WO2021128919A1 - Cst1在预防和/或治疗肝脏免疫失调疾病中的应用 - Google Patents

Cst1在预防和/或治疗肝脏免疫失调疾病中的应用 Download PDF

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WO2021128919A1
WO2021128919A1 PCT/CN2020/112234 CN2020112234W WO2021128919A1 WO 2021128919 A1 WO2021128919 A1 WO 2021128919A1 CN 2020112234 W CN2020112234 W CN 2020112234W WO 2021128919 A1 WO2021128919 A1 WO 2021128919A1
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cst1
protein
ifn
receptor
another preferred
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PCT/CN2020/112234
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French (fr)
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程新
徐艺林
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中国科学院分子细胞科学卓越创新中心
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Priority to EP20905087.1A priority Critical patent/EP4088735A4/en
Priority to US17/789,378 priority patent/US20230043161A1/en
Publication of WO2021128919A1 publication Critical patent/WO2021128919A1/zh

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    • 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
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • G01N33/5044—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 involving specific cell types
    • G01N33/5073—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin

Definitions

  • the present invention relates to the field of biomedicine. Specifically, the present invention relates to the application of CST1 in the prevention and/or treatment of liver immune disorders.
  • Acute Liver Failure is an acute syndrome with high mortality. It is characterized by rapid necrosis and apoptosis of a large number of liver parenchymal cells in a short period of time, and loss of original liver function. It is accompanied by complications such as disorders of the immune system in the liver, hepatic encephalopathy and possible multiple organ failure.
  • Hepatitis viruses such as hepatitis A, hepatitis B and hepatitis E (Hepatitis A, B&E); caused by medication or poisoning, such as Chinese herbal medicine (Chinese Herbal Medicine), acetaminophen (Acetaminophen) ); Other factors such as Ischemic Hepatocellular Injury, Hypoxic Hepatitis and so on.
  • liver transplantation has many problems and is still not a perfect treatment for acute liver failure.
  • the shortage of donor livers leads to patients who can receive liver transplantation in time.
  • defects such as rejection reactions and the need for lifelong immunosuppression still exist after liver transplantation.
  • MSCs Mesenchymal Stem Cells
  • the purpose of the present invention is to provide a new protein drug with good curative effect on liver immune disorders, such as acute liver failure.
  • the purpose of the present invention is to provide a medicine and treatment plan based on a specific protein CST1 for the treatment of acute liver failure.
  • the first aspect of the present invention provides a use of the CST1 gene, or its protein or its promoter, for preparing a composition or preparation, and the composition or preparation is used to prevent and/or treat liver immune disorders.
  • the liver immune disorders include liver immune disorders caused by excessive activation of IFN- ⁇ signals.
  • the liver immune disorders include acute liver failure and acute liver injury.
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the mammal includes a human or non-human mammal.
  • the non-human mammals include rodents (such as mice, rats, rabbits) and primates (such as monkeys).
  • the promoter refers to a substance that can increase the activity and/or content of the CST1 gene or its protein in vivo or in vitro; the substance can be a synthetic or natural compound, protein, or nucleoside Sour etc.
  • the CST1 promoter includes a substance that promotes the expression of CST1.
  • the CST1 promoter includes a CST1 protein promoter and/or a CST1 gene promoter.
  • the promotion of CST1 expression or activity refers to increasing the expression or activity of CST1 gene or protein by ⁇ 20%, preferably, ⁇ 50%, more preferably, ⁇ 70%.
  • the CST1 promoter is selected from the group consisting of small molecule compounds, CST1 expression vectors, or a combination thereof.
  • the vector for expressing CST1 includes a viral vector.
  • the viral vector is selected from the group consisting of adeno-associated viral vectors, lentiviral vectors, or a combination thereof.
  • the protein includes a full-length protein or protein fragment.
  • the CST1 gene or its protein is derived from mammals, more preferably from rodents (such as mice, rats), primates and humans.
  • the CST1 gene or its protein is derived from endoderm stem cells or definitive endoderm.
  • the CST1 protein also includes derivatives of CST1 protein.
  • the derivative of the CST1 protein includes a modified CST1 protein, a protein molecule whose amino acid sequence is homologous to the natural CST1 protein and has the activity of the natural CST1 protein, a dimer or multimer of the CST1 protein, A fusion protein containing the amino acid sequence of the CST1 protein.
  • the modified CST1 protein is a PEGylated CST1 protein.
  • the "protein molecule whose amino acid sequence is homologous to the natural CST1 protein and has natural CST1 protein activity” means that its amino acid sequence has ⁇ 85% homology with the CST1 protein, preferably ⁇ 90% homology, more preferably ⁇ 95% homology, and best ⁇ 98% homology; protein molecules with natural CST1 protein activity.
  • the CST1 protein is selected from the following group:
  • (C) Compared with the amino acid sequence shown in SEQ ID NO.:1, the sequence has a homology of ⁇ 90%, preferably ⁇ 95%, more preferably ⁇ 98%, and most preferably ⁇ 99% derived from CST1 protein ⁇ , or active fragments thereof.
  • the CST1 gene encodes CST1 protein.
  • the CST1 gene is selected from the following group:
  • the nucleotide sequence has a homology of ⁇ 95% (preferably ⁇ 98%) with the sequence shown in SEQ ID NO.: 2 and a polynucleotide encoding the polypeptide shown in SEQ ID NO.: 1;
  • the composition includes a pharmaceutical composition.
  • the pharmaceutical composition contains (a) the CST1 gene, or its protein or its promoter; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes stem cells that do not express the CST1 gene or its protein.
  • the stem cells include embryonic stem cells and mesenchymal stem cells.
  • the stem cells include human embryonic stem cells.
  • 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 60-99wt% of the total weight of the pharmaceutical composition, preferably 75-99wt%, more preferably 80-99wt% %.
  • the composition also includes other drugs for preventing and/or treating liver immune disorders.
  • composition or preparation can be used alone or in combination in the prevention and/or treatment of liver immune disorders.
  • the combined use includes: combined use with other drugs for preventing and/or treating liver immune disorders.
  • the second aspect of the present invention provides the use of an inhibitor of IFN- ⁇ or its receptor for preparing a composition or preparation for preventing and/or treating liver immune disorders.
  • the inhibitor is selected from the group consisting of antibodies, small molecule compounds, or combinations thereof.
  • the inhibitor of IFN- ⁇ or its receptor inhibits the binding of IFN- ⁇ to its receptor.
  • the inhibitor of IFN- ⁇ or its receptor reduces the efficiency of IFN- ⁇ binding to its receptor.
  • the inhibitor of IFN- ⁇ or its receptor reduces the binding efficiency of IFN- ⁇ protein and its receptor protein, and reduces the ratio of IFN- ⁇ receptor protein to IFN- ⁇ protein.
  • the inhibitor of IFN- ⁇ or its receptor reduces the ratio of IFN- ⁇ receptor protein to IFN- ⁇ protein by ⁇ 30%, preferably, ⁇ 50%, more preferably , ⁇ 70%.
  • the IFN- ⁇ protein includes a full-length protein or protein fragment.
  • the IFN- ⁇ includes active fragments of IFN- ⁇ or derivatives thereof.
  • the homology of the active fragment or its derivative with IFN- ⁇ is at least 90%, preferably 95%, more preferably 98%, 99%.
  • the active fragment or derivative thereof has at least 80%, 85%, 90%, 95%, 100% IFN- ⁇ activity.
  • the IFN- ⁇ protein also includes derivatives of IFN- ⁇ protein.
  • the derivative of the IFN- ⁇ protein includes a modified IFN- ⁇ protein, a protein molecule whose amino acid sequence is homologous to the natural IFN- ⁇ protein and has natural IFN- ⁇ protein activity, and IFN- ⁇ Protein dimers or multimers, fusion proteins containing the amino acid sequence of IFN- ⁇ protein.
  • the "protein molecule with an amino acid sequence that is homologous to the natural IFN- ⁇ protein and has natural IFN- ⁇ protein activity” means that its amino acid sequence is more than 85% identical to that of the IFN- ⁇ protein.
  • Source preferably ⁇ 90% homology, more preferably ⁇ 95% homology, and most preferably ⁇ 98% homology; protein molecules with natural IFN- ⁇ protein activity.
  • the IFN- ⁇ is derived from mammals; preferably, it is derived from humans, mice, rats, or rabbits; more preferably, it is derived from humans.
  • the IFN- ⁇ receptor is selected from the following group: IFNGR2, IFNGR1, or a combination thereof.
  • the IFN- ⁇ receptor includes a full-length protein or protein fragment.
  • the IFN- ⁇ receptor includes an active fragment of IFN- ⁇ receptor or a derivative thereof.
  • the homology between the active fragment or its derivative and the IFN- ⁇ receptor is at least 90%, preferably 95%, more preferably 98%, 99%.
  • the active fragment or derivative thereof has at least 80%, 85%, 90%, 95%, 100% IFN- ⁇ activity.
  • the IFN- ⁇ receptor also includes IFN- ⁇ receptor derivatives.
  • the derivative of the IFN- ⁇ receptor includes a modified IFN- ⁇ receptor, and the amino acid sequence is homologous to the natural IFN- ⁇ receptor and has a natural IFN- ⁇ receptor.
  • Active protein molecules, dimers or multimers of IFN- ⁇ receptors, fusion proteins containing IFN- ⁇ receptor amino acid sequences are included in the derivative of the IFN- ⁇ receptor.
  • the "protein molecule whose amino acid sequence is homologous to the receptor of natural IFN- ⁇ and has the receptor activity of natural IFN- ⁇ ” means that its amino acid sequence is compared with that of the receptor of IFN- ⁇ . It has ⁇ 85% homology, preferably ⁇ 90% homology, more preferably ⁇ 95% homology, and best ⁇ 98% homology; and has a natural IFN- ⁇ receptor Active protein molecule.
  • the IFN- ⁇ receptor is derived from mammals; preferably, it is derived from humans, mice, rats, or rabbits; more preferably, it is derived from humans.
  • the composition includes a pharmaceutical composition.
  • the pharmaceutical composition contains (a) an inhibitor of IFN- ⁇ or its receptor; 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 60-99wt% of the total weight of the pharmaceutical composition, preferably 75-99wt%, more preferably 80-99wt% %.
  • the composition also includes other drugs for preventing and/or treating liver immune disorders.
  • composition or preparation can be used alone or in combination in the prevention and/or treatment of liver immune disorders.
  • the combined use includes: combined use with other drugs for preventing and/or treating liver immune disorders.
  • the third aspect of the present invention provides a cell preparation, including:
  • a first pharmaceutical composition containing (a) a first active ingredient, the first active ingredient being the CST1 gene, or its protein or its promoter; or IFN- ⁇ or its receptor Inhibitors of the body, and a pharmaceutically acceptable carrier;
  • the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions, or the same pharmaceutical composition.
  • the stem cells include embryonic stem cells and mesenchymal stem cells.
  • the stem cells include human embryonic stem cells.
  • the cell preparation is a liquid preparation.
  • the cells in the cell preparation are basically ( ⁇ 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) or all of them do not express the CST1 gene , Or its protein stem cell composition.
  • the component (a1) accounts for 0.5-10 wt% of the total weight of the cell preparation, preferably 1-10 wt%, more preferably 2-10 wt%.
  • the component (a2) accounts for 90-99 wt% of the total weight of the cell preparation, preferably 90-98 wt%, more preferably 90-97 wt%.
  • the weight ratio of the first active ingredient and the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.
  • the concentration of the component (a1) is 100-1000ug/ml, preferably, 200-1000ug/ml, more preferably, 300-1000ug/ml.
  • the concentration of the component (a2) is 1.0 ⁇ 10 6 -1.0 ⁇ 10 8 /ml, preferably 5.0 ⁇ 10 6 -1.0 ⁇ 10 8 /ml, more preferably 1.0 ⁇ 10 7 -1.0 ⁇ 10 8 pieces/ml.
  • the carrier is selected from the following group: infusion carrier and/or injection carrier, preferably, the carrier is one or more carriers selected from the group: physiological saline, glucose Salt water, or a combination thereof.
