WO2018223364A1 - Crebzf在治疗、预防和诊断代谢性疾病中的应用 - Google Patents

Crebzf在治疗、预防和诊断代谢性疾病中的应用 Download PDF

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WO2018223364A1
WO2018223364A1 PCT/CN2017/087688 CN2017087688W WO2018223364A1 WO 2018223364 A1 WO2018223364 A1 WO 2018223364A1 CN 2017087688 W CN2017087688 W CN 2017087688W WO 2018223364 A1 WO2018223364 A1 WO 2018223364A1
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crebzf
gene
protein
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mouse
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李于
张菲菲
胡志敏
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中国科学院上海生命科学研究院
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • G01N2800/04—Endocrine or metabolic disorders

Definitions

  • the present invention relates to the use of CREBZF for the treatment, prevention and diagnosis of metabolic diseases.
  • metabolic syndrome represented by obesity, type 2 diabetes, fatty liver, hyperglycemia and hyperlipidemia
  • MS metabolic syndrome
  • high blood lipids caused by disorders of fat metabolism are important for other chronic diseases such as fatty liver, obesity, coronary heart disease and atherosclerosis. Therefore, finding a drug target for effectively preventing and controlling metabolic disorders has important practical significance for the prevention and treatment of metabolic diseases, and at the same time finding a way to monitor obesity, non-alcoholic fatty liver (NAFLD), high insulin, hyperglycemia, hyperlipidemia, etc.
  • NAFLD non-alcoholic fatty liver
  • the molecular markers related to metabolic syndrome which are representative, will play a positive role in clinical molecular diagnosis and treatment and prevention of diseases.
  • Obesity and type 2 diabetes are closely related to the development of a series of metabolic syndromes, including insulin resistance, fatty liver, hyperglycemia, and dyslipidemia. At the same time, obesity and type 2 diabetes can accelerate clinical and cardiovascular-related diseases. process. Excessive accumulation of triglycerides in the liver is a prominent feature of fatty liver and is closely related to insulin resistance. Increased fatty acid de novo synthesis and excessive accumulation of triglycerides in the liver can further accelerate fat metabolism. The pathogenesis of the liver.
  • CREBZF also known as ZF, Zhangfei
  • ATF/CREB Activation Transcription Factor/cAMP Response Element-Binding protein
  • HCF-1 cytokine interacts to inhibit herpes simplex virus replication [Akhova, O., M. Bainbridge and V. Misra, The Neuronal Host Cell Factor-Binding Protein Zhangfei Inhibits Herpes Simplex Virus Replication, Journal of Virology, 2005.79 (23): p.14708-14718].
  • CREBZF differs from other ATF/CREB family transcription factors in that it does not directly bind DNA in the form of homodimers; an unfolded protein response (unfolded protein response) that regulates endoplasmic reticulum response.
  • UPR can act as a co-transcription factor and ATF4 to form a heterodimer to play a regulatory role [Hogan, MR, GPCockram and R.Lu, Cooperative interaction of Zhangfei and ATF4in transactivation of the cyclic AMP response element, FEBS Letters, 2006. 580(1): p. 58-62].
  • the first aspect of the invention provides the use of the following agents in the manufacture of a medicament for slowing weight gain in a subject, reducing blood glucose in a subject, improving liver fat deposition in a subject, increasing glucose tolerance in a subject, and/or slowing lipid accumulation in a subject's liver cells:
  • the subject's weight gain, hyperglycemia, hyperlipidemia, liver fat deposition, low glucose tolerance, and/or hepatocyte lipid accumulation are caused by the subject's high-fat, high-sugar diet .
  • the medicament is for reducing the incidence of diabetes, cardiovascular disease, obesity, and/or fatty liver or treating such metabolic diseases.
  • the agent that reduces expression of a subject CREBZF protein is an agent that inhibits or decreases the expression level of a CREBZF gene.
  • the agent that inhibits or reduces the expression level of the CREBZF gene is selected from the group consisting of:
  • the agent that reduces the activity of the CREBZF protein expressed by the subject is a specific antibody to the CREBZF protein or a small molecule compound having inhibitory activity.
  • a second aspect of the invention provides a nucleic acid sequence selected from the group consisting of
  • nucleic acid sequence comprising a CREBZF gene, a LoxP site, and a Frt-Neo-Frt site;
  • the nucleic acid sequence can further comprise a homologous arm sequence.
  • the nucleic acid sequence contains a LoxP site, a CREBZF gene, a Frt-Neo-Frt site, and a LoxP site in sequence from 5' to 3'.
  • the nucleic acid sequence contains, from 5' to 3', a homology arm, a LoxP site, a CREBZF gene, a Frt-Neo-Frt site, a LoxP site, and a homology arm.
  • the homologous arm and the LoxP site, between the LoxP site and the CREBZF gene, between the Frt site and the LoxP site, and between the LoxP and the homology arm site may have any Selected connection sequence.
  • sequences of the respective genes, sites, and ligation sequences are set forth in SEQ ID NO:9.
  • a third aspect herein provides a vector comprising a nucleic acid sequence described herein.
  • the vector is for homologous recombination.
  • Also provided in a fourth aspect herein is a genetically engineered host cell transformed into a vector described herein and comprising in its genome a LoxP site comprising a 5' to 3' sequence as described herein. , the nucleic acid sequence of the CREBZF gene, the Frt-Neo-Frt site, and the LoxP site.
  • the host cell is a non-human mammalian cell.
  • the host cell is a rodent cell.
  • the host cell is a human somatic cell or an embryonic stem cell and a somatic cell of a non-human mammal.
  • a fifth aspect of the invention also provides a method of constructing a transgenic mouse, the method comprising:
  • the construction method further comprises mating the homozygous knockout mouse obtained in step (5) with the homozygous control mouse obtained in the step, thereby expanding the homozygous knockout mouse Population.
  • the transgenic mouse is characterized by no expression of CREBZF protein by systemic or liver, or decreased expression of CREBZF protein compared to control animals, or expression of inactive CREBZF protein or reduced activity of CREBZF protein.
  • the sixth aspect of the invention also provides a transgenic mouse which does not express CREBZF protein in the whole body or liver, or has a reduced expression of CREBZF protein or an inactive CREBZF protein or activity compared to a control animal. Reduced CREBZF protein.
  • the seventh aspect of the invention further provides a CREBZF gene or protein as a target for screening drugs for treating or preventing metabolic diseases or symptoms, or for clinical use for diagnosing insulin resistance, type 2 diabetes, hyperlipemia, The progression of metabolic syndrome such as obesity and fatty liver.
  • the eighth aspect herein provides the use of an agent for detecting a CREBZF gene or protein for the preparation of a kit for diagnosing a metabolic disease or for determining the progression of a metabolic disease.
  • a ninth aspect of the invention provides a test kit comprising a reagent for detecting a CREBZF gene and/or a protein.
  • Figure 1 Construction and identification of CREBZF liver-specific knockout mice.
  • A Build with Flox The CREBZF allele mouse and CREBZF liver-specific knockout mouse pattern
  • B identified CREBZF Flox mice using F1 and R1 PCR, respectively, to generate a target fragment of 248 bp; the target fragment size for identifying Alb-Cre is 100bp.
  • FIG. 2 Construction and identification of CREBZF liver-specific knockout mice.
  • A Body weight of mice after four months of experimental treatment. * represents a significant difference compared with the chow diet group P ⁇ 0.05, # represents a significant difference compared with the CREBZF LKO group and the CREBZF WT group in the HFHS group, P ⁇ 0.05, the same below.
  • C Body composition analysis of mice, body weight ratio of fat and lean meat (Fat Mass, Lean Mass) in each group.
  • FIG. 3 CREBZF LKO mice can improve liver fat deposition in mice induced by high-fat and high-glucose diets and reduce triglyceride levels in serum and liver of mice.
  • A Liver HE staining and oil red O staining in mice of different treatment groups, and staining of all mouse sections was observed by observing more than 8 sheets.
  • B Triglyceride and total cholesterol test results in mouse liver (P ⁇ 0.05).
  • C Detection results of triglyceride and total cholesterol in mouse plasma (P ⁇ 0.05).
  • FIG. 4 Effect of CREBZF liver-specific knockout mice on blood glucose and glucose tolerance in obese mice induced by high fat and high glucose diet.
  • A Hepatic fasting blood glucose was observed in mice of different treatment groups (P ⁇ 0.05).
  • B, C Glucose tolerance test (GTT) in the normal diet group and the high fat and high sugar diet induced group of mice.
  • Figure 5 Effect of CREBZF liver-specific knockout mice on lipid-related genes in obese mice induced by high-fat and high-glucose diets.
  • A The expression levels of SREBP-1 and FAS in the liver of obese mice induced by normal diet and high-sugar and high-fat diet.
  • B The expression level of Ising-2a mRNA in the liver of obese mice induced by high glucose and high fat diet (P ⁇ 0.05).
  • C High glucose and high fat diet induced the level of SREBP-1c mRNA in the liver of obese mice (P ⁇ 0.05).
  • D High glucose and high fat diet induced mRNA levels of key genes related to fatty acid and triglyceride synthesis in the liver of obese mice (P ⁇ 0.05).
  • This paper explores the role of CREBZF in the liver in the case of overnutrition by constructing a mouse model of liver-specific knockout of CREBZF through a disease model induced by a high-fat, high-sucrose diet. And biological functions.
  • liver-specific knockout CREBZF significantly slowed the weight gain of mice induced by high-fat and high-glucose diet, lowered blood sugar and blood lipids, improved liver fat deposition, increased glucose tolerance, and slowed lipid accumulation in liver cells. Therefore, by using certain techniques or drugs, CREBZF as a target, knocking down or inhibiting its activity will effectively reduce the incidence of diabetes, cardiovascular disease, obesity and fatty liver.
  • this article involves CREBZF as a target to slow down the weight gain induced by high-fat and high-glucose diets, reduce blood sugar and blood lipids in patients, improve liver fat deposition, increase glucose tolerance in subjects, and/or slow down lipid accumulation in liver cells.
  • CREBZF CREBZF
  • CREBZF is a basic leucine transcription factor of the Activation Transcription Factor/cAMP Response Element-Binding protein (ATF/CREB) family.
  • ATF/CREB Activation Transcription Factor/cAMP Response Element-Binding protein
  • CREBZF various basic leucine zipper transcription factors, defined by the art as CREBZF, such as CREBZF from different species. Such different species include, but are not limited to, mammals and rodents, such as rodents.
  • the coding gene of human CREBZF is located on human chromosome 11, the specific chromosome segment is 11q14.1, and the accession numbers in NCBI are AF 039942.1 (gene sequence) and AAD 28325.1 (amino acid sequence).
