WO2018094964A1 - 一种以凋亡信号调节激酶1n端二聚化为靶点筛选用于治疗脂肪性肝炎药物的方法 - Google Patents

一种以凋亡信号调节激酶1n端二聚化为靶点筛选用于治疗脂肪性肝炎药物的方法 Download PDF

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WO2018094964A1
WO2018094964A1 PCT/CN2017/081936 CN2017081936W WO2018094964A1 WO 2018094964 A1 WO2018094964 A1 WO 2018094964A1 CN 2017081936 W CN2017081936 W CN 2017081936W WO 2018094964 A1 WO2018094964 A1 WO 2018094964A1
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ask1
kinase
myc
apoptosis signal
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李红良
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武汉大学
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Priority claimed from CN201611054149.3A external-priority patent/CN106589079B/zh
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
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    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Definitions

  • the present invention belongs to the field of biotechnology, and in particular, the present invention relates to the establishment of a screening model targeting N-terminal dimerization of Apoptosis signal-regulated kinase 1 (ASK1), and screening
  • ASK1 N-terminal dimerization inhibitor is used in the preparation of a medicament for preventing, alleviating and/or treating steatohepatitis diseases.
  • Apoptosis signal-regulated kinase 1 is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family.
  • the ASK1 protein contains 1375 amino acid residues and has a molecular weight of approximately 160 kDa [1] .
  • the protein structure of ASK1 is divided into five parts, wherein amino acid 46-277 at the N-terminus is a thioredoxin (Trx) binding region, amino acid 297-324 is an N-terminal coiled-coil domain, and amino acid 384-655 is a TRAF binding region.
  • the amino acid number 687-945 is the kinase domain of ASK1, while the amino acid number 1239-1295 is the C-terminal coiled-coil domain [2] .
  • ASK1 undergoes homo-oligomerization reaction through the coiled-coil domain of the C-terminus to form a high molecular weight complex with a molecular weight of approximately 1500-2000 kDa, while Trx binds to the Nx-terminal Trx binding region of ASK1 to form an ASK1 signalsome.
  • the specific binding of Trx inhibits the binding of TRAF2, TRAF6 and ASK1, thereby inhibiting the activity of ASK1.
  • Trx When stimulated by TNF- ⁇ , LPS, endoplasmic reticulum pressure, oxidative stress, etc., Trx is rapidly activated and separated from ASK1, and TRAF2 and TRAF6 are recruited and bound to the TRAF binding region of ASK1, and the kinase domain of ASK1 is Phosphorylation activates ASK1 and induces the SEK1-JNK and MKK3/MKK6-p38 signaling cascades [3] .
  • ASK1 plays an important regulatory role in a variety of diseases, which can promote AngII-induced cardiac hypertrophy, cardiac remodeling, interstitial fibrosis and coronary remodeling [4] ; can promote neurons after cerebral ischemia and reperfusion - The death of microglia, which worsens the development of stroke disease [5] ; ASK1 is significantly activated in the model of acute renal injury induced by ischemia-reperfusion [6] . Based on the activation mechanism of ASK1 and its regulation of various diseases, various studies have been devoted to the development of drugs that can regulate the ASK1 signal cascade to achieve the purpose of treating diseases.
  • Nonalcoholic steatohepatitis is a pathological manifestation of hepatocyte lipid accumulation, hepatic steatosis, and inflammation.
  • Disease due to its high incidence worldwide, has become a major health problem for humans. It can further develop into liver cirrhosis, hepatocellular carcinoma and liver failure, which can damage digestive system function, reduce human immunity, weaken detoxification function, affect hormone metabolism, etc., seriously affecting people's health and quality of life, and also giving society Bring a heavy burden.
  • the treatment is mainly limited to the treatment of its complications, such as insulin resistance, hyperglycemia, obesity, etc., the treatment effect is very limited [7-10] . Therefore, how to effectively treat steatohepatitis, how to effectively screen drugs that can effectively treat steatohepatitis has become an urgent problem to be solved at this stage.
  • the object of the present invention is to overcome the deficiencies of the prior art and to provide a use of apoptosis signal-regulating kinase 1 to screen for the prevention, alleviation and/or treatment of steatohepatitis by targeting apoptosis signal-regulated kinase 1 N-terminal dimerization.
  • a drug that inhibits the N-terminal dimerization of an apoptotic signaling regulatory kinase is to overcome the deficiencies of the prior art and to provide a use of apoptosis signal-regulating kinase 1 to screen for the prevention, alleviation and/or treatment of steatohepatitis by targeting apoptosis signal-regulated kinase 1 N-terminal dimerization.
  • a drug that inhibits the N-terminal dimerization of an apoptotic signaling regulatory kinase is to overcome the deficiencies of the prior art and to provide a use of apoptosis signal-regulating kinase 1 to
  • N-terminal dimerization of apoptotic signal-regulated kinase 1 refers to the binding of two ASK1 monomers to each other via a coiled-coil domain at their N-terminus to form a dimer. Specifically, under normal conditions, ASK1 binds to each other through its C-terminal coiled-coil domain to form a homodimer. At this time, the small molecule Trx in vivo binds to the N-terminus of ASK1, blocks the N-terminal polymerization of ASK1, and inhibits ASK1 activation. Under the stimulation of reactive oxygen species and other factors, Trx undergoes an oxidation reaction and then dissociates from ASK1, causing ASK1 N-terminus to form a dimer, resulting in ASK1 activation.
  • ASK1 apoptotic signal-regulated kinase 1
  • Steatohepatitis is a liver disease secondary to inflammation and fibrosis in hepatic steatosis and cell damage, and is classified into nonalcoholic steatohepatitis and alcoholic steatohepatitis according to its etiology.
  • the steatohepatitis in the present invention mainly refers to nonalcoholic steatohepatitis, and the histopathological changes of the liver are similar to those of alcoholic steatohepatitis, but the patient has no clear history of drinking. Nonalcoholic steatohepatitis will further develop into cirrhosis, hepatocellular carcinoma and liver failure.
  • a method for screening for a drug for preventing, alleviating and/or treating steatohepatitis by targeting apoptosis signal-mediated kinase 1 N-terminal dimerization comprises the steps of:
  • the candidate substance if the candidate substance inhibits the N-terminal dimerization of the apoptosis signal-regulating kinase, the candidate substance is a potential substance for preventing, alleviating and/or treating steatohepatitis.
  • Step (a) comprises: in the test set, adding the candidate substance to the system comprising the 1N terminus of the apoptosis signal-regulating kinase; and/or
  • Step (b) comprises: detecting the dimerization of the 1N-terminus of the apoptosis signal-regulating kinase in the system of the test group, and comparing the control group with the apoptosis-signaling kinase without adding the candidate substance 1N end system;
  • test results in the test group show that the apoptosis signal-regulating kinase 1 N-terminal dimerization is inhibited, it indicates that the candidate substance is a potential substance for preventing, alleviating and/or treating steatohepatitis.
  • Step (a) includes:
  • amino acid sequence encoding the N-terminal 1-478 aa of the apoptosis signal-regulating kinase is as shown in SEQ NO: 1, and the DNA sequence is shown in SEQ NO: 2;
  • Step (b) includes: the successfully transfected cells are tested for firefly luciferase and Renilla luciferase RLU after 24 hours of culture, and the ratios of the two are calculated, and the degree of polypeptide inhibition between the different samples is compared according to the obtained ratio.
  • the firefly luciferase reporter gene is expressed at a high level when the above cells continue to be transfected into the pG5luc vector; if the apoptosis signal regulates the N-terminal dimerization of the kinase 1 Inhibited, the firefly luciferase reporter gene on the pG5luc vector is not expressed;
  • the Dual-Luciferase dual luciferase reporter assay system can analyze the N-terminal dimerization of apoptosis signal-regulated kinase 1;
  • transfection refers to the process of transferring or transporting a biologically functional nucleic acid into a cell and maintaining the biological function of the nucleic acid in the cell.
  • a biologically functional nucleic acid refers to a pBIND vector linked to a DNA encoding an apoptotic signal-regulating kinase 1N-term 1-78aa, and a pACT linked to a DNA encoding an apoptosis signal-regulated kinase 1N-1-678aa.
  • the animal cells used in the step (a) of the above screening method are selected from the group consisting of HEK-293T, L02, Hela, Huh7, Hepg2, A549, 3T3, MEFs, H9C2.
  • HEK-293T human embryonic kidney cell
  • GNHu43 human embryonic kidney cell
  • Hela a human cervical cancer cell
  • TCHu187 Keratin immunoperoxidase staining was positive. Detected by mycoplasma, STR detection.
  • Huh7 a human hepatoma cell, was purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHu182.
  • Hepg2 a human hepatoma cell, was purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHu72.
  • A549 a human non-small cell lung cancer cell, was purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHu150.
  • 3T3 mouse embryonic cells, purchased from the Chinese Academy of Sciences Cell Bank, catalog number GNM6.
  • MEFs mouse embryonic fibroblasts, were purchased from the Chinese Academy of Sciences Cell Bank, catalog number SCSP-101.
  • H9C2 rat cardiomyocytes, purchased from the Chinese Academy of Sciences Cell Bank, catalog number GNR5.
  • a clonal cell line of BD1X rat embryonic heart tissue was subcloned into a H9c2 (2-1) cell line.
  • the animal cells used in the step (a) of the above screening method are selected from the group consisting of HEK-293T.
  • a regulating signal based on the prevention of apoptosis terminated dimer into 1N kinase target, alleviation and / or treatment of cells steatohepatitis model for drug screening, selection of mammalian two-hybrid system CheckMate TM
  • the DNA encoding the apoptotic signal-regulating kinase 1 N-1-678 aa was ligated into the pBIND vector encoding the DNA-binding domain and the pACT vector of the transcriptional activation domain, respectively, and the two vectors were transfected into
  • a cell model for drug screening for prevention, alleviation and/or treatment of steatohepatitis is constructed, and the animal cells used are selected from the group consisting of HEK-293T, L02, Hela, Huh7, Hepg2, A549, 3T3, MEFs, H9C2.
  • Step (a) includes:
  • Step (b) includes: the cells successfully transfected are detected by immunoprecipitation and Western blot after 24 hours of culture, and the contents of HA-ASK1 N and Myc-ASK1 N in the protein solution after immunoprecipitation are detected;
  • Co-immunoprecipitation is a method developed by the specific binding of an antigenic protein and an antibody and the phenomenon that the "protein A/G" of a bacterial protein specifically binds to an FC fragment of an antibody (immunoglobulin).
  • protein A/G is pre-bound on argarose beads to react with the antigen-containing solution and antibody, and the prorein A/G on the beads can achieve the purpose of adsorbing antigen.
  • the antigen of interest can be separated from other antigens from a solution containing the antigen of interest.
  • the antibody is selected from an antibody HA (anti-HA), the antigen of interest is HA, and the solution containing the antigen is transfected with HA-ASK1 N , Myc-ASK1 N , HA-ASK1 N + Myc-ASK1, respectively.
  • anti-HA binds the antigen of interest to protein A/G agarose beads.
  • Western blot uses anti-Myc as the primary antibody, if the HA-ASK1 N , Myc-ASK1 N and a candidate substance of psi-flag-Peptide are simultaneously transfected with HA-ASK1 N or Myc-ASK1
  • the control group of N was the same, and no significant Myc protein band was detected, and the candidate substance was a potential substance for preventing, alleviating and/or treating steatohepatitis.
  • the animal cells used in the step (a) of the above screening method are selected from the group consisting of HEK-293T, L02, Hela, Huh7, Hepg2, A549, 3T3, MEFs, H9C2.
  • the animal cells used in the step (a) of the above screening method are selected from the group consisting of HEK-293T.
  • a cell model for preventing, alleviating and/or treating a drug for steatohepatitis based on apoptosis signal-regulated kinase 1 N-terminal dimerization which respectively encodes said apoptosis
  • the DNA of the signal-regulated kinase 1N-1-678 amino acid (SEQ NO: 2) was ligated into the pcDNA5-HA and pcDNA5-Myc plasmids, and the HA-ASK1 N and Myc-ASK1 N plasmids were constructed and transfected into animal cells separately or simultaneously.