  • the cell preparation also includes other drugs for preventing and/or treating liver immune disorders.
  • the fourth aspect of the present invention provides a medicine kit including:
  • the first container, and the active ingredient in the first container (a1) CST1 gene, or its protein or its promoter; or inhibitor of IFN- ⁇ or its receptor, or containing the active ingredient (a) Drugs; and
  • An optional second container, and the active ingredient (a2) in the second container is a stem cell that does not express the CST1 gene or its protein, or a drug containing the active ingredient (a2).
  • first container and the second container are the same or different containers.
  • the medicine in the first container is a single preparation containing the CST1 gene, or its protein or its promoter.
  • the medicine in the second container is a unilateral preparation containing stem cells that do not express the CST1 gene or its protein.
  • 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 liver immune disorders.
  • the preparation containing the active ingredient (a1) CST1 gene, or its protein or its promoter; or inhibitor of IFN- ⁇ or its receptor, or containing other agents for prevention and/or treatment includes capsules, tablets, suppositories, or intravenous injections.
  • the concentration of the promoter; or the inhibitor of IFN- ⁇ or its receptor is 0.1-100 mg/kg body weight, preferably 0.1-50 mg/kg body weight, more preferably 1-20 mg/kg body weight.
  • the concentration of the stem cells that do not express the CST1 gene or its protein is 0.1-100 mg/kg body weight , Preferably 0.1-50 mg/kg body weight, more preferably 1-20 mg/kg body weight.
  • the fifth aspect of the present invention provides a method for screening potential therapeutic agents for the prevention and/or treatment of liver immune disorders, including:
  • the "significantly higher” means that E1/E2 ⁇ 2, preferably, ⁇ 3, more preferably, ⁇ 4.
  • the cells include human embryonic stem cells and human endoderm stem cells.
  • the cell is a cell cultured in vitro.
  • 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 the mammal's liver immune disorders.
  • the mammal includes a human or a non-human mammal.
  • the non-human mammals include rodents, primates, and preferably include mice, rats, rabbits, and monkeys.
  • the sixth aspect of the present invention provides a method for screening potential therapeutic agents for the prevention and/or treatment of liver immune disorders, including:
  • the method includes step (b): administering the candidate compound determined in step (a) to a mammal, thereby determining its effect on the mammal's liver immune disorders.
  • the mammal includes a human or a non-human mammal.
  • the non-human mammals include rodents, primates, and preferably include mice, rats, rabbits, and monkeys.
  • the "significantly lower” means that E1/E2 ⁇ 1/2, preferably, ⁇ 1/3, more preferably ⁇ 1/4.
  • the method is non-diagnostic and non-therapeutic.
  • the cells include human embryonic stem cells, human endoderm stem cells, and human mesenchymal stem cells.
  • the cell is a cell cultured in vitro.
  • the seventh aspect of the present invention provides a method for preventing and/or treating liver immune disorders, including the steps:
  • the administration includes oral administration.
  • the subject includes humans or non-human mammals.
  • the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.
  • the dosage of the CST1 gene, its protein or its promoter; or IFN- ⁇ or its receptor inhibitor is 0.1-100 mg/kg body weight, preferably 1-50 mg/ kg body weight, preferably 5-20 mg/kg body weight.
  • the frequency of administration of the CST1 gene, or its protein or its promoter; or IFN- ⁇ or its receptor inhibitor is one injection every 7-30 days, preferably, 14-30 days One injection.
  • the administration time of the CST1 gene, its protein or its promoter; or IFN- ⁇ or its receptor inhibitor is 7 to 30 days, and preferably, 14-30 days. Inject once a day.
  • Figure 1 shows that in the cell line hEnSCs expressing CST1, transplanting hEnSCs into the liver of acute liver failure rats can effectively treat acute liver failure. It can greatly improve the survival rate of rats and protect liver function. Compared with cell lines that are known to have curative effects on acute liver failure but do not express CST1, mesenchymal stem cells, hEnSCs have considerable or even better curative effects.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • Blood Ammonia blood ammonia
  • C Single-cell sequencing heatmap (Heatmap).
  • the sequencing library method is Smart-seq2
  • the sequencing platform is the Illumina second-generation sequencing platform.
  • the gene pointed to by the red arrow is the potential key factor CST1 that we think.
  • Figure 2 shows that in a cell line that does not express CST1 and cannot treat acute liver failure: over-expression of CST1 in embryonic stem cells can give it a certain therapeutic effect on acute liver failure. It is manifested in the improvement of survival rate and the protection of liver function.
  • Rat Primary Hepatocyte Rat-derived hepatocytes.
  • FIG. 3 shows that CST1 protein can significantly reduce the expression of ISGs (Interferon stimulated genes) downstream genes activated by IFN- ⁇ in macrophages (Mac, Macrophage). The reason for this inhibition is that CST1 can interfere IFN- ⁇ binds to its receptor IFNGR2, thereby blocking the expression of ISGs genes that further activate IFNGR2 downstream.
  • ISGs Interferon stimulated genes
  • IFN- ⁇ antibody is used as an immunoprecipitation antibody to separate IFN- ⁇ and IFNGR2 bound to it at the same time.
  • CST1 is added to this separation system, the amount of IFN- ⁇ bound to IFN- ⁇ is reduced, which is reflected in western blotting
  • the detected IFNGR2/IFN- ⁇ ratio decreased, indicating that CST1 affected the combination of IFN- ⁇ and IFNGR2 and reduced the binding efficiency of the two.
  • CST1 protein gene, or its protein, or its promoter; or inhibitor of IFN- ⁇ or its receptor can significantly prevent and/or treat Liver immune disorders.
  • the present invention also unexpectedly discovered that the CST1 protein gene, or its protein, or its promoter can also (a) improve the survival rate of mammals; and/or (b) significantly reduce serum alanine aminotransferase , Aspartate aminotransferase, and/or blood ammonia levels; and/or (c) protect liver parenchymal cell function; and/or (d) inhibit the pro-inflammatory activation effect of IFN- ⁇ on macrophages; and/or (e) Inhibit the expression of a large number of ISGs downstream of IFN- ⁇ ; and/or (f) reduce the binding efficiency of IFN- ⁇ and IFNGR2.
  • the present invention also unexpectedly discovered that overexpression of CST1 can make non-curative cells obtain a certain therapeutic effect, which shows that the CST1 protein itself and cell therapy based on CST1 overexpression have great potential for the treatment of acute liver failure.
  • the inventor completed the present invention.
  • the term “about” means that the value can vary from the recited value by no more than 1%.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
  • administration refers to the use of any of various methods and delivery systems known to those skilled in the art to physically introduce the product of the present invention into a subject, including intravenous, intramuscular, subcutaneous, and peritoneal Internal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • CST1 protein As used herein, the terms “CST1 protein”, “Cystatin SN protein”, and “cysteine protease inhibitor SN” are used interchangeably.
  • the present invention relates to a CST1 protein and variants thereof.
  • the amino acid sequence of the CST1 protein is shown in SEQ ID NO.:1.
  • the CST1 protein or its promoter of the present invention can (a) prevent and/or treat liver immune disorders; and/or (b) improve the survival rate of mammals; and/or (c) significantly reduce serum alanine aminotransferase and aspartate The level of transaminase, and/or blood ammonia.
  • the present invention also includes 50% or more of the sequence shown in SEQ ID NO. 1 of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.
  • SEQ ID NO.: 1 is human CST1 protein, and the accession number is NM_001898.2.
  • the "same or similar function” mainly refers to: "(a) prevent and/or treat liver immune disorders; and/or (b) improve the survival rate of mammals; and/or (c) significantly reduce serum glutathione The level of transaminase, aspartate transaminase, and/or blood ammonia".
  • the protein of the present invention can be a recombinant protein, a natural protein, or a synthetic protein.
  • the protein of the present invention can be a natural purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plant, insect, and mammalian cells) using recombinant technology.
  • a prokaryotic or eukaryotic host for example, bacteria, yeast, higher plant, insect, and mammalian cells
  • the protein of the present invention may be glycosylated or non-glycosylated.
  • the protein of the present invention may also include or not include the initial methionine residue.
  • the present invention also includes CST1 protein fragments and analogs with CST1 protein activity.
  • fragment and analogs refer to a protein that substantially maintains the same biological function or activity as the natural CST1 protein of the present invention.
  • the mutein fragment, derivative or analogue of the present invention may be (i) a mutein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acids
  • the residue may or may not be encoded by the genetic code, or (ii) a mutein with a substitution group in one or more amino acid residues, or (iii) a mature mutein and another compound (such as an extended mutein) Half-life compounds, such as polyethylene glycol) fused to form a mutant protein, or (iv) additional amino acid sequence fused to the mutant protein sequence to form a mutant protein (such as leader sequence or secretory sequence or used to purify the mutant protein)
  • the sequence or proprotein sequence, or the fusion protein formed with the antigen IgG fragment According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • conservatively substituted amino acids are preferably generated by amino acid substitution
  • the present invention also includes that the natural CST1 protein of the present invention has 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99 %) Homologous polypeptides or proteins with the same or similar functions.
  • the protein variant can be obtained by substituting, deleting or adding at least one amino acid by several (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10) Derivative sequences, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • the function of the protein is usually not changed, and the addition of one or several amino acids to the C-terminal and/or ⁇ terminal usually does not change the function of the protein.
  • the present invention includes that the difference between the natural CST1 protein analog and the natural CST1 protein can be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both.
  • Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other known biological techniques.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.
  • Modified (usually without changing the primary structure) forms include: chemically derived forms of proteins in vivo or in vitro, such as acetic acid or carboxylation. Modifications also include glycosylation, such as those that undergo glycosylation modifications during protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). In addition, the mutant protein of the present invention can also be modified.
  • Modified (usually not changing the primary structure) forms include: in vivo or in vitro chemically derived forms of mutein such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modifications during the synthesis and processing of the mutant protein or during further processing steps. This modification can be accomplished by exposing the mutein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes mutant proteins that have been modified to increase their resistance to proteolysis or optimize their solubility.
  • the invention also provides a polynucleotide sequence encoding the CST1 protein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes: DNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded.
  • a polynucleotide encoding a mature polypeptide includes: a coding sequence that only encodes the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequences) of the mature polypeptide and non-coding sequences.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention.
  • the variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but it will not substantially change the function of the encoded polypeptide. .
  • the DNA sequence encoding the human CST1 protein encodes the ECM1 protein shown in SEQ ID NO.:1, and the polynucleotide sequence encoding the CST1 protein is shown in SEQ ID NO.:2.
  • SEQ ID NO.: 2 is the nucleotide sequence of the human CST1 gene.
  • the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing the nucleotide sequence in segments and then performing overlap extension PCR.
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions (or stringent conditions).
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, and more Fortunately, hybridization occurs when more than 95%.
  • proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, are purified to homogeneity.
  • the full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • primers can be designed according to the relevant 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 is used as a template to amplify the relevant sequences. 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 is usually done by 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 fragments with very long sequences.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the method of using PCR technology to amplify DNA/RNA is preferably used to obtain the polynucleotide of the present invention.
  • the RACE method RACE-cDNA end rapid amplification method
  • the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods.
  • the amplified DNA/RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.
  • the CST1 promoter includes substances that can increase the activity and/or content of the CST1 gene or its protein in vivo or in vitro.
  • the expression of CST1 can be increased by the following methods: the tissue itself secretes a large amount of CST1 protein or artificially overexpresses the CST1 protein, or artificially delivers the CST1 protein (for example, using a viral vector, such as an adeno-associated virus vector) or a CST1 promoter.
  • a viral vector such as an adeno-associated virus vector
  • the CST1 promoter is not particularly limited, as long as it can promote the expression of CST11 or enhance the activity of CST1 protein, it is within the protection scope of the present invention.
  • the CST1 promoter includes a small molecule compound.
  • IFN- ⁇ or its receptor protein and “IFN- ⁇ or its receptor polypeptide” are used interchangeably, and both refer to a protein or polypeptide having the amino acid sequence of IFN- ⁇ or its receptor. They include IFN- ⁇ or its receptor protein with or without starting methionine. In addition, the term also includes full-length IFN- ⁇ or its receptor and fragments thereof.
