，糖尿病小鼠其他损伤标志物(包括血液8-OHdG；心脏p53和p21，Bax/Bcl-2和Cleaved Caspase-3)的表达也大大减弱。 考虑到体重下降是1型糖尿病发生多器官损伤后的标志性体征，故在上述变化的基础上，我们还发现胰岛素的降血糖效应促进了糖尿病小鼠减轻的体重逐步得到恢复(图14F)。以上结果表明，胰岛素治疗对于心脏膜上LRP5/6下调和糖尿病损伤实现的逆转在时间上的一致性进一步暗示，糖尿病心脏损伤与受到血糖严格调控的血液Dkk1和膜上LRP5/6的表达水平密切相关。
实施例15 Dkk1通过诱导LRP5/6膜内吞引起糖尿病心脏损伤
为了进一步论证糖尿病小鼠心脏LRP5/6膜内吞及DNA损伤与Dkk1水平上调的相关性，我们使用能阻止Dkk1内吞膜上LRP5/6的抑制剂MDC，观察膜上Dkk1-LRP5/6轴在糖尿病心脏损伤中的作用。如图15A所示，我们发现STZ诱导的糖尿病心脏膜上LRP5/6下调能够被MDC抑制，而总体LRP5/6及细胞核或总体β-catenin表达水平保持不变，说明Dkk1特异地介导了糖尿病心脏LRP5/6的膜内吞，但不影响经典Wnt通路。同时，如图15B和15C所示，我们分别通过免疫荧光染色和Western Blotting检测显示，与胰岛素对DNA损伤的拯救类似，糖尿病心脏的γH2AX激活也能被MDC所抑制。此外，MDC还阻止了糖尿病小鼠血液中8-OHdG的上升(图15D)。这些结果表明糖尿病小鼠的DNA损伤主要受到Dkk1诱导的膜上LRP5/6下调的调控。如图15C和15E所示，MDC还抑制了糖尿病心脏中p53，p21，Bax/Bcl-2和Cleaved Caspase-3的上调，以及糖尿病小鼠体重的减轻。心脏HE染色表明，STZ造模引起糖尿病心肌结构显著的损伤，包括心肌纤维的排列混乱和断裂，及组织间隙空间的扩大，以上这些变化均可以被胰岛素(阳性对照)和MDC阻止(图15F)。重要的是，MDC在阻止上述损伤的同时没有改变糖尿病小鼠的血糖浓度(图15G)，表明高血糖引起的糖尿病损伤本质上归因于膜上LRP5/6的下调。这些结果强烈提示Dkk1通过诱导膜上LRP5/6下调导致糖尿病心脏损伤的可能性。为了证明Dkk1与心脏损伤的直接关系，我们使用Dkk1纯化蛋白注射心肌，结果表明Dkk1可以不影响β-catenin信号而直接诱导心脏膜上LRP5/6的下调和γH2AX，p53和p21的上调，并且这些改变也可以被MDC预处理所逆转(图15H)，这说明Dkk1能够直接下调膜上LRP5/6来诱导心脏DNA损伤和细胞周期停滞。此外，如图15I，我们在Dkk1纯化蛋白联合BIM-46187局部注射小鼠心脏的实验中发现，BIM-46187(G蛋白一般抑制剂)同样能够有效抑制Dkk1通过LRP5/6膜内吞诱导的心脏损伤，包括γH2AX，p53和p21，该结果表明Dkk1-LRP5/6轴介导的心脏DNA损伤和细胞周期停滞可能与膜上LRP5/6下调造成的GPCR信号激活有关。
总之，上述结果初步提出了一种LRP5/6不依赖β-catenin调控糖尿病心脏损伤的分子机制，即糖尿病的高血糖表型诱导血液Dkk1上调，后者导致膜上LRP5/6的下调，即Dkk1-LRP5/6轴发生了改变，进而影响了GPCR信号的稳定，引起心脏DNA损伤，细胞周期停滞，甚至凋亡等稳态失衡的表现，最终形成了糖尿病的心脏损伤。
实施例16 Leptin-/-小鼠膜上LRP5/6下调导致糖尿病心脏损伤
