WO2013020372A1 - 防治胰岛素抵抗和糖尿病的方法和试剂 - Google Patents

防治胰岛素抵抗和糖尿病的方法和试剂 Download PDF

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WO2013020372A1
WO2013020372A1 PCT/CN2012/001063 CN2012001063W WO2013020372A1 WO 2013020372 A1 WO2013020372 A1 WO 2013020372A1 CN 2012001063 W CN2012001063 W CN 2012001063W WO 2013020372 A1 WO2013020372 A1 WO 2013020372A1
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socs
seq
jak2
stat3
amyloid
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PCT/CN2012/001063
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French (fr)
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翟琦巍
张一�
乐颖影
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中国科学院上海生命科学研究院
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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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin

Definitions

  • Diabetes ⁇ i 1-.3 ⁇ 4 is divided into I ⁇ ⁇ ⁇ and 2 ⁇ .
  • ⁇ ⁇ i two kinds of H 1 saccharide ⁇ ⁇ by Ding - ⁇
  • IH. No. SRTEBP- lc can inhibit the rotation of 1RS-2; r, y*-i3 ⁇ 4 IRS-2 ⁇ & white underwater 3 ⁇ 4., 2 15 white matter translation modification, inhibition; Vitt, Example i/lltt island and IRS can PKC-S6K I. ERK. JK and ⁇ 3 ⁇ phosphorylation of tyrosine, inhibiting island 4 1 ⁇ 2 4., promoting ⁇ island ⁇ ⁇ sway inhibition 3 ⁇ 4 « * ⁇ , a ⁇ ⁇ PTPIB, PT, ⁇ acid «
  • SOCS cytokine signaling
  • AD Alzheimer's disease
  • memory loss a progressive neurodegenerative disease characterized by memory loss, cognitive dysfunction, and behavioral abnormalities. Its incidence varies with age. Increase and increase.
  • AD Alzheimer's disease
  • amyloid plaques or senile plaques
  • neurofilaments formed by highly phosphorylated microtubule-associated protein tau ⁇ white.
  • a ⁇ is a normal metabolite of cells that can pass through the blood-brain barrier with the help of its receptors.
  • the concentration of ⁇ in the blood and cerebrospinal fluid of healthy people is in an equilibrium state.
  • a large body of evidence supports ⁇ as the main virulence factor of AD.
  • the currently widely accepted Amyloid cascade hypothesis suggests that ⁇ plays a major role in the development and progression of AD, and the imbalance of ⁇ production and clearance in the brain causes ⁇ to accumulate in the brain. Damage to neurons and their synaptic function ultimately leads to cognitive impairment.
  • ⁇ is mainly produced by neurons in the brain.
  • ⁇ is formed by the digestion of ⁇ by ⁇ -secretase and ⁇ -secretase. Due to the different cleavage sites of ⁇ -secretase, two forms are mainly produced: ⁇ 40 and ⁇ 42.
  • ⁇ 42 has a strong aggregation ability and is therefore highly toxic. Both ⁇ and ⁇ -secretase distributions are relatively broad and are expressed in many tissues.
  • ⁇ -secretase was originally thought to be highly expressed only in neurons in the brain, but recent studies have shown that ⁇ -secretase is also expressed in glial cells and other peripheral cells, such as fat cells and hepatocytes, suggesting that peripheral tissues It is also possible to produce ⁇ .
  • ⁇ can be detected in peripheral tissues. Joachim et al. first reported that ⁇ deposition was detected in non-neural tissues such as blood vessels, skin, subcutaneous tissue, and intestine of AD patients, and ⁇ deposition was also detected in non-neural tissues in some normal elderly individuals. Lee et al. reported that ⁇ deposition was detected in both normal human and visceral fat. In addition, ⁇ can also be detected in the lesions of inclusion body myositis. These deposited ⁇ may be produced by the brain and then transported to the periphery via the circulatory system, or it may be produced by peripheral cells. However, the current study only detects peripheral ⁇ , and their function is not unclear. The ⁇ concentration in plasma itself is not suitable as a biomarker for AD ( Liu, JK , et al., J Alzheimers Dis. 2010 ;20(4): 1233-42. ).
  • amyloid proteins In addition to ⁇ , a variety of amyloid proteins have been discovered, which cause a series of lesions in abnormal deposition in various organs and tissues. As of 2010, there are 27 known amyloid proteins, of which amyloid-like peptide (IAPP) is recognized as closely related to diabetes. IAPP is a terminal product of islet ⁇ -cell secretion, containing 37 amino acids. However, it is quite different from ⁇ in sequence and shear mechanism. The pathological anatomical examination of the cadaver found that 90% of patients with type 2 diabetes had sputum deposition in the islets, and the number of ⁇ cells decreased, and the degree of islet amyloidosis was consistent with the degree of diabetes, indicating that ⁇ is associated with the onset of type 2 diabetes.
  • IAPP amyloid-like peptide
  • Human sputum can induce ⁇ cell apoptosis, and the two are dose-related. Homozygous obese mice transferred to the human sputum gene soon showed a large number of sputum degeneration deposits in the islets after treatment with sucrose, rouge diet, growth hormone or glucocorticoids. The level of ⁇ -cell apoptosis was greater than the level of replication, and the number decreased. The final development is type 2 diabetes. These evidences suggest that degeneration and deposition of sputum in islets cause beta cell apoptosis, and a decrease in number is one of the pathogenesis of type 2 diabetes. But there is no prior evidence that ⁇ , which is amyloid, is in insulin. The target tissue, such as the liver, induces another characteristic insulin resistance of type 2 diabetes, or ⁇ has a similar function to sputum in the pathogenesis of type 2 diabetes.
  • the object of the present invention is to provide a method and a formula for controlling insulin resistance and diabetes.
  • inhibition of JAK2/STAT3-SOCS-1 signaling pathway activation inhibition of JAK2/STAT3-SOCS-1 signaling pathway expression or activity, or inhibition of amyloid ⁇ expression or activity inhibition
  • the JAK2/STAT3-SOCS-1 signaling pathway protein comprises: JAK2 ⁇ white,
  • STAT3 protein or SOCS-1 is white.
  • the activation of the JAK2/STAT3-SOCS-1 signaling pathway is: activation of the JAK2/STAT3-SOCS-1 signaling pathway by amyloid ⁇ ( ⁇ ) ⁇ .
  • amyloid white ⁇ ( ⁇ ) comprises: a full-length amyloid ⁇ white ⁇ , or a ruthenium fragment thereof. More preferably, the protein fragment is selected from the group consisting of: A ⁇ 40, A ⁇ 42, A ⁇ 25-35 and the like.
  • the inhibitor includes (but is not limited to):
  • interfering molecule such as a small interfering RNA molecule or an antisense nucleotide
  • JAK2/STAT3-SOCS-1 signaling pathway an interfering molecule (such as a small interfering RNA molecule or an antisense nucleotide) that specifically interferes with the JAK2/STAT3-SOCS-1 signaling pathway;
  • a binding molecule such as an antibody or a ligand that specifically binds to the JAK2/STAT3-SOCS-1 signaling pathway; or AG490 that inhibits JAK2; or
  • An interfering molecule (such as a small interfering RNA molecule or an antisense nucleotide) that specifically interferes with the expression of amyloid ⁇ or its precursor;
  • spinning molecule such as an antibody or ligand that specifically binds to amyloid ⁇
  • the interfering molecule is siRNA, the sequence of which is selected from one or more of SEQ ID NO: 2-SEQ ID NO: 7.
  • the interfering molecule has the following structure:
  • Seq S ij is a sequence complementary to Seq ⁇ ;
  • X is a spacer sequence located between Seq J ⁇ and Seq ii , and the spacer sequence is not complementary to Seq Eft and Seq S fi .
  • the interfering molecule is an adenovirus expression vector.
  • the interfering molecules can form the following structure:
  • Seq ⁇ r ⁇ Seq Sia and X are defined as above,
  • the binding molecule is an antibody against amyloid ⁇ .
  • the anti-amyloid beta-beta antibody comprises (but is not limited to): 3F5 BA27 BC05
  • an inhibitor which inhibits JAK2/STAT3-SOCS-1 signaling pathway activation or inhibits JAK2/STAT3-SOCS-1 signaling pathway expression or activity which is an siRNA whose sequence is selected from the group consisting of SEQ ID NO: 2-SEQ ID NO: 7 and SEQ ID NO 36
  • a method of screening for a potential substance for preventing or ameliorating insulin resistance or diabetes comprising:
  • step (1) comprises: adding a candidate substance to a system comprising amyloid ⁇ and JAK2/STAT3-SOCS-1 signal pathways;
  • Step (2) comprises: detecting a change in a JAK2/STAT3-SOCS-1 signaling pathway or a pathway protein, which is compared with a control group, wherein the control group is amyloid-free white beta and does not add the candidate substance a system of JAK2/STAT3-SOCS-1 signaling pathways; If the candidate substance is statistically (eg, significantly inhibited by more than 20%, preferably 50°/. or more; more preferably 80% or more), inhibiting the amyloid ⁇ white ⁇ on the JAK2/STAT3-SOCS-1 signaling pathway or pathway ⁇ For white activation, the candidate substance is a potential substance for preventing or alleviating insulin resistance or diabetes.
  • the system comprising the amyloid ⁇ and JAK2/STAT3-SOCS-1 signaling pathways is selected from the group consisting of: a cell system (or a cell culture system), a subcellular system, a solution system, an animal system Or organizational system.
  • the system comprising the amyloid ⁇ and JAK2/STAT3-SOCS-1 signaling pathways is: a system comprising intracellular amyloid ⁇ and JAK2/STAT3-SOCS-1 signaling pathways. Or a system obtained by treating cells containing the JAK2/STAT3-SOCS-1 signaling pathway with amyloid beta.
  • the system comprising the amyloid ⁇ white ⁇ and JAK2/STAT3-SOCS-1 signaling pathway is a cell of a target organ of insulin such as a hepatocyte, a muscle cell or an adipocyte.
  • the method further comprises: performing further cell experiments and/or animal tests on the obtained potential substances to further select and determine from the candidate substances useful for controlling or alleviating insulin resistance or diabetes. substance.
  • the candidate substance is selected from, but not limited to, an interfering molecule, a nucleic acid inhibitor, a binding molecule (eg, an antibody) designed for a protein in the JAK2/STAT3-SOCS-1 signaling pathway or signaling pathway. Or ligand), small molecule compounds.
  • amyloid ⁇ white ⁇ is provided (including: full-length amyloid ⁇ white ⁇ , or a ruthenium fragment thereof. More preferably, the leucorrhizal fragment is selected from the group consisting of: ⁇ 40, ⁇ 42, ⁇ 25 -35, etc.) Use as a marker for blood (including: plasma or blood stasis) for detecting insulin resistance or diabetes.
  • an agent for specifically recognizing amyloid ⁇ preferably for recognizing amyloid ⁇ in blood (including plasma or blood sputum), for preparing insulin for detection Reagent or kit for resistance or diabetes.
  • the agent for specifically recognizing amyloid ⁇ white ⁇ is selected from the group consisting of:
  • binding molecule such as an antibody or a ligand that specifically binds to amyloid ⁇ white ⁇ ;
  • the binding molecule that specifically binds to amyloid ⁇ white ⁇ is an antibody selected from the group consisting of: 3F5, 1H3, 6C8, 10C1 1 , 7B 10 , BA27 or BC05.
  • kits for detecting insulin resistance or diabetes in blood comprising: an agent that specifically recognizes amyloid ⁇ .
  • ⁇ induces insulin resistance in primary liver cells and ⁇ 4 ⁇ cells.
  • mice hepatic primary cells were given ⁇ 25-35 (a, b) and ⁇ 42 (c) treatment on the insulin signaling pathway, the dose and time given are shown.
  • the phosphorylation of mouse primary cells InsR, Akt and GSK-3p was analyzed by immunoblotting. Among them, (a) cells were incubated with the indicated dose of ⁇ 25-35 for 60 h, (b) cells were incubated with 10 ⁇ ⁇ 25-35 for the indicated time, (c) cells were incubated with 10 ⁇ ⁇ 42 for 60 h; 100 nM insulin was given for 20 min.
  • a, b, HepG2 cells were given the effect of ⁇ 25-35 treatment on the insulin signaling pathway, and the dose and time of administration were shown.
  • Phosphorylation of InsR, Akt and GSK-30 in HepG2 cells was analyzed by immunoblotting. Among them, (a) was incubated with the indicated dose of ⁇ 25-35 for 60 h, (b) for 10 ⁇ 25-35 for the specified time; then with or without 100 nM insulin for 20 min.
  • WT mice littermate wild type mice
  • b, c, d, 12 weeks and 16 weeks of feeding b
  • body fat content c
  • lean body mass content d
  • Figure 7 Changes in important inflammatory factors and their downstream gene expression in liver, muscle and fat.
  • Figure 9. ⁇ upregulates SOCS-1 mRNA and chalk levels in primary liver cells.
  • ⁇ induces insulin resistance by up-regulating SOCS-1 to induce hepatocytes.
  • a Inhibition of SOCS-1 expression in primary hepatocytes by two different siRNAs.
  • b Phosphorylation levels of InsR and Akt in primary hepatocytes treated with ⁇ 25-35 (10 ⁇ ) or not treated with ⁇ 25-35 for 60 h in the presence of the indicated siRNA, wherein the amount of SOCS-1 RNAi 1 was used. It is 40 nM; the amount of SOCS-1 RNAi 2 is 40 nM.
  • c (b) quantification of the phosphorylation levels of InsR and Akt, wherein the amount of SOCS-1 RNAi 1 is 40 nM; the amount of SOCS-1 RNAi 2 is 40 nM. Data are expressed as mean and standard deviation, *P ⁇ 0.05.
  • a, b, c mouse primary hepatocytes were treated with the indicated dose of ⁇ 25-35 for 36 h (a), 10 ⁇ 25-35 for the indicated time (b) or 10 ⁇ ⁇ 42 for 36 h (c), phosphorylated STATl ( Immunoblot analysis of protein levels of Tyr701) phosphorylated STAT3 (Tyr705) and phosphorylated JAK2 (Tyrl007/1008).
  • d Tyrosine residue phosphorylation levels of STAT3 and JAK2 in the liver of APP/PS1 mice and wild-type control mice at 20 weeks.
  • RNAi down-regulation of STAT3 or JAK2(a, c) or AG490 (b) by RNAi inhibits JAK2 from inhibiting the increase in SOCS-1 levels by ⁇ .
  • the amount of STAT3 RNAi 1 is 40 nM
  • the amount of STAT3 RNAi 2 is 40 nM
  • the amount of AG490 is 0 ⁇
  • the amount of JAK2 RNAi 1 is 40 nM
  • the amount of JAK2 RNAi 2 is 40 nM.
  • the phosphorylation level of InsR and Akt was reduced by down-regulating STAT3 or JAK2 by RNAi (a, e) or JAK2 by AG490(c)
  • the amount of STAT3 RNAi 1 is 40 nM
  • the amount of STAT3 RNAi 2 is 40 nM
  • the amount of AG490 is ⁇ ⁇
  • the amount of JAK2 RNAi 1 is 40 nM
  • the amount of JAK2 RNAi 2 is 40 nM.
  • Quantitative analysis of phosphorylated InsR and Akt levels in b, d, f, (a, c, e) data expressed as mean and standard deviation, *P ⁇ 0.05.
  • Glucose tolerance experiments showed (A and B) that the glucose tolerance of APP/PS1 mice injected with JAK2 siRNA-derived adenovirus was improved.
  • Insulin resistance experiments showed (C and D) that APP/PS1 mouse insulin with adenovirus injected with JAK2 siRNA was significantly improved.
  • the phosphorylation levels of InsR and Akt in APP/PS 1 mice injected with JAK2 siRNA-derived adenovirus were significantly higher than those of APP/PS1 mice (E and F) injected with control virus.
  • the inventors have conducted extensive research to provide a new way for the diagnosis of insulin resistance and type 2 diabetes in patients with insulin resistance or diabetes.
  • the present invention also discloses for the first time that amyloid ⁇ ( ⁇ ) can induce peripheral tissue to produce insulin resistance by activating JAK2/STAT3-SOCS-1 signaling pathway (ie, stimulating JAK2/STAT3-dependent upregulation of SOCS-1) And realized. Therefore, the JAK2/STAT3-SOCS-1 pathway or its pathway can be used as a target for screening drugs to study drugs for preventing or alleviating insulin resistance or diabetes.
  • Reagents or kits for detecting insulin resistance or diabetes can be used as a target for screening drugs to study drugs for preventing or alleviating insulin resistance or diabetes.
  • ⁇ or its ⁇ white fragments can be used as markers for diagnosing insulin resistance or diabetes: (0 for insulin resistance or diabetes typing, identification Diagnosis, and/or susceptibility analysis; (ii) assessment of insulin resistance or diabetes medications, drug efficacy, prognosis, and selection of appropriate treatments in the relevant population; (iii) early assessment of the risk of insulin resistance or diabetes in the relevant population, Early monitoring of early prevention and treatment. For example, people with insulin resistance or diabetes caused by elevated levels of ⁇ in blood (plasma or serum) can be isolated for more targeted treatment.
  • the present invention provides the use of A ⁇ (including its precursor white or chalky fragments) or a gene encoding the same for the preparation of a reagent or kit for diagnosing (or detecting) insulin resistance or diabetes.
  • ⁇ including its precursor protein or ⁇ white fragment
  • expression and deposition all of which are encompassed by the present invention.
  • available techniques such as ELISA, Western blotting, Southern blotting, DNA sequence analysis, PCR, etc., can be used in combination.
  • the present invention also provides an agent for detecting the presence or absence and expression of ⁇ (including its precursor leucovorin or blushing fragment) in an analyte (blood).
  • a primer for specifically amplifying a gene of A ⁇ (including a precursor white or a white fragment thereof) or a probe specifically recognizing the A ⁇ gene can be used to determine the presence of the A ⁇ gene.
  • a binding molecule (such as an antibody or a ligand) that specifically binds to ⁇ white can be used to determine the expression of ⁇ protein.
  • the agent is an antibody (monoclonal antibody or polyclonal antibody) which specifically binds to ⁇ or its ⁇ white fragment.
  • an antibody monoclonal antibody or polyclonal antibody
  • Methods for detecting the expression of ⁇ white in an analyte using an antibody that specifically binds to ⁇ ⁇ white are well known to those skilled in the art.
  • the presence of ⁇ or a protein fragment thereof in the blood can be detected by ELISA.
  • ELISA techniques are well known to those skilled in the art.