  • the IFN- ⁇ or its receptor protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutant and its functionally active fragments.
  • IFN- ⁇ or its receptor is a type II interferon and a binding receptor of type II interferon on the cell surface, including, but not limited to, IFN- ⁇ , IFNGR1, IFNGR2.
  • the IFN- ⁇ receptor is selected from the group consisting of IFNGR2, IFNAR1, IFNAR2 (wherein IFNAR1 and IFNAR2 are type I interferon receptors), or a combination thereof.
  • IFN- ⁇ is human IFN- ⁇ protein, and the accession number is P01579.
  • the IFN- ⁇ receptor is a human IFN- ⁇ receptor protein, and the accession numbers are as follows:
  • IFNGR2 P38484;
  • IFNGR1 P15260.
  • 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 IFN- ⁇ or its acceptor nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and use a commercially available cDNA library or according to those skilled in the art.
  • the cDNA library prepared by the conventional method is used 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 is usually done by 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 fragments with very long sequences.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This 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) of the present invention encoding human IFN- ⁇ or its receptor polypeptide, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • IFN- ⁇ or its receptor 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 IFN- ⁇ or its receptor encoding DNA sequence and appropriate transcription/translation control signals can be used to construct an expression vector containing IFN- ⁇ or its receptor encoding DNA sequence and appropriate 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, etc.
  • 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 prokaryotic organism 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 .
  • the transformation can also be carried out 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 the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is 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 a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • IFN- ⁇ or its receptor inhibitor and pharmaceutical composition IFN- ⁇ or its receptor inhibitor and pharmaceutical composition
  • the IFN- ⁇ or its receptor inhibitor (or antagonist) that can be used in the present invention includes any substance that can inhibit the expression and/or activity of IFN- ⁇ or its receptor gene or its encoded protein.
  • IFN- ⁇ or its receptor inhibitors also include inhibitors that inhibit the binding of IFN- ⁇ to its receptors (including inhibitors that inhibit the binding efficiency of IFN- ⁇ to its receptors or reduce IFN- ⁇ protein Inhibitor of the ratio of receptor protein).
  • the inhibitor of IFN- ⁇ and its receptor includes small molecule compounds, IFN- ⁇ and its receptor antibodies, IFN- ⁇ and its receptor nucleic acid antisense RNA, siRNA, shRNA, miRNA, or IFN- ⁇ Inhibitors of its receptor activity.
  • the methods and steps for inhibiting IFN- ⁇ and its receptor include neutralizing its protein with antibodies to IFN- ⁇ and its receptor, and using shRNA or siRNA or CRISPR carried by a virus (such as adeno-associated virus).
  • the reagent performs gene silencing of IFN- ⁇ and its receptor.
  • the inhibition rate of IFN- ⁇ and its receptor 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, Methods such as fluorescence quantitative PCR or Western blot control and detect the inhibition rate of IFN- ⁇ and its receptors.
  • the inhibitors of IFN- ⁇ and its receptor proteins of the present invention can inhibit IFN- ⁇ and their receptors when administered (administered) therapeutically.
  • 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 value 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 infusion 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 injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • 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 aseptic conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram-10 mg/kg body weight per day.
  • the stem cell of the present invention is a stem cell that does not express the CST1 gene or its protein.
  • the stem cells of the present invention include, but are not limited to, embryonic stem cells and mesenchymal stem cells.
  • composition (cell preparation)
  • the present invention also provides a pharmaceutical composition, which contains an effective amount of (a) CST1 gene, or its protein or its promoter, or IFN- ⁇ or its receptor inhibitor; (b) optionally does not express CST1 Stem cells of genes, or their proteins, and pharmaceutically acceptable carriers.
  • components (a) and (b) can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, such as physiological saline, where the pH is usually about 5-8, preferably, The pH is about 7-8.
  • a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium such as physiological saline, where the pH is usually about 5-8, preferably, The pH is about 7-8.
  • the term "effective amount” or “effective dose” refers to an amount that can produce function or activity on humans and/or animals and can be accepted by humans and/or animals.
  • the effective amount of the component (a) is: 100-1000ug/ml, preferably, 200-1000ug/ml, more preferably, 300-1000ug/ml;
  • the effective amount of component (b) is 1.0 ⁇ 10 6 -1.0 ⁇ 10 8 pieces/ml, preferably 5.0 ⁇ 10 6 -1.0 ⁇ 10 8 pieces/ml, more preferably 1.0 ⁇ 10 7 -1.0 ⁇ 10 8 pieces/ml /ml.
  • the effective amount of components (a) and (b) are injected at one time.
  • pharmaceutically acceptable ingredients are substances that are suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances that have a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent, including.
  • the pharmaceutically acceptable carrier that can be used is not particularly limited. It can be one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient Purity and sufficiently low toxicity.
  • “Compatibility” here means that the components in the composition can be blended with the adipose-derived mesenchymal progenitor cells of the present invention without significantly reducing its therapeutic effect.
  • Examples of pharmaceutically acceptable carriers of the present invention include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • an optimized vector can also be designed according to the properties of adipose-derived mesenchymal progenitor cells.
  • the carrier is preferably an infusion carrier and/or an injection carrier.
  • the pharmaceutical composition of the present invention contains safe and effective amounts of components (a) and (b), and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be made into an injection form, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods.
  • the pharmaceutical composition should be manufactured under aseptic conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be made into a sustained-release preparation.
  • the effective amount of the components (a) and (b) of the present invention may vary with the mode of administration and the severity of the disease to be treated.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (for example, through clinical trials).
  • the factors include, but are not limited to: the pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, and the like.
  • the pharmaceutical composition of the present invention is preferably an intravenous injection reagent.
  • the concentration of component (a) is: 100-1000ug/ml, preferably, 200-1000ug/ml, more preferably, 300-1000ug/ml
  • the effective amount of the component (b) is 1.0 ⁇ 10 6 -1.0 ⁇ 10 8 pieces/ml, preferably 5.0 ⁇ 10 6 -1.0 ⁇ 10 8 pieces/ml, more preferably 1.0 ⁇ 10 7 -1.0 ⁇ 10 8 pcs/ml.
  • the injection method of the pharmaceutical composition is not particularly limited, and it can be a single injection preparation or a combination of preparations for multiple injections. In a preferred embodiment of the present invention, the pharmaceutical composition is a single injection.
  • the pharmaceutical composition is preferably an intravenous injection preparation.
  • the present invention also provides the use of the active ingredients of the present invention or corresponding drugs to (a) prevent and/or treat liver immune disorders; and/or (b) improve the survival rate of mammals; and/or (c) significantly reduce Serum alanine aminotransferase, aspartate aminotransferase, and/or blood ammonia levels; and/or (c) protect liver parenchymal cell function; and/or (d) inhibit the pro-inflammatory activation of IFN- ⁇ on macrophages; and/ Or (e) inhibit the expression of a large number of ISGs downstream of IFN- ⁇ ; and/or (f) a method for reducing the efficiency of IFN- ⁇ binding to IFNGR2, which includes administering an effective amount of the active ingredient to the mammal (a) CST1 gene, Or its protein or its promoter; or an inhibitor of IFN- ⁇ or its receptor, or administration of a pharmaceutical composition containing the active ingredient (a); and optionally (b) not expressing the CST1 gene or its protein Or administer a
  • the active ingredient of the present invention can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the form of tablets: , Pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspending agent), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or Parenteral administration is in the form of a sterile injectable solution or suspension (containing about 0.05-5% suspension in an isotonic medium).
  • these pharmaceutical preparations may contain about 0.01-99%, more preferably about 0.1%-90% by weight of the active ingredient mixed with the carrier.
  • the two active ingredients or pharmaceutical compositions of the present invention can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration .
  • routes of administration include oral administration, intramuscular administration or intravenous administration.
  • the preferred pharmaceutical composition is a liquid composition, especially an injection.
  • the two active ingredients or drugs of the present invention can also be used in combination with other drugs for preventing and/or treating liver immune disorders.
  • the present invention found for the first time that the CST1 protein gene, or its protein, or its promoter or inhibitor of IFN- ⁇ or its receptor can significantly prevent and/or treat liver immune disorders.
  • the present invention finds for the first time that the CST1 protein gene, or its protein, or its promoter or inhibitor of IFN- ⁇ or its receptor can also (a) improve the survival rate of mammals; and/or (b) significantly Reduce the level of serum alanine aminotransferase, aspartate aminotransferase, and/or blood ammonia; and/or (c) protect liver parenchymal cell function; and/or (d) inhibit the pro-inflammatory activation of IFN- ⁇ on macrophages; and /Or (e) inhibit the expression of a large number of ISGs downstream of IFN- ⁇ ; and/or (f) reduce the binding efficiency of IFN- ⁇ and IFNGR2.
  • the present invention proves for the first time the therapeutic effect of CST1 protein on acute liver failure, and CST1 protein is an easy-to-produce protein product with a wide range of potential applications.
  • Human endoderm stem cells were obtained from a cell line independently established by our laboratory. The method of line establishment has been published in articles (Cheng, X., Ying, L., Lu, L., Galvao, AM, Mills, JA, Lin, HC, Kotton ,DN,Shen,SS,Nostro,MC,Choi,JK,et al. (2012).Self-renewing endodermal progenitor lines generated from human pluripotent stem cells.Cell Stem Cell 10,371-384.).
  • hEnSCs human endoderm stem cells
  • hEnSCs In vitro maintenance of hEnSCs can be carried out by culturing and subcultured in a conditioned medium with Matrigel and a certain amount of irradiated mouse embryonic fibroblast (MEF) (3X10 5 /10 cm-dish) matrix. , Carry out quantity amplification.
  • the conditioned medium consists of Serum Free Medium (SFD) as the basic medium and added a certain amount of cytokines: bFGF (FGF2) (10ng/ml), EGF (10ng/ml), VEGF (10ng/ml) ml) and BMP4 (10ng/ml) and the small molecule compound Kartogenin (1.5 ⁇ M/ml).
  • 1L SFD consists of 250ml Ham's F12, 750ml Homemade IMDM, 5ml 10% BSA, 5ml N2 supplement, and 10ml B27 supplement. Change the medium every 2 days, and passage once every 5 days. The passage is generally carried out at a ratio of 1:4 or 1:5. The cells need to be digested for 5 minutes and then pipetted into single cells, mixed with MEF, and then spread to a new one. On the petri dish, plate with 1.5-2 X 10 6 /10 cm-dish.
  • the hES strain is H9, which is a stable human embryonic stem cell line established in the laboratory.
  • the establishment method refers to the following documents (Cheng, X., Ying, L., Lu, L., Galvao, AM, Mills, JA, Lin, HC , Kotton, DN, Shen, SS, Nostro, MC, Choi, JK, et al. (2012). Self-renewing endodermal progenitor lines generated from human pluripotent stem cells. Cell Stem Cell 10, 371-384.).
  • the cell culture medium components are: basal medium DMEM/F12 50:50 (Invitrogen), 15% KOSR (current lot: 771421), 1x NEAA, 1x P/S, 1x L-Glu, 1x Sodium Bicarbonate (8ml from stock) ), 1x Sodium Pyruvate (2.5ml from stock), 1:550 dilution 2ME (55mM stock).
  • Cystatin SN (CST1) protein is highly expressed in the definitive endoderm stage of human embryonic development. Therefore, we use the cDNA made from the definitive endoderm induced by hES in vitro differentiation, clone the CST1 gene from the cDNA and connect it to the lentiviral vector Go to pWPI.1. Then we used the 293FT cell line as the cell line for packaging CST1 overexpression virus, and transfected the pWPI.1-CST1 plasmid and the packaging plasmids pspAX2 and pMD2.G into 293FT by the calcium transfer method. After waiting for 2 days, the supernatant was collected and packaged.
  • a good virus is added to the hES culture system and infected for 2 days (1:1 virus supernatant: hES medium).
  • hES medium 1:1 virus supernatant: hES medium.
  • puromycin 1ug/ml was added to hES medium to sieve pWPI.1-CST1 hES cells, until the medium was changed, a large number of cell deaths were not seen, and the remaining cells were CST1 overexpressing hES series, referred to as CST1 + ES.