  • the drug screening model for preventing, alleviating and/or treating steatohepatitis is constructed, and the animal cells used are selected from the group consisting of HEK-293T, L02, Hela, Huh7, Hepg2, A549, 3T3, MEFs, H9C2.
  • a polypeptide Peptide1 for preventing, alleviating and/or treating steatohepatitis which is screened by apoptotic signal-regulated kinase 1 N-terminal dimerization, has an amino acid sequence such as Seq No. 20 Shown, or a pharmaceutically acceptable salt thereof.
  • a polynucleotide molecule which encodes the polypeptide Peptide1, the nucleotide sequence of which is shown in Seq No. 24.
  • a pharmaceutical composition comprising:
  • the present invention utilizes a liver-targeted gene therapy vector of adeno-associated virus AAV8, which mediates the screening of the latent substance Peptide1, which is overexpressed in the liver tissue of cynomolgus monkeys, and is induced by a high-fat diet (diet induced obesity, DIO).
  • AAV8-GFP-Peptide1 The function of Peptide1 was studied. It was found that there was no significant difference in body weight and BMI between the AAV8-GFP-Peptide1 group and the AAV8-GFP-Peptide1 group.
  • liver function The determination of blood lipids and the activity of enzymes (ALT, AST) in response to liver function showed that the serum levels of triglyceride and low-density cholesterol in the serum of AAV8-GFP-Peptide1 group were significantly decreased, the content of high-density cholesterol was increased, and the activity of ALT was significantly decreased. Liver histopathological staining showed that lipid accumulation in liver tissue of AAV8-GFP-Peptide1 group was significantly reduced, and liver fatty degeneration was significantly reduced. This indicates that in macaques, the polypeptide Peptide1 is able to inhibit the development of steatohepatitis.
  • a method of modulating hepatic cell steatosis comprising: modulating an intracellular apoptosis signal-regulated kinase 1 N-terminal dimerization.
  • the present invention is apparent by playing a model of hepatic steatosis induced by ASK1 in a high-fat diet.
  • the mechanism of promoting action for the first time, found that N-terminal dimerization of ASK1 has an important regulatory effect on hepatic cell steatosis, which can promote the development of hepatic steatosis.
  • Targeting N-terminal dimerization of ASK1 a novel drug for preventing, alleviating and/or treating steatohepatitis can be screened by detecting whether a candidate substance inhibits N-terminal dimerization of ASK1.
  • Figure 1 is the result of the ratio of firefly luciferase to Renilla luciferase RLU;
  • FIG. 2 is a Western blot analysis of a lysate of HEK-293T cells transfected with different plasmids and a protein solution obtained by immunoprecipitation of the lysate;
  • Figure 3 shows the results of Western blot analysis of the ASK1-JNK1 signaling pathway in L02 cells transfected with different polypeptide plasmids after 1 h of PA stimulation;
  • Figure 4 is a quantitative analysis of oil red O staining and intracellular triglyceride content of L02 cells transfected with different polypeptide plasmids after 12 hours of PA stimulation, where a is the oil red O staining diagram and b is the triglyceride content determination result statistics.
  • Figure 5 shows the results of detection of Peptide1 overexpression in the liver of cynomolgus monkeys mediated by adeno-associated virus AAV8.
  • a is the result of virus transfection efficiency in the liver tissues of the saline control group, the AAV8-GFP control group and the AAV8-GFP-Peptide1 group;
  • b is the AAV8-GFP control group and the AAV8-GFP-Peptide1 group in the portal vein virus vector The detection results of Peptide1 expression in liver tissues of cynomolgus macaques;
  • Figure 6 is a graph showing the results of body weight and BMI index of AAV8-GFP and AAV8-GFP-Peptide1 groups.
  • a is the weight result map
  • b is the BMI index result map (n.s.: p>0.05vs AAV8-GFP group);
  • Fig. 7 is the AAV8-GFP and AAV8-GFP-Peptide1 group cynomolgus monkey blood lipid content and liver function test results
  • a is the result of determination of serum triglyceride content
  • b is the result of determination of serum total cholesterol content
  • c is the result of determination of serum high-density cholesterol content
  • d is the result of determination of serum low-density cholesterol content
  • e is alanine aminotransferase (ALT)
  • f is the results of the determination of aspartate aminotransferase (AST) content (ns: p> 0.05 vs AAV8-GFP group, **: p ⁇ 0.01 vs AAV8-GFP group);
  • Figure 8 is a graph showing the results of HE and oil red O staining of liver tissues of AAV8-GFP and AAV8-GFP-Peptide1 groups.
  • HEK-293T cells were cultured in DMEM high glucose medium (containing 10% FBS, 1% penicillin-streptomycin); L02 cells were cultured in DMEM high glucose medium (containing 10% FBS, 1% penicillin-streptomycin) In the medium, ensure the air humidity in the incubator, 5% CO 2 content, and culture at 37 °C.
  • ASK1 Gene PCR amplified cDNA fragment obtained ASK1 N1 (NCBI BC088829);
  • upstream primer 2 5'-GGAGCACCAGAATGACATCAGGAAAGCTCG-3'
  • downstream primer 2 5'-GC TCTAGA TCAATCATATTCATAGTCATACTCCAGC-3'
  • PCR amplification of ASK1 (NCBI: BC088829) gene cDNA to obtain ASK1 N2 fragment
  • upstream primer 1 5'-CG GGATCC GGATGAGCACGGAGGCGGACGA- 3 '
  • downstream primer 2 5'-GC TCTAGA TCAATCATATTCATAGTCATACTCCAGC- 3'
  • ASK1 N1 ASK1 N2 mixed template overlapping PCR fragments amplified ASK1 N;
  • 3pcDNA TM 5 / FRT mammalian expression vector (Thermo, V601020) to perform a PCR using the following primers linearized, and treated with HindIII (NEB, R0104L) and BamH I and;
  • 3pcDNA TM 5 / FRT mammalian expression vector (Thermo, V601020) to perform a PCR using the following primers linearized, and digested with HindIII and BamH I;
  • the annealing product was ligated with the digested linearized vector to obtain a pcDNA5-Myc vector.
  • the synthesized DNA fragment was digested with NheI (NEB, R0131L) and MfeI (NEB, R0589L) and ligated with pSicoR (Addgene, #67884) plasmid digested with NheI+EcoRI (NEB, R0101L) to obtain psi- Flag carrier.
  • HA-ASK1 N vectors using the upstream primer: 5'-CG GGATCC ATGAGCACGGAGGCGGACGA-3 ', downstream primer: 5'-T GCGGCCGC TCAATCATATTCATAGT CATACTCCAGC- 3', amplification ASK1 (NCBI: BC088829) gene the N-terminal sequence, The amplified product and the pcDNA5-HA vector were digested with restriction endonucleases BamHI and NotI (NEB, R0189L) to obtain a HA-ASK1 N vector.
  • BamHI and NotI NEB, R0189L
  • Myc-ASK1 N vector using the upstream primer: 5'-GA AGATCT ATGAGCACGGAGGCGGACGA-3', downstream primer: 5'-CATG CCATGG TCAATCATATTCATAGTCATACTCCAGC-3', amplifying the N-terminal sequence of ASK1 (NCBI:BC088829) gene, will be expanded
  • the resulting product and the pcDNA5-Myc vector were digested with restriction endonucleases BgIII (NEB, R0144L) and NcoI (NEB, R0193L) to obtain a Myc-ASK1 N vector.
  • amino acid sequence of the polypeptide used is as follows:
  • the 5 psi-Flag vector and the amplified target gene were digested with BamH I+Xba I enzyme, respectively, and ligated to obtain a psi-flag-peptides (Flag-peptides) vector.
  • the ASK1 N mammalian two-hybrid assay vector constructed above was packaged in HEK-293T cells for 72 hours, and the cell culture medium was collected for infection;
  • HEK-293T adherent cells were plated into 6-well plates.
  • the number of cells in the case of lentivirus infection is about 2 ⁇ 10 5 /well;
  • the original medium was replaced with 2 ml of fresh medium containing 6 ⁇ g/ml polybrene, and an appropriate amount of virus suspension was added;
  • the medium containing the virus was replaced with fresh medium, and the plasmid of the polypeptide to be screened was transfected.
  • a 0.5 ⁇ g psi-Flag-Peptides + 0.5 ⁇ g pG5luc plasmid was added to 200 ⁇ l of serum-free DMEM medium, vortexed and mixed;
  • the above transfection solution was added to a 24-well culture dish plated with a stable expression of ASK1 N dimerization mammalian two-hybrid screening system, shaken and mixed, and cultured at 37 ° C for 24 h.
  • 1 lysing cells the target cells cultured for a period of time are exhausted to the cell culture medium, and directly added to the reporter cell lysate; after fully lysing, centrifugation at 10,000-15,000 g for 3-5 min, and the supernatant is taken for measurement;
  • Renilla luciferase assay buffer a Renilla luciferase assay buffer
  • Renilla luciferase assay substrate 100X
  • the measurement interval is set to 2s, and the measurement time is set to 10s;
  • the HA-ASK1 N and Myc-ASK1 N plasmids constructed above were transfected into HEK-293T cells separately or simultaneously.
  • the psi-flag-Peptides plasmid was then transfected into HEK-293T cells containing both HA-ASK1 N and Myc-ASK1 N plasmids, respectively.
  • the cells were added to the lysate and protein samples were quantified using the BCA Protein Assay Kit.
  • the prepared gel plate is placed in the electrophoresis tank, and the electrophoresis liquid and the electrophoresis liquid are added.
  • Each electrophoresis tank needs 200 ml of electrophoresis liquid, and the electrophoresis liquid needs to be filled with 2/3 of the volume of the electrophoresis tank.
  • the protein sample was loaded into the SDS-PAGE gel sample well, and electrophoresis was started after the spotting was completed.
  • the negative (black side) of the transfer tank should be placed with the negative (black side) of the splint and filled with the transfer liquid to submerge the gel.
  • the film transfer tank is connected to the power supply, the voltage is set to 250V, and the current is set to 0.2A. Start electrophoresis transfer, the starting voltage should be greater than 100V, if it is lower than 100V, the current can be adjusted to the required voltage and transferred for 1.5h.
  • the protein film was placed in a pre-prepared TBS, and the transfer liquid on the film was washed away.
  • the protein membrane was placed in a blocking solution and shaken slowly on a shaker at room temperature for 1-4 h.
  • Sealing machine seal the film into the hybrid bag, add a primary antibody, seal, and leave as much air as possible.
  • AAV8 is a liver-targeted gene therapy vector.
  • AAV8 is used to mediate the overexpression of Peptide1 in the liver of cynomolgus monkeys to study the effect of Peptide1 overexpression on nonalcoholic steatohepatitis.
  • T2A-EGFP fragment from plasmid PX458 (Addgene, 48138) using primers PX458-HindIII-F (CCC AAGCTT GGTACCACTAGTGTCGACgaattcGGCAGTGGAGAGGG) and PX458-BgLII-R (GGA AGATCT TTACTTGTACAGCTCGTCCATGCC), ligated to use HindIII and BgLII (NEB) , R0144L)
  • PX458-HindIII-F CCC AAGCTT GGTACCACTAGTGTCGACgaattcGGCAGTGGAGAGGG
  • PX458-BgLII-R GGA AGATCT TTACTTGTACAGCTCGTCCATGCC
  • MCS-Oligo1-SacI CtctagactcgagaccggtCTTAAGGCTAGCGATATCGGAT CCAAGCTTGGTAC
  • MCS-Oligo2-KpnI CAAGCTTGGATCCGATATCGCTAGCCTTAAGACCGGTCTCGAGTCTAGAGCT
  • the pAAV-Peptide1-T2A-EGFP/pAAV-MCS-T2A-EGFP plasmid was diluted to 5 ⁇ 10 1 to 5 ⁇ 10 8 concentration gradients according to 9.1 ⁇ 10 11 1000 bp double-stranded DNA molecules. Into the standard.
  • the HEK-293T (pACT-ASK1 N + pBIND-ASK1 N ) cells stably expressing the ASK1 N dimerization detection plasmid were divided into 5 groups, labeled as A, B, C, D, and E, respectively.