肥胖被认为是导致2型糖尿病发病的高危因素，近年来，随着肥胖人数急剧增加，2型糖尿病的患病率也呈上升趋势。如前所述，Leptin -/-小鼠显示出肥胖表型，并伴有严重的高血糖，因此，我们将其作为模拟2型糖尿病的小鼠模型来考察LRP5/6在其中的相关作用。如图16A和16B所示，和同窝出生的Leptin +/-或WT小鼠相比，5周龄的Leptin -/-小鼠体重和血糖均显著增加。如图16C所示，6周龄的Leptin -/-小鼠血糖浓度达到最高水平时，其心脏的膜上LRP5/6显著下调，并且γH2AX，p53和p21的表达也轻微上调，但没有发生β-catenin的核移位。值得注意的是，4周龄的Leptin -/-，Leptin +/-和WT同窝出生的小鼠之间心脏膜上LRP5/6的表达水平相似(图16D)，同时，4周龄的Leptin -/-与WT小鼠的血糖水平也很相似，但Leptin -/-小鼠的体重水平已经显著增加(图16A和16B)，表明6周龄Leptin -/-小鼠膜上LRP5/6下调是由高血糖而不是高体重(肥胖因素)导致的。此外，如图16E所示，ELISA检测显示出6周龄Leptin -/-小鼠血液Dkk1水平显著上调，表明Dkk1可能参与诱导了2型糖尿病心脏膜上LRP5/6下调和DNA损伤。
考虑到糖尿病患者的糖耐量普遍受损，所以我们对6周龄Leptin -/-小鼠做了葡萄糖耐量测试。结果如图16F所示，注射葡萄糖后Leptin -/-小鼠的血糖维持较高水平长达2h，但WT小鼠的血糖在15min内立即降低，这表明Leptin -/-小鼠体内血糖的清除存在延迟。有趣的是，如图16H，16I和16J所示，体内注射葡萄糖(每2h一次，持续24h)能明显诱导Leptin -/-小鼠心脏膜上LRP5/6的下调，γH2AX激活和血液8-OHdG的上调，及心脏p53，p21和Bax/Bcl-2的上调，而核内β-catenin没有明显变化。为了验证这些变化是否与Dkk1有关，首先，我们通过ELISA检测发现注射葡萄糖比注射PBS的Leptin -/-小鼠血液Dkk1水平明显升高(图16G)；其次，MDC显著逆转了由注射葡萄糖引起的心脏膜上LRP5/6的下调及上述损伤分子指标的上调(图16H，16I和16K)。这些结果表明高血糖直接上调的血液Dkk1在促进膜上LRP5/6内吞和糖尿病心脏损伤中的有害作用，同时还提供了一种新的机制， 可以很好地解释限制代谢综合征患者的葡萄糖摄入对于改善糖尿病损伤的重要性。
总之，上述1型和2型糖尿病小鼠模型的体内结果共同表明，高血糖可诱导血液Dkk1的上调和细胞膜上LRP5/6的下调，进而导致糖尿病小鼠心脏的DNA损伤应答，细胞周期停滞，甚至凋亡。因此，LRP5/6在细胞膜上的完整性对于维持组织器官稳态是至关重要的。此外，MDC借助其稳固膜上LRP5/6表达的作用可能有望成为1型和2型糖尿病患者器官损伤的临床治疗药物。
实施例17 LRP5/6基因敲除加重了糖尿病心脏损伤
细胞实验表明，敲低LRP5/6基因能够促使不良培养环境(H 2O 2)中的HUVEC细胞DNA损伤显著加重，可见LRP5/6的存在的确有助于维持细胞稳态。类似地，为了进一步探讨LRP5/6不依赖β-catenin维持成年机体稳态的功能，我们对他莫昔芬诱导4周后的全身LRP5/6 -/-或β-catenin -/-小鼠进行STZ诱导的糖尿病造模，以考察体内LRP5/6的缺失能否加重糖尿病引起的心脏损伤。如图17A所示，STZ诱导1周的全身LRP5/6 -/-的糖尿病小鼠心脏分子损伤的程度比全身非敲除的糖尿病小鼠要更加严重，包括显著上调的γH2AX，p53，p21，Bax/Bcl-2以及Cleaved Caspase-3(图17A)；并且，全身LRP5/6 -/-的糖尿病小鼠在STZ诱导2周时发生了明显的心脏物理功能受损(包括EF％和FS％的减少，图17B和17E)。