WO2021180047A1 - 一种gp73抑制剂在制备治疗糖尿病的药物中的应用 - Google Patents

一种gp73抑制剂在制备治疗糖尿病的药物中的应用 Download PDF

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WO2021180047A1
WO2021180047A1 PCT/CN2021/079613 CN2021079613W WO2021180047A1 WO 2021180047 A1 WO2021180047 A1 WO 2021180047A1 CN 2021079613 W CN2021079613 W CN 2021079613W WO 2021180047 A1 WO2021180047 A1 WO 2021180047A1
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glucagon
diabetes
antibody
seq
inhibitor
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PCT/CN2021/079613
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English (en)
French (fr)
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林长青
孙志伟
高琦
郄霜
徐磊
李靖
林建波
朱恒奇
郑飞
刘雪超
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北京舜景生物医药技术有限公司
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Priority to EP21767174.2A priority Critical patent/EP4116420A4/en
Priority to JP2022543685A priority patent/JP7383163B2/ja
Publication of WO2021180047A1 publication Critical patent/WO2021180047A1/zh
Priority to US17/930,144 priority patent/US20230021840A1/en

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Definitions

  • the invention relates to the field of biomedicine, and in particular to the application of a GP73 inhibitor in the preparation of a medicine for treating diabetes.
  • Diabetes mellitus is a metabolic disease characterized by high blood sugar, which is mainly divided into four categories: type I diabetes, type II diabetes, gestational diabetes and other special types of diabetes.
  • Type I diabetes is insulin-dependent diabetes mellitus, which is mainly caused by islet ⁇ -cell damage mediated by autoimmune response, accounting for 5% to 10% of the total number of diabetic patients.
  • the occurrence and development of type I diabetes are divided into 6 stages: 1 genetic susceptibility; 2 certain environmental factors initiate autoimmune response; 3 autoimmune response period, insulin secretion function is still normal; 4 autoimmune response persists, insulin secretion The function is progressively reduced; 5 Diabetes appears clinically, but part of the insulin secretion function is still retained; 6 Pancreatic ⁇ -cells are completely destroyed.
  • Type II diabetes is mainly caused by the body's lack of insulin and insulin resistance, accounting for 90% to 95% of the total number of patients.
  • the occurrence and development of type II diabetes is divided into 4 stages: 1 genetic susceptibility; 2 hyperinsulinemia and/or insulin resistance; 3 impaired glucose tolerance; 4 clinical diabetes.
  • glucagon is a necessary condition for the onset of diabetes.
  • the main evidence is: 1In the case of insulin deficiency, glucagon increases the production of liver glucose and ketone bodies; 2Various types of Diabetic patients with poor blood sugar control have hyperglucagonemia; 3In glucagon receptor-deficient mice, destroying all ⁇ cells does not cause diabetes; 4In glucagon receptor-deficient mice, pancreas Infusion of anti-insulin serum causes obvious hyperglucagonemia, and insulin in the pancreatic islets has a continuous paracrine inhibitory effect on the secretion of glucagon.
  • Glucagon is mainly a peptide hormone secreted by islet ⁇ cells.
  • Glucagon is a linear polypeptide composed of 29 amino acids and has a molecular weight of 3485 Daltons.
  • Glucagon interacts with its receptor (GCGR) to promote glycogenolysis and gluconeogenesis through cAMP, AMPK, and JNK signaling pathways to increase the concentration of blood glucose.
  • GCGR receptor for Glucagon
  • the liver, brain, gastrointestinal tract, kidney, adipose tissue, heart and other organs are the target organs of glucagon, but the main target organ for blood sugar raising is the liver.
  • diabetes is a pancreatic disease caused by insulin deficiency, insulin resistance, and glucagon excess.
  • drugs for the treatment of diabetes they still focus on insulin, which is difficult to change the gradual deterioration of blood sugar control. More than one-third of patients' blood sugar is not well controlled.
  • the onset of diabetes is the result of a variety of genetic susceptibility and a variety of environmental factors, leading to the heterogeneity of the disease and progressive pathological changes, resulting in the existing treatment methods have significant efficacy limitations.
  • diabetic complications and comorbidities also limit the application of some drugs.
  • some drugs will lose the therapeutic effect, so the continuous development of new diabetes treatment drugs has great practical significance.
  • Golgi protein 73 (Golgi protein 73, GP73) is a type II transmembrane protein located in the Golgi apparatus, also known as Golgi membrane protein 1 (GOLM1) or Golgi phosphor protein 2 (Golgi phosphor protein 2) , GOLPH2). It is located on chromosome 9 and has a length of 3042bp. There are two methionine codons in the same reading frame in the gene, 10 codons apart from each other, and 400 or 391 amino acid products are transcribed respectively.
  • GOLM1 Golgi membrane protein 1
  • GOLPH2 Golgi phosphor protein 2
  • the structure of GP73 is mainly divided into five parts: the cytoplasmic domain at positions 1-12 at the N-terminal, the transmembrane domain at positions 12-35, the coiled-coil domain at positions 36-205, and the amorphous domain at positions 206-348. And the acidic fragment region at positions 349-401. Except that the amorphous domain is a variable region, several other domains are highly conserved.
  • PC proprotein convertase
  • GP73 is almost not expressed in normal liver tissues, but in liver diseases caused by various reasons, almost all liver cells are expressed, especially around connective tissues and nodules in liver cirrhosis.
  • the expression level of GP73 protein in serum and liver tissue is significantly up-regulated, which is 3-5 times that of normal tissues. Therefore, serum GP73 is considered to be an effective serological tumor marker for the diagnosis of liver cancer.
  • the expression of GP73 is significantly up-regulated in prostate tissue of patients with esophageal cancer, breast cancer, and prostate cancer, as well as in urine, bladder cancer, and cervical cancer.
  • GP73 Although the abnormally high expression of GP73 is closely related to a variety of tumors, the biological function of GP73 is not yet fully understood. The function of soluble GP73 outside the cell is poorly understood. There is only one report showing that GP73 in serum mediates the transmission of endoplasmic reticulum stress between hepatocytes and immune cells. This cascade of amplification effects induces tumors. The recruitment of macrophages causes immune tolerance in the tumor microenvironment.
  • GP73 plays a key role in blood glucose regulation.
  • soluble GP73 can specifically bind to glucagon to form a complex, and enhance the blood glucose raising function and gluconeogenesis function of glucagon , Prolong the half-life of glucagon; found that soluble GP73 can activate the production of sugar in the liver and/or kidney and the gluconeogenesis signaling pathway in a way that does not rely on glucagon; the inventor also found that soluble GP73 can cause mice Fasting blood glucose increases, and induces impaired glucose tolerance and pyruvate tolerance; based on the above-discovered regulation of GP73 on blood glucose, the inventors also proved through animal experiments that GP73 inhibitors can reduce blood glucose levels and glycosylated hemoglobin levels in diabetic mice , And has a protective effect on pancreatic islet ⁇ cells, and has a therapeutic effect on diabetes.
  • the first aspect of the present invention is to provide an application of a GP73 inhibitor in the preparation of medicines for the treatment of diabetes and its complications.
  • the second aspect of the present invention is to provide a medicament for the treatment of diabetes and its complications.
  • the medicament includes a GP73 inhibitor as an active ingredient.
  • the third aspect of the present invention is to provide a method for treating diabetes and its complications, including the following steps: administering an effective dose of a GP73 inhibitor to a subject suffering from diabetes.
  • the diabetes includes: type I diabetes, type II diabetes, and gestational diabetes.
  • the treatment of diabetes includes any one or more of the following: (1) reducing fasting and/or postprandial blood sugar; (2) improving glucose tolerance; (3) ) Protect pancreatic islet ⁇ cells and/or pancreatic ⁇ cells; (4) Reduce the glucose-increasing ability and/or gluconeogenesis ability of glucagon; (5) Shorten the half-life of glucagon. (6) Reduce GP73's own non-insulin-dependent glycemic effect and gluconeogenesis function.
  • the treatment of diabetic complications includes any one or more of the following: diabetic nephropathy, diabetic eye complications, diabetic foot, diabetic peripheral neuropathy; among them: diabetic eye Partial complications include one or more of the following: diabetic retinopathy, uveitis associated with diabetes, and diabetic cataract.
  • the GP73 inhibitor includes: polypeptides, proteins, nucleic acid sequences, or small molecule compounds that down-regulate the level, activity, function, and/or stability of GP73; optionally, The polypeptide, protein, nucleic acid sequence or small molecule compound that down-regulates the level, activity, function and/or stability of GP73 has one or more of the following properties: (1) It can inhibit the transcription of the gene encoding GP73 and cut it correctly And/or translation; (2) inhibit or hinder the binding of GP73 to receptors and/or ligands in the body; (3) inhibit or hinder the interaction of GP73 with specific interacting molecules in the body; (4) shorten GP73 Half-life in the body.
  • the GP73 inhibitor includes: anti-GP73 monoclonal antibody or an antibody fragment containing its antigen binding site, an anti-GP73 monoclonal antibody or a fusion protein of an antibody fragment containing its antigen binding site, which specifically inhibits the nucleic acid sequence of GP73 One or more of.
  • the GP73 is selected from one or more of the following: natural or recombinant full-length GP73, GP73 fragments, existing in vivo or isolated in vitro, GP73 mutant or modified GP73; optionally, the GP73 is selected from full-length GP73 or GP73 that does not include amino acids 1-55.
  • the anti-GP73 monoclonal antibody is selected from: monoclonal antibodies produced by hybridoma cells, monoclonal antibodies screened by antibody libraries, and monoclonal antibodies produced by single-cell PCR One or more of genetically engineered monoclonal antibodies, heterologous antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, Nanobodies, and Heavy chain antibodies.
  • the type of the antibody fragment is selected from: Fab, Fab-SH, Fv, scFv, F(ab′) 2 , DsFv, Diabody, Minibody, Tribody, Sc One or more of (Fv) 2 , [Sc(Fv) 2 ] 2 and (ScFv-SA) 4.
  • the nucleic acid that specifically inhibits GP73 includes one or more of siRNA, shRNA, microRNA, antisense oligonucleotide, miRNA, and nucleic acid aptamer
  • the siRNA that specifically inhibits GP73 is selected from one or more nucleotide sequences shown in SEQ ID NO: 1-SEQ ID NO: 9, or selected from those shown in SEQ ID NO: 1-SEQ ID NO: 9 Any one nucleotide sequence has at least 60%, 70%, 80%, 90% homology; further alternatively, the siRNA that specifically inhibits GP73 is selected from the nucleotide sequence shown in SEQ ID NO: 4 or with Sequences with at least 60%, 70%, 80%, 90% homology.
  • the drugs for the treatment of diabetes and its complications also include other drugs for the treatment of diabetes, and the methods for the treatment of diabetes and its complications are combined with a GP73 inhibitor and a drug for the treatment of diabetes.
  • Other drugs include other drugs for the treatment of diabetes, and the methods for the treatment of diabetes and its complications are combined with a GP73 inhibitor and a drug for the treatment of diabetes.
  • the other drugs for the treatment of diabetes are selected from insulin, dimethyl biguanide, sulfonylurea hypoglycemic agents, alpha glycosidase inhibitors, thiazolidinediones, One or more of dipeptidyl peptidase 4 (DPP4) inhibitors, glucagon-like peptide-1 (GLP-1) analogs, and SGLT2 (sodium glucose cotransporter-2) inhibitors.
  • DPP4 dipeptidyl peptidase 4
  • GLP-1 glucagon-like peptide-1
  • SGLT2 sodium glucose cotransporter-2
  • the medicine further includes at least one pharmaceutically acceptable excipient.
  • the use of the drugs is one or more of intravenous injection, intramuscular injection, subcutaneous injection, and oral administration.
  • the subject is selected from one or more of humans, mice, rats, monkeys, rabbits, pigs, and dogs.
  • the fourth aspect of the present invention is to provide an application of a GP73 inhibitor in the preparation of drugs for inhibiting glucagon.
  • the fifth aspect of the present invention is to provide a drug for inhibiting glucagon, and the drug includes a GP73 inhibitor.
  • the sixth aspect of the present invention is to provide a method for inhibiting glucagon, including the following steps: administering an effective dose of a GP73 inhibitor to a subject who needs to inhibit glucagon.
  • the inhibition of glucagon includes any one or more of the following: (1) shortening the half-life of glucagon; (2) reducing glucagon The glycemic ability and/or gluconeogenesis ability of the vegetarian.
  • the GP73 inhibitor includes: polypeptides, proteins, nucleic acid sequences, or small molecule compounds that down-regulate the level, activity, function, and/or stability of GP73; optionally, The polypeptide, protein, nucleic acid sequence or small molecule compound that down-regulates the level, activity, function and/or stability of GP73 has one or more of the following properties: (1) It can inhibit the transcription of the gene encoding GP73 and cut it correctly And/or translation; (2) inhibit or hinder the binding of GP73 to receptors and/or ligands in the body; (3) inhibit or hinder the interaction of GP73 with specific interacting molecules in the body; (4) shorten GP73 Half-life in the body.
  • the GP73 inhibitor includes: anti-GP73 monoclonal antibody or an antibody fragment containing its antigen binding site, an anti-GP73 monoclonal antibody or a fusion protein of an antibody fragment containing its antigen binding site, which specifically inhibits the nucleic acid sequence of GP73 One or more of.
  • GP73 is selected from one or more of the following: natural or recombinant full-length GP73, GP73 fragments, GP73 mutations, existing in vivo or isolated in vitro, Or modified GP73; alternatively, the GP73 is selected from full-length GP73 or GP73 that does not include amino acids 1-55.
  • the anti-GP73 monoclonal antibody is selected from: monoclonal antibodies produced by hybridoma cells, monoclonal antibodies screened by antibody libraries, and monoclonal antibodies produced by single-cell PCR One or more of genetically engineered monoclonal antibodies, heterologous antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, Nanobodies, and Heavy chain antibodies.
  • the type of the antibody fragment is selected from: Fab, Fab-SH, Fv, scFv, F(ab′) 2 , DsFv, Diabody, Minibody, Tribody, Sc One or more of (Fv) 2 , [Sc(Fv) 2 ] 2 and (ScFv-SA) 4.
  • the nucleic acid that specifically inhibits GP73 includes one or more of siRNA, shRNA, microRNA, antisense oligonucleotide, miRNA, and nucleic acid aptamer
  • the siRNA that specifically inhibits GP73 is selected from one or more nucleotide sequences shown in SEQ ID NO: 1-SEQ ID NO: 9, or selected from those shown in SEQ ID NO: 1-SEQ ID NO: 9 Any one nucleotide sequence has at least 60%, 70%, 80%, 90% homology; further optionally, the siRNA that specifically inhibits GP73 is selected from the nucleotide sequence shown in SEQ ID NO: 4 or with Sequences with at least 60%, 70%, 80%, 90% homology.
  • the medicine further includes at least one pharmaceutically acceptable excipient.
  • the use of the drugs is one or more of intravenous injection, intramuscular injection, subcutaneous injection, and oral administration.
  • the subject is selected from one or more of humans, mice, rats, monkeys, rabbits, pigs, and dogs.
  • the seventh aspect of the present invention is to provide a GP73-glucagon complex, wherein: GP73 is combined with glucagon.
  • the GP73 is selected from one or more of the following: natural or recombinant full-length GP73 that is present in the body or isolated in vitro, GP73 fragment, GP73 mutant or modified GP73; optionally, the GP73 is selected from full-length GP73 or GP73 that does not include amino acids 1-55.
  • the species source of the GP73 is selected from one or more of human, mouse, rat, monkey, rabbit, pig, and dog.
  • the eighth aspect of the present invention is to provide a method for determining the binding epitope of GP73 and glucagon, which includes the following steps: using complex crystal analysis method, epitope determining site excision method (Epitope Excision), hydrogen tritium One or more of Hydrogen/Deuterium Exchange and Peptide-Panning method determine the binding epitope of GP73 and glucagon.
  • the ninth aspect of the present invention is to provide a method for determining the inhibitory effect of a GP73 inhibitor in inhibiting the formation of the GP73-glucagon complex, which includes any one of the following two methods:
  • the source of candidate GP73 inhibitors includes one or more selected from the following: Hybridoma cells, B cells, memory B cells, antibody libraries, compound libraries, GP73 analogs, glucagon analogs.
  • the method for determining the strength of the inhibitory effect of GP73 inhibitors in inhibiting the formation of GP73-glucagon complex includes methods selected from the following One or more of: surface plasmon resonance (SPR) assay, micro thermophoresis (MST) assay, competitive ELISA.
  • SPR surface plasmon resonance
  • MST micro thermophoresis
  • the tenth aspect of the present invention is to provide an application of a reagent for detecting GP73 in preparing a reagent for detecting diabetes.
  • the eleventh aspect of the present invention is to provide a reagent for detecting diabetes, the reagent including a reagent for detecting GP73.