  • the pG5luc plasmid was added without adding the target polypeptide plasmid, and the B, C, D, and E groups were transfected with the psi-flag-Peptide 1, 2, 3, 4 plasmid and the pG5luc plasmid, respectively, to the above HEK-293T cells, ie, 5 groups.
  • the cells were cultured at 37 ° C for 24 h, after which the cells were collected and subjected to Luciferase fluorescence detection.
  • Group A does not add psi-flag-Peptide plasmid, N-terminus of ASK1. Normal dimerization, firefly luciferase RLU value divided by Renilla luciferase RLU value of about 1.9; when psi-flag-Peptide1 plasmid was added (group B), firefly luciferase RLU value divided by Renilla luciferase RLU The value was significantly lower than that of the no-polypeptide group, ie, Peptide1 inhibited the N-terminal dimerization of ASK1; and when psi-flag-Peptide 2/3/4 (groups C, D, and E) were added, the ratio was compared. There was no significant change in group A, ie Peptide 2, 3, 4 did not affect the N-terminal dimerization of ASK1.
  • HEK-293T cells were divided into 7 groups, numbered A, B, C, D, E, F, G.
  • Group A and group B cells were transfected with HA-ASK1 N or Myc-ASK1 N plasmids respectively, C, D, Cells in groups E, F and G were simultaneously transfected with two plasmids, HA-ASK1 N and Myc-ASK1 N , and positive cells were screened 48 hours later.
  • the resulting cells are as follows:
  • Group A HEK-293T cells (HA-ASK1 N )
  • Group B HEK-293T cells (Myc-ASK1 N )
  • Groups C, D, E, F, G HEK-293T cells (HA-ASK1 N + Myc-ASK1 N )
  • the target polypeptide plasmid was transiently transfected, and only the same amount of the transfection solution containing the target polypeptide plasmid was added to the cells of group A, B, and C, and the cells of group D, E, F, and G were respectively added with psi-flag-Peptide1.
  • the transfection solution of the /psi-flag-Peptide2/psi-flag-Peptide3/psi-flag-Peptide4 plasmid, ie, 7 groups were:
  • protein A/G agarose beads are bound to HA-ASK1 N in cell lysate by HA antibody. If the N-terminus of ASK1 is normally dimerized, the protein obtained after immunoprecipitation is loaded. HA-ASK1 N and Myc-ASK1 N were contained in the solution. The results of Western blot analysis showed that there were two bands of HA-ASK1 N and Myc-ASK1 N. If the N-terminal dimerization of ASK1 was inhibited, the content of Myc-ASK1 N was significant. Reduced or not detected.
  • L02 cells were cultured in culture dishes, numbered A, B, C, D, E, cultured at 37 ° C to a cell density of 70%, group A cells transfected with psi-flag plasmid as control, B, C, D
  • the E group was transfected with psi-Flag-Peptides 1, 2, 3, and 4 respectively.
  • a part of the cell culture medium was stimulated with palmitate (PA) for 1 h, and another part of the cell culture medium of each group.
  • PA palmitate
  • BSA was added for 1 h as a control.
  • the cells were collected, and the expression levels of each protein involved in the ASK1-JNK1 signaling pathway were analyzed by Western blot. The results are shown in Fig.
  • Example 4 Inhibition of N-terminal dimerization of ASK1 promotes cell lipid metabolism and inhibits steatohepatitis.
  • L02 cells were cultured in culture dishes, numbered A, B, C, D, E.
  • Group A cells were transfected with psi-flag plasmid as control, and groups B, C, D, and E were transfected with psi-flag-.
  • Oil red O staining was performed, and the content of intracellular triglyceride was measured by Triglyceride assay kit (colorimetric method) (Cayman, 10010303), and the detection value of triglyceride in group A BSA control group was 1 Calculate the relative values of the results of the remaining groups to obtain the relative content of triglycerides in each group.
  • Oil red O staining steps are as follows:
  • a is the oil red O staining diagram. It can be seen that a large number of cells in group A are red, indicating that there is obvious lipid deposition in the cells after 12 hours of PA stimulation; cell staining results in group C, D and E and group A Similarly, in the B group, when Peptide1 was expressed in the cells, after being stimulated by PA for 12 hours, the cells stained with oil red O were less, and the intracellular staining area was smaller.
  • b is the measurement result of intracellular relative triglyceride content. The content of triglyceride in the cells treated with BSA was lower, and there was no significant difference in the triglyceride content of the 5 groups.
  • Example 5 Construction of a liver-exposed Peptide1 overexpressing cynomolgus macaque model mediated by adeno-associated virus (AAV8-Peptide1)
  • the adeno-associated virus vector is injected into the cynomolgus monkey through the portal vein, and Peptide1 is overexpressed in the liver.
  • Crab-equivalent macaque high-fat diet (purchased from Beijing Huakang Biotechnology Co., Ltd., #5043, calorie percentage: protein: 17.86%; carbohydrate: 58.8%; fat: 22.34%) was randomly divided into three groups after feeding for 2 days ( Each group of 6-8 was injected with AAV8-GFP-Peptide1, AAV8-GFP, and physiological saline, respectively.
  • Portal vein injection requires open surgery, fasting for 10h-14h before surgery, and water for 6h.
  • intramuscular injection of atropine 0.05mg/kg to reduce the secretion of intraoperative glands
  • about 10min intramuscular injection of Shumining II 0.1ml / kg.
  • the body weight was weighed, skin was prepared, and the macaques were fixed on the operating table in the supine position.
  • the ECG monitor was connected to monitor blood oxygen saturation, blood pressure and heart rate, and the operation area was disinfected with iodine for 3 times.
  • Surgery and assistants wash hands and disinfect the surgical gown, disinfect the towel for the experimental monkey, and prepare for surgery.
  • the skin was layered along the midline of the abdomen, the abdominal cavity was opened, and the portal vein was slowly injected into the portal vein with a buffer containing 1 ml of AAV8-GFP-Peptide1 virus vector (titer 5.88E+08 Tu/ml, serotype AAV2/8).
  • the hemostasis is pressed with a sterile gauze block, and the suture is prepared after the absence of bleeding.
  • intravenous intravenous infusion 4 million units of penicillin to prevent infection.
  • the abdominal cavity was closed by stratification, and the abdominal band was worn to prevent the incision tension from being excessively enlarged and the intra-abdominal hemorrhage was reduced after surgery (AAV8-GFP and saline groups were injected with the same volume of the same amount of AAV8-GFP virus and the same volume of physiological saline, respectively, and the method was the same as AAV8).
  • - GFP-Peptide1 group is the same).
  • the tissue was removed by needle biopsy and the overexpression of the adeno-associated virus-mediated polypeptide Peptide1 was detected by immunofluorescence and Western blot.
  • Crab-eating macaques are kept for 30 weeks through high-fat diet #5043 and supply 150g of seasonal fruit daily. All animals are free to drink water.
  • All experimental monkeys were tested for physiological indicators every two weeks, including body weight, body temperature, respiration, heart rate, blood pressure, waist circumference, hip circumference, and sitting height. Abdominal ultrasound testing was performed every 4 weeks. At the 0th week and the 30th week, blood was collected from 5ml of separated serum, and the liver lipids (triglyceride, total cholesterol, high-density lipoprotein, low-density lipoprotein) and liver function (ALT, AST) were detected. The tissue removed by needle biopsy was used to detect the overexpression of adeno-associated virus-mediated Peptide1 by GFP fluorescence detection and Western blot.
  • liver tissue Frozen sections of liver tissue were prepared, and the green fluorescence intensity of GFP was observed under a fluorescence microscope to determine the infection efficiency of adeno-associated virus.
  • the expression of Peptide1 in liver tissue was detected by Western blot analysis.
  • the tissue taken out by the living body puncture was embedded in a freezing machine, and after embedding, it was sliced with a cryostat (slice thickness: 5 ⁇ m), and the tissue piece was approached with a slide glass, and attached to the glass slide.
  • the frozen sections were placed under a fluorescence microscope to observe the green fluorescence intensity.
  • liver tissue sample was taken at -80 ° C and placed in dry ice. 3-4 steel balls were placed in each EP tube and pre-cooled in dry ice. Use the ophthalmic scissors to cut the desired sample into the corresponding EP tube, weigh and record the weight of each sample.
  • the sample was ground in a -80 ° C pre-cooled grinder adapter with a grinding parameter set to 30 Hz/s for 90 s.
  • the SDS-PAGE gel was configured and subjected to loading, electrophoresis, membrane transfection, blocking, antibody incubation and protein detection.
  • the primary antibody used in this experiment was anti-Flag (Sigma, #F3165) and the secondary antibody was Biotin Affini Pure Goat Anti-Mouse IgG (H+L) (Abbkine, A21210).
  • Frozen and paraffin sections were prepared and stained with oil red O and HE.
  • the liver tissue specimen taken out by the living body puncture is fixed in the formaldehyde fixing solution, and the fixed part of the liver tissue is taken in the marked embedding frame, and rinsed in a small flow water for more than 30 minutes.
  • 1 dehydration 75% alcohol (45min) ⁇ 75% alcohol (45min) ⁇ 85% alcohol (45min) ⁇ 85% alcohol (45min) ⁇ 95% alcohol (45min) ⁇ 95% Alcohol (45min) ⁇ absolute alcohol (1h) ⁇ absolute alcohol (1h); 2 transparent: xylene (1h) ⁇ xylene (1h); 3 waxing (65°C): paraffin (1h) ⁇ paraffin (1h) ).
  • the embedded frame containing the tissue is loaded into the basket of the machine to start the above procedure.
  • the tissue embedding frame is taken out and sent to the pathological chamber to embed the tissue, and the machine is cleaned for use.
  • liver tissue paraffin sections were placed in a 65 ° C oven (30 min) ⁇ xylene (5 min ⁇ 3 times) ⁇ 100% alcohol (1 min) ⁇ 90% alcohol (1 min) ⁇ 70% alcohol (1 min) ⁇ distilled water wash ⁇ hematoxylin ( 5min) ⁇ wash water to remove the floating color on the slice ⁇ 1% hydrochloric acid alcohol (1 to 3 seconds) ⁇ tap water washing ⁇ Scott blue liquid (sodium bicarbonate 0.35g, magnesium sulfate 2g, distilled water 100ml) (1min) ⁇ The number of tap water washing ⁇ Yihong (1min) ⁇ distilled water to wash the floating color on the slice ⁇ 70% alcohol ⁇ 90% alcohol ⁇ 100% alcohol (30s ⁇ 3 times) ⁇ xylene (2min ⁇ 3 times) ⁇ in When the xylene is not dry, seal the film and take a picture.
  • the frozen liver tissue sections were air-dried in a fume hood for 30 min, and fixed with 4% paraformaldehyde for 10 min. It was washed in double distilled water for 10 min to remove paraformaldehyde on the surface of the tissue.
  • the fluorescence detection results are shown in Fig. 5a.
  • the liver tissues of AAV8-GFP and AAV8-Peptide1 groups showed obvious green fluorescence under fluorescence microscope, and there was no significant difference in fluorescence intensity between the two groups, indicating that AAV8 virus vector was in the liver tissues of two groups of macaques.
  • the transfection rate is the same.
  • the expression of Peptide1 polypeptide by Western blot was shown in Figure 5b.
  • liver histopathological staining are shown in Fig. 8.
  • the liver sections of the AAV8-GFP group after 30 weeks of high-fat diet feeding were stained by HE and vacuolized and fused into flakes, and the morphology of liver cells was destroyed.
  • AAV8- The vacuolation of GFP-Peptide1 group was significantly reduced compared with AAV8-GFP group (Fig. 8).
  • the results of oil red O staining showed that the AAV8-GFP group had a large area of red around the hepatic portal vein, suggesting a large amount of lipid deposition, while AAV8- The red area of the GFP-Peptide1 group was significantly reduced, and the amount of lipid deposition was decreased (Fig. 8).
  • Peptide1 inhibits the development of typhoid fever in cynomolgus monkeys.