  • D-Galactosamine hydrochloride D-GalN
  • D-GalN D-Galactosamine hydrochloride
  • the separation method is based on Percoll TM density gradient centrifugation for separation.
  • D-GalN-induced male acute hepatic failure rats two days after the induction of D-GalN administration, anesthetized them in a sterile environment, opened the abdominal cavity and perfused the liver.
  • the perfusion needle was inserted through the portal vein of the rat, and the inferior vena cava opening was used as the outlet for the perfusion fluid, and the peristaltic pump was used to continuously and stably perfuse the fluid.
  • First perfuse 1X Hank's Balanced Salt Solution (HBSS) 100ml, about 10 minutes after perfusion, to flush out the blood in the liver.
  • HBSS Hank's Balanced Salt Solution
  • rat liver For a rat, add 20ml of RPMI-1640 medium containing 0.1% Collagenase IV + 0.01% DNase I + 5% Fetal Bovine Serum (FBS) + 1% Penicillin-Streptomycin (P/S) to the chopped liver. Slowly shake in a 37°C water bath for one hour. After digestion, filter out undigested tissue with a 100 ⁇ m filter.
  • RPMI-1640 medium containing 0.1% Collagenase IV + 0.01% DNase I + 5% Fetal Bovine Serum (FBS) + 1% Penicillin-Streptomycin (P/S)
  • Recombinant protein IFN- ⁇ IFNG
  • CST1 was added to the culture system at a final concentration of 500ng/ml.
  • the medium was RPMI-1640+10%FBS+1%P/S, cultured 3 After days, the medium was aspirated, and the adherent cells were lysed with RNA extraction lysate, RNA was extracted, and the expression of ISGs in macrophages was detected under different conditions.
  • Co-immunoprecipitation was performed using Thermo Fisher’s Pierce TM Classic Magnetic IP/Co-IP kit. The same amount of macrophage protein lysate ( ⁇ 1000ug) was used in each experiment.
  • the immunoprecipitation product was obtained by western blot and IFN- ⁇ (IFNG) and IFNGR2 antibody detection, grayscale analysis used ImageJ software for analysis.
  • liver function indexes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and blood ammonia (Blood Ammonia) have been significantly reduced, which indicates that transplantation of hEnSCs has a good protective effect on the liver function of rats with acute liver failure. ( Figure 1B).
  • hEnSCs transplantation has a good effect in animal experiments for acute liver failure, in order to be beneficial to the treatment of acute liver failure, the molecular mechanism behind hEnSCs treatment of acute liver failure is more worthy of study.
  • hEnSCs that is, endoderm stem cells
  • ES human embryonic stem cells
  • Hepatoblast human hepatoblast
  • Hepatocyte human hepatocytes
  • Figure 1C the targets of screening are those that have immunomodulatory or anti-apoptosis effects, and have positive effects on liver function, such as protection.
  • the CST1 gene the full name Cystatin SN, is a secreted protein. It is preliminarily speculated that CST1 may play an immunomodulatory role in the treatment of hEnSCs.
  • hEnSCs may be a key molecule in the treatment of acute liver failure
  • the cell line is human embryonic stem cells (ES), and then the lentivirus is used to package the CST1 overexpression plasmid vector and infected with ES to establish an ES cell line overexpressing CST1, namely CST1 + ES. Then we tested whether the CST1 gene was overexpressed in the established cell line, and compared its expression with hEnSCs, ES, and MSC. As shown in Figure 2A, CST1+ES overexpressed CST1 gene, but the expression level was still not as high as hEnSCs, while ES and MSCs did not express CST1.
  • ES human embryonic stem cells
  • CST1 + ES to treat acute liver failure in rats, and compared it with the known effective cell line hEnSCs, Hepatocyte, and the known ineffective cell line ES. The comparison is still evaluated in terms of survival rate and liver function indicators. Among the 5 rats with acute liver failure transplanted with CST1 + ES, 3 survived within a week, while all 5 rats with normal ES died ( Figure 2B). Compared with hEnSCs and Hepatocyte, it still has a better therapeutic effect, but CST1 + ES does have a significant improvement in the therapeutic effect compared to ordinary ES.
  • CST1 + ES In terms of liver function indicators, although CST1 + ES is not as good as hEnSCs and Hepatocyte, it has lower serum ALT and AST than ordinary ES. This shows that CST1 + ES also has a certain protective effect on liver function. In view of the fact that the expression of CST1 in CST1 + ES is still not as good as hEnSCs, but good therapeutic effects have been obtained, which also reflects the importance of CST1 for the therapeutic effect of hEnSCs.
  • CST1 + ES In terms of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicators, CST1 + ES also has a significant decrease compared to ordinary ES, which means that CST1 + ES has a certain liver function due to the expression of CST1 The role of protection ( Figure 2C).
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • CST1 protein has a good therapeutic effect on acute liver failure in rats, and the therapeutic effect of CST1 protein on acute liver failure has never been reported.
  • Simply overexpression of CST1 can make cells that have no therapeutic effect have a certain effect, which shows that the CST1 protein itself and cell therapy based on CST1 overexpression have great potential for the treatment of acute liver failure.
  • the present invention not only discovers that CST1 has immunomodulatory effects on animals with acute liver failure and increases survival rate in transplanted animals, but also explores the mechanism of CST1 immunomodulation in the liver of animals, and explains the immune function of CST1 in the liver. Some mechanisms of regulation.
  • ISGs Interferon- ⁇
  • IFN ⁇ Interferon- ⁇
  • ISGs Interferon- ⁇ and other types of interferon signal-activated downstream genes (Interferon stimulated genes, ISGs) are collectively referred to as ISGs. . ISGs are highly activated in liver macrophages in rats with acute liver failure.
  • IFN- ⁇ receptor has two different subunits IFNGR1 and IFNGR2. As shown in the left panel of Figure 3B, using Co-Immunoprecipitation technology, it is found that IFN- ⁇ binds to one of its receptor subunits IFNGR2.
  • IFN- ⁇ (IFNG) antibodies are used to co-precipitate proteins that can bind to IFN- ⁇ in the protein lysate of rat macrophages.
  • IFN- ⁇ is used as a protein receptor for IFN- ⁇ , which can naturally interact with IFN- ⁇ is bound and co-precipitated, and then detected by western blotting; but if CST1 recombinant protein is added to rat macrophage protein lysis (5ug is added to 1000ug protein lysis solution), IFN- ⁇ and IFNGR2 are co-precipitated.