  • the diabetes includes: type I diabetes, type II diabetes, and gestational diabetes.
  • the reagent for detecting GP73 includes a reagent for detecting soluble GP73 in serum.
  • the twelfth aspect of the present invention is to provide an application of a GP73 inhibitor in the preparation of drugs for gluconeogenesis signal pathway inhibitors.
  • the thirteenth aspect of the present invention is to provide a medicament for inhibiting the gluconeogenesis signal pathway, and the medicament includes a GP73 inhibitor.
  • the fourteenth aspect of the present invention is to provide a method for inhibiting the gluconeogenesis signaling pathway, including the following steps: administering an effective dose of a GP73 inhibitor to a subject who needs to inhibit the gluconeogenesis signaling pathway.
  • the inhibiting gluconeogenesis signaling pathway includes any one or more of the following: (1) inhibiting hepatocyte gluconeogenesis to produce sugar; (2) down-regulating sugar The expression levels of key xenobiotic enzymes Pcx, Pck1, G6pc; (3) Down-regulate the phosphorylation level and kinase activity of PKA.
  • the GP73 inhibitor includes: polypeptides, proteins, nucleic acid sequences, or small molecule compounds that down-regulate the level, activity, function, and/or stability of GP73; optionally, The polypeptide, protein, nucleic acid sequence or small molecule compound that down-regulates the level, activity, function and/or stability of GP73 has one or more of the following properties: (1) It can inhibit the transcription of the gene encoding GP73 and cut it correctly And/or translation; (2) inhibit or hinder the binding of GP73 to receptors and/or ligands in the body; (3) inhibit or hinder the interaction of GP73 with specific interacting molecules in the body; (4) shorten GP73 Half-life in the body.
  • the GP73 inhibitor includes: anti-GP73 monoclonal antibody or an antibody fragment containing its antigen binding site, an anti-GP73 monoclonal antibody or a fusion protein of an antibody fragment containing its antigen binding site, which specifically inhibits the nucleic acid sequence of GP73 One or more of.
  • GP73 is selected from one or more of the following: natural or recombinant full-length GP73, GP73 fragments, GP73 mutations, existing in vivo or isolated in vitro, Or modified GP73; alternatively, the GP73 is selected from full-length GP73 or GP73 that does not include amino acids 1-55.
  • the anti-GP73 monoclonal antibody is selected from: monoclonal antibodies produced by hybridoma cells, monoclonal antibodies screened by antibody libraries, and monoclonal antibodies produced by single-cell PCR One or more of genetically engineered monoclonal antibodies, heterologous antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, Nanobodies, and Heavy chain antibodies.
  • the type of the antibody fragment is selected from: Fab, Fab-SH, Fv, scFv, F(ab′) 2 , DsFv, Diabody, Minibody, Tribody, Sc One or more of (Fv) 2 , [Sc(Fv) 2 ] 2 and (ScFv-SA) 4.
  • the nucleic acid that specifically inhibits GP73 includes one or more of siRNA, shRNA, microRNA, antisense oligonucleotide, miRNA, and nucleic acid aptamer
  • the siRNA that specifically inhibits GP73 is selected from one or more nucleotide sequences shown in SEQ ID NO: 1-SEQ ID NO: 9, or selected from those shown in SEQ ID NO: 1-SEQ ID NO: 9 Any one nucleotide sequence has at least 60%, 70%, 80%, 90% homology; further alternatively, the siRNA that specifically inhibits GP73 is selected from the nucleotide sequence shown in SEQ ID NO: 4 or with Sequences with at least 60%, 70%, 80%, 90% homology.
  • the medicine further includes at least one pharmaceutically acceptable excipient.
  • the use of the drugs is one or more of intravenous injection, intramuscular injection, subcutaneous injection, and oral administration.
  • the subject is selected from one or more of humans, mice, rats, monkeys, rabbits, pigs, and dogs.
  • GP73 plays a key role in blood glucose regulation.
  • soluble GP73 can specifically bind to glucagon to form a complex, and enhance the blood glucose raising function and sugar of glucagon.
  • GP73 can activate the production of liver sugar and the activation of gluconeogenesis signaling pathways in a glucagon-independent way; the inventors also found that soluble GP73 can cause mice Fasting blood glucose increases, and induces impaired glucose tolerance and pyruvate tolerance; based on the above-discovered regulation of GP73 on blood glucose, the inventors also proved through animal experiments that GP73 inhibitors can reduce blood glucose levels and glycosylated hemoglobin levels in diabetic mice , And has a protective effect on pancreatic islet ⁇ cells, and has the effect of treating diabetes, such as by blocking and/or neutralizing GP73 by anti-GP73 monoclonal antibody, or treating diabetes by down-regulating the level of GP73, or by RNA interference The method specifically reduces the expression of GP73 to treat diabetes.
  • GP73 inhibitors can also be used to prepare drugs for inhibiting glucagon.
  • the inventors proved the existence of the GP73-glucagon complex, which has guiding significance for the further study of the interaction between GP73 and glucagon.
  • the inventor found that the expression level of GP73 in diabetic people is significantly higher than that in healthy people, so GP73 can be used as a marker for diabetes detection.
  • Figure 1A shows the gender and age matching of the healthy population and the diabetic population in Example 1 of the present invention. The results show that there is no significant difference in the distribution of gender and age between the two groups of people; Figure 1B shows this The soluble GP73 protein level in serum of healthy people and diabetic people in Example 1 of the invention showed that the level of serum soluble GP73 protein in diabetic people was significantly higher than that in healthy people on physical examination (P ⁇ 0.01).
  • FIG. 2A shows the fasting blood glucose levels of the recombinant mouse soluble GP73 (rmsGP73) injection experimental group and the PBS control group in Example 2 of the present invention.
  • the results show that the fasting blood glucose of the rmsGP73 injection experimental group is significantly higher than that of the PBS control Group (*, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001)
  • Figure 2B shows the intraperitoneal glucose tolerance of mice in the rmsGP73 injection experimental group and the PBS control group in Example 2 of the present invention IPGTT level, the results showed that the mice in the rmsGP73 injection experimental group showed abnormal IPGTT (*, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001)
  • Figure 2C shows rmsGP73 in Example 2 of the present invention The pyruvate tolerance PTT levels of the mice in the injection experimental group and the PBS control group showed that the PTT of the mice in the rmsGP73 injection experimental group was abnormal (
  • Figure 3A shows the fluorescence distribution of the fluorescent dye Cy7 control group and Cy7-labeled rmsGP73 protein (rmsGP73-Cy7) experimental group in Example 3 of the present invention after intravenous injection of mice. The results show that 30 minutes after rmsGP73 protein injection, The accumulation in liver and kidney is obvious;
  • Figure 3B shows the distribution of fluorescence in various organs after intravenous injection of Cy7 control group and rmsGP73-Cy7 experimental group in Example 3 of the present invention. The dyes are widely distributed differently.
  • the rmsGP73 protein mainly aggregates in the liver and kidney;
  • Figure 3C shows the fluorescence intensity values of various organs after intravenous injection of mice in the Cy7 control group and the rmsGP73-Cy7 experimental group in Example 3 of the present invention.
  • the results show , The fluorescence intensity values in the liver, kidney and spleen in the rmsGP73-Cy7 group were significantly higher than those in the control group.
  • Figure 4A shows the binding and dissociation curve of recombinant human soluble GP73 (rhsGP73) and glucagon (GCG) measured by Reichert 4SPR in Example 4 of the present invention.
  • Figure 4B shows the binding and dissociation curve of recombinant mouse soluble GP73 (rmsGP73) and GCG in Reichert 4SPR in Example 4 of the present invention.
  • Figure 4C shows the binding and dissociation curve of recombinant rat soluble GP73 (rrsGP73) and GCG in Example 4 of the present invention.
  • Figure 4D shows the binding and dissociation curve of recombinant monkey soluble GP73 (rMsGP73) and GCG in Reichert4SPR in Example 4 of the present invention.
  • Figure 4E shows the in vivo binding capacity of soluble GP73 and GCG in the co-immunoprecipitation experiment in Example 4 of the present invention. The results show that there is a specific interaction between soluble GP73 and GCG in mouse serum, and this interaction follows fasting. The extension of time gradually strengthened.
  • Figure 5A shows the effect of rmsGP73 on the half-life of GCG determined in Example 5 of the present invention.
  • the results show that rmsGP73 can significantly prolong the half-life of GCG (*, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001);
  • Figure 5B shows the blood glucose of mice in the GCG experimental group, rmsGP73 experimental group, and rmsGP73+GCG experimental group in Example 5 of the present invention.
  • the results show that the recombinant mouse soluble GP73 protein can significantly promote GCG in mice.
  • Figure 6A shows the effect of different concentrations of rmsGP73 on the sugar production of primary hepatocytes in mouse primary hepatocytes in Example 6 of the present invention under the condition of serum culture. The results show that rmsGP73 promotes mouse primary hepatocytes in a dose-dependent manner.
  • Fig. 6B shows the effect of different concentrations of rmsGP73 on the sugar production of primary hepatocytes under serum-free culture conditions of mouse primary hepatocytes in Example 6 of the present invention. The results show that the rmsGP73 protein is in the absence of serum.
  • FIG. 6C shows the effect of anti-GP73 antibody 6B6 on rmsGP73 of mouse primary hepatocytes in Example 6 of the present invention under serum-free culture conditions.
  • the results show that 6B6 can specifically block the sugar production of primary hepatocytes promoted by rmsGP73
  • Figure 6D shows the effect of mouse primary hepatocytes in Example 6 of the present invention. Under serum-free culture conditions, the effect of rmsGP73 on the rate-limiting enzymes of gluconeogenesis in primary hepatocytes.
  • Figure 7A shows the fasting blood glucose levels of the mouse IgG and anti-GP73 antibody 6B6 30mg/kg injection group in Example 7 of the present invention every week after the injection. The results show that the high dose of 6B6 significantly reduces the blood glucose of type I diabetic mice.
  • Figure 7B shows the injection of mouse IgG and 6B6 antibody at different doses of 7.5, 15, 30 mg/kg in Example 7 of the present invention
  • the level of glycosylated hemoglobin HbA1c in the fourth week after the injection showed that the middle and high doses of anti-GP73 antibody 6B6 had a significant reduction effect on glycosylated hemoglobin in type I diabetic mice (*, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001).
  • Figure 8A is a three-color immunofluorescence staining diagram of mice in the STZ+IgG group and STZ+6B6 group in Example 8 of the present invention.
  • the results show that the anti-GP73 antibody 6B6 has obvious effects on pancreatic islet ⁇ cells and ⁇ cells in type I diabetic mice Protective effect;
  • Fig. 8B-C is a count chart of islet ⁇ cells and islet ⁇ cells of STZ+IgG and STZ+6B6 mice in Example 8 of the present invention.
  • Figure 9A shows the immunoblotting image of 9 GP73 siRNA transfected into H22 cells in Example 9 of the present invention. The results show that different sequences have different knockdown effects on the level of endogenous GP73 protein in the cell. Sequence 4 was selected as a candidate siRNA; Figure 9B shows the fasting blood glucose values of mice in the control (Ctr siRNA) group and the GP73 siRNA group in Example 9 of the present invention at the fourth week after injection.
  • GP73 refers to Golgi transmembrane glycoprotein 73 (GP73), also known as GOLM1 (Golgi membrane protein 1) or GOLPH2 (Golgi phosphorprotein 2).
  • PC proprotein convertase
  • GP73 generally refers to: natural or recombinant full-length GP73, GP73 fragments, GP73 mutants, or modified GP73 that are present in the body or isolated in vitro; alternatively, the GP73 is selected from the group consisting of Long GP73 or GP73 that does not include amino acids 1-55.
  • GP73 When the word "GP73" is collocated with other words, it also has the meaning defined here.
  • GP73 in GP73 inhibitor, anti-GP73 antibody, and anti-GP73 monoclonal antibody all have the meaning defined here.
  • GP73 inhibitor refers to any polypeptide, protein, nucleic acid sequence or small molecule compound that can down-regulate GP73 level (including gene level or protein level), activity, function and/or stability; optionally
  • the polypeptide, protein, nucleic acid sequence or small molecule compound that down-regulates the level, activity, function, and/or stability of GP73 has one or more of the following properties: (1) It can inhibit the transcription of the gene encoding GP73 and cut correctly. Cut and/or translation; (2) inhibit or hinder the binding of GP73 to receptors and/or ligands in the body; (3) inhibit or hinder the interaction of GP73 with specific interacting molecules in the body; (4) shorten The half-life of GP73 in the body.
  • GP73 inhibitors include but are not limited to anti-GP73 antibodies (including anti-GP73 monoclonal antibodies, bispecific antibodies, multispecific antibodies, and antibody fusion proteins), siRNA that specifically inhibits GP73, shRNA that specifically inhibits GP73, and specific inhibition The microRNA of GP73, the antisense oligonucleotide that specifically inhibits GP73, the nucleic acid aptamer that specifically inhibits GP73, the small molecule compound that specifically inhibits GP73, and the small molecule compound that specifically inhibits GP73.
  • antibody refers to an immunoglobulin molecule composed of four polypeptide chains, and the four polypeptide chains refer to two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • the term "monoclonal antibody” refers to an antibody that is highly uniform and only directed against a specific epitope, which can be achieved by known techniques such as hybridoma technology, antibody library technology, transgenic mouse technology or single-cell PCR technology. To prepare.
  • chimeric antibody refers to the use of DNA recombination technology to insert the light and heavy chain variable region genes of a heterologous monoclonal antibody into an expression vector containing the constant region of a human antibody to transform mammalian cells to express chimeric
  • the variable regions of the light and heavy chains in the antibody molecule expressed in this way are heterologous, while the constant regions are of human origin, so that nearly two-thirds of the entire antibody molecule is of human origin.
  • the antibody produced in this way reduces the immunogenicity of the heterologous antibody while retaining the ability of the parent antibody to specifically bind to the antigen.
  • the term "humanized antibody” refers to the fact that the FR in the variable region of the chimeric antibody still retains a certain degree of immunogenicity.
  • genetic engineering technology is used on the basis of the chimeric antibody.
  • the term "fully human antibody” refers to the transfer of all human antibody-encoding genes to genetically engineered animals with antibody gene deletion through transgenic or chromosome technology, so that the animals can express human antibodies to achieve a fully human source of antibodies.
  • the purpose or monoclonal antibodies obtained through screening of the human antibody library, or human monoclonal antibodies obtained through single-cell PCR technology.
  • antigen-binding fragment of an antibody refers to a part of a full-length antibody, usually a target binding region or a variable region.
  • treatment refers to the ability to produce beneficial or desired results, including but not limited to: prevention, alleviation, amelioration or cure of one or more symptoms, reduction of the severity of the disease, and survival compared to expected survival extend.
  • the term "effective dose” refers to an amount sufficient to effectively deliver the active ingredient for the treatment of diseases when the active ingredient is administered by the method of the embodiment of the present invention, and it can also be a single or multiple administration of the active ingredient.
  • the effective dose can be determined by the participating clinician as a person skilled in the art through known techniques and observations under similar circumstances.
  • the participating clinicians should consider a variety of factors, including but not limited to: mammalian species; size, age, and general health; specific diseases involved The degree of involvement or severity of the disease; the response of the individual patient; the specific compound administered; the mode of administration; the bioavailability properties of the administered preparation; the selected dosing regimen; the use of concomitant drug therapy; and others The relevant situation.
  • pharmaceutically acceptable excipients can be pharmaceutical carriers, excipients and other additives used in conventional formulations, such as common antibody drug excipients.
  • the detection method of soluble GP73 in human serum is as follows: a simple random sampling method is used to select 190 diabetic patients from the Endocrinology Department of multiple physical examination centers at the same time, and 75 health examinations are randomly selected by multiple physical examination centers at the same time. The one for comparison. Both patients and healthy people were given intravenous blood samples overnight on an empty stomach, and the serum was stored in a low-temperature refrigerator at -20°C. After all the samples were collected, a kit based on magnetic particle chemiluminescence immunoassay (purchased from Hotview Bio) was used to detect the level of soluble GP73 in human serum.
  • the mouse glucose detection method is as follows: all blood samples are collected from the tail, and the glucose oxidase method is used to determine the blood glucose using an automatic blood glucose meter (ACCU-CHEK; Roche); normal mice and diabetic children The rats were fasted for 6 hours, and the fasting blood glucose was measured; the random blood glucose level was measured at 9 o'clock in the morning; when the blood glucose level was greater than 35 mM (the upper limit of blood glucose meter detection), the value was recorded as 35 mM.