  • the coding gene of mouse (Mus musculus) CREBZF is located on chromosome 7, the specific chromosome segment is 7E1, the amino acid sequence of the amino acid sequence in NCBI is NP_660133, and the gene number is 233490.
  • subject refers to various needs including, but not limited to, mammals and rodents, such as humans and mice.
  • Reducing expression of a subject CREBZF protein includes, but is not limited to, inhibiting expression of a gene encoding a CREBZF protein or expressing the level of the gene.
  • an agent which inhibits or decreases the expression level of the CREBZF gene may be administered, and the reagent includes an agent which inhibits the transcriptional activity of the CREBZF gene, an agent which inhibits the transcription level of the CREBZF mRNA, and an agent which promotes degradation of CREBZF mRNA, and the CREBZF gene is targeted against the CREBZF gene.
  • siRNA an agent that inhibits the translation of CREBZF mRNA, and a specific An agent that recognizes a nucleic acid that directs the CREBZF gene and cleaves it to reduce its expression level.
  • CREBZF gene expression can be inhibited or reduced by the administration of siRNA to the CREBZF gene.
  • inhibition or reduction of CREBZF gene expression can be achieved by knocking out the CREBZF full gene by administration of a targeting vector.
  • the agent which lowers the activity of the CREBZF protein may be a specific antibody such as CREBZF or a small molecule compound having an inhibitory activity.
  • the activity can also be reduced by introducing a mutation into the CREBZF protein.
  • the activity of the CREBZF protein refers in particular to its activity in mediating metabolic diseases as described herein.
  • mutations that result in a decrease or loss of their corresponding activity are introduced in the functional domain of the CREBZF protein.
  • the mutation may be an insertion, deletion or substitution of one or several or even more (eg, more than 10, more than 20, more than 30) amino acids.
  • a mutation in the functional domain of the CREBZF protein encoded thereby causes a decrease or loss of its associated biological activity can be achieved by administering an agent that acts on the CREBZF gene.
  • Such agents alter the sequence of the CREBZF gene, resulting in the presence of a corresponding mutation in the encoded CREBZF protein, resulting in reduced activity, or loss of activity.
  • the CREBZF gene in wild-type cells can be replaced by a mutated CREBZF gene by homologous recombination techniques, resulting in expression of a weakly active or inactive CREBZF protein.
  • this article includes the pharmaceutical use of the following reagents:
  • the agent for reducing the expression of the CREBZF protein of the subject includes the agent for inhibiting the expression of the CREBZF gene or reducing the expression level thereof as described above, and the agent for reducing the activity of the CREBZF protein expressed by the subject includes the agent for reducing the activity of the CREBZF protein as described above.
  • metabolic diseases mediated by CREBZF proteins include, but are not limited to, insulin resistance, diabetes (especially type 2 diabetes), hyperlipidemia, obesity, and fatty liver syndromes.
  • the symptoms may include weight gain induced by a high-fat, high-sugar diet, hyperglycemia, hyperlipidemia, liver fat deposition, low glucose tolerance, and/or hepatocyte lipid accumulation. It should be understood that it is possible to determine whether a subject's blood glucose is hyperglycemia, whether blood lipids are hyperlipidemia, and whether glucose tolerance is low glucose tolerance, using art recognized standards.
  • the metabolic disease or condition is caused by overnutrition.
  • these agents can be used to prepare preventive and/or therapeutic metabolic syndromes such as hypoglycemic agents, hypolipidemic drugs, diet pills, and/or reduced fatty liver.
  • the medicament may contain, in addition to a therapeutically or prophylactically effective amount of the agent, a pharmaceutically acceptable carrier or excipient.
  • the drugs described herein are liver targeted drugs.
  • reducing the activity of the CREBZF protein comprises reducing the activity of the CREBZF protein by at least 30%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90, compared to its corresponding wild-type protein. %, even completely inactive.
  • Also included herein is a method of treating or preventing a metabolic syndrome or a symptom described herein, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the following agents:
  • an animal model preferably a rodent model, especially a rodent.
  • the animal model is characterized in that the CREBZF protein is not expressed in the system or liver, or the expression level of the CREBZF protein is decreased, or the inactive CREBZF protein or the reduced activity CREBZF protein is expressed as compared with the control animal.
  • the animal model is a liver-specific knockout CREBZF transgenic mouse.
  • the liver-specific knockout CREBZF transgenic mice can be constructed by hybridization of a CREBZF allele mouse with a Flox site and an Albumin-Cre transgenic mouse (Alb-Cre TG). Specifically, the transgenic mouse in which the liver-specific knockout CREBZF can be constructed as follows:
  • step (2) transferring the plasmid of step (1) into mouse embryonic stem cells, and screening for embryonic stem cell clones obtained by homologous recombination;
  • the allele mouse with the Flox site CREBZF obtained in the step (4) is Albumin-Cre transgenic mice were crossed to obtain first generation heterozygous mice, and then heterozygous mice were used for second generation selfing to obtain homozygous knockout mice, ie, the mouse model.
  • the step (5) further obtains a homozygous control mouse
  • the construction method further comprising mating the homozygous knockout mouse obtained in the step (5) with the homozygous control mouse, Thereby expanding the population of homozygous knockout mice.
  • Ella Cre (No. 003724, name B6.FVB-Tg (EIIa-cre) C5379Lmgd/J, from Jackson Corporation) is used to hybridize to the CREBZF allele mouse with a Flox site, A mouse that knocked out CREBZF whole body (CREBZF KO) was constructed.
  • the sequence of the CREBZF gene can be as shown in nucleotide sequence 5383-7516 of SEQ ID NO: 9; the homology arm region can be as SEQ ID NO: 9 positions 142-5312 and 9661-12661
  • the base sequence is shown;
  • the LoxP site can be represented by nucleotide sequences 5329-5362 and 9598-9631 of SEQ ID NO: 9;
  • the Frt-Neo-Frt site can be 7517 of SEQ ID NO: 9.
  • the base sequence of -7579 is shown, wherein the Neo box is located at the 7564-9545 position.
  • the plasmids of step (1) of the above construction method can be constructed using techniques and sequences well known in the art.
  • the plasmid may also contain other regulatory elements well known in the art to facilitate integration of the CREBZF gene, such as in the genome of an embryonic stem cell.
  • the CREBZF allele mice bearing the Flox site can be constructed using a variety of experimental mice well known in the art, including but not limited to C57BL/6J mice.
  • nucleic acid sequence comprising a CREBZF gene, a LoxP site, and a Frt-Neo-Frt site.
  • the nucleic acid sequence comprises, in order from 5' to 3', a LoxP site, a CREBZF gene, a Frt-Neo-Frt site, and a LoxP site.
  • the nucleic acid sequence may further comprise a homologous arm sequence.
  • the nucleic acid sequence comprises, from 5' to 3', a homology arm, a LoxP site, a CREBZF gene, a Frt-Neo-Frt site, a LoxP site, and a homology arm.
  • an optional linker sequence can be provided between the homology arm and the LoxP site, between the LoxP site and the CREBZF gene, between the Frt site and the LoxP site, and between the LoxP and homology arm sites.
  • the sequence of these linker sequences may be as shown in SEQ ID NO: 9 of the 5313-5328 base sequence, the 5363-5382 base sequence, the 9580-9597 base sequence or the 9632-9660 base sequence. .
  • the nucleic acid sequences herein may be included in a plasmid or vector.
  • the plasmid or vector is for use A plasmid or vector that is homologously recombined.
  • the plasmid or vector may also contain other elements that facilitate homologous recombination in the host cell.
  • the plasmids or vectors described herein can be constructed using methods well known in the art.
  • a host cell can be transformed with a plasmid or vector herein to obtain a genetically engineered host cell that has been transferred into a vector described herein and has its sequence from 5' to 3' as described herein.
  • a nucleic acid sequence comprising a LoxP site, a CREBZF gene, a Frt-Neo-Frt site, and a LoxP site.
  • the host cell may be a mammalian cell and a rodent cell, and may be an embryonic stem cell, a pluripotent stem cell, or a somatic cell. In certain embodiments, the host cell is not a human embryonic stem cell.
  • the host cell is a non-human mammalian cell, including embryonic stem cells and somatic cells thereof, such as embryonic stem cells and somatic cells that can be rodent. In certain embodiments, the host cell is a human somatic cell. In certain embodiments, the host cell is a primary hepatocyte, adipocyte or mouse embryonic fibroblast (MEF) cell.
  • embryonic stem cells and somatic cells thereof such as embryonic stem cells and somatic cells that can be rodent.
  • the host cell is a human somatic cell.
  • the host cell is a primary hepatocyte, adipocyte or mouse embryonic fibroblast (MEF) cell.
  • a CREBZF gene or protein as a target in the screening of a medicament for the treatment or prevention of a metabolic disease or condition, including but not limited to screening for the prevention and/or treatment of diabetes, cardiovascular disease, obesity and/or fatty liver.
  • a drug, or a drug for reducing body weight, blood sugar, blood fat, liver fat deposition, and/or hepatocyte lipid accumulation and or increasing glucose tolerance can be used as a molecular target to screen for a molecule capable of inhibiting its activity or expression level, and the molecule may be a novel small molecule compound or a known compound.
  • the screening can be performed by a luciferase reporter gene assay, a glucose uptake assay, or a fatty acid synthesis assay using cells expressing or not expressing CREBZF, such as primary hepatocytes, adipocytes, or mouse embryonic fibroblasts (MEF) cells.
  • a luciferase reporter gene assay such as primary hepatocytes, adipocytes, or mouse embryonic fibroblasts (MEF) cells.
  • CREBZF-Luc which has been successfully cloned, can be used to perform Qualcomm in primary hepatocytes, adipocytes or MEF cells into which the promoter reporter gene has been transferred by luciferase reporter gene assay.
  • a reagent for inhibiting the transcriptional activity of CREBZF-Luc is selected as a reagent for inhibiting the expression of the CREBZF gene.
  • SEQ ID NO: 10 is the nucleotide sequence of the expression vector (pGL3-basic-mCREBZF-LUC) obtained after cloning the CREBZF promoter reporter gene into pGL3-basic, wherein the promoter sequence of positions 27-2126.
  • CREBZF + / + and CREBZF - / - mouse primary hepatocytes, adipocytes, or MEF cells can be utilized, using fluorescently labeled 2-NBDG (2-[N-(7-nitrobenzene-2-oxine) -1,3-oxadiazol-4-yl)amino]-2-deoxy-D-glucose, Invitrogen), high-throughput screening by glucose uptake assay, selected to increase glucose uptake in CREBZF + / + cells, There is no such reagent in CREBZF - / - cells as a potential inhibitor of CREBZF gene expression or a decrease in its expression level.