  • specific primers or probes for the ⁇ gene is a technique well known to those skilled in the art, for example, to prepare a probe which specifically binds to a specific site on the ⁇ gene, and not to other than the ⁇ gene.
  • the gene specifically binds and the probe carries a detectable signal.
  • a pair of primers can be prepared which specifically amplify the ⁇ gene.
  • the invention also provides a kit for detecting the presence or absence and expression of an A ⁇ gene in an analyte, the kit comprising: an antibody or a ligand specifically binding to A ⁇ ; a primer for specifically amplifying the A ⁇ gene; Or a probe that specifically recognizes the ⁇ gene.
  • kit may further include various other reagents required for antigen-antibody reaction, hybridization, color development, DNA extraction, PCR, etc., including but not limited to: color developing solution, enzyme standard solution, coating Liquid, washing solution, extract, amplification solution, hybridization solution, enzyme, control solution, and the like.
  • kit may further include instructions for use and/or protein, nucleic acid sequence analysis software, and the like.
  • the "(signal) pathway” refers to a signal system formed by a series of chalk or gene interactions or interactions that generally cause some cellular events to occur.
  • the JAK2/STAT3-SOCS-1 signaling pathway includes, but is not limited to, the JA2 gene (and/or its encoded sputum), the STAT3 gene (and/or its encoded sputum), SOCS-1 ( And / or its coded white).
  • the nucleotide sequence of the JAK2 gene is shown, for example, in GenBank accession ⁇ NM_004972; its ⁇ white amino acid sequence is shown, for example, in GenBank Accession No. NP-004963.
  • the nucleotide sequence of the STAT3 gene is shown, for example, in GenBank Accession Nos. NM-139276, NM_213662, NM-003150; its chalky amino acid sequence is shown, for example, in GenBank Accession No. NP-644805, NP 998827, NP-003141. .
  • the nucleotide sequence of the SOCS-1 gene is shown, for example, in GenBank Accession No. NM_003745; its chalk amino acid sequence is shown, for example, in GenBank Accession No. NP 003736.
  • the SOCS White Family consists of eight members: CIS and SOCS-1 - SOCS-7. Intracellular SOCS protein expression Often lower, but can be significantly up-regulated when stimulated by cytokines. The relationship between SOCS protein and insulin signaling is the most studied with SOCS-1 and SOCS-3. SOCS-1 knockout mice have very low blood glucose, and their embryonic fibroblasts differentiate into adipocytes. (Kawazoe, Y., et al., Signal transducer and activator of transcription (ST AT)-induced STAT inhibitor 1 (SSI) -l)/suppressor of cytokine signaling 1 (SOCSl) inhibits insulin signal transduction pathway through modulating insulin receptor substrate 1 (IRS-1) phosphorylation.
  • ST AT Signal transducer and activator of transcription
  • SSI STAT inhibitor 1
  • SOCSl insulin receptor substrate 1
  • the above-described chalk or coding gene When used as a target for screening or artificially established a screening system, the above-described chalk or coding gene may be naturally occurring, such as may be purified and isolated from a mammal; or may be recombinantly produced, such as may be conventional Genetic recombination technology to produce recombinant chalk. In addition, any form of variation that does not affect the biological activity of these chalks is available, such as derivatives or variants whose function has not changed.
  • ⁇ can up-regulate the inhibition of the insulin pathway in mouse hepatic primary cells, and the expression of SOCS-1 in the liver of APP/PS1 mice is also elevated.
  • Down-regulation of SOCS-1 ⁇ expression in mouse liver primary cells alleviated the inhibition of insulin signaling pathway by A ⁇ .
  • ⁇ also activates JAK2/STAT3 in primary liver cells, and JAK2/STAT3 activity is also higher in the liver of APP/PS1 mice.
  • Down-regulation or inhibition of JAK2/STAT3 in mouse liver primary cells by RNAi or inhibitor can alleviate the up-regulation of SOCS-1 by ⁇ and the inhibition of insulin signaling pathway.
  • the present invention provides a method for screening for potential substances for preventing or alleviating insulin resistance or diabetes, contacting candidate substances with a system containing ⁇ and JAK2/STAT3-SOCS-1 signaling pathways; screening for inhibition of ⁇ to JAK2/STAT3-SOCS -1 A signalling pathway or pathway-activated substance that is a potential substance for the prevention or alleviation of insulin resistance or diabetes.
  • inhibitor refers to a statistically significant “inhibition”. Namely: Significantly “suppress”. For example, compared with the control group's chalky activity, chalky expression, protein interaction, significant "inhibition", more than 20%, better than 50%; better 80% or more.
  • the system comprising the A ⁇ and JAK2/STAT3-SOCS-1 pathways is selected from the group consisting of: a cell system (or cell culture system), a subcellular system, a solution system, an animal system, or a tissue system.
  • a cell system or cell culture system
  • the system comprising the JAK2/STAT3-SOCS-1 pathway is a hepatocyte.
  • the method further comprises: performing further cell experiments and/or animal tests on the obtained potential substances to further select and determine substances useful for controlling or alleviating insulin resistance or diabetes from the candidate substances. .
  • oligonucleotide hybridization techniques e. g., probes
  • PCR polypurine chain reaction
  • polyacrylamide gel electrophoresis polyacrylamide gel electrophoresis
  • immunoblotting e.g., Western blotting
  • Substances initially screened by the above methods can constitute a screening sputum so that one can finally screen out substances that can be truly useful for preventing or alleviating insulin resistance or diabetes.
  • the present invention also provides a latent substance obtainable by the screening method for preventing or alleviating insulin resistance or diabetes.
  • the substance is a substance that inhibits the activation of the JAK2/STAT3-SOCS-1 signaling pathway induced by A ⁇ , or is an inhibitor of JAK2/STAT3-SOCS-1 signaling pathway or pathway chalking.
  • the present invention provides an inhibition of JAK2/STAT3-SOCS-1 signaling pathway activation, inhibition of JAK2/STAT3-SOCS-1 signaling pathway expression or activity or inhibition of amyloid leukosin beta
  • an inhibitor of expression or activity for the preparation of a composition for the prevention or alleviation of insulin resistance or diabetes.
  • an inhibitor that inhibits activation of the JAK2/STAT3-SOCS-1 signaling pathway, inhibits JAK2/STAT3-SOCS-1 signaling pathway protein expression or activity, or inhibits the expression or activity of amyloid leukosin beta includes antagonism Agent, lowering agent, retarder, blocker, etc.
  • the inhibitor of JAK2/STAT3-SOCS-1 signaling pathway activation, inhibition of JAK2/STAT3-SOCS-1 signaling pathway expression or activity or inhibition of amyloid ⁇ expression or activity means any reduction of JAK2 /STAT3-SOCS-1 signaling pathway protein activity, decreased JAK2/STAT3-SOCS-1 signaling pathway gene or chalky stability, down-regulated JTAK2/STAT3-SOCS-1 signaling pathway chalky expression, decreased JAK2/STAT3-
  • the SOCS-1 signaling pathway is a potent effective time, or a substance that inhibits the transcription and translation of the JAK2/STAT3-SOCS-1 signaling pathway gene, which can be used in the present invention as a means for down-regulating the JAK2/STAT3-SOCS-1 signaling pathway.
  • the inhibitor is: an interfering molecule that specifically interferes with JAK2/STAT3-SOCS-1 signaling pathway protein expression; or a binding molecule that specifically binds to the JAK2/STAT3-SOCS-1 signaling pathway; or inhibits JAK2 AG490; or an interfering molecule that specifically interferes with the expression of amyloid ⁇ white ⁇ or its precursor ⁇ white; or a binding molecule that specifically binds to amyloid ⁇ white ⁇
  • the inhibitor is an antibody specific for amyloid ⁇ .
  • the antibody may be a monoclonal antibody or a polyclonal antibody. Immune animals can be immunized with amyloid beta, such as rabbits, small
  • the antibodies may also be monoclonal antibodies, and such monoclonal antibodies may be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol. 6: 511, 1976; Kohler et al, Eur. J. Immunol. 6: 292, 1976; Hammerling et al, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981).
  • the inhibitor is a small interfering RNA molecule (siRNA).
  • siRNA small interfering RNA
  • siRNA refers to a short-segment double-stranded RNA molecule that is capable of degrading specific mRNAs with mRNAs of homologous complementary sequences, a process that is RNA interference (RNA interference) process.
  • Small interfering RNA can be prepared in the form of a double-stranded nucleic acid comprising a sense strand and an antisense strand which form a double strand only under hybridization conditions.
  • a double-stranded RNA complex can be prepared from a sense strand and an antisense strand separated from each other.
  • a complementary sense strand and an antisense strand are chemically synthesized, which can then be hybridized to produce a synthetic double-stranded RNA complex.
  • a small interfering RNA molecule having good effects is provided, and the small interfering RNA molecule has a good effect of suppressing the signal pathway of J AK2 / ST AT3 - SOCS-1.
  • the small interfering RNA molecule is a small interfering RNA molecule having the nucleotide sequence shown in SEQ ID NOS. 2-7.
  • the sense strand and the antisense strand contained in the small interfering RNA can be prepared by one or more expression cassettes encoding the sense strand and the antisense strand.
  • the sense and antisense strands are encoded by a single expression cassette, they can be cleaved from the resulting transcript to form isolated sense and antisense strands, which are then hybridized to produce double-stranded small interfering RNA.
  • the small interfering RNA can be delivered to the cells by the use of appropriate transfection reagents, or can be delivered to the cells using a variety of techniques known in the art.
  • the present invention also provides a method for preparing a medicament for preventing or alleviating insulin resistance or diabetes, the method comprising: synthesizing and/or purifying a substance obtained by the aforementioned screening for controlling or alleviating insulin resistance or diabetes, for use as a control Or a drug that relieves insulin resistance or diabetes.
  • the obtained substances useful for controlling or alleviating insulin resistance or diabetes can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about pH 6. -8, although the pH may vary depending on the nature of the substance being formulated and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intramuscular, intravenous, or subcutaneous administration.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, 2002, or according to the manufacturer's recommended conditions. . Percentages and parts are by weight unless otherwise stated.
  • HepG2 human liver cancer cells; purchased from ATCC.
  • H4IIE Rat liver cancer cells; purchased from ATCC. Population sample collection and diagnostic evaluation.
  • the fasting blood glucose was judged as: fasting blood glucose concentration greater than or equal to 6.1 mmol/L and less than 7.0 mmol/L, and for measuring postprandial blood glucose for 2 hours, the blood glucose concentration must be less than 7.8.
  • Mm ol / L; diabetes is judged as: fasting blood glucose concentration greater than or equal to 7.0 mmol / L or 2 hours postprandial blood glucose concentration greater than or equal to 1 1.1 mmol / L.
  • studies have been conducted to combine impaired fasting blood glucose and diabetes into a hyperglycemia group.
  • mice and their littermate wild type controls were purchased from the Institute of Model Animals of Nanjing University, and the words were raised in the animal house of the Institute of Nutritional Sciences of the Chinese Academy of Sciences. All mice were housed according to the management of the experimental animals and the regulations of the Sichuan Committee, and the Slack Company provided sufficient water and standard feed. Main reagents and kits
  • ⁇ 42 Apeptide, article number AP3006;
  • Collagenase Sigma, ⁇ C0130;
  • Serine/threonine phosphatase inhibitor mixture Sigma, P5726;
  • Protease inhibitor mixture Sigma, goods ⁇ P8340; Amplex® Red Glucose ⁇ Glucose Oxidase Assay Kit: Invitrogen, A22189; Lipofectamine 2000: Invitrogen, Cat. No. 11668019;
  • Insulin for injection Lilly France S.A.S., trade name, Uberin. Common name, recombinant human insulin injection.
  • ⁇ 40 ELlSAkit Covance, SIG-38950, where the primary antibody combined with ⁇ 40 is BA27;
  • ⁇ 42 ELlSAkit Covance, article number SIG-38952, wherein the primary antibody combined with ⁇ 42 is BC05;
  • DMEM Invitrogen, goods ⁇ 12100-046;
  • FBS Invitrogen, article number 16000-044;
  • M-MLV Reverse Transcription Kit
  • PVDF membrane Millipore. Antibody
  • Tyr 1150/1151 refers to the 1150th or 1151th Tvr
  • Thr308 refers to the 308th Thr phosphorylation
  • Micropipettes were purchased from Gilson, ultra-low temperature refrigerators were purchased from Thermo Scientific Forma, PCR machines, vertical electrophoresis tanks, wet transfer membranes and power supplies were purchased from Bio-Rad, refrigerated centrifuges were purchased from Eppendorf, and high-speed centrifugation was performed.
  • the machine was purchased from Beckman, the high-speed pulper was purchased from Qiagen, the precision balance and pH meter were purchased from Mettler Toledo, the 0.22 ⁇ , 0.45 ⁇ needle filter was purchased from Millipore, and the ChemiDoc chemiluminescence imaging system was purchased from Wealtec.
  • the enzyme-linked immunosorbent assay was purchased from Molecular Device, and the X-ray and X-ray film processor was purchased from Kodak.
  • FreeStyle blood glucose tester and blood glucose test strip purchased from TheraSense
  • the minispec mq20 nuclear magnetic resonance (NMR) was purchased from Bruker
  • HITACHI 7020 automatic biochemical analyzer was purchased from HITACHI
  • ABI 7900HT real-time quantitative PCR was purchased from Applied Biosystems the company. Isolation and culture of mouse liver primary cells
  • the digested liver was placed in a 10 ml culture dish pre-cooled with 20 ml of collagen-free pre-chilled perfusate P2, and then cut up and pipetted repeatedly and filtered through a 70 ⁇ pore size cell sieve to 50 ml of centrifugation.
  • 4'C 50g was centrifuged for 2 minutes, then resuspended, then washed 3 times with collagenase-free pre-cooled perfusate P2, then 15 ml containing 20 U/ml penicillin, 20 g/ml streptomycin, 10%
  • the FBS DMEM was resuspended.
  • HepG2 H4UE cell lines were cultured in DMEM medium containing 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin and 25 mM glucose.
  • the cell culture incubator was kept at 37 ° C and contained 5% carbon dioxide. Cells generally per Passage once every 2-3 days, keep the density before passage more than 80%. Cells were seeded into 12-well plates 16-24 hours prior to use. Preparation, pretreatment
  • Preparation of ⁇ 25-35 Weigh the appropriate amount of ⁇ 25-35 powder, dissolve it in PBS to a final concentration of 1 mM, and store it in a -80 ⁇ refrigerator for later use.
  • Incubation Reference method (Coraci, I S. , et al., CD36 , a class B scavenger receptor, is expressed on microglia in Alzheimer's disease brains and can mediate production of reactive oxygen species in response to beta-amyloid fibrils. Am J Pathol, 2002. 160( 1 ): p. 101 -12), stored 1 mM ⁇ 25-35 was incubated in a 37 ° C, 5% CO 2 incubator for 2 days.
  • Preparation of ⁇ 42 Weigh the appropriate amount of ⁇ 42 powder, dissolve it in deionized water to a final concentration of 2 mM, and store it in a - 8 CTC refrigerator for later use. Incubation: Reference method (Jana, M., CA Palencia, and K. Pahan, Fibrillar amyloid-beta peptides activate microglia via TLR2: implications for Alzheimer's disease. J Immunol, 2008. 181 (10): p. 7254-62) The stored 2 mM ⁇ 42 was incubated in a 37 ° C, 5% CO 2 incubator for 5 days before use. Cell processing and transfection
  • the well-plated 12-well plates were plated overnight and replaced with blood-free DMEM containing 25 mM glucose while adding the indicated concentrations of ⁇ 25-35 or ⁇ 42. AG490 was added 24 hours after the addition of ⁇ 25-35. If insulin stimulation is required, cells are stimulated by adding 100 ⁇ ⁇ insulin for 20 minutes.
  • Transfected cells are required and cells are seeded into 12-well plates 16-24 hours prior to transfection. Transfection with Lipofectamine 2000. ⁇ treatment was added 48 hours after the end of transfection.
  • SDS-PAGE loading buffer 50 mM Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT
  • Tissue RIPA lysate (20 ⁇ 8 / ⁇ 1 pepstatin, 5 ⁇ ⁇ / ⁇ 1 leupeptin, 20 ⁇ / ⁇ aprotinin, 1 mM PMSF and 50 nM trichostatin A) and a mixture of phosphatase inhibitors (20) mM Tris, pH 7.5, 100 mM KC1, 0.1% Nonidet P-40, 1 mM EDTA, 10% glycerol, 50 mM NaF, 10 mM sodium pyrophosphate) After lysis, the whitening concentration was measured, the same amount of sputum white ( ⁇ 60 ⁇ 8 ) Add an appropriate amount of 5 ⁇ loading buffer to heat ⁇ for about 10 minutes.
  • the supernatant was applied to a 9% or 12% SDS polyacrylamide gel, electrophoresed, wet-transduced, and the PVDF membrane was blocked with 5% skim milk powder in TBST for 1 hour. After the end of the blocking, the cells were washed 3 times with TBST at room temperature and medium speed for 5 minutes each time, and then added to the primary antibody prepared in TBST containing 5% BSA, and incubated at room temperature for 3 hours or overnight at 4 °C.
  • the primary antibody is usually added with 0.02 to 0.05% sodium azide bacteriostatic for repeated use.
  • the TBST was washed three times on the shaker at room temperature and medium speed for 5 minutes each time.
  • the secondary antibody in 3 (1: 1000 ⁇ 1:5000), incubate for 1 to 3 hours at room temperature, and then wash the membrane 3 times with ti TBST.
  • H4IIE cells cultured in a 12-well plate treated with the indicated drug were washed three times with PBS, and 400 ⁇ l of a sugar-free DMEM medium containing 20 mM sodium lactate and 2 mM sodium pyruvate was added to each well, requiring insulin stimulation. Stimulate by adding 50 nM insulin. After 3 hours, the culture solution was collected, and the glucose level in the culture solution was measured using an Amplex® Red Glucose lucose Oxidase Assay kit, and the chalk concentration after cell lysis in each well was measured as an internal standard. Plasma separation and AP40/42 determination of blood samples from the population
  • Peripheral venous blood add EDTA and mix anticoagulation.
  • the plasma sample was centrifuged at 2,700 g for 4 min at 4 ° C, and the upper plasma fraction was collected and stored in a -80 ⁇ refrigerator. ⁇ 40/42 in plasma was measured using a Covance ELISA kit according to the instructions.