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Abstract

提供了一种以凋亡信号调节激酶1N端二聚化为靶点筛选用于治疗脂肪性肝炎药物的方法，一种基于凋亡信号调节激酶1N端二聚化为靶点的预防、缓解和/或治疗脂肪性肝炎药物筛选的细胞模型，凋亡信号调节激酶1在制备抑制肝脏脂肪变性的组合物中的用途，一种预防、缓解和/或治疗脂肪性肝炎的多肽peptide1，以及含有其的组合物。

Description

一种以凋亡信号调节激酶1N端二聚化为靶点筛选用于治疗脂肪性肝炎药物的方法
本申请以申请号为201611054148.9和201611054149.3的申请为优先权，其中涉及的多肽peptide1和201611054149.3的申请中的多肽S1是同一个多肽。
技术领域
本发明属于生物技术领域，具体地说，本发明涉及一种以凋亡信号调节激酶1(ASK1,Apoptosis signal-regulated kinase 1)N端二聚化为靶点的筛选模型的建立，并将筛选得到的ASK1N端二聚化抑制剂应用于制备预防、缓解和/或治疗脂肪性肝炎疾病的药物中。
背景技术
凋亡信号调节激酶1(ASK1,Apoptosis signal-regulated kinase 1)是有丝***原激活的蛋白激酶激酶激酶(MAPKKK)家族成员之一。ASK1蛋白包含1375个氨基酸残基，分子量约为160kDa[1]。ASK1的蛋白结构分为5部分，其中N端的46-277号氨基酸为硫氧还蛋白(Trx)结合区，297-324号氨基酸为N端卷曲螺旋结构域，384-655号氨基酸为TRAF结合区，687-945号氨基酸为ASK1的激酶结构域，而1239-1295号氨基酸则为C端卷曲螺旋结构域[2]。在正常状态下，ASK1通过C端的卷曲螺旋结构域进行同源寡聚反应，形成分子量约为1500-2000kDa的高分子量复合体，同时Trx结合在ASK1的N端的Trx结合区，形成ASK1信号小体。Trx的特异性结合可抑制TRAF2、TRAF6与ASK1结合，从而抑制ASK1的活性。当受到如TNF-α、LPS、内质网压力、氧化压力等刺激时，Trx迅速被激活并与ASK1分离，TRAF2、TRAF6被募集并结合到ASK1的TRAF结合区上，ASK1的激酶结构域被磷酸化，使ASK1激活，诱导SEK1-JNK及MKK3/MKK6-p38信号级联反应[3]。ASK1对于多种疾病均具有重要的调控作用，其可促进AngⅡ诱导的心肌肥厚、心脏重构、间质纤维化以及冠状动脉重构等[4]；可促进脑缺血再灌注后神经元-小神经胶质细胞的死亡，从而恶化脑卒中疾病的发展[5]；在缺血再灌注诱导的急性肾损伤模型中，ASK1被显著激活[6]。基于ASK1的激活机制以及其对多种疾病的调控作用，目前已有多种研究致力于 研发可调控ASK1信号级联反应的药物，以达到治疗疾病的目的。
肝脏是机体的一个主要的调控糖代谢以及脂肪代谢的组织器官，非酒精性脂肪性肝炎(Nonalcoholic steatohepatitis(NASH))是一种以肝细胞脂质聚集、肝脏脂肪变性以及炎症反应为病理表现的疾病，由于其在世界范围内的高发病率，已经成为人类的一个主要的健康问题。其可进一步发展成为肝硬化、肝细胞癌及肝功能衰竭，可损害消化***功能、降低人体免疫力、减弱解毒功能、影响激素代谢等，严重影响了人们的身体健康和生活质量，也给社会带来沉重负担。现阶段，对于其治疗主要集中在对于其并发症，如胰岛素抵抗、高血糖、肥胖等的治疗上，治疗效果非常有限[7-10]。因此，如何有效治疗脂肪性肝炎，如何高效的筛选能有效治疗脂肪性肝炎这一疾病的药物成为现阶段亟待解决的问题。
参考文献
[1]Ichijo H,Nishida E,Irie K,et al.Induction of apoptosis by ASK1,a mammalian MAPKKK that activates SAPK/JNK and p38signaling pathways[J].Science,1997,275(5296):90-94.
[2]Fujino G,Noguchi T,Matsuzawa A,et al.Thioredoxin and TRAF family proteins regulate reactive oxygen species-dependent activation of ASK1through reciprocal modulation of the N-terminal homophilic interaction of ASK1[J].Molecular and cellular biology,2007,27(23):8152-8163.
[3]T.Noguchi,K.Takeda,A.Matsuzawa,K.Saegusa,H.Nakano,J.Gohda,J.Inoue,H.Ichijo,Recruitment of tumor necrosis factor receptorassociated factor family proteins to apoptosis signal-regulating kinase 1signalosome is essential for oxidative stress-induced cell death,J.Biol.Chem.280(2005)37033–37040.
[4]Izumiya Y,Kim S,Izumi Y,et al.Apoptosis signal-regulating kinase 1plays a pivotal role in angiotensin II–induced cardiac hypertrophy and remodeling[J].Circulation Research,2003,93(9):874-883.
[5]Shi Y,Pu H,Hu X,et al.Aberrant Activation of ASK1Mediates Proinflammatory and Neurotoxic Microglial Responses After Cerebral Ischemia/Reperfusion[J].Stroke,2016,47(Suppl 1):A147-A147.
[6]Terada Y,Inoshita S,Kuwana H,et al.Important role of apoptosis signal-regulating kinase 1in ischemic acute kidney injury[J].Biochem Bioph Res CO, 2007,364(4):1043-1049.
[7]Review,T.,et al.World Gastroenterology Organisation global guidelines:Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.J Clin Gastroenterol48,467-473(2014).
[8]Yki-Jarvinen,H.Non-alcoholic fatty liver disease as a cause and a consequence of metabolic syndrome.Lancet Diabetes Endocrinol 2,901-910(2014).
[9]Bhatia,L.S.,Curzen,N.P.,Calder,P.C.&Byrne,C.D.Non-alcoholic fatty liver disease:a new and important cardiovascular risk factor？Eur Heart J 33,1190-1200(2012).
[10]Czech,M.P.Obesity Notches up fatty liver.Nat Med 19,969-971(2013).
发明内容
本发明的目的是克服现有技术的缺陷，提供一种凋亡信号调节激酶1的用途，以凋亡信号调节激酶1N端二聚化为靶点筛选预防、缓解和/或治疗脂肪性肝炎的药物，所述的药物能够抑制凋亡信号调节激酶1N端二聚化。
凋亡信号调节激酶1(ASK1)“N端二聚化”指的是两个ASK1单体通过位于其N端的卷曲螺旋结构域相互结合形成二聚合体。具体的说，正常状态下，ASK1通过其C末端卷曲螺旋结构域相互结合,形成同源二聚体。此时，体内小分子Trx结合在ASK1的N端，阻断ASK1的N端聚合，抑制ASK1激活。而在活性氧等因素刺激下，Trx发生氧化反应，然后从ASK1上解离，引起ASK1N端结合形成二聚合体，导致ASK1激活。
脂肪性肝炎是一种继发于肝脂肪变性和细胞损伤的炎症和纤维化状态的肝脏疾病，按其病因分为非酒精性脂肪性肝炎和酒精性脂肪性肝炎。本发明中的脂肪性肝炎主要指非酒精性脂肪性肝炎，其肝脏组织病理学变化与酒精性脂肪性肝炎相似，但患者无明确饮酒史。非酒精性脂肪性肝炎进一步将发展为肝硬化、肝细胞癌及肝功能衰竭。
在本发明的第二方面，提供一种以凋亡信号调节激酶1N端二聚化为靶点筛选预防、缓解和/或治疗脂肪性肝炎的药物的方法，包括如下步骤：
(a)将含有凋亡信号调节激酶1N端的体系和候选物质进行接触；所述的含有凋亡信号调节激酶1N端的体系是含有凋亡信号调节激酶1N端的细胞，或是含 含有凋亡信号调节激酶1N端的溶液；
(b)观察候选物质对于凋亡信号调节激酶1N端二聚化的影响；
其中，若所述候选物质可抑制凋亡信号调节激酶1N端二聚化，则表明该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
在另一优选例中，包括如下步骤：
步骤(a)包括：在测试组中，将候选物质加入到含有凋亡信号调节激酶1N端的体系中；和/或
步骤(b)包括：检测测试组的体系中凋亡信号调节激酶1N端的二聚化作用，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凋亡信号调节激酶1N端的体系；
其中，如果测试组中检测结果显示凋亡信号调节激酶1N端二聚化受抑制，就表明该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
在另一优选例中，
步骤(a)包括：
(1)选用CheckMateTM哺乳动物双杂交***，分别将编码凋亡信号调节激酶1N端1-678aa的DNA连接至编码DNA结合结构域的pBIND载体以及转录激活结构域的pACT载体中，并将两种载体转染至动物细胞中，构建凋亡信号调节激酶1N端二聚化哺乳动物双杂交筛选***；
其中，编码凋亡信号调节激酶1N端1-678aa的氨基酸序列如SEQ NO：1所示，DNA序列如SEQ NO：2所示；
(2)将候选多肽的核苷酸片段分别连接至psi-Flag载体中，将候选多肽质粒psi-flag-Peptides与pG5luc质粒同时转染上述已构建的用于凋亡信号调节激酶1N端二聚化筛选的动物细胞；
步骤(b)包括：转染成功的细胞在培养24小时后检测萤火虫荧光素酶以及Renilla荧光素酶的RLU，并计算二者比值，根据得到的比值来比较不同样品间多肽抑制作用的程度。
若凋亡信号调节激酶1的N端正常二聚化，则当上述细胞继续转染入pG5luc载体后，萤火虫荧光素酶报告基因高水平表达；若凋亡信号调节激酶1的N端二聚化被抑制，则pG5luc载体上的萤火虫荧光素酶报告基因不表达；通过 Dual-Luciferase双荧光素酶报告基因检测***可分析凋亡信号调节激酶1的N端二聚化情况；
其中，转染是指将具有生物功能的核酸转移或运送到细胞内，并使核酸在细胞内维持其生物功能的过程。在本专利的实施方案中，具有生物功能的核酸指连接有编码凋亡信号调节激酶1N端1-678aa的DNA的pBIND载体、连接有编码凋亡信号调节激酶1N端1-678aa的DNA的pACT载体、pG5luc载体、pcDNA5-HA质粒、pcDNA5-Myc质粒、HA-ASK1N质粒、Myc-ASK1N质粒或连接有候选多肽核苷酸片段的psi-Flag载体。在另一优选例中，上述筛选方法的步骤(a)所用动物细胞选自HEK-293T、L02、Hela、Huh7、Hepg2、A549、3T3、MEFs、H9C2。
HEK-293T，人胚肾细胞，购自中国科学院细胞库，目录号GNHu43。
L02，人肝细胞系，购自中国科学院细胞库，目录号GNHu6。
Hela，人***细胞，购自中国科学院细胞库，目录号TCHu187。角蛋白免疫过氧化物酶染色阳性。已通过支原体检测，STR检测。
