WO2021134842A1 - Zc3h12b基因或蛋白的用途及一种肝脏疾病动物模型的建立方法 - Google Patents

Zc3h12b基因或蛋白的用途及一种肝脏疾病动物模型的建立方法 Download PDF

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WO2021134842A1
WO2021134842A1 PCT/CN2020/072609 CN2020072609W WO2021134842A1 WO 2021134842 A1 WO2021134842 A1 WO 2021134842A1 CN 2020072609 W CN2020072609 W CN 2020072609W WO 2021134842 A1 WO2021134842 A1 WO 2021134842A1
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zc3h12b
liver
protein
bile duct
gene
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关桂君
常宇阳
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上海海洋大学
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Definitions

  • the present invention relates to the field of biomedical technology, in particular to the new functions of genes or proteins and the establishment of animal models of related diseases. Specifically, it relates to the use of ZC3H12B genes or proteins and methods for establishing animal models for simulating liver diseases.
  • Liver diseases are divided into two parts: non-neoplastic and neoplastic liver diseases, but non-neoplastic liver diseases (common pathogenic infections such as hepatitis viruses or parasites, chemical or alcoholic liver injury, and genetic developmental abnormalities such as cholelithiasis and bile duct malformations) , Because of the microenvironment where hepatocytes, bile duct epithelial cells and stem cells in the liver sinusoids are constantly stimulated by bile secreted by the bile duct epithelium, the natural macrophages (Kupffer cells, KC) of the liver sinusoids and other lymphocytes that regulate the immune system The influence of secreted cytokines leads to hepatocellular fatty lesions, fibrosis, cirrhosis, and even liver tumor carcinogenesis at the local injury site of the liver.
  • non-neoplastic liver diseases common pathogenic infections such as hepatitis viruses or parasites, chemical or alcoholic liver injury, and
  • HCC Hepatocellular Carcinoma
  • ICC Intrahepatic Cholangiocarcinoma
  • the pathogenic factors of ICC include inflammation of the bile duct caused by pathogenic infections such as hepatitis virus and parasites, chemical carcinogens, and genetic factors.
  • pathogenic infections such as hepatitis virus and parasites, chemical carcinogens, and genetic factors.
  • pathogenic infections such as hepatitis virus and parasites
  • chemical carcinogens such as hepatitis virus and parasites
  • genetic factors pathogenic mechanism and exact etiology are still unknown, and have shown an upward trend in the world in recent years.
  • ICC has no obvious clinical symptoms in the early stage, early diagnosis and timely treatment are of great special significance.
  • current research mainly focuses on sex hormones and cytokines.
  • treatment programs for the estrogen androgen receptor pathway and inflammation intervention have not achieved the expected clinical results.
  • the Zc3h12 protein family is a type of CCCH-type zinc finger protein that acts on immune response and inflammation. It is characterized by a CCCH-type zinc finger domain (related to DNA or RNA binding), a PIN Zc3h12 functional domain, and an RNase activity In the region, there is also an independent ubiquitin-related functional domain (UBA) that is highly conserved in the ZC3H12 family ( Figure 2-3).
  • the Zc3h12 protein family includes 4 members: ZC3H12A, ZC3H12B, ZC3H12C and ZC3H12D. Although the amino acid sequences of these four proteins are highly homologous, their tissue distribution is very different. The function of the ZC3H12 protein family is not fully understood.
  • Zc3h12b For Zc3h12b, Wawro M., etc. (Wawro M., Wawro K., Kochan J., Solecka A., Sowinska W., Lichawska-Cieslar A., et al., Zc3h12b/MCPIP2, a new active of the ZC3H12 .RNA.25(2019)840-856.) discloses a human and mammalian Zc3h12b, which has proinflammatory interleukin-6 (IL-6) mRNA), which exists in the cytoplasm to form particles ( granule-like structure), the function of regulating mRNA transcription and protein translation to stop the cell cycle in the G2 phase.
  • IL-6 interleukin-6
  • the purpose of the present invention is to provide the use of ZC3H12B gene or protein against the deficiencies in the prior art, and to provide a method for establishing an animal model of liver disease based on this.
  • ZC3H12B gene or protein or their up-regulators are provided for preparing and treating intrahepatic bile duct cystadenoma (biliary cystadenoma, BCA), intrahepatic bile duct cystadenocarcinoma (biliary cystadenocarcinoma, BCAC) or with them Related to the application of fatty liver or liver cancer drugs.
  • BCA biliary cystadenoma
  • BCAC biliary cystadenocarcinoma
  • the upregulator is selected from small molecule compounds or biological macromolecules.
  • a pharmaceutical composition for the treatment of intrahepatic bile duct cystadenoma, intrahepatic bile duct cystadenocarcinoma, or their related fatty liver or liver cancer comprising ZC3H12B gene or protein The up-regulating agent, and a pharmaceutically acceptable carrier.
  • ZC3H12B gene or protein is provided as a diagnostic marker in the preparation of diagnostic reagents or kits for intrahepatic bile duct cystadenoma, intrahepatic bile duct cystadenocarcinoma, or their related fatty liver or liver cancer application.
  • a reagent for detecting ZC3H12B gene or protein content is provided in the preparation of diagnostic reagents or kits for intrahepatic cholangiocystadenomas, intrahepatic cholangiocystadenocarcinoma, or fatty liver or liver cancer related to them. application.
  • a method for screening drugs for the treatment of intrahepatic bile duct cystadenoma, intrahepatic bile duct cystadenocarcinoma, or their related fatty liver or liver cancer by using ZC3H12B gene or protein as a target include:
  • the candidate substance can increase the expression of the ZC3H12B gene or protein, it indicates that the candidate substance is a desired potential substance, and vice versa, it indicates that the candidate substance is an unneeded potential substance.
  • a method for establishing an animal model of liver disease which includes the step of knocking down the expression of the animal's Zc3h12b gene or protein.
  • the liver disease is selected from intrahepatic bile duct cystadenoma, intrahepatic bile duct cystadenocarcinoma, or their related fatty liver or liver cancer.
  • the animal is selected from lower to higher vertebrates other than humans.
  • an animal model of liver disease established according to any one of the above methods is provided, and the use is selected from:
  • the present invention uses gene editing technology for the first time to knock out the zc3h12b gene of the medaka targeted to create a zc3h12b deleted medaka, produce a series of frameshift mutations, and delete the heterozygotes and homozygotes of the Zc3h12b protein product.
  • These heterozygotes and homozygotes all showed different degrees of liver disease, and with the increase of months, local fatty liver, bile duct hyperplasia and fibrosis appeared, and the 6-month-old medaka showed significant fatty liver and local cyst necrosis.
  • the liver sinusoids of the zc3h12b knockout medaka had obvious lymphocytic infiltration, an abnormal increase in the number of macrophages, hepatobiliary hyperplasia and fusion, and hepatocellular lipopathy.
  • the immunohistochemical reaction not only detected smooth muscle actin SMA positive cells, but also detected GPC3, SMA, CK19 and other human hepatobiliary tumor cell markers positive cells.
  • the number of MMP9 positive macrophages increased significantly, accompanied by iron particle deposition, etc. Similar to the symptoms of human liver cancer. Based on:
  • Medaka with zc3h12b deletion can be used as an animal model for studying intrahepatic cholangiocystadenoma, intrahepatic cholangiocystadenocarcinoma or their pathological process, for in-depth study of CCC3H type zinc finger protein Zc3h12b regulating and activating the inflammatory response of macrophages, and The inherent lipopolysaccharide metabolism of other tissues (gonads, brain, etc.) other than the liver-the molecular mechanism of macrophage immune response, provides a research platform in living animals. Because Chinese medicine refers to "liver cancer" as "fat” disease, the zc3h12b knockout medaka is named "fat medaka”.