  • primary hepatocytes, adipocytes, or MEF cells of CREBZF + / + and CREBZF - / - mice can be used to perform high-temperature fluorescent acid synthesis experiments by BODIPY (D3922, Molecular Probes, Carlsbad, Calif, USA). Flux screening, selection of agents capable of inhibiting lipid synthesis or accumulation in CREBZF + / + cells, but not in CREBZF - / - cells, as a potential inhibitor of CREBZF gene expression or a decrease in its expression level.
  • agents include, but are not limited to, small molecule compounds, siRNAs, and polypeptides.
  • CREBZF - / - mice can be derived from the liver-specific knockout CREBZF transgenic mice described herein and the CREBZF whole body knockout mice, while the CREBZF - / - mouse fat Cells and MEF cells can be derived from mice that have been knocked out of CREBZF.
  • the CREBZF gene or protein can also be used as a molecular marker for clinical diagnosis of the progression of metabolic diseases such as insulin resistance, type 2 diabetes, hyperlipemia, obesity and fatty liver.
  • the invention relates to the use of an agent for detecting a CREBZF gene or protein for the preparation of a kit for diagnosing a metabolic disease or for determining the progression of a metabolic disease.
  • Such reagents include, but are not limited to, various primers and probes for detecting the CREBZF gene, and/or specific antibodies for detecting CREBZF proteins, and the like, and such reagents are included in the preparation of samples containing CREBZF genes or proteins and are implemented.
  • Other reagents used in the assay, such as solvents, etc. include, but are not limited to, various reagents required for performing PCR and the like.
  • a detection kit comprising the reagents for detecting CREBZF genes and/or proteins as described above, including but not limited to primers and probes required for amplification and detection of the CREBZF gene, and CREBZF Protein specific antibody. Rapid detection of CREBZF levels by kits, as measured by their level, can be used as an alternative indicator of metabolic disease judgment.
  • the risk of the subject having a metabolic disease may be initially determined to be increased or May have a metabolic disease.
  • CREBZF allele mice with a Flox site were made by Saiye Co., Ltd. (Cyagen, China). Briefly, an open reading frame (ORF) sequence of 1062 bp base of CREBZF gene and a LoxP site and a Frt-Neo-Frt site and a homologous arm region on both sides were constructed in vitro (SEQ ID NO: 9: The base sequences of positions 142-5312 and 9661-12661 are homology arm regions; the base sequences of positions 5329-5362 and 9598-9631 are LoxP sites; and the base sequence of 5383-7516 is CKO region; the nucleotide sequence of 7517-7579 is a Frt-Neo-Frt site, wherein the Neo box is located at positions 7564-9545; the rest is the linker sequence of the vector backbone; the exon sequence is at positions 5383-7516 and 96
  • Neo probe-F TCATCTCACCTTGCTCCTGC (SEQ ID NO: 1)
  • Neo probe-R AAGGCGATAGAAGGCGATGC (SEQ ID NO: 2)
  • the genome of the cells was detected by Southern blotting, and the ES cell clones with homologous recombination were screened out.
  • the ES cell clones screened for homologous recombination were injected into the blastocyst of C57BL/6 (B6), and the injected capsules were injected. Embryos were transferred to the pseudopregnant mother (C57BL/6J) oviduct according to standard procedures. After the chimeric mice were born, the chimera and chimera were judged according to the coat color, and the male chimeric mice and Wild type female mice were mated with C57BL/6J.
  • a mouse of interest containing a Flox site at both ends of CREBZF CKO was obtained by Flp-mediated homologous recombination, that is, the mouse with the Flox site CREBZF allele.
  • liver-specific knockout CREBZF-bearing mice Liver-specific knockout CREBZF-bearing mice (CREBZF LKO) were passed through the Flox site CREBZF allele mouse and Albumin-Cre transgenic mice (Alb-Cre TG) , obtained from the Jackson Company of the United States, the mouse name is B6.Cg-Tg (Alb-cre) 21Mgn/J, mouse number: 003574), and the first generation heterozygous mice were obtained, and then the heterozygous mice were used.
  • the second generation was selfed, and a small amount of homozygous knockout mouse CREBZF LKO (CREBZF Flox +/+ Cre +/- ) and homozygous control mouse CREBZF WT (CREBZF Flox +/+ Cre -/- ) were obtained.
  • CREBZF Flox +/+ Cre +/- homozygous knockout mouse CREBZF LKO
  • CREBZF Flox +/+ Cre +/-/- homozygous control mouse CREBZF WT
  • F1 GCTTGCAGTTTAGAGAGAAACAGC (SEQ ID NO: 3)
  • R1 CAGCCAGAGTATCGCGAGATTC (SEQ ID NO: 4)
  • R2 TTTCCAACTTCTCAAGTGGTGAAC (SEQ ID NO: 6)
  • PCR was performed using F1 and R1 primers, and the desired fragments were 248 bp (wild type) and 318 bp (CREBZF mutant containing Flox site), and PCR was carried out using F2 and R2 primers, respectively, and the resulting fragment was 157 bp (wild type). And 285 bp (CREBZF mutant containing a Flox site).
  • the primers used to detect Alb-Cre are:
  • CREBZF LKO Eight-week-old male liver-specific CREBZF knockout mice (CREBZF LKO) with C57Bl/6 background were randomly divided into four groups, two of which were given normal diet (Chow) induction, and the corresponding two groups were given high fat and high glucose respectively. (HFHS) diet feeding.
  • High-fat and high-sugar feed (HFHS) consumed by mice was purchased from Research Diet, USA.
  • NMR nuclear magnetic resonance
  • microplate reader Tecan-200-plate reader Infinite 200PRO
  • 4 ° C / -20 ° C refrigerator from Haier ultra-low temperature refrigerator purchased from Thermo Scientific Forma
  • micropipette Purchased from Rainin or Eppendorf
  • vertical electrophoresis tank, wet transfer membrane and supporting power supply were purchased from Bio-Rad
  • refrigerated centrifuge was purchased from Eppendorf
  • PCR instrument was purchased from Bio-Rad
  • real-time fluorescent quantitative PCR system ABI The 7500 Fast Real-Time PCR System was purchased from Applied Biosystems.
  • mice were kept in the SPF animal room. After 22 days of weaning, the mice were divided according to the male and female mice. The mice were allowed to eat and drink at will (standard food provided by Shanghai Experimental Animal Center). The animal room maintained a temperature of 22 ⁇ 3 ° C, a humidity of 35 ⁇ 5%, and a 12-hour day and night cycle. All experiments were performed using 8-week-old mice.
  • mice were tested for fasting blood glucose three months after the experimental treatment.
  • mice The body composition of the mice in a conscious, free-diet state, including fat mass, was determined using a nuclear-magnetic resonance system (NMR) spectrometer (Bruker Corp). ), lean tissue mass (Lean Mass) and body fluids Body Fluid, etc.
  • NMR nuclear-magnetic resonance system
  • mice were deeply comatose by isoflurane anesthesia, then the pleural cavity was opened with a 1 ml syringe, and the blood was placed in a blood collection tube containing an anticoagulant, and centrifuged at 4 ° C, 3000 rpm for 15 min, and the supernatant was taken. After taking the blood, the ventral side of the mouse was photographed. Quickly pick up the liver and weigh it after taking a picture. The liver hepatic lobe was taken for formaldehyde fixation and cryopreservation (OCT) embedding. The liver pathological changes of the experimental mice were observed. The remaining liver tissues were used for Western Blot experiments and RT-PCR experiments. Open the mouse abdominal cavity to remove all epididymal fat and photograph and weigh.
  • OCT formaldehyde fixation and cryopreservation
  • liver-specific CREBZF knockout mice The construction process of liver-specific CREBZF knockout mice is as described above and shown in Fig. 1(A).
  • the results of the identification of mice that specifically knock out CREBZF by liver are shown in Fig. 1(B).
  • PCR was carried out using primers SEQ ID NO: 3 and 4, and the target fragments produced were 248 bp (wild type) and 318 bp (CREBZF genotype mice containing a Flox site), respectively, and the target fragment size of the identified Alb-Cre was 100 bp.
  • mice can slow diet-induced weight gain in mice
  • mice were performed using age-matched eight-week-old mice (CREBZF WT and CREBZF LKO) and fed a normal normal diet (Chow Diet) and a high fat and high sucrose diet (HFHS Diet).
  • the body weight of each group of mice was measured four months after the experiment. It was found that the experimental group CREBZF liver-specific knockout mice can slow the weight gain induced by the high-fat and high-glucose diet (Fig. 2, A).
  • the body weight of the mice was measured every week. Under normal diet, there was no significant difference in body weight between CREBZF LKO mice and CREBZF WT mice. In the case of HFHS feeding, the growth curve of CREBZF LKO mice was significantly lower.
  • CREBZF liver-specific knockout mice improve liver fat deposition in mice induced by high-fat and high-glucose diet, and reduce triglyceride and cholesterol levels in serum and liver of mice.
  • HE staining hematoxylin-eosin staining
  • triglyceride (TG) and total cholesterol (TC) assay kits were used to measure triglycerides and total cholesterol in liver and serum of mice.
  • TC total cholesterol
  • the results showed that there was no significant difference in liver and serum triglyceride and cholesterol levels between the experimental group and the control group under normal diet (Chow Diet), but in a mouse model induced by high fat and high glucose diet.
  • the liver and serum triglyceride levels in the CREBZF LKO group were significantly lower than those in the control group (P ⁇ 0.05) (Fig. 3, B, C).
  • the total cholesterol level in the liver and serum of the CREBZF LKO group was significantly lower than that of the control group (P ⁇ 0.05) (Fig. 3, B, C).
  • liver fat deposition induced by a high-fat and high-glucose diet can be improved, and triglyceride and cholesterol levels in serum and liver of mice can be lowered.
  • CREBZF liver-specific knockout mice significantly improved fat deposition, liver and serum triglyceride levels and cholesterol levels in the abdominal and liver of obese mice induced by high-fat and high-glucose diets.
  • CREBZF liver-specific knockdown in improving the metabolism of obese mice induced by high-fat diet, we extracted mouse liver tissue protein for Western Blot test, extracted liver tissue RNA, reversed cDNA, and performed RT- PCR experiment.
  • the CREBZF liver-specific knockout group significantly reduced the expression level of the activated form of N-SREBP-1 (Sterol Regulatory Element Binding Proteins1) when fed with high-fat high-sugar (HFHS), while regulating fat.