Huh7，人肝癌细胞，购自中国科学院细胞库，目录号TCHu182。
Hepg2，人肝癌细胞，购自中国科学院细胞库，目录号TCHu72。
A549，人非小细胞肺癌细胞，购自中国科学院细胞库，目录号TCHu150。
3T3，小鼠胚胎细胞，购自中国科学院细胞库，目录号GNM6。
MEFs，小鼠胚胎成纤维细胞，购自中国科学院细胞库，目录号SCSP-101。
H9C2，大鼠心肌细胞，购自中国科学院细胞库，目录号GNR5。BD1X大鼠胚胎心脏组织的克隆细胞株亚克隆了H9c2(2-1)细胞株。
在另一优选例中，上述筛选方法的步骤(a)所用动物细胞选自HEK-293T。
在本发明的第三方面，提供一种基于凋亡信号调节激酶1N端二聚化为靶点的预防、缓解和/或治疗脂肪性肝炎药物筛选的细胞模型，选用CheckMateTM哺乳动物双杂交***，分别将编码凋亡信号调节激酶1N端1-678aa的DNA(SEQ NO：2)连接至编码DNA结合结构域的pBIND载体以及转录激活结构域的pACT载体中，并将两种载体转染至动物细胞中，构建成预防、缓解和/或治疗脂肪性肝炎药物筛选细胞模型，所用动物细胞选自HEK-293T、L02、Hela、Huh7、Hepg2、A549、3T3、MEFs、H9C2。
在另一优选例中，
步骤(a)包括：
(1)分别将所述的编码凋亡信号调节激酶1N端1-678氨基酸的DNA连接至pcDNA5-HA以及pcDNA5-Myc质粒中，构建HA-ASK1N和Myc-ASK1N质粒，并分别或同时转染动物细胞；
(2)将候选多肽的DNA分别连接至psi-Flag载体中，将psi-flag-Peptides质粒分别转染至同时含有HA-ASK1N和Myc-ASK1N质粒的动物细胞中；
步骤(b)包括：转染成功的细胞在培养24h后通过免疫共沉淀及Western blot实验检测免疫沉淀反应后的蛋白溶液中HA-ASK1N和Myc-ASK1N的含量；
免疫共沉淀是指利用抗原蛋白质和抗体的特异性结合以及细菌蛋白质的“protein A/G”特异性地结合到抗体(免疫球蛋白)的FC片段的现象而开发出来的方法。目前多用protein A/G预先结合在argarose beads上，使之与含有抗原的溶液及抗体反应后，beads上的prorein A/G就能达到吸附抗原的目的。通过低速离心，可以从含有目的抗原的溶液中将目的抗原与其它抗原分离。
在本专利的实施方案中，抗体选用HA抗体(anti-HA)，目的抗原即为HA，含有抗原的溶液指分别转染HA-ASK1N、Myc-ASK1N、HA-ASK1N+Myc-ASK1N、HA-ASK1N+Myc-ASK1N+psi-flag-Peptide1、HA-ASK1N+Myc-ASK1N+psi-flag-Peptide2、HA-ASK1N+Myc-ASK1N+psi-flag-Peptide3或HA-ASK1N+Myc-ASK1N+psi-flag-Peptide4 24h后的HEK-293T细胞裂解液的上清液。anti-HA将目的抗原与protein A/G琼脂糖珠相连。
Western blot采用anti-Myc作为一抗，如果同时转染HA-ASK1N、Myc-ASK1N和某一候选物质的psi-flag-Peptide的动物细胞组与仅转染HA-ASK1N或Myc-ASK1N的对照组相同，未检测到明显Myc蛋白条带，则该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
在另一优选例中，上述筛选方法的步骤(a)所用动物细胞选自HEK-293T、L02、Hela、Huh7、Hepg2、A549、3T3、MEFs、H9C2。
在另一优选例中，上述筛选方法的步骤(a)所用动物细胞选自HEK-293T。
在本发明的第四方面，提供一种基于凋亡信号调节激酶1N端二聚化为靶点 的预防、缓解和/或治疗脂肪性肝炎药物筛选的细胞模型，分别将所述的编码凋亡信号调节激酶1N端1-678氨基酸的DNA(SEQ NO：2)连接至pcDNA5-HA以及pcDNA5-Myc质粒中，构建HA-ASK1N和Myc-ASK1N质粒，并分别或同时转染动物细胞，构建成预防、缓解和/或治疗脂肪性肝炎药物筛选模型，所用动物细胞选自HEK-293T、L02、Hela、Huh7、Hepg2、A549、3T3、MEFs、H9C2。在另一优选例中，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，以选出抑制凋亡信号调节激酶1N端二聚化的物质，用于预防、缓解和/或治疗脂肪性肝炎。
本发明的第五方面，提供一种以凋亡信号调节激酶1N端二聚化为靶点筛选出来的预防、缓解和/或治疗脂肪性肝炎的多肽Peptide1，其氨基酸序列如Seq No.20所示，或其药学上可接受的盐。
本发明的第六方面，提供了一种多核苷酸分子，它编码多肽Peptide1，其核苷酸序列如Seq No.24所示。
本发明的第七方面，提供了一种药物组合物，它含有：
(a)上述多肽Peptide1或其药学上可接受的盐；和
(b)药学上可接受的载体或赋形剂。
本发明利用腺相关病毒AAV8这种肝脏靶向的基因治疗载体，介导筛选到的潜在物质多肽Peptide1在食蟹猕猴肝脏组织中过表达，通过高脂饮食诱导的肥胖模型(diet induced obesity，DIO)研究Peptide1的功能，结果发现与AAV8-GFP对照组食蟹猕猴相比，AAV8-GFP-Peptide1组猕猴体重、BMI指标无显著差异。通过血脂含量测定以及反应肝功能的酶(ALT、AST)活性测定结果表明AAV8-GFP-Peptide1组猕猴血清中甘油三脂和低密度胆固醇含量显著降低，高密度胆固醇含量增加，ALT酶活性显著降低；肝脏组织病理染色结果表明AAV8-GFP-Peptide1组猕猴肝脏组织中脂质蓄积明显减少，肝脏脂肪变性显著减轻。这表明在猕猴中，多肽Peptide1能够抑制脂肪性肝炎的发生。
在本发明的第八方面，提供一种调节肝脏细胞脂肪变性的方法，所述方法包括：调节细胞内凋亡信号调节激酶1N端二聚化。
本发明的主要优点在于：
本发明通过对ASK1在高脂饮食诱导的肝脏脂肪变性模型中起到的明显的 促进作用的机制研究，首次发现ASK1的N端二聚化对于肝脏细胞脂肪变性具有重要的调控作用，可促进肝脏脂肪变性的发展。以ASK1的N端二聚化为靶点，通过检测候选物质是否抑制ASK1的N端二聚化，可以筛选新型预防、缓解和/或治疗脂肪性肝炎的药物。
附图说明
图1是萤火虫荧光素酶与Renilla荧光素酶RLU的比值结果；
图2是转染不同质粒的HEK-293T细胞的裂解液以及裂解液经免疫共沉淀后得到的蛋白溶液的Western blot检测结果；
图3是转染不同多肽质粒的L02细胞经PA刺激1h后细胞内ASK1-JNK1信号通路的Western blot检测结果；
图4是转染不同多肽质粒的L02细胞经PA刺激12h后油红O染色图及细胞内甘油三酯含量定量检测结果，其中a为油红O染色图，b为甘油三酯含量测定结果统计图；
图5是腺相关病毒AAV8介导的食蟹猕猴肝脏中Peptide1过表达效率检测结果
a为生理盐水对照组、AAV8-GFP对照组和AAV8-GFP-Peptide1组食蟹猕猴肝脏组织中病毒转染效率结果；b为AAV8-GFP对照组和AAV8-GFP-Peptide1组在门静脉注射病毒载体后食蟹猕猴肝脏组织Peptide1表达情况检测结果；
图6是AAV8-GFP和AAV8-GFP-Peptide1组食蟹猕猴体重以及BMI指标结果图
a为体重结果图，b为BMI指标结果图(n.s.:p＞0.05vs AAV8-GFP组)；图7是AAV8-GFP和AAV8-GFP-Peptide1组食蟹猕猴血脂含量以及肝功能测定结果图
a为血清甘油三酯含量测定结果图，b为血清总胆固醇含量测定结果图，c为血清高密度胆固醇含量测定结果图，d为血清低密度胆固醇含量测定结果图，e为谷丙转氨酶(ALT)含量测定结果图，f为谷草转氨酶(AST)含量测定结果图(n.s.:p＞0.05vs AAV8-GFP组，**：p＜0.01vs AAV8-GFP组)；
图8是AAV8-GFP和AAV8-GFP-Peptide1组食蟹猕猴肝脏组织HE和油红O染色结果图。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的 实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容。
实验方法
(1)细胞培养
HEK-293T细胞培养于DMEM高糖培养基(含10％FBS，1％青霉素-链霉素)中；L02细胞培养于DMEM高糖培养基(含10％FBS，1％青霉素-链霉素)中，保证培养箱内的空气湿度，5％的CO2含量，37℃培养。
(2)载体构建
1)哺乳动物双杂交检测载体pACT-ASK1N和pBIND-ASK1N的构建
利用CheckMateTM哺乳动物双杂交***(Promega,E2440)，构建ASK1N二聚化检测载体：
①利用上游引物1：5’-CGGGATCCGGATGAGCACGGAGGCGGACGA-3’，下游引物1：5’-TGATGTCATTCTGGTGCTCCTCGCCCTCGC-3’，从ASK1(NCBI：BC088829)基因cDNA中PCR扩增得到ASK1N1片段；
②利用上游引物2：5’-GGAGCACCAGAATGACATCAGGAAAGCTCG-3’，下游引物2：5’-GCTCTAGATCAATCATATTCATAGTCATACTCCAGC-3’，从ASK1(NCBI：BC088829)基因cDNA中PCR扩增得到ASK1N2片段；
③利用上游引物1：5’-CGGGATCCGGATGAGCACGGAGGCGGACGA-3’，下游引物2：5’-GCTCTAGATCAATCATATTCATAGTCATACTCCAGC-3’，从ASK1N1和ASK1N2混合模板中，搭桥PCR扩增得到ASK1N片段；
④pACT载体和pBIND载体分别利用BamH I(NEB，R0136L)+Xba I(NEB，R0145L)酶切后与扩增得到的目标基因连接，得到pACT-ASK1N和pBIND-ASK1N载体；
2)pcDNA5-HA、pcDNA5-Myc、psi-Flag骨架载体的构建
pcDNA5-HA的构建：
①合成含有HA标签序列的两条互补引物：
Figure PCTCN2017081936-appb-000001
②两条引物退火得到带有粘性末端的产物；
③pcDNATM5/FRT哺乳动物表达载体(Thermo,V601020)用下列引物做PCR 线性化，并用HindIII(NEB，R0104L)和BamH I酶切；
Figure PCTCN2017081936-appb-000002
④退火产物与酶切的线性化载体连接得到pcDNA5-HA载体。
pcDNA5-Myc的构建：
①合成含有Myc标签序列的两条互补引物：
Figure PCTCN2017081936-appb-000003
②两条引物退火得到带有粘性末端的产物；
③pcDNATM5/FRT哺乳动物表达载体(Thermo,V601020)用下列引物做PCR线性化，并用HindIII和BamH I酶切；
Figure PCTCN2017081936-appb-000004
④退火产物与酶切的线性化载体连接得到pcDNA5-Myc载体。
psi-Flag的构建:
合成如下序列：
Figure PCTCN2017081936-appb-000005
合成的DNA片段用NheI(NEB，R0131L)和MfeI(NEB，R0589L)双酶切，并与用NheI+EcoRI(NEB，R0101L)双酶切的pSicoR(Addgene,#67884)质粒连接，得到psi-Flag载体。
3)用于免疫共沉淀验证目标多肽对ASK1N端二聚化的抑制作用的HA-ASK1N和Myc-ASK1N载体的构建
HA-ASK1N载体的构建：利用上游引物：5’-CGGGATCCATGAGCACGGAGGCGGACGA-3’，下游引物：5’-TGCGGCCGCTCAATCATATTCATAGT CATACTCCAGC-3’，扩增ASK1(NCBI：BC088829)基因N端序列，将扩增得到的产物以及pcDNA5-HA载体用限制性内切酶BamHI和NotI(NEB，R0189L)酶切连接，得到HA-ASK1N载体。