  • the animal model of the present invention can better simulate the clinical occurrence of intrahepatic bile duct cystadenoma or intrahepatic bile duct cystadenocarcinoma, and is also more helpful for in-depth discussion
  • Intrahepatic bile duct cystadenoma and intrahepatic bile duct cystadenocarcinoma have gender differences at the genetic level, and the single gene knockout method is simple and easy to operate.
  • ZC3H12B gene or protein can be used as a therapeutic target for intrahepatic bile duct cystadenoma, intrahepatic bile duct cystadenocarcinoma or their related fatty liver or liver cancer.
  • ZC3H12B gene or protein can be used as a diagnostic marker for intrahepatic bile duct cystadenoma, intrahepatic bile duct cystadenocarcinoma, or their related fatty liver or liver cancer.
  • the ZC3H12B gene or protein can be used as a target to screen drugs for the treatment of intrahepatic bile duct cystadenomas, intrahepatic bile duct cystadenocarcinoma, or their related fatty liver or liver cancer.
  • FIG. 1 zc3h12b knockout medaka: hepatobiliary hyperplasia and fusion, balloon-like steatosis of hepatocytes (***); lymphocyte infiltration in hepatic sinusoids.
  • Figure 2-3 The molecular structure and functional domain of Medaka Zc3h12b protein is highly evolutionarily conserved.
  • Figure 2. The amino acid sequence of the Zc3h12b protein of medaka is the same as the Zc3h12b of birds, mammals, mice and humans, in the functional domain (the green box is the first box: UBA related to ubiquitin, and the red box is the second box: PINZc3h12 , The orange box is the third box: CCCH-type zinc finger domain, and the blue box is the fourth box: RNase active region).
  • the amino acid sequence is highly homologous and conserved.
  • Zc3h12b protein belongs to the Zc3h12 family member.
  • Phylogenetic tree analysis shows Zc3h12A, -B,- C, -D are members of the four Zc3h12 families.
  • Figures 4-7 The strategy of using the CRISPR/CAS9 editing system to target the Zc3h12b gene of the medaka to obtain five mutant medakas with partial or complete deletion of Zc3h12b.
  • Figure 4. The sequence number and structure of medaka zc3h12b gene, in which the yellow highlight is the gRNA sequence of two target sites on the exon 1 of medaka zc3h12b;
  • Figure 5. PCR genotype identification of wild type and knockout type;
  • Figure 6. DNA sequencing of wild-type and knock-out genomic PCR products confirmed; Figure 7.
  • Antibodies prepared with N-terminal peptides, Western blot analysis of wild-type and knock-out liver Zc3h12b protein can detect wild-type Zc3h12b (OlaX-201, 845aa, predicted 94.57kDa; OlaX-202, 833aa, predicted 93.37kDa) band, attenuated in knockout liver (Mut3), or no protein expression (Mut1), confirm the Zc3h12b protein after knockout Was partially or completely destroyed. All five mutants cannot express the complete Zc3h2b protein.
  • Figure 8 Morphological comparison and tissue section analysis of wild-type and knock-out livers.
  • Normal male and female livers ab) are crimson or brown, with soft texture; knockout homozygous livers cd) are pale yellow to milky white, with thick texture and nodules;
  • normal female liver slices e-e') liver sinusoids are regular and dense;
  • knock Excluding liver slices f-f' have a large number of bile duct hyperplasia and fusion, cholestasis, which is consistent with the obvious shrinkage of the gallbladder in the solid liver.
  • e-f low magnification
  • e'-f high magnification observation.
  • FIG. 9 Immunohistochemical analysis of anti-human autoimmune antibody SMA.
  • Anti-SMA immunohistochemistry results of normal female (A-A'), male (B-B') and knockout (C-C') liver slices at low and high magnification showed that a large number of hyperplastic bile ducts in the knockout liver Surrounded by many SMA-positive cells, similar to the phenotype of SMA antibody-positive cells surrounding a large number of bile duct hyperplasia in a mouse liver cancer model induced by lithocholic acid feeding (P Fickert et al., American Journal of Pathology 2006).
  • FIG 10 Immunohistochemical analysis of anti-human cytokeratin 19 (CK19).
  • the negative control does not add anti-human CK19, and there is no brown signal (B"NC).
  • the symptoms of CK19 antibody-positive cells surrounding the bile duct hyperplasia in a mouse liver cancer model induced by lithocholic acid feeding are similar (PFickert et al., American Journal of Pathology 2006).
  • FIG 11 Multicolor immunofluorescence reaction of liver cancer marker antibody.
  • the anti-GPC3 and MMP9 reaction in normal male liver is weak and difficult to detect, but there are a large number of anti-human GPC3, MMP9, CK19 and SMA positive cells in the zc3h12b knockout medaka liver, suggesting that these cells may have become cancerous.
  • Anti-GPC3 green, arrow
  • anti-MMP9, anti-CK19, anti-SMA red, arrow
  • Figure 12 The abnormal activation of macrophages in the liver of the zc3h12b knockout medaka. Prussian blue staining and eosin negative staining showed that normal males (low magnification A, high magnification A') and females (low magnification B, high magnification B') have a small amount of iron-containing sinusoidal macrophages (KC) in the liver sinusoids. These KCs proliferate abnormally in the liver of the zc3h12b knockout medaka (low power C, high power C').
  • KC iron-containing sinusoidal macrophages
  • Zc3h12b is a member of the Zc3h12 protein family, which is a type of CCCH zinc finger protein that acts on immune response and inflammatory response.
  • the Zc3h12b protein used may be naturally occurring, for example, it may be isolated or purified from lower to higher vertebrates.
  • the Zc3h12b protein can also be artificially prepared, for example, the recombinant Zc3h12b protein can be produced according to conventional genetic engineering recombination techniques.
  • the Zc3h12b protein includes a full-length Zc3h12b protein or a biologically active fragment thereof.
  • the amino acid sequence of the Zc3h12b protein formed by the substitution, deletion or addition of one or more amino acid residues is also included in the present invention.
  • the Zc3h12b protein or biologically active fragments thereof include a part of conservative amino acid substitution sequence, and the amino acid substitution sequence does not affect its activity or retain part of its activity.
  • Appropriate replacement of amino acids is a technique well known in the art, which can be easily implemented and ensures that the biological activity of the resulting molecule is not changed. These techniques have made those skilled in the art realize that, generally speaking, changing a single amino acid in a non-essential region of a polypeptide does not substantially change the biological activity. See Watson et al., Molecular Biology of The Gene, Fourth Edition, 1987, TheBenjamin/CummingsPub.Co.P224.
  • any biologically active fragment of Zc3h12b protein can be applied to the present invention.
  • the biologically active fragment of the Zc3h12b protein means that as a polypeptide, it can still maintain all or part of the functions of the full-length Zc3h12b protein.
  • the biologically active fragment retains at least 50% of the activity of the full-length Zc3h12b protein.
  • the active fragment can maintain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length Zc3h12b protein.
  • the present invention can also use a modified or improved Zc3h12b protein, for example, a Zc3h12b protein that has been modified or improved in order to promote its half-life, effectiveness, metabolism, and/or protein effectiveness.