  • the cells in each well of 12-well plate were lysed with 1 ml of Trizol, and about 100 mg of tissue was lysed with 1 ml of Trizol, allowed to stand at room temperature for 5 minutes, then 0.2 ml of chloroform was added, shaken vigorously for 15 seconds, and allowed to stand at room temperature for 2-3 minutes, 12 , OOOxg After centrifugation at 4 V for 15 minutes, remove the sputum into a new centrifuge tube (approx. 0.45 ml), add 0.5 ml of isopropyl alcohol and mix at room temperature. After 0 minutes, centrifuge at 12, OOOxg, 4 Torr for 10 minutes.
  • Real-time PCR was performed in a 384-well plate in a three-fold format. Each well contains 5 ⁇ Power SYBR® Green PCR Master Mix (ABI) and 1 ⁇ template DNA (the cDNA obtained by reverse transcription is diluted 10-60 times depending on the gene abundance), the concentration of the forward and reverse primers Both were 0.5 ⁇ , and ABI's 7900 system was used to monitor fluorescence in real time.
  • the PCR reaction conditions were 50 ° C 2 min, 95 ° C for 10 min, followed by 95 ° C for 15 s and 60 ° C for 1 min. 40 cycles. To confirm that there was no non-specific amplification, each PCR product was analyzed using a melting curve, and agarose electrophoresis was also performed to ensure product specificity. The same plate was used to detect actin as an internal reference for comparing the relative expression levels of other genes.
  • Conventional PCR and qRT-PCR primers for various genes are shown in the real-time quantitative PCR section of Table 1.
  • the PCR primers used are shown in Table 1.
  • Transgenic mouse genotype identification PCR Basic phenotypic analysis of mice
  • mice All experimental control mice were littermates of the same sex as APPswe/PSENdE9 transgenic mice. The mice were weighed weekly at 10-19 weeks of age. The mice were measured for food intake every two days from 12 weeks to 16 weeks, and the average daily food intake was obtained. The body fat mass and lean mass of the mice were measured by nuclear magnetic resonance using a Minispec Mq7.5 Analyzer (Bruker, Germany), and then one body weight was normalized to obtain body fat content and lean body mass content. Glucose tolerance test (GTT)
  • mice were starved for 15 hours. After detecting fasting blood glucose, the glucose solution was intraperitoneally injected (according to the body weight of the mice, 2 g/kg), and then the blood glucose meter was used to detect 15, 30, 60 and 120 after the injection. Minutes the concentration of glucose in the blood of the tail vein of mice. Blood glucose meter (FreeStyle, Alameda, California, USA)
  • ITT insulin tolerance test
  • mice were fasted for 4 hours before the experiment, after detecting fasting blood glucose, 0.75 U/kg of human insulin injection was injected according to body weight, and mice were detected by blood glucose meter at 15, 30, 60 and 120 minutes after injection. Blood sugar level. Detection of plasma insulin levels, blood lipid levels and AP40/42 in mice
  • Plasma sample is blood 4 ° C
  • Plasma insulin levels were measured according to the instructions using the radioimmunoassay kit of the Beijing Institute of Biological Products.
  • the plasma levels of triglyceride, total cholesterol, high-density lipid ⁇ white 1ST alcohol and low-density lipid ⁇ white cholesterol are HITACHI 7020 automatic biochemical points Analyzer measurement.
  • ⁇ 40/42 in plasma was measured according to the instructions of Covance's EL1SA kit. Detection of insulin sensitivity in mouse liver and muscle
  • mice The 20-week-old mice were weighed for 16 hours and weighed. After anesthesia, the abdominal cavity was snoring. First, a piece of liver and one upper thigh muscle mass were taken, and liquid nitrogen was quickly frozen into an untreated control. Subsequently, 2 U/kg of human insulin injection was injected from the portal vein according to body weight, and the remaining liver was taken after 5 minutes, and the muscle on the other side of the thigh was taken 10 minutes later. The removed tissue was immediately frozen into liquid nitrogen, and after the tissue collection was completed, the sample was transferred to a -80 °C refrigerator. Subsequent whitening and western blot experiments were performed to detect the activation level of the key protein in the insulin signaling pathway. Data analysis and statistics
  • Example 1 induces hepatocytes to produce insulin resistance
  • the inventors selected hepatocytes to detect the effect of ⁇ on the insulin signaling pathway.
  • Hepatocytes were first treated with the ⁇ 25-35 fragment, which is the toxic core of full-length ⁇ 42 (Pike, CJ, et al., Structure-activity analyses of beta-amyloid peptides contributions of the beta 25-35 region to aggregation and neurotoxicity. J Neurochem, 1995 64(1): p. 253-65).
  • Western blot analysis showed that ⁇ 25-35 inhibited the insulin signaling pathway in mouse hepatic primary cells after insulin stimulation with dose and time effects, which were expressed as phosphoric acid of Insulin receptor (InsR), Akt, Glycogen synthase kinase 3p (GSK3p). Decline in the level ( Figures la and lb).
  • the present inventors also used full-length ⁇ 42 to treat mouse primary liver cells, and found that ⁇ 42 also inhibited insulin activation of insulin signaling pathway, Western blot analysis showed that phosphorylation levels of InsR, Akt, and GSK3p also decreased (Fig. Lc)
  • Fig. Lc One of the important functions of insulin is to inhibit the gluconeogenesis of hepatocytes.
  • insulin reduced the gluconeogenesis of the control group by about 60%.
  • the inhibitory effect of insulin on gluconeogenesis was gradually decreased with the increase of the concentration of ⁇ 25-35 (Gall I d).
  • the inventors also obtained similar results in the human liver cancer cell line HepG2.
  • Western blot analysis showed that ⁇ 25-35 inhibited the insulin signaling pathway of HepG2 cells stimulated by insulin with dose and time effects, which showed a decrease in phosphorylation levels of insulin receptors (InsR), Akt, and GSK3P (Gall 2a and 2b).
  • InsR insulin receptors
  • Akt Akt
  • GSK3P Gall 2a and 2b
  • ⁇ can induce insulin resistance in cells cultured in vitro, and insulin resistance in the body can cause hyperglycemia, so the inventors detected plasma of people with normal blood sugar and high blood sugar population (including clinically diagnosed patients with impaired fasting glucose and diabetes) In the Ap 7] flat.
  • ELISA results showed that plasma levels of ⁇ 40/42 were significantly higher in hyperglycemic people than in those with normal blood glucose (Figure 3).
  • ⁇ can induce insulin resistance in hepatocytes and plasma ⁇ levels in hyperglycemic patients.
  • mice are commonly used double transgenic AD mouse models. It is a ⁇ -secretase PS1 that triggers the expression of a mouse/human chimeric amyloid ⁇ white precursor protein ⁇ containing a Swedish mutation ( ⁇ 594/5 ⁇ ) and a deletion ⁇ of exon 9 by a ⁇ white promoter ( PSENdE9)
  • PSENdE9 ⁇ white promoter
  • the present inventors observed some basic phenotypes of APP/PS 1 mice at 10-20 Ml age and found that APP/PS 1 mice were 10-19 weeks old compared to littermate control mice (Fig. 4a), 12 Weeks and 16 weeks of ingestion (ffl 4b), body fat content (Cell 4c), lean body mass (
  • the present inventors examined changes in glucose tolerance and insulin sensitivity of APP/PS 1 mice and littermate wild type mice. Glucose tolerance experiments showed that glucose tolerance in APP/PS1 mice was slightly impaired at 10 weeks, and more severe impaired glucose tolerance was observed by 18 weeks, indicating a phenotype with increased glucose tolerance due to age (Fig.
  • the inventors examined the activation of insulin-induced downstream signaling pathways in mouse liver and muscle.
  • the results showed that the phosphorylation levels of InsR and Akt in the liver tissue of APP/PS 1 mice were significantly lower than those in the littermate control mice after insulin stimulation (Fig. 6a, 6b), indicating insulin in the liver tissue of APP/PS1 mice.
  • the downstream signal is damaged.
  • similar phenomena to liver tissue were observed in muscle tissue.
  • Akt phosphorylation in APP/PS1 mice was also significantly lower than in littermate control mice (Fig. 6c, 6d), indicating that insulin signaling was also inhibited in muscle.
  • hypoglycemic effect of A ⁇ antibody The present inventors administered intraperitoneal injection to a 3 month old male APP/PS1 mouse using ⁇ antibody 3F5 (purchased from Yes Biotech Laboratories Ltd, titer 1: 50-1:500) at a dose of 10 per dose. Mg/kg. The male APP/PS 1 mice were injected with the same dose of IgG once a week.
  • mice tested for 4 hours of starvation showed that the blood glucose of APP/PS1 mice decreased from 152.3 mg / dl to 127 mg / dl, indicating that the antibody 3F5 treatment of ⁇ has a significant hypoglycemic effect.
  • the present inventors used a ⁇ antibody 1H3 (purchased from Yes Biotech Laboratories Ltd, titer 1:50-1:500), 6C8 (purchased from Yes Biotech Laboratories Ltd, titer) for 3 month old male APP/PSI mice. 1:50-1:500)
  • the dose is 10 mg/kg each time.
  • Male J/PS1 mice were injected with the same dose of I g G every J injection.
  • a ⁇ can up-regulate the expression level of SOCS-1 in mouse liver and liver primary cells.
  • the inventors' research found that sputum/PS1 mice showed severe insulin resistance with increasing age, but the mechanism of this phenomenon is still unclear. Since the inflammatory response plays an important role in the ⁇ -induced nerve injury, and the inflammatory response is also a major cause of insulin resistance, the inventors suspected that ⁇ might induce insulin resistance by activating the inflammatory response of peripheral tissues. .
  • the present inventors examined the expression of some important inflammatory factors of liver, muscle and adipose tissue and the downstream factor of the signaling pathway in the target organs of insulin in APP/PS1 mice and their littermate control mice by quantitative PCR. happening.
  • the present inventors have found that the expression of IL- ⁇ , IL-8 in S0CS-1 and S0CS-3, GP130 in the liver, and fat in the liver and muscle of APP/PS mice is significantly increased.
  • the remaining inflammatory factors and their downstream chalky expression did not change significantly (Fig. 7a, 7b, 7c), indicating that there was no significant inflammatory response in the insulin target organs of APP/PS mice.
  • SOCS chalk was originally found to be an inhibitor of the cytokine signaling pathway, and the inventors focused on the role of S0CS in the insulin signaling pathway.
  • Western blot analysis showed that SOCS-1 ⁇ white expression in the liver of APP/PS1 mice was significantly up-regulated compared with littermate control mice, while SOCS-3 ⁇ white expression was unchanged (Fig. 8a, 8b).
  • Example 5 ⁇ induces insulin resistance by up-regulating SOCS-1 to induce hepatocytes
  • the inventors further examined whether ⁇ -induced insulin resistance is dependent on up-regulation of SOCS-1.
  • SOCS-1 siRNA-1 and SOCS-I siRNA-2 insulin is stimulated by insulin-stimulated cells in primary liver cells after SOC ⁇ 25-35 (10a)
  • Inhibition of the signaling pathway was attenuated by a significant response to the phosphorylation levels of InsR and Akt (Fig. 10b, 10c).
  • JAK2/STAT3 signaling pathway mediates ⁇ -induced SOCS-1 upregulation and insulin resistance
  • SOCS sputum is usually expressed low in resting cells, and after stimulation by cytokines, it induces SOCS gene transcription mainly by activating JAK/STAT signaling pathway (Ronn, S.G., N. Billestrup, and T.
  • Tyk2/STAT3 signaling mediates beta-amyloid-induced neuronal cell death: implications in
  • Alzheimer's disease J Neurosci, 2010. 30(20): p. 6873-8 ⁇ , Chiba, T., et al., Amyloid-beta causes memory impairment by disturbing the JAK2/STAT3 axis in hippocampal neurons.
  • the level of chemistry was significantly higher than that of the control mice (Gel, l ie), indicating that STAT3 and JAK2 in the liver of APP/PS 1 mice were in a relatively high activity state, and this state was also likely to be induced by ⁇ . .
  • the present inventors applied the antibody 1H3 of Chuanxiong ⁇ (purchased from Yes Biotech Laboratories Ltd, titer 1:50-1:500), 6C8 (purchased from Yes Biotech Laboratories Ltd, to the male APP/PS1 mouse of 3 months old).
  • the titer was 1:50-1:500) and the intraperitoneal injection was administered at a dose of 10 mg/lcg each time.
  • the male APP/PS1 mice were injected with the same dose of IgG once a week.
  • the inventors examined whether the up-regulation of S0CS-1 by ⁇ is mediated by STAT3 and ⁇ 2.
  • the inventors designed two different siRNA sequences targeting STAT3 (see Table 3. STAT3 siRNA-1 and STAT3 siRNA-2) and found that when STAT3 is down-regulated, ⁇ is inhibited by up-regulation of SOCS-1 (Fig. 12a).
  • Inhibition of JAK2 by the inhibitor AG490 or down-regulation of JAK2 by two different RNAi sequences see Table 3, JAK2 siRNA-1 and JAK2 siRNA-2
  • the upregulation of dry SOCS-1 by ⁇ was inhibited (Fig. 12b, 12c) .
  • a ⁇ induces insulin resistance by activating the JAK2/STAT3 signaling pathway.
  • STAT3 protein expression was reduced by RNAi
  • the inhibitory effect of A ⁇ on insulin signaling pathway was significantly attenuated, and the phosphorylation levels of InsR and Akt induced by A ⁇ were significantly reduced (Fig. 13a, 13b).
  • inhibition of JAK2 by inhibitors or down-regulation of JAK2 by RNAi significantly attenuated the inhibition of insulin-induced insulin signaling pathway activation, and ⁇ -induced phosphorylation of InsR and Akt was also significantly reduced (Gen 13c, 01063
  • the inventors In order to verify in vivo that ⁇ -induced insulin resistance is dependent on the dry JAK2/STAT3/SOCS-1 signaling pathway, the inventors have packaged an siRNA adenovirus that specifically down-regulates JAK2 expression (for the sequence 5'-GCAAACCAGGAATGCTCA-3', SEQ ID NO: 36) Overexpression in the liver of APP/PS1 mice by tail vein injection. As a result, adenovirus overexpressing JAK2 siRNA down-regulated the level of JAK2 (JAK2i), and the level of STAT3 was not affected.
  • the JAK2 siRNA adenoviral vector (Ad-JAK2i) construction method is as follows: Adenovirus is constructed according to
  • GC-3' (SEQ ID NO: 38), post-synthesis annealing, was inserted into a U6-entry vector (Invitrogen). Subsequent recombination with the pAd/BLOCK-iTT DEST plasmid. The plasmid obtained after the completion of the recombination was linearized with Pac 1 endonuclease and then transfected into 293A cells for the second recombination, and the recombinant adenovirus was obtained about 0 days. Purification of adenovirus was purified by CsCl gradient ultracentrifugation.
  • the LacZi siRNA adenoviral vector (Ad-LacZi) construction method is specifically as follows: The oligonucleotide for the bacterial LacZ gene interference fragment provided by the Invitrogen adenovirus kit is annealed and inserted into the U6- en tiy vector. The remaining steps are consistent with the JAK2 siRNA adenoviral vector construction method.
  • the present inventors studied changes in glucose tolerance and insulin sensitivity of ⁇ /PS1 mice infected with JAK2 siRNA-derived adenovirus.
  • Glucose tolerance experiments showed an increase in glucose tolerance in APP/PS 1 mice injected with JAK2 siRNA-derived adenovirus (Fig. 18A and B).
  • Insulin resistance experiments showed significant improvement in insulin in APP/PS1 mice injected with JAK2 siRNA-derived adenovirus (Fig. 18C and D).
  • the present inventors examined the activation of insulin-induced downstream signaling pathway in the liver of APP/PS 1 mice infected with JAK2 siRNA-derived adenovirus.
  • Test group the above cell model treated with the candidate substance
  • Control group The above cell model that was not treated with the candidate substance.
  • the candidate substance was a substance that could prevent or alleviate insulin resistance or diabetes.
  • the cell model was treated with SEQ ID NO. 4-7 (in turn, candidate substance 1-4), and siRNA of SEQ ID NO: 1 (candidate 5) as candidate substances.
  • candidate substance 1-4 is a substance which can potentially prevent or alleviate insulin resistance or diabetes; and the candidate substance 5 has no effect.
  • Example 9 Test kit
  • a kit for detecting insulin resistance comprising:
  • some other containers are respectively provided with a color developing solution, an enzyme standard solution, a coating liquid, and a washing liquid.
  • the present inventors first studied the correlation between ⁇ and insulin resistance at the cellular level, and found that ⁇ can inhibit insulin-stimulated pathways in insulin-stimulated cells of mice, and reduce the inhibitory effect of insulin on hepatocyte gluconeogenesis.
  • the inventors found that ⁇ in the plasma of patients with hyperglycemia was significantly elevated, and insulin resistance was an important cause of hyperglycemia, suggesting that ⁇ is associated with insulin resistance in vivo.
  • APP/PS1 mice overexpressing A ⁇ in plasma showed impaired glucose tolerance and insulin resistance symptoms with increasing age, and activation of insulin signaling pathway in liver and muscle of APP/PS 1 mice was inhibited, indicating APP/PS 1 There is indeed insulin resistance in the liver and muscle of mice, suggesting that overexpression of ⁇ in plasma can directly induce insulin resistance. Finally, the inventors found that ⁇ exerts its insulin-inducing effect by activating JAK2/STAT3 signaling pathway to up-regulate SOCS-1. The above studies show that abnormal elevation of ⁇ can induce insulin resistance.
  • the experiment of the present inventors selected APP/PS 1 mice of less than or equal to 20 weeks of age, in which the amyloid deposit in the brain of the mouse was not obvious, and the cognitive ability of the mouse was not impaired, indicating that it was produced at this time. Insulin resistance is not due to Alzheimer's disease, and suggests that peripheral insulin resistance may be caused by an increase in peripheral ⁇ . Similar to some widely accepted mouse models of type 2 diabetes, APP/PS 1 began to exhibit similar symptoms of human type 2 diabetes such as hyperinsulinemia and hyperglycemia at around 10 weeks, and is a potential new type 2 Diabetic animal model.
  • the deposition of ⁇ in the brain of AD patients is an important pathological feature.
  • the level of ⁇ in plasma of AD patients has been reported to be significantly increased, but there have been no significant changes reported. The reasons for these differences in research results may come from experimental design, such as the age of the test subject, the severity of the disease, and so on. There were no significant changes in peripheral ⁇ levels in many AD patients, which partly explained that only a small proportion of AD patients developed impaired fasting glucose and type 2 diabetes.
  • the anti-A ⁇ antibody in the plasma of diabetic patients is significantly higher than that of the control population, it is suggested that the ⁇ in the blood plasma of diabetic patients may also increase, but whether the ⁇ level in the plasma of diabetic patients has not changed. See the report.