  • ACCU-CHEK automatic blood glucose meter
  • the detection method of the glucose metabolism experiment is as follows: in the glucose, insulin and pyruvate tolerance test, the mice are fasted for 12 hours, and then intraperitoneally injected with D-glucose (Sigma, article number: G8270, 1.5g/ kg body weight), tail vein injection of insulin (Sigma, product number: I9278, 0.75U/kg body weight) or sodium pyruvate (Sigma, product number: P2256, 2g/kg body weight); after injection at 0 minutes, 15 minutes, 30 minutes, At 45 minutes, 60 minutes, 120 minutes, and 24 hours, mice were subjected to tail vein blood sampling, blood glucose was measured, and intraperitoneal injection glucose tolerance (IPGTT), pyruvate tolerance (PTT) and insulin glucose tolerance (ITT) were determined.
  • D-glucose Sigma, article number: G8270, 1.5g/ kg body weight
  • tail vein injection of insulin Sigma, product number: I9278, 0.75U/kg body weight
  • sodium pyruvate Sigma, product number: P
  • the detection method of the GP73 glycemic test is as follows: select male C57BL/6N mice, fast for 6 hours, and inject rmsGP73 (80 ⁇ g/kg body weight) into the tail vein; Minutes, 60 minutes, 90 minutes, and 120 minutes were used to collect blood from the tail vein of the mice to measure blood glucose.
  • the detection method of the mouse in vivo imaging experiment is as follows: select male C57BL/6N mice, fast overnight, and shave their whole body one day in advance; label rmsGP73 with Cy7 dye, and use a 96-well plate to remove Cy7 The fluorescence intensity of the dye control and the rmsGP73-Cy7 protein are adjusted to the same.
  • the Cy7 dye and rmsGP73-Cy7 with adjusted fluorescence intensity are injected into the mice through the tail vein (ie the Cy7 control group and the rmsGP73-Cy7 experimental group, each group has 4 Only, the injection dose is 200ul/mouse), the mouse is anesthetized in an anesthesia box, and the mouse is imaged in vivo in the in vivo imaging instrument at 0 minutes and 30 minutes after the injection, and the fluorescence distribution in the mouse is observed; then the mouse is quickly put to death
  • the white fat, muscle, liver, spleen, pancreas, kidney, and brain of the mouse were taken out and placed neatly into a mouse imager to observe the fluorescence intensity of each organ.
  • the detection method of the micro thermal surge experiment is as follows: the recombinant soluble GP73 with His tag and the RED-tris-NTA marker are incubated for 30 minutes in the dark at room temperature; 15 concentration gradients are prepared in a PCR tube Mix different concentrations of GCG and labeled GP73 protein and incubate at room temperature for 30 minutes. Use a capillary tube to draw the above-mentioned mixed liquid and inject it into the capillary column cartridge in turn; use a micro thermal surge instrument (NanoTemper), select NT115 mode The binding of GP73 protein and GCG was detected, and the affinity value was calculated according to the fitted curve.
  • the detection method of the OpenSPR experiment is as follows: AmineSensorChips (AmineSensorChips, article number: SEN-AU-100-3-AMINE, lot:#SAB0122, Nicoya) is coupled with recombinant soluble GP73, pancreas with different concentration gradients.
  • Glucagon (HY-P0082, lot: #34006) is the mobile phase, and its binding and dissociation curves are measured by openSPR (OpenSPR-XT, a product of Nicoya), and the affinity value is obtained by curve fitting.
  • the detection method of the immunoprecipitation experiment is as follows: blood is collected from the retro-orbital venous plexus, the mouse blood is collected and centrifuged to obtain mouse serum; anti-mouse GP73 antibody (Santa Cruz, catalog number: sc-365817) ), anti-glucagon antibody (Abcam, product number: ab92517), RRX ⁇ S/T antibody (CST company, product number: 9624), PKA-p antibody (CST company, product number: 5661), ⁇ -Tubulin antibody (Sigma Aldrich, Product number: T9026) or PKA (CST company, product number: 5842) antibody.
  • the method for detecting the half-life of glucagon is as follows: Female C57BL/6N mice are injected with GCG (1 ⁇ g/kg body weight) or GCG (1 ⁇ g/kg body weight) and recombinant mouse soluble GP73 protein (1mg /kg body weight) (incubate at room temperature for 10 minutes). After injection, at 0 minutes, 1 minute, 3 minutes, 5 minutes, 10 minutes, 20 minutes, and 30 minutes, blood was collected from the retro-orbital venous plexus.
  • GCG (purchased from MCE, article number: HY-P0082, lot number: 34006); recombinant mouse soluble GP73 protein rmsGP73, expressed in HEK293 cells in our laboratory (ie, recombinant mouse soluble GP73 in Example 2); protease inhibitor ( Protease Inhibotor cocktail I, purchased from Millipore, article number: 20-201); DPP4 inhibitor (purchased from Millipore, article number: DPP4-010), Aprotinin (purchased from Sigma, article number: A6106).
  • protease inhibitor Protease Inhibotor cocktail I, purchased from Millipore, article number: 20-201
  • DPP4 inhibitor purchased from Millipore, article number: DPP4-010
  • Aprotinin purchased from Sigma, article number: A6106
  • the immunofluorescence staining method is as follows: after the mouse is sacrificed, the stripped pancreatic tissue is fixed with 10% (v/v) formalin and paraffin section specimens are prepared, and paraffin sections with a thickness of 5 ⁇ m are performed; immunofluorescence After the sections are deparaffinized to water, block for 30 minutes, and incubate with anti-mouse insulin antibody (Abcam, catalog number: ab181547) or anti-glucagon antibody (Abcam, catalog number: ab10988) for 1 hour, then repeat washing three times; add the corresponding After incubating the fluorescent secondary antibody for 1 hour at room temperature, it was washed and stained with DAPI; the image was taken under a confocal fluorescence microscope (Zeiss LSM710) or an automatic digital slide scanner (3D HISTECH); 5 to 10 equidistant pancreas were taken from each pancreas Section imaging, at least 3 mice in each group, at least three fields of stained positive cells, and the total number of cells is
  • Example 1 The level of soluble GP73 in serum of diabetic population is significantly higher than that of healthy population
  • the GP73 protein was expressed and purified through the mammalian cell expression system HEK293 cells to obtain genetically recombinant mouse soluble GP73 protein (rmsGP73) (NCBI Reference Sequence: NP_001030294.1).
  • This protein lacks amino acids 1-55 of GP73, and is the main form of soluble GP73 in the blood (the recombinant soluble GP73 protein used in the mouse experiments in the examples of the present invention is the recombinant mouse soluble GP73 protein, referred to as rmsGP73).
  • mice in the injection experimental group were significantly higher than that of the control group (as shown in Figure 2A).
  • the mice in the rmsGP73 injection experimental group showed abnormal glucose tolerance (IPGTT) (as shown in Figure 2B) and abnormal pyruvate tolerance (PTT), which represents the ability of gluconeogenesis ( As shown in Figure 2C), the insulin glucose tolerance test (ITT), which represents insulin sensitivity, is not significantly different from the control mice (as shown in Figure 2D).
  • IPGTT abnormal glucose tolerance
  • PTT abnormal pyruvate tolerance
  • ITT insulin glucose tolerance test
  • ITT insulin glucose tolerance test
  • GIP Gastric inhibitory peptide
  • the central nervous system is an important blood glucose level sensor, which directly or indirectly regulates the secretion of glucagon through the vagus nerve and or cholinergic nerve.
  • the ratio of insulin to glucagon (I/G) in the blood is a key factor in controlling liver glycogen and gluconeogenesis.
  • a high ratio of I/G indicates that energy is in a sufficient state, glycogen synthesis increases, and gluconeogenesis is inhibited. On the contrary, glycogen is decomposed, and gluconeogenesis is enhanced.
  • Hyperglucagonemia is seen in all types of diabetes.
  • the secretion of glucagon is regulated by endogenous insulin, and the loss of this regulation mechanism causes the body to secrete more glucagon, raise blood sugar, and cause diabetes.
  • glucagon is a key factor in the occurrence of hyperglycemia.
  • Normal mice lacking glucagon receptors show symptoms of hypoglycemia, while diabetic mice lacking glucagon receptors ( db/db) does not show symptoms of hyperinsulinism or hyperglycemia.
  • the direct and indirect effects of insulin are impaired, and the enhancement of glucagon signal further aggravates glycogen degradation and gluconeogenesis, which leads to an increase in glucose and blood sugar.
  • Glucagon receptor gene knock-out mice type I diabetes model mice
  • GCGR adenovirus glucagon Receptor
  • glucagon may be a major contributor to hyperglycemia. All research results indicate that glucagon is a key factor in the development of diabetes, and reducing the activity of glucagon will make the treatment of diabetes go further.
  • Recombinant human soluble GP73 (rhsGP73) can specifically bind to GCG.
  • Recombinant mouse (rmsGP73), rat (rrsGP73) and monkey (rMsGP73) soluble GP73 can also specifically bind to GCG (as shown in Figure 4B, C, D and Table 1). All kinds of soluble GP73 are prepared by our company using mammalian cell expression and purification.
  • Monkey soluble GP73 NCBI Reference Sequence: XP_011769391.1; Rat soluble GP73: NCBI Reference Sequence: XP_001056825.3; Mouse soluble GP73: NCBI Reference Sequence: NP_001030294.1; Human soluble GP73: NCBI Reference Sequence: NP_057632.2.
  • Table 1 The affinity (KD) of recombinant soluble GP73 and GCG of different species
  • mice In order to confirm the in vivo binding activity of soluble GP73 and GCG, C57BL/6N mice fasted for different periods of time were taken from blood, and then co-immunoprecipitation experiment was performed.
  • Mouse anti-GP73 monoclonal antibody F12 was purchased from Santa Cruz Company.
  • Recombinant soluble GP73 prolongs the half-life of plasma GCG and promotes the glycemic effect of GCG
  • rmsGP73 can cause elevated fasting blood glucose and impaired glucose tolerance in mice, and sGP73 can specifically bind to GCG, suggesting that sGP73 is likely to function as a partner molecule for GCG in the body to avoid rapid degradation of GCG. Or deamidation and loss of function. For this reason, we conducted an experimental study on the effect of rmsGP73 on the half-life of glucagon.
  • mice used in the experiment were C57BL/6N mice (6-8 weeks old, weight 20-25g, male), 6 mice in each group.
  • the blood glucose meter and blood glucose test paper used to detect blood glucose were purchased from Roche.
  • GCG experimental group mice were injected with GCG (purchased from MCE) through the tail vein at a dose of 100 ng/mouse, and blood glucose was measured at 0, 15, 30, 60, 90, and 120 minutes;
  • rmsGP73 experimental group Mice were injected with rmsGP73 protein via tail vein at a dose of 100 ⁇ g/mouse, and blood glucose was measured at 0, 15, 30, 60, 90, 120 minutes respectively;
  • rmsGP73+GCG experimental group the recombinant mouse soluble GP73 100 ⁇ g/mouse was combined with GCG 100ng/mouse was incubated in vitro for 10 minutes at room temperature and then injected into the tail vein of mice. The blood glucose of the mice was measured at 0, 15, 30, 60, 90, and 120 minutes.
  • mice are C57BL/6N mice (6-8 weeks old, weight 20-25g, male), each group has 6 mice at 0, 15, 30, 60, 90 , 120 minutes of blood sampling to test the blood glucose of the mice.
  • mice were injected with GCG through the tail vein, their blood glucose levels increased sharply, and rmsGP73 significantly promoted the glucose-increasing ability of GCG (as shown in Figure 5B).
  • the glycemic ability of rmsGP73 to promote GCG was blocked by the specific anti-GP73 antibody 6B6 (as shown in Figure 5C).
  • the anti-GP73 monoclonal antibody 6B6 used in the present invention is obtained by immunizing animals with human soluble GP73 antigen and screening hybridoma clones with mouse soluble GP73 antigen.
  • the anti-GP73 monoclonal antibody 6B6 of the present invention was prepared by the following method: the pure human soluble GP73 recombinant antigen (NP_057632.2) was immunized against Balb/c mice, and the spleen cells of the immunized mice and mice Myeloma cells were fused with SP2/0, and mouse soluble GP73 antigen (NP_001030294.1) was used to screen GP73-specific hybridoma monoclonal to establish a stable cell line (this hybridoma cell 6B6G6 (abbreviated as 6B6) was deposited in Chaoyang, Beijing, China The General Microbiology Center of China Microbial Culture Collection Management Committee at No.
  • Example 6 Recombinant soluble GP73 promotes activation of hepatic gluconeogenesis signaling pathway and liver sugar production
  • mice In order to study the direct effects of soluble GP73 on liver gluconeogenesis and liver sugar production, we starved mice for 12 hours, extracted mouse primary hepatocytes, cultured the cells with 10% serum low-sugar DMEM medium, and plated them on Cultivate in 6 cell culture dishes in a cell incubator at 37°C. In the case of serum-free culture, the cells are replaced with serum-free medium after the cells adhere to the wall, and the culture is continued overnight. The experimental cells were cultured with serum and serum-free.
  • the cells were washed twice with PBS, replaced with sugar-free DMEM medium, added with pyruvate and lacto-gluconeogenic raw materials, 6 dishes of cells were divided into control group (with PBS), 3 dishes and experimental group (with GP73 purified protein 16nM) 3 dishes, placed in a 37°C cell incubator, sucked 20ul medium supernatant at 0h, 1h, 2h, 4h, 8h to test the glucose content, and finally collect the cells to test the protein content, and the glucose/protein yields per cell protein.
  • rhsGP73 protein recombinant human soluble GP73 protein
  • CREB transcription factor response element binding protein
  • mice C57BL/6N mice (male, 8 weeks old, weight 20-25g), set up four groups of PBS, rmsGP73, GCG, rmsGP73+GCG, each Group 3 rats; PBS, rmsGP73 (100 ⁇ g/mouse), GCG (100ng/mouse) and rmsGP73+GCG (rmsGP73 100 ⁇ g/mouse and GCG 100ng/mouse, incubated together at room temperature in vitro for 10 minutes and then injected), injected 48 After hours, the mice were sacrificed to separate the liver tissues and perform immunoblotting experiments. Rabbit anti-pCREB polyclonal antibody (purchased from Abcam Company, ab32096) and rabbit anti-CREB monoclonal antibody (purchased from CST Company, #9197) were used in western blotting.
  • mice were injected with intraperitoneal STZ to induce type I diabetes mouse model (6-8 weeks old, weight 20-25g, male), after 10 days of stable period , The mice were randomly assigned to the control mouse IgG group, the 7.5, 15, 30 mg/kg dose 6B6 group, a total of 4 groups, each with 7 mice (the specific grouping situation is shown in Table 3); daily administration through the tail vein until At the end of the experiment, mouse body weight and fasting blood glucose were monitored every 7 days.
  • mice in the 6B6 antibody treatment group were not significantly different from that of the control mice (as shown in Table 4); the 6B6 antibody has a significant effect on reducing fasting blood glucose in mice, and has a dose-dependent effect (as shown in Table 5 and Shown in Figure 7A).
  • Table 3 Treatment grouping of type I diabetic mice with anti-GP73 antibody 6B6
  • Table 5 Fasting blood glucose of type I diabetic mice with anti-GP73 antibody 6B6 for different time
  • HbA1c glycosylated hemoglobin
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • blood lipids The HbA1c detection kit was purchased from Crystal Chem (Cat. No. 80310), and it was detected by TECAN SPARK multifunctional microplate reader.
  • the test kits for liver function and blood lipids were purchased from Rierda Company (article numbers: ALT191230 and AST200218), and were tested with RIELE's Photometer L100 biochemical analyzer.
  • HbA1c In the serum of diabetic control mice treated with IgG, HbA1c averaged 8.78 ⁇ 1.7%; in diabetic mice treated with medium-dose and high-dose 6B6 antibody, HbA1c levels were 6.99 ⁇ 1.6 and 6.71 ⁇ 1.5( Table 6 and Figure 7B) show that the anti-GP73 antibody 6B6 reduces HbA1c levels in STZ-induced T1D mice in a dose-dependent manner.
  • the results of liver function and blood lipids showed that after 4 weeks of continuous injection of the antibody, the medium dose of the antibody significantly reduced the level of AST in mice (Table 6), suggesting that our antibody may have a certain protective effect on STZ-induced liver injury.
  • the levels of triglyceride and cholesterol in the antibody-treated mice were also significantly lower than those in the control group (Table 6).
  • mice were fasted overnight (male, 8-10 weeks old, 20-22 g), and received a single intraperitoneal STZ (175 mg/kg) injection. After the 10-day stabilization period, based on the animal's body weight and fasting blood glucose, the mice were randomly assigned to the mouse IgG control group and the 24 mg/kg dose 6B6 experimental group, with 3 mice in each group, using a computer-generated random process. The drug was administered through the tail vein every day until the end of the experiment.
  • mice were killed 4 weeks after the administration, and the islet tissue was obtained, then formalin-fixed, paraffin sections were prepared, and three-color immunofluorescence staining was used. Among them, DAPI blue is the nucleus, insulin-staining ⁇ cells are green, and glucagon-staining ⁇ cells are red.