Myc-ASK1N载体的构建：利用上游引物：5’-GAAGATCTATGAGCACGGAGGCGGACGA-3’，下游引物：5’-CATGCCATGGTCAATCATATTCATAGTCATACTCCAGC-3’，扩增ASK1(NCBI：BC088829)基因N端序列，将扩增得到的产物以及pcDNA5-Myc载体用限制性内切酶BgIII(NEB，R0144L)和NcoI(NEB，R0193L)酶切连接，得到Myc-ASK1N载体。
4)构建候选多肽质粒
所用多肽氨基酸序列如下：
Figure PCTCN2017081936-appb-000006
①利用上游引物：5’-CGGGATCCCTCCATAATGGGAGAAG-3’，下游引物：5’-GCTCTAGAAATGCAATCGATTATC-3’，从cFLIP(NCBI：NM_003879.5)基因cDNA中PCR扩增得到Peptide1片段；
②利用上游引物：5’-CGGGATCCATGTCTGCTGAAGTC-3’，下游引物：5’-GCTCTAGATCACAGTTCAGCCAAGTC-3’，从cFLIP(NCBI：NM_003879.5)基因cDNA中PCR扩增得到Peptide2片段；
③利用上游引物：5’-CGGGATCCATGGTGAGCAAGGGCG-3’，下游引物：5’-GCTCTAGATCACCAGGGCACGGGCAG-3’，从GFP(NCBI：KX130867.1)基因DNA中PCR扩增得到Peptide3片段；
④利用上游引物：5’-CGGGATCCGGCGTGCAGTGCTTC-3’，下游引物：5’-GCTCTAGATCACTCGATGCGGTTCAC-3’，从GFP(NCBI：KX130867.1)基 因DNA中PCR扩增得到Peptide4片段；
⑤psi-Flag载体与扩增得到的目标基因分别利用BamH I+Xba I酶酶切后连接，得到psi-flag-peptides(Flag-peptides)载体。
(3)构建ASK1N二聚化哺乳动物双杂交筛选***
①将上述构建得到的ASK1N哺乳动物双杂交检测载体在HEK-293T细胞中包装病毒72h，收集细胞培养液，用于感染；
②慢病毒感染前18-24h，将HEK-293T贴壁细胞铺到6孔板中。使细胞在慢病毒感染时的数量为2×105/孔左右；
③第二天，用含有6μg/ml polybrene的2ml新鲜培养基替换原培养基，加入适量病毒悬液；
④继续培养48h后，用新鲜培养基替换含有病毒的培养基，转染待筛选多肽质粒。
(4)目标多肽质粒瞬时转染
①按照以下步骤配置转染溶液：
a取0.5μg psi-Flag-Peptides+0.5μg pG5luc质粒加至200μl无血清的DMEM培养基中，涡旋震荡混匀；
b轻微涡旋震荡混匀TurboFect转染试剂，加入2μl至a的培养基中，立即吹打混匀，室温静置20min；
②将上述转染溶液加入至铺有稳定表达ASK1N二聚化哺乳动物双杂交筛选***的24孔培养皿中，摇晃混匀，37℃培养24h。
(5)Luciferase检测荧光
①裂解细胞：将培养了一段时间后的目标细胞吸尽细胞培养液，直接加入报告基因细胞裂解液；充***解后，10,000-15,000g离心3-5min，取上清液用于测定；
②溶解萤火虫荧光素酶检测试剂和Renilla荧光素酶检测缓冲液，并达到室温，将Renilla荧光素酶检测底物(100X)置于冰浴或冰盒上备用；
③按照每个样品需100μl的量，取适量Renilla荧光素酶检测缓冲液，按照1:100加入Renilla荧光素酶检测底物(100X)配制成Renilla荧光素酶检测工作液；
④开启荧光测定仪(Promega，
Figure PCTCN2017081936-appb-000007
20/20Luminometer)，将测定间 隔设为2s，测定时间设为10s；
⑤每个样品测定时，取样品20-100μl，加入100μl萤火虫荧光素酶检测试剂，用枪打匀混匀后测定RLU(relative light unit)，以报告基因细胞裂解液为空白对照；
⑥完成测定萤火虫荧光素酶步骤后，加入100μl Renilla荧光素酶检测工作液，用枪打匀混匀后测定RLU(relative light unit)；
⑦在以Renilla荧光素酶为内参的情况下，用萤火虫荧光素酶测定得到的RLU值除以Renilla荧光素酶测定得到的RLU值。根据得到的比值来比较不同样品间多肽抑制作用的程度。
(6)免疫共沉淀
①将上述构建的HA-ASK1N和Myc-ASK1N质粒，分别或同时转染HEK-293T细胞。之后将psi-flag-Peptides质粒分别转染至同时含有HA-ASK1N和Myc-ASK1N质粒的HEK-293T细胞中。
②转染24h后收获细胞，加入适量细胞裂解缓冲液(含蛋白酶抑制剂)，冰上裂解30min，细胞裂解液于4℃，用最大转速离心30min后取上清液；
③取少量裂解液以备Western blot分析，剩余裂解液加1μg anti-HA抗体(Sigma，#H6908)，4℃缓慢摇晃孵育过夜；
④取10μl protein A/G琼脂糖珠(11719394001and 11719386001,Roche)，用适量裂解缓冲液洗3次，每次3,000rpm离心3min；
⑤将预处理过的10μl protein A/G琼脂糖珠加入到和抗体孵育过夜的细胞裂解液中4℃缓慢摇晃孵育2-4h，使抗体与protein A/G琼脂糖珠偶连；
⑥免疫沉淀反应后，在4℃以3,000rpm速度离心3min，将琼脂糖珠离心至管底；将上清小心吸去，琼脂糖珠用1ml裂解缓冲液洗3-4次；最后加入15μl的2×SDS上样缓冲液，沸水煮5min；
⑦SDS-PAGE,Western blot分析。
(7)Western blot
1)蛋白质提取
细胞加入裂解液，运用BCA Protein Assay Kit定量收集蛋白样品。
2)上样与电泳
①将配制好的凝胶板架在电泳槽中，加入电泳内液和电泳外液，每个电泳槽需200ml电泳内液，电泳外液需要灌满电泳槽体积的2/3。
②把蛋白样品上样到SDS-PAGE胶加样孔内，点样完成后开始电泳。
3)转膜
①按要求配制转膜液于4℃预冷。
②按8cm×5.9cm裁剪好PVDF膜并在一角剪个缺口作为膜的左上角，使用前在甲醇中浸泡15s后放入转膜液中备用。
③将夹板左右摊开，负极向右。黑色的一边为负极，白色为正极，两边各铺上2张海绵和5张滤纸(海绵和滤纸预先用转移缓冲液浸湿)。
④取出凝胶板中的凝胶，去除多余部分，用转膜液洗涤凝胶，将凝胶平铺在负极的滤纸上，赶走气泡，将PVDF膜覆盖其上，剪缺口的地方应与大Marker的一角对齐，赶走气泡后覆盖上左边的滤纸及海绵(不能有气泡)，夹上夹板。
⑤将夹板放入转膜槽中，转膜槽的负极(黑色面)应与夹板的负极(黑色面)放在一起，灌满转膜液以淹没凝胶。
⑥转膜槽接通电源，电压设为250V，电流设为0.2A。开始电泳转移，起始电压应大于100V，如果低于100V，可适当调高电流至所需电压，转移1.5h。
⑦转移结束后，取出PVDF膜。
4)封闭
把蛋白膜放置到预先准备好的TBS中，洗去膜上的转膜液。蛋白膜放入封闭液中，在摇床上缓慢摇动，室温封闭1-4h。
5)一抗孵育
①用TBST洗涤蛋白膜3次，每次5min。
②封口机将薄膜封入杂交袋中，加上一抗，封口，尽可能不留空气。
③将杂交袋放入4℃摇床中，过夜。
6)二抗孵育
①将薄膜取出用TBST洗涤3次，每次5min，回收一抗。
②将膜放入对应的加有二抗的二抗稀释液中，避光孵育1h。
7)蛋白检测
孵育后用TBST洗涤3次，每次5min。利用Bio-Rad Chemi Doc XRS+凝胶 成像***检测目的条带。
(8)构建多肽Peptide1过表达腺相关病毒载体***(AAV8-GFP-peptide1/AAV8-GFP)
AAV8为一种肝脏靶向的基因治疗载体，本发明中使用AAV8介导Peptide1在食蟹猴肝脏中的过表达，以研究Peptide1过表达对非酒精性脂肪性肝炎的影响。
(1)用引物PX458-HindIII-F(CCCAAGCTTGGTACCACTAGTGTCGACgaattcGGCAGTGGAGAGGG)和PX458-BgLII-R(GGAAGATCTTTACTTGTACAGCTCGTCCATGCC)从质粒PX458(Addgene，48138)中扩增T2A-EGFP片段，连接至使用HindIII和BgLII(NEB，R0144L)双酶切的AAV载体pAAV-MCS中，构建pAAV-T2A-EGFP载体。为了便于以后使用，再合成MCS-Oligo1-SacI(CtctagactcgagaccggtCTTAAGGCTAGCGATATCGGAT CCAAGCTTGGTAC)和MCS-Oligo2-KpnI(CAAGCTTGGATCCGATATCGCTAGCCTTAAGACCGGTCTCGAGTCTAGAGAGCT)，通过变性，融合之后连入经SacI(NEB，R0156L)和KpnI(NEB，R0142L)酶切的上述pAAV-T2A-EGFP载体中，构建pAAV-MCS-T2A-EGFP载体。
(2)利用上游引物：5’-GCTCTAGAgccaccATGCTCCATAATGGGAGAAG-3’，下游引物：5’-CGGGATCCCTTGTCATCGTCGTCCTTGTAATCAATGCAATCGATTATC-3’，从cFLIP(NCBI：NM_003879.5)基因cDNA中PCR扩增得到Peptide1的编码序列；将Peptide1的编码序列用XbaI和BamHI双酶切，然后克隆到经同样酶切的pAAV-MCS-T2A-EGFP载体中，得到pAAV-Peptide1-T2A-EGFP载体，此载体中Peptide1片段与EGFP在同一读码框内，在表达Peptide1肽段的同时表达EGFP，便于切片观察。
(3)将三质粒转染***(pAAV-Peptide1-T2A-EGFP，pAAV-Helper和pAAV-2/8)用PEI(Polysciences cat#24765)共转染至AAV293细胞中，转染72h后收集细胞，超声裂解后去除细胞碎片(AAV8-GFP对照组三质粒转染***为：pAAV-MCS-T2A-EGFP，pAAV-Helper和pAAV-2/8)。
(4)用氯化铯梯度离心的方法纯化病毒液，用1×PBS+5％Sorbitol-in Slide-A-Lyzer dialysis cassettes透析纯化的病毒液，去除氯化铯，获得腺相关病毒 AAV8-GFP-Peptide1和AAV8-GFP。
(5)荧光定量PCR检测病毒滴度，具体步骤如下：
a.取10μl纯化后的病毒液，加入100μl DNase裂解液(10mM Tris·Cl,pH7.5、10mM MgCl2、2mM CaCl2、50U/ml DNase I)，37℃孵育1h。
b.加入0.5M的EDTA，混合均匀后70℃孵育10min，加入终浓度为50μg/ml的蛋白酶K，50℃过夜孵育。
c.在99℃下孵育10min，彻底灭活蛋白酶K。加入灭菌双蒸水至1ml，取出2μL补水至400μL用作荧光定量PCR的模板。
d.按照9.1×1011个1000bp的双链DNA分子计算，将pAAV-Peptide1-T2A-EGFP/pAAV-MCS-T2A-EGFP质粒稀释至5×101至5×108八个浓度梯度，制成标准品。
e.分别取2μL样品和标准品，用GFP特异性的引物Forward5’-AGCAGCACGACTTCTTCAAGTCC；和Reverse 5’-TGTAGTTGTACTCCA GCTTGTGC做荧光定量PCR。根据标准品指定标准曲线，计算出病毒中的载体拷贝数(病毒滴度)。
【实施例1】筛选可抑制ASK1的N端二聚化的多肽
稳定表达ASK1N二聚化检测质粒的HEK-293T(pACT-ASK1N+pBIND-ASK1N)细胞分为5组，分别标记为A、B、C、D、E，其中A组在目标多肽瞬时转染时只添加pG5luc质粒而不添加目标多肽质粒，B、C、D、E组分别用psi-flag-Peptide1、2、3、4质粒与pG5luc质粒转染上述HEK-293T细胞，即5组分别为：
A:HEK-293T细胞(pACT-ASK1N+pBIND-ASK1N)+pG5luc
B:HEK-293T细胞(pACT-ASK1N+pBIND-ASK1N)+psi-flag-Peptide1+pG5luc
C:HEK-293T细胞(pACT-ASK1N+pBIND-ASK1N)+psi-flag-Peptide2+pG5luc