  • SOCS up-regulates SOCS-1 by activating JAK7STAT.
  • SOCS was originally found to be a suppressor of the cytokine signaling pathway.
  • SOCS protein also inhibits the insulin signaling pathway, and some cytokine-induced insulin resistance requires SOCS involvement.
  • ⁇ can also induce glial cells to express cytokines such as ⁇ - ⁇
  • the present inventors did not detect IL- ⁇ , IL-6, TNF-ct and other inflammatory factors in the peripheral tissues of APP/PS 1 mice.
  • Increased expression, and cytokine-induced SOCS gene transcription is often broad, a cytokine can induce multiple SOCS gene expression (Fasshauer, M., et al., Insulin resistance-inducing cytokines differentially regulate SOCS mRNA expression via growth factor - and Jak/Stat-signaling pathways in 3T3-L1 adipocytes. J Endocrinol, 2004. 181(]): p. 129-38).
  • a ⁇ activates ERK in hepatic primary cells (data not shown), whereas ERK phosphorylates the serine residue of STAT3 to regulate STAT3 activity, and the integration of these intracellular signals may determine that A ⁇ up-regulates SOCS- Specificity of 1.

Abstract

本发明公开了防治胰岛素抵抗和糖尿病的方法和试剂。本发明揭示了淀粉样蛋白β(Aβ)能诱导外周组织产生胰岛素抵抗，其是通过激活JAK2/STAT3-SOCS-1信号通路实现的。

Description

防治 岛 抵抗和糖尿 ¾的 u试剂
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PTEN. SHTP ④佶' '）迎路' I1的 白和其抑制 ί&白的 例如炎忭细胞 W子 1L-6 等能上调细胞因子信号通路的抑制子 (Suppressor of cytokine signaling, SOCS)的蚩白表 达， SOCS能结合到胰岛素受体抑制信号转 。
阿尔茨海默病 (AD)是老年痴呆病中最常见的一种， 是一种以记忆能力减退、 认知功 能障碍、 行为异常为特征的慢性进行性神经退行性疾病， 其发病率随年龄增大而增加。
AD 的主要病理特征是大脑内出现以沉积的 Λβ 为中心的淀粉样斑块 （amyloid plaque),或称老年斑 （senile plaque)和由高度磷酸化的微管相关蛋白 tau蚩白形成的神经 丝缠结 （neurofibrillary tangle)。 Αβ是细胞一种正常的代谢产物， 能在其受体帮助下透过 血脑屏障。 健康人血液和脑脊液中的 Αβ浓度处于一种平衡状态。 大量证据支持 Αβ是 AD 的主要致病因子。 目前被广泛接受的淀粉样蚩白级联假说 （Amyloid cascade hypothesis)认为， Αβ在 AD的发生、 发展中起着主 作用， 脑中 Αβ的生成和清除的失 衡， 使得 Αβ在脑中积累， 进而 致神经元和其突触功能的损伤， 最终造成认知的损害。
目前认为 Αβ主要由脑中的神经元产生。 Αβ由 ΑΡΡ经过 β-分泌酶和 γ-分泌酶酶解形 成。 由于 γ-分泌酶的切割位点不同， 主要产生两种形式： Αβ40和 Αβ42。 Αβ42的聚集能 力较强因而毒性也较大。 ΑΡΡ 和 γ-分泌酶分布都相对广泛， 在很多组织中都有表达。 β- 分泌酶最初认为只在脑中的神经元高表达， 但近来有研究显示， β-分泌酶也表达在神经 胶质细胞和其它外周的细胞， 例如脂肪细胞和肝细胞， 这提示外周组织也可能产生 Αβ。
研究表明， 在外周组织能检测到 Αβ。 Joachim等最先报道在 AD病人的血管、皮肤、 皮下组织和肠等非神经组织检测到 Αβ沉积， 在一部分正常的老年个体中也能在非神经 组织检测到 Αβ的沉积。 Lee等报道在正常人皮下脂肪和内脏脂肪中都能检测到 Αβ的沉 积。 另外， 包涵体肌炎的病灶处也能检测到 Λβ。 这些沉积的 Αβ， 可能是由脑中产生， 再经过循环系统运送到外周， 也有可能是由外周的细胞产生的。 但目前的研究都只检测 到外周 Αβ， 而它们有什么功能还不淸楚， 血浆中的 Αβ浓度本身也并不适合作为 AD的 生物标记 ( Liu, J K , et al., J Alzheimers Dis. 2010;20(4): 1233-42. ) 。
除 Αβ外目前还发现多种淀粉样蛋白 (Amyloid protein) , 其在各种器官和组织 的异常沉积会造成系列病变。 截至 2010年， 已知的淀粉样蛋白有 27种， 其中公 认为与糖尿病有密切关系的是胰岛淀粉样多肽 (IAPP)， IAPP是胰岛 β细胞分泌的 一种止常产物， 含 37个氨基酸， 但与 Αβ在序列和剪切机制上大为不同。 尸体病 理解剖检验发现， 90%的 2型糖尿病患者胰岛中有 ΙΑΡΡ沉积，伴 β细胞数量减少， 且胰岛淀粉样变性程度与糖尿病的病变程度一致， 说明 ΙΑΡΡ 与 2型糖尿病发病 相关。 人源 ΙΑΡΡ可诱 β细胞凋亡， 且二者呈剂量相关性。 转入人源 ΙΑΡΡ基因的纯 合子肥胖小鼠在卨糖、 卨脂饮食、 生长激素或糖皮质激素处理后胰岛内很快出现 大量 ΙΑΡΡ变性沉积， β细胞凋亡水平大于复制水平， 数量下降， 最终发展为 2型 糖尿病。 这些证据表明， ΙΛΡΡ在胰岛中变性沉积， 导致 β细胞凋亡， 数量减少是 2型糖尿病致病机制之一。但没有在先证据显示， 同为淀粉样蛋白的 Αβ在胰岛素 的靶组织如肝脏等能诱导 2型糖尿病的另一特征胰岛素抵抗， 或者 Αβ在 2型糖 尿病的发病中具有与 ΙΑΡΡ类似的功能。
流行病学的研究显示， 糖尿病患者相对于正常人有高约两倍的摧患 AD的风险。 虽 然其机理目前仍不清楚， 有人认为糖尿病能影响 Αβ的代谢。例如 Ho等报道在 AD模型 小鼠中用高脂诱 胰岛素抵抗能显著增加 Αβ40和 Λβ42的生成， 同时脑中老年斑的沉 积加剧， 学习记忆能力的损害也恶化。 Cao等报道在 AD模型小鼠中用糖水诱 胰岛素 抵抗， 脑中老年斑的沉积也加剧， 主要是由于脑中沉积的 Αβ42增高了约 2倍， 水迷宫 实验结果也显示了学习记忆能力的损害恶化。但是 Takeda等的研究表明， AD模型小鼠 与 2型糖尿病模型小鼠杂交的后代， 显示学习记忆能力的损害发生加剧， 脑中 Αβ的总 量没有变化， 而是显示脑血管出现炎性并伴随淀粉样血管病变。
综上， 尽管现有技术中对于糖尿病或胰岛素抵抗与 AD的关系有过研究， 然而还没 有明确的、 可信的结论， 在机理也不明确。 因此， 本领域还需要进一步地研究 Αβ与糖 尿病或胰岛素抵抗的相关性， 并找到有用的、 适于药物幵发的靶点。 发明内容
本发明的目的在于提倂防治胰岛素抵抗和糖尿病的方法和式剂。
在本发明的第一方面， 提供抑制 JAK2/STAT3-SOCS- 1 信号通路激活， 抑制 JAK2/STAT3-SOCS-1 信号通路蚩白表达或活性， 或抑制淀粉样蚩白 β的表达或活性的 抑制剂的用途， 用于制备防治或缓解胰岛素抵抗或糖尿病的药物。
在另一优选例中， 所述的 JAK2/STAT3- SOCS- 1 信号通路蛋白包括： JAK2 蚩白，
STAT3蛋白或 SOCS-1蚩白。
在另一优选例中，所述的 JAK2/STAT3-SOCS-1信号通路激活是： 淀粉样蚩白 β(Αβ) ^致的 JAK2/STAT3-SOCS- 1信号通路激活。
在另一优选例中， 所述的淀粉样萤白 β(Αβ)包括： 全长的淀粉样蚩白 β, 或其蚩白片 段。 更佳地， 所述的蛋白片段选自： Αβ40、 Αβ42、 Αβ25-35等。
在另一优选例中， 所述的抑制剂包括 (但不限于)：
特异性干扰 JAK2/STAT3-SOCS- 1信号通路蚩白表达的干扰分子 (如小干扰 RNA分 子或反义核苷酸)； 或
特异性与 JAK2/STAT3-SOCS- 1信号通路蚩白结合的结合分子 (如抗体或配体)； 或 抑制 JAK2的 AG490; 或
特异性干扰淀粉样蛋白 β或其前体蚩白表达的干扰分子 (如小干扰 RNA分子或反义 核苷酸)； 或
特异性与淀粉样蛋白 β结合的纺仓分子 (如抗体或配体)； 或
β-分泌酶抑制剂、 y-分泌酶抑制剂和调节剂或抗淀粉样蚩白聚集剂。 在另一优选例中，所述的干扰分子是 siRNA,其序列选自 SEQ ID NO: 2-SEQ ID NO: 7的一种或多种。
在另一优选例中， 所述的干扰分子具有以下结构：
Seq X— Seq
其中， Seq 的核苷酸序列如 SEQ ID NO. 36所示； Seq S ij为与 Seq ^互补的序列；
X为位于 Seq J ^和 Seq ii之间的间隔序列， 并且所述间隔序列与 Seq Eft和 Seq S fi 不互补。
在另一优选例中， 所述的干扰分子是腺病毒表达载体。
在另一优选例中， 所述的干扰分子可形成以下结构：
Seq正向一^ s
' ' X
Seq反向
其中，
Seq ^r^ Seq Sia和 X的定义如上述，
II 表示在 Seq 和 Seq S ia之间形成的氢键。
在另一优选例中， 所述的结合分子是抗淀粉样蛋白 β的抗体。
在另一优选例中，所述的抗淀粉样蚩白 β的抗体包括 (但不限于)： 3F5 BA27 BC05
1H3 , 6C8 solanezumab (Eli Lilly and Company), Bapineuzumab (JANSSEN Alzheimer Immunotherapy Research & Development , LLC; Elan Pharmaceuticals, Inc., Wyeth) , MABT5102A (Genentech, Inc.) , Ponezumab (Pfizer, Inc.), Intravenous Immunoglobulin (Baxter Healthcare) 最近己经发现，人免疫球蚩白（Ig)包含结合 Αβ的抗体 (Weksler et al., 2005), 静脉注射 Ig有助于促进 Αβ的清除。
在本发明的另一方面， 提供抑制 JAK2/STAT3- SOCS-1 信 通路激活或抑制 JAK2/STAT3-SOCS- 1信号通路蚩白表达或活性的抑制剂，其是 siRNA,其序列选自 SEQ ID NO: 2-SEQ ID NO: 7和 SEQ ID NO 36
在本发明的另一方面， 提供一种筛选防治或缓解胰岛素抵抗或糖尿病的潜在物质的 方法， 所述方法包括：
(1)将候选物质与含有淀粉样蛋白 β和 JAK2/STAT3-SOCS- 1信号通路的体系接触；
(2)筛选出抑制淀粉样蚩白 β对 JAK2/STAT3-SOCS- 1信号通路或通路蛋白的激活的 物质， 所述物质是防治或缓解胰岛素抵抗或糖尿病的潜在物质。
在另一优选例中， 步骤 （1) 包括： 向含有淀粉样蛋白 β和 JAK2/STAT3-SOCS- 1信 号通路的体系中添加候选物质； 和
步骤 (2)包括： 检测 JAK2/STAT3-SOCS- 1 信号通路或通路蛋白的变化， 并与对照组 比较， 其中所述的对照组是不添加所述候选物质的、 含有淀粉样蚩白 β 和 JAK2/STAT3-SOCS-1信号通路的体系； 若候选物质在统计学上 (如显著抑制 20%以上， 较佳的 50°/。以上； 更佳的 80%以上) 抑制淀粉样蚩白 β对 JAK2/STAT3-SOCS- 1信 通路或通路蚩白的激活， 则该候选物质 是防治或缓解胰岛素抵抗或糖尿病的潜在物质。
在另一优选例中， 所述的含有淀粉样蚩白 β和 JAK2/STAT3-SOCS- 1 信号通路的体 系选自： 细胞体系（或细胞培养物体系）、 亚细胞体系、 溶液体系、 动物体系或组织体系。
在另一优选例中， 所述的含有淀粉样蚩白 β和 JAK2/STAT3-SOCS- 1 信号通路的体 系是： 细胞内包含淀粉样蚩白 β和 JAK2/STAT3-SOCS- 1信号通路的体系； 或以淀粉样 蛋白 β处理包含 JAK2/STAT3- SOCS-1信^通路的细胞后获得的体系。
在另一优选例中， 所述的含有淀粉样蚩白 β和 JAK2/STAT3-SOCS- 1 信 通路的体 系是胰岛素的靶器官的细胞如肝细胞、 肌肉细胞或脂肪细胞。
在另一优选例中， 所述的方法还包括： 对获得的潜在物质迸行进一步的细胞实验和 / 或动物试验， 以从候选物质中进一步选择和确定对于防治或缓解胰岛素抵抗或糖尿病有 用的物质。
在另一优选例中， 所述的候选物质选自 (但不限于)： 针对 JAK2/STAT3-SOCS- 1 信号 通路或信号通路中的蛋白设计的干扰分子、 核酸抑制物、 结合分子 (如抗体或配体)、 小 分子化合物。
在本发明的另一方面， 提供淀粉样蚩白 β (包括： 全长的淀粉样蚩白 β, 或其蚩白片 段。 更佳地， 所述的蚩白片段选自： Αβ40、 Αβ42、 Λβ25-35等） 用作检测胰岛素抵抗或 糖尿病的血液 (包括： 血浆或血淸)标志物的用途。
在本发明的另一方面，提供一种特异性识别淀粉样蚩白 β (较佳地识别血液 (包括： 血 浆或血淸)中的淀粉样蛋白 β)的试剂的用途， 用于制备检测胰岛素抵抗或糖尿病的试剂 或试剂盒。
在另一优选例中， 所述的特异性识别淀粉样蚩白 β的试剂选自：
特异性结合淀粉样蚩白 β的结合分子 (如抗体或配体)；
特异性扩增淀粉样蚩白 β或其前体蚩白的编码基因或切割前体蚩白的蚩白酶的编码 基因的引物； 或
特异性识别淀粉样蚩白 β或其前体蚩白的编码基因或切割前体蚩白的蚩白酶的编码 基因的探针。
在另一优选例中， 所述的特异性结合淀粉样蚩白 β的结合分子是抗体， 选自： 3F5， 1H3 , 6C8 , 10C1 1 , 7B 10 , BA27或 BC05。
在本发明的另一方面， 提供一种检测血液 (包括： 血浆或血清)中胰岛素抵抗或糖尿 病的试剂盒， 所述的试剂盒中含有： 特异性识别淀粉样蛋白 β的试剂。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的。 附阁说明
1、 Αβ诱 肝原代细胞和 Η4ΙΙΕ细胞胰岛素抵抗。
a, b, c， 小鼠肝原代细胞给予 Αβ25-35 (a, b)和 Αβ42 (c)处理后对胰岛素信号通路 的影响，给予的剂量和时间见图。免疫印迹法分析了小鼠肝原代细胞 InsR， Akt和 GSK-3p 的磷酸化。其中，（a)为细胞用指定剂量的 Αβ25- 35孵育 60h，（b)为细胞用 10 μΜ Αβ25-35 孵育指定的时间， （c)为细胞用 10 μΜ Αβ42孵育 60 h; 之后给予或不给予 100 nM胰岛 素刺激 20 min。
d, 大鼠肝癌细胞株 H4IIE中糖质新生情况。 细胞用指定剂量的 Λβ25- 35孵育 60h, 然后用 50nM胰岛素再处理 3h。数据以平均值和标准差表示， **Ρ<0.01, *** < 0.001ο 图 2、 Αβ诱^ HepG2细胞胰岛素抵抗。
a, b， HepG2细胞给予 Αβ25-35处理后对胰岛素信号通路的影响， 给予的剂量和时 间见图。 免疫印迹法分析了 HepG2细胞 InsR， Akt和 GSK-30的磷酸化。 其中， （a)为用 指定剂量的 Αβ25-35孵育 60h， （b)为 10 μΜΑβ25-35孵育指定时间； 之后给予或不给予 100 nM 胰岛素处理 20min。
图 3、 高血糖病人血浆中 Αβ水平显著升高。
ELISA检测正常血糖和高血糖人群的血浆中 Αβ40/42水平。 正常血糖 η = 26; 高血 糖 η = 35, 数据以平均值和标准误表示。
4、 APP/PS1小鼠一些基本表型正常。
a, 雄性 APP/PS1小鼠 (n = 9)和同窝野生型 (WT)小鼠 (n = 8) 10-19周（w)的体重。 b, c, d, 12周和 16周的摄食 (b)、 体脂含量 (c)、 瘦体重含量 (d)。
e, f， 20周龄雄性八？？/1^1小鼠 (n = 4)和同窝野生型 (WT)小鼠 (n = 6)血脂中重要的 主要脂质指标 (e；)， 谷丙转氨酶和天冬氨酸转氨酶水平 (f)。 数据以平均值和标准差表示。
5、 APP/PS1小鼠显示随年龄加重的糖耐量受损和胰岛素抵抗症状。
a，b,10周或 18周雄性 APP/PS1小鼠 (每一年龄 n= 14- 16)和同窝野生型 (WT)小鼠 (每 一年龄 η= Π-19)的葡萄糖耐受试验 Pgkg—
c, d, 13周或 19周雄性 APP/PS1小鼠 (每一年龄 n= 16)和同窝野生型 (WT)小鼠 (每 一年龄 n = 17-20)的葡萄糖耐受试验 (0.75 U kg1)^
e, f, 葡萄糖耐受试验 GTT(e)和胰岛素耐受试验 ITT(f)的曲线下面积 (AUC)。
g, 饥饿和喂食状态下 APP/PS1小鼠 （《 = 6, 4)和 WT同窝小鼠 （《 = 6， 6)血浆胰岛 素水平。
数据以平均值和标准差表示， * <0.05， **P<0.01, *** < 0.001c
6、 APP/PS1小鼠肝脏和肌肉胰岛素刺激后的信兮激活受损。
a， 20周的 APP/PS1 小鼠和同窝野生型 (WT)小鼠的肝中胰岛素刺激的 InsR和 Akt 的磷酸化。 b， （a)中 InsR和 Akt的磷酸化水平的定量。 c， 20周的 APP/PS1小鼠和同窝 野生型 (WT)小鼠的肌肉中胰岛素刺激的 Akt的磷酸化。 d， （c)中 Akt的磷酸化水平的定 量。 数据以平均值和标准差表示， ** < 0.01。
图 7、 肝脏、 肌肉和脂肪中重要炎性因子及其下游基因表达变化。
a, b, c， 20周的 APP/PS1小鼠 (n = 4)和野生型 (WT)对照 (n = 4)的肝 (a)， 肌肉（b)和 白色脂肪组织 (WAT)中炎性因子和相关基因表达的定量 PCR分析。数据以平均值和标准 差表示， 与对照组相比， * < 0.05。
8、 APP/PS1小鼠肝脏中 SOCS-1蛋¾表达上调。
a, 20周的 APP/PS1小鼠 (n = 3)和野生型 (WT)对照 (n = 3)的肝 SOCS- 1、 SOCS-3蚩 白表达的免疫印迹分析。 b, a中 SOCS-l、 SOCS-3蛋白表达的定量分析。 数据以平均值 和标准差表示， **P<0.01。