  • mice Male, 8-10 weeks old, 20-22g
  • the animals were fasted for 6 hours, and the fasting blood glucose was detected.
  • the blood glucose value was greater than 11.3mM is a successful model.
  • they were randomly divided into the Ctr siRNA group and the GP73 siRNA experimental group, with 6 mice in each group, injected every 5 days.
  • the injection method was first tail vein injection, 4nM each time, and then 4nM intraperitoneally. Four weeks after the first injection, the mice were sacrificed and the blood glucose and glycosylated hemoglobin were tested.
  • mice in the GP73 siRNA injection experimental group showed a significant improvement in glucose tolerance ( Figure 9C-D) and an increase in insulin sensitivity ( Figure 9E-F).
  • HbA1c averaged 4.39 ⁇ 0.67%
  • HbA1c levels were 3.37 ⁇ 0.53%, respectively (Table 9), indicating that GP73 Specific siRNA can reduce HbA1c levels in T2D mice induced by high fat.
  • Table 8 Fasting blood glucose of mice with type II diabetes mellitus antibody treatment at different times
  • Table 9 Levels of glycosylated protein in mice with type II diabetes mellitus antibody treatment at different times
  • the present invention proves that the increase of serum GP73 level is closely related to human diabetes, which is an attractive target for the treatment of diabetes.
  • GP73 interacts with glucagon, which is dependent on and independent of glucagon. It promotes gluconeogenesis in the liver and/or kidneys. Therefore, inhibiting GP73 is an effective strategy for the treatment of diabetes.
  • the embodiment of the present invention provides an application of a GP73 inhibitor in the preparation of a medicine for the treatment of diabetes.
  • the application adjusts blood glucose.
  • the inventors have also proved through animal experiments that the GP73 inhibitor can reduce the blood glucose level of diabetic mice and Glycated hemoglobin level, has a protective effect on pancreatic islet ⁇ cells, and has a therapeutic effect on diabetes.

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Abstract

涉及一种GP73抑制剂在制备治疗糖尿病的药物中的应用。发现了GP73对血糖调节发挥关键作用，特别是，已发现可溶性GP73可以特异性结合胰高血糖素形成复合物，并增强胰高血糖素的升血糖功能和糖异生功能，延长胰高血糖素的半衰期；发现可溶性GP73可以通过不依赖胰高血糖素的方式，激活肝脏和/或肾脏糖的产生以及糖异生信号通路；基于上述发现的GP73对血糖的调节作用，还通过动物实验证明了：GP73抑制剂可以降低糖尿病小鼠血糖水平及糖化血红蛋白水平，并对胰岛β细胞具有保护作用，起到治疗糖尿病的效果。

Description

一种GP73抑制剂在制备治疗糖尿病的药物中的应用
交叉引用
本发明要求在中国专利局提交的、申请号为202010154792.3、发明名称为“一种GP73抑制剂在制备治疗糖尿病的药物中的应用”的中国专利申请的优先权，该申请的全部内容通过引用结合在本发明中。
技术领域
本发明涉及生物医药领域，尤其涉及一种GP73抑制剂在制备治疗糖尿病的药物中的应用。
背景技术
据国际糖尿病联盟估计，2017年全球20～79岁成年人糖尿病患病率约为8.8％，约有4.25亿人，其中，400万人死于糖尿病，占全球死亡人数总量的10.7％。中国作为全球糖尿病大国，患者人数约1.144亿人。这一数字逐年递增，预计到2045年，全球将会有至少6.29亿人处于20～79岁的糖尿病患者。
糖尿病是一种以高血糖为特征的代谢性疾病，主要分为I型糖尿病、II型糖尿病、妊娠糖尿病和其他特殊类型糖尿病等4类。I型糖尿病为胰岛素依赖性糖尿病，主要是由自身免疫反应介导的胰岛β细胞损伤引起，占糖尿病患者总数的5％～10％。I型糖尿病发生发展分为6个阶段：①遗传学的易感性；②某些环境因素启动自身免疫反应；③自身免疫反应活动期，胰岛素分泌功能尚正常；④自身免疫反应持续存在，胰岛素分泌功能进行性降低；⑤临床上出现糖尿病，但尚保留部分胰岛素分泌功能；⑥胰岛β细胞完全破坏。II型糖尿病主要由机体缺乏胰岛素以及胰岛素抵抗引起，占总患者人数的90％～95％。II型糖尿病发生发展分为4个阶段:①遗传学易感性；②高胰岛素血症和/或胰岛素抵抗；③糖耐量减低；④临床糖尿病。
目前研究结果表明，胰高血糖素(Glucagon,GCG)过量是糖尿病发病的必要条件，主要证据有：①在胰岛素缺乏情况下，胰高血糖素增加肝葡萄糖和酮体产生；②各种类型的血糖控制不良的糖尿病患者，存在高胰高血糖素血症；③在胰高血糖素受体缺陷小鼠，破坏所有β细胞并不导致糖尿病；④在胰高血糖素受体缺陷小鼠体内胰腺灌注抗胰岛素血清引起明显高胰高血糖素血症，胰岛内胰岛素对胰高血糖素的分泌有持续的旁分泌抑制作用。胰高血糖素主要是由胰岛α细胞分泌的肽类激素，由29个氨基酸组成的直链多肽，分子量为3485道尔顿。胰高血糖素与其受体(GCGR)相互作用，通过cAMP、AMPK和JNK等信号通路促进糖原分解和糖异生而升高血液中葡萄糖的浓度。肝脏、大脑、胃肠道、肾脏、脂肪组织、心脏等器官是胰 高血糖素的靶器官，但升血糖作用的主要靶器官为肝脏。
因此，糖尿病是胰岛素缺乏和胰岛素抵抗、胰高血糖素过量双激素紊乱性胰腺疾病。目前治疗糖尿病的药物虽然种类比较多，但仍以围绕胰岛素为主，难以改变血糖控制的逐渐恶化，三分之一以上的患者血糖并未得到很好的控制。糖尿病的发病是多种遗传易感性和多种环境因素共同作用的结果，导致了该疾病的异质性和进行性病理改变，致使现有治疗方法对其有显著的疗效局限性。另外，糖尿病并发症和合并症，也限制了一些药物的应用。同时，随着用药时间的延长，一些药物将失去治疗效果，所以不断开发新的糖尿病治疗药物，具有重大的现实意义。
高尔基体蛋白73(Golgi protein 73，GP73)是定位于高尔基体的Ⅱ型跨膜蛋白，又被称为高尔基体膜蛋白1(Golgi membrane protein 1，GOLM1)或者高尔基体磷蛋白2(Golgi phosphorprotein 2，GOLPH2)。它位于第9号染色体，长度为3042bp。其基因内有两个同读码框的甲硫氨酸密码子，相互间隔10个密码子，分别转录出400或391个氨基酸产物。GP73结构主要分为5个部分：N端1-12位的胞质结构域、12-35位的跨膜结构域、36-205位的卷曲螺旋结构域、206-348位的无定形结构域以及349-401位的酸性片段区。除了无定形结构域是可变区之外，其他几个结构域都有极高的保守性。GP73的第55位氨基酸附近有一个前蛋白转化酶(proprotein convertase，PC)的切割位点，全长GP73被PC切割后从高尔基体释放，分泌进入血液循环系统，释放入血的GP73片段被称为可溶性GP73。GP73在正常肝组织中几乎不表达，但在各种原因引发的肝脏疾病中，几乎所有肝细胞均有表达，特别是结缔组织周边和肝硬化结节部位表达尤其强烈。70％以上的肝癌病人，GP73蛋白在血清以及肝组织中的表达水平均显著上调，为正常组织的3-5倍。因此，血清GP73被认为是有效诊断肝癌的血清学肿瘤标志物。除此之外，GP73在食管癌、乳腺癌、前列腺癌病人的前列腺组织以及尿液、膀胱癌和宫颈癌等多种肿瘤中的表达均显著上调。
虽然GP73的异常高表达与多种肿瘤密切相关，但是GP73的生物学功能尚不十分清楚。可溶性GP73在细胞外的功能知之甚少，目前仅有一篇报道，显示血清中的GP73介导了内质网应激在肝细胞-免疫细胞之间的传递，这种级联放大效应诱发了肿瘤巨噬细胞的募集，造成了肿瘤微环境中的免疫耐受。
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
发明目的
本发明实施例中发明人发现GP73对血糖调节发挥关键作用，特别是，已发现可溶性GP73可以特异性结合胰高血糖素形成复合物，并增强胰高血糖素的升血糖功能和糖异生功能，延长胰高血糖素的半衰期；发现可溶性GP73可以通过不依赖胰高血糖素的方式，激活肝脏和/或肾脏中糖的产生以及糖异生信号通路；发明人还发现可溶性GP73可以导致小鼠空腹血糖升高，并诱发糖耐量异常及丙酮酸耐量异常；基于上述发现的GP73对血糖的调节作用，发明人还通过动物实验证明了：GP73抑制剂可以降低糖尿病小鼠血糖水平及糖化血红蛋白水平，并对胰岛β细胞具有保护作用，起到治疗糖尿病的效果。
本发明提供了以下技术方案：
本发明的第一方面在于，提供了一种GP73抑制剂在制备治疗糖尿病及其并发症的药物中的应用。
本发明的第二方面在于，提供了一种用于治疗糖尿病及其并发症的药物，所述药物包括作为活性成分的GP73抑制剂。
本发明的第三方面在于，提供了一种用于治疗糖尿病及其并发症的方法，包括以下步骤：向患有糖尿病的受试者施用有效剂量的GP73抑制剂。
上述应用、药物、方法在一种可能的实现方式中，所述糖尿病包括：I型糖尿病、II型糖尿病、妊娠糖尿病。
上述应用、药物、方法在一种可能的实现方式中，所述治疗糖尿病包括以下的任意一种或多种：(1)降低空腹和/或餐后血糖；(2)改善糖耐量；(3)保护胰岛α细胞和/或胰岛β细胞；(4)降低胰高血糖素的升糖能力和/或糖异生能力；(5)缩短胰高血糖素的半衰期。(6)降低GP73自身的非胰岛素依赖的升糖作用及糖异生的功能。
上述应用、药物、方法在一种可能的实现方式中，治疗糖尿病并发症包括以下的任意一种或多种：糖尿病肾病、糖尿病眼部并发症、糖尿病足、糖尿病周围神经病变；其中：糖尿病眼部并发症包括以下的一种或多种：糖尿病性视网膜病变、与糖尿病相关的葡萄膜炎、糖尿病性白内障。
上述应用、药物、方法在一种可能的实现方式中，所述GP73抑制剂包括：下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物；可选地，所述下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物具有以下性质中的一种或多种：(1)能抑制编码GP73的基因转录、正确剪切和/或翻译；(2)抑制或阻碍GP73与机体内的受体和/或配体结合；(3)抑制或阻碍GP73与机体内的特异性相互作用分子的相 互作用；(4)缩短GP73在机体内的半衰期。进一步可选地，GP73抑制剂包括：抗GP73单克隆抗体或包含其抗原结合部位的抗体片段，抗GP73单克隆抗体或包含其抗原结合部位的抗体片段的融合蛋白，特异性抑制GP73的核酸序列中的一种或多种。
上述应用、药物、方法在一种可能的实现方式中，所述GP73选自以下的一种或多种：机体内存在的或体外分离得到的、天然的或重组的全长GP73、GP73片段、GP73突变体或被修饰过的GP73；可选地，所述GP73选自全长GP73或不包括1-55位氨基酸的GP73。
上述应用、药物、方法在一种可能的实现方式中，所述抗GP73单克隆抗体选自：杂交瘤细胞生产的单克隆抗体、抗体库筛选的单克隆抗体、单细胞PCR生产的单克隆抗体、基因工程改造的单克隆抗体、异源性抗体、嵌合抗体、人源化抗体、全人源抗体、纳米抗体(Nanobody)、重链抗体(Heavy chain antibody)中的一种或多种。
上述应用、药物、方法在一种可能的实现方式中，所述抗体片段的种类选自：Fab、Fab-SH、Fv、scFv、F(ab′) 2、DsFv、Diabody、Minibody、Tribody、Sc(Fv) 2、[Sc(Fv) 2] 2、(ScFv-SA) 4中的一种或多种。
上述应用、药物、方法在一种可能的实现方式中，所述特异性抑制GP73的核酸包括siRNA、shRNA、microRNA、反义寡核苷酸、miRNA、核酸适配体中的一种或多种；可选地，特异性抑制GP73的siRNA选自SEQIDNO:1-SEQ ID NO:9所示的核苷酸序列一条或多条，或选自与SEQIDNO:1-SEQ ID NO:9所示的任意一条核苷酸序列至少有60％、70％、80％、90％同源性的序列；进一步可选地，特异性抑制GP73的siRNA选自SEQIDNO:4所示的核苷酸序列或与其具有至少60％、70％、80％、90％同源性的序列。
上述应用、药物、方法在一种可能的实现方式中，治疗糖尿病及其并发症的药物中还包括治疗糖尿病的其他药物，治疗糖尿病及其并发症的方法中联合使用GP73抑制剂和治疗糖尿病的其他药物。
上述应用、药物、方法在一种可能的实现方式中，所述治疗糖尿病的其他药物选自胰岛素、二甲基双胍、磺脲类降糖药、α糖苷酶抑制剂、噻唑烷二酮类、二肽基肽酶4(DPP4)抑制剂、胰高血糖素样肽-1(GLP-1)类似物、SGLT2(钠葡萄糖共转运蛋白-2)抑制剂中的一种或多种。
上述应用、药物在一种可能的实现方式中，所述药物还包括至少一种药学上可接受的辅料。
上述应用、药物、方法在一种可能的实现方式中，所述药物的使用方式为静脉注射、肌肉注射、皮下注射、口服给药中的一种或多种。
上述方法在一种可能的实现方式中，所述受试者选自人、小鼠、大鼠、猴、兔子、猪、狗 中的一种或多种。
本发明的第四方面在于，提供了一种GP73抑制剂在制备抑制胰高血糖素的药物中的应用。
本发明的第五方面在于，提供了一种用于抑制胰高血糖素的药物，所述药物包括GP73抑制剂。
本发明的第六方面在于，提供了一种用于抑制胰高血糖素的方法，包括以下步骤：向需要抑制胰高血糖素的受试者施用有效剂量的GP73抑制剂。
上述应用、药物、方法在一种可能的实现方式中，所述抑制胰高血糖素包括以下的任意一种或多种：(1)缩短胰高血糖素的半衰期；(2)降低胰高血糖素的升糖能力和/或糖异生能力。