  • the modified or improved Zc3h12b protein may be a conjugate of the Zc3h12b protein, or it may include substituted or artificial amino acids.
  • any variation that does not affect the biological activity of the Zc3h12b protein can be used in the present invention.
  • the up-regulator of Zc3h12b includes promoters, agonists and the like. Any substance that can increase the activity of the Zc3h12b protein, maintain the stability of the Zc3h12b protein, promote the expression of the Zc3h12b protein, promote the secretion of the Zc3h12b protein, extend the effective time of the Zc3h12b protein, or promote the transcription and translation of the Zc3h12b can be used in the present invention.
  • the Zc3h12b protein upregulator includes (but is not limited to): an expression vector or expression construct that can express (preferably overexpression) Zc3h12b after being transformed into a cell.
  • the expression vector contains a gene cassette, and the gene cassette contains a gene encoding Zc3h12b and an expression control sequence operatively connected to it.
  • the term "operably connected” or “operably connected to” refers to a situation in which certain parts of a linear DNA sequence can regulate or control the activity of other parts of the same linear DNA sequence. For example, if the promoter controls the transcription of the sequence, then it is operably linked to the coding sequence.
  • the present invention provides the application of ZC3H12B gene or protein or their upregulators in the preparation of drugs for treating intrahepatic bile duct cystadenoma, intrahepatic bile duct cystadenocarcinoma, or fatty liver or liver cancer related to them.
  • Cholangiocarcinoma is a malignant tumor of the liver, which can be divided into two categories: primary and secondary. In the present invention, it preferably refers to primary intrahepatic bile duct cystadenocarcinoma, that is, liver malignant tumor originating from hepatobiliary epithelium.
  • the etiology can include hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, aflatoxin, drinking water pollution, alcohol, liver cirrhosis, sex hormones, nitrosamines, trace elements, autoimmune liver disease, etc. Wait.
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • the present invention also provides the application of ZC3H12B gene or protein as a diagnostic marker in the preparation of diagnostic reagents or kits for intrahepatic bile duct cystadenoma, intrahepatic bile duct cystadenocarcinoma, or their related fatty liver or liver cancer.
  • the reagent for detecting ZC3H12B gene or protein content can be used to prepare diagnostic reagents or kits for intrahepatic cholangiocystadenomas, intrahepatic cholangiocystadenocarcinoma, or fatty liver or liver cancer related to them.
  • test sample By analyzing the expression of the Zc3h12b protein or its coding gene in the test sample (sample), the subject's disease status can be known, and the diagnosis or prognosis of the disease can be provided.
  • the sample to be tested or the sample to be tested is the tissue or body fluid of the patient.
  • nucleic acid detection includes (but are not limited to): gene chip technology, probe hybridization technology, polymerase chain reaction (PCR), Northern blot and other methods.
  • PCR polymerase chain reaction
  • the detection of protein can be done by means of mass spectrometry equipment, etc., or can be done by methods such as Western Blot or ELISA.
  • the present invention also provides a method for screening drugs for the treatment of intrahepatic cholangiocystadenoma, intrahepatic cholangiocystadenocarcinoma or their related fatty liver or liver cancer by using ZC3H12B gene or protein as a target, said method comprising:
  • the candidate substance can increase the expression of the ZC3H12B gene or protein, it indicates that the candidate substance is a desired potential substance, and vice versa, it indicates that the candidate substance is an unneeded potential substance.
  • the Zc3h12b-expressing system may be a cell (or cell culture) system, and the cell may be a cell that endogenously expresses Zc3h12b; or it may be a cell that expresses Zc3h12b recombinantly.
  • the system for expressing Zc3h12b can also be a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as animal models, preferably low or higher vertebrate models, such as fish, mice, rabbits, sheep, monkeys, etc.) Wait.
  • a control group in order to make it easier to observe changes in the expression of Zc3h12b during screening, a control group may also be set, and the control group may be a system expressing Zc3h12b without adding the candidate substance.
  • a method for establishing an animal model of liver disease includes the steps of knocking down the expression of the zc3h12b gene or protein of the animal.
  • the liver disease may be intrahepatic cholangiocystadenoma, intrahepatic cholangiocystadenocarcinoma, or fatty liver or liver cancer related to them.
  • the animals are selected from lower to higher vertebrates other than humans, including fish, amphibians, reptiles, birds, and mammals including rats, dogs, rabbits, monkeys and humans.
  • the animal model of the present invention can be used as a good platform for the study of molecular mechanism of intrahepatic cholangiocystadenoma, intrahepatic cholangiocystadenocarcinoma or their related fatty liver or liver cancer diseases.
  • the pharmaceutical composition of the present invention may contain the active agent described herein and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are generally safe and non-toxic, and in a broad sense can include any known substances used in the pharmaceutical industry for preparing pharmaceutical compositions, such as fillers, diluents, coagulants, binders, lubricants, Glidants, stabilizers, colorants, wetting agents, disintegrants, etc.
  • the main consideration is the mode of administration of the pharmaceutical composition, and those skilled in the art are familiar with this technology.
  • the content of the active agent in the medicament of the present invention can be determined according to different therapeutic uses.
  • compositions can be prepared according to known pharmaceutical procedures, such as Remington's Pharmaceutical Sciences (17th edition, edited by Alfonoso R. Gennaro, Mack Publishing Company), Easton, Pennsylvania (1985)) There is a detailed record in the book.
  • the medicament of the present invention can be in various suitable dosage forms, including but not limited to capsules, granules, tablets, pills, oral liquids or injections and the like.
  • the zc3h12b gene was knocked out by microinjecting medaka one-cell stage fertilized eggs.
  • the specific method is as follows:
  • zc3h12bCrispr1 GCATGCCACTGAGGAGTCGG (SEQ ID NO:1)
  • zc3h12bCrispr2 GGGAGAAACTAGGCCGGTCG (SEQ ID NO: 2)
  • the zc3h12bgRNA1 and zc3h12bgRNA2 were respectively subjected to PCR (94°C3min 1 round; 94°C30s, 65°C30s, 72°C1min (34 rounds); 72°C5min 1 round), and SgRNA-scaffold 5'-AAAAGCACCGACTCGGTGCCATTTTTTCAAGTTGATAACGGCTACTTGCAATTTTTTCAAGTTGATAACGGCTACTTGAA 5) Connect.
  • the PCR product was purified by QIAquick PCR Pruification Kit, and then used T7In Vitro Transcription Kit (Thermo Fisher, USA) was used for in vitro transcription to obtain synthetic gRNA.
  • Mixed with Cas9 protein GeneScript Biotechnology, China
  • microinjected to detect the targeted knockout efficiency after embryo injection, and finally focus on the above two target sites.
  • gRNA1 100ng/ ⁇ l
  • Cas9 protein 100ng/ ⁇ l
  • gRNA1 100ng/ ⁇ l
  • gRNA2 100ng/ ⁇ l
  • Cas9 protein 100ng/ ⁇ l
  • the primers Fw9 and Rv10 can specifically amplify the exon1 fragment of medaka zc3h12b, which is used to distinguish wild type and zc3h12b knockout type.
  • Table 1 The overall survival rate and germline transmission efficiency of zc3h12b target gene knockout medaka
  • the medaka is raised in a water circulation system at 26-28°C, with a light-dark cycle time of 14/10 hours, and is handled in strict accordance with the guidance of the Laboratory Animal Research Committee of Shanghai Ocean University.