图 9、 Αβ在肝原代细胞中上调 SOCS-1的 mRNA和蚩白水平。
3，1),(;,(1,定量？0 (3)和免疫印迹(1)，（；，（1)分析小鼠肝原代细胞中 SOCS- 1和 SOCS-3 的表达。 细胞以 ΙΟ μΜ Αβ25-35处理指定的时间 (a， b), 以指定剂量的 Αβ25-35 (c)或 10 μΜ Αβ42 (d) 处理 60 h。 数据以平均值和标准差表示， *尸<0.05， **Ρ<0.0\α
m 10、 Αβ通过上调 SOCS-1诱 肝细胞产生胰岛素抵抗。
a, 两种不同的 siRNA对原代肝细胞中 SOCS-1表达的抑制。 b, 在指定的 siRNA存 在下， 给予 Αβ25-35(10 μΜ)或不给于 Αβ25-35处理 60 h的原代肝细胞中 InsR和 Akt的 磷酸化水平， 其中， SOCS-1 RNAi 1的用量是 40 nM; SOCS-1 RNAi 2的用量是 40 nM。 c，（b)中 InsR和 Akt的磷酸化水平的定量，其中， SOCS- 1 RNAi 1的用量是 40 nM;SOCS-l RNAi 2的用量是 40 nM。 数据以平均值和标准差表示， *尸<0.05。
1 Αβ能诱 STAT3和 JAK2的磷酸化激活。
a， b， c, 小鼠原代肝细胞以指定剂量的 Αβ25- 35 处理 36 h(a)， 10μΜΑβ25-35处 理指定时间 (b)或 10 μΜ Αβ42 处理 36 h(c), 磷酸化 STATl (Tyr701) 磷酸化 STAT3 (Tyr705)和磷酸化 JAK2(Tyrl007/1008)的蛋白水平的免疫印迹分析。 d， 20周的 APP/PS1 小鼠和野生型对照小鼠肝中 STAT3和 JAK2的酪氨酸残基磷酸化水平。 e， （d)中 STAT3 和 JAK2的酪氨酸残基磷酸化水平的定量分析。
图 12、 Αβ(10 μΜ)诱^ SOCS-1上调依赖于 STAT3和 JAK2。
a, b, c， 通过 RNAi下调 STAT3或 JAK2(a, c)或 AG490 (b)抑制 JAK2阻止由 Αβ 介^的 SOCS-1蚩白水平的上升。 其中， STAT3 RNAi 1的用量是 40nM; STAT3 RNAi 2的用量是 40 nM; AG490的用量是】0 μΜ; JAK2 RNAi 1的用量是 40 nM; JAK2 RNAi 2的用量是 40 nM。
图 13、 Αβ诱^的胰岛素抵抗依赖于 STAT3和 JAK2。
a, c, e, 在 Λβ(10 μΜ)诱^的胰岛素抵抗状态下， 通过 RNAi (a, e)下调 STAT3或 JAK2或通过 AG490(c)抑制 JAK2使得 InsR和 Akt的磷酸化水平降低得到了显著回复； 其中， STAT3 RNAi 1的用量是 40 nM; STAT3 RNAi 2的用量是 40 nM; AG490的用量 是 ΙΟ μΜ; JAK2 RNAi 1的用量是 40 nM; JAK2 RNAi 2的用量是 40 nM。 b, d, f， (a, c， e)中磷酸化的 InsR和 Akt水平的定量分析， 数据以平均值和标准差表示， *P < 0.05。
14、 Α β的抗体 1H3、 6C8的降糖作用， 以抗体 IgG作为对照。
15、 6C8抗体注射 9个月后 (每周注射一次)， 检测饥饿 4小时的小鼠血糖 (A)、 胰 岛素 (B)水平以及 HOMA-IR, 显示 APP/PS1小鼠的血糖显著下降更为显著。
16、 注射 Α β的抗体 1Η3(Α、 Β)和 6C8(C、 D)， 能显著降低 APP/PS1小鼠肝脏中 的 STAT3和 JAK2活性和 SOCS-1的表达。 B、 D图分别是 A、 C图的灰度统计结果。
图 17、 下调 JAK2蛍白水平能显著降低 JAK2的磷酸化水平， STAT3的磷酸化水平 也显著下调， SOCS-1的蛋白表达也显著下降。 B图是 A图的灰度统计结果。
18、 葡萄糖耐受实验显示 (A和 B)， 注射了 JAK2 siRNA的腺病毒的 APP/PS1小 鼠糖耐量有显示提高。 胰岛素耐受实验显示 (C和 D)， 注射了 JAK2 siRNA的腺病毒的 APP/PS1小鼠胰岛素明显改善。胰岛素刺激后，注射了 JAK2 siRNA的腺病毒的 APP/PS 1 小鼠肝脏组织 InsR和 Akt磷酸化水平显著高于注射对照病毒的 APP/PS1小鼠 (E和 F)。 具体实施方式
本发明人经过广泛的研究， 首次 i正实了胰岛素抵抗或糖尿病患者外周血中淀粉样蚩 白或蚩白片段含量显著上升，为胰岛素抵抗和 2型糖尿病的诊断提供了新的途径。并且， 本发明还首次揭示了淀粉样蚩白 β (Λβ) 能诱 外周组织产生胰岛素抵抗， 其是通过激 活 JAK2/STAT3-SOCS-1信号通路 （即诱 JAK2/STAT3依赖的 SOCS-1上调)而实现的。 因此， 可以以 JAK2/STAT3-SOCS-1通路或其通路蚩白作为筛选药物的靶点， 研究防治 或缓解胰岛素抵抗或糖尿病的药物。 检测胰岛素抵抗或糖尿病的试剂或试剂盒
基于本发明人的上述新发现， 可以将 Αβ或其蚩白片段 (包括 Αβ40、 Αβ42、 Αβ25-35 等)作为诊断胰岛素抵抗或糖尿病的标志物： （0 进行胰岛素抵抗或糖尿病的分型、 鉴别 诊断、和 /或易感性分析；（ii) 评估相关人群的胰岛素抵抗或糖尿病治疗药物、药物疗效、 预后，以及选择合适的治疗方法；（iii) 早期评估相关人群胰岛素抵抗或糖尿病患病风险， 早期监测早期防治。 比如， 可分离出由 Αβ 在血液 (血浆或血清)含量升高而 致胰岛素 抵抗或糖尿病的人群， 从而可进行更有针对性地治疗。
因此， 本发明提供了 Αβ (包括其前体蚩白或蚩白片段)或其编码基因的用途， 用于制 备诊断 (或检测)胰岛素抵抗或糖尿病的试剂或试剂盒。
可采用各种本领域已知的技术来检测 Αβ (包括其前体蛋白或蚩白片段)的存在与否以 及表达、 沉积情况， 这些技术均包含在本发明中。 例如可用己有的技术如 ELISA 法、 Western印迹法、 Southern印迹法、 DNA序列分析、 PCR等， 这些方法可结合使用。 本发明还提供了用于在分析物 (血液)中检测 Αβ (包括其前体蚩白或蚩白片段)的存在 与否以及表达情况的试剂。优选的，当进行基因水平的检测时，可以采用特异性扩增 Αβ (包 括其前体蚩白或蚩白片段)基因的引物； 或特异性识别 Αβ基因的探针来确定 Αβ基因的 存在与否； 当进行蚩白水平的检测时， 可以采用特异性结合 Αβ蚩白的结合分子 (如抗体 或配体)来确定 Αβ蛋白的表达情况。
作为本发明的优选方式， 所述的试剂是抗体 (单抗或多抗)，其可特异性结合 Αβ或其 蚩白片段。 利用特异性结合 Αβ蚩白的抗体来检测分析物中 Αβ蚩白表达情况的方法是本 领域人员熟知的技术。 例如， 采用所述的抗体， 可通过 ELISA方法检测到血液中 Αβ或其 蛋白片段的存在情况。 ELISA技术是本领域技术人员所熟知的。
针对 Αβ基因的特异性引物或探针的设计是本领域人员熟知的技术， 例如， 制备一种探 针， 其可与 Αβ基因上特定位点发生特异性结合， 而不与 Αβ基因以外的其它基因特异性结 合， 且所述探针带有可检测信号。 或制备一对引物， 其可特异性扩增出 Αβ基因。
本发明还提供了用于在分析物中检测 Αβ基因的存在与否以及表达情况的试剂盒， 该 试剂盒包括： 特异性结合 Αβ蚩白的抗体或配体； 特异性扩增 Αβ基因的引物或特异性识 别 Αβ基因的探针。
此外， 所述的试剂盒中还可包括川于抗原抗体反应、 杂交、 显色、 提取 DNA、 PCR 等所需的其它各种试剂， 包括但不限于： 显色液、 酶标液、 包被液、 洗涤液、 抽提液、 扩增液、 杂交液、 酶、 对照液等。
此外， 所述的试剂盒中还可包括使用说明书和 /或蛋白、 核酸序列分析软件等。 信号通路及相关蚩白
如本发明所用， 所述的 "（信号)通路" 是指由一系列蚩白或基因发生相互制约或相 互作用而形成的信号系统， 其一般会 ^致一些细胞事件的发生。 所述的 JAK2/STAT3-SOCS- 1信号通路包括 (但不限于）： JA 2基因 (和 /或其编码的蚩白）、 STAT3 基因 (和 /或其编码的蚩白）、 SOCS- 1 (和 /或其编码的蚩白）。
所述的 JAK2基因的核苷酸序列例如如 GenBank登录兮 NM_004972所示； 其蚩白 氨基酸序列例如如 GenBank登录号 NP— 004963所示。
所述的 STAT3基因的核苷酸序列例如如 GenBank登录号 NM— 139276， NM_213662， NM— 003150所示； 其蚩白氨基酸序列例如如 GenBank登录号 NP— 644805， NP 998827 , NP— 003 141所示。
所述的 SOCS- 1基因的核苷酸序列例如如 GenBank登录号 NM_003745所示； 其蚩 白氨基酸序列例如如 GenBank登录号 NP 003736所示。
SOCS蚩白家族包括 8个成员： CIS和 SOCS-1— SOCS-7。细胞内 SOCS蛋白表达通 常较低，但在受细胞因子刺激时能明显上调。 SOCS蛋白与胰岛素信号的关系以 SOCS- 1 和 SOCS-3的研究最多。 SOCS- 1敲除小鼠血糖很低，其胚胎成纤维细胞分化为脂肪细胞 的曾 力增 虽 (Kawazoe， Y.， et al. , Signal transducer and activator of transcription (ST AT)- induced STAT inhibitor 1 (SSI-l)/suppressor of cytokine signaling 1 (SOCSl) inhibits insulin signal transduction pathway through modulating insulin receptor substrate 1 (IRS-1) phosphorylation. J Exp Med, 2001. 1 3(2): p. 263-9), 提示胰岛素信号在这种 SOCS-1敲除细胞中的增强。通过腺病毒，直接在小鼠肝脏中过表达 SOCS-1或 SOCS-3 , 都能诱 明显的胰岛素抵抗。 SOCS-1和 SOCS-3在 2型糖尿病的模型小鼠 ί όΑΛ小鼠的 肝脏中也是高表达的。 这些研究结果提示， SOCS 蚩白在某些因素 致的胰岛素抵抗中 发挥了重要作用。
当用于作为筛选的靶标时或人为建立筛选系统时， 以上的蚩白或编码基因可以是天 然存在的， 比如其可被纯化和分离自哺乳动物； 也可以是重组制备的， 比如可以根据常 规的基因重组技术来生产重组的蚩白。 此外， 任何不影响这些蚩白的生物活性的变化形 式都是可用的， 如它们的功能未发生改变的衍生物或变异体。 用途及筛选方法
本发明人研究显示， Αβ能在小鼠肝原代细胞中上调胰岛素通路的抑制蚩白 S0CS-1 , 并且 APP/PS1小鼠肝脏中 SOCS-1表达也升高。 在小鼠肝原代细胞中下调 SOCS- 1蚩 β 表达能缓解 Αβ对胰岛素信号通路的抑制。 Αβ还能在肝原代细胞中激活 JAK2/STAT3 , 并且 APP/PS1小鼠肝脏中 JAK2/STAT3活性也更高。通过 RNAi或抑制剂在小鼠肝原代 细胞中下调或抑制 JAK2/STAT3 ,能缓解 Αβ对 SOCS-1的上调及对胰岛素信号通路转^ 的抑制。
基于本发明人的新发现，对 Αβ与 JAK2/STAT3-SOCS-1通路的研究有着多方面的用 途， 所述的用途包括 (但不限于)： 筛选抑制 Αβ对该信号通路的激活的物质， 该物质可 用于制备防治或缓解胰岛素抵抗或糖尿病的药物。
因此， 本发明提供了筛选防治或缓解胰岛素抵抗或糖尿病的潜在物质的方法， 将候 选物质与含有 Αβ 和 JAK2/STAT3-SOCS-1 信号通路的体系接触； 筛选出抑制 Αβ 对 JAK2/STAT3-SOCS-1 信号通路或通路蚩白的激活的物质， 所述物质是防治或缓解胰岛 素抵抗或糖尿病的潜在物质。
如本文所用， 所述的 "抑制"是指具有统计学意义的 "抑制" 。 即： 显著地 "抑制" 。 如与对照组的蚩白活性、 蚩白表达、 蛋白相互作用相比， 显著 "抑制" 、 20%以上， 较 佳的 50%以上； 更佳的 80%以上。
所述的含有 Αβ和 JAK2/STAT3-SOCS- 1 通路的体系选自： 细胞体系（或细胞培养物 体系）、 亚细胞体系、 溶液体系、 动物体系或组织体系。 可以是： 细胞内包含 Αβ 和 JAK2/STAT3-SOCS-1信^通路的体系； 或以 Αβ处理包含 JAK2/STAT3- SOCS-1信号通 路的细胞后获得的体系。 例如， 所述的含有 JAK2/STAT3-SOCS-1通路的体系是肝细胞。
作为本发明的优选方式， 所述的方法还包括： 对获得的潜在物质进行进一步的细胞 实验和 /或动物试验， 以从候选物质中进一步选择和确定对于防治或缓解胰岛素抵抗或 糖尿病有用的物质。
当进行筛选时， 可以采用本领域熟知的各种技术来确定蚩白或其编码基因的变化情 况以及相互作用情况。
可以采用多种常规的技术来鉴定系统中基因的转录或蚩白表达情况。 这些技术包括 但不限于： 寡核苷酸杂交技术 (如探针）， 多聚嗨链反应 (PCR)， 聚丙烯酖胺凝胶电泳， 免 疫印迹（如 Western印迹）等。
通过上述方法初步筛选出的物质可构成一个筛选厍， 以便于人们最终可以从中筛选 出能够对于防治或缓解胰岛素抵抗或糖尿病真正有用的物质。
本发明还提供了采用所述筛选方法获得的可用于防治或缓解胰岛素抵抗或糖尿病 的潜在物质。所述物质是抑制 Λβ 致的 JAK2/STAT3-SOCS- 1信号通路激活的物质， 或 是 JAK2/STAT3-SOCS- 1信兮通路或通路蚩白的抑制剂。
基于本发明人的上述新发现， 本发明提供了一种抑制 JAK2/STAT3-SOCS-1 信号通 路激活、 抑制 JAK2/STAT3-SOCS-1信号通路蚩白表达或活性或抑制淀粉样蚩白 β的表 达或活性的抑制剂的用途， 用于制备防治或缓解胰岛素抵抗或糖尿病的组合物。
如本文所用， 所述的抑制 JAK2/STAT3-SOCS- 1 信号通路激活、 抑制 JAK2/STAT3-SOCS-1 信号通路蛋白表达或活性或抑制淀粉样蚩白 β的表达或活性的抑 制剂包括了拮抗剂、 下调剂、 阻滞剂、 阻断剂等。
所述的抑制 JAK2/STAT3-SOCS- 1信号通路激活、 抑制 JAK2/STAT3-SOCS-1信^通 路蚩白表达或活性或抑制淀粉样蛋白 β的表达或活性的抑制剂是指任何可降低 JAK2/STAT3-SOCS-1信号通路蛋白的活性、降低 JAK2/STAT3-SOCS-1信号通路基因或 蚩 白的稳定性、 下调 JTAK2/STAT3-SOCS- 1 信号通路蚩 白 的表达、 减少 JAK2/STAT3-SOCS-1信号通路蚩白有效作用时间、或抑制 JAK2/STAT3- SOCS-1信号通 路基因的转录和翻译的物质， 这些物质均可用于本发明， 作为对于下调 JAK2/STAT3-SOCS-1 信号通路有用的物质， 从而可用于防治或缓解胰岛素抵抗或糖尿 病。 例如， 所述的抑制剂是： 特异性干扰 JAK2/STAT3-SOCS-1信号通路蛋白表达的干 扰分子； 或特异性与 JAK2/STAT3-SOCS- 1信号通路蚩白结合的结合分子； 或抑制 JAK2 的 AG490; 或特异性干扰淀粉样蚩白 β或其前体蚩白表达的干扰分子； 或特异性与淀粉 样蚩白 β结合的结合分子
作为本发明的一种优选方式， 所述的抑制剂是一种特异性抗淀粉样蛋白 β的抗体。 所述的抗体可以是单克隆抗体或多克隆抗体。 可用淀粉样蛋白 β免疫动物， 如家兔， 小
- I I - 鼠， 大鼠等来生产多克隆抗体； 多种佐剂可川于增强免疫反应， 包括但不限于弗氏佐剂 等。 与之相似的， 表达淀粉样蚩白 β或其具有抗原性的片段的细胞可用来免疫动物来生 产抗体。 所述的抗体也可以是单克隆抗体， 此类单克隆抗体可以利用杂交瘤技术来制备 (见 Kohler等人， Nature 256； 495, 1975 ; Kohler等人， Eur.J. Immunol. 6: 511 , 1976； Kohler等人， Eur.J. Immunol. 6： 292, 1976； Hammerling等人， In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N Y， 1981)。
作为本发明的一种优选方式， 所述的抑制剂是一种小干扰 RNA分子 （siRNA)。 如 本文所用， 所述的 "小干扰 RNA (small interfering RNA， siRNA)"是指一种短片段双链 RNA分子， 能够以同源互补序列的 mRNA为靶目标降解特定的 mRNA， 这个过程就是 RNA干扰 （RNA interference) 过程。
小干扰 RNA可以制备成双链核酸的形式， 它含有一个正义链和一个反义链， 这两 条链仅在杂交的条件下形成双链。 一个双链 RNA复合物可以由相互分离的正义链和反 义链来制备。 因此， 举例来讲， 互补的正义链和反义链是化学合成的， 其后可通过退火 杂交， 产生合成的双链 RNA复合物。
作为本发明的特别优选的方式， 提供了一种效果良好的小干扰 RNA分子， 所述的 小干扰 RNA分子具有良好的抑制 J AK2/ST AT3-SOCS- 1信号通路的效果。所述的小干扰 RNA分子是具有 SEQ ID NO. 2-7所示的核苷酸序列的小干扰 RNA分子。
此外， 小干扰 RNA包含的正义链和反义链可通过一个或多个编码正义链和反义链 的表达盒来制备。 当正义链和反义链由一个单独的表达盒编码时， 它们可以从生成的转 录物中断裂形成分离的正义链和反义链， 其后杂交生成双链小干扰 RNA。
所述的小干扰 RNA可通过采用适当的转染试剂被输送到细胞内， 或还可采用本领 域已知的多种技术被输送到细胞内。
本发明还提供了一种制备防治或缓解胰岛素抵抗或糖尿病的药物的方法， 所述方法 包括： 合成和 /或纯化前述筛选获得的对于防治或缓解胰岛素抵抗或糖尿病有用的物质， 作 为用于防治或缓解胰岛素抵抗或糖尿病的药物。
可将获得的对于防治或缓解胰岛素抵抗或糖尿病有用的物质配制于无毒的、 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)： 肌内、 静脉内、 或皮下给药。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明 而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件如 J.萨姆布鲁克等编著， 分子克隆实验指南， 科学出版社， 2002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。
除非另行定义， 文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义 相同。 此外， 任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。 文中所 述的较佳实施方法与材料仅作示范之用。
I. 材料和方法
哺乳动物细胞株
HepG2: 人肝癌细胞； 购自 ATCC。
H4IIE: 大鼠肝癌细胞； 购自 ATCC。 人群样品收集与诊断评估 .