  • HFHS high-fat high-sugar
  • FAS Fatty Acid Synthase
  • liver-specific knockout of CREBZF significantly slowed the weight gain of mice induced by high-fat and high-glucose diet, decreased blood sugar and blood lipids, improved liver fat deposition, increased glucose tolerance, and liver pathological sections also indicated liver-specific knockdown.
  • CREBZF significantly slows hepatocyte lipid accumulation.
  • liver-specific knockdown of CREBZF can increase the level of Insig-2a mRNA and decrease the level of lipid synthesis-related gene SREBP-1, while CREBZF liver-specific knockout is active in the high-fat high-sugar (HFHS) group.
  • HFHS high-fat high-sugar
  • N-SREBP-1 Sterol Regulatory Element Binding Proteins1
  • FAS fatty acid synthase
  • CREBZF In the case of knocking out the liver CREBZF, in addition to the purpose of achieving hypoglycemic, it also has other effects of improving metabolism. Therefore, inhibition of CREBZF by liver targeting is expected to obtain a hypoglycemic agent having "Glucose Plus" efficacy. Therefore, CREBZF can be used as a drug target to study hypoglycemic drugs. In addition to its simple hypoglycemic effect, it also improves metabolism, and the application prospects of this drug will be great. In addition, through the role of the liver to adipose tissue, it has the effect of effectively losing weight. At present, among obese patients, the effect of lipid-lowering on adipose tissue alone is not obvious. The experimental results in this paper indicate that the specific knockdown of CREBZF in the liver can significantly reduce the body fat content of mice, which is targeted to the fat by the liver, and to a certain extent, achieves high-efficiency lipid-lowering. The role.
  • a specific drug target can be designed for CREBZF, and CREBZF is used as a molecular target to design a small molecule drug to inhibit its activity or expression level, thereby making it a preventive and adjuvant treatment for excessive diet-induced metabolism.
  • a new drug for the disease can be used as a molecular target to screen existing drugs, and a potential therapeutic drug with unknown molecular mechanisms can be selected to better treat insulin resistance, type 2 diabetes, hyperlipidemia, Drugs for metabolic syndrome such as obesity and fatty liver.
  • the clinical level of CREBZF in the liver can be used as a potential molecular indicator for judging the progression of the disease, and it can also be developed.
  • the CREBZF-related test kit is used to quickly detect the level of CREBZF through the kit and measure its level as an alternative indicator for its judgment.

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Abstract

本发明提供了减少对象CREBZF蛋白表达的试剂，和/或降低对象所表达CREBZF蛋白活性的试剂在制备减缓对象体重增加，降低对象血糖血脂，改善对象肝脏脂肪沉积，增加对象葡萄糖耐受性，和/或减缓对象肝细胞脂质堆积的药物中的用途；以及构建CREBZF敲除转基因小鼠的方法。

Description

CREBZF在治疗、预防和诊断代谢性疾病中的应用 技术领域
本发明涉及CREBZF在治疗、预防和诊断代谢性疾病中的应用。
背景技术
随着现代人们生活水平的提高和膳食结构的改变，以肥胖、二型糖尿病、脂肪肝、高血糖、高血脂症等为代表的代谢综合征(metabolism syndrome，MS)发病率逐年升高并且呈现上升的趋势，由于脂肪代谢紊乱引起的高血脂是脂肪肝、肥胖、冠心病和动脉粥状硬化等其他慢性病的重要。因此寻找到有效预防和控制代谢紊乱的药物靶点，对于防治代谢病具有重要的现实意义，同时找到一种能够监测肥胖、非酒精性脂肪肝(NAFLD)、高胰岛素、高血糖、高血脂等为代表的代谢综合征相关的分子指标，对于疾病的临床分子诊断和治疗以及预防将会起到积极地作用。
肥胖和二型糖尿病和一系列代谢综合征的发生发展紧密相关，其中包括胰岛素抵抗，脂肪肝，高血糖，血脂异常，同时肥胖和二型糖尿病的发生也会加速临床中与心血管相关疾病的进程。肝脏中过度累积甘油三酯是脂肪肝的显著特点，并且和胰岛素抵抗具有密切的相关性，肝脏中增加的脂肪酸从头合成和过多的甘油三酯累积所造成的代谢紊乱也会进一步的加速脂肪肝的发病进程。
CREBZF(也被称作为ZF、Zhangfei)属于Activation Transcription Factor/cAMP Response Element-Binding protein(ATF/CREB)家族的碱性亮氨酸转录因子，最初对它的研究是发现它可以通过与单纯疱疹病毒中的HCF-1细胞因子发生相互作用来抑制单纯疱疹病毒复制〔Akhova,O.,M.Bainbridge和V.Misra，The Neuronal Host Cell Factor-Binding Protein Zhangfei Inhibits Herpes Simplex Virus Replication，Journal of Virology,2005.79(23):p.14708-14718〕。CREBZF和其它ATF/CREB家族的转录因子不同的是，它并不以同源二聚体的形式直接结合DNA；在调控内质网应急的未折叠蛋白反应(unfolded protein response， UPR)时，它可以作为一个共转录因子和ATF4形成异源二聚体来发挥调控作用〔Hogan,M.R.、G.P.Cockram和R.Lu,Cooperative interaction of Zhangfei and ATF4in transactivation of the cyclic AMP response element，FEBS Letters,2006.580(1):p.58-62〕。之前已有人对CREBZF在生化分子功能进行了初步的研究〔Xie,Y.-B.、B.Nedumaran和H.-S.Choi，Molecular characterization of SMILE as a novel corepressor of nuclear receptors.Nucleic Acids Research,2009.37(12):p.4100-4115；Xie,Y.-B.等，Transcriptional Corepressor SMILE Recruits SIRT1to Inhibit Nuclear Receptor Estrogen Receptor-related ReceptorγTransactivation.Journal of Biological Chemistry,2009.284(42):p.28762-28774〕，但是对于CREBZF在营养过剩的情况下是如何调控代谢的机制研究还知之甚少。
发明内容
本文第一方面提供以下试剂在制备减缓对象体重增加，降低对象血糖血脂，改善对象肝脏脂肪沉积，增加对象葡萄糖耐受性，和/或减缓对象肝细胞脂质堆积的药物中的用途：