  • the tail fins are cut to extract DNA, and FW9 and RV10 primers are used.
  • PCR was performed to identify the zc3h12b genotype.
  • PCR product ligated to In Easy (Promega, USA) vector the plasmid was extracted and sequenced to identify the DNA sequence. The confirmed heterozygous male and female individuals were paired with each other, and the progeny genotype identification showed that they conformed to the Mendelian law of inheritance, and finally the homozygous zc3h12b editing knockout was
  • FW9 and RV10 primers were used for specific PCR amplification of the local region of exon 1 of zc3h12b in genomic DNA, and wild-type, heterozygous and homozygous were identified.
  • Five types of mutations were identified: Mut 1 deleted a base sequence of 214 bp; Mut 2 deleted a base sequence of 68 bp; Mut 3 inserted a base sequence of 137 bp; Mut 4 increased by 1 bp; Mut 5 increased A 4bp base.
  • electrophoresis detection and comparison of wild-type and knock-out PCR amplification products the genotypes of wild-type and multiple mutant individuals are obtained (Figure 4-7).
  • the results are shown in Figure 8.
  • the control wild-type male and female livers are light brown (a and b in Figure 8), and the knockout homozygous liver is slightly yellow or milky white, and there are obvious nodules and cystic masses in the liver ( Figure 8 c, d, c'and d'); the control wild-type gallbladder is green (a” and b” in Fig. 8), while the gallbladder of knockout homozygous is significantly atrophied, and cholestasis is distributed in the liver (c” and b” in Fig. 8).
  • hepatic sinusoids and hepatocytes are arranged evenly and orderly, and there is no cholestasis in the bile ducts (e and e'in Figure 8), while there are obvious cysts in the knockout liver, many of which can be seen under high magnification Bile duct hyperplasia, irregular fusion, cholestasis in the bile duct, hepatocyte necrosis and shedding (figure 8 f and f').
  • Cytokeratin 19 is one of the main antibodies for differential diagnosis of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC).
  • HCC hepatocellular carcinoma
  • ICC intrahepatic cholangiocarcinoma
  • Prussian blue staining method combined with the negative staining of eosin, we detected a small number of Prussian blue-positive (iron-containing particles) macrophages in the normal male and female livers, and a large number of iron-containing particles in the zc3h12b knockout liver Macrophage proliferation was activated (Figure 12).
  • the present invention establishes a fat medaka that mimics human intrahepatic bile duct cystadenoma, intrahepatic bile duct cystadenocarcinoma, or their related fatty liver or liver cancer, and its liver cholestasis, Bile duct hyperplasia and fusion, fatty inflammation and macrophages are activated, human liver and gallbladder tumor marker positive cells increase and other liver cancerous shapes, which can be intrahepatic bile duct cystadenoma, intrahepatic bile duct cystadenocarcinoma, or fatty liver or liver cancer related to them
  • the research provides live animal models.

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Abstract

ZC3H12B基因或蛋白的用途及一种肝脏疾病动物模型的建立方法。利用基因编辑技术，靶向敲除青鳉的zc3h12b基因，建立了缺失Zc3h12b蛋白产物的青鳉，均表现出不同程度的肝胆管增生融合和肝细胞脂肪变性，纤维化等肝脏病变，并伴随着月龄增长呈现显著脂肪肝，且局部囊肿坏死，其肝窦有明显的淋巴细胞浸润，巨噬细胞数目异常增多，还检测到人肿瘤标志物CK19，SMA和GPC3阳性的细胞，提示ZC3H12B可作为肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的治疗靶标和生物标志物，zc3h12b缺失青鳉可作为研究肝内胆管囊腺瘤、肝内胆管囊腺癌或两者病变过程的动物模型。

Description

ZC3H12B基因或蛋白的用途及一种肝脏疾病动物模型的建立方法 技术领域