D:HEK-293T细胞(pACT-ASK1N+pBIND-ASK1N)+psi-flag-Peptide3+pG5luc
E:HEK-293T细胞(pACT-ASK1N+pBIND-ASK1N)+psi-flag-Peptide4+pG5luc
添加转染溶液后，细胞于37℃培养24h，之后收集细胞，进行Luciferase荧光检测。
荧光检测结果如图1所示，A组不添加psi-flag-Peptide质粒，ASK1的N端 正常二聚化，萤火虫荧光素酶RLU值除以Renilla荧光素酶RLU值约为1.9；当添加psi-flag-Peptide1质粒时(B组)，萤火虫荧光素酶RLU值除以Renilla荧光素酶RLU值相比于无多肽组显著降低，即Peptide1抑制了ASK1的N端二聚化；而当分别添加psi-flag-Peptide 2/3/4(C、D、E组)时，比值结果相比于A组无显著变化，即Peptide2、3、4不影响ASK1的N端二聚化。
【实施例2】免疫共沉淀验证多肽对ASK1的N端二聚化的抑制作用
HEK-293T细胞分为7组，编号为A、B、C、D、E、F、G，A组以及B组细胞分别只转染HA-ASK1N或Myc-ASK1N质粒，C、D、E、F、G组细胞同时转染HA-ASK1N和Myc-ASK1N两种质粒，48小时后筛选阳性细胞。所得细胞如下：
A组：HEK-293T细胞(HA-ASK1N)
B组：HEK-293T细胞(Myc-ASK1N)
C、D、E、F、G组：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)
之后进行目标多肽质粒瞬时转染，A、B、C三组细胞中只加入同等量的不含目标多肽质粒的转染溶液，D、E、F、G组细胞分别加入含psi-flag-Peptide1/psi-flag-Peptide2/psi-flag-Peptide3/psi-flag-Peptide4质粒的转染溶液，即7组分别为：
A：HEK-293T细胞(HA-ASK1N)
B：HEK-293T细胞(Myc-ASK1N)
C：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)
D：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)+psi-flag-Peptide1
E：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)+psi-flag-Peptide2
F：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)+psi-flag-Peptide3
G：HEK-293T细胞(HA-ASK1N+Myc-ASK1N)+psi-flag-Peptide4
转染24h后收获细胞，进行免疫共沉淀分析，验证目标多肽对ASK1的N端二聚化的影响。Western blot分析分析中所用一抗信息：HA(Sigam，#H6908)，Myc(MBL，#M192-3)，Flag(Sigma，#F3165)，所需二抗信息：HRP AffiniPure Goat Anti-Rabbit IgG(H+L)(Jackson，#111-035-003)，Biotin AffiniPure Goat Anti-Mouse IgG(H+L)(Abbkine，A21210)。
在免疫共沉淀过程中，protein A/G琼脂糖珠通过HA抗体与细胞裂解液中的HA-ASK1N相结合，若ASK1的N端正常二聚化，则免疫沉淀反应后得到的上样蛋白溶液中含有HA-ASK1N以及Myc-ASK1N，Western blot分析结果可见HA-ASK1N和Myc-ASK1N两条条带；若ASK1的N端二聚化被抑制，则Myc-ASK1N含量显著降低或检测不到。
Western blotting分析结果如图2所示，细胞裂解液的Western blotting分析显示在B-G组细胞中，Myc-ASK1N均正常表达，且表达量一致；在D-G组细胞中，不同多肽表达含量一致。免疫共沉淀后Western blotting结果表明A组可见清晰HA-ASK1N条带；B组无明显条带；C组可清晰检测到两条条带；E、F、G组的检测结果与C组相同，即ASK1的N端二聚化不被抑制；而在D组中，当含有Peptide1时，Myc-ASK1N的检测条带明显变弱，HA-ASK1N条带强弱不变，这一结果进一步验证了Peptide1可抑制ASK1的N端二聚化。
【实施例3】抑制ASK1的N端二聚化可抑制ASK1-JNK1信号通路
通过Western blot分析来检测ASK1的N端二聚化被抑制后对细胞内JNK1信号通路的影响。所需一抗信息：p-ASK1(Cell Signaling Technology，#3765)，ASK1(GeneTex，#GTX107921)，p-MKK7(Aviva Systems Biology，#OAAF05547)，MKK7(Cell Signaling Technology，#4172)，p-JNK1(NOVUS，#NB100-82009)，JNK1(Abcam，#ab199380)，Flag(Sigma，#F3165)；所需二抗信息：HRP AffiniPure Goat Anti-Rabbit IgG(H+L)(Jackson,#111-035-003),Biotin AffiniPure Goat Anti-Mouse IgG(H+L)(Abbkine,A21210)。
L02细胞分5组于培养皿中培养，编号为A、B、C、D、E，37℃培养至细胞密度为70％，A组细胞转染psi-flag质粒作为对照，B、C、D、E组分别转染psi-Flag-Peptides1、2、3、4，转染24h后，各组的一部分细胞培养基中加棕榈酸酯(PA)刺激1h，同时各组的另一部分细胞培养基中加入BSA处理1h作为对照。收集细胞，Western blot分析细胞内ASK1-JNK1信号通路中涉及的各蛋白表达含量变化。结果如图3所示，其中Flag蛋白表达含量在各组中无明显变化，说明B、C、D、E 4组在多肽质粒转染后多肽表达量基本一致。与其他组相比，仅B组p-ASK1、p-MKK7、p-JNK1蛋白含量相比于其BSA对照组未见升高，即ASK1的N端二聚化被抑制后，会抑制MKK7、JNK1的磷酸化，抑制 ASK1-JNK1信号通路。
【实施例4】抑制ASK1的N端二聚化可促进细胞脂质代谢，抑制脂肪性肝炎。
L02细胞分5组于培养皿中培养，编号为A、B、C、D、E，A组细胞转染psi-flag质粒作为对照，B、C、D、E组分别转染psi-flag-Peptides1、2、3、4。转染24h后，各组一部分细胞的培养基中加入PA处理12h，同时另一部分用BSA处理12h作为对照(Ctl)。之后进行油红O染色，并用甘油三酯(Triglyceride)分析试剂盒(比色法)(Cayman，10010303)检测细胞内甘油三酯的含量，以A组BSA对照组细胞甘油三酯检测值为1，计算其余各组检测结果的相对值，得到各组细胞甘油三酯相对含量。油红O染色步骤如下：
(1)样品组和对照组用1×PBS洗涤2次，加入300μl 3％多聚甲醛固定20min；
(2)加入1×PBS洗涤2次后，加入60％异丙醇漂洗10s；
(3)加入1×PBS洗涤2次，通风橱吹干；
(4)每孔500μl加入油红O染色1h；
(5)加入1x PBS洗涤2次，60％异丙醇进行分选，再加入1x PBS洗2次；镜检，拍照；
结果如图4所示，a为油红O染色图，可见A组大量细胞呈红色，表明PA刺激12h后，细胞内出现明显的脂质沉积；C、D、E组细胞染色结果与A组相似，均呈现大面积红色；而在B组中，当细胞内表达Peptide1时，经PA刺激12h后，被油红O染红的细胞较少，且细胞内着色面积较小。b为细胞内相对甘油三酯含量测定结果图，经BSA处理的对照组细胞内甘油三酯含量均较低，且5组细胞甘油三酯含量无显著性差异，而经PA处理12h后，相比于各组的对照组，细胞内甘油三酯含量显著增加，但转染Flag-Peptide1的B组细胞内甘油三酯含量的增加程度显著低于其他组。上述结果表明Peptide1可通过抑制ASK1的N端二聚化抑制肝细胞脂肪变性，ASK1的N端二聚化对筛选治疗脂肪性肝炎的药物具有重要意义，其可作为一个新的治疗靶点，应用于脂肪性肝炎疾病的治疗中。
【实施例5】腺相关病毒(AAV8-Peptide1)介导的肝脏Peptide1过表达食蟹猕猴模型的构建
腺相关病毒载体通过门静脉注射至食蟹猕猴体内，在肝脏中过表达Peptide1。
食蟹猕猴高脂饲料(购自北京华阜康生物科技有限公司，#5043，热量百分比：蛋白质:17.86％；碳水化合物:58.8％；脂肪:22.34％)喂养2d后，随机分为三组(每组6-8只，分别注射AAV8-GFP-Peptide1、AAV8-GFP、生理盐水)。门静脉注射需要开腹手术，术前禁食10h-14h，禁水6h。术前先肌肉注射阿托品0.05mg/kg(减少术中腺体的分泌)，约10min后肌肉注射舒眠宁Ⅱ0.1ml/kg。待实验猴麻醉后称量体重，备皮，将猕猴以仰卧位固定于手术台上，连接心电监护仪，监测血氧饱和度、血压、心率，用碘酒消毒手术区域3遍。手术及助手洗手消毒穿手术衣，给实验猴消毒铺巾，准备手术。沿腹中线分层划开皮肤，打开腹腔，找到门静脉后缓慢将含有AAV8-GFP-Peptide1病毒载体(滴度5.88E+08Tu/ml，血清型AAV2/8)1ml的缓冲液注射入门静脉内，注射完成后用无菌小纱布块按压止血，检查无出血情况后准备缝合。同时静脉匀速滴注青霉素400万单位，预防感染。分层关闭腹腔，穿好腹带防止手术后切口张力过大和减少腹腔内出血(AAV8-GFP、生理盐水组分别注射同等体积的含同等量AAV8-GFP病毒以及同等体积的生理盐水，其方法与AAV8-GFP-Peptide1组相同)。高脂饲养20周后，穿刺活检术取出组织通过免疫荧光以及Western blot检测腺相关病毒介导的多肽Peptide1的过表达。
食蟹猕猴通过高脂饲料#5043饲养30周，并每天供应150g时令水果。所有动物均自由饮水。
【实施例6】食蟹猕猴肝脏过表达Peptide1抑制高脂饲养引起的脂肪性肝炎发生
所有实验猴每两周检测生理指标，包括体重，体温，呼吸，心率，血压，腰围，臀围，坐高等指标。每4周进行腹部超声检测。第0周及30周时采集血液5ml分离血清后进行空腹肝脏脂质(甘油三酯、总胆固醇、高密度脂蛋白、低密度脂蛋白)及肝功能(ALT、AST)检测。通过穿刺活检术取出组织通过GFP荧光检测以及Western blot检测腺相关病毒介导的Peptide1的过表达。
1、荧光检测以及肝脏组织Western blot分析
制作肝脏组织冰冻切片，在荧光显微镜下观察GFP的绿色荧光强度，以确定腺相关病毒的感染效率；肝脏组织Western blot分析检测肝脏组织中Peptide1的表达情况。
(1)肝脏组织冰冻切片
将活体穿刺取出的组织置于冰冻机中包埋，包埋完成后使用冰冻切片机切片(切片厚度5μm)，用载玻片接近组织片，将其粘贴于载玻片上。冰冻切片置于荧光显微镜下观察绿色荧光强度。
(2)Western blot检测Peptide1表达
1)蛋白质提取
①于-80℃取出肝脏组织样本，放入干冰中。每个EP管放入3-4颗钢珠，放入干冰中预冷。用眼科剪剪下所需样本放入对应的EP管，称重并记录每个样本的重量。
②裂解液中加入PMSF，混匀，加入相应量的裂解液到样品中，迅速摇匀。
③于-80℃预冷研磨仪适配器中研磨样品，研磨参数设置为30Hz/s，90s。
④研磨结束后，冰上放置10min，取出钢珠。
⑤超声裂解仪裂解样本5KHz/次，每次1s，间隔1s，重复10次。超声完成后冰上放置10min。
⑥样本放入4℃预冷的离心机中离心，4℃，12000rpm/min离心30min。
⑦吸取上清转移到新的EP管中，4℃，14000rpm/min离心10min。
⑧吸取上清转移到新的EP管中继续离心，4℃，14000rpm/min离心5min。准确吸取清液并利用BCA Protein Assay Kit(PierecTM，23225)进行蛋白定量。
2)根据前文所述步骤，配置SDS-PAGE凝胶并进行上样、电泳、转膜、封闭、抗体孵育及蛋白检测。本次实验中所用一抗为anti-Flag(Sigma，#F3165)，二抗为Biotin AffiniPure Goat Anti-Mouse IgG(H+L)(Abbkine，A21210)。
2、生理生化指标检测
(1)生理指标检测