在获得了中国科学院上海生命科学研究院营养科学研究所伦理道德委员会的批准 以及受试者知情同意的情况下进行试验。 正常血糖组和高血糖组空腹外周静脉血样品和 空腹血糖、血脂数据从上海市徐汇区中心医院获得。 正常血糖组共 26人 (男性 17人， 女 性 9人， 平均年龄 58.6岁， SD = 5.5)。 高血糖患者共 35人 (男性 21人， 女性 14人， 平 均年龄 59.6岁， SD = 6.7)。 按照 WHO于 1999年颁发的标准， 空腹血糖受损的判定为： 空腹血糖浓度大于或等于 6.1 mmol/L并小于 7.0 mmol/L,并且对于测定 2小时餐后血糖 的情况， 血糖浓度必须小于 7.8 mmol/L;糖尿病的判定为： 空腹血糖浓度大于或等于 7.0 mmol/L或餐后 2小时血糖浓度大于或等于 1 1.1 mmol/L。 本发明中把空腹血糖受损和糖 尿病合并为高血糖组进行研究。 正常组血糖平均值为 5.3 mmol/L, SD = 0.5。 高血糖组 血糖平均值为 8.1 mmol/L, SD = 2.5。 小鼠品系和词养条件
APPswe/PSENdE9小鼠及其同窝野生型对照均购自南京大学模式动物研究所， 词养 于中 科学院营养科学研究所动物房。 所有小鼠均按照实验动物管理和使川委员会的规 程进行饲养， 由斯莱克公司提供充足的水源和标准饲料。 主要试剂和试剂盒
Αβ25-35 : Sigma公司， 货号 A4559;
Αβ42: Apeptide公司， 货号 AP3006;
AG490.- Sigma公司， 货号 T3434;
胶原酶： Sigma公司， 货兮 C0130;
胶原： Millipore公司， 货号 08-1 15 ;
酪氨酸磷酸酶抑制剂混合液： Sigma公司， 货号 P2850;
丝氨酸 /苏氨酸磷酸酶抑制剂混合液： Sigma公司， 货 P5726;
蛋白酶抑制剂混合液： Sigma公司， 货^ P8340; Amplex® Red Glucose^Glucose Oxidase Assay Kit: Invitrogen公司, 货 A22189; Lipofectamine 2000： Invitrogen公司， 货号 11668019;
注射用胰岛素： Lilly France S.A.S.公司， 商品名， 优泌林。 通用名， 重组人胰岛素 注射液。
胰岛素放射免疫分析盒； 北京北方生物技术硏究所；
Αβ40 ELlSAkit: Covance公司， 货 SIG-38950, 其中结合 Αβ40的一抗是 BA27;
Αβ42 ELlSAkit: Covance公司， 货号 SIG-38952, 其中结合 Αβ42的一抗是 BC05;
DMEM: Invitrogen公司， 货^■ 12100-046;
无糖 DMEM: Invitrogen公司， 货号 11966-025;
FBS: Invitrogen公司， 货号 16000-044;
Trizo Invitrogen公司， 货^ 15596-018;
核酸分析电泳用琼脂糖： Biowest公司；
反转录试剂盒 (M- MLV): Promega公司；
SYBR Green PCR mixture: AB1公司；
蚩白印迹化学发光试剂 SuperSignal West Pico: Pierce公司；
PVDF膜： Millipore公司。 抗体
抗体名称 来源 公 司 货 号 稀释比例
Tubulin 小鼠 T6074 J: 10000
Sigma
β-actin 小鼠 A5316 1:10000
Phospho-lnsulin Receptor β
(Tyrl 150/1151)
或称为 p-InsR-Tyrll50或 1151 兔 Cell signaling 3024 1.1000 (Tyr 1150/1151指第 1150或 1151位 Tvr
发生磷酸化）
Insulin Receptor β 兔 3025 1:1000
Phospho-Akt (Scr473)
或称为 p-Akt-Scr473 兔 9271 1:1000 (Scr473指第 473位 Scr发生磷酸化）
Phospho-Akt (Thr308)
或称为 p-Akt-Thr308 兔 9275 1:1000 (Thr308指第 308位 Thr发生磷酸化）
Phospho-GSK-3p (Scr9)
兔 9336 1:1000 或称为 p-GSK3p-Scr!
SOCS-1 兔 3950 1:1000 SOCS-3 兔 2923 1:1000
Phospho-Statl (Tyr701) 兔 9167 1:1000
Phospho-Stat3 (Tyr705) 兔 9135 1:1000
Phospho-Jak2 (Tyrl 007/1008) 兔 3776 1:1000
STAT3 兔 9132 1:1000
JA 2 兔 3773 1:1000
HRP-anti-mousc IgG 山羊 Jackson 115-035-146 1:10000
HRP-anti-rabbit IgG 山羊 ImmunoRcscarch 111-035-144 1:2000 实验仪器
微量移液器购自 Gilson公司， 超低温冰箱购自 Thermo Scientific Forma公司， PCR 仪、 垂直电泳槽、湿转转膜仪和配套电源购自 Bio- Rad公司， 冷冻离心机购自 Eppendorf 公司， 高速离心机购自 Beckman公司， 高速勾浆机购自 Qiagen公司， 精密天平及 pH计 购自 Mettler Toledo公司， 0.22μπι、 0.45μηι针头式过滤器购自 Millipore公司， ChemiDoc 化学发光成像系统购自 Wealtec公司， 酶联免疫检测仪购自 Molecular Device公司， X 光片及 X光片洗片机购自 Kodak公司。 FreeStyle血糖检测仪及血糖试纸，购自 TheraSense 公司， The minispec mq20型核磁共振仪 (NMR)购自 Bruker公司， HITACHI 7020全自动 生化分析仪购自 HITACHI公司， ABI 7900HT实时定量 PCR仪购自 Applied Biosystems 公司。 小鼠肝原代细胞分离与培养
10周龄的 C57BU6J雄鼠麻醉后打开腹腔， 从下腔静脉以 7ml/min的速率灌入 50ml 在 37°C预热的灌流液 PI (含有 4.8 mMKCl, 1.2 mM MgS04> 1.2 mM KH2P04, 7.455 mM HEPES, 7 mg/ml NaCl, 2 mg/ml NaHC03, 0.1 mM EGTA, 3.6 mg/ml葡萄糖）， 之后再 灌入 50mL 37'C预热的灌流液 P2(含有 4.8 mM KC1， 1.2 mM MgS04, 1.2 mM KH2P04. 7.455 mM HEPES, 7 mg/ml NaCl， 2 mg/ml NaHC03, 3.6 mg/ml葡萄糖， 1.372 mM CaCl2， 0.66 mg/ml胶原酶)进行消化。 消化好的肝脏置于预先放入 20 ml不含胶原酶的预冷灌流 液 P2的 10 cm培养皿中，剪碎后用移液管反复吹打并用 70 μηι孔径细胞筛网过滤至 50 ml 的离心管中。 4'C 50g离心 2分钟后重悬， 随即用不含胶原酶的预冷灌流液 P2洗 3遍， 再用 15 ml含有 20 U/ml的青霉素， 20 g/ml的链霉素， 10% FBS的 DMEM培液重悬。 台盼蓝染色确定细胞存活率并进行细胞计数后用含有 10%FBS的 DMEM培液稀释， 按 照 5χΙ05细胞每孔铺于用胶原包被过的 12孔板。过夜培养后，更换培养液用于后续试验。
HepG2、 H4IIE细胞培养
HepG2, H4UE细胞株培养于含 10%FBS、 100U/ml青霉素、 100 g/ml链霉素和 25 mM葡萄糖的 DMEM培养液中。细胞培养箱保持 37'C， 并含 5%二氧化碳。 细胞一般每 2-3天传代一次， 保持传代前的密度超过 80%。 细胞在使用前 16- 24小时接种到 12孔板 中。 的配制、 预处理
Αβ25-35的配制： 称取适量 Αβ25-35粉末， 溶于 PBS中至终浓度为 1 mM， 分装后储 存于 -80Ό冰箱备用。孵育： 参照文献方法 (Coraci , I S. , et al.， CD36 , a class B scavenger receptor, is expressed on microglia in Alzheimer's disease brains and can mediate production of reactive oxygen species in response to beta-amyloid fibrils. Am J Pathol, 2002. 160( 1 ): p. 101 -12), 将储存的 1 mM Αβ25-35在 37°C、 5%C02培养箱中孵育 2天后使用。
Αβ42的配制： 称取适量 Λβ42粉末， 溶于去离子水中至终浓度为 2 mM, 分装后储存 于- 8CTC冰箱备用。孵育： 参照文献方法 (Jana, M.， C.A. Palencia, and K. Pahan, Fibrillar amyloid-beta peptides activate microglia via TLR2: implications for Alzheimer's disease. J Immunol, 2008. 181 (10): p. 7254-62), 将储存的 2 mM Αβ42在 37'C、 5%C02培养箱中 孵育 5天后使用。 细胞处理和转染
铺好细胞的 12孔板， 过夜培养后， 换无血淸的含有 25 mM葡萄糖的 DMEM培液， 同时加入指定浓度的 Αβ25-35或 Αβ42。 AG490在加入 Αβ25-35后 24小时加入。 需要胰 岛素刺激的， 加入 100 ηΜ胰岛素刺激细胞 20分钟后收集细胞样品。
需要转染的细胞，细胞在转染前 16-24小时接种到 12孔板中。采用 Lipofectamine 2000 转染。 转染结束后 48小时加入 Αβ处理。
Western blot
细胞用 SDS- PAGE上样缓冲液 (50 mM Tris-Cl , pH 6.8 , 2%SDS , 10%甘油， 100 mM DTT)裂解， 100'C煮沸 10分钟变性。组织用加了蚩白酶抑制剂（10 μ8/ηι1 pepstatin, 5 μ§/ιη1 leupeptin, 20 μ /ιηΐ aprotinin, 1 mM PMSF和 50 nM trichostatin A)和磷酸酶抑制剂混合 物的 RIPA裂解液 (20 mM Tris, pH 7.5 , 100 mM KC1, 0.1% Nonidet P-40， 1 mM EDTA, 10%glycerol, 50 mM NaF , 10 mM sodium pyrophosphate)裂解后， 进行蚩白浓度测定， 相同量的蚩白（〜60 μ8)加入适量 5Χ上样缓冲液 100Γ加热约 10分钟。 细胞或组织样品 离心后取上清上样于 9%或 12% SDS聚丙烯酰胺凝胶， 电泳， 湿转， PVDF膜用含 5% 脱脂奶粉的 TBST溶液封闭 1小时。 封闭结束后， 用 TBST室温、 中速在摇床上洗涤 3 次， 每次 5分钟， 随后加入配制在含 5%BSA的 TBST中的一抗， 室温孵育 3小时或者 4°C过夜。 一抗中一般加入 0.02〜0.05%的叠氮钠抑菌以便重复使用。 一抗结合完成后， 用 TBST室温、中速在摇床上洗漆 3次，每次 5分钟。加入配制在含 5%脱脂奶粉的 TBST 3 中的二抗 （1 : 1000〜 1 :5000), 室温孵育 1〜3小时， 再 ti TBST洗膜 3次。 加入蚩白印迹 化学发光试剂 SuperSignal West Pico反应 2〜5分钟， 用保鲜膜包好压片， 先依次以 5、 15、 30、 60秒曝光显影曝光在 Kodak X-Omat胶片上， 再根据结果调整曝光时间或者显 影时间。 葡萄糖输出实验
经过指定药物处理过的培养在 12孔板内的 H4IIE细胞， 用 PBS洗三遍， 每孔加入 400 μ1含有 20 mM乳酸钠， 2 mM丙酮酸钠的无糖 DMEM培养液， 需要胰岛素刺激的， 同时加入 50 nM胰岛素刺激。 3小时后收集培养液， 用 Amplex® Red Glucose lucose Oxidase Assay 试剂盒测定培养液中的葡萄糖水平， 同吋测定每孔细胞裂解后的蚩白浓 度作为内标。 人群血液样本的血浆分离和 AP40/42测定
外周静脉血， 加入 EDTA混匀抗凝。 血浆样品是将血液 4°C 2700g离心 10 min, 收 集上层血浆部分， 分装保存在 -80Ό冰箱。 血浆中的 Λβ40/42用 Covance公司的 ELISA 试剂盒按照说明书测定。
RNA抽提， 反转录
12孔板每孔细胞用 1 ml Trizol裂解， 组织约 100 mg用 lml Trizol裂解， 室温放置 5 分钟， 然后加入 0.2 ml的氯仿， 剧烈摇晃 15秒后， 室温静置 2-3分钟， 12 , OOOxg, 4 V离心 15分钟后， 取上淸至新的离心管中（约 0.45 ml), 再加入 0.5 ml的异丙醇混勾， 室温放置】 0分钟后， 12， OOOxg, 4Ό离心 10分钟， 沉淀用 1 ml 75%的酒精洗条后， 去 上淸， 晾干残留乙醇， 将沉淀溶干适量 DEPC水中， 检测纯度及浓度。 用 Takara公司的 RNase-free DNase I处理抽提得到的总 RNA, 以除去可能的 DNA污染， 然后按照 DNase I的说明书纯化经过消化的总 RNA, 再检测其纯度及浓度。 OD260/280 > 1.9即符合使用 标准。 取 2-4 μ 总 RNA, 按照 M-MLV reverse transcriptase (Promega) 反转录系统提供 的说明书进行反转录， 引物选用 random hexamer。 反转录得到 cDN A， Hi于后续的 PCR 检测。 实时定量 PCR
Real-time PCR用 384孔板， 以三复孔方式进行。 每孔含有 5 μΐ Power SYBR®Green PCR Master Mix (ABI)和 1 μΐ 模板 DNA (反转录获得的 cDNA根据基因的表达丰度稀释 10-60倍不等）， 正向和反向引物的浓度均为 0.5 μΜ， 用 ABI公司的 7900 system来实时 监测荧光。 PCR反应条件为 50'C 2 min, 95 °C 10 min,随后是 95 °C 15 s和 60 °C 1 min, 40个循环。 为了确定没有非特异性扩增， 每一 PCR产物都用熔解曲线进行分析， 同时 也进行琼脂糖电泳以确保产物的特异性。 同一块板检测 actin作为内参， 用于比较其它 基因的相对表达量。 各种基因的常规 PCR及 qRT- PCR引物见表 1实时定量 PCR部分。
表 1 基因 正向 反向 转基因小鼠基因型鉴定 PCR
GACTGACCACTCGACCAGGTTCTG CTTGTAAGTTGGATTCTCATATCCG
APPs c
(SEQ ID NO: 8) (SEQ ID NO: 9)
GTGGATAACCCCTCCCCCAGCCTAGACC AATAGAGAACGGCAGGAGCA
PSENdE9
(SEQ ID NO: 10) (SEQ ID NO: 11)
实时定量 PCR
TGTCCACCTTCCAGCAGATGT AGCTCAGTAACAGTCCGCCTAGA
actin
(SEQ ID NO: 12) (SEQ ID NO: 13)
CTGCGGCTTCTATTGGGGAC AAAAGGCAGTCGAAGGTCTCG
SOCS-1
(SEQ ID NO: 14) (SEQ ID NO: 15)
ATGGTCACCCACAGCAAGTTT TCCAGTAGAATCCGCTCTCCT
SOCS-3
(SEQ ID NO: 16) (SEQ ID NO: 17)
CGAAGACTACAGTTCTGCCATT GACGTTTCAGAGGTTCTCAGAG
IL-la
(SEQ ID NO: 18) (SEQ ID NO: 19)
GCAACTGTTCCTGAACTCAACT ATCTTTTGGGGTCCGTCAACT
IL-Ιβ
(SEQ ID NO: 20) (SEQ ID NO: 21)
TAGTCCTTCCTACCCCAATTTCC TTGGTCCTTAGCCACTCCTTC
IL-6
(SEQ ID NO: 22) (SEQ ID NO: 23)
CCCTCACACTCAGATCATCTTCT GCTACGACGTGGGCTACAG
T F-a
(SEQ ID NO: 24) (SEQ ID NO: 25)
GTGCTACTGGGGCTCATTTGT GGAGTAAGAGGACACTTGCGAAT
IL-1R1
(SEQ ID NO: 26) (SEQ ID NO: 27)
CCTGAGACTCAAGCAGAAATGG AGAAGGAAGGTCGGCTTCAGT
IL-6Ra
(SEQ ID NO: 28) (SEQ ID NO: 29)
CCGTGTGGTTACATCTACCCT CGTGGTTCTGTTGATGACAGTG
GP-130
(SEQ ID NO: 30) (SEQ ID NO: 31) AACCAACAGGCAGGCTTTAGT CATGACGGATCGGGTCCTTC
IL-8R
(SEQ ID NO: 32) (SEQ ID NO: 33)
CAATACCATTGACCTGCCGAT GAGCGACTCAAACTGCCCT
STAT3
(SEQ ID NO: 34) (SEQ ID NO. 35) 小鼠基因型鉴定
幼鼠年龄为 22-23天时断奶， 分笼， 进行基因型鉴定。 将剪刀用酒精灯烧烫， 迅速 剪取 5 mm左右小鼠尾巴尖端， 力 tl 200 μΐ 50 mM NaOH, 100°C加热 1小时， 充分振荡至 小鼠尾巴松散， 加 15μ1 l M Tris-HCl (pH8.0)， 振荡混匀， 离心使残渣沉淀， 得到基因组 DNA即可用于 PCR鉴定基因型。 使用的 PCR引物见表 1转基因小鼠基因型鉴定 PCR。 小鼠基本表型分析
所有实验对照组小鼠都是和 APPswe/PSENdE9转基因小鼠相同性别的同窝小鼠。小 鼠在 10- 19周龄每周测量体重。小鼠在 12周到 16周中，每两天测量一次小鼠的进食量， 得到每天摄食量的平均值。小鼠的体脂重量 （fat mass)和瘦体重 (lean mass)通过核磁共振 用 Minispec Mq7.5 Analyzer (Bruker, Germany)测出， 然后 1 本重标准化后得到体脂含量 和瘦体重含量值。 葡萄糖耐受实验 （GTT, glucose tolerance test)
葡萄糖耐受性实验前， 将小鼠饥饿 15小时， 检测空腹血糖后， 腹腔注射葡萄糖溶 液 （根据小鼠体重， 2 g/kg), 然后分别用血糖仪检测注射后 15、 30、 60和 120分钟小鼠 尾静脉血液中葡萄糖的浓度。 血糖用血糖仪 （FreeStyle, Alameda, California, USA)检
胰岛素耐受实验 (ITT, insulin tolerance test)
胰岛素耐受性实验， 实验前小鼠禁食 4小时， 检测空腹血糖后， 根据体重注射 0.75 U/kg 的人胰岛素注射液， 分别用血糖仪检测注射后 15 , 30, 60和 120分钟小鼠的血糖 水平。 小鼠血浆胰岛素水平、 血脂含量和 AP40/42的检测
小鼠眼眶取血。 检测空腹胰岛素水平须将小鼠饥饿 16 h。 血浆样品是将血液 4°C
2700g离心 10 min, 收集上层血浆部分， 保存在 -8CTC冰箱。 血浆胰岛素水平用北京北方 生物制品研究所的放射性免疫试剂盒， 根据说明书进行检测。 甘油三酯、 总胆固醇、 高 密度脂蚩白胆 1ST醇和低密度脂蚩白胆固醇的血浆水平是用 HITACHI 7020全自动生化分 析仪测定。 血浆中的 Αβ40/42用 Covance公司的 EL1SA试剂盒按照说明说测定。 小鼠肝脏、 肌肉中胰岛素敏感性的检测
20周龄的小鼠饥饿 16小时后称重， 麻醉后打幵腹腔， 先取一片肝和一侧大腿上肌 肉 块， 迅速冻入液氮作为未处理的对照。随后按照体重从门静脉注射 2 U/kg的人胰岛 素注射液， 5分钟后取余下肝脏， 10 min后取另一侧大腿上肌肉。 取下的组织立即冻入 液氮， 组织采集完毕后将样品转移至 -80 °C冰箱。 后续进行蚩白抽提和 western blot实验 检测胰岛素信 通路关键蚩白的激活水平。 数据分析和统计
统计学分析采用 GraphPad 5.0软件，除特别标注夕卜，所有定量数据均以平均值 (Mean) ± 标准偏差 (SD)表示。 两组间差异的显著性分析采用非成对 Student's t-test。 多因素比 较用 Two-Way ANOVA进行差异的显著性分析。 P < 0.05代表具有统计学意义的显著性 差异， 尸< 0.01代表差异极显著。
II. 实施例
实施例 1、 Ap诱 肝细胞产生胰岛素抵抗
由于外周血液循环中的 Αβ主要是由肝脏吸收和代谢 (Ghiso, J.， et al. , Systemic catabolism of Alzheimer's Abeta40 and Abeta42. J Biol Chem, 2004. 279(44): p.