本发明涉及生物医学技术领域，尤其涉及基因或蛋白的新功能及相关疾病动物模型的建立，具体地说，涉及ZC3H12B基因或蛋白的用途及模拟肝脏疾病的动物模型的建立方法。
背景技术
肝脏疾病分为非肿瘤性和肿瘤性肝病两部分，但非肿瘤性肝病(常见的肝炎病毒或寄生虫等病原性感染，化学或酒精性肝损伤，以及胆石症，胆管畸形等遗传发育异常)，会因为肝窦中肝细胞，胆管上皮细胞及干细胞所在的微环境，不断受到胆管上皮分泌的胆汁刺激，肝窦固有的巨噬细胞(Kupffer cell，KC)及其它调控自身免疫系统的淋巴细胞分泌的细胞因子的影响，导致肝脏局部损伤部位的肝细胞脂肪性病变，纤维化，进而肝硬化，甚至肝肿瘤性癌变。流行病学调查资料显示，肝细胞肝癌(Hepatocellular Carcinoma，HCC)和肝内胆管细胞癌(Intrahepatic Cholangiocarcinoma，ICC)在男性与女性发病比例方面有所不同，HCC的男性与女性的发病比例约为2:1-4:1，ICC发病率方面女性略高于男性(3:2)。ICC是起源于肝胆管上皮细胞，肝细胞，干细胞，以及胆管旁腺体细胞的胆管腺癌。其发病率仅次于肝细胞癌(HCC)的肝脏原发恶性肿瘤。ICC致病因素包括肝炎病毒、寄生虫等病原性感染的胆管炎症，化学致癌物以及遗传等因素，但其致病机制和确切病因仍然不明，且近年来全球都呈现上升趋势。因ICC早期无明显临床症状，早期诊断和及时治疗都具有重大特殊意义。对于ICC肝癌性别差异的解释，目前的研究主要围绕于性激素和细胞因子展开，然而近年来，针对雌雄激素受体通路和炎症干预的治疗方案在临床上并未取得预期的治疗结果。一方面，归因于现有的很多研究成果来源于化学诱导的肝癌发生动物模型，不能完全模拟临床上的HBV和HCV感染导致的ICC，尤其是遗传因素导致的ICC发生过程；另一方面，由于ICC中细胞种类不同，性别差异的原因和机制并不完全清楚，ICC动物模型都是利用药物所致，缺乏基因组层面的深入研究。因此，基因和蛋白分子的ICC动物模型，对肝癌致病机制以及其分子基因层面的性别差异研究，快速诊断技术的研发及靶向药物的设计和筛选等都具有重要的基础科学和临床应用价值。
Zc3h12蛋白家族是一类作用于免疫应答和炎症反应的CCCH型锌指蛋白，其特点是含有一个CCCH型锌指结构域(与DNA或RNA结合相关)，一个PIN Zc3h12功能域，一个RNA酶活性区域，还有一个独立的ZC3H12家族保守性很高的泛素相关功能域 (UBA)(图2-3)。Zc3h12蛋白家族包括4个成员：ZC3H12A、ZC3H12B、ZC3H12C和ZC3H12D。虽然这四种蛋白氨基酸序列同源性很高，但组织分布差异性很大。ZC3H12蛋白家族功能还不完全明了。针对Zc3h12b，Wawro M.等(Wawro M.,Wawro K.,Kochan J.,Solecka A.,Sowinska W.,Lichawska-Cieslar A.,et al.,Zc3h12b/MCPIP2,a new active member of the ZC3H12 family.RNA.25(2019)840-856.)公开了一种人和哺乳动物的Zc3h12b，具有结合前炎性白介素6(proinflammatory interleukin-6(IL-6)mRNA)，存在于细胞质中形成微粒(granule-like structure)，调控mRNA转录和蛋白翻译，使细胞周期停滞在G2期的功能。然而，截至本专利申请日之前，没有Zc3h12b与肝脏疾病，尤其是ICC及其相关的脂肪肝和肝癌的关系报道，也没有在小鼠或其他任何动物中敲除Zc3h12b得到肝脏疾病动物模型的报道。
发明内容
本发明的目的是针对现有技术中的不足，提供ZC3H12B基因或蛋白的用途，并基于此提供一种肝脏疾病动物模型的建立方法。
在本发明的第一方面，提供了ZC3H12B基因或蛋白或它们的上调剂在制备治疗肝内胆管囊腺瘤(biliary cystadenoma，BCA)、肝内胆管囊腺癌(biliary cystadenocarcinoma，BCAC)或与它们相关的脂肪肝或肝癌的药物中的应用。
作为本发明的一种优选实施方式，所述上调剂选自小分子化合物或生物大分子。
在本发明的第二方面，提供了一种治疗肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的药物组合物，所述药物组合物包含ZC3H12B基因或蛋白的上调剂，及药学上可接受的载体。
在本发明的第三方面，提供了ZC3H12B基因或蛋白作为诊断标志物在制备肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的诊断试剂或试剂盒中的应用。
在本发明的第四方面，提供了检测ZC3H12B基因或蛋白含量的试剂在制备肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的诊断试剂或试剂盒中的应用。
在本发明的第五方面，提供了一种以ZC3H12B基因或蛋白作为靶点筛选治疗肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的药物的方法，所述的方法包括：
用候选物质处理表达ZC3H12B基因或蛋白的体系；和
检测所述体系中ZC3H12B基因或蛋白的表达；
若所述候选物质可提高ZC3H12B基因或蛋白的表达，则表明该候选物质是需要的潜在物质，反之则表明该候选物质是非需要的潜在物质。
在本发明的第六方面，提供了一种肝脏疾病动物模型的建立方法，包括敲降动物的Zc3h12b基因或蛋白的表达的步骤。
作为本发明的一种优选实施方式，所述肝脏疾病选自肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌。
作为本发明的另一优选实施方式，所述动物选自除人以外的低等至高等脊椎动物。
在本发明的第七方面，提供了按照如上任一所述的方法建立的肝脏疾病动物模型的用途，所述用途选自：
a)研究人或/和动物zc3h12b调控巨噬细胞介导的脂肪肝发生及肝癌样变化相关的分子机制；
b)研究人或动物肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的分子致病机制；
c)研究肝内胆管囊腺瘤或肝内胆管囊腺癌肝实质萎陷坏死及纤维化癌变的致病机理和机制；
d)筛选影响人或动物的雌雄肝脏分化发育，及肝内胆管囊腺瘤或肝内胆管囊腺癌在发生率上受内在雌雄性别差异或外源环境因子的调控机制。
本发明优点在于：
本发明首次利用基因编辑技术，靶向敲除青鳉的zc3h12b基因，建立了zc3h12b缺失的青鳉，产生了一系列移码突变，缺失Zc3h12b蛋白产物的杂合子和纯合子。这些杂合子和纯合子均表现出不同程度的肝脏病变，并伴随着月龄增长，局部出现脂肪肝，胆管增生纤维化，6月龄青鳉呈现显著脂肪肝，且局部囊肿坏死。对照正常组，zc3h12b敲除青鳉的肝窦有明显的淋巴细胞浸润、巨噬细胞数目异常增多、肝胆管增生融合和肝细胞脂肪病变。免疫组化反应不仅检测到了平滑肌肌动蛋白SMA阳性细胞，还检测到了GPC3，SMA，CK19等人肝胆肿瘤细胞标志物阳性的细胞，MMP9阳性巨噬细胞数量显著增多，并伴有铁颗粒沉积等类似人肝癌的症状。基于此：
1、zc3h12b缺失青鳉可作为研究肝内胆管囊腺瘤、肝内胆管囊腺癌或它们的病变过程的动物模型，为深入研究CCC3H型锌指蛋白Zc3h12b调控激活巨噬细胞的炎症反应，以及肝脏以外其他组织(性腺和脑等)固有的脂多糖代谢-巨噬细胞免疫应答分子机制，提供活体动物研究平台。因中医称“肝癌”为“肥气”病，因此命名zc3h12b敲除青鳉为“肥气青鳉”。本发明的动物模型相比于现有技术中化学诱导的肝癌发生动物模型，能良好的模拟临床上的肝内胆管囊腺瘤或肝内胆管囊腺癌发生过程，也更有助于深入探讨肝内胆管囊腺瘤及肝内胆管囊腺癌在基因层面的性别差异，且单基因敲除的方式简单易 操作。
2、ZC3H12B基因或蛋白可作为肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的治疗靶标。