(1)减少对象CREBZF蛋白的表达的试剂；和/或
(2)降低对象所表达的CREBZF蛋白的活性的试剂。
在一个或多个实施方案中，所述对象的体重增加、高血糖、高血脂、肝脏脂肪沉积、低葡萄糖耐受性和/或肝细胞脂质堆积由该对象的高脂高糖饮食所致。
在一个或多个实施方案中，所述药物用于降低糖尿病、心血管疾病、肥胖和/或脂肪肝的发病机率或治疗这些代谢性疾病。
在一个或多个实施方案中，减少对象CREBZF蛋白的表达的试剂为抑制CREBZF基因表达或降低其表达水平的试剂。
在一个或多个实施方案中，抑制CREBZF基因表达或降低其表达水平的试剂选自：
(a)抑制该CREBZF基因转录活性的试剂；
(b)抑制该CREBZF mRNA的转录水平的试剂；
(c)促进该CREBZF mRNA降解的试剂；
(d)针对该CREBZF基因的siRNA；
(e)抑制CREBZF mRNA的翻译的试剂；
(f)特异性识别CREBZF基因的导向核酸并进行剪切以降低其表达水平的试剂；和
(g)用于部分或全部敲除CREBZF基因的试剂
在一个或多个实施方案中，所述降低对象所表达的CREBZF蛋白的活性的试剂为CREBZF蛋白的特异性抗体或具有抑制活性的小分子化合物。
本文第二方面提供一种核酸序列，选自：
(1)含有CREBZF基因、LoxP位点和Frt-Neo-Frt位点的核酸序列；和
(2)(1)所述序列的互补序列。
在一个或多个实施方案中，所述核酸序列还可进一步地含有同源臂序列。
在一个或多个实施方案中，所述核酸序列从5’到3’依次含有LoxP位点、CREBZF基因、Frt-Neo-Frt位点和LoxP位点。
在一个或多个实施方案中，所述核酸序列从5’到3’依次含有同源臂、LoxP位点、CREBZF基因、Frt-Neo-Frt位点、LoxP位点和同源臂。
在一个或多个实施方案中，所述同源臂与LoxP位点之间、LoxP位点与CREBZF基因、Frt位点与LoxP位点之间以及LoxP与同源臂位点之间可具有任选的连接序列。
在一个或多个实施方案中，所述各基因、位点和连接序列的序列如SEQ ID NO:9所示。
本文第三方面提供一种载体，其含有本文所述的核酸序列。
在一个或多个实施方案中，所述载体用于同源重组。
本文第四方面还提供一种经基因工程改造的宿主细胞，所述宿主细胞转入了本文所述的载体，并在其基因组中含有本文所述的从5’到3’依次含有LoxP位点、CREBZF基因、Frt-Neo-Frt位点和LoxP位点的核酸序列。
在一个或多个实施方案中，所述宿主细胞为非人哺乳动物细胞。
在一个或多个实施方案中，所述宿主细胞为啮齿类动物的细胞。
在一个或多个实施方案中，所述宿主细胞为人的体细胞或非人哺乳动物的胚胎干细胞及体细胞。
本文第五方面还提供一种构建转基因小鼠的方法，所述方法包括：
(1)提供本文所述的载体；
(2)将所述载体转入小鼠胚胎干细胞中，筛选获得同源重组的胚胎干细胞克隆；
(3)将步骤(2)获得的胚胎干细胞注射到小鼠囊胚中，并将该囊胚转入假孕母鼠中，获得雄性嵌合体小鼠；
(4)将步骤(3)获得的雄性嵌合体小鼠与野生型雌性小鼠交配，获得带有Flox位点的CREBZF等位基因小鼠；
(5)将步骤(4)获得的带有Flox位点CREBZF的等位基因小鼠与Albumin-Cre或Ella Cre转基因小鼠杂交，得到第一代杂合子小鼠，然后采用杂合子小鼠进行第二代自交，从而获得纯合的敲除小鼠，即所述转基因小鼠。
在一个或多个实施方案中，该构建方法还包括使步骤(5)获得的纯合的敲除小鼠与该步骤获得的纯合的对照小鼠交配，从而扩大纯合的敲除小鼠的种群。
在一个或多个实施方案中，该转基因小鼠的特征是，全身或肝脏不表达CREBZF蛋白、或与对照动物相比CREBZF蛋白的表达量降低、或表达无活性的CREBZF蛋白或活性降低的CREBZF蛋白。
因此，本文第六方面也提供一种转基因小鼠，所述转基因小鼠的全身或肝脏不表达CREBZF蛋白、或与对照动物相比CREBZF蛋白的表达量降低、或表达无活性的CREBZF蛋白或活性降低的CREBZF蛋白。
本文第七方面还提供CREBZF基因或蛋白作为靶点在筛选治疗或预防代谢性疾病或症状的药物中的应用，或用作分子指标在临床上用诊断胰岛素抵抗、2型糖尿病、高血脂症、肥胖和脂肪肝等代谢综合征的病程发展。
本文第八方面提供检测CREBZF基因或蛋白的试剂在制备用于诊断代谢性疾病或判断代谢性疾病病程发展的试剂盒中的应用。
本文第九方面提供一种检测试剂盒，所述检测试剂盒含有检测CREBZF基因和/或蛋白的试剂。
附图说明
图1：CREBZF肝特异性敲除的小鼠的构建以及鉴定。(A)构建带有Flox 位点CREBZF等位基因小鼠以及CREBZF肝特异性敲除的小鼠模式图(B)鉴定CREBZF Flox小鼠使用F1和R1 PCR分别产生的目的片段是248bp；鉴定Alb-Cre的目标片段大小为100bp。
图2：CREBZF肝特异性敲除的小鼠的构建以及鉴定。(A)四个月实验处理后小鼠体重。*代表与chow diet组相比有显著性差异P<0.05，#代表与HFHS组的CREBZF LKO组和CREBZF WT组相比有显著性差异P<0.05，下同。(B)实验处理期间小鼠体重变化。Bar代表标准误(实验中小组中小鼠数量为n＝8，8，12，12)。(C)小鼠体成分分析，各组小鼠脂肪与瘦肉(Fat Mass，Lean Mass)所占体重比。
图3：CREBZF LKO小鼠可以改善高脂高糖饮食诱导的小鼠肝脏脂肪沉积，降低小鼠血清和肝脏中甘油三酯水平。(A)不同处理组小鼠肝HE染色和油红O染色，所有的小鼠切片的染色都是观察大于8张的结果得出。(B)小鼠肝脏中甘油三酯和总胆固醇检测结果(P<0.05)。(C)小鼠血浆中甘油三酯和总胆固醇检测结果(P<0.05)。
图4：CREBZF肝脏特异性敲除的小鼠对高脂高糖饮食诱导肥胖小鼠血糖以及葡萄糖的耐受性的影响。(A)不同处理组小鼠肝禁食血糖(P<0.05)。(B，C)正常饮食组和高脂高糖饮食诱导的小鼠组的葡糖糖耐受性测试(GTT)。
图5：CREBZF肝脏特异性敲除的小鼠对高脂高糖饮食诱导肥胖小鼠脂质相关基因的影响。(A)正常饮食和高糖高脂饮食诱导肥胖小鼠肝脏中SREBP-1、FAS的表达水平。(B)高糖高脂饮食诱导肥胖小鼠肝中Ising-2a mRNA的表达水平(P<0.05)。(C)高糖高脂饮食诱导肥胖小鼠肝脏中SREBP-1c的mRNA的水平(P<0.05)。(D)高糖高脂饮食诱导肥胖小鼠肝脏中脂肪酸和甘油三酯合成相关的关键基因的mRNA的水平(P<0.05)。
具体实施方式
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
本文通过构建肝脏特异性敲除CREBZF的小鼠模型，通过高脂肪高蔗糖饮食诱导的疾病模型来探究肝脏中CREBZF在营养过剩的情况下中所起的作用 和生物学功能。本文发现，肝脏特异性敲除CREBZF显著的减缓高脂高糖饮食诱导的小鼠体重增加，降低血糖血脂，改善小鼠肝脏脂肪沉积，增加葡萄糖耐受性，减缓肝细胞脂质堆积。所以通过一定的技术或者药物手段，以CREBZF为靶点，敲减或者抑制其活性，将能够有效降低糖尿病、心血管疾病、肥胖和脂肪肝的发病机率。
因此，本文涉及以CREBZF为靶点减缓对象高脂高糖饮食诱导的体重增加，降低对象血糖血脂，改善对象肝脏脂肪沉积，增加对象葡萄糖耐受性，和/或减缓对象肝细胞脂质堆积，以有效降低糖尿病、心血管疾病、肥胖和/或脂肪肝的发病机率或治疗这些代谢性疾病。
本文中，CREBZF是Activation Transcription Factor/cAMP Response Element-Binding protein(ATF/CREB)家族的碱性亮氨酸转录因子。本文包括各种被本领域定义为CREBZF的碱性亮氨酸拉链转录因子，例如来自不同物种的CREBZF。所述不同物种包括但不限于哺乳动物和啮齿类动物，例如鼠类。人CREBZF的编码基因位于人第11号染色体上，具体的染色体区段是11q14.1，在NCBI中的登陆号为AF 039942.1(基因序列)和AAD 28325.1(氨基酸序列)。小鼠(Mus musculus)CREBZF的编码基因位于其第7号染色体上，具体的染色体区段是7E1，其氨基酸序列在NCBI中的登陆号为NP_660133，基因编号是233490。
本文中，“对象”指各种需要包括但不限于哺乳动物和啮齿类动物，例如人和鼠。
本文通过抑制CREBZF蛋白的活性而实现上述目的。可通过如下方式实现CREBZF蛋白活性的抑制：
(1)减少对象CREBZF蛋白表达；和/或
(2)降低对象所表达的CREBZF蛋白的活性；
减少对象CREBZF蛋白表达包括但不限于抑制CREBZF蛋白的编码基因的表达或将其该基因的表达水平。例如，可给予抑制CREBZF基因表达或降低其表达水平的试剂，这些试剂包括：抑制该CREBZF基因转录活性的试剂，抑制该CREBZF mRNA的转录水平的试剂，促进CREBZF mRNA降解的试剂，针对该CREBZF基因的siRNA，抑制CREBZF mRNA的翻译的试剂，和特异 性识别CREBZF基因的导向核酸并进行剪切以降低其表达水平的试剂。在某些实施方案中，可通过给予CREBZF基因的siRNA来抑制CREBZF基因表达或降低其表达水平。在其他实施方案中，可通过给予打靶载体将CREBZF全基因敲除，从而实现CREBZF基因表达的抑制或降低。降低CREBZF蛋白活性的试剂可以是例如CREBZF的特异性抗体或具有抑制活性的小分子化合物。
在某些实施方案中，也可通过在CREBZF蛋白中引入突变而使其活性降低。本文中，CREBZF蛋白的活性尤其是指其在本文中所述的介导代谢性疾病的活性。因此，在某些实施方案中，在CREBZF蛋白的功能域中引入致其相应活性减弱或丧失的突变。突变可以是1个或数个甚至更多(例如10个以上、20个以上、30个以上)个氨基酸的插入、缺失或取代。可通过给予作用于CREBZF基因的试剂，使其所编码的CREBZF蛋白的功能域中存在导致其相关生物学活性减弱或丧失的突变。这类试剂可改变CREBZF基因的序列，导致所编码的CREBZF蛋白存在相应的突变，从而具有减弱的活性，或活性丧失。例如，可通过同源重组技术用突变的CREBZF基因替换野生型细胞中的CREBZF基因，从而导致其表达除弱活性或无活性的CREBZF蛋白。
因此，本文包括以下试剂的制药用途：
(1)减少对象CREBZF蛋白的表达的试剂；和/或
(2)降低对象所表达的CREBZF蛋白的活性的试剂。