  • the ratio of IFNGR2 protein to IFN- ⁇ protein detected by western blotting is shown in the right panel of Figure 3B. This shows that CST1 protein interferes with the binding between IFN- ⁇ and IFN- ⁇ , and CST1 weakens IFN- ⁇ The efficiency of binding to IFNGR2, thereby reducing the expression of ISGs downstream of IFN- ⁇ , and inhibiting the pro-inflammatory activation of macrophages, thereby achieving its immunomodulatory effect.
  • the inventor not only discovered the therapeutic effect of hEnSCs cell level on acute liver failure, but also discovered that CST1 is a key factor for hEnSCs in the treatment of acute liver failure.
  • CST1 is a rare secreted protein that has been studied.
  • CST1 is a cysteinase inhibitor, which is abundant in saliva and tears, but is not expressed in most human tissues. .
  • CST1 has a certain effect on anti-allergic and antibacterial effects, but its effect on acute liver failure has never been reported. Therefore, the present invention discovered the therapeutic effect of CST1 on acute liver failure, and invented a method for the treatment of acute liver failure based on CST1.
  • CST1 is not expressed in most tissues of the human body, the therapeutic molecular mechanisms of cells that are known to have therapeutic effects on acute liver failure, such as human mesenchymal stem cells (MSCs), are different from this, which also shows that CST1 is one of the same in treating acute liver failure.
  • MSCs human mesenchymal stem cells
  • CST1 Simply overexpressing the CST1 gene can make ES cells that have no therapeutic effect to obtain good curative effect, so CST1 combined with some cells that have therapeutic effect, such as MSCs, may achieve better therapeutic effect.

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Abstract

本发明提供了CST1在预防和/或治疗肝脏免疫失调疾病中的应用。具体地，本发明提供了一种CST1基因、或其蛋白或其促进剂的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗肝脏免疫失调疾病。

Description

CST1在预防和/或治疗肝脏免疫失调疾病中的应用 技术领域
本发明涉及生物医药领域，具体地，本发明涉及CST1在预防和/或治疗肝脏免疫失调疾病中的应用。
背景技术
急性肝衰竭(Acute Liver Failure,ALF)是一种高死亡率的急性综合征，它的特征是大量的肝实质细胞在较短的一段时间内快速的坏死凋亡，失去原有的肝功能，并伴随着肝内免疫系统失调，肝性脑病以及可能导致的多器官衰竭等并发症。
引起急性肝衰竭的病因有以下三类，由肝炎病毒引起的如甲肝乙肝和戊肝(Hepatitis A,B&E)；由用药当或中毒引起，如中草药(Chinese Herbal Medicine)，对乙酰氨基酚(Acetaminophen)；其他因素如缺血性肝细胞损伤(Ischemic Hepatocellular Injury)，缺氧性肝炎(Hypoxic Hepatitis)等等。
目前急性肝衰竭的唯一有效的治疗手段是肝移植(Liver Transplantation),但肝移植存在着许多问题仍然不是急性肝衰竭的完美治疗方法，诸如供体肝脏的紧缺，导致能够及时接受肝移植的病人很少，肝脏移植后仍然存在的排异反应和需要终生免疫抑制等缺陷。
作为肝移植的代替疗法，目前有体外人工肝和细胞治疗两种策略，而细胞治疗是比较新兴且简易可行的疗法，目前在动物水平的实验已经有相当多的报道。如间充质干细胞(Mesenchymal Stem Cells，MSCs)移植治疗急性肝衰竭被证实有一定疗效，但MSCs治疗急性肝衰的分子机制研究仍仅限于免疫调节方面，发现相关的免疫调节因子是一些已知对于免疫有调节作用的因子，且如果单独使用，对急性肝衰的治疗缓解作用非常有限。
因此，本领域迫切需要开发对于肝脏免疫失调疾病，比如急性肝衰竭有着良好疗效的新蛋白质药物。
发明内容
本发明的目的是提供一种对于肝脏免疫失调疾病，比如急性肝衰竭有着良 好疗效的新蛋白质药物。
本发明的目的在于提供一种基于一种特定蛋白CST1治疗急性肝衰竭的药物和治疗方案。
本发明第一方面提供了一种CST1基因、或其蛋白或其促进剂的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗肝脏免疫失调疾病。
在另一优选例中，所述肝脏免疫失调疾病包括IFN-γ信号过度激活引起的肝脏免疫失调疾病。
在另一优选例中，所述肝脏免疫失调疾病包括急性肝衰竭、急性肝损伤。
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)提高哺乳动物的生存率；
(b)显著降低血清谷丙转氨酶、谷草转氨酶、和/或血氨的水平；
(c)保护肝实质细胞功能；
(d)抑制IFN-γ对于巨噬细胞的促炎激活作用；
(e)抑制IFN-γ下游的大量ISGs的表达；
(f)减弱IFN-γ与IFNGR2结合的效率。
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、兔)、灵长类动物(如猴)。
在另一优选例中，所述促进剂是指能够在体内或体外提高CST1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、核苷酸等。
在另一优选例中，所述CST1促进剂包括促进CST1表达的物质。
在另一优选例中，所述CST1促进剂包括CST1蛋白促进剂和/或CST1基因促进剂。
在另一优选例中，所述促进CST1表达或活性指将CST1基因或蛋白的表达或活性提高≥20％，较佳地，≥50％，更佳地，≥70％。
在另一优选例中，所述CST1促进剂选自下组：小分子化合物、表达CST1的载体、或其组合。
在另一优选例中，所述表达CST1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，所述的蛋白包括全长蛋白或蛋白片段。
在另一优选例中，所述的CST1基因、或其蛋白来源于哺乳动物，更佳地来源于啮齿动物(如小鼠、大鼠)、灵长动物和人。
在另一优选例中，所述的CST1基因、或其蛋白来源于内胚层干细胞、定型内胚层。
在另一优选例中，所述CST1蛋白还包括CST1蛋白的衍生物。
在另一优选例中，所述CST1蛋白的衍生物包括经修饰的CST1蛋白、氨基酸序列与天然CST1蛋白同源且具有天然CST1蛋白活性的蛋白分子、CST1蛋白的二聚体或多聚体、含有CST1蛋白氨基酸序列的融合蛋白。
在另一优选例中，所述经修饰的CST1蛋白是PEG化的CST1蛋白。
在另一优选例中，所述“氨基酸序列与天然CST1蛋白同源且具有天然CST1蛋白活性的蛋白分子”是指其氨基酸序列与CST1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CST1蛋白活性的蛋白分子。
在另一优选例中，所述CST1蛋白选自下组：
(A)氨基酸序列如SEQ ID NO.:1所示的多肽；
(B)将SEQ ID NO.:1所示的氨基酸序列经过一个或几个(通常为1-60个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸残基的取代、缺失或添加而形成的CST1蛋白衍生物，或其活性片段；
(C)序列与SEQ ID NO.:1所示的氨基酸序列相比，同源性≥90％，较佳地≥95％，更佳地≥98％，最佳地≥99％的CST1蛋白衍生物，或其活性片段。
在另一优选例中，所述的CST1基因编码CST1蛋白。
在另一优选例中，所述的CST1基因选自下组：
(a)编码如SEQ ID NO.:1所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:2所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:2所示序列的同源性≥95％(较佳地≥98％)，且编码SEQ ID NO.:1所示多肽的多核苷酸；
(d)与(a)-(c)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)CST1基因、或其蛋白或其促进 剂；和(b)药学上可接受的载体。
在另一优选例中，所述药物组合物还包括不表达CST1基因、或其蛋白的干细胞。
在另一优选例中，所述干细胞包括胚胎干细胞、间充质干细胞。
在另一优选例中，所述干细胞包括人类胚胎干细胞。
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60-99wt％，较佳地75-99wt％，更佳地80-99wt％。
在另一优选例中，所述组合物还包括其他的预防和/或治疗肝脏免疫失调疾病的药物。
在另一优选例中，所述组合物或制剂在预防和/或治疗肝脏免疫失调疾病的应用中，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：与其它的预防和/或治疗肝脏免疫失调疾病的药物联合使用。
本发明第二方面提供了一种IFN-γ或其受体的抑制剂的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗肝脏免疫失调疾病。
在另一优选例中，所述抑制剂选自下组：抗体、小分子化合物、或其组合。
在另一优选例中，所述IFN-γ或其受体的抑制剂抑制IFN-γ与其受体的结合。
在另一优选例中，所述IFN-γ或其受体的抑制剂降低IFN-γ与其受体结合的效率。
在另一优选例中，所述IFN-γ或其受体的抑制剂降低IFN-γ蛋白与其受体蛋白的结合效率，降低所述IFN-γ受体蛋白与IFN-γ蛋白的比例。
在另一优选例中，所述IFN-γ或其受体的抑制剂将IFN-γ受体蛋白与IFN-γ蛋白的比例下降了≥30％，较佳地，≥50％，更佳地，≥70％。
在另一优选例中，所述IFN-γ蛋白包括全长蛋白或蛋白片段。
在另一优选例中，所述IFN-γ包括IFN-γ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IFN-γ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IFN-γ活性。
在另一优选例中，所述IFN-γ蛋白还包括IFN-γ蛋白的衍生物。
在另一优选例中，所述IFN-γ蛋白的衍生物包括经修饰的IFN-γ蛋白、氨基酸序列与天然IFN-γ蛋白同源且具有天然IFN-γ蛋白活性的蛋白分子、IFN-γ蛋白的二聚体或多聚体、含有IFN-γ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IFN-γ蛋白同源且具有天然IFN-γ蛋白活性的蛋白分子”是指其氨基酸序列与IFN-γ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IFN-γ蛋白活性的蛋白分子。
在另一优选例中，所述的IFN-γ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IFN-γ的受体选自下组：IFNGR2、IFNGR1、或其组合。
在另一优选例中，所述IFN-γ的受体包括全长蛋白或蛋白片段。
在另一优选例中，所述IFN-γ的受体包括IFN-γ受体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IFN-γ受体的同源性至少为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IFN-γ活性。
在另一优选例中，所述IFN-γ的受体还包括IFN-γ的受体的衍生物。
在另一优选例中，所述IFN-γ的受体的衍生物包括经修饰的IFN-γ的受体、氨基酸序列与天然IFN-γ的受体同源且具有天然IFN-γ的受体活性的蛋白分子、IFN-γ的受体的二聚体或多聚体、含有IFN-γ的受体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IFN-γ的受体同源且具有天然IFN-γ的受体活性的蛋白分子”是指其氨基酸序列与IFN-γ的受体相比具有≥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IFN-γ的受体活性的蛋白分子。
在另一优选例中，所述的IFN-γ的受体来源于哺乳动物；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)IFN-γ或其受体的抑制剂；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60-99wt％，较佳地75-99wt％，更佳地80-99wt％。
在另一优选例中，所述组合物还包括其他的预防和/或治疗肝脏免疫失调疾病的药物。
在另一优选例中，所述组合物或制剂在预防和/或治疗肝脏免疫失调疾病的应用中，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：与其它的预防和/或治疗肝脏免疫失调疾病的药物联合使用。
本发明第三方面提供了一种细胞制剂，包括：
(a1)第一药物组合物，所述第一药物组合物含有(a)第一活性成分，所述第一活性成分为CST1基因、或其蛋白或其促进剂；或IFN-γ或其受体的抑制剂，以及药学上可接受的载体；
(a2)任选的第二药物组合物，所述第二药物组合物含有(b)第二活性成分，所述第二活性成分为不表达CST1基因、或其蛋白的干细胞；以及药学上可接受的载体。
在另一优选例中，所述的第一药物组合物和第二药物组合物为不同的药物组合物，或同一药物组合物。
在另一优选例中，所述干细胞包括胚胎干细胞、间充质干细胞。
在另一优选例中，所述干细胞包括人类胚胎干细胞。
在另一优选例中，所述细胞制剂为液体制剂。