3、ZC3H12B基因或蛋白可作为肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的诊断标志物。
4、可以ZC3H12B基因或蛋白作为靶点筛选治疗肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的药物。
附图说明
附图1：zc3h12b敲除型肥气青鳉：肝胆管增生融合，肝细胞气球样脂肪变性(***)；肝窦内淋巴细胞浸润。
附图2-3：青鳉Zc3h12b蛋白的分子结构功能域具有高度进化保守性。图2.青鳉Zc3h12b蛋白氨基酸序列同鸟类，哺乳类小鼠和人的Zc3h12b，在功能结构域(绿色框即第一个框：泛素相关的UBA，红色框即第二个框：PINZc3h12，橙色框即第三个框：CCCH型锌指结构域，蓝色框即第四个框：RNA酶活性区域)上的氨基酸序列，高度同源保守。图3.用软件Mega X的MUSCLE多序列比对，并用软件MegaX的Neighbor-joining tree绘制进化树，得到青鳉Zc3h12b蛋白属于Zc3h12家族成员的结论，系统进化树分析显示了Zc3h12A，-B，-C，-D四大Zc3h12家族成员。
附图4-7：利用CRISPR/CAS9编辑系统对青鳉Zc3h12b基因靶向敲除的策略，得到5种部分或完全缺失Zc3h12b的突变型青鳉。图4.青鳉zc3h12b基因序号和结构，其中黄色亮标是青鳉zc3h12b外显子1上2个靶位点gRNA序列；图5.野生型和敲除型的PCR基因型鉴定；图6.野生型和敲除型基因组PCR产物的DNA测序确认；图7.用N-末端多肽制备的抗体，对野生型及敲除后的肝脏Zc3h12b蛋白的Westernblot(免疫印迹)分析，能检测到野生型Zc3h12b(OlaX-201，845aa，预测94.57kDa；OlaX-202，833aa，预测93.37kDa)的条带，敲除型肝脏中减弱(Mut3)，或无蛋白表达(Mut1)，确认敲除后Zc3h12b蛋白被部分或全部破坏。所有5种突变型都不能表达完整的Zc3h2b蛋白。
附图8：野生型和敲除型肝脏的形态比较和组织切片分析。正常雌雄肝脏a-b)呈绛红或褐色，质地柔和；敲除型纯合子肝脏c-d)呈淡黄色偏乳白，质地厚实有结节；正常雌性肝脏切片e-e’)肝窦有规则密布；敲除型肝脏切片f-f’)中有大量胆管增生及融合，胆汁淤积，同实体肝脏中胆囊明显缩小一致。e-f)低倍，e’-f”)高倍观察。
附图9：抗人自身免疫抗体SMA的免疫组化分析。低倍和高倍的正常雌性(A-A’)、雄性(B-B’)及敲除型(C-C’)肝脏切片的抗SMA免疫组化结果显示，敲除型肝脏的大 量增生胆管周围，有许多SMA阳性细胞包围，类似于石胆酸喂食诱导的小鼠肝癌模型中SMA抗体阳性细胞围绕着大量胆管增生的表型(P Fickert et al.,American Journal of Pathology 2006)。
附图10：抗人细胞角蛋白19(CK19)的免疫组化分析。正常雌性、敲除型雌性及敲除型雄性(低倍A，B，C；高倍A’，B’，C’)肝脏切片的抗人CK19免疫组化反应。正常肝脏中只有少许CK19阳性细胞，几乎检测不到，而敲除型肝脏中有大量CK19阳性细胞，聚集在增生胆管及附近组织中。阴性对照不加抗人CK19，没有褐色信号(B”NC)。同石胆酸喂食诱导的小鼠肝癌模型CK19抗体阳性细胞围绕胆管增生的症状相似(PFickert et al.,American Journal ofPathology 2006)。
附图11：肝癌标志抗体的多色免疫荧光反应。正常雄性肝脏中抗GPC3和MMP9反应微弱难以检测到，但在zc3h12b敲除的青鳉肝脏中有大量抗人GPC3，MMP9，CK19和SMA的阳性细胞，提示这些细胞可能已经癌变。抗GPC3(绿色，箭头)，抗MMP9，抗CK19，抗SMA(红色，箭)，细胞核用4’,6-二脒基-2-苯吲哚(4’,6-diamidino-2-phenylindole，DAPI)负染。
附图12：zc3h12b敲除青鳉肝脏中巨噬细胞异常激活。普鲁士蓝染色及伊红负染显示，正常雄性(低倍A，高倍A’)和雌性(低倍B，高倍B’)肝窦中有少量含铁颗粒的肝窦巨噬细胞(KC)，这些KC在zc3h12b敲除青鳉的肝脏(低倍C，高倍C’)中异常增生。
具体实施方式
Zc3h12b
Zc3h12b是Zc3h12蛋白家族成员之一，该蛋白家族是一类作用于免疫应答和炎症反应的CCCH型锌指蛋白。
在本发明中，所用的Zc3h12b蛋白可以是天然存在的，比如其可被分离或纯化自低等至高等脊椎动物。此外，所述的Zc3h12b蛋白也可以是人工制备的，比如可以根据常规的基因工程重组技术来生产重组Zc3h12b蛋白。
任何适合的Zc3h12b蛋白均可用于本发明。所述的Zc3h12b蛋白包括全长的Zc3h12b蛋白或其生物活性片段。
经过一个或多个氨基酸残基的取代、缺失或添加而形成的Zc3h12b蛋白的氨基酸序列也包括在本发明中。Zc3h12b蛋白或其生物活性片段包括一部分保守氨基酸的替代序列，所述经氨基酸替换的序列并不影响其活性或保留了其部分的活性。适当替换氨基酸是本领域公知的技术，所述技术可以很容易地被实施并且确保不改变所得分子的生物活 性。这些技术使本领域人员认识到，一般来说，在一种多肽的非必要区域改变单个氨基酸基本上不会改变生物活性。见Watson等，Molecular Biology of The Gene，第四版，1987，TheBenjamin/CummingsPub.Co.P224。
任何一种Zc3h12b蛋白的生物活性片段都可以应用到本发明中。在这里，Zc3h12b蛋白的生物活性片段的含义是指作为一种多肽，其仍然能保持全长的Zc3h12b蛋白的全部或部分功能。优选的，所述的生物活性片段至少保持50％的全长Zc3h12b蛋白的活性。在更优选的条件下，所述活性片段能够保持全长Zc3h12b蛋白的60％、70％、80％、90％、95％、99％、或100％的活性。
本发明也可采用经修饰或改良的Zc3h12b蛋白，比如，可采用为了促进其半衰期、有效性、代谢、和/或蛋白的效力而加以修饰或改良的Zc3h12b蛋白。所述经过修饰或改良的Zc3h12b蛋白可以是一种Zc3h12b蛋白的共轭物，或其可包含被取代的或人工的氨基酸。
也就是说，任何不影响Zc3h12b蛋白的生物活性的变化形式都可用于本发明中。
同样地，天然存在的、分离纯化的、人工制备的、修饰或改良的但仍具备Zc3h12b蛋白编码能力的Zc3h12b基因或其片段均可用于本发明。
上调剂
如本文所用，所述Zc3h12b的上调剂包括了促进剂、激动剂等。任何可提高Zc3h12b蛋白的活性、维持Zc3h12b蛋白的稳定性、促进Zc3h12b蛋白的表达、促进Zc3h12b蛋白的分泌、延长Zc3h12b蛋白有效作用时间、或促进Zc3h12b的转录和翻译的物质均可用于本发明。
作为本发明的优选方式，所述的Zc3h12b蛋白的上调剂包括(但不限于)：在转入细胞后可表达(优选过表达)Zc3h12b的表达载体或表达构建物。通常，所述表达载体包含一基因盒，所述的基因盒含有编码Zc3h12b的基因及与之操作性相连的表达调控序列。所述的“操作性相连”或“可操作地连于”指这样一种状况，即线性DNA序列的某些部分能够调节或控制同一线性DNA序列其它部分的活性。例如，如果启动子控制序列的转录，那么它就是可操作地连于编码序列。
用途
本发明提供ZC3H12B基因或蛋白或它们的上调剂在制备治疗肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的药物中的应用。
胆管癌(cholangiocarcinoma)是一种肝脏恶性肿瘤，可分为原发性和继发性两大类。本发明中，优选指代原发性肝内胆管囊腺癌，即起源于肝胆上皮的肝脏恶性肿瘤。其病 因可包括乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)感染、黄曲霉素、饮水污染、酒精、肝硬化、性激素、亚硝胺类物质、微量元素、自身免疫性肝病等等。