45897-908), 本发明人选用肝细胞检测 Αβ对胰岛素信号通路的影响。
先使用 Αβ25-35片段处理肝细胞， 这个片段是全长 Αβ42的毒性核心 (Pike, C.J. , et al.， Structure-activity analyses of beta-amyloid peptides contributions of the beta 25-35 region to aggregation and neurotoxicity. J Neurochem , 1995 64(1): p. 253-65)。 Western blot 实验显示， Αβ25-35能抑制小鼠肝原代细胞受胰岛素刺激后的胰岛素信号通路并具有剂 量和时间效应， 表现为 Insulin receptor (InsR)、 Akt、 Glycogen synthase kinase 3p(GSK3p) 的磷酸化水平的下降 (图 l a和 lb)。
本发明人同时也使用了全长的 Αβ42处理小鼠肝原代细胞， 发现 Αβ42也能抑制胰 岛素对胰岛素信号通路的激活， Western blot实验显示 InsR、 Akt、 GSK3p的磷酸化水平 也下降了（图 lc) . 胰岛素的重要功能之一是抑制肝细胞的糖质新生。 在大鼠肝癌细胞株 H4IIE中， 胰岛素能将对照组的糖质新生降低约 60%。 但在加入 Αβ的实验组， 随着加 入 Αβ25-35浓度的升高， 胰岛素对糖质新生的抑制作用也逐渐降低 (阁 I d) ,
本发明人在人肝癌细胞株 HepG2中也得到了类似的结果。 Western blot实验显示 Αβ25-35能抑制 HepG2细胞受胰岛素刺激后的胰岛素信号通路并具剂量效应和时间效 应， 表现为胰岛素受体 (InsR)、 Akt、 GSK3P的磷酸化水平的下降（阁 2a和 2b)。 以上数据表明， Αβ能在培养的肝细胞中诱 胰岛素抵抗。 实施例 2、 高血糖病人血浆中 ΑΡ水平显著升高
Αβ能诱 体外培养的细胞胰岛素抵抗， 而体内胰岛素抵抗又能 致高血糖血症， 于是本发明人检测了血糖正常的人群以及高血糖人群 (包括临床确诊空腹血糖受损病人 和糖尿病病人)血浆中的 Ap 7]平。 ELISA结果显示， 高血糖人群血浆中 Αβ40/42水平均 显著高于血糖正常的人群 (图 3)。
此结果提示， 血浆中 Αβ40/42升高与高血糖血症相关， 而胰岛素抵抗又是高血糖血 症的重要诱因， 提示血浆中 Αβ40/42升高与胰岛素抵抗相关。 实施例 3、 过表达 Αβ的 APPswe/PSENdE9(APP/PSl)小鼠显示胰岛素抵抗的症状 1、 APP/PS1小鼠血浆中 Αβ显著升高
如前述实施例所述， 在细胞水平 Αβ能在肝细胞中诱^胰岛素抵抗并且高血糖病人 血浆 Αβ水平提高， 这些发现促使本发明人进一歩在动物水平研究 Αβ能否诱 胰岛素 抵抗。
本发明人选取了 AD的模型小鼠 APP/PS 1作为研究对象。 APP/PS1小鼠是常用的双 转基因 AD小鼠模型。 它是由朊蚩白启动子启动表达含瑞典突变 （ΚΜ594/5Ν)的小鼠 /人 嵌合淀粉样蚩白前体蛋白 ΑΡΡ和缺失外显子 9的剪切 ΑΡΡ的 γ-分泌酶 PS1 (PSENdE9) 两个质粒共同注射入小鼠受精卵而建立的 (Jankowsky, J L , et al. , Co-expression of multiple transgenes in mouse CNS: a comparison of strategies. Biomol Eng， 2001. 17(6): p. 157-65)。 文献报道在 6个月大的这种小鼠的脑中能观察到少量的淀粉样沉淀， 同时脑中 也能检测到微量的聚集状的 Αβ40和 Αβ42，而其认知能力没有受到损害 (Savonenko, A , et al. , Neurobiol Dis, 2005. 18(3): p. 602-17)。 在小于 6个月的小鼠脑中并没有检测到泶 集状的 Αβ40和 Ap42(Garcia-Alloza, M. , et al. , Neurobiol Dis, 2006 24(3): p. 516-24； Jankowsky , J L.， et al. , Hum Mol Genet, 2004. 13(2): p. 159-70), 而 20周龄转基因小 鼠血液中的 Αβ已经显著增加了 (Burgess, B丄.， et al. , Elevated plasma triglyceride levels precede amyloid deposition in Alzheimer ' s disease mouse models with abundant A beta in plasma. Neurobiol Dis, 2006. 24(1): p. 1 14-27)。本发明人通过 ELISA实验也检测到 Αβ40 和 Αβ42在 20周龄的 APP/PS1小鼠血浆中与同窝对照小鼠比显著增加 (表 2)。 已有研究 指出， 2-3月大的 APP/PS1转基因小鼠血浆中 Αβ即有显著增加 (Takeda, S.， et al.， Biochem Biophys Res Commun, 2009. 385(2): p. 193-7)。
Figure imgf000022_0001
Ap40(pg/ml) N.D. 263.6 ± 42.9
Ap42(pg/ml) N D. 136.8 ± 37.1
2、 APP/PS1小鼠一些基本表型正常
本发明人观察了 APP/PS 1小鼠 10-20 Ml龄的一些基本表型，发现 APP/PS 1小鼠和同 窝对照小鼠相比， 10-19周的体重 (图 4a), 12周和 16周的摄食 (ffl 4b)、体脂含量 (阁 4c) 瘦体重含量 (|¾ 4d)， 20周龄血脂中重要的主要脂质指标 (图 4e)都没有显著差异。 由于外 周的 Αβ主要是由肝脏吸收和代谢 (Ghiso, J , et al. , Systemic catabolism of Alzheimer's Abeta40 and Abeta42. J Biol Chem, 2004. 279(44): p. 45897-908), 本发明人同时也检测了 20周龄小鼠肝功能的重要指标谷丙转氨酶和天冬氨酸转氨酶 (图 4f 发现也没有显著变 化， 提示外周 Αβ的显著升高对肝脏功能没有造成明显损伤。
以上结果显示， APP/PS1小鼠在 10-20周龄时的一些基本表型是正常的。
3、 APP/PS1小鼠显示随年龄增长而加重的糖耐量受损和胰岛素抵抗症状
本发明人检测了 APP/PS 1小鼠和同窝野生型小鼠的糖耐量和胰岛素敏感性的变化。 葡萄糖耐受实验显示， APP/PS1小鼠糖耐量在 10周时有轻微受损， 到 18周时显示更加 严重的糖耐量受损， 表明有随年齡增加而糖耐量损伤加重的表型 (图 5a, 5b, 5e) o 胰岛 素耐受实验显示， APP/PS1 小鼠胰岛素敏感性的损害更显著， 表现为 13 周龄时胰岛素 耐受已有显著损伤， 到 19周时损伤更加显著 (阁 5c, 5d, 5f) o 20周时测定 APP/PS1小 鼠和同窝对照小鼠饥饿和喂食状态下的胰岛素水平发现， APP/PS 1的胰岛素水平均显著 高于同窝对照小鼠， 显示由于胰岛素抵抗引起的胰岛素代偿性分泌升高 (图 5g)。
以上结果显示， APP/PS1小鼠随年龄增长呈现糖耐量受损和胰岛素抵抗加重的趋势。
4、 肌肉的胰岛素抵抗情况
为了确认 APP/PS1小鼠肝脏和肌肉中是否出现胰岛素抵抗，本发明人在小鼠肝脏和 肌肉中检测了胰岛素诱 的下游信^通路的激活。 结果显示， 胰岛素刺激后， APP/PS 1 小鼠肝脏组织的 InsR和 Akt的磷酸化水平要显著低于同窝对照小鼠 (图 6a, 6b), 表明在 APP/PS1小鼠肝脏组织中胰岛素下游的信号转 受损。 相一致的是， 在肌肉组织中也观 察到了与肝脏组织类似的现象。 胰岛素刺激后， APP/PS1小鼠肌肉的 Akt磷酸化水平也 显著低于同窝对照小鼠 (图 6c, 6d)， 显示肌肉中胰岛素信号转^也受到抑制。
以上结果显示， APP/PSI小鼠肝脏和肌肉中确实存在着胰岛素抵抗现象。
5、 A β的抗体的降糖作用 本发明人对 3 个月大的雄性 APP/PS1 小鼠使用 Αβ的抗体 3F5(购自 Yes Biotech Laboratories Ltd， 效价 1： 50- 1： 500)进行腹腔注射治疗， 给药量为每次 10 mg/kg。 每周注 射一次， 对照的雄性 APP/PS 1小鼠注射同剂量 IgG。
注射 3个月后，检测饥饿 4小时的小鼠血糖显示， APP/PS1小鼠的血糖从 152.3mg/dl 降低到 127mg/dl， 说明 Αβ的抗体 3F5治疗有显著的降糖效果。
本发明人对 3个月大的雄性 APP/PSI 小鼠使用 Αβ的抗体 1H3 (购自 Yes Biotech Laboratories Ltd, 效价 1:50-1:500), 6C8 (购自 Yes Biotech Laboratories Ltd, 效价 1:50-1:500)进行腹腔注射治疗， 给药量为每次 10 mg/kg。 每 J注射一次， 对照的雄性 APP/PS1小鼠注射同剂量 IgG。
1H3和 6C8抗体注射 3个月后， 检测饥饿 4小时的小鼠血糖显示， APP/PS1小鼠的 血糖显著下降 (如图 14所示)， 说明 Αβ的抗体 1H3, 6C8治疗有显著的降糖效果。
6C8抗体注射 9个月后， 检测饥饿 4小时的小鼠血糖和胰岛素水平显示， APP/PS1 小鼠的血糖显著下降更为显著 (阁 15A), 并且小鼠的血浆胰岛素也显著下降 (图 15Β)。根 据小鼠空腹血糖和空腹胰岛素计算出的稳态模型的胰岛素抵抗指数 (homeostasi s model assessment-estimated insulin resistance, HOMA-IR)也显示 A β的抗体 6C8治疗能显著提 高 APP/PS1小鼠的胰岛素敏感性 (阁 15C)。 实施例 4、 Αβ通过上调 SOCS-1诱 胰岛素抵抗
1、 Αβ能上调小鼠肝脏和肝原代细胞中的 SOCS-1表达水平
本发明人的研究发现，ΑΡΡ/PSl小鼠显示出随着年龄增加而严重的胰岛素抵抗现象， 但 致这种现象的机制还不太清楚。由于炎性反应在 Αβ诱 的神经损伤中起重要作用， 并且炎性反应同样也是胰岛素抵抗的一个重耍诱因， 因此本发明人猜想 Αβ是否可能通 过激活外周组织的炎性反应来诱 胰岛素抵抗的。 本发明人通过定量 PCR 检测了 APP/PS1小鼠及其同窝对照组小鼠胰岛素的靶器官 ——肝脏、 肌肉和脂肪组织的一些重 要炎性因子以及介 其信号通路的下游因子的基因表达情况。 本发明人发现， APP/PS 小鼠肝脏和肌肉中 S0CS-1和 S0CS-3, 肝脏中 GP130, 脂肪中 IL-Ια, IL-8基因表达显 著升高。其余的炎性因子和其下游蚩白表达没有明显变化 (图 7a, 7b, 7c),说明在 APP/PS 小鼠的胰岛素靶器官中没有明显的炎性反应。
SOCS蚩白最初被发现是细胞因子信号通路的抑制因子，本发明人重点研究了 S0CS 蚩白在胰岛素信号通路中的作用。 Western blot实验显示，与同窝对照小鼠相比， APP/PS1 小鼠肝脏中的 SOCS-1蚩白表达显著上调了， 而 SOCS-3蚩白表达没有变化 (图 8a， 8b)。
以上结果提示， S0CS-1参与 APP/PS1小鼠的胰岛素抵抗形成。 为了验证 S0CS-1蛋白表达的上调是否是由 Αβ S接诱^的，本发明人用 Αβ处理小 鼠肝原代细胞。 定量 PCR结果显示 Αβ25-35能上调 SOCS-1基因转录并具有时间效应， 而 SOCS-3基因表达没有变化 (图 9a)。 Western blot也显示， Αβ25-35能上调 SOCS-1蚩 白表达水平， 并且存在剂量效应和时间效应， 而 SOCS-3的蚩白表达水平则不受影响 (图 9b , 9c)。 全长的 Αβ42同样也能上调 SOCS- 1蛋白表达， 并且也不影响 SOCS-3的蚩白 水平（阁 9d) u
以上结果显示， Αβ能直接上调 SOCS- 1基因转录和蚩白表达。 实施例 5、 Αβ通过上调 SOCS-1诱^肝细胞产生胰岛素抵抗
本发明人进一步检测了 Αβ诱 的胰岛素抵抗是否依赖于 SOCS-1 的上调。 通过两 条不同的 RNAi序列（见表 3 , SOCS- 1 siRNA- l和 SOCS- I siRNA-2)来下调 SOCS- 1以后 ( 10a) , Αβ25-35对肝原代细胞受胰岛素刺激后的胰岛素信号通路的抑制作用都得到缓 解， 表现为 InsR、 Akt磷酸化水平的显著回复 (图 10b , 10c)。
表 3
Figure imgf000025_0001
以上结果显示， Αβ主要通过上调 SOCS-1诱 ^肝细胞产生胰岛素抵抗。
JAK2/STAT3信号通路介 了 Αβ诱 的 SOCS-1上调和胰岛素抵抗
Αβ能诱 STAT3和 JAK2的磷酸化激活
己知 SOCS蚩白在静息状态的细胞中通常表达较低， 在受细胞因子刺激后， 主要通 过激活 JAK/STAT 信号通路诱 SOCS 基因转录 (Ronn, S.G. , N. Billestrup, and T.