值得一提的是，相较于全身非敲除的糖尿病小鼠，在全身β-catenin -/-的糖尿病小鼠中上述心脏损伤分子指标和心脏物理功能并没有出现显著变化(图17B和17E)。此外，与全身非敲除的糖尿病小鼠相比，全身LRP5/6 -/-的糖尿病小鼠在STZ诱导1周时还出现了较为明显的体重下降(图17C)，但两者的血糖水平并没有差异(图17D)。上述结果共同表明，完全缺失LRP5/6能够明显加重糖尿病心脏和机体的损伤，并且这一现象不受β-catenin信号和血糖水平的影响；也进一步证明LRP5/6自身具有不依赖β-catenin维持成年机体稳态的一般生物学功能。
实施例18 LRP6胞外段(LRP6N)阻止了糖尿病心脏损伤
鉴于外源性LRP6N蛋白能够在体外阻止LRP5/6敲低诱导的细胞DNA损伤这一事实，我们进一步考察了LRP6N是否也可以预防体内的糖尿病心脏损伤。首先，如图18A所示，我们使用转座子技术构建了携带LRP6N基因片段的转基因小鼠，并通过条件性Cre-loxP重组酶系统诱导小鼠全身过表达LRP6N蛋白(带myc标签)。在他莫昔芬诱导4周后的LRP6N/Tg小鼠心脏中，除了LRP6N过表达外，没有观察到 其他分子的改变(图18B)。然而，如图18C和18D所示，LRP6N/Tg小鼠阻止了STZ造模引起的糖尿病心脏γH2AX，p53，p21，Bax/Bcl-2和Cleaved Caspase-3以及血液8-OHdG的上调(图18C和18D)。同时，LRP6N/Tg小鼠也抑制了STZ造模诱导的内源性膜上LRP5/6下调(图18C)。类似地，如图18E和18F所示，通过小鼠尾静脉注射LRP6N质粒(带myc标签)，也减弱了糖尿病心脏γH2AX，p53，p21，Bax/Bcl-2和Cleaved Caspase-3的增加(图18E和18F)。此外，如图18G所示，和各自的对照组相比，小鼠内源性LRP5/6或β-catenin的敲除以及外源性LRP6N的过表达(如转基因或体内转染质粒)均不会改变机体的血糖浓度，表明体内LRP5/6或β-catenin表达的变化对血糖代谢不会产生任何影响(图18G)。值得强调的是，由于分泌型LRP6N蛋白是在细胞膜表面发挥保护作用，所以，这些体内结果进一步表明糖尿病心脏损伤直接归因于膜上LRP5/6的下调，而不依赖β-catenin信号和血糖水平的调控；同时也表明LRP6N能够取代内源性LRP5/6阻止糖尿病心脏损伤的发生。
如图18H所示，通过LRP6N/Tg小鼠和LRP6N质粒尾静脉注射的小鼠体内过表达LRP6N，能够有效阻止STZ造模诱导的糖尿病体重减轻(图18H)，这些结果提示LRP6N具有普遍预防糖尿病损伤的能力。如前所述，1型糖尿病小鼠多器官受损所导致的消瘦表型同样能够被胰岛素和MDC所抑制。由于LRP6N过表达和MDC干预不会改变血糖水平，因此，可溶性LRP6N蛋白和MDC可以作为潜在的针对糖尿病损伤的治疗方案，即使在高血糖条件下对机体和器官仍然能够起到很好地保护作用。
实施例19 糖尿病小鼠膜上LRP5/6下调特异激活心脏β-arrestin1/2信号的传导
由于高血糖影响了糖尿病心脏膜上LRP5/6的表达，因此，我们也想知道高血糖是否也影响了GPCR-β-arrestin1/2信号。首先，如图19A和19B所示，我们发现了β-arrestin1/2在STZ诱导的1型糖尿病心脏中的移位(包括膜上β-arrestin1/2的下调，浆内β-arrestin1/2及核内β-arrestin1的上调)(图19A)，并且胰岛素治疗能够逆转这种移位(图19B)，表明高血糖可以影响β-arrestin1/2信号。