本发明还提供ZC3H12B基因或蛋白作为诊断标志物在制备肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的诊断试剂或试剂盒中的应用，根据本发明，检测ZC3H12B基因或蛋白含量的试剂可用于制备肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的诊断试剂或试剂盒。
通过分析待测样品(样本)中Zc3h12b蛋白或其编码基因的表达情况，从而得知受试者的患病状况，为疾病的诊断或预后提供依据。所述的待测样品或待测样本是患者的组织或体液。
可采用各种技术来检测Zc3h12b的表达情况，这些技术均包含在本发明中。检测核酸可用的已有技术如(但不限于)：基因芯片技术、探针杂交技术、聚合酶链反应(PCR)、Northern Blot等方法。检测蛋白可借助于质谱分析仪器等，或可通过Western Blot或ELISA等方法。
本发明还提供以ZC3H12B基因或蛋白作为靶点筛选治疗肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的药物的方法，所述的方法包括：
用候选物质处理表达ZC3H12B基因或蛋白的体系；和
检测所述体系中ZC3H12B基因或蛋白的表达；
若所述候选物质可提高ZC3H12B基因或蛋白的表达，则表明该候选物质是需要的潜在物质，反之则表明该候选物质是非需要的潜在物质。
所述的表达Zc3h12b的体系可以是细胞(或细胞培养物)体系，所述的细胞可以是内源性表达Zc3h12b的细胞；或可以是重组表达Zc3h12b的细胞。所述的表达Zc3h12b的体系还可以是亚细胞体系、溶液体系、组织体系、器官体系或动物体系(如动物模型，优选低或高等脊椎动物模型，如鱼、鼠、兔、羊、猴等)等。在本发明的优选方式中，在进行筛选时，为了更易于观察到Zc3h12b的表达的改变，还可设置对照组，所述的对照组可以是不添加所述候选物质的表达Zc3h12b的体系。
动物模型
基于本发明的发现，提供一种肝脏疾病动物模型的建立方法，包括敲降动物的zc3h12b基因或蛋白的表达的步骤。所述肝脏疾病可以是肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌。所述动物选自除人以外的低等至高等脊椎动物，包括鱼、两栖类、爬行类、鸟类、及哺乳类包括鼠、狗、兔、猴和人。
本发明的动物模型可作为肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝 或肝癌疾病分子机制研究的良好平台。
药物组合物
本发明的药物组合物可含有本文所述的活性试剂和药学上可接受的载体。药学上可接受的载体通常是安全、无毒的，且广义上可包括制药产业中用于制备药物组合物的任何已知物质，如填充剂、稀释剂、凝结剂、黏合剂、润滑剂、助流剂、稳定剂、着色剂、润湿剂、崩解剂等。在选择适用于投递合成肽的赋形剂时，主要需考虑此药物组合物的给药方式，本领域技术人员熟知此项技术。可根据不同的治疗用途确定本发明药物中所述活性试剂的含量。可根据已知的药学程序来制备上述药物组合物，譬如《雷明顿制药科学》(Remington’s Pharmaceutical Sciences，第17版，AlfonosoR.Gennaro编，麦克出版公司(Mack Publishing Company)，伊斯顿，宾夕法尼亚(1985))一书中有详细的记载。本发明的药物可以是各种合适的剂型，包括但不限于胶囊剂、颗粒剂、片剂、丸剂、口服液或注射液等。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1
1.zc3h12b基因敲除及鉴定
利用CRISPR/Cas9系统，通过显微注射青鳉一细胞期的受精卵，进行zc3h12b基因敲除。具体方法如下：
(1)通过enseble网站(http://asia.ensembl.org/index.html)获取青鳉(Japanese medaka HdrR，Oryzias latipes)zc3h12b基因序列。在crisprscan网站(http://crisprscan.org/)上，根据青鳉zc3h12b基因序列，检索CRISPR/CAS9编辑系统的靶向敲除位点，同时通过对青鳉转录组BLAST比对检索，选择没有预测到任何其他非特异性结合的候选位点。靶位点选择在启动子之后靠近第一个ATG的位置附近。在靶序列前加上T7启动子序列，后端加上gRNA scaffold序列，同时为确保T7启动子的效率，靶位点5’端前两个碱基应为GG，若无则将C改成G，本项目实际靶位点如下：
zc3h12bCrispr1：GCATGCCACTGAGGAGTCGG(SEQ ID NO:1)
zc3h12bCrispr2：GGGAGAAACTAGGCCGGTCG(SEQ ID NO:2)
由上海生工进行合成，合成序列分别为：
(a)zc3h12bgRNA1：
5’- TAATACGACTCACTATAGGATGCCACTGAGGAGTCGG GTTTTAGAGCTAGAAATA GC(SEQ ID NO:3)
(b)zc3h12bgRNA2：
5’- TAATACGACTCACTATAGGGAGAAACTAGGCCGGTCG GTTTTAGAGCTAGAAATA GC(SEQ ID NO:4)
将zc3h12bgRNA1和zc3h12bgRNA2分别通过PCR(94℃3min 1轮；94℃30s，65℃30s，72℃1min(34轮)；72℃5min 1轮)，与SgRNA-scaffold 5’-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC(SEQ ID NO:5)相连。将PCR产物通过QIAquick PCR Pruification Kit试剂盒进行纯化，之后用
Figure PCTCN2020072609-appb-000001
T7In Vitro Transcription Kit(Thermo Fisher，USA)试剂盒进行体外转录，得到合成gRNA。同Cas9蛋白(金斯瑞生物科技，中国)混合后显微注射，检测胚胎注射后的靶向敲除效率，最终聚焦以上两个靶位点。
(2)显微注射一细胞期受精卵，分两批次注射，显微注射液组成分别为：
1)gRNA1(100ng/μl)，加Cas9蛋白(100ng/μl)；
2)gRNA1(100ng/μl)和gRNA2(100ng/μl)，加Cas9蛋白(100ng/μl)。
(3)靶向敲除的结果鉴定
1)引物合成
Fw9：5’-GACTTAGACGGAGAAGACCATATTAG(SEQ ID NO:6)
Rv10：5’-CGCACCAATTCAGCAAGAAC(SEQ ID NO:7)
引物Fw9和Rv10能特异扩增青鳉zc3h12b的exon1片段，用于鉴别野生型和zc3h12b敲除型。
2)取注射后5天的鱼卵提取DNA，用引物Fw9和Rv10进行PCR，PCR产物直接测序检测靶位点的突变效率。对照正常野生型(WT)，敲除胚胎的PCR直接测序结果显示，在靶位点处有明显的重叠峰出现，说明靶位点被编辑，即gRNA/Cas9系统的编辑效果良好。
3)将注射后的鱼卵养至成鱼，剪尾鳍提取DNA，并用FW9和RV10引物进行PCR扩增，PCR产物经DNA测序确认重叠峰的有无，进一步确认被检测的成鱼个体在zc3h12b基因上是否发生基因编辑。结果见表1。两批次显微注射，共注射受精卵134枚，囊胚存活率76％，幼苗孵化率66％，最终得到5条F0种鱼(Founder：1雌2雄3条单靶点的种鱼；1雌1雄两条双靶点的具有生殖细胞传递子代能力的种鱼)。
表1 zc3h12b靶基因敲除青鳉的总存活率和种系传递效率
Figure PCTCN2020072609-appb-000002
1)以被注射受精卵数目为分母得出的存活百分比。
2)以鱼苗数目为分母得出种鱼百分比。
2.遗传杂交，选育得到不同zc3h12b敲除类型的鱼
青鳉饲养在26-28℃的水循环系统中，光暗循环时间为14/10小时，严格按照上海海洋大学实验动物研究委员会的指导进行处理。取同时注射gRNA1和gRNA2双靶点，且发生基因编辑的成鱼作为种鱼，将其分别与野生型的雌雄进行配对，产生的子代饲养至成鱼，剪尾鳍提取DNA，使用FW9和RV10引物进行PCR鉴定zc3h12b基因型。PCR产物连接到