Mandrup-Poulsen, Diabetes and suppressors of cytokine signaling proteins. Diabetes, 2007.
56(2): p. 541-8)0 而 Αβ又能在神经细胞中激活 STAT3和 JAK2/Tyk2(Wan， J.， et al. ,
Tyk2/STAT3 signaling mediates beta-amyloid-induced neuronal cell death: implications in
Alzheimer's disease. J Neurosci , 2010. 30(20)： p. 6873-8 ·, Chiba, T.， et al. , Amyloid-beta causes memory impairment by disturbing the JAK2/STAT3 axis in hippocampal neurons.
Mol Psychiatry, 2009. 14(2): p. 206-22), 因此本发明人检测了 Αβ是否能在肝原代细胞中 激活 STAT3和 JAK2。 Western blot实验显示， Λβ25- 35能以剂量依赖地诱 STAT3酪 氨酸残基磷酸化， 而 STAT1的酪氨酸残基磷酸化水平则不受 Αβ25-35调控， 说明 Αβ能 特异性地激活 STAT3。 Αβ处理后， 〗ΛΚ2的酪氨酸残基磷酸化变化呈现和 STAT3类似 的趋势 (图 l la)。 进一步的研究显示， Αβ25-35诱 STAT3和 JAK2的酪氨酸残基磷酸 化具有良好的时间效应，随着诱 时间的延长 STAT3和 JAK2的酪氨酸残基磷酸化水平 总体上逐渐升高 (图 l lb)。 全长的 Αβ42同样也能诱 STAT3和 JAK2的酪氨酸残基磷 酸化 (图 l lc)o 同时， 本发明人还检测发现 APP/PS1小鼠肝脏组织的 STAT3和 JAK2酪 氨酸残基磷酸化水平要显著高于同窝对照小鼠 (阁 l i d, l ie), 说明 APP/PS 1小鼠肝脏中 的 STAT3和 JAK2处于相对高活性状态， 并且该状态也很可能是由 Αβ诱 产生的。
以上结果表明， Αβ 能在肝原代细胞诱^ STAT3 和 JAK2 的磷酸化激活， 而且
APP/PS1小鼠肝脏中的 STAT3和 JAK2活性也较高。
本发明人对 3个月大的雄性 APP/PS1小鼠使川 Α β的抗体 1H3 (购自 Yes Biotech Laboratories Ltd , 效价 1 :50-1 :500) , 6C8 (购自 Yes Biotech Laboratories Ltd , 效价 1 :50-1 :500)进行腹腔注射治疗， 给药量为每次 10 mg/lcg。 每周注射一次， 对照的雄性 APP/PS1小鼠注射同剂量 IgG。
分别注射 1H3 3个月后， 注射 6C8 9个月后检测 APP/PS1小鼠肝脏中的 STAT3和 JAK2活性和 S0CS-1 的表达， 发现注射 Α β的抗体能显著降低 APP/PS1小鼠肝脏中的 STAT3和 JAK2活性和 S0CS-1的表达， 如阁 16所示。 实施例 6、 Αβ诱 的 SOCS-1上调依赖于 STAT3和 JAK2
基于 JAK/STAT信号通路对于 S0CS表达的重要调控作用， 本发明人检测了 Αβ对 S0CS-1的上调是否是由 STAT3和 ΜΚ2所介 的。 本发明人设计了 2条针对 STAT3 的不同 siRNA序列 (见表 3. STAT3 siRNA-1和 STAT3 siRNA-2)，发现当 STAT3下调时， Αβ对于 SOCS- 1的上调作用被抑制 (图 12a)。 用抑制剂 AG490抑制 JAK2或通过两条不 同的 RNAi序列（见表 3 , JAK2 siRNA-1和 JAK2 siRNA-2)来下调 JAK2， Αβ对干 SOCS-1 的上调作用被抑制 (图 12b, 12c)。
以上结果显示， Αβ对 SOCS- 1的上调是依赖于 STAT3和 JAK2的。 实施例 7、 Αβ诱 的胰岛素抵抗依赖于 STAT3和 JAK2
最后，本发明人检测了 Αβ是否通过激活 JAK2/STAT3信^通路来诱^胰岛素抵抗。 当通过 RNAi降低 STAT3蛋白表达后，Αβ对胰岛素信 通路激活的抑制作用显著缓解， Αβ 致的 InsR和 Akt的磷酸化水平降低得到了显著 Μ复 (图 13a, 13b)。 类似地， 通过 抑制剂抑制 JAK2或通过 RNAi下调 JAK2, Αβ对胰岛素诱 的胰岛素信号通路激活的 抑制作用显著缓解，Αβ ^致的 InsR和 Akt的磷酸化水平降低也得到了显著回复 (阁 13c, 01063
13d, 13e, 13f)。
以上结果显示， STAT3和 JAK2介^了 Αβ诱^的胰岛素抵抗。
为了在体内验证 Αβ诱^的胰岛素抵抗依赖干 JAK2/STAT3/SOCS-1信号通路， 本 发 明人包装 了 特异性下调 JAK2 表达的 siRNA 腺病毒（针对序列为 5'-GCAAACCAGGAATGCTCA- 3', SEQ ID NO: 36)通过尾静脉注射使其在 APP/PS1小 鼠肝脏内过表达。结果，过表达 JAK2 siRNA的腺病毒能下调 JAK2的蚩白水平 (JAK2i)， STAT3的蚩白水平未受影响。 与预期一致， 下调 JAK2蚩白水平能显著降低 JAK2的磷 酸化水平， STAT3的磷酸化水平也显著下调， SOCS-1 的蚩白表达也显著下降了， 如图 17所示。 LacZi用于作为实验对照， 可沉默细菌的 LacZ基因。
JAK2 siRNA 腺病毒载体（Ad-JAK2i)构建方法具体如下： 腺病毒的构建按照
Invitrogen公司的 BLOCK-iT™ 腺病毒 RNAi表达系统的说明书构建。 简要步骤如下， 设 计了针对 JAK2编码序列 5'-GCAAACCAGGAATGCTCA-3'(SEQ ID NO: 36)的寡核苷酸：
GC-3' (SEQ ID NO: 38), 合成后退火， 插入到 U6-entry 载体 (Invitrogen)中。 随后与 pAd/BLOCK-iTT DEST质粒进行重组。 重组完成后得到的质粒用 Pac 1内切酶线性化后 转染 293A细胞进行第二次重组， 转染 】0天左右得到重组腺病毒。 腺病毒的纯化利用 CsCl梯度超速离心进行纯化。
LacZi siRNA腺病毒载体 (Ad- LacZi)构建方法具体如下： 用 Invitrogen的腺病毒试剂 盒提供的针对细菌 LacZ基因干扰片段的寡核苷酸， 退火， 插入到 U6-entiy载体中。 余 下步骤与 JAK2 siRNA腺病毒载体构建方法一致。
进一步本发明人研究了注射了 JAK2 siRNA的腺病毒的 ΛΡΡ/PSl小鼠的的糖耐量和 胰岛素敏感性的变化。 葡萄糖耐受实验显示， 注射了 JAK2 siRNA的腺病毒的 APP/PS 1 小鼠糖耐量有显示提高 （图 18A和 B)。 胰岛素耐受实验显示， 注射了 JAK2 siRNA的腺 病毒的 APP/PS1小鼠胰岛素明显改善（图 18C和 D)。为了确认 APP/PS 1小鼠肝脏胰岛素 抵抗的改善， 本发明人在注射了 JAK2 siRNA的腺病毒的 APP/PS 1小鼠肝脏中检测了胰 岛素诱 的下游信号通路的激活。 结果显示， 胰岛素刺激后， 注射了 JAK2 siRNA的腺 病毒的 APP/PS1小鼠肝脏组织的 InsR和 Akt的磷酸化水平要显著高于注射了对照病毒 的 APP/PS1小鼠（图 18E和 F)， 表明在 APP/PS1小鼠肝脏中下调 JAK2能调高 APP/PS1 小鼠的胰岛素敏感性。 提示 Αβ在体内的诱^胰岛素抵抗效应是由 JAK2信号通路介^ 的。 实施例 8、 筛选方法
细胞模型： 前面所述的小鼠原代肝细胞， 其中包含 JAK2/STAT3信号通路及其下游 的 SOCS-1 , 并以 ΙΟ μΜ Αβ42处理小鼠原代肝细胞 36 h。
测试组： 用候选物质处理的上述细胞模型；
对照组： 不用候选物质处理的上述细胞模型。
如果与对照组相比， 测试组中的 Αβ42对 JAK2/STAT3-SOCS-1信号通路的激活显 著下降 50%以上， 则说明该候选物质是潜在可防治或缓解胰岛素抵抗或糖尿病的物质。
釆用上述方法， 将由 SEQ ID NO. 4- 7(依次为候选物质 1-4)， 以及 SEQ ID NO: 1的 siRNA (候选物质 5)作为候选物质， 处理所述细胞模型。 结果发现， 候选物质 1-4是潜在 可防治或缓解胰岛素抵抗或糖尿病的物质； 而候选物质 5没有作用。 实施例 9. 检测试剂盒
一种检测胰岛素抵抗的试剂盒， 该试剂盒中含有：
容器 1 , 以及装于该容器中特异性结合 Αβ的抗体；
以及， 其它一些容器， 其中分别装有显色液、 酶标液、 包被液、 洗涤液。
以及， 说明检测方法的使用说明书。 讨论
本发明人先在细胞水平研究了 Αβ与胰岛素抵抗的相关性， 发现 Αβ能抑制小鼠肝 原代细胞受胰岛素刺激后的胰岛素信 通路， 降低胰岛素对肝细胞糖质新生的抑制作 用。 在人群样本中本发明人发现高血糖病人血浆中的 Αβ显著升高， 由于胰岛素抵抗是 高血糖的重要诱因， 提示体内 Αβ与胰岛素抵抗相关。 血浆中过表达 Λβ的 APP/PS1小 鼠显示随年龄增长而加重的糖耐量受损和胰岛素抵抗症状， APP/PS 1小鼠肝脏和肌肉中 胰岛素信号通路的激活受到抑制，表明 APP/PS 1小鼠肝脏和肌肉中确实存在着胰岛素抵 抗现象， 提示血浆中 Αβ 的过表达能直接诱 胰岛素抵抗。 最后本发明人研究发现 Αβ 通过激活 JAK2/STAT3信号通路上调 SOCS- 1而产生其诱 胰岛素抵抗的作用。 以上研 究说明， 外 l Αβ的异常升高能诱 胰岛素抵抗。
本发明人的实验选择的是小于或等于 20周龄的 APP/PS 1小鼠， 此时小鼠脑内淀粉 样沉淀很不明显， 小鼠的认知能力也没有受损， 显示此时产生的胰岛素抵抗不是由于老 年痴呆 致的， 并提示外周的胰岛素抵抗有可能由外周 Αβ的升高引起。 与广泛接受的 一些 2型糖尿病的小鼠模型相似， APP/PS 1在 10周左右开始表现出高胰岛素血症和高 血糖等人类 2型糖尿病的类似症状， 是一种潜在的新的 2型糖尿病动物模型。
AD病人脑中 Αβ的沉积是一个重要的病理特征， AD病人血浆中 Αβ的水平有研究 报道是显著升高了， 但也有报道没有显著变化。 造成这些研究结果不同的原因可能来自 实验设计， 例如测试对象的年龄、 疾病的严重程度不同等。 很多 AD病人外周 Αβ水平 没有显著变化也部分解释了只有小部分的 AD病人发展出空腹血糖受损和 2型糖尿病。 另一方面， 尽管有报道显示糖尿病患者血浆中抗 Αβ的抗体比起对照人群显著升髙， 提 示糖尿病人血桨中 Αβ有可能也升高了， 但糖尿病人血浆中 Αβ水平是否有变化还未见 报道。 本发明人的研究发现在高血糖人群 (包括空腹血糖受损和糖尿病)血浆中 Αβ 的显 著升高， 提示血浆中的 Αβ水平可能成为胰岛素抵抗和 2型糖尿病的一个潜在生物标志 物。
本发明人的研究发现 Αβ通过激活 JAK7STAT继而上调 SOCS-l。 SOCS蚩白最初被 发现是细胞因子信兮通路的抑制子， 细胞因子通过激活 JAK/STAT信号通路上调 SOCS 蚩白， SOCS蚩白结合到 JAK蚩白， 抑制信号通路的转 形成一个负反馈通路， 从而调 控细胞因子产生的生物效应。 后续研究发现 SOCS蛋白也能抑制胰岛素信号通路， 有些 细胞因子诱 的胰岛素抵抗需要 SOCS 蚩白参与。 虽然 Αβ 也能诱 胶质细胞表达 ΤΝΡ-α等细胞因子，但是本发明人并没有在 APP/PS 1小鼠外周组织中检测到 IL-Ιβ, IL-6, TNF-ct等炎性因子的表达升高， 而且细胞因子诱 的 SOCS基因转录往往比较宽泛， 一 种细胞因子能诱 多个 SOCS基因表达 (Fasshauer, M. , et al.， Insulin resistance-inducing cytokines differentially regulate SOCS mRNA expression via growth factor- and Jak/Stat-signaling pathways in 3T3-L1 adipocytes. J Endocrinol, 2004. 181(】）： p. 129-38)。 本发明人的研究发现， Αβ处理肝原代细胞时， 只有 SOCS- 1的转录被激活。这些结果都 提示 Αβ不是通过上调一些炎性因子诱 S0CS-1表达的。 另一方面， Αβ有较强的促氧 化应激能力， 例如. Αβ 能诱 人和小鼠小胶质细胞和巨噬细胞产生活性氧分子 (reactive oxygen species, R0S)。 ROS又能激活 JAK/STAT信号通路。这些结果提示 Αβ有可能是 通过产生 ROS继而激活 JAK/STAT/SOCS信号通路的。 本发明人还发现 Αβ在肝原代细 胞能激活 ERK (数据未列出），而 ERK能磷酸化 STAT3的丝氨酸残基调控 STAT3的活性， 这些细胞内信号的整合有可能决定了 Αβ上调 S0CS-1的特异性。
胰岛素抵抗发生发展过程非常复杂， 不同因素引起的胰岛素抵抗虽然最终都可能^ 致糖尿病， 但针对糖尿病诱因不同应采取不同的治疗方法， 因此阐明胰岛素抵抗的机理 对于 2型糖尿病的治疗具有指 ^性意义。 本发明人在小鼠体外和体内的研究结果都显示 外周 Αβ能通过激活 JAK2/STAT3/SOCS- 1信号通路诱 胰岛素抵抗。 这些结果加深了 对胰岛素抵抗和 2型糖尿病致病机理的理解， 提示外周 Αβ水平有可成为胰岛素抵抗和 2型糖尿病的一个生物标记物。 降低 Αβ的生成、 下调外周 Αβ、 抑制 Αβ激活的信号通 路等都有可能成为治疗胰岛素抵抗和 2型糖尿病的重要策略。 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

1. 抑制 JAK2/STAT3-SOCS-1信号通路激活， 抑制 JAK2/STAT3-SOCS-1信号通路 蛋白表达或活性， 或抑制淀粉样蚩白 P的表达或活性的抑制剂的用途， 用于制备防治或 缓解胰岛素抵抗或糖尿病的药物。
2. 如权利要求 1所述的用途， 其特征在于， 所述的抑制剂包括：
特异性干扰； IAK2/STAT3-SOCS- 1信 通路蚩白表达的干扰分子； 或
特异性与 JAK2/STAT3-SOCS-1信号通路蛋白结合的结合分子； 或
抑制 JAK2的 AG490; 或
特异性干扰淀粉样蚩白 β或其前体蚩白表达的干扰分子； 或
特异性与淀粉样蚩白 β结合的结合分子； 或
β-分泌酶抑制剂、 Υ-分泌嗨抑制剂和调节剂或抗淀粉样蚩白聚集剂。
3. 如权利要求 2所述的用途， 其特征在于， 所述的千扰分子是 siRNA, 其序列选自 SEQ ID NO: 2-SEQ ID NO: 7的一种或多种； 或
所述的干扰分子具有以下结构：
Seq (¾-X-Seq
其中， Seq 的核苷酸序列如 SEQ ID NO: 36所示； Seq ^为与 Seq ^互补的序列； X为位于 Seq 和 Seq ^之间的间隔序列， 并且所述间隔序列与 Seq ^和 Seq ^ 不互补。
4. 如权利要求 2所述的用途， 其特征在于， 所述的结合分子是抗淀粉样蚩白 β的抗 体或抑制淀粉样蛋白 β的合成、 剪切、 聚集和沉积的分子。
5. 如权利要求 1或 4所述的用途，其特征在于，所述的抗体包括：3F5 , BA27 BC05 , 1H3 , 6C8 , Solanezumab , Bapineuzumab , MABT5102A , Ponezumab , Intravenous Immunoglobulin
6. 抑制 JAK2/STAT3-SOCS-1信号通路激活或抑制 JAK2/STAT3-SOCS-1信号通路 蛋白表达或活性的抑制剂，其是 siRNA,其序列选自 SEQ ID NO: 2-SEQ ID NO: 7和 SEQ ID NO: 36
7. 淀粉样蛋白 β用作检测胰岛素抵抗或糖尿病的血液标志物的用途。
8. 一种特异性识别淀粉样蛋白 β的试剂的用途，用于制备检测胰岛素抵抗或糖尿病 的试剂或试剂盒。
9. 如权利要求 8所述的用途， 其特征在于， 所述的特异性识别淀粉样蚩白 β的试剂 选自- 特异性结合淀粉样蚩白 β的结合分子；
特异性扩增淀粉样蚩白 β或其前体蚩白的编码基因或切割前体蛋白的蚩白酶的编码 基因的引物； 或
特异性识别淀粉样蛋白 β或其前体蚩白的编码基因或切割前体蚩白的蚩白酶的编码 基因的探针。
10. 一种检测血液中胰岛素抵抗或糖尿病的试剂盒， 其特征在于， 所述的试剂盒中 含有： 特异性识别淀粉样蛋白 β的试剂。
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