其次，如图19C和19D所示，高血糖引起的β-arrestin1/2移位改变也能通过MDC干预或LRP6N/Tg小鼠的LRP6N过表达得到阻止(图19C和19D)，表明高血糖是通过影响Dkk1-LRP5/6轴(上调Dkk1和下调膜上LRP5/6)诱导了心脏β-arrestin1/2的移位。支持这一观点的是，Dkk1直接注射心脏诱导的膜 上β-arrestin1/2下调，浆内β-arrestin1/2及核内β-arrestin1上调，能够被MDC(图19E)或BIM-46187(图19F)所抑制，提示Dkk1通过下调心脏膜上LRP5/6(Dkk1-LRP5/6轴)直接影响了GPCR-β-arrestin1/2信号的传导。如图19G所示，因为单独使用胰岛素，MDC或者体内过表达LRP6N都没能诱导心脏β-arrestin1/2的移位，这些结果进一步支持高血糖通过上调Dkk1和下调膜上LRP5/6特异地影响GPCR-β-arrestin1/2信号的观点。
如前所述，LRP5/6 -/-而不是β-catenin -/-小鼠也能诱导心脏β-arrestin1/2的移位改变(图12A)，这为LRP5/6调控β-arrestin1/2提供了直接证据。由于G蛋白一般抑制剂BIM-46187能够通过抑制GPCR信号紊乱显著阻止Dkk1直接内吞膜上LRP5/6诱导的心脏β-arrestin1/2移位改变(图19F)及DNA损伤(图15I)，因此，LRP5/6的缺失可以破坏众多GPCR的稳态，引起下游β-arrestin1/2的信号传导，后者促进了DNA损伤反应，严重的DNA损伤甚至会发生凋亡，最终导致器官功能受损。
值得注意的是，LRP5/6和胰岛素受体(insulin receptor，IR)都是单次跨膜蛋白，并且胰岛素受体被认为在糖尿病的发展中起到关键作用，它属于受体酪氨酸激酶(receptor tyrosine kinase，RTKs)家族，由两个α亚基(IR-α)和两个β亚基(IR-β)通过二硫键连接而成。两个α亚基位于细胞膜外侧，其上有胰岛素的结合位点；两个β亚基是受体的跨膜部分，起信号转导作用。为了探究LRP5/6在糖尿病心脏损伤中的保护作用是否与胰岛素受体有关，我们考察了糖尿病及其各种干预条件下胰岛素受体的表达情况。如图19A-E所示，糖尿病小鼠或Dkk1蛋白直接注射的心脏细胞膜上和细胞浆内的胰岛素受体并没有发生类似LRP5/6及β-arrestin1/2的移位改变。此外，单独使用胰岛素，MDC或者体内过表达LRP6N也没有诱导心脏膜上或浆内胰岛素受体的移位(图19G)。因此，这些结果强烈暗示膜上LRP5/6下调是通过特异地调控GPCR-β-arrestin1/2信号来参与糖尿病心脏损伤，而不受胰岛素受体的影响。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种DKK1基因或其编码蛋白抑制剂的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗肿瘤恶病质与糖尿病伴随疾病。
  2. 如权利要求1所述的用途，其特征在于，所述组合物或制剂还用于选自下组的一种或多种用途：
    (a)降低肿瘤细胞内β-arrestin2的含量；
    (b)抑制哺乳动物中的IκB和NFκB的激活；
    (c)抑制p53和Bax/Bcl-2的上调；
    (d)抑制肌肉蛋白标志物的下调；
    (e)抑制哺乳动物肌肉组织中的细胞因子的上调。
  3. 一种药物组合物，其特征在于，包括：
    (a1)用于预防和/或治疗(a)肿瘤恶病质与糖尿病伴随疾病的第一活性成分，所述第一活性成分包括：DKK1基因或其编码蛋白抑制剂；