上述应用、药物、方法在一种可能的实现方式中，所述GP73抑制剂包括：下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物；可选地，所述下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物具有以下性质中的一种或多种：(1)能抑制编码GP73的基因转录、正确剪切和/或翻译；(2)抑制或阻碍GP73与机体内的受体和/或配体结合；(3)抑制或阻碍GP73与机体内的特异性相互作用分子的相互作用；(4)缩短GP73在机体内的半衰期。进一步可选地，GP73抑制剂包括：抗GP73单克隆抗体或包含其抗原结合部位的抗体片段，抗GP73单克隆抗体或包含其抗原结合部位的抗体片段的融合蛋白，特异性抑制GP73的核酸序列中的一种或多种。
上述应用、药物、方法在一种可能的实现方式中，GP73选自以下的一种或多种：机体内存在的或体外分离得到的、天然的或重组的全长GP73、GP73片段、GP73突变体或被修饰过的GP73；可选地，所述GP73选自全长GP73或不包括1-55位氨基酸的GP73。
上述应用、药物、方法在一种可能的实现方式中，所述抗GP73单克隆抗体选自：杂交瘤细胞生产的单克隆抗体、抗体库筛选的单克隆抗体、单细胞PCR生产的单克隆抗体、基因工程改造的单克隆抗体、异源性抗体、嵌合抗体、人源化抗体、全人源抗体、纳米抗体(Nanobody)、重链抗体(Heavy chain antibody)中的一种或多种。
上述应用、药物、方法在一种可能的实现方式中，所述抗体片段的种类选自：Fab、Fab-SH、Fv、scFv、F(ab′) 2、DsFv、Diabody、Minibody、Tribody、Sc(Fv) 2、[Sc(Fv) 2] 2、(ScFv-SA) 4中的一种或多种。
上述应用、药物、方法在一种可能的实现方式中，所述特异性抑制GP73的核酸包括siRNA、shRNA、microRNA、反义寡核苷酸、miRNA、核酸适配体中的一种或多种；可选地，特异性抑制GP73的siRNA选自SEQIDNO:1-SEQ ID NO:9所示的核苷酸序列一条或多条，或选自与 SEQIDNO:1-SEQ ID NO:9所示的任意一条核苷酸序列至少有60％、70％、80％、90％同源性的序列；进一步可选地，特异性抑制GP73的siRNA选自SEQIDNO:4所示的核苷酸序列或与其具有至少60％、70％、80％、90％同源性的序列。
上述应用、药物在一种可能的实现方式中，所述药物还包括至少一种药学上可接受的辅料。
上述应用、药物、方法在一种可能的实现方式中，所述药物的使用方式为：静脉注射、肌肉注射、皮下注射、口服给药中的一种或多种。
上述方法在一种可能的实现方式中，所述受试者选自人、小鼠、大鼠、猴、兔子、猪、狗中的一种或多种。
本发明的第七方面在于，提供了一种GP73-胰高血糖素复合物，其中：GP73与胰高血糖素相结合。
上述GP73-胰高血糖素复合物在一种可能的实现方式中，所述GP73选自以下的一种或多种：机体内存在的或体外分离得到的、天然的或重组的全长GP73、GP73片段、GP73突变体或被修饰过的GP73；可选地，所述GP73选自全长GP73或不包括1-55位氨基酸的GP73。
上述GP73-胰高血糖素复合物在一种可能的实现方式中，所述GP73的种属来源选自人、小鼠、大鼠、猴、兔子、猪、狗中的一种或多种。
本发明的第八方面在于，提供了一种确定GP73与胰高血糖素结合表位的方法，其包括以下步骤：利用复合物结晶解析法、表位决定部位切除法(Epitope Excision)、氢氚交换法(Hydrogen/Deuterium Exchange)、Peptide-Panning法中的一种或多种确定GP73与胰高血糖素的结合表位。
本发明的第九方面在于，提供了一种确定GP73抑制剂在抑制GP73-胰高血糖素复合物形成中的抑制作用强弱的方法，其包括以下两种方法中的任意一种：
方法1：
将候选GP73抑制剂与GP73孵育后，再将二者的混合物和/或复合物与胰高血糖素结合；
比较GP73在与候选GP73抑制剂孵育前后与胰高血糖素结合的能力；
方法2：
利用计算机模拟比较GP73在与候选GP73抑制剂孵育前后与胰高血糖素结合的能力。
上述确定GP73抑制剂在抑制GP73-胰高血糖素复合物形成中的抑制作用强弱的方法在一种可能的实现方式中，候选GP73抑制剂的来源包括选自以下的一种或多种：杂交瘤细胞、B细胞、记忆B细胞、抗体库、化合物库、GP73类似物、胰高血糖素类似物。
上述确定GP73抑制剂在抑制GP73-胰高血糖素复合物形成中的抑制作用强弱的方法在一 种可能的实现方式中，确定GP73与胰高血糖素结合的能力的方法包括选自以下方法的一种或多种：表面等离子共振(SPR)测定法、微量热泳动(MST)测定法，竞争ELISA法。
本发明的第十方面在于，提供一种检测GP73的试剂在制备糖尿病检测试剂中的应用。
本发明的第十一方面在于，提供一种用于检测糖尿病的试剂，所述试剂包括检测GP73的试剂。
上述应用、试剂在一种可能的实现方式中，所述糖尿病包括：I型糖尿病、II型糖尿病、妊娠糖尿病。
上述应用、试剂在一种可能的实现方式中，检测GP73的试剂包括检测血清中可溶性GP73的试剂。
本发明的第十二方面在于，提供了一种GP73抑制剂在制备糖异生信号通路抑制剂的药物中的应用。
本发明的第十三方面在于，提供了一种用于抑制糖异生信号通路的药物，所述药物包括GP73抑制剂。
本发明的第十四方面在于，提供了一种用于抑制糖异生信号通路的方法，包括以下步骤：向需要抑制糖异生信号通路的受试者施用有效剂量的GP73抑制剂。
上述应用、药物、方法在一种可能的实现方式中，所述抑制糖异生信号通路包括以下的任意一种或多种：(1)抑制肝细胞糖异生产生糖；(2)下调糖异生关键酶Pcx、Pck1、G6pc表达水平；(3)下调PKA磷酸化水平和激酶活性。
上述应用、药物、方法在一种可能的实现方式中，所述GP73抑制剂包括：下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物；可选地，所述下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物具有以下性质中的一种或多种：(1)能抑制编码GP73的基因转录、正确剪切和/或翻译；(2)抑制或阻碍GP73与机体内的受体和/或配体结合；(3)抑制或阻碍GP73与机体内的特异性相互作用分子的相互作用；(4)缩短GP73在机体内的半衰期。进一步可选地，GP73抑制剂包括：抗GP73单克隆抗体或包含其抗原结合部位的抗体片段，抗GP73单克隆抗体或包含其抗原结合部位的抗体片段的融合蛋白，特异性抑制GP73的核酸序列中的一种或多种。
上述应用、药物、方法在一种可能的实现方式中，GP73选自以下的一种或多种：机体内存在的或体外分离得到的、天然的或重组的全长GP73、GP73片段、GP73突变体或被修饰过的GP73；可选地，所述GP73选自全长GP73或不包括1-55位氨基酸的GP73。
上述应用、药物、方法在一种可能的实现方式中，所述抗GP73单克隆抗体选自：杂交瘤 细胞生产的单克隆抗体、抗体库筛选的单克隆抗体、单细胞PCR生产的单克隆抗体、基因工程改造的单克隆抗体、异源性抗体、嵌合抗体、人源化抗体、全人源抗体、纳米抗体(Nanobody)、重链抗体(Heavy chain antibody)中的一种或多种。
上述应用、药物、方法在一种可能的实现方式中，所述抗体片段的种类选自：Fab、Fab-SH、Fv、scFv、F(ab′) 2、DsFv、Diabody、Minibody、Tribody、Sc(Fv) 2、[Sc(Fv) 2] 2、(ScFv-SA) 4中的一种或多种。
上述应用、药物、方法在一种可能的实现方式中，所述特异性抑制GP73的核酸包括siRNA、shRNA、microRNA、反义寡核苷酸、miRNA、核酸适配体中的一种或多种；可选地，特异性抑制GP73的siRNA选自SEQIDNO:1-SEQ ID NO:9所示的核苷酸序列一条或多条，或选自与SEQIDNO:1-SEQ ID NO:9所示的任意一条核苷酸序列至少有60％、70％、80％、90％同源性的序列；进一步可选地，特异性抑制GP73的siRNA选自SEQIDNO:4所示的核苷酸序列或与其具有至少60％、70％、80％、90％同源性的序列。
上述应用、药物在一种可能的实现方式中，所述药物还包括至少一种药学上可接受的辅料。
上述应用、药物、方法在一种可能的实现方式中，所述药物的使用方式为：静脉注射、肌肉注射、皮下注射、口服给药中的一种或多种。
上述方法在一种可能的实现方式中，所述受试者选自人、小鼠、大鼠、猴、兔子、猪、狗中的一种或多种。
有益效果
(1)本发明实施例中发明人发现GP73对血糖调节发挥关键作用，特别是，已发现可溶性GP73可以特异性结合胰高血糖素形成复合物，并增强胰高血糖素的升血糖功能和糖异生功能，延长胰高血糖素的半衰期；发现可溶性GP73可以通过不依赖胰高血糖素的方式，激活肝脏糖的产生以及糖异生信号通路的激活；发明人还发现可溶性GP73可以导致小鼠空腹血糖升高，并诱发糖耐量异常及丙酮酸耐量异常；基于上述发现的GP73对血糖的调节作用，发明人还通过动物实验证明了：GP73抑制剂可以降低糖尿病小鼠血糖水平及糖化血红蛋白水平，并对胰岛β细胞具有保护作用，起到治疗糖尿病的效果，如通过以下方式：通过抗GP73单克隆抗体阻断和/或中和GP73，或者通过下调GP73水平治疗糖尿病，或者通过RNA干扰的方式特异性降低GP73的表达量以治疗糖尿病。
(2)本发明实施例中发明人首次发现GP73与胰高血糖素的相互结合及其对胰高血糖素功效的影响，因此还可以将GP73抑制剂用于制备抑制胰高血糖素的药物。本发明实施例中发明 人证明了GP73-胰高血糖素复合物的存在，对于进一步研究GP73与胰高血糖素的相互作用具有指导意义。
(3)本发明实施例中发明人首次发现可溶性GP73可以通过不依赖胰高血糖素的方式，激活肝脏糖的产生以及糖异生信号通路的激活。
(4)本发明实施例中发明人发现GP73在糖尿病人群中的表达水平显著地高于健康人群，因此可以将GP73作为标志物用于糖尿病的检测。
附图说明
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定。在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
图1A显示的是本发明实施例1中健康人群和糖尿病人群的入组人群性别和年龄的匹配情况，结果显示，两组人群在性别和年龄方面的分布无显著差异；图1B显示的是本发明实施例1中健康人群和糖尿病人群的血清中可溶性GP73蛋白水平，结果显示，血清可溶性GP73蛋白在糖尿病人群中的水平显著高于健康体检人群(P<0.01)。
图2A显示的是本发明实施例2中重组小鼠可溶性GP73(rmsGP73)注射实验组和PBS对照组小鼠的空腹血糖水平，结果显示，rmsGP73注射实验组小鼠的空腹血糖明显高于PBS对照组(*,P<0.05；**,P<0.01；***,P<0.001)；图2B显示的是本发明实施例2中rmsGP73注射实验组和PBS对照组小鼠的腹腔内糖耐量IPGTT水平，结果显示，rmsGP73注射实验组小鼠呈现IPGTT异常(*,P<0.05；**,P<0.01；***,P<0.001)；图2C显示的是本发明实施例2中rmsGP73注射实验组和PBS对照组小鼠的丙酮酸耐量PTT水平，结果显示，rmsGP73注射实验组小鼠PTT异常(*,P<0.05；**,P<0.01；***,P<0.001)；图2D显示的是本发明实施例2中rmsGP73注射实验组和PBS对照组小鼠的胰岛素耐量ITT水平，结果显示，rmsGP73注射实验组小鼠与对照组无显著性差异(*,P<0.05；**,P<0.01；***,P<0.001)。
图3A显示的是本发明实施例3中荧光染料Cy7对照组、Cy7标记的rmsGP73蛋白(rmsGP73-Cy7)实验组静脉注射小鼠后的荧光分布情况，结果显示，rmsGP73蛋白注射30分钟后，在肝脏和肾脏中聚积明显；图3B显示的是本发明实施例3中Cy7对照组、rmsGP73-Cy7实验组静脉注射小鼠后的荧光在各脏器的分布情况，结果显示，与未标记的Cy7染料的广泛分布不同，rmsGP73蛋白主要聚集在肝脏及肾脏；图3C显示的是本发明实施例3中Cy7对照组、rmsGP73-Cy7实验组静脉注射小鼠后各脏器的荧光强度值，结果显示，rmsGP73-Cy7组的肝脏、 肾脏以及脾脏中的荧光强度值明显高于对照组。
图4A显示的是本发明实施例4中Reichert 4SPR测定重组人可溶性GP73(rhsGP73)和胰高血糖素(GCG)的结合解离曲线。图4B显示的是本发明实施例4中Reichert 4SPR测定重组小鼠可溶性GP73(rmsGP73)和GCG的结合解离曲线。图4C显示的是本发明实施例4中重组大鼠可溶性GP73(rrsGP73)和GCG的结合解离曲线。图4D显示的是本发明实施例4中Reichert4SPR测定重组猴可溶性GP73(rMsGP73)和GCG的结合解离曲线。图4E显示的是本发明实施例4中免疫共沉淀实验可溶性GP73和GCG的体内结合能力，结果显示，小鼠血清中的可溶性GP73与GCG存在特异性相互作用，这种相互作用随着禁食时间的延长而逐渐增强。
图5A显示的是本发明实施例5中测定rmsGP73对GCG的半衰期的影响，结果显示，rmsGP73可以显著延长GCG的半衰期(*,P<0.05；**,P<0.01；***,P<0.001)；图5B显示的是本发明实施例5中GCG实验组、rmsGP73实验组和rmsGP73+GCG实验组的小鼠血糖情况，结果显示，重组小鼠可溶性GP73蛋白在小鼠体内能够显著促进GCG的升糖能力(*,P<0.05；**,P<0.01；***,P<0.001)；图5C显示的是本发明实施例5中rmsGP73+GCG+IgG实验组和rmsGP73+GCG+6B6实验组的小鼠血糖情况，结果显示，rmsGP73在小鼠体内促进GCG的升糖能力可以被特异性的抗GP73抗体6B6阻断(*,P<0.05；**,P<0.01；***,P<0.001)。
图6A显示的是本发明实施例6中小鼠原代肝细胞在有血清培养条件下，不同浓度的rmsGP73对原代肝细胞产糖的影响，结果显示，rmsGP73以剂量依赖性方式促进小鼠原代肝细胞产糖；图6B显示的是本发明实施例6中小鼠原代肝细胞在无血清培养条件下，不同浓度的rmsGP73对原代肝细胞产糖的影响，结果显示，rmsGP73蛋白在无任何其他激素的辅助下，以剂量依赖性方式直接促进原代肝细胞产糖；图6C显示的是本发明实施例6中小鼠原代肝细胞在无血清培养条件下，抗GP73抗体6B6对rmsGP73蛋白促进小鼠原代肝细胞糖产糖的影响，结果显示，6B6可以特异性阻断rmsGP73促进的原代肝细胞产糖；图6D显示的是本发明实施例6中小鼠原代肝细胞在无血清培养条件下，rmsGP73对原代肝细胞中糖异生限速酶的影响，结果显示，rmsGP73促进三种糖异生关键酶(Pck1,Pcx和G6pc)的表达上调；图6E是本发明实施例6中的针对HepG2细胞系，rhsGP73对糖异生信号通路的关键激酶PKA酶活性的影响，结果显示，与阳性对照物IBMX(Sigma Aldrich,货号：I5879)相似，rhsGP73促进PKA的磷酸化水平(PKA-p)以及其激酶活性(RRXρS/T)；图6F是本发明实施例6中PBS对照组、GCG实验组、rmsGP73实验组和rmsGP73+GCG实验组,小鼠肝脏组织的CREB-p，CREB和α-Tubulin的免疫印迹图，结果显示，rmsGP73可以直接促进糖异生信号通路的激活、协同增强GCG的肝脏糖异生能力。
图7A显示的是本发明实施例7中小鼠IgG和抗GP73抗体6B6 30mg/kg注射组在注射后每周的空腹血糖水平，结果显示，6B6高剂量对I型糖尿病小鼠血糖具有显著的降低作用(*,P<0.05；**,P<0.01；***,P<0.001)；图7B显示的是本发明实施例7中小鼠IgG和6B6抗体7.5、15、30mg/kg不同剂量注射组在注射后第四周的糖化血红蛋白HbA1c水平，结果显示，抗GP73抗体6B6中剂量和高剂量对I型糖尿病小鼠糖化血红蛋白具有显著的降低作用(*,P<0.05；**,P<0.01；***,P<0.001)。
图8A是本发明实施例8中STZ+IgG组和STZ+6B6组小鼠的三色免疫荧光染色图，结果显示，抗GP73抗体6B6对I型糖尿病小鼠胰岛α细胞和β细胞具有明显的保护作用；图8B-C是本发明实施例8中STZ+IgG组和STZ+6B6组小鼠的胰岛α细胞和胰岛β细胞的计数图，结果显示，抗GP73抗体6B6对I型糖尿病小鼠胰岛α细胞和β细胞具有显著的保护作用(*,P<0.05；**,P<0.01；***,P<0.001)；图8D是本发明实施例8中STZ+IgG组和STZ+6B6组小鼠的胰岛β细胞/α细胞的比值图，结果显示，抗GP73抗体6B6对I型糖尿病小鼠β细胞/α细胞比值无显著性影响。