Figure PCTCN2020072609-appb-000003
Easy(Promega，USA)载体中，提取质粒并测序鉴定DNA序列。将确认的杂合子雌雄个体进行相互配对，子代基因型鉴定显示符合孟德尔遗传定律，最终得到zc3h12b编辑敲除的纯合子。
使用FW9和RV10引物进行基因组DNA中特异性PCR扩增zc3h12b的外显子1局部区域，进行野生型、杂合子和纯合子的鉴定。鉴定得到了5种突变类型：Mut 1缺失了214bp的碱基序列；Mut 2缺失了68bp的碱基序列；Mut 3插入了137bp的碱基序列；Mut 4增加了1bp的碱基；Mut 5增加了4bp的碱基。依据敲除突变型碱基的增加或减少，电泳检测并比较野生型和敲除型PCR扩增产物，得到野生型和多种突变型个体的基因型(图4-7)。
3.野生型和zc3h12b基因敲除的青鳉肝脏实体及组织学形态观察
性成熟青鳉(3月龄以上)的解剖及实体观察。取野生型成鱼，雌雄各3尾；杂合子雄性2条，纯合子5条进行实体解剖，用4％多聚甲醛(PFA)常规固定肝脏和性腺等组织，并包埋至石蜡中进行组织切片(6微米厚)(Leica RM2265自动切片仪，德国)，苏木精伊红(HE)染色，观察野生型和zc3h12b基因敲除个体的肝脏等组织学形态变化。
结果见图8，对照野生型雌雄肝脏，呈淡褐色(图8中a和b)，敲除型纯合子肝脏略显淡黄色或乳白色，肝脏中有明显结节和囊性肿块(图8中c、d、c’和d’)；对照野生型胆囊呈绿色(图8中a”和b”)，而敲除纯合子胆囊萎缩明显，胆汁淤积分布在肝脏中 (图8中c”和d”)；正常肝脏切片中肝窦肝细胞排列均匀有序，胆管中没有胆汁淤积(图8中e和e’)，而敲除型肝脏中有明显的囊腔，高倍下可以看到许多胆管增生，不规则融合，胆管中有胆汁淤积，肝细胞坏死脱落(图8中f和f’)。
4.肝癌表面抗体的免疫组化分析
表2 使用的4种抗体信息
抗体 制备商 免疫源
α-SMA(ab5694) Abcam(UK) 兔抗人SMA多克隆抗体
α-CK19(ab15463) Abcam(UK) 兔抗人CK19多克隆抗体
α-MMP9(BA2202) Boster(USA) 兔抗人MMP3多抗
α-GPC3(ab129381) Abcam(UK) 鼠抗人GPC3单抗
用兔抗人α-SMA(或兔抗人CK19，兔抗人MMP3和鼠抗人GPC3等)抗体作为1st抗体(1:100)，山羊抗兔(或鼠)IgG-偶联HRP(MBL，日本)作为二抗(2nd抗体，1:1000)，对正常组和敲除组的肝脏切片进行免疫组化或/和免疫荧光分析，并用过氧化物酶的生色底物二氨基联苯胺(DAB，丹麦)化学反应显色，或用酪酰胺荧光信号放大技术TSA TMPlus Fluorescence System(PerkinElmer Life Science，美国)，在激光共聚焦显微镜(Leica DMi8 TCS SP8，德国)下观察和拍摄图像。
在野生型6月龄的正常肝脏切片中，有少量细胞呈微弱的α-SMA阳性反应(图9中A和A’)，且雄性局部脂肪颗粒附近的细胞有较多的α-SMA阳性细胞(图9中B和B’)，而6月龄的敲除型肝脏中，在胆管增生区域周围，α-SMA阳性细胞异常增生增多(图9中C和C’)，阴性对照的片子不加一抗，因而检测不到上述信号(图9中A”、B”和C”)，证明信号是一抗的特异抗体免疫反应。
细胞角蛋白19(CK19)是鉴别诊断肝细胞性肝癌(HCC)和肝内胆管细胞癌(ICC)的主要抗体之一。我们发现，在3月龄至6月龄的敲除型雌雄肝脏中，在胆管周围有不同程度的抗人CK19阳性细胞(图10)，提示zc3h12b敲除引起胆汁淤积和ICC的可能性大。
人肝胆肿瘤细胞标志物基质金属蛋白酶-9(MMP9)和膜性硫酸乙酰肝素多糖蛋白(GPC3)，在zc3h12b敲除的青鳉肝脏切片中，也都明显高于正常肝脏对照组的表达(图11)。从分布上，可以看到有部分细胞同时表达GPC3/MMP9、GPC3/CK19(图11中B和C)，而GPC3同SMA在不同细胞中表达(图11中D-D’,箭指α-SMA阳性细胞，箭头α-GPC3阳性细胞)。
5.zc3h12b敲除的肝脏中有大量铁沉积的巨噬细胞激活
普鲁士蓝染铁沉积巨噬细胞方法：肝脏石蜡切片，二甲苯常规脱蜡，经系列梯度酒精递减还原水，去离子水冲洗三次后，用现配的20％盐酸+10％亚铁氰化钾1:1混合，室温避光染色2小时，4度放置过夜。次日恢复室温半小时，蒸馏水洗三次(5分钟/次)后，伊红染色2-3分钟，酒精梯度脱水，还原至二甲苯，中性树胶封片后，显微镜观察拍照(尼康NI-S-E)。
肝巨噬细胞通过释放各种促或抗炎症因子，来调节和维持肝脏局部微环境，是肝脏内部重要的天然免疫细胞。用普鲁士蓝染铁法，结合伊红细胞负染，我们检测到在正常雌雄肝脏中有少量普鲁士蓝阳性(含铁颗粒)的巨噬细胞，而在zc3h12b敲除型肝脏中有大量含铁颗粒的巨噬细胞增殖被激活(图12)。
综上所述，本发明通过敲除zc3h12b，建立了模拟人肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的肥气青鳉，其肝脏中胆汁淤积，胆管增生融合，脂肪炎症及巨噬细胞被激活，人肝胆肿瘤标志物阳性细胞增多等肝脏癌变形状，可以为肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的研究提供活体动物模型。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (10)

  1. ZC3H12b基因或蛋白或它们的上调剂在制备治疗肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述上调剂选自小分子化合物或生物大分子。
  3. 一种治疗肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的药物组合物，其特征在于，所述药物组合物包含ZC3H12B基因或蛋白的上调剂，及药学上可接受的载体。
  4. ZC3H12B基因或蛋白作为诊断标志物在制备肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的诊断试剂或试剂盒中的应用。
  5. 检测ZC3H12B基因或蛋白含量的试剂在制备肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的诊断试剂或试剂盒中的应用。
  6. 一种以ZC3H12B基因或蛋白作为靶点筛选治疗肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的药物的方法，其特征在于，所述的方法包括：
    用候选物质处理表达ZC3H12B基因或蛋白的体系；和
    检测所述体系中ZC3H12B基因或蛋白的表达；
    若所述候选物质可提高ZC3H12B基因或蛋白的表达，则表明该候选物质是需要的潜在物质，反之则表明该候选物质是非需要的潜在物质。
  7. 一种肝脏疾病动物模型的建立方法，其特征在于，包括敲降动物的ZC3H12B基因或蛋白的表达的步骤。
  8. 根据权利要求7所述的应用，其特征在于，所述肝脏疾病为肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌。
  9. 根据权利要求7所述的应用，其特征在于，所述动物选自除人以外的低等至高等脊椎动物。
  10. 根据权利要求7-9任一所述方法建立的肝脏疾病动物模型的用途，其特征在于，所述用途选自：
    a)研究人或/和动物zc3h12b调控巨噬细胞介导的脂肪肝发生及肝癌样变化相关的分子机制；
    b)研究人或动物Zc3h12b引起的肝内胆管囊腺瘤、肝内胆管囊腺癌或与它们相关的脂肪肝或肝癌的分子致病机制；
    c)研究Zc3h12b引起的肝内胆管囊腺瘤或肝内胆管囊腺癌肝实质萎陷坏死及纤维化癌变的致病机理和机制；
    d)筛选影响人或动物的雌雄肝脏分化发育，及肝内胆管囊腺瘤或肝内胆管囊腺癌在发生率上的内在雌雄性别差异或外源环境因子。
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