减少对象CREBZF蛋白的表达的试剂包括前文所述的抑制CREBZF基因表达或降低其表达水平的试剂，而降低对象所表达的CREBZF蛋白的活性的试剂包括前文所述的降低CREBZF蛋白活性的试剂。
这类试剂可用于预防和/或治疗由CREBZF蛋白介导的代谢性疾病或症状。本文中，由CREBZF蛋白介导的代谢性疾病包括但不限于胰岛素抵抗、糖尿病(尤其是2型糖尿病)、高血脂症、肥胖和脂肪肝等代谢综合征。所述症状可包括高脂高糖饮食诱导的体重增加，高血糖，高血脂，肝脏脂肪沉积，低葡萄糖耐受性，和/或肝细胞脂质堆积等。应理解的是，可采用本领域公认标准判断对象血糖是否为高血糖，血脂是否为高血脂，以及葡萄糖耐受是否为低葡萄糖耐受。在某些方面，所述代谢性疾病或症状是营养过剩引起。因此，可使用这些试剂制备降糖药、降血脂药、减肥药和/或降脂肪肝等预防和/或治疗代谢综 合征或减轻其症状的药物，用于预防和/或治疗糖尿病、心血管疾病、肥胖和/或脂肪肝，或用于降低体重、血糖、血脂、肝脏脂肪沉积、肝细胞脂质堆积和/或增加葡萄糖耐受性。
所述药物除含有治疗或预防有效量的所述试剂外，还可含有药学上可接受的载体或赋形剂。在某些实施方案中，本文所述的药物是肝靶向的药物。
本文中，降低CREBZF蛋白的活性包括使CREBZF蛋白的活性与其对应的野生型蛋白相比降低至少30％，例如至少40％、至少50％、至少60％、至少70％、至少80％、至少90％，甚至完全无活性。
本文也包括治疗或预防本文所述的代谢性综合征或所述的症状的方法，所述方法包括给予有此需要的对象治疗或预防有效量的下述试剂：
(1)减少对象CREBZF蛋白的表达的试剂；和/或
(2)降低对象所表达的CREBZF蛋白的活性的试剂。
本文也提供一种动物模型，该动物模型优选为啮齿类动物模型，尤其是鼠类。该动物模型的特征是，全身或肝脏中不表达CREBZF蛋白、或与对照动物相比CREBZF蛋白的表达量降低、或表达无活性的CREBZF蛋白或活性降低的CREBZF蛋白。在某些实施方案中，该动物模型是肝特异性敲除CREBZF的转基因小鼠。
可利用带有Flox位点的CREBZF等位基因小鼠和Albumin-Cre转基因小鼠(Alb-Cre TG)杂交来构建所述肝特异性敲除CREBZF的转基因小鼠。具体而言，可如下构建所述肝特异性敲除CREBZF的转基因小鼠：
(1)构建带有CREBZF基因、LoxP位点、Frt-Neo-Frt位点和两侧同源臂的质粒；
(2)将步骤(1)的质粒转入小鼠胚胎干细胞中，筛选获得同源重组的胚胎干细胞克隆；
(3)将步骤(2)获得的胚胎干细胞注射到小鼠囊胚中，并将该囊胚转入假孕母鼠中，获得雄性嵌合体小鼠；
(4)将步骤(3)获得的雄性嵌合体小鼠与野生型雌性小鼠交配，获得带有Flox位点的CREBZF等位基因小鼠；
(5)将步骤(4)获得的带有Flox位点CREBZF的等位基因小鼠与 Albumin-Cre转基因小鼠杂交，得到第一代杂合子小鼠，然后采用杂合子小鼠进行第二代自交，从而获得纯合的敲除小鼠，即所述小鼠模型。
在某些实施方案中，所述步骤(5)还获得纯合的对照小鼠，该构建方法还包括使步骤(5)获得的纯合的敲除小鼠与纯合的对照小鼠交配，从而扩大纯合的敲除小鼠的种群。
在某些实施方案中，利用Ella Cre(编号003724，名称B6.FVB-Tg(EIIa-cre)C5379Lmgd/J，来自Jackson公司)与所述带有Flox位点的CREBZF等位基因小鼠杂交，构建全身敲除CREBZF的小鼠(CREBZF KO)。
作为示范性的例子，CREBZF基因的序列可如SEQ ID NO:9的第5383－7516位碱基序列所示；同源臂区域可如SEQ ID NO:9第142－5312位和第9661－12661位碱基序列所示；LoxP位点可如SEQ ID NO:9第5329－5362位和第9598－9631位碱基序列所示；Frt-Neo-Frt位点可如SEQ ID NO:9第7517－7579位碱基序列所示，其中Neo盒子位于第7564－9545位。应理解，可利用本领域熟知的技术和序列来构建上述构建方法步骤(1)的质粒。质粒中还可含有其它本领域熟知的调控元件，以利于该CREBZF基因整合如胚胎干细胞的基因组中。另外，可利用本领域熟知的各种实验小鼠构建所述带有Flox位点的CREBZF等位基因小鼠，这类小鼠包括但不限于C57BL/6J小鼠。
可采用类似的方法构建其它动物模型。
在某些方面，本文也提供一种核酸序列，所述核酸序列含有CREBZF基因、LoxP位点和Frt-Neo-Frt位点。在某些实施方案中，所述核酸序列从5’到3’依次含有LoxP位点、CREBZF基因、Frt-Neo-Frt位点和LoxP位点。所述核酸序列还可进一步地含有同源臂序列。在这些实施方案中，所述核酸序列从5’到3’依次含有同源臂、LoxP位点、CREBZF基因、Frt-Neo-Frt位点、LoxP位点和同源臂。在某些实施方案中，同源臂与LoxP位点之间、LoxP位点与CREBZF基因、Frt位点与LoxP位点之间以及LoxP与同源臂位点之间可具有任选的连接序列。这些连接序列的序列可如本文SEQ ID NO:9第5313-5328位碱基序列、第5363-5382位碱基序列、第9580-9597位碱基序列或第9632-9660位碱基序列所示。
本文的核酸序列可包含于质粒或载体中。特别优选地，该质粒或载体为用 于同源重组的质粒或载体。质粒或载体中还可含有其它利于在宿主细胞中同源重组的元件。可采用本领域周知的方法构建本文所述的质粒或载体。
可使用本文的质粒或载体转化宿主细胞，以获得经基因工程改造的宿主细胞，该宿主细胞转入了本文所述的载体，并在其基因组中有本文所述的从5’到3’依次含有LoxP位点、CREBZF基因、Frt-Neo-Frt位点和LoxP位点的核酸序列。所述宿主细胞可以是哺乳动物细胞和啮齿类动物细胞，可以是胚胎干细胞、多能干细胞或体细胞。在某些实施方案中，所述宿主细胞不是人胚胎干细胞。在某些实施方案中，所述宿主细胞是非人哺乳动物细胞，包括其胚胎干细胞和体细胞，例如可以是啮齿类动物的胚胎干细胞和体细胞。在某些实施方案中，所述宿主细胞是人的体细胞。在某些实施方案中，所述宿主细胞是原代肝细胞、脂肪细胞或小鼠胚胎成纤维(MEF)细胞。
本文还涉及CREBZF基因或蛋白作为靶点在筛选治疗或预防代谢性疾病或症状的药物中的应用，包括但不限于筛选用于预防和/或治疗糖尿病、心血管疾病、肥胖和/或脂肪肝的药物，或用于降低体重、血糖、血脂、肝脏脂肪沉积和/或肝细胞脂质堆积和或增加葡萄糖耐受性的药物。具体而言，可以CREBZF蛋白或基因作为分子靶标，筛选出能抑制其活性或者表达水平的分子，该分子可以是新的小分子化合物，也可以是已知的化合物。可利用表达或不表达CREBZF的细胞，如原代肝细胞、脂肪细胞或小鼠胚胎成纤维(MEF)细胞通过荧光素酶报告基因实验、葡萄糖吸收实验或脂肪酸合成实验进行所述筛选。例如，可利用已经克隆成功的CREBZF启动子报告基因(CREBZF-Luc)，通过荧光素酶报告基因实验在转入了该启动子报告基因的原代肝细胞、脂肪细胞或者MEF细胞中进行高通量筛选，选择能够抑制CREBZF-Luc转录活性的试剂，作为抑制CREBZF基因表达的试剂。SEQ ID NO：10为本文将CREBZF启动子报告基因克隆到pGL3-basic中后获得的表达载体(pGL3-basic-mCREBZF-LUC)的核苷酸序列，其中，第27-2126位启动子序列。或者，可利用CREBZF+/+和CREBZF-/-小鼠的原代肝细胞、脂肪细胞或者MEF细胞，利用荧光标记的2-NBDG(2-[N-(7-硝基苯-2-噁-1,3-二唑-4-基)氨基]-2-脱氧-D-葡萄糖，Invitrogen)，通过葡萄糖吸收实验进行高通量筛选，选择能够在CREBZF+/+细胞中增加葡萄糖吸收，而在CREBZF-/-细胞中没有该作 用的试剂，作为潜在的抑制CREBZF基因表达或降低其表达水平的试剂。又或者，可利用CREBZF+/+和CREBZF-/-小鼠的原代肝细胞、脂肪细胞或者MEF细胞，通过BODIPY(D3922，Molecular Probes,Carlsbad,Calif,USA)荧光标记的脂肪酸合成实验进行高通量筛选，选择能够在CREBZF+/+细胞中抑制脂质合成或者积累，而在CREBZF-/-细胞中没有该作用的试剂，作为潜在的抑制CREBZF基因表达或降低其表达水平的试剂。上述这类试剂包括但不限于小分子化合物、siRNA和多肽。应理解的是，CREBZF-/-小鼠的原代肝细胞可来自本文所述的肝特异性敲除CREBZF的转基因小鼠和全身敲除CREBZF的小鼠，而CREBZF-/-小鼠的脂肪细胞和MEF细胞则可来自全身敲除CREBZF的小鼠。
CREBZF基因或蛋白还可用作分子指标，在临床上用于诊断胰岛素抵抗、2型糖尿病、高血脂症、肥胖和脂肪肝等代谢性疾病的病程发展。因此，在某些实施方案中，本文涉及检测CREBZF基因或蛋白的试剂在制备用于诊断代谢性疾病或判断代谢性疾病病程发展的试剂盒中的应用。这类试剂包括但不限于用于检测CREBZF基因的各种引物和探针，和/或用于检测CREBZF蛋白的特异性抗体等，这类试剂和包括在制作含CREBZF基因或蛋白的样品以及实施检测过程中使用的其它试剂，如溶剂等，包括但不限于实施PCR等所需的各种试剂。
因此，本文也提供一种检测试剂盒，所述检测试剂盒含有前文所述的检测CREBZF基因和/或蛋白的试剂，包括但不限于扩增和检测CREBZF基因所需的引物和探针以及CREBZF蛋白的特异性抗体。通过试剂盒快速检测CREBZF的水平，衡量其水平的高低，可作为代谢性疾病判断的一个备选指标。通常，若检测到对象CREBZF基因的表达水平和/或CREBZF蛋白的活性高于正常人群CREBZF基因的表达水平和/或CREBZF蛋白的活性，则可初步判断该对象患有代谢性疾病的风险增加或可能患有代谢性疾病。
下文将以具体实施例的方式产生本发明。应理解，这些实施例仅仅是阐述性的，并非意图用以限制本发明的范围。实施例中所用到的方法和材料，除非另有说明，否则为本领域常规的方法和材料。
一、材料与仪器
1、实验动物和食物