在另一优选例中，所述的细胞制剂中的细胞基本上(≥90％、95％、96％、97％、98％、99％、99.5％、99.9％)或全部由不表达CST1基因、或其蛋白的干细胞构成。
在另一优选例中，所述的细胞制剂中，所述组分(a1)占所述细胞制剂总重量的0.5-10wt％，较佳地1-10wt％，更佳地2-10wt％。
在另一优选例中，所述的细胞制剂中，所述组分(a2)占所述细胞制剂总重量的90-99wt％，较佳地90-98wt％，更佳地90-97wt％。
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，所述的细胞制剂中，所述组分(a1)的浓度为100-1000ug/ml，较佳地，200-1000ug/ml，更佳地，300-1000ug/ml。在另一优选例中，所述的细胞制剂中，所述组分(a2)的浓度为1.0×10 6-1.0×10 8个/ml，较佳地5.0×10 6-1.0×10 8个/ml，更佳地1.0×10 7-1.0×10 8个/ml。
在另一优选例中，所述的载体选自下组：输液剂载体和/或注射剂载体，较佳地，所述的载体是选自下组的一种或多种载体：生理盐水、葡萄糖盐水、或其组合。
在另一优选例中，所述细胞制剂还包括其他预防和/或治疗肝脏免疫失调疾病的药物。
本发明第四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)CST1基因、或其蛋白或其促进剂；或IFN-γ或其受体的抑制剂，或含有活性成分(a)的药物；和
(ii)任选的第二容器，以及位于该第二容器中的活性成分(a2)不表达CST1基因、或其蛋白的干细胞,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CST1基因、或其蛋白或其促进剂的单方制剂。
在另一优选例中，所述的第二容器的药物是含不表达CST1基因、或其蛋白的干细胞的单方制剂。
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/或治疗肝脏免疫失调疾病的说明。
在另一优选例中，所述含有活性成分(a1)CST1基因、或其蛋白或其促进剂；或IFN-γ或其受体的抑制剂的制剂或含有其他的用于预防和/或治疗肝脏免疫失调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)CST1基因、或其蛋白或其促进剂；或IFN-γ或其受体的抑制剂的制剂中，所述CST1基因、或其蛋白或其促进剂；或IFN-γ或其受体的抑制剂的浓度为0.1-100mg/kg体重，较佳地0.1-50mg/kg 体重，更佳地1-20mg/kg体重。在另一优选例中，所述含有活性成分(a2)不表达CST1基因、或其蛋白的干细胞的制剂中，所述不表达CST1基因、或其蛋白的干细胞的浓度为0.1-100mg/kg体重，较佳地0.1-50mg/kg体重，更佳地1-20mg/kg体重。
本发明第五方面提供了一种筛选预防和/或治疗肝脏免疫失调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CST1基因的细胞一段时间T1，检测测试组所述培养体系中的CST1基因的表达量E1；
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CST1基因的表达量E2；和
(b)对E1和E2进行比较，如果E1显著高于E2，则表示所述测试化合物是肝脏免疫失调疾病的潜在治疗剂。
在另一优选例中，所述“显著高于”指E1/E2≥2，较佳地，≥3，更佳地，≥4。
在另一优选例中，所述细胞包括人胚胎干细胞、人内胚层干细胞。
在另一优选例中，所述细胞为体外培养的细胞。
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肝脏免疫失调疾病的影响。
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。
本发明第六方面提供了一种筛选预防和/或治疗肝脏免疫失调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，培养细胞一段时间T1，检测测试组所述培养体系中所述IFN-γ与其受体的结合情况；
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IFN-γ与其受体的结合情况；
(b)如果所述测试组中的所述IFN-γ与其受体的结合效率E1显著低于所述对照组中的所述IFN-γ与其受体的结合效率E2，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肝脏免疫失调疾病的影响。
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E1/E2≤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，所述细胞包括人胚胎干细胞、人内胚层干细胞、人间充质干细胞。
在另一优选例中，所述细胞为体外培养的细胞。
本发明第七方面提供了一种预防和/或治疗肝脏免疫失调疾病的方法，包括步骤：
给需要的对象，施用CST1基因、或其蛋白或其促进剂；或IFN-γ或其受体的抑制剂、本发明第二方面所述的细胞制剂、或本发明第三方面所述的药盒。
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CST1基因、或其蛋白或其促进剂；或IFN-γ或其受体的抑制剂的施用剂量为0.1-100mg/kg体重，较佳地为1-50mg/kg体重，最佳地为5-20mg/kg体重。
在另一优选例中，所述CST1基因、或其蛋白或其促进剂；或IFN-γ或其受体的抑制剂的施用频率为7－30天注射一次，较佳地，14-30天注射一次。
在另一优选例中，所述CST1基因、或其蛋白或其促进剂；或IFN-γ或其受体的抑制剂的施用时间为7天－30天注射一次，较佳地，14-30天注射一次。
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显示了在表达CST1的细胞系hEnSCs中，移植hEnSCs到急性肝衰竭大鼠的肝脏中能够有效治疗急性肝衰竭。表现在能够大大提升大鼠生存率，保护肝功能。相比于已知对急性肝衰竭有疗效但不表达CST1的细胞系，间充质干细胞，hEnSCs有着相当乃至更好的疗效。
A.hEnSC移植治疗急性肝衰竭的在大鼠实验上的一周生存曲线，每一组20只大鼠。图例代表的组别：Normal：健康大鼠；ALF：急性肝衰大鼠；ALF+hBM-MSCs：移植了人骨髓来源的间充质干细胞治疗急性肝衰的大鼠；ALF+hEnSC：移植了hEnSC细胞治疗的大鼠。
B.移植了hEnSCs治疗急性肝衰竭的大鼠的肝功能指标在第2、3天的变化。ALT：谷丙转氨酶；AST：谷草转氨酶；Blood Ammonia：血氨。
C.单细胞测序热图(Heatmap)。测序建库方法为Smart-seq2，测序平台为Illumina二代测序平台。红色箭头指向的基因是我们认为的潜在关键因子CST1。
图2显示了在不表达CST1且不能治疗急性肝衰竭的细胞系：胚胎干细胞中过表达CST1，可以使之获得一定的治疗急性肝衰竭的疗效。表现在生存率的提升和肝功能的保护。
A.CST1的RNA表达水平。
B.移植了不同种类细胞的急性肝衰竭大鼠在一周内的生存曲线，每组5只大鼠。Rat Primary Hepatocyte：大鼠源代肝细胞。
C.移植不同种类细胞的急性肝衰竭大鼠在第三天的血清ALT、AST的指标。不同组别之间的显著性用“*”的数量标示，“ns”代表无显著性。
图3显示了CST1蛋白可以显著降低巨噬细胞(Mac，Macrophage)中IFN-γ激活的下游基因ISGs(Interferon stimulated genes,干扰素激活基因)的表达量，这种抑制的原因是因为CST1能干扰IFN-γ与其受体IFNGR2的结合，从而阻断进一步激活IFNGR2下游的ISGs基因的表达。
A.巨噬细胞中ISGs在加入CST1后的表达变化。体外培养的巨噬细胞中在加入IFN-γ激活下游ISGs表达时，加入CST1可以显著抑制ISGs的表达上升。
B.免疫共沉淀实验鉴定CST1对于IFN-γ与其受体IFNGR2结合效率的影响。IFN-γ抗体用于作为免疫共沉淀的抗体，同时分离IFN-γ和与其结合的IFNGR2，当CST1加入了这个分离体系中，与IFN-γ结合的 IFNGR2的量减少了，反应在western蛋白印迹检测到的IFNGR2/IFN-γ的比值减小，说明了CST1影响了IFN-γ与IFNGR2的结合，降低了两者的结合效率。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次意外地发现CST1蛋白基因、或其蛋白、或其促进剂；或IFN-γ或其受体的抑制剂可显著预防和/或治疗肝脏免疫失调疾病，此外，本发明还意外的发现，CST1蛋白基因、或其蛋白、或其促进剂还可(a)提高哺乳动物的生存率；和/或(b)显著降低血清谷丙转氨酶、谷草转氨酶、和/或血氨的水平；和/或(c)保护肝实质细胞功能；和/或(d)抑制IFN-γ对于巨噬细胞的促炎激活作用；和/或(e)抑制IFN-γ下游的大量ISGs的表达；和/或(f)减弱IFN-γ与IFNGR2结合的效率。此外，本发明还意外发现，过表达CST1就可以使无疗效的细胞获得一定的疗效，这说明了CST1蛋白本身和基于CST1过表达的细胞治疗对于急性肝衰竭的治疗都有着不小的潜力。在此基础上，本发明人完成了本发明。
术语说明
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
如本文所用，术语“给予”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内，肌内，皮下，腹膜内，脊髓或其它肠胃外给药途径，例如通过注射或输注。
CST1蛋白及其编码核酸
如本文所用，术语“CST1蛋白”“Cystatin SN蛋白”、“半胱氨酸蛋白酶抑制SN”可互换使用。
本发明涉及一种CST1蛋白及其变体，在本发明的一个优选例中，所述CST1蛋白的氨基酸序列如SEQ ID NO.:1所示。本发明的CST1蛋白或其促进剂可(a)预防和/或治疗肝脏免疫失调疾病；和/或(b)提高哺乳动物的生存率；和/或(c)显著降低血清谷丙转氨酶、谷草转氨酶、和/或血氨的水平。
本发明还包括与本发明的SEQ ID NO.:1所示序列具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的具有相同或相似功能的多肽或蛋白。
其中，SEQ ID NO.:1为人源CST1蛋白，登录号为NM_001898.2。
所述“相同或相似功能”主要是指：“(a)预防和/或治疗肝脏免疫失调疾病；和/或(b)提高哺乳动物的生存率；和/或(c)显著降低血清谷丙转氨酶、谷草转氨酶、和/或血氨的水平”。
本发明的蛋白可以是重组蛋白、天然蛋白、合成蛋白。本发明的蛋白可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的蛋白可以是糖基化的，或可以是非糖基化的。本发明的蛋白还可包括或不包括起始的甲硫氨酸残基。
本发明还包括具有CST1蛋白活性的CST1蛋白片段和类似物。如本文所用，术语“片段”和“类似物”是指基本上保持本发明的天然CST1蛋白相同的生物学功能或活性的蛋白。
本发明的突变蛋白片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的突变蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的突变蛋白，或(iii)成熟突变蛋白与另一个化合物(比如延长突变蛋白半衰期的化合物，例如聚乙二醇)融合所形成的突变蛋白，或(iv)附加的氨基酸序列融合到此突变蛋白序列而形成的突变蛋白(如前导序列或分泌序列或用来纯化此突变蛋白的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。本发明中，保守性替换的氨基酸最好根据表I进行氨基酸替换而产生。
表I
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还包括与本发明的天然CST1蛋白具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的具有相同或相似功能的多肽或蛋白。在蛋白质变体可以经过若干个(通常为1-60个，较佳地1-30个，更佳地1-20个，最佳地1-10个)取代、缺失或添加至少一个氨基酸所得的衍生序列，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能，在C末端和/或\末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。本发明包括天然CST1蛋白类似物与天然CST1蛋白的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白的类似物包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分了生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述例举的代表性蛋白。
修饰(通常不改变一级结构)形式包括：体内或体外蛋白的化学衍生形式如乙酸化或羧基化。修饰还包括糖基化，如那些在蛋白质合成和加工中进行糖基化修饰。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。此外，还可以对本发明突变蛋白进行修饰。修饰(通常不改变一级结构)形式包括：体内或体外的突变蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在突变蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的突变蛋白。这种修饰可以通过将突变蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的突变蛋白。
本发明还提供了编码CST1蛋白的多核苷酸序列。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括：DNA、基因组DNA或人工合成的DNA，DNA可以是单链的或是双链的。编码成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
在本发明优选例中，编码人CST1蛋白的DNA序列编码SEQ ID NO.:1所示的ECM1蛋白，编码CST1蛋白的多核苷酸序列如SEQ ID NO.:2所示。
其中，SEQ ID NO.:2为人源CST1基因的核苷酸序列。
根据本文所述的核苷酸序列，本技术领域人员可方便地用各种已知方法制得本发明的编码核酸。这些方法例如但不限于：PCR，DNA人工合成等，具体的方法可参见J.萨姆布鲁克，《分子克隆实验指南》。作为本发明的一种实施方式，可通过分段合成核苷酸序列再进行重叠延伸PCR的方法来构建本发明的编码核酸序列。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少 70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的蛋白和多核苷酸优选以分离的形式提供，更佳地，被纯化至均质。