    (a2)预防和/或治疗(a)肿瘤恶病质与糖尿病伴随疾病的第二活性成分，所述第二活性成分包括：其他的用于预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的药物；和
    (b)药学上可接受的载体。
  4. 一种药盒，其特征在于，包括：
    (i)第一容器，以及位于该第一容器中的活性成分(a1)DKK1基因或其编码蛋白抑制剂，或含有活性成分(a)的药物；和
    (ii)第二容器，以及位于该第二容器中的活性成分(a2)其他的用于预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的药物,或含有活性成分(a2)的药物。
  5. 一种体外降低肿瘤细胞内β-arrestin2的含量的方法，其特征在于，包括步骤：
    在DKK1基因或其编码蛋白抑制剂存在的条件下，培养肿瘤细胞，从而降低肿瘤细胞内β-arrestin2的含量。
  6. 一种权利要求3所述的药物组合物或权利要求4所述药盒的用途，其特征在于，用于制备用于预防和/或治疗肿瘤恶病质与糖尿病伴随疾病的药物。
  7. 一种筛选肿瘤恶病质与糖尿病伴随疾病的潜在治疗剂的方法，其特征在于， 包括：
    (a)在测试组中，在培养体系中，在测试化合物的存在下，培养表达DKK1基因或其蛋白的细胞一段时间T1，检测测试组所述培养体系中的DKk1基因或其蛋白的表达量E1和/或活性A1；
    并且在不存在所述测试化合物且其他条件相同的对照组中，检测对照组所述培养体系中DKK1基因或其蛋白的表达量E2和/或活性A2；和
    (b)对E1和E2进行比较，如果E1显著低于E2，则表示所述测试化合物是肿瘤恶病质与糖尿病伴随疾病的潜在治疗剂；或
    对A1和A2进行比较，如果A1显著低于A2，则表示所述测试化合物是肿瘤恶病质与糖尿病伴随疾病的潜在治疗剂。
  8. 如权利要求7所述的方法，其特征在于，所述的方法包括步骤(c)：将步骤(b)中所确定的潜在治疗剂施用于哺乳动物，从而测定其对哺乳动物的肿瘤恶病质与糖尿病伴随疾病的影响。
  9. 一种筛选肿瘤恶病质与糖尿病伴随疾病的潜在治疗剂的方法，其特征在于，包括：
    (a)在测试组中，在培养体系中，在测试化合物的存在下，培养肿瘤细胞一段时间T1，检测测试组所述培养体系中所述DKK1与LRP6复合物的形成情况；
    并且在不存在所述测试化合物且其他条件相同的对照组中，检测对照组所述培养体系中所述DKK1与LRP6复合物的形成情况；
    (b)如果所述测试组中的所述述DKK1与LRP6复合物的形成数量Q1显著低于所述对照组中的所述述DKK1与LRP6复合物的形成数量Q2，则表示所述测试化合物是候选化合物。
  10. 如权利要求9所述的方法，其特征在于，所述的方法包括步骤(c)：将步骤(b)中所确定的候选化合物施用于哺乳动物，从而测定其对哺乳动物的肿瘤恶病质与糖尿病伴随疾病的影响。
  11. 一种确定肿瘤恶病质与糖尿病伴随疾病的治疗方案的方法，其特征在于，包括：
    a)提供来自受试者的测试样品；
    b)检测测试样品中DKK1蛋白的水平；和
    c)基于所述样品中的DKK1蛋白水平来确定(a)肿瘤恶病质与糖尿病伴随疾病的治疗方案。
PCT/CN2020/086807 2019-07-04 2020-04-24 Dkk1抑制剂在预防和/或治疗肿瘤恶病质与糖尿病伴随疾病中的应用 WO2021000640A1 (zh)

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