带有Flox位点CREBZF等位基因小鼠的产生：带有Flox位点的CREBZF等位基因小鼠是由赛业公司制作(Cyagen，China)。简而言之，在体外构建一个CREBZF基因1062bp个碱基的开放阅读框(ORF)序列以及一个LoxP位点和一个Frt-Neo-Frt位点和两侧的同源臂区域(SEQ ID NO:9：第142－5312位和第9661－12661位碱基序列为同源臂区域；第5329－5362位和第9598－9631位碱基序列为LoxP位点；第5383－7516位碱基序列为CKO区域；第7517－7579位碱基序列为Frt-Neo-Frt位点，其中Neo盒子位于第7564－9545位；其余为载体骨架的连接序列；外显子序列在第5383－7516位和第9661－12661位)，克隆到目标质粒上(基因敲除质粒ploxPFNeoFloxP由赛业公司构建保存，原始质粒来源于pGEM-T easy vector，购自Promega公司)，通过限制性内切酶Swal把质粒线性化，然后通过电穿孔仪把体外构建好的含有目的基因片段的序列导入到胚胎干细胞中(AB1，129/SvEv)，通过药物抗性筛选ES细胞克隆。使用以下Neo探针：
Neo probe-F：TCATCTCACCTTGCTCCTGC(SEQ ID NO:1)
Neo probe-R：AAGGCGATAGAAGGCGATGC(SEQ ID NO:2)
采用Southern blot技术对细胞的基因组进行检测，筛选出同源重组的ES细胞克隆，并将筛选出同源重组的ES细胞克隆注射到C57BL/6(B6)的囊胚中，将注射好的囊胚按照标准程序转移到假孕的母鼠(C57BL/6J)输卵管中，待嵌合体的小鼠出生之后，根据毛色判断是否为嵌合体以及嵌合体的程度大小，并把雄性嵌合体小鼠和野生型的雌性小鼠C57BL/6J交配。通过Flp介导的同源重组得到在CREBZF CKO(Conditional Knockout)两端含有Flox位点的目的小鼠，即所述带有Flox位点CREBZF等位基因小鼠。
肝特异性敲除CREBZF的小鼠的产生：肝特异性敲除CREBZF的小鼠(CREBZF LKO)是通过带有Flox位点CREBZF等位基因小鼠和Albumin-Cre转基因小鼠(Alb-Cre TG，获自美国Jackson公司，小鼠品名为B6.Cg-Tg(Alb-cre)21Mgn/J，小鼠货号为：003574)杂交，得到第一代杂合子小 鼠，然后采用杂合子小鼠进行第二代自交，得到少量纯合的敲除小鼠CREBZF LKO(CREBZF Flox+/+Cre+/-)和纯合的对照小鼠CREBZF WT(CREBZF Flox+/+Cre-/-)。通过得到的敲除小鼠和对照小鼠交配，扩大种群数量，得到足够数量的可供实验的小鼠。基因型鉴定CREBZF Flox的引物有两对，分别如下：
F1：GCTTGCAGTTTAGAGAGAAACAGC(SEQ ID NO:3)
R1：CAGCCAGAGTATCGCGAGATTC(SEQ ID NO:4)
F2：TTGACAATAAGTATTGAGGCATGCG(SEQ ID NO:5)
R2：TTTCCAACTTCTCAAGTGGTGAAC(SEQ ID NO:6)
使用F1和R1引物进行PCR，分别产生的目的片段是248bp(野生型)和318bp(含有Flox位点的CREBZF突变型)，使用F2和R2引物进行PCR，分别产生的目的片段是157bp(野生型)和285bp(含有Flox位点的CREBZF突变型)。用来检测Alb-Cre的引物是：
正向:GCGGTCTGGCAGTAAAAACTATC(SEQ ID NO:7)
反向：GTGAAACAGCATTGCTGTCACTT(SEQ ID NO:8)
C57Bl/6背景的八周龄雄性肝特异性CREBZF敲除的小鼠(CREBZF LKO)被随机分为四组，其中两组给予正常饮食(Chow)诱导，另外对应两组分别给予高脂高糖(HFHS)饮食饲养。
小鼠食用的高脂高糖饲料(HFHS)购买于美国Research Diet公司。
2、主要试剂、仪器和设备
主要试剂：甘油三酯检测试剂盒(Infinity Triglycerides Reagent，TG)、胆固醇检测试剂盒(Infinity Cholesterol Reagent，TC)购自Thermo Scientific公司；Ultrasensitive Mouse Insulin ELISA试剂盒(Mercodia)；血糖仪Freestyle和血糖试纸(Abbott)：重组人胰岛素注射液(Eli Lilly and Company)；葡萄糖(Sigma)。
使用仪器和设备：核磁共振(NMR)体成分分析(Bruker磁共振脂肪含量测量仪Mq7.5)；酶标仪(Tecan-200-酶标仪Infinite 200PRO)；4℃/-20℃冰箱购自海尔公司；超低温冰箱购自Thermo Scientific Forma公司；微量移液器 购自Rainin或者Eppendorf公司；垂直电泳槽、湿转转膜仪和配套电源均购自Bio-Rad公司；冷冻离心机购自Eppendorf公司；PCR仪购自Bio-Rad公司；实时荧光定量PCR系统ABI 7500Fast Real-Time PCR System购自Applied Biosystems公司。
3、动物分组
将八周龄的CREBZF LKO雄性小鼠和匹配的CREBZF WT雄性小鼠进行随机分组，分组情况如表1：
表1：小鼠分组情况及处理方式
Figure PCTCN2017087688-appb-000001
4、实验小鼠模型构建
小鼠一直饲养在SPF级动物房中，在22天断奶后，依据小鼠雌雄进行分笼，小鼠可任意进食及饮水(上海实验动物中心提供的标准食物)。动物房保持温度22±3℃，湿度35±5％，12小时昼夜周期。所有实验都是选用8周龄小鼠进行实验。
5、小鼠指标测定和小鼠组织收集
(1)小鼠每周指标测定：每个星期测定小鼠的体重、进食量，并记录数据。
(2)小鼠实验处理三个月后测其空腹血糖。
(3)小鼠身体组分分析：利用核磁共振系统mini-spec nuclear magnetic resonance(NMR)spectrometer(Bruker Corp)测定小鼠在清醒、自由饮食状态下的的身体组分，包括脂肪量(Fat Mass)、瘦组织量(Lean Mass)和体液 成分(Body Fluid)等。
(4)小鼠处死：实验四个月后将小鼠处死收取组织。
处死方法：采用异氟烷麻醉致小鼠深度昏迷，然后打开胸腔用1ml注射器进行心尖取血，血液放入含有抗凝剂的采血管中，4℃、3000rpm离心15min，取上清。取血后对小鼠腹侧面拍照。快速摘取其肝脏，拍照后称重。取肝脏肝大叶部分进行甲醛固定和冰冻(OCT)包埋，用于观察实验小鼠肝脏病理性变化，剩余肝脏组织用于Western Blot实验和RT-PCR实验。打开小鼠腹腔取出所有附睾脂肪，拍照称重。
二、实验结果
1、成功的构建了肝特异性CREBZF敲除的小鼠
肝特异性CREBZF敲除的小鼠的构建过程如前文和图1(A)所示。肝特异性敲除CREBZF的小鼠的鉴定结果如图1(B)所示。使用引物SEQ ID NO:3和4进行PCR，分别产生的目的片段是248bp(野生型)和318bp(含有Flox位点的CREBZF基因型小鼠)，鉴定Alb-Cre的目标片段大小为100bp。使用引物SEQ ID NO:5和6进行PCR分别产生的目的片段是157bp(野生型)和285bp(含有Flox位点的CREBZF基因型小鼠)。
2、CREBZF肝脏特异性敲除的小鼠可以减缓饮食诱导的小鼠体重增加
采用年龄匹配的八周龄小鼠(CREBZF WT和CREBZF LKO)进行实验，按照分组分别给予普通正常饮食(Chow Diet)和高脂高蔗糖饲料饮食(HFHS Diet)喂养。实验四个月后测定各组小鼠的体重。结果发现实验组CREBZF肝特异性敲除小鼠可以减缓高脂高糖饮食诱导的体重增加(图2，A)。实验过程中，每周对小鼠体重进行测定，在正常饮食情况下，CREBZF LKO小鼠和CREBZF WT小鼠的体重没有明显区别；在HFHS喂养情况下CREBZF LKO小鼠的体重的生长曲线明显低于对照组(图2，B)。同时我们采用核磁共振体成分分析仪(NMR)对各组小鼠的每只小鼠的脂肪量，瘦组织量进行活体分析测试。结果发现CREBZF LKO小鼠在高脂高蔗糖饲料(HFHS Diet)喂养情况下可以显著地降低脂肪量所占的体重比(图2，C)。这说明肝脏特异性敲除的 小鼠可以减缓饮食诱导的小鼠体重增加，同时也能减少体脂含量。同时我们在实验期间，每周对小鼠进食量进行测定和计算，结果显示实验组和对照组的小鼠进食量没有显著性差异(结果未显示在图中)，说明实验组小鼠体重的差异并不是由于进食量引起的。
3、CREBZF肝脏特异性敲除的小鼠改善高脂高糖饮食诱导的小鼠肝脏脂肪沉积，降低小鼠血清和肝脏中甘油三酯和胆固醇水平
首先，我们把经过4％多聚甲醛48h固定的肝脏组织进行石蜡包埋切片，然后用苏木精-伊红染色(HE染色)观察小鼠肝脏病理变化。HE染色结果显示，在正常饮食喂养的情况下，小鼠肝索结构清晰，肝细胞以中央静脉为中心呈放射状分布，且肝细胞细胞中无明显的脂肪堆积，细胞核清晰，胞浆均匀等，并且实验组和对照组没有显著性差别(图3，A)。在高脂喂养的情况下，对照组小鼠(CREBZF WT)的HE切片中肝脏肝索结构不明显，肝细胞中出现大量空泡(脂滴)，但是实验组小鼠(CREBZF LKO)肝细胞空泡明显少于对照组，肝细胞中脂滴堆积的情况得到明显改善(图3，A)。通过观察脂滴的数量与大小可以看出，实验组小鼠(CREBZF LKO)在高脂高糖饮食情况下可以显著的降低肝脏脂肪沉积。同时我们也通过对OCT包埋的肝组织进行肝脏冰冻切片，之后对切片进行油红染色(Oil Red staining)，得到了相同的实验结果(图3，A)。所有实验结果都是观察大于8只不同小鼠的切片得出。
其次，我们利用甘油三酯(TG)和总胆固醇(TC)检测试剂盒对小鼠肝脏和血清中的甘油三酯和总胆固醇进行测定。结果显示，在正常饮食(Chow Diet)喂养下，实验组小鼠和对照组小鼠肝脏和血清中甘油三酯和胆固醇水平没有显著性差异，但是在高脂高糖饮食诱导的小鼠模型中，CREBZF LKO组小鼠肝脏和血清中甘油三酯水平与对照组相比均显著降低(P<0.05)(图3，B、C)。同时我们也发现CREBZF LKO组小鼠肝脏和血清中总胆固醇水平与对照组相比均显著降低(P<0.05)(图3，B、C)。由此说明，在肝脏中特异性敲除CREBZF的情况下可以改善由高脂高糖饮食诱导的小鼠肝脏脂肪沉积，降低小鼠血清和肝脏中甘油三酯和胆固醇水平。
4、CREBZF肝脏特异性敲除的小鼠对高脂高糖饮食诱导肥胖小鼠血糖以及葡萄糖的耐受性的影响
以上实验证明，CREBZF肝脏特异性敲除的小鼠对高脂高糖饮食诱导的肥胖小鼠腹部和肝脏中脂肪沉积、肝脏和血清甘油三酯水平和胆固醇水平有显著的改善作用。同时我们也测定了各组小鼠对高脂高糖饮食诱导的肥胖的血糖以及葡萄糖的耐受性情况。