图9A显示的是本发明实施例9中9条GP73 siRNA转染H22细胞后的免疫印迹图，结果显示，不同序列对细胞内源GP73蛋白水平的敲低作用效率不同，选取4号序列作为候选siRNA；图9B显示的是本发明实施例9中对照(Ctr siRNA)组和GP73 siRNA组在注射后第四周小鼠的空腹血糖值，结果显示，GP73 siRNA对II型糖尿病型小鼠空腹血糖具有显著的降低作用(*,P<0.05；**,P<0.01；***,P<0.001)；图9C显示的是本发明实施例9中Ctr siRNA组和GP73 siRNA组在注射后第四周的IPGTT水平，结果显示，GP73 siRNA对II型糖尿病小鼠IPGTT具有明显的改善作用(*,P<0.05；**,P<0.01；***,P<0.001)；图9D显示的是本发明实施例9中GP73 siRNA对II型糖尿病小鼠IPGTT的AUC值(area under the curve)的影响差异显著(*,P<0.05；**,P<0.01；***,P<0.001)；图9E显示的是本发明实施例9中Ctr siRNA组和GP73 siRNA组在注射后第四周的ITT水平，结果显示，GP73 siRNA对II型糖尿病小鼠ITT具有明显的改善作用；(*,P<0.05；**,P<0.01；***,P<0.001)；图9F显示的是本发明实施例9中GP73 siRNA对II型糖尿病小鼠ITT的AUC值的影响差异显著(*,P<0.05；**,P<0.01；***,P<0.001)。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在一些实施例中，对于本领域技术人员熟知的原料、元件、方法、手段等未作详细描述，以便于凸显本发明的主旨。
除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。
一、名词解释
本发明中，“GP73”一词是指高尔基体跨膜糖蛋白73(GP73)，又叫GOLM1(Golgi membrane protein 1))或者GOLPH2(Golgi phosphorprotein 2)。GP73的第55位氨基酸附近有一个前蛋白转化酶(Proprotein convertase，PC)的切割位点，全长GP73被PC切割后可以从高尔基体释放，分泌进入血液循环系统，称为可溶性GP73。本发明中，GP73泛指：机体内存在的或体外分离得到的、天然的或重组的全长GP73、GP73片段、GP73突变体或被修饰过的GP73；可选地，所述GP73选自全长GP73或不包括1-55位氨基酸的GP73。当“GP73”一词与其他词语搭配时，其同样具有此处定义的含义，如：GP73抑制剂、抗GP73抗体、抗GP73单克隆抗体中的GP73均具有此处定义的含义。
本发明中，“GP73抑制剂”一词是指任何可以下调GP73水平(包括基因水平或蛋白质水平)、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物；可选地，所述下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物具有以下性质中的一种或多种：(1)能抑制编码GP73的基因转录、正确剪切和/或翻译；(2)抑制或阻碍GP73与机体内的受体和/或配体结合；(3)抑制或阻碍GP73与机体内的特异性相互作用分子的相互作用；(4)缩短GP73在机体内的半衰期。GP73抑制剂包括但不限于抗GP73抗体(包括抗GP73单克隆抗体、双特异性抗体、多特异性抗体、抗体融合蛋白)，特异性抑制GP73的siRNA，特异性抑制GP73的shRNA、特异性抑制GP73的microRNA、特异性抑制GP73的反义寡核苷酸，特异性抑制GP73的核酸适配体，特异性抑制GP73的小分子化合物，特异性抑制GP73的小分子化合物。
本发明中，“抗体”一词指由四条多肽链组成的免疫球蛋白分子，四条多肽链指通过二硫键互相连接的两条重(H)链和两条轻(L)链。
本发明中，“单克隆抗体”一词指高度均一、仅针对某一特定抗原表位的抗体，其可以通过已知的例如杂交瘤技术、抗体库技术、转基因小鼠技术或单细胞PCR技术来制备。
本发明中，“嵌合抗体”一词指利用DNA重组技术，将异源单抗的轻、重链可变区基因插入 含有人抗体恒定区的表达载体中，转化哺乳动物细胞表达出嵌合抗体，这样表达的抗体分子中轻重链的可变区是异源的，而恒定区是人源的，这样整个抗体分子的近2/3部分都是人源的。这样产生的抗体，减少了异源性抗体的免疫原性，同时保留了亲本抗体特异性结合抗原的能力。
本发明中，“人源化抗体”一词指由于嵌合抗体的可变区中的FR仍残留一定的免疫原性，为减少异源成分，利用基因工程技术在嵌合抗体的基础上，用人FR替代异源FR，形成人源化程度更高的抗体，即除了CDR是异源的外，其余全是人源结构，使人源化抗体获得鼠源单抗的抗原结合特异性，同时减少其异源性；或通过表面重塑等技术将异源性抗体的大部分氨基酸序列用人源序列取代，同时基本保留亲本异源单克隆抗体的亲和力和特异性，又降低了其异源性，有利应用于人体的单克隆抗体。
本发明中，“全人源抗体”一词指通过转基因或转染色体技术，将人类编码抗体的基因全部转移至基因工程改造的抗体基因缺失动物中，使动物表达人类抗体，达到抗体全人源的目的；或通过人抗体库筛选获得的单克隆抗体，或通过单细胞PCR技术获得的人单克隆抗体。
本文中，“抗体的抗原结合片段”一词指全长抗体的一部分，通常是靶结合区或可变区。
本文中，纳米抗体、重链抗体、Fab、Fab-SH、Fv、scFv、F(ab′) 2、DsFv、Diabody、Minibody、Tribody、Sc(Fv) 2、[Sc(Fv) 2] 2、(ScFv-SA) 4等未具体解释的名词也都具有本领域常规的含义。
本文中，“治疗”一词指能产生有益或期望的结果，包括但不限于：一种或多种症状的预防、减轻、改善或治愈，病症程度的缩减，与预期存活期相比存活期延长。
本文中，“有效剂量”一词指当通过本发明实施例的方法给予所述有效成分时，足以有效传递用于治疗疾病的活性成分的量，也可以是有效成分经单次或多次施用于患者而给所诊断或所治疗的患者提供预期效应的量或剂量。有效剂量可由所参与的临床医师作为本领域技术人员通过已知技术以及在类似情形下所得的观察结果而确定。在确定所施用有效成分的有效量或剂量时，所参与的临床医师应考虑多种因素，所述因素包括但不限于：哺乳动物的种属；体积、年龄及一般健康；所涉及的具体疾病；该疾病的涉入程度或严重程度；个体患者的响应；所施用的具体化合物；给药模式；所施用制剂的生物利用度性质；所选择的给药方案；伴随药物疗法的使用；以及其它相关的情形。
本文中，“药学上可接受的辅料”可以是常规制剂用的药用载体、赋形剂及其它添加剂，如常见的抗体药物的辅料。
二、检测方法或实验方法
1、本发明中，人血清中的可溶性GP73的检测方法如下：采用单纯随机抽样的方法从多个体检中心内分泌科抽取同时期190例糖尿病患者，同时期多个体检中心随机选取75例健康体 检者作对照。患者和健康人群均隔夜空腹后静脉采血，血清存放于-20℃低温冰箱中。全部样品收集齐全后，采用基于磁微粒化学发光免疫分析法的试剂盒(购自热景生物)，进行人血清中的可溶性GP73水平的检测。
2、本发明中，小鼠葡萄糖的检测方法如下：所有血液样本均采自尾部，葡萄糖氧化酶法测定血糖，采用全自动血糖仪(ACCU-CHEK；罗氏公司)检测；正常小鼠和糖尿病小鼠均禁食6小时，测定空腹血糖；测量随机血糖水平于上午9点进行；当血糖水平大于35mM时(血糖仪检测上限)，记录为35mM值。
3、本发明中，糖代谢实验的检测方法如下：在葡萄糖、胰岛素和丙酮酸耐受性试验中，小鼠禁食12小时后，腹腔注射D-葡萄糖(Sigma，货号：G8270，1.5g/kg体重)、尾静脉注射胰岛素(Sigma，货号：I9278，0.75U/kg体重)或丙酮酸钠(Sigma，货号：P2256，2g/kg体重)；注射后于0分钟、15分钟、30分钟、45分钟、60分钟、120分钟、24h对小鼠进行尾静脉采血，测定血糖，确定腹腔注射糖耐量(IPGTT)、丙酮酸耐量(PTT)和胰岛素糖耐量(ITT)。
4、本发明中，GP73升糖实验的检测方法如下：选取雄性C57BL/6N小鼠，禁食6小时后，尾静脉注射rmsGP73(80μg/kg体重)；注射后于0分钟、15分钟、30分钟、60分钟、90分钟、120分钟对小鼠进行尾静脉采血，测定血糖。
5、本发明中，小鼠活体成像实验的检测方法如下：选取雄性C57BL/6N小鼠，禁食过夜，提前一天将小鼠全身剃毛；将rmsGP73用Cy7染料标记，用96孔板将Cy7染料对照及rmsGP73-Cy7蛋白的荧光强度调成一致，将荧光强度调整好的Cy7染料和rmsGP73-Cy7通过尾静脉注射进入小鼠体内(即Cy7对照组和rmsGP73-Cy7实验组，每组各4只，注射剂量为200ul/只)，将小鼠放入麻醉箱中麻醉，注射后于0分钟和30分钟在活体成像仪器中进行老鼠活体成像，观察小鼠体内的荧光分布情况；然后迅速处死小鼠，取出白色脂肪、肌肉、肝、脾、胰腺、肾、脑，摆放整齐放入小鼠成像仪中观察其各个脏器的测定荧光强度。
6、本发明中，微量热涌动实验的检测方法如下：将带有His标签的重组可溶性GP73与RED-tris-NTA标记物室温中避光孵育30分钟；在PCR管中配制15个浓度梯度的GCG，将不同浓度的GCG与标记的GP73蛋白混匀室温孵育30分钟，用毛细管分别吸取上述混合液体，依次注入毛细柱卡槽里；利用微量热涌动仪器(NanoTemper公司)，选择NT115模式进行GP73蛋白与GCG的结合的检测，并根据拟合曲线计算亲和力数值。
7、本发明中，OpenSPR实验的检测方法如下：通过氨基芯片(AmineSensorChips，货号：SEN-AU-100-3-AMINE，lot:#SAB0122，Nicoya公司)偶联重组可溶性GP73，不同浓度梯度的 胰高血糖素(HY-P0082，lot:#34006)为流动相，通过openSPR(OpenSPR-XT，Nicoya公司产品)测定其结合及解离曲线，曲线拟合得出亲和力数值。
8、本发明中，免疫共沉淀实验的检测方法如下：采用眼眶后静脉丛采血方式，收集小鼠血液后离心，获得小鼠血清；加入抗小鼠GP73抗体(Santa Cruz，货号：sc-365817)、抗胰高血糖素抗体(Abcam，货号：ab92517)、RRXρS/T抗体(CST公司，货号：9624)、PKA-p抗体(CST公司，货号：5661)、α-Tubulin抗体(Sigma Aldrich，货号：T9026)或者PKA(CST公司，货号：5842)抗体任意一种，于4℃摇床孵育1小时后，加入琼脂糖珠(Protein A/G PLUS-Agarose，Santa Cruz，货号：sc-2003)继续孵育2小时；结合琼脂糖珠的蛋白加入SDS裂解液，沸水浴10分钟，离心后取上清进行SDS-PAGE；电泳结束后利用半干式转印系统将凝胶上的蛋白质转移到PVDF支持膜上，5％的脱脂牛奶封闭液中室温摇床孵育封闭1小时；根据二抗的种属特异性，分别加入HRP标记的山羊抗小鼠二抗(中杉金桥，货号：ZB-2305)或HRP标记的山羊抗兔二抗(中杉金桥，货号：ZB-2301)，室温摇床孵育1小时；取适量的ECL显色液均匀地滴至PVDF膜上，利用Tanon 5200全自动化学发光成像分析系统进行成像。
9、本发明中，检测胰高血糖素半衰期的方法如下：雌性C57BL/6N小鼠从尾静脉注射GCG(1μg/kg体重)或者GCG(1μg/kg体重)与重组小鼠可溶性GP73蛋白(1mg/kg体重)的混合物(室温孵育10分钟)，分别注射后于0分钟、1分钟、3分钟、5分钟、10分钟、20分钟、30分钟时，采用眼眶后静脉丛采血方式收集小鼠血液；采集管中按照所需浓度加入3种蛋白酶抑制剂(DPP4、Protease inhibitor cocktail和Aprotinin)迅速混匀，室温静置一小时，以3000rpm离心10分钟，获得小鼠血清；GCG的浓度采用
Figure PCTCN2021079613-appb-000001
RAT METABOLIC MAGNETIC BEAD PANEL KIT 96 Well Plate Assay试剂盒检测(Millipore，货号RMHMAG-84K)；所得数据进行非房室模型的药代动力学参数计算，之后依次计算出半衰期数值。
GCG(购自MCE，货号：HY-P0082，批号：34006)；重组小鼠可溶性GP73蛋白rmsGP73，本实验室HEK293细胞表达产品(即实施例2中的重组小鼠可溶性GP73)；蛋白酶抑制剂(Protease Inhibotor cocktail I，购自Millipore，货号：20-201)；DPP4抑制剂(购自Millipore，货号：DPP4-010)，Aprotinin(购自Sigma，货号：A6106)。
10、本发明中，免疫荧光染色的方法如下：小鼠处死后，剥离的胰腺组织用10％(v/v)福尔马林固定并制备石蜡切片标本，进行5μm厚度的石蜡切片；免疫荧光切片脱蜡至水后，封闭30分钟，与抗小鼠胰岛素抗体(Abcam，货号：ab181547)或者抗胰高血糖素抗体(Abcam，货号：ab10988)共同孵育1小时后，重复清洗三次；加入相应的荧光二抗室温孵育1小时后， 进行清洗和DAPI染色；图像在共焦荧光显微镜(Zeiss LSM710)或自动数字幻灯片扫描仪(3D HISTECH)下拍摄；取每个胰腺5～10个等距切片成像，每组至少3只小鼠，染色阳性细胞至少三个视野，总细胞数目不少于200个。
实施例1.糖尿病人群血清中可溶性GP73水平显著高于健康体检人群
实验方法：为比较糖尿病人群与健康人群中，血清中可溶性GP73的水平是否有差异，我们对多个体检中心门诊体检的75例健康体检人群以及由内分泌科诊断为糖尿病的190例糖尿病患者，进行了隔夜空腹后血清可溶性GP73水平的检测。这些入组人群，进行了性别和年龄的匹配，其分布在健康人群和糖尿病人群中无显著差异(如图1A所示)。
实验结果：在健康人群中，可溶性GP73的平均血清浓度为52.81ng/ml(3.5-146ng/ml)；糖尿病人群中，可溶性GP73的平均血清浓度为68.82ng/ml(19.36-198ng/ml)，两组具有显著统计学差异(P<0.01)(如图1B所示)。以上结果显示，糖尿病人群血清中可溶性GP73水平显著高于健康体检人群。
实施例2.重组可溶性GP73调节糖代谢
实验方法：首先通过哺乳动物细胞表达系统HEK293细胞，进行GP73蛋白的表达纯化，获得基因重组小鼠可溶性GP73蛋白(rmsGP73)(NCBI Reference Sequence:NP_001030294.1)。该蛋白缺失了GP73的1-55位氨基酸，是血液中可溶性GP73的主要存在形式(本发明实施例中涉及的小鼠实验所用的重组可溶性GP73蛋白都为该重组小鼠可溶性GP73蛋白，简称为rmsGP73)。我们采用300ng/只剂量rmsGP73和PBS，分别通过尾静脉注射C57BL/6N小鼠(即实验组和PBS对照组，每组各10只小鼠)后，检测注射后24小时、48小时小鼠的空腹血糖(血糖仪和血糖检测试纸购自罗氏公司)，按检测方法或实验方法中第3部分进行腹腔注射糖耐量(IPGTT)、丙酮酸耐量(PTT)和胰岛素糖耐量(ITT)检测。
实验结果：注射实验组小鼠的空腹血糖明显高于对照组(如图2A所示)。重要的是，在代谢实验中，与对照组相比，rmsGP73注射实验组小鼠呈现糖耐量(IPGTT)异常(如图2B所示)以及代表糖异生能力的丙酮酸耐量(PTT)异常(如图2C所示)，而代表胰岛素敏感性的胰岛素糖耐量试验(ITT)与对照组小鼠无显著差别(如图2D所示)。
实施例3.重组可溶性GP73在小鼠肝脏和肾脏聚集
实验方法：按检测方法或实验方法中第5部分进行荧光染料Cy7对照组、Cy7标记的 rmsGP73蛋白(rmsGP73-Cy7)实验组静脉注射小鼠后的荧光分布情况、荧光强度检测。
实验结果：小鼠活体成像显示，与Cy7对照组相比较，rmsGP73-Cy7主要聚积在肝脏和肾脏(如图3A)。取其脏器后发现，与Cy7对照组相比较，rmsGP73-Cy7在肝脏及肾脏的荧光强度最强(如图3B)。对小鼠的各脏器的荧光强度进行分析处理，发现rmsGP73-Cy7的肝脏、肾脏、脾脏的荧光强度显著高于Cy7对照组(如图3C)。