本发明多核苷酸全长序列通常可以通过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有关核苷酸序列，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，或其衍生物)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(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
CST1促进剂
在本发明中，CST1促进剂包括能够在体内或体外提高CST1基因或其蛋白的活性和/或含量的物质。
其中，可通过下述方法增加CST1的表达量：组织自身分泌大量CST1蛋白或人工过表达CST1蛋白、或者人工输送CST1蛋白(比如，用病毒载体，如腺相关病毒载体)或者CST1促进剂。
在本发明中，所述CST1促进剂没有特别限制，只要能够促进CST11的表 达或者增强CST1蛋白活性都在本发明的保护范围内。
在一优选实施方式中，所述CST1促进剂包括小分子化合物。
IFN-γ或其受体
在本发明中，术语“IFN-γ或其受体蛋白”、“IFN-γ或其受体多肽”可互换使用，都指具有IFN-γ或其受体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IFN-γ或其受体蛋白。此外，该术语还包括全长的IFN-γ或其受体及其片段。本发明所指的IFN-γ或其受体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。
IFN-γ或其受体是二型干扰素和二型干扰素在细胞表面的结合受体，包括，但并不限于，IFN-γ、IFNGR1、IFNGR2。
在一优选实施方式中，IFN-γ受体选自下组：IFNGR2、IFNAR1、IFNAR2(其中IFNAR1、IFNAR2为一型干扰素受体)、或其组合。
在本发明中，IFN-γ为人源IFN-γ蛋白，登录号为P01579。
IFN-γ受体为人源IFN－γ受体蛋白，登录号分别如下所示：
IFNGR2:P38484；
IFNGR1:P15260。
在得到了IFN-γ或其受体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IFN-γ或其受体核苷酸序列，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IFN-γ或其受体多肽的多核苷酸(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，IFN-γ或其受体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IFN-γ或其受体编码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。
IFN-γ或其受体的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IFN-γ或其受体发生相互作用的物质，尤其是抑制剂等。
可用于本发明的IFN-γ或其受体抑制剂(或拮抗剂)包括任何可以抑制IFN-γ或其受体基因或其编码蛋白的表达和/或活性的物质。
在本发明中，IFN-γ或其受体抑制剂还包括抑制IFN-γ与其受体的结合的抑制剂(包括抑制降低IFN-γ与其受体结合的效率的抑制剂或降低IFN-γ蛋白与受体蛋白的比例的抑制剂)。
例如，所述IFN-γ与其受体的抑制剂包括小分子化合物、IFN-γ与其受体的抗体、IFN-γ与其受体的核酸的反义RNA、siRNA、shRNA、miRNA、或IFN-γ与其受体的活性抑制剂。
在一种优选的实施方式中，抑制IFN-γ与其受体的方法和步骤包括利用IFN-γ与其受体的抗体中和其蛋白，利用病毒(如腺相关病毒)携带的shRNA或siRNA或CRISPR试剂进行IFN-γ与其受体的基因的沉默。
对IFN-γ与其受体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IFN-γ与其受体的抑制率进行控制和检测。
本发明IFN-γ与其受体蛋白的抑制剂(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IFN-γ与其受体蛋白的表达和/或活性、或抑制IFN-γ与其受体的结合、或降低IFN-γ与其受体结合的效率、或降低IFN-γ蛋白与受体蛋白的比例，从而预防和/或治疗肝脏免疫失调疾病。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。
干细胞
在本发明中，本发明的干细胞为不表达CST1基因或其蛋白的干细胞。
在一优选实施方式中，本发明的干细胞包括但并不限于，胚胎干细胞、间充质干细胞。
药物组合物(细胞制剂)
本发明还提供了一种药物组合物，它含有有效量的(a)CST1基因、或其蛋白或其促进剂或IFN-γ或其受体的抑制剂；(b)任选的不表达CST1基因、或其蛋白的干细胞以及药学上可接受的载体。
通常，可将组分(a)和(b)配制于无毒的、惰性的和药学上可接受的水性载体介质中，如生理盐水中，其中pH通常约为5-8，较佳地，pH约为7-8。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。在本发明的优选实施例中，所述组分(a)的有效量为：100-1000ug/ml，较佳地，200-1000ug/ml，更佳地，300-1000ug/ml；所述组分(b)的有效量为1.0×10 6-1.0×10 8个/ml，较佳地5.0×10 6-1.0×10 8个/ml，更佳地1.0×10 7-1.0×10 8个/ml。优选地，所述的有效量的组分(a)和(b)一次性注射完毕。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无 过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括。在本发明中，可以使用的药学上可接受的载体并没有特别的限制，可以是一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的脂肪间充质祖细胞相互掺和，而不明显降低其治疗效果。本发明药学上可以接受的载体部分例子有生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末，适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。除了上述的常规载体外，也可以根据脂肪间充质祖细胞的性质设计优化的载体。所述的载体优选为输液剂载体和/或注射剂载体。
本发明的药物组合物含有安全有效量的组分(a)和(b)，以及药学上可接受的载体。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。通常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。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的药物制剂还可制成缓释制剂。
本发明所述组分(a)和(b)的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。
本发明的药物组合物优选为静脉注射试剂。在另一优选例中，所述的静脉注射试剂中，组分(a)的浓度为：100-1000ug/ml，较佳地，200-1000ug/ml，更佳地，300-1000ug/ml，所述组分(b)的有效量为1.0×10 6-1.0×10 8个/ml，较佳地5.0×10 6-1.0×10 8个/ml，更佳地1.0×10 7-1.0×10 8个/ml。所述的药物组合物的注射方式没有特别限制，可以是单次注射制剂，也可以是多次注射的制剂组合。在本发明的一种优选实施例中，所述的药物组合物为单次注射剂。
在本发明中，所述的药物组合物优选为静脉注射制剂。
治疗方法
本发明还提供了用本发明的活性成分或相应的药物来(a)预防和/或治疗肝脏免疫失调疾病；和/或(b)提高哺乳动物的生存率；和/或(c)显著降低血清谷丙转氨酶、谷草转氨酶、和/或血氨的水平；和/或(c)保护肝实质细胞功能；和/或(d)抑制IFN-γ对于巨噬细胞的促炎激活作用；和/或(e)抑制IFN-γ下游的大量ISGs的表达；和/或(f)减弱IFN-γ与IFNGR2结合的效率的方法，它包括给哺乳动物施用有效量的活性成分(a)CST1基因、或其蛋白或其促进剂；或IFN-γ或其受体的抑制剂，或者施用含有所述活性成分(a)的药物组合物；和任选的(b)不表达CST1基因、或其蛋白的干细胞，或者施用含有所述活性成分(b)的药物组合物。
当本发明的活性成分被用于上述用途时，可与一种或多种药学上可接受的载体或赋形剂混合，如溶剂、稀释剂等，而且可以用如下形式口服给药：片剂、丸剂、胶囊、可分散的粉末、颗粒或悬浮液(含有如约0.05-5％悬浮剂)、糖浆(含有如约10-50％糖)、和酏剂(含有约20-50％乙醇)，或者以无菌可注射溶液或悬浮液形式(在等渗介质中含有约0.05-5％悬浮剂)进行非肠胃给药。例如，这些药物制剂可含有与载体混合的约0.01-99％，更佳地约为0.1％-90％(重量)的活性成分。
本发明的两种活性成分或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)：肌内、腹膜内、静脉内、皮下、皮内、口服、瘤内或局部给药。优选的给药途径包括口服给药、肌内给药或静脉内给药。
从易于给药的立场看，优选的药物组合物是液态组合物，尤其是注射剂。
此外，本发明的两种活性成分或药物还可与其他预防和/或治疗肝脏免疫失调疾病的药物联合使用。
本发明的主要优点包括：
(1)本发明首次发现，CST1蛋白基因、或其蛋白、或其促进剂或IFN-γ或其受体的抑制剂可显著预防和/或治疗肝脏免疫失调疾病。
(2)本发明首次发现，CST1蛋白基因、或其蛋白、或其促进剂或IFN-γ或其受体的抑制剂还可(a)提高哺乳动物的生存率；和/或(b)显著降低血清谷丙转氨酶、谷草转氨酶、和/或血氨的水平；和/或(c)保护肝实质细胞功能；和/或(d)抑制IFN-γ对于巨噬细胞的促炎激活作用；和/或(e)抑制IFN-γ下游的大量ISGs的表达；和/或(f)减弱IFN-γ与IFNGR2结合的效率。
(3)本发明首次发现，过表达CST1就可以使无疗效的细胞获得一定的疗效，这说明了CST1蛋白本身和基于CST1过表达的细胞治疗对于急性肝衰竭的治疗都有着不小的潜力。
(4)本发明首次证实了CST1蛋白对于急性肝衰竭的治疗疗效，并且CST1蛋白是一个易于生产的蛋白产品，其潜在用途很广泛。
下面结合具体实施，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，否则百分比和份数按重量计算。
实施例中用到的材料和试剂如无特殊说明，均为市售产品。
人类内胚层干细胞获自本实验室自主建成的细胞系，建系方法已发表文章(Cheng,X.,Ying,L.,Lu,L.,Galvao,A.M.,Mills,J.A.,Lin,H.C.,Kotton,D.N.,Shen,S.S.,Nostro,M.C.,Choi,J.K.,et al.(2012).Self-renewing endodermal progenitor lines generated from human pluripotent stem cells.Cell Stem Cell 10,371-384.)。
通用方法
(1)人类内胚层干细胞(hEnSCs)的体外维持与扩增
hEnSCs的体外维持可以通过在表面铺有Matrigel和一定量的辐照后的小鼠胚胎成纤维细胞(Mouse Embryonic Fibroblast，MEF)(3X10 5/10cm-dish)基质的条件培养基中进行培养和传代，进行数量扩增。该条件培养基由无血清培养基(Serum Free Medium,SFD)作为基础培养基，添加了一定量的细胞因子：bFGF(FGF2)(10ng/ml),EGF(10ng/ml),VEGF(10ng/ml)和BMP4(10ng/ml)以及小分子化合物Kartogenin(1.5μM/ml)。1L SFD由250ml Ham’s F12,750ml Homemade IMDM，5ml 10％BSA，5ml N2 supplement,10ml B27 supplement组成。每2天换一次培养基，每5天传代一次，传代一般以1:4或1:5的比例进行，细胞需要消化5min后用移液管吹打成单细胞与MEF混合再铺到新的培养皿上，以1.5-2 X 10 6/10cm-dish进行铺板。
(2)人多能干细胞/胚胎干细胞(Human Pluripotent Stem Cells/Human Embryonic Stem Cells；hPSCs/hES)的体外维持与扩增
hES的品系为H9，是实验室建立的稳定的人胚胎干细胞系，建立方法参照如下文献(Cheng,X.,Ying,L.,Lu,L.,Galvao,A.M.,Mills,J.A.,Lin,H.C.,Kotton,D.N.,Shen,S.S.,Nostro,M.C.,Choi,J.K.,et al.(2012).Self-renewing endodermal progenitor lines generated from human pluripotent stem cells.Cell Stem Cell 10,371-384.)。培养hES需要在组织培养皿中预先铺好一层用培养基1:6稀释的Matrigel，并预先加入8X10 5/10cm-dish的辐照后的小鼠胚胎成纤维细胞(Mouse Embryonic Fibroblast，MEF)，然后再种入细胞。细胞的培养基成分为：基础培养基DMEM/F12 50:50(Invitrogen)，15％KOSR(current lot:771421)，1x NEAA，1x P/S，1x L-Glu，1x Sodium Bicarbonate(8ml from stock)，1x Sodium Pyruvate(2.5ml from stock)，1:550 dilution 2ME(55mM stock)。每日换培养基，直至细胞密度到达～90％进行传代。传代时先吸取培养基，加入TRYPLE Express w/Phenol Red消化2min，吸出TRYPLE，在加入hES培养基，用软细胞刮(Soft Cell Scrapers)轻轻刮下细胞，以1:3-1:6的比例传入新的组织培养皿中。
(3)CST1基因慢病毒质粒的克隆与慢病毒的包装，感染hES与筛选CST1过表达的hES流程
Cystatin SN(CST1)蛋白在人的胚胎发育定型内胚层阶段高表达，因此我们取用hES体外诱导分化得到的定型内胚层制成的cDNA，从该cDNA中克隆CST1基因，并连接到慢病毒载体pWPI.1中去。然后我们利用293FT细胞系作为包装CST1过表达病毒的细胞系，采用钙转法将pWPI.1-CST1质粒与包装质粒pspAX2与pMD2.G按比例转染到293FT中，等待2天后收集上清里包装好的病毒，加入hES培养体系中感染2天(1:1病毒上清：hES培养基)。hES感染4天后，在hES培养基中加入1ug/ml浓度的puromycin药筛感染了pWPI.1-CST1的hES细胞，直至换液时不见有大量细胞死亡，剩下的细胞即为CST1过表达的hES系，简称CST1 +ES。
(4)hEnSCs细胞移植动物体内的方法
实验采用WISTAR品系野生型雄性大鼠(Rat)作为实验对象，均是通过肝 门静脉(Portal Vein)注射状态活率良好hEnSC(或hES，或CST1 +ES)的单细胞悬液(PBS为溶液)，注射数量为1X 10 7/Rat。移植手术前需用气体麻醉动物并在手术中用气体麻醉维持，手术中用手术器械打开动物腹腔，开口尽量小，翻开肝门静脉上的肠子，用胰岛素针顺肝门静脉进入肝脏方向进针注射单细胞悬液，抽针后立即用棉签按压进针位置止血，止血后将肠子归位，缝合动物腹腔肌肉和皮肤。动物苏醒后注意观察，无菌环境饲养以防止手术后造成感染。