在饮食诱导实验处理三月后，我们对各组小鼠禁食16h，测定其空腹血糖。结果发现，在正常饮食情况下实验组和对照组小鼠血糖没有显著性变化(图4，A)。在HFHS饮食诱导的情况下实验组小鼠(CREBZF LKO)空腹血糖与对照组小鼠(CREBZF WT)相比显著降低(P<0.05)(图4，A)，同时通过对小鼠进行葡萄糖耐受实验(GTT)，实验结果表明在正常饮食情况下实验组和对照组小鼠葡萄糖耐受性没有显著性变化(图4，B)。在HFHS饮食诱导的情况下实验组小鼠葡萄糖耐受性相比对照组显著增加(P<0.05)(图4，B、C，这说明在肝脏中特异性敲除CREBZF可以显著改善由高脂高糖饮食诱导的葡萄糖耐受性降低的表型。这些结果证明，肝脏中特异性敲除CREBZF不仅可以改善高脂高糖饮食诱导的高血脂，而且也可以起到调节血糖稳态的作用。
5、CREBZF肝脏特异性敲除的小鼠对高脂高糖饮食诱导肥胖小鼠脂质相关基因的影响
为了研究CREBZF肝脏特异性敲除对改善高脂饮食诱导的肥胖小鼠代谢的分子机制，我们提取小鼠肝脏组织蛋白进行Western Blot测试分析，同时提取肝组织RNA，反转得到cDNA，进行RT-PCR实验。如图5(A)所示，CREBZF肝脏特异性敲除组在喂养高脂高糖(HFHS)时激活形式的N-SREBP-1(Sterol Regulatory Element Binding Proteins1)表达量显著降低，同时它调控脂肪生成的下游基因脂肪酸合成酶(Fatty Acid Synthase,FAS)的表达也显著减少。该结果表明CREBZF肝脏特异性敲除之后可以通过降低脂肪酸合成而降低脂肪肝。另外，我们也通过RT-PCR检测发现，肝脏中CREBZF效率可以达到80％，在肝脏特异性敲除CREBZF之后，可以显著性的增加Insig-2a的mRNA的水平(P<0.05)(图5，B)，这也导致了SREBP-1c的mRNA的水平的显著减少(P<0.05) (图5，C)。同时我们也检测了脂肪酸和甘油三酯合成相关的关键基因的mRNA的水平，包括ACLY、ACC1、FAS、SCD1、GPAT1、DGAT和DGAT2，结果表明在肝脏特异性敲除CREBZF之后这些关键基因的mRNA的水平都显著性降低(P<0.05)(图5，D)。这些结果表明，在肝脏中特异性敲除CREBZF可以降低HFHS饮食诱导的脂肪肝以及降低脂肪酸合成相关基因的表达，这可能是通过增加Insig-2a的表达水平来抑制肝脂肪的生成。
三、讨论
本文首次成功构建了肝特异性敲除CREBZF的小鼠，这为之后更好的进行CREBZF在体内生物学功能研究起到了非常关键的作用，尤其是在研究胰岛素抵抗、2型糖尿病、高血脂症、肥胖和脂肪肝等代谢综合征模型的研究中起到非常重要的作用。利用该肝特异性敲除CREBZF的小鼠，本文发现了碱性亮氨酸拉链(bZIP)转录因子CREBZF在营养过剩引起的代谢性疾病中的作用。在本文之前，关于CREBZF的研究主要集中在其转录调控方面的分子调控机制，及其在生长和增殖方面的作用，而本文通过首次构建得到的肝特异性敲除的CREBZF小鼠模型研究其在代谢调控方面的作用和机制，为其在代谢方面进行更加深入的研究提供了新的思路。
本文发现肝脏特异性敲除CREBZF显著的减缓高脂高糖饮食诱导的小鼠体重增加，降低血糖血脂，改善小鼠肝脏脂肪沉积，增加葡萄糖耐受性，同时肝脏病理切片也表明肝脏特异性敲除CREBZF显著的减缓肝细胞脂质堆积。并且还发现肝脏特异性敲除CREBZF可以通过增加Insig-2a的mRNA的水平和降低脂质合成相关基因SREBP-1的水平，同时CREBZF肝脏特异性敲除喂养高脂高糖(HFHS)组的激活形式是N-SREBP-1(Sterol Regulatory Element Binding Proteins1)表达量显著降低，它调控脂肪生成的下游基因脂肪酸合成酶(fatty acid synthase,FAS)的表达也显著减少，这说明敲除CREBZF可以通过抑制脂肪酸从头合成来降低脂肪肝。
在敲除肝脏CREBZF情况下，除了能够达到降糖的目的，同时它也附带其他改善代谢的作用。因而通过肝脏靶向抑制CREBZF有望获得具有“Glucose Plus”功效的降糖药。因此，可以CREBZF作为药物靶点，研究出降糖的药物， 它将除了能够单纯的降糖之外，同时它还能够改善代谢，这种药物的应用前景会很大。另外，通过肝脏到脂肪组织的作用，具有高效减肥的效果。目前在肥胖的病人当中，单纯针对脂肪组织的降脂，效果并不明显。本文的实验结果表明，在在肝脏中特异性的敲低CREBZF，能够显著的降低小鼠体脂含量，这种通过肝脏进而靶向到脂肪的作用，从一定程度上来说，达到了高效降脂的作用。
根据本文的研究结果，可针对CREBZF设计出特异性的药物靶点，以CREBZF作为分子靶标，设计小分子药物来抑制其活性或者表达水平，从而使之成为预防和辅助治疗过量饮食引起的代谢性疾病的一种新药。另外也可以通过CREBZF的活性或表达水平作为分子靶标来筛选现有的药物，从众多未知分子机制的潜在治疗药物中筛选出一种能够更好的治疗胰岛素抵抗、2型糖尿病、高血脂症、肥胖和脂肪肝等代谢综合征的药物。在临床诊断方面，对待胰岛素抵抗、2型糖尿病、高血脂症、肥胖和脂肪肝等代谢综合征的病人，肝脏中CREBZF的临床水平可以作为判断其病程发展的一个潜在分子指标，同时也可以开发出CREBZF相关的检测试剂盒，通过试剂盒快速检测CREBZF的水平，衡量其水平的高低，作为其病症判断的一个备选指标。

Claims (10)

  1. 以下试剂在制备减缓对象体重增加，降低对象血糖血脂，改善对象肝脏脂肪沉积，增加对象葡萄糖耐受性，和/或减缓对象肝细胞脂质堆积的药物中的用途：
    (1)减少对象CREBZF蛋白的表达的试剂；和/或
    (2)降低对象所表达的CREBZF蛋白的活性的试剂。
  2. 如权利要求1所述的用途，其特征在于，
    所述对象的体重增加、高血糖、高血脂、肝脏脂肪沉积、低葡萄糖耐受性和/或肝细胞脂质堆积由该对象的高脂高糖饮食所致；和/或
    所述药物用于降低糖尿病、心血管疾病、肥胖和/或脂肪肝的发病机率或治疗这些代谢性疾病。
  3. 如权利要求1或2所述的用途，其特征在于，所述减少对象CREBZF蛋白的表达的试剂为抑制CREBZF基因表达或降低其表达水平的试剂，优选选自：
    (a)抑制该CREBZF基因转录活性的试剂；
    (b)抑制该CREBZF mRNA的转录水平的试剂；
    (c)促进该CREBZF mRNA降解的试剂；
    (d)针对该CREBZF基因的siRNA；
    (e)抑制CREBZF mRNA的翻译的试剂；
    (f)特异性识别CREBZF基因的导向核酸并进行剪切以降低其表达水平的试剂；和
    (g)用于部分或全部敲除CREBZF基因的试剂；和
    所述降低对象所表达的CREBZF蛋白的活性的试剂为CREBZF蛋白的特异性抗体或具有抑制该CREBZF蛋白的活性的小分子化合物。
  4. 一种核酸序列，选自：
    (1)含有CREBZF基因、LoxP位点和Frt-Neo-Frt位点的核酸序列；和
    (2)(1)所述序列的互补序列。
  5. 如权利要求4所述的核酸序列，其特征在于，
    所述核酸序列从5’到3’依次含有LoxP位点、CREBZF基因、Frt-Neo-Frt位点和LoxP位点；或
    所述核酸序列还含有同源臂，从5’到3’依次含有同源臂、LoxP位点、CREBZF基因、Frt-Neo-Frt位点、LoxP位点和同源臂；
    任选地，所述同源臂与LoxP位点之间、LoxP位点与CREBZF基因、Frt位点与LoxP位点之间以及LoxP与同源臂位点之间具有连接序列；
    优选地，所述CREBZF基因的序列如SEQ ID NO:9的第5383－7516位碱基序列所示；所述同源臂区域分别如SEQ ID NO:9第142－5312位和第9661－12661位碱基序列所示；所述LoxP位点如SEQ ID NO:9第5329－5362位所示；Frt-Neo-Frt位点如SEQ ID NO:9第7517－7579位碱基序列所示；
    优选地，所述核酸序列如SEQ ID NO:9所示，或者为SEQ ID NO:9的互补序列。
  6. 一种载体，其特征在于，所述载体含有权利要求4或5所述的核酸序列；优选地，所述载体为用于同源重组的载体。
  7. 一种经基因工程改造的宿主细胞，其特征在于，所述宿主细胞转入了权利要求6所述的载体，并在其基因组中同源重组有从5’到3’依次含有LoxP位点、CREBZF基因、Frt-Neo-Frt位点和LoxP位点的核酸序列。
  8. 一种构建转基因小鼠的方法，所述方法包括：
    (1)提供权利要求6所述的载体；
    (2)将所述载体转入小鼠胚胎干细胞中，筛选获得同源重组的胚胎干细胞克隆；
    (3)将步骤(2)获得的胚胎干细胞注射到小鼠囊胚中，并将该囊胚转入 假孕母鼠，获得雄性嵌合体小鼠；
    (4)将步骤(3)获得的雄性嵌合体小鼠与野生型雌性小鼠交配，获得带有Flox位点的CREBZF等位基因小鼠；
    (5)将步骤(4)获得的带有Flox位点CREBZF的等位基因小鼠与Albumin-Cre或Ella Cre转基因小鼠杂交，得到第一代杂合子小鼠，然后采用杂合子小鼠进行第二代自交，从而获得纯合的敲除小鼠，即所述转基因小鼠；
    任选地，所述构建方法还包括使步骤(5)获得的纯合的敲除小鼠与该步骤获得的纯合的对照小鼠交配，从而扩大纯合的敲除小鼠的种群；
    其中，所述转基因小鼠的特征是，全身或肝脏不表达CREBZF蛋白、或与对照小鼠相比CREBZF蛋白的表达量降低、或表达无活性的CREBZF蛋白或活性降低的CREBZF蛋白。
  9. CREBZF基因或蛋白作为靶点在筛选治疗或预防代谢性疾病的药物中的应用，或在筛选减缓对象体重增加，降低对象血糖血脂，改善对象肝脏脂肪沉积，增加对象葡萄糖耐受性，和/或减缓对象肝细胞脂质堆积的药物中的应用；或
    检测CREBZF基因或蛋白的试剂在制备用于诊断代谢性疾病或判断代谢性疾病病程发展的试剂盒中的应用；任选地，所述试剂包括用于检测CREBZF基因的引物和探针，和/或用于检测CREBZF蛋白的特异性抗体。
  10. 一种检测试剂盒，其特征在于，所述检测试剂盒含有检测CREBZF基因和/或蛋白的试剂；
    任选地，所述试剂包括用于检测CREBZF基因的引物和探针，和/或用于检测CREBZF蛋白的特异性抗体。
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