实施例4.重组可溶性GP73与胰高血糖素特异性相互作用
血糖水平降低到一定的阈值时，胰高血糖素迅速分泌。一些氨基酸，如谷氨酰胺、精氨酸和丙氨酸，以及血浆中游离脂肪酸是胰高血糖素分泌的强刺激剂。抑胃肽(GIP)在低血糖情况下促进胰高血糖素的分泌。胰岛素、γ-氨基丁酸、瘦素和生长抑素等直接抑制胰高血糖素的分泌，胰高血糖素样肽1(GLP-1)间接抑制胰高血糖素的分泌。同时，中枢神经系统是重要的血糖水平感受器，通过迷走神经和或胆碱能神经，直接或间接调节胰高血糖素的分泌。血液中胰岛素与胰高血糖素的比例(I/G)是控制肝脏糖原和糖异生的关键因素，高比例的I/G表明能量处于充足状态，糖原合成增加，抑制糖异生。反之，糖原进行分解，糖异生增强。
高胰高血糖素血症见于各类型糖尿病。胰高血糖素的分泌受内源性胰岛素调节，这一调节机制的丧失导致了机体分泌更多的胰高血糖素，升高血糖，引发糖尿病。在II型糖尿病模型中，胰高血糖素是高血糖发生的关键因素，缺乏胰高血糖素受体的正常小鼠呈现出低血糖的症状，而缺乏胰高血糖素受体的糖尿病小鼠(db/db)不表现出高胰岛素或高血糖症状。在II型糖尿病中，胰岛素的直接和间接作用受损，而胰高血糖素信号的增强进一步加剧了糖原降解以及糖异生，从而导致了葡萄糖的增多和血糖的升高。临床研究也显示，胰高血糖素不合时宜的分泌是II型糖尿病患者高血糖的主要因素。II型糖尿病患者，在低胰岛素分泌和胰岛素抵抗的同时，伴有空腹胰高血糖素水平增高，餐后胰高血糖素抑制功能减弱，以及细胞对血糖和胰岛素抑制胰高血糖素分泌的敏感性降低等胰高血糖素调节失常，因此II型糖尿病被普遍认为是胰岛素缺乏和胰岛素抵抗，胰高血糖素过量双激素紊乱性胰腺疾病。胰高血糖素受体基因敲出(GCGR-/-)小鼠(I型糖尿病模型小鼠)，破坏几乎所有的β细胞，也不会出现糖尿病的临床表现，而通过腺病毒胰高血糖素受体(GCGR)表达载体，恢复肝细胞表达GCGR后，小鼠血糖水平迅速升高。对于长期患有I型糖尿病并有残留胰岛细胞的患者，胰高血糖素可能是高血糖的一个重大贡献者。所有的研究结果都表明，胰高血糖素是糖尿病发展的关键因素，降低胰高血糖素活性，将使糖尿病的治疗更进一步。
4.1不同种属重组可溶性GP73和GCG的亲和力测定
实验方法：为研究GP73与胰高血糖素(GCG，购自MCE公司，HY-P0082)的相互作用，我们首先采用Reichert 4SPR(Life Sciences公司)、微量热涌动实验(microscale thermophoresis，MST，NanoTemper公司)和OpenSPR(OpenSPR-XT，Nicoya公司)，测定了重组可溶性GP73和GCG的结合活性。
实验结果：重组人可溶性GP73(rhsGP73)可与GCG特异性结合，Reichert 4SPR、MST和OpenSPR三种方法所测得到的亲和力分别为KD＝2.83μM、KD＝2.45μM和KD＝2.80μM(如图4A和表1所示)。重组小鼠(rmsGP73)、大鼠(rrsGP73)及猴(rMsGP73)的可溶性GP73也同样可与GCG特异性结合(如图4B、C、D和表1所示)。各种属可溶性GP73均为本公司采用哺乳动物细胞表达纯化制备。猴可溶性GP73：NCBI Reference Sequence:XP_011769391.1；大鼠可溶性GP73：NCBI Reference Sequence:XP_001056825.3；小鼠可溶性GP73：NCBI Reference Sequence:NP_001030294.1；人可溶性GP73：NCBI Reference Sequence:NP_057632.2。
表1：不同种属重组可溶性GP73和GCG的亲和力(KD)
Figure PCTCN2021079613-appb-000002
注：NA为未检测
4.2可溶性GP73和GCG的体内结合活性检测
实验方法：为证实可溶性GP73和GCG的体内结合活性，禁食不同时间的C57BL/6N小鼠采血后，进行免疫共沉淀实验。鼠抗GP73单抗F12购自Santa cruz公司。
实验结果：血清中的GP73沉淀物结合GCG(图4E)。有意思的是，GP73-GCG的相互作用条带逐渐变深，说明GP73-GCG复合物随着禁食时间的延长而逐渐增加(如图4E所示)。
实施例5.重组可溶性GP73延长血浆GCG的半衰期，并促进GCG的升糖作用
前述研究结果表明，rmsGP73可以造成小鼠空腹血糖升高和糖耐量异常，而且sGP73可与GCG特异性结合，提示我们sGP73在体内很可能对GCG行使伴侣分子的功能，以避免GCG被迅速降解和或脱酰胺而失去功能。为此，我们进行了rmsGP73对胰高血糖素半衰期的影响的实验研究。
实验方法：用C57BL/6N小鼠12只(8周龄，重量20-25g，雌性)，分为GCG组及 GCG+rmsGP73两组，每组6只。实验方法详见检测方法或试验方法第9部分。
实验结果：重组可溶性GP73可以延长血浆胰高血糖素的半衰期(如图5A和表2所示)。
表2：重组可溶性GP73对血浆胰高血糖素半衰期的影响
组别 半衰期(min)
GCG 4.10±0.64
GCG+rmsGP73 5.72±1.83*
与GCG组相比，*,P<0.05；**,P<0.01；***,P<0.001
为研究可溶性GP73对GCG的活性的影响，设GCG、rmsGP73、rmsGP73+GCG三个实验组进行升糖实验。实验所用小鼠为C57BL/6N小鼠(6-8周龄，重量20-25g，雄性)，每组6只，检测血糖所用血糖仪和血糖检测试纸购自罗氏公司。
实验方法：GCG实验组：小鼠尾静脉注射GCG(购自MCE公司)，剂量为100ng/只，分别在0、15、30、60、90、120分钟检测小鼠的血糖；rmsGP73实验组：小鼠尾静脉注射rmsGP73蛋白，剂量为100μg/只，分别在0、15、30、60、90、120分钟检测小鼠的血糖；rmsGP73+GCG实验组：将重组小鼠可溶性GP73 100μg/只与GCG 100ng/只体外共同室温孵育10分钟后共同注入小鼠尾静脉，分别在0、15、30、60、90、120分钟检测小鼠的血糖。
为进一步确定可溶性GP73蛋白特异性靶向GCG的升糖能力，我们在rmsGP73与GCG共同孵育的过程中，加入抗GP73特异性抗体6B6或者小鼠IgG，重复以上实验，即设GCG+rmsGP73+IgG、rmsGP73+GCG+6B6二个实验组，小鼠为C57BL/6N小鼠(6-8周龄，重量20-25g，雄性)，每组6只，分别在0、15、30、60、90、120分钟采血检测小鼠的血糖。
实验结果：小鼠尾静脉注射GCG后，血糖水平急剧升高，rmsGP73显著促进了GCG的升糖能力(如图5B所示)。rmsGP73促进GCG的升糖能力被特异性的抗GP73抗体6B6阻断(如图5C所示)。
本发明使用的抗GP73单克隆抗体6B6是通过使用人可溶性GP73抗原对动物免疫、采用小鼠可溶性GP73抗原进行筛选杂交瘤克隆而获得。具体而言，本发明的抗GP73单克隆抗体6B6由下述方法制备：人可溶性GP73重组抗原纯品(NP_057632.2)对Balb/c小鼠进行免疫，取免疫小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2/0融合，再用小鼠可溶性GP73抗原(NP_001030294.1)筛选GP73特异的杂交瘤单克隆，建立稳定细胞株(该杂交瘤细胞6B6G6(简称6B6)保藏于位于中国北京市朝阳区北辰西路1号院3号的中国微生物菌种保藏管理委员会普通微生物中心，在该保藏中心登记入册的编号为CGMCC NO.18165，保藏日期为2019年6月20日，建议的分类命名为：小鼠杂交瘤细胞)。将培养的该单克隆细胞注射至小鼠腹腔， 收集腹水，纯化单克隆抗体。
实施例6.重组可溶性GP73促进肝脏糖异生信号通路的激活和肝脏产糖
实验方法：为研究可溶性GP73对肝脏糖异生和肝脏产糖的直接作用，我们将小鼠饥饿12h后，提取小鼠原代肝细胞，用含10％血清低糖DMEM培养液培养细胞，铺板于6个细胞培养皿中，37℃细胞培养箱中培养。在无血清培养情况下是在细胞贴壁后更换成无血清培养基，继续培养过夜。实验用细胞采用有血清和无血清两种培养方式。试验前，细胞用PBS洗涤2遍，更换成无糖DMEM培养基，加入丙酮酸、乳酸糖异生原料，6皿细胞分成对照组(加PBS)3皿和实验组(加GP73纯化蛋白16nM)3皿，置于37℃细胞培养箱中培养，分别于0h、1h、2h、4h、8h吸取20ul培养基上清检测葡萄糖含量，最后收取细胞检测蛋白含量，葡萄糖/蛋白得到单位细胞蛋白的产糖量；3个6孔板按0nM、4nM、8nM、16nM、32nM、63nM的浓度梯度添加GP73纯化蛋白，置于37度细胞培养箱中培养，4h后收取培养基上清检测葡萄糖含量，收取细胞检测蛋白含量，葡萄糖/蛋白得到单位细胞蛋白的产糖量。
为研究rmsGP73蛋白是否能够影响小鼠原代肝细胞糖异生关键酶的表达，重复上述实验，获取细胞后，PBS洗涤两遍，裂解细胞，提取细胞RNA，反转录成cDNA，根据糖异生关键酶Pcx、Pxk1、G6pc设计上下游引物，进行qRT-PCR实验。
为研究重组人可溶性GP73蛋白(rhsGP73蛋白)是否能够影响肝细胞糖异生关键激酶PKA的磷酸化水平和酶活性，用HepG细胞加入rhsGP73不同时间后，获取细胞裂解液，进行免疫印迹实验。
实验结果：PBS对照组和rmsGP73实验组，在有血清培养情况下，小鼠原代肝细胞糖异生产糖量不同，rmsGP73实验组产糖量多于对照组，且随着剂量的增加逐渐增大，直到饱和(如图6A所示)。PBS对照组和rmsGP73实验组，在无血清培养情况下，小鼠原代肝细胞糖异生产糖量不同，rmsGP73的实验组产糖量多于对照组，且随着剂量的增加逐渐增大，直到饱和(如图6B所示)。6B6能够特异性抑制GP73促进原代肝细胞糖异生产生糖(如图6C所示)。与PBS对照组相比，rmsGP73组的糖异生关键酶Pcx(phosphoenolpyruvate carboxykinase 1)、Pck1(pyruvate carboxylase)、G6pc(glucose-6-phosphatase)表达水平明显上调(如图6D所示)。与PBS对照组相比，rhsGP73组的PKA磷酸化水平(PKA-p)和激酶活性(RRXρS/T)明显上调(如图6E所示)。
糖异生程序的复杂的调控网络中，转录因子效应元件结合蛋白CREB(cAMP-responsive element binding protein,CREB)是非常重要的调节因子，在肝脏中它们通过胰高血糖素 -cAMP-PKA信号通路促进糖异生，因此，CREB的磷酸化水平代表着糖异生的状态。
实验方法：为研究rmsGP73在小鼠体内对糖异生的影响，C57BL/6N小鼠(雄性，8周龄，体重为20-25g)，设PBS、rmsGP73、GCG、rmsGP73+GCG四组，每组3只；分别尾静脉注射PBS、rmsGP73(100μg/只)、GCG(100ng/只)以及rmsGP73+GCG(rmsGP73 100μg/只和GCG 100ng/只，体外室温共同孵育10分钟后注射)，注射48小时后，处死小鼠分离肝脏组织，进行免疫印迹实验。免疫印迹实验中使用兔抗pCREB多抗(购自Abcam公司，ab32096)和兔抗CREB单抗(购自CST公司，#9197)。
实验结果：单独注射GCG可以增强CREB的磷酸化水平，rmGP73显著增强了GCG激活的CREB磷酸化，说明rmsGP73通过与GCG相互作用，增强其肝脏糖异生能力，促进了GCG的活性(如图6F所示)。同时，rmsGP73单独处理，依然能够促进肝脏糖异生，再次提示我们可溶性GP73以依赖和不依赖于GCG两种方式，参与糖异生信号通路的激活。
实施例7.抗GP73抗体对Ⅰ型糖尿病小鼠的降糖作用
实验方法：为进一步研究6B6抗体的体内降糖效果，采用C57BL/6N小鼠腹腔STZ注射诱发Ⅰ型糖尿病小鼠模型(6-8周龄，重量20-25g，雄性)，在稳定期10天后，将小鼠随机分配为对照小鼠IgG组、7.5、15、30mg/kg剂量6B6组共4组，每组7只(具体分组情况如表3所示)；每日通过尾静脉给药直至实验结束，每7天进行小鼠体重和空腹血糖监测。
实验结果：6B6抗体治疗组小鼠的体重与对照组小鼠没有显著差异(如表4所示)；6B6抗体对小鼠具有显著的降低空腹血糖作用，并具有剂量依赖作用(如表5和图7A所示)。
表3：I型糖尿病小鼠抗GP73抗体6B6治疗分组情况
组别 治疗 剂量(mg/kg/d) 注射次数 给药途径 动物数量
1 IgG 15 1次/每天 静脉 7
2 6B6 7.5 1次/每天 静脉 7
3 6B6 15 1次/每天 静脉 7
4 6B6 30 1次/每天 静脉 7
表4：I型糖尿病小鼠抗GP73抗体6B6治疗不同时间小鼠的体重
Figure PCTCN2021079613-appb-000003
Figure PCTCN2021079613-appb-000004
表5：I型糖尿病小鼠抗GP73抗体6B6治疗不同时间小鼠的空腹血糖
Figure PCTCN2021079613-appb-000005
与IgG组相比，*,P<0.05；**,P<0.01；***,P<0.001
实验方法：治疗4周后，采血测定糖化血红蛋白(HbA1c)、谷丙转氨酶(ALT)、谷草转氨酶(AST)和血脂的水平。HbA1c检测试剂盒购自Crystal Chem公司(货号：80310)，采用TECAN SPARK多功能酶标仪检测。肝功和血脂检测试剂盒购自瑞尔达公司(货号：ALT191230和AST200218)，采用RIELE公司的Photometer L100生化分析仪检测。
实验结果：IgG治疗的糖尿病对照组小鼠血清中，HbA1c平均为8.78±1.7％；在中剂量和高剂量6B6抗体治疗的糖尿病小鼠中，HbA1c水平均分别为6.99±1.6和6.71±1.5(表6和图7B)，表明抗GP73抗体6B6对STZ-诱导的T1D小鼠以剂量依赖的方式降低HbA1c水平。肝功和血脂结果表明，抗体连续注射4周后，中剂量抗体显著降低了小鼠AST的水平(表6)，提示我们抗体对STZ诱发的肝损伤可能具有一定的保护作用。除此之外，抗体治疗组小鼠甘油三脂和胆固醇水平也显著低于对照组小鼠(表6)。
表6：抗GP73抗体6B6治疗Ⅰ型糖尿病小鼠后相关生化指标变化
Figure PCTCN2021079613-appb-000006
与IgG组相比，*,P<0.05；**,P<0.01；***,P<0.001
实施例8.抗GP73抗体对Ⅰ型糖尿病小鼠的胰岛保护作用
实验方法：为进一步研究GP73阻断对STZ诱导造成的胰岛损伤的保护效果，我们采用STZ腹腔注射诱发的Ⅰ型糖尿病小鼠模型。在此实施例中，将C57BL/6N小鼠禁食过夜(雄性，8-10周龄，20-22g)，进行单次腹腔STZ(175mg/kg)注射。在10天稳定期后，基于动物的体重和空腹血糖，使用计算机产生的随机过程将小鼠随机分配为小鼠IgG对照和24mg/kg剂量6B6实验组共2组，每组3只。每日通过尾静脉给药直至实验结束。给药后4周杀死小鼠，获取胰岛组织后，进行福尔马林固定，制备石蜡切片，采用三色免疫荧光染色。其中，DAPI蓝色为细胞核、胰岛素染色阳性的β细胞为绿色、胰高血糖素染色阳性的α细胞为红色。
实验结果：抗GP73抗体阻断后，胰岛α、β细胞数目分别增加了1倍以上(图8A-C)，说明抗GP73抗体6B6对Ⅰ型糖尿病小鼠胰岛α细胞和β细胞均具有明显的保护作用。β细胞/α细胞比值从20％上升到23％左右，略有升高，但没有统计学显著性差异(图8D)。
实施例9.特异性阻断GP73的RNAi对Ⅱ型糖尿病小鼠的降糖作用
为进一步确定阻断GP73具有降糖效果，我们构建了由高脂饮食诱发的Ⅱ型糖尿病模型，采用GP73特异性RNAi oligos敲低的方式，研究其对Ⅱ型糖尿病小鼠的降糖作用。
实验方法：我们首先合成了针对鼠GP73不同位点共9条RNAi oligos(表7，SEQ ID NO.1-9)，通过转染H22小鼠肝脏细胞，转染24小时后，收取细胞进行免疫印迹实验。
实验结果：No.4 RNAi oligos序列对细胞内源GP73具有较好的敲低效率(图9A)。随后，我们在吉玛基因公司合成了3′端胆固醇修饰、两端硫代骨架修饰以及全链甲氧基修饰的No.4 GP73 RNAi oligos(GP73 siRNA)，这种化学修饰后的siRNA在体内的稳定性为3-6天，序列被打乱的RNAi oligos作为对照(Ctr siRNA；表7)。