(5)动物急性肝衰的诱导方法
实验采用化合物D-半乳糖苷盐酸盐(D-Galactosamine hydrochloride,D-GalN)腹腔注射来诱导急性肝衰竭，注射前动物需禁食一天，注射后24小时后进行细胞移植。大鼠的注射用量为1.4g/kg，注射后大鼠在一周内有着很高的死亡率，死亡高峰期在第二，三天。
(6)hEnSCs治疗急性肝衰疗效检测方法
主要分为两项指标：(a)动物的生存率变化，通过观察绘制多只大鼠的一周内的生存曲线变化来判断。(b)动物血清肝功能指标的变化检测，通过心脏采血，采集一周内大鼠的血样对血清AST，ALT以及血氨(Blood Ammonia)，凝血酶原时间(Prothrombin Time)进行检测，比较治疗与不治疗组的肝功能差异。
(7)大鼠巨噬细胞分离方法：
该分离方法基于Percoll TM密度梯度离心进行分离。我们将D-GalN诱导的雄性急性肝衰大鼠，在D-GalN给药诱导两天后，麻醉至于无菌环境中，打开腹腔对肝脏进行灌流。灌流针从大鼠门静脉扎入，下腔静脉开口作为灌流液出口，利用蠕动泵持续稳定灌流液体。先灌流1X Hank's Balanced Salt Solution(HBSS)100ml，约10min灌流完，冲洗掉肝内的血液。然后剪下大鼠肝脏，用1X HBSS洗干净表面血液，用剪刀将肝脏剪碎。对于一只大鼠，剪碎后的肝脏加入20ml含有0.1％Collagenase IV+0.01％DNaseI+5％Fetal Bovine Serum(FBS)+1％Penicillin-Streptomycin(P/S)的RPMI-1640培养基中，37℃水浴缓慢震荡消化一小时。消化后用100μm的滤筛过滤掉未消化组织。300g离 心10min，用20ml 25％percoll溶液重悬，缓慢加入盛有20ml 50％percoll的离心管中，然后放到离心机中，4℃离心，转速1260g，离心时间15min，升降速都调到最低档0档。离心后小心吸出25％percoll与50％percoll之间的一层细胞，然后加入RPMI培养基，再离心洗去残留Percoll溶液，用RPMI+10％FBS+1％P/S重悬，以2.5 X 10 5/well的密度至于六孔板(6-well-dish)中37℃培养半小时，洗去未贴壁细胞，换上新的培养基(同前)，所得贴壁细胞即为巨噬细胞。
(8)巨噬细胞培养方法：
分离所得原代巨噬细胞，立即开始实验。重组蛋白IFN-γ(IFNG)以100ng/ml终浓度加入培养体系中，CST1以500ng/ml终浓度加入培养体系中，培养基为RPMI-1640+10％FBS+1％P/S，培养3天后，吸去培养基，用RNA提取裂解液裂解贴壁细胞，提取RNA，检测不同条件下巨噬细胞内ISGs的表达。
(9)免疫共沉淀:
免疫共沉淀使用了Thermo Fisher的Pierce TM Classic Magnetic IP/Co-IP试剂盒进行，每次实验使用了相同量的巨噬细胞蛋白裂解液(～1000ug)，免疫沉淀的所得产物使用western blot方法及IFN-γ(IFNG)和IFNGR2的抗体检测，灰度分析使用了ImageJ软件进行分析。
实施例1人类内胚层干细胞(hEnSCs)对于急性肝衰竭治疗疗效以及有效成分的分析
为了寻找肝移植的代替疗法治疗急性肝衰竭，而细胞治疗是具有潜力的疗法之一，因此我们在尝试了多种具有潜力的细胞类型后，寻找到了一种新型的，且对急性肝衰竭有良好治疗效果的干细胞系，即人类内胚层干细胞(hEnSCs)。通过在急性肝衰竭大鼠身上的动物实验，我们发现通过肝门静脉移植hEnSCs，可以在一周内显著提高动物的生存率，效果还略优于已知对急性肝衰有一定疗效的间充质干细胞(MSCs)(图1A)。且肝功能指标谷丙转氨酶(ALT)、谷草转氨酶(AST)以及血氨(Blood Ammonia)都有着显著的降低，这表明了移植hEnSCs对于急性肝衰大鼠的肝功能起到了很好的保护作用(图1B)。
既然hEnSCs移植对于急性肝衰竭在动物实验上有着良好的效果，那么为 了有利于治疗急性肝衰竭疾病，hEnSCs治疗急性肝衰竭背后的分子机制更加值得研究。
为了了解hEnSCs治疗急性肝衰竭的独特的分子机制，本发明尝试寻找仅在hEnSCs中特异性高表达的基因，从而筛选可能的关键治疗因子。通过比较hEnSCs，即内胚层干细胞，与其分化路径上下游的细胞：人胚胎干细胞(ES)，人肝母细胞(Hepatoblast)，人肝细胞(Hepatocyte)的单细胞转录组测序数据进行比较，找出了一些hEnSCs特异性高表达的基因(图1C)。而在这些基因中，筛选的对象是具有免疫调节或抗凋亡等作用，对于肝功能有保护等积极作用的基因。通过大量筛选，筛到了CST1基因，全称Cystatin SN，是一个分泌型的蛋白，初步推测CST1可能会在hEnSCs的治疗中起到免疫调节的作用。
实施例2 CST1蛋白对于急性肝衰竭的疗效验证实验
为了验证我们认为可能是hEnSCs治疗急性肝衰竭的关键分子，我们决定在一个不表达且对急性肝衰竭无治疗作用的细胞系中过表达CST1基因，如果该细胞系能够获得一定治疗急性肝衰竭的疗效，那么则说明了CST1基因对于急性肝衰竭有着一定治疗效果。
细胞系是人胚胎干细胞(ES)，然后采用慢病毒包装CST1过表达质粒载体，并感染ES，建立CST1过表达的ES细胞系，即CST1 +ES。然后检测了CST1基因是否在建立的细胞系中过表达，并比较其与hEnSCs，ES，MSC中的表达量。如图2A，CST1+ES过表达CST1基因，但表达量仍然不及hEnSCs高，而ES与MSCs中不表达CST1。
接着我们采用了CST1 +ES去治疗大鼠急性肝衰竭，并与已知有效果的细胞系hEnSCs，Hepatocyte，已知没有效果的细胞系ES进行比较。比较仍然是在生存率和肝功能指标两方面评估。在移植了CST1 +ES的5只急性肝衰大鼠中，一周内有3只存活，而普通ES则5只大鼠全部死亡(图2B)。相比hEnSCs与Hepatocyte仍然有着更好的治疗效果，但是CST1 +ES确实相比普通ES，治疗效果有着显著的提升。而在肝功能指标方面，CST1 +ES虽然比不上hEnSCs与Hepatocyte，却相较普通的ES有着更低的血清ALT、AST。这说明了CST1 +ES对于肝功能也有着一定的保护作用。鉴于CST1 +ES中的CST1表达量仍然不及hEnSCs，但已经获得了不错的治疗效果，这也反应了CST1对于hEnSCs治疗疗效的重要性。在谷丙转氨酶(ALT)和谷草转氨酶(AST)指标上，CST1 +ES相 比于普通ES，也有着显著的降低，这代表着CST1 +ES由于CST1的表达，使之具有了一定的肝功能保护的作用(图2C)。
综上所述，可以确定CST1蛋白对于急性肝衰竭在大鼠身上有着良好的治疗效果，而CST1蛋白对于急性肝衰竭的治疗作用是从未被报道过的。单纯过表达CST1就可以使无疗效的细胞获得一定的疗效，这说明了CST1蛋白本身和基于CST1过表达的细胞治疗对于急性肝衰竭的治疗都有着不小的潜力。
实施例3 CST1作用机理的研究
本发明不仅发现了CST1在移植动物体内对于急性肝衰竭的动物有着免疫调节、增加存活率的作用，而且对于CST1在动物肝内免疫调节的机制进行了一些探究，并阐释了CST1在肝内免疫调节的一些机制。
已知肝内的巨噬细胞(Macrophage)在肝脏内大量存在，且在急性肝衰竭发病过程中起着重要的作用。巨噬细胞在急性肝衰竭过程中呈现较强的促炎作用，会招募其他的免疫细胞如单核细胞，嗜中性粒细胞，辅助性T细胞等等，加重肝脏的负担，造成更加严重的免疫反应，从而加重急性肝衰病情。而干扰素-γ(Interferon-γ，IFNγ)是巨噬细胞的主要促炎激活因子之一，干扰素-γ以及其他类型干扰素信号激活的下游基因(Interferon stimulated genes,ISGs)，统称为ISGs。ISGs在急性肝衰大鼠肝脏巨噬细胞中是高度激活的。
如图3A所示，将急性肝衰竭大鼠肝脏中的巨噬细胞提取出来，在体外进行培养，加入IFN-γ信号，可以显著提高ISGs的RNA水平表达量，模拟体内巨噬细胞的免疫状态。而当加入了500ng/ml浓度的CST1重组蛋白进入巨噬细胞的培养体系中培养3天后，IFN-γ对于巨噬细胞内ISGs的激活作用被大大减弱，图中列出的高表达的ISGs中有Ifit2、Ifit3、Mx2、Irf7、Eif2ak2、Ifitm3、Isg15和Sp100都显著性的下降了。这个实验从细胞层面证明了，CST1可以抑制IFN-γ对于巨噬细胞的促炎激活作用，表现在其可以抑制IFN-γ下游的大量ISGs的表达。
不仅如此，还从分子层面上对于CST1的作用进行了研究。基于CST1能够抑制IFN-γ信号下游的ISGs的表达，那么我们怀疑CST1可能能够影响或者阻断IFN-γ信号通路中的某个环节。因此我们对IFN-γ基因的受体进行了研究，IFN-γ受体有两种不同的亚基IFNGR1和IFNGR2。如图3B左图，利用免疫共沉 淀技术(Co-Immunoprecipitation)，发现IFN-γ与其受体其中一种亚基IFNGR2的结合，在体系中加入了CST1重组蛋白后，结合效率变低了，降低了约50％，如图3B右图所示。具体地，是用IFN-γ(IFNG)的抗体在大鼠巨噬细胞的蛋白裂解液中去共沉淀与IFN-γ能够结合的蛋白，IFNGR2作为IFN-γ的蛋白受体，自然地能和IFN-γ结合，被共沉淀下来，然后用western印记检测到；但如果CST1重组蛋白加入到了大鼠巨噬细胞蛋白裂解中去(5ug加入到1000ug蛋白裂解液中)，IFN-γ与IFNGR2共沉淀后，western印记检测到的IFNGR2蛋白与IFN-γ蛋白的比例，如图3B右图，这说明，CST1蛋白对于IFN-γ与IFNGR2之间的结合有着干扰作用，CST1是通过减弱IFN-γ与IFNGR2结合的效率，从而减弱IFN-γ下游的ISGs表达，达到抑制巨噬细胞促炎激活，从而达到其免疫调节作用的。
讨论
寻找肝移植的替代疗法一直是治疗急性肝衰研究领域的热点，而细胞治疗目前是一种非常具有潜力的治疗策略。但是无论是哪种治疗策略，治疗急性肝衰都依赖于治疗疗效背后的分子机制研究，没有确切的分子机制和有效治疗因子，对于疗法的应用与优化是无法进行的。
发明人不仅发现了hEnSCs细胞水平对于急性肝衰竭的治疗作用，还进一步发现了CST1是hEnSCs治疗急性肝衰竭的关键因子。
CST1是一个被研究的很少的分泌型蛋白，在已知的文献中，CST1是一个半胱氨蛋白酸酶抑制剂，在唾液和泪液中丰富存在，在人的大多数组织里却不表达。CST1对于抗过敏、抗菌有着一定的作用，但是对于急性肝衰竭的疗效从未被人报道过。因此，本发明发现了CST1对于急性肝衰的治疗作用，并发明了基于CST1的急性肝衰竭治疗方法。
由于CST1不表达于人体大部分组织，已知对急性肝衰有治疗作用的细胞如人间充质干细胞(MSCs)等的治疗分子机制都与此不同，这也说明了CST1治疗急性肝衰竭是一种新颖而独特的疗法。
单纯过表达CST1基因就可以使得无治疗疗效的ES细胞获得不错的疗效，那么CST1配合一些本身就具有治疗疗效的细胞如MSCs，可能会获得更好的治疗疗效。
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 (12)

  1. 一种CST1基因、或其蛋白或其促进剂的用途，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/或治疗肝脏免疫失调疾病。
  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)提高哺乳动物的生存率；和/或
    (b)显著降低血清谷丙转氨酶、谷草转氨酶、和/或血氨的水平；和/或
    (c)保护肝实质细胞功能；
    (d)抑制IFN-γ对于巨噬细胞的促炎激活作用；
    (e)抑制IFN-γ下游的大量ISGs的表达；
    (f)减弱IFN-γ与IFNGR2结合的效率。
  3. 如权利要求1所述的用途，其特征在于，所述CST1促进剂选自下组：小分子化合物、表达CST1的载体、或其组合。
  4. 如权利要求1所述的用途，其特征在于，所述的CST1基因、或其蛋白来源于内胚层干细胞、定型内胚层。
  5. 如权利要求1所述的用途，其特征在于，所述CST1蛋白选自下组：
    (A)氨基酸序列如SEQ ID NO.:1所示的多肽；
    (B)将SEQ ID NO.:1所示的氨基酸序列经过一个或几个(通常为1-60个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸残基的取代、缺失或添加而形成的CST1蛋白衍生物，或其活性片段；
    (C)序列与SEQ ID NO.:1所示的氨基酸序列相比，同源性≥90％，较佳地≥95％，更佳地≥98％，最佳地≥99％的CST1蛋白衍生物，或其活性片段。
  6. 一种IFN-γ或其受体的抑制剂的用途，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/或治疗肝脏免疫失调疾病。
  7. 一种细胞制剂，其特征在于，包括：
    (a1)第一药物组合物，所述第一药物组合物含有(a)第一活性成分，所述第一活性成分为CST1基因、或其蛋白或其促进剂或其促进剂；或IFN-γ或其受体的抑制剂，以及药学上可接受的载体；
    (a2)任选的第二药物组合物，所述第二药物组合物含有(b)第二活性成分，所述第二活性成分为不表达CST1基因、或其蛋白的干细胞；以及药学上可接受的 载体。
  8. 如权利要求7所述的细胞制剂，其特征在于，所述的细胞制剂中，所述组分(a1)的浓度为100-1000ug/ml，较佳地，200-1000ug/ml，更佳地，300-1000ug/ml。
  9. 如权利要求7所述的细胞制剂，其特征在于，所述的细胞制剂中，所述组分(a2)的浓度为1.0×10 6-1.0×10 8个/ml，较佳地5.0×10 6-1.0×10 8个/ml，更佳地1.0×10 7-1.0×10 8个/ml。
  10. 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)CST1基因、或其蛋白或其促进剂；或IFN-γ或其受体的抑制剂，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)不表达CST1基因、或其蛋白的干细胞,或含有活性成分(a2)的药物。
  11. 一种筛选预防和/或治疗肝脏免疫失调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CST1基因的细胞一段时间T1，检测测试组所述培养体系中的CST1基因的表达量E1；
    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CST1基因的表达量E2；和
    (b)对E1和E2进行比较，如果E1显著高于E2，则表示所述测试化合物是肝脏免疫失调疾病的潜在治疗剂。
  12. 一种筛选预防和/或治疗肝脏免疫失调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，培养细胞一段时间T1，检测测试组所述培养体系中所述IFN-γ与其受体的结合情况；
    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IFN-γ与其受体的结合情况；
    (b)如果所述测试组中的所述IFN-γ与其受体的结合效率E1显著低于所述对照组中的所述IFN-γ与其受体的结合效率E2，则表示所述测试化合物是候选化合物。
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