禁食10-14h，禁水6h，盐酸***10mg/kg肌肉注射麻醉，检测以上生理指标并记录相关实验数据。
(2)生化指标检测
实验前禁食10-14h，禁水6h，盐酸***10mg/kg肌肉注射麻醉，静脉采血后收集血液5ml，分离血清后送公司(武汉迪安)检测脂质及肝功能情况。
3、肝脏组织病理染色相关实验
制作冰冻、石蜡切片，并进行油红O和HE染色。
(1)肝脏脱水，透明，浸蜡
活体穿刺取出的肝脏组织标本置于甲醛固定液中固定，取固定好的部分肝脏组织于标记的包埋框内，在小流量流水冲洗30min以上。按照以下流程在机器上设置以下程序，①脱水：75％酒精(45min)→75％酒精(45min)→85％酒精(45min)→85％酒精(45min)→95％酒精(45min)→95％酒精(45min)→无水酒精(1h)→无水酒精(1h)；②透明：二甲苯(1h)→二甲苯(1h)；③浸蜡(65℃)：石蜡(1h)→石蜡(1h)。待组织冲洗完毕后，将包含组织的包埋框装进机器篮筐内，启动上述程序。上述程序完成后，取出组织包埋框送病理室包埋组织，同时清洗机器备用。
(2)肝脏组织切片
使用切片机切片(切片厚度5μm)。
(3)肝脏组织苏木精-伊红(HE)染色
将肝脏组织石蜡切片放入65℃烘箱(30min)→二甲苯中(5min×3次)→100％酒精(1min)→90％酒精(1min)→70％酒精(1min)→蒸馏水洗→苏木素(5min)→自来水洗去切片上的浮色→1％盐酸酒精(1至3秒)→自来水洗数下→Scott促蓝液(碳酸氢钠0.35g，硫酸镁2g，蒸馏水100ml)(1min)→自来水洗数下→伊红(1min)→蒸馏水洗去切片上的浮色→70％酒精一下→90％酒精一下→100％酒精(30s×3次)→二甲苯(2min×3次)→在二甲苯未干时封片，拍照。
(4)肝脏组织油红O染色
①将冰冻肝脏组织切片在通风橱中风干30min，4％多聚甲醛固定10min。置于双蒸水中稍洗10min，以除去组织表面的多聚甲醛。
②以60％异丙醇处理1min。
③用油红O(公司sigma，货号O0625，浓度0.5g/100ml 100％异丙醇)染色30min。
④之后以60％异丙醇漂洗1分钟×3次，直至背景干净。
⑤用Mayer’s苏木素染液(5滴)淡染细胞核。
⑥水漂洗，稀碳酸锂水溶液中促蓝，充分水洗，水洗至细胞核蓝化。
⑦用甘油明胶封片，拍照。
4、检测结果
荧光检测结果如图5a，AAV8-GFP以及AAV8-Peptide1组猕猴肝脏组织在荧光显微镜下均可见明显绿色荧光，且两组之间荧光强度无显著差异，说明AAV8病毒载体在两组猕猴肝脏组织中转染率相同。肝脏组织Western blot检测Peptide1多肽表达结果如图5b所示，可见在AAV8-GFP中未见Western blot条带，即未检测到Peptide1表达，而在AAV8-Peptide1组中可见明显条带，即AAV8-Peptide1门静脉注射组猕猴肝脏中Peptide1多肽表达显著。
猕猴体重及BMI测定结果如图6所示。AAV8-GFP以及AAV8-GFP-Peptide1组食蟹猕猴从实验开始直至高脂饲养30周，两组动物体重均无显著差异(图6a)，且其BMI结果也无明显差异(图6b)。
血脂以及ASL、AST检测结果如图7所示。在高脂饲料饲养30周后，AAV8-GFP-Peptide1组猕猴血清甘油三酯含量显著低于AAV8-GFP对照组(图7a)；两组动物血清总胆固醇含量无明显差异(图7b)，但AAV8-GFP-Peptide1组猕猴血清高密度胆固醇含量高于AAV8-GFP组(图7c)，低密度胆固醇含量低于AAV8-GFP组(图7d)。两组猕猴AST酶含量无显著差异但AAV8-GFP-Peptide1组ALT酶含量显著低于AAV8-GFP组(图7e、f)。这些结果表明，多肽Peptide1抑制了高脂饮食引起的肝功能的恶化以及脂肪性肝炎的发生。
肝脏组织病理染色结果如图8所示，高脂饲料饲养30周后的AAV8-GFP组猕猴肝脏切片经HE染色后可见明显空泡化且融合连成片状，肝脏细胞形态被破坏，AAV8-GFP-Peptide1组空泡化较AAV8-GFP组明显减轻(图8上)；油红O染色结果可见AAV8-GFP组猕猴的肝门静脉周围呈大面积红色，提示有大量脂质沉积，而AAV8-GFP-Peptide1组红色面积显著减少，脂质沉积量降低(图8下)。病理染色结果表明Peptide1过表达显著减轻了食蟹猕猴由高脂饮食导致的肝脏脂肪变性以及肝脏脂质沉积。Peptide1可抑制食蟹猕猴脂肪性肝炎疾病的发生。
上述结果显示，Peptide1的过表达可显著减轻HFD诱导下发生的脂肪性肝炎病变。Peptide1对改善脂肪性肝炎具有显著的作用。Peptide1可有望成为一种治疗脂肪性肝炎的新型药物。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替 代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (19)

  1. 一种凋亡信号调节激酶1的用途，其特征在于，以凋亡信号调节激酶1N端二聚化为靶点筛选预防、缓解和/或治疗脂肪性肝炎的药物。
  2. 如权利要求1所述的用途，其特征在于，所述的药物能够抑制凋亡信号调节激酶1N端二聚化。
  3. 一种以凋亡信号调节激酶1N端二聚化为靶点筛选预防、缓解和/或治疗脂肪性肝炎的药物的方法，其特征在于，所述方法包括步骤：
    (a)将含有凋亡信号调节激酶1N端的体系和候选物质进行接触；所述的含有凋亡信号调节激酶1N端的体系是含有凋亡信号调节激酶1N端的细胞，或是含含有凋亡信号调节激酶1N端的溶液；
    (b)观察候选物质对于凋亡信号调节激酶1N端二聚化的影响；
    其中，若所述候选物质可抑制凋亡信号调节激酶1N端二聚化，则表明该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
  4. 如权利要求3所述的方法，其特征在于，
    步骤(a)包括：在测试组中，将候选物质加入到含有凋亡信号调节激酶1N端的体系中；和/或
    步骤(b)包括：检测测试组的体系中凋亡信号调节激酶1N端的二聚化作用，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凋亡信号调节激酶1N端的体系；
    其中，如果测试组中检测结果显示凋亡信号调节激酶1N端二聚化受抑制，就表明该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
  5. 如权利要求3所述的方法，其特征在于，
    步骤(a)包括：
    (1)构建含有凋亡信号调节激酶1N端的细胞：选用CheckMateTM哺乳动物双杂交***，分别将编码凋亡信号调节激酶1N端1-678号氨基酸的DNA连接至编码DNA结合结构域的pBIND载体以及转录激活结构域的pACT载体中，并将两种载体转染至动物细胞中,；其中，编码凋亡信号调节激酶1N端1-678号的氨基酸序列如SEQ NO：1所示，核苷酸序列如SEQ NO：2所示；
    (2)将候选短肽的核苷酸片段分别连接至psi-Flag载体中，将候选短肽质粒psi-flag-Peptides与pG5luc质粒同时转染上述已构建的用于凋亡信号调节激酶 1N端二聚化筛选的动物细胞；
    步骤(b)包括：转染成功的细胞在培养24小时后检测萤火虫荧光素酶以及Renilla荧光素酶的RLU，并计算二者比值，根据得到的比值来比较不同样品间短肽抑制作用的程度；若凋亡信号调节激酶1的N端正常二聚化，则当上述细胞继续转染入pG5luc载体后，萤火虫荧光素酶报告基因高水平表达；若凋亡信号调节激酶1的N端二聚化被抑制，则pG5luc载体上的萤火虫荧光素酶报告基因不表达。
  6. 如权利要求5所述的方法，其特征在于，步骤(a)所用动物细胞选自HEK-293T、L02、Hela、Huh7、Hepg2、A549、3T3、MEFs、H9C2。
  7. 如权利要求6所述的方法，其特征在于，步骤(a)所用动物细胞选自HEK-293T。
  8. 一种基于凋亡信号调节激酶1N端二聚化为靶点的预防、缓解和/或治疗脂肪性肝炎药物筛选的细胞模型，其特征在于，选用CheckMateTM哺乳动物双杂交***，分别将编码凋亡信号调节激酶1N端1-678aa的DNA，其核苷酸序列如SEQ NO：2所示，连接至编码DNA结合结构域的pBIND载体以及转录激活结构域的pACT载体中，并将两种载体转染至动物细胞中。
  9. 根据权利要求8所述的细胞模型，其特征在于，所述动物细胞为HEK-293T细胞。
  10. 如权利要求3所述的方法，其特征在于，
    步骤(a)包括：
    (1)构建含有凋亡信号调节激酶1N端的细胞：分别将权利要求5所述的编码凋亡信号调节激酶1N端1-678氨基酸的DNA连接至pcDNA5-HA以及pcDNA5-Myc质粒中，构建HA-ASK1N和Myc-ASK1N质粒，并分别或同时转染动物细胞；
    (2)将候选短肽的DNA分别连接至psi-Flag载体中，将psi-flag-Peptides质粒分别转染至同时含有HA-ASK1N和Myc-ASK1N质粒的动物细胞中；
    步骤(b)包括：转染成功的细胞在培养24h后通过免疫共沉淀及Western blot实验检测免疫沉淀反应后的蛋白溶液中HA-ASK1N和Myc-ASK1N的含量；
    免疫共沉淀采用anti-HA将目标蛋白与protein A/G琼脂糖珠相连，Western blot采用anti-Myc作为一抗，如果同时转染HA-ASK1N、Myc-ASK1N和某一候 选物质的psi-flag-Peptide的动物细胞组与仅转染HA-ASK1N或Myc-ASK1N的对照组相同，未检测到明显Myc蛋白条带，则该候选物质是预防、缓解和/或治疗脂肪性肝炎的潜在物质。
  11. 如权利要求10所述的方法，其特征在于，步骤(a)所用动物细胞选自HEK-293T、L02、Hela、Huh7、Hepg2、A549、3T3、MEFs、H9C2。
  12. 如权利要求11所述的方法，其特征在于，步骤(a)所用动物细胞选自HEK-293T。
  13. 一种基于凋亡信号调节激酶1N端二聚化为靶点的预防、缓解和/或治疗脂肪性肝炎药物筛选的细胞模型，其特征在于，分别将所述的编码凋亡信号调节激酶1N端1-678氨基酸的DNA，其核苷酸序列如SEQ NO：2所示，连接至pcDNA5-HA以及pcDNA5-Myc质粒中，构建HA-ASK1N和Myc-ASK1N质粒，并分别或同时转染动物细胞，所用动物细胞选自HEK-293T、L02、Hela、Huh7、Hepg2、A549、3T3、MEFs、H9C2。
  14. 如权利要求3-7、10-12任意一项所述的方法，其特征在于，所述的方法还包括步骤：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以选出抑制凋亡信号调节激酶1N端二聚化的物质，用于预防、缓解和/或治疗脂肪性肝炎。
  15. 一种凋亡信号调节激酶1的用途，其特征在于，用于制备抑制肝脏脂肪变性的组合物。
  16. 一种调节肝脏细胞脂肪变性的方法，其特征在于，所述方法包括：调节细胞内凋亡信号调节激酶1N端二聚化。
  17. 一种预防、缓解和/或治疗脂肪性肝炎的多肽Peptide1，其氨基酸序列如Seq No.20所示，或其药学上可接受的盐。
  18. 一种多核苷酸分子，其特征在于，它编码权利要求17所述的多肽Peptide1，其核苷酸序列如Seq No.24所示。
  19. 一种药物组合物，其特征在于，它含有：
    (a)权利要求17所述的多肽Peptide1或其药学上可接受的盐；和
    (b)药学上可接受的载体或赋形剂。
PCT/CN2017/081936 2016-11-25 2017-04-25 一种以凋亡信号调节激酶1n端二聚化为靶点筛选用于治疗脂肪性肝炎药物的方法 WO2018094964A1 (zh)

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CN201611054149.3A CN106589079B (zh) 2016-11-25 2016-11-25 一种多肽s1及其在制备治疗脂肪肝和/或ⅱ型糖尿病药物中的应用
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