实验方法：在此基础上，我们选择C57BL/6N小鼠(雄性，8-10周龄，20-22g)，高脂饲料饲养20周后，动物禁食6小时，检测空腹血糖，血糖值大于11.3mM为造模成功。按照空腹血糖随机分为Ctr siRNA组和GP73 siRNA实验组，每组6只，每5天注射一次，注射方式为先尾静脉注射，每次注射4nM，然后再腹腔注射4nM。自第1次注射后4周，将小鼠处死，进行血糖和糖化血红蛋白等指标的检测。
实验结果：GP73 siRNA注射组，有效的降低了高脂诱导的Ⅱ型糖尿病小鼠的空腹血糖(表8和图9B)。重要的是，与Ctr siRNA组相比，GP73 siRNA注射实验组小鼠呈现显著的糖耐量 改善(图9C-D)以及胰岛素敏感性的提高(图9E-F)。在治疗4周后，Ctr siRNA治疗的糖尿病对照组小鼠血清中，HbA1c平均为4.39±0.67％，GP73 siRNA治疗的糖尿病小鼠中，HbA1c水平均分别3.37±0.53％(表9)，表明GP73特异性siRNA在高脂诱导的T2D小鼠中可以降低HbA1c水平。肝功和血脂结果表明，GP73 siRNA连续注射4周后，显著降低了小鼠血清中AST、ALT和胆固醇水平，提示我们GP73阻断具有降低血脂、保护肝功的作用(表9)。
表7：GP73 siRNA的序列
GP73 siRNA 序列5’‐3’
Ctr AUCACACCAACACAGGUCCTT
SEQ ID No:1 275‐CCUGGUGGCCUGUGUUAUUTT
SEQ ID No:2 553‐GCGAGAAGCUCAUUCGAGATT
SEQ ID No:3 950‐GCAGAAUGAGGAAACCAAUTT
SEQ ID No:4 995‐CCAACAGGCAUCCAUCCAATT
SEQ ID No:5 1198‐CAGGAGAUGAAUACGACAUTT
SEQ ID No:6 1263‐GCAGGGAAUGACAGAAAUATT
SEQ ID No:7 1430‐UGUGAAAUGGACAGCGAAATT
SEQ ID No:8 1802‐GCUCUUACCGUCAGCAUAATT
SEQ ID No:9 2108‐GCACCUAUGGUCUGUGUUUTT
表8：II型糖尿病小鼠抗体治疗不同时间小鼠的空腹血糖
Figure PCTCN2021079613-appb-000007
与Ctr siRNA组相比，*,P<0.05；**,P<0.01；***,P<0.001
表9：II型糖尿病小鼠抗体治疗不同时间小鼠的糖化蛋白等水平
组别 HbA1c ALT AST TG CHO
Ctrl siRNA 4.39±0.67 77±22 243±65 1.27±0.37 5.62±0.97
GP73 siRNA 3.37±0.53* 56±10* 186±34** 1.26±0.16 4.74±1.43*
与Ctr siRNA组相比，*,P<0.05；**,P<0.01；***,P<0.001
本发明中证明了血清GP73水平的升高与人类糖尿病密切相关，其是治疗糖尿病的一个有 吸引力的靶点，GP73与胰高血糖素相互作用，以胰高血糖素依赖和不依赖两种方式，促进肝脏和/或肾脏的糖异生，因此，抑制GP73是治疗糖尿病的有效策略。
工业实用性
本发明实施例提供的一种GP73抑制剂在制备治疗糖尿病的药物中的应用，通过该应用对对血糖进行调节，发明人还通过动物实验证明了：GP73抑制剂可以降低糖尿病小鼠血糖水平及糖化血红蛋白水平，并对胰岛β细胞具有保护作用，起到治疗糖尿病的效果。

Claims (31)

  1. 一种GP73抑制剂在制备治疗糖尿病及其并发症的药物中的应用。
  2. 一种用于治疗糖尿病及其并发症的药物，其特征在于：所述药物包括作为活性成分的GP73抑制剂。
  3. 一种用于治疗糖尿病及其并发症的方法，其特征在于：包括以下步骤：向患有糖尿病的受试者施用有效剂量的GP73抑制剂。
  4. 根据权利要求1所述的应用、权利要求2所述的药物或权利要求3所述的方法，其特征在于：所述糖尿病包括：I型糖尿病、II型糖尿病、妊娠糖尿病。
  5. 根据权利要求1所述的应用、权利要求2所述的药物或权利要求3所述的方法，其特征在于：所述治疗糖尿病包括以下的任意一种或多种：(1)降低空腹和/或餐后血糖；(2)改善糖耐量；(3)保护胰岛α细胞和/或胰岛β细胞；(4)降低胰高血糖素的升糖能力和/或糖异生能力；(5)缩短胰高血糖素的半衰期；(6)降低GP73自身的非胰岛素依赖的升糖作用及糖异生的功能。
  6. 根据权利要求1所述的应用、权利要求2所述的药物或权利要求3所述的方法，其特征在于：治疗糖尿病并发症包括以下的任意一种或多种：糖尿病肾病、糖尿病眼部并发症、糖尿病足、糖尿病周围神经病变；其中：糖尿病眼部并发症包括以下的一种或多种：糖尿病性视网膜病变、与糖尿病相关的葡萄膜炎、糖尿病性白内障。
  7. 根据权利要求1所述的应用、权利要求2所述的药物或权利要求3所述的方法，其特征在于：所述GP73抑制剂包括：下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物；可选地，所述下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物具有以下性质中的一种或多种：(1)能抑制编码GP73的基因转录、正确剪切和/或翻译；(2)抑制或阻碍GP73与机体内的受体和/或配体结合；(3)抑制或阻碍GP73与机体内的特异性相互作用分子的相互作用；(4)缩短GP73在机体内的半衰期；进一步可选地，GP73抑制剂包括：抗GP73单克隆抗体或包含其抗原结合部位的抗体片段，抗GP73单克隆抗体或包含其抗原结合部位的抗体片段的融合蛋白，特异性抑制GP73的核酸序列中的一种或多种。
  8. 根据权利要求5所述的应用、药物或方法，其特征在于：所述GP73选自以下的一种或多种：机体内存在的或体外分离得到的、天然的或重组的全长GP73、GP73片段、GP73突变体或被修饰过的GP73；可选地，所述GP73选自全长GP73或不包括1-55位氨基酸的GP73。
  9. 根据权利要求5所述的应用、药物或方法，其特征在于：抗GP73单克隆抗体选自：杂 交瘤细胞生产的单克隆抗体、抗体库筛选的单克隆抗体、单细胞PCR生产的单克隆抗体、基因工程改造的单克隆抗体、异源性抗体、嵌合抗体、人源化抗体、全人源抗体、纳米抗体(Nanobody)、重链抗体(Heavy chain antibody)中的一种或多种；
    和/或，所述抗体片段的种类选自：Fab、Fab-SH、Fv、scFv、F(ab′) 2、DsFv、Diabody、Minibody、Tribody、Sc(Fv) 2、[Sc(Fv) 2] 2、(ScFv-SA) 4中的一种或多种；
    和/或，所述特异性抑制GP73的核酸包括siRNA、shRNA、microRNA、反义寡核苷酸、miRNA、核酸适配体中的一种或多种；可选地，特异性抑制GP73的siRNA选自SEQ ID NO:1-SEQ ID NO:9所示的核苷酸序列一条或多条，或选自与SEQ ID NO:1-SEQ ID NO:9所示的任意一条核苷酸序列至少有60％、70％、80％、90％同源性的序列；进一步可选地，特异性抑制GP73的siRNA选自SEQIDNO:4所示的核苷酸序列或与其具有至少60％、70％、80％、90％同源性的序列。
  10. 根据权利要求1所述的应用、权利要求2所述的药物或权利要求3所述的方法，其特征在于：治疗糖尿病及其并发症的药物中还包括治疗糖尿病的其他药物；可选地，所述治疗糖尿病的其他药物选自胰岛素、二甲基双胍、磺脲类降糖药、α糖苷酶抑制剂、噻唑烷二酮类、二肽基肽酶4(DPP4)抑制剂、胰高血糖素样肽-1(GLP-1)类似物、SGLT2抑制剂中的一种或多种。
  11. 一种GP73抑制剂在制备抑制胰高血糖素的药物中的应用。
  12. 一种用于抑制胰高血糖素的药物，其特征在于：所述药物包括GP73抑制剂。
  13. 一种用于抑制胰高血糖素的方法，其特征在于：包括以下步骤：向需要抑制胰高血糖素的受试者施用有效剂量的GP73抑制剂。
  14. 根据权利要求11所述的应用、权利要求12所述的药物或权利要求13所述的方法，其特征在于：所述抑制胰高血糖素包括以下的任意一种或多种：(1)缩短胰高血糖素的半衰期；(2)降低胰高血糖素的升糖能力和/或糖异生能力。
  15. 根据权利要求11所述的应用、权利要求12所述的药物或权利要求13所述的方法，其特征在于：所述GP73抑制剂包括：下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物；可选地，所述下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物具有以下性质中的一种或多种：(1)能抑制编码GP73的基因转录、正确剪切和/或翻译；(2)抑制或阻碍GP73与机体内的受体和/或配体结合；(3)抑制或阻碍GP73与机体内的特异性相互作用分子的相互作用；(4)缩短GP73在机体内的半衰期；进一步可选地，GP73抑制剂包括：抗GP73单克隆抗体或包含其抗原结合部位的抗体片段， 抗GP73单克隆抗体或包含其抗原结合部位的抗体片段的融合蛋白，特异性抑制GP73的核酸序列中的一种或多种。
  16. 根据权利要求11所述的应用、权利要求12所述的药物或权利要求13所述的方法，其特征在于：GP73选自以下的一种或多种：机体内存在的或体外分离得到的、天然的或重组的全长GP73、GP73片段、GP73突变体或被修饰过的GP73；可选地，所述GP73选自全长GP73或不包括1-55位氨基酸的GP73。
  17. 根据权利要求11所述的应用、权利要求12所述的药物或权利要求13所述的方法，其特征在于：所述抗GP73单克隆抗体选自：杂交瘤细胞生产的单克隆抗体、抗体库筛选的单克隆抗体、单细胞PCR生产的单克隆抗体、基因工程改造的单克隆抗体、异源性抗体、嵌合抗体、人源化抗体、全人源抗体、纳米抗体(Nanobody)、重链抗体(Heavy chain antibody)中的一种或多种；
    和/或，所述抗体片段的种类选自：Fab、Fab-SH、Fv、scFv、F(ab′) 2、DsFv、Diabody、 Minibody、Tribody、Sc(Fv) 2、[Sc(Fv) 2] 2、(ScFv-SA) 4中的一种或多种；
    和/或，所述特异性抑制GP73的核酸包括siRNA、shRNA、microRNA、反义寡核苷酸、miRNA、核酸适配体中的一种或多种；可选地，特异性抑制GP73的siRNA选自SEQIDNO:1-SEQ ID NO:9所示的核苷酸序列一条或多条，或选自与SEQ ID NO:1-SEQ ID NO:9所示的任意一条核苷酸序列至少有60％、70％、80％、90％同源性的序列；进一步可选地，特异性抑制GP73的siRNA选自SEQ ID NO:4所示的核苷酸序列或与其具有至少60％、70％、80％、90％同源性的序列。
  18. 一种GP73-胰高血糖素复合物，其特征在于：GP73与胰高血糖素相结合；所述GP73选自以下的一种或多种：机体内存在的或体外分离得到的、天然的或重组的全长GP73、GP73片段、GP73突变体或被修饰过的GP73；可选地，所述GP73选自全长GP73或不包括1-55位氨基酸的GP73；进一步可选地，所述GP73的种属来源选自人、小鼠、大鼠、猴、兔子、猪、狗中的一种或多种。
  19. 一种确定GP73与胰高血糖素结合表位的方法，其特征在于：包括以下步骤：利用复合物结晶解析法、表位决定部位切除法、氢氚交换法、Peptide-Panning法中的一种或多种确定GP73与胰高血糖素的结合表位。
  20. 一种确定GP73抑制剂在抑制GP73-胰高血糖素复合物形成中的抑制作用强弱的方法，其特征在于：包括以下两种方法中的任意一种：
    方法1：
    将候选GP73抑制剂与GP73孵育后，再将二者的混合物和/或复合物与胰高血糖素结合；
    比较GP73在与候选GP73抑制剂孵育前后与胰高血糖素结合的能力；
    方法2：
    利用计算机模拟比较GP73在与候选GP73抑制剂孵育前后与胰高血糖素结合的能力。
  21. 根据权利要求20所述的方法，其特征在于：候选GP73抑制剂的来源包括选自以下的一种或多种：杂交瘤细胞、B细胞、记忆B细胞、抗体库、化合物库、GP73类似物，胰高血糖素类似物；
    和/或，确定GP73与胰高血糖素结合的能力的方法包括选自以下方法的一种或多种：表面等离子共振(SPR)测定法、微量热泳动(MST)测定法，竞争ELISA法。
  22. 一种血清可溶性GP73检测试剂在制备糖尿病检测试剂中的应用。
  23. 根据权利要求22所述的应用，其特征在于：所述糖尿病包括：I型糖尿病、II型糖尿病、妊娠糖尿病。
  24. 根据权利要求22所述的应用，其特征在于：检测GP73的试剂包括检测血清中可溶性GP73的试剂。
  25. 一种GP73抑制剂在制备糖异生信号通路抑制剂的药物中的应用。
  26. 一种用于抑制糖异生信号通路的药物，其特征在于：所述药物包括GP73抑制剂。
  27. 一种用于抑制糖异生信号通路的方法，其特征在于：包括以下步骤：向需要抑制糖异生信号通路的受试者施用有效剂量的GP73抑制剂。
  28. 根据权利要求25所述的应用、权利要求26所述药物或权利要求27所述方法，其特征在于：所述抑制糖异生信号通路包括以下的任意一种或多种：(1)抑制肝细胞糖异生产生糖；(2)下调糖异生关键酶Pcx、Pck1、G6pc表达水平；(3)下调PKA磷酸化水平和激酶活性。
  29. 根据权利要求25所述的应用、权利要求26所述药物或权利要求27所述方法，其特征在于：所述GP73抑制剂包括：下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物；可选地，所述下调GP73水平、活性、功能和/或稳定性的多肽、蛋白质、核酸序列或小分子化合物具有以下性质中的一种或多种：(1)能抑制编码GP73的基因转录、正确剪切和/或翻译；(2)抑制或阻碍GP73与机体内的受体和/或配体结合；(3)抑制或阻碍GP73与机体内的特异性相互作用分子的相互作用；(4)缩短GP73在机体内的半衰期；进一步可选地，GP73抑制剂包括：抗GP73单克隆抗体或包含其抗原结合部位的抗体片段，抗GP73单克隆抗体或包含其抗原结合部位的抗体片段的融合蛋白，特异性抑制GP73的核酸序列 中的一种或多种。
  30. 根据权利要求25所述的应用、权利要求26所述药物或权利要求27所述方法，其特征在于：GP73选自以下的一种或多种：机体内存在的或体外分离得到的、天然的或重组的全长GP73、GP73片段、GP73突变体或被修饰过的GP73；可选地，所述GP73选自全长GP73或不包括1-55位氨基酸的GP73。
  31. 根据权利要求25所述的应用、权利要求26所述药物或权利要求27所述方法，其特征在于：所述抗GP73单克隆抗体选自：杂交瘤细胞生产的单克隆抗体、抗体库筛选的单克隆抗体、单细胞PCR生产的单克隆抗体、基因工程改造的单克隆抗体、异源性抗体、嵌合抗体、人源化抗体、全人源抗体、纳米抗体(Nanobody)、重链抗体(Heavy chain antibody)中的一种或多种；
    和/或，所述抗体片段的种类选自：Fab、Fab-SH、Fv、scFv、F(ab′) 2、DsFv、Diabody、 Minibody、Tribody、Sc(Fv) 2、[Sc(Fv) 2] 2、(ScFv-SA) 4中的一种或多种；
    和/或，所述特异性抑制GP73的核酸包括siRNA、shRNA、microRNA、反义寡核苷酸、miRNA、核酸适配体中的一种或多种；可选地，特异性抑制GP73的siRNA选自SEQIDNO:1-SEQ ID NO:9所示的核苷酸序列一条或多条，或选自与SEQ ID NO:1-SEQ ID NO:9所示的任意一条核苷酸序列至少有60％、70％、80％、90％同源性的序列；进一步可选地，特异性抑制GP73的siRNA选自SEQ ID NO:4所示的核苷酸序列或与其具有至少60％、70％、80％、90％同源性的序列。
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