CN117487816B - Znf709基因在制备治疗pbc的药物中的应用 - Google Patents
Znf709基因在制备治疗pbc的药物中的应用 Download PDFInfo
- Publication number
- CN117487816B CN117487816B CN202311455183.1A CN202311455183A CN117487816B CN 117487816 B CN117487816 B CN 117487816B CN 202311455183 A CN202311455183 A CN 202311455183A CN 117487816 B CN117487816 B CN 117487816B
- Authority
- CN
- China
- Prior art keywords
- pbc
- znf709
- gene
- nets
- bile duct
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 101150070435 ZNF709 gene Proteins 0.000 title claims abstract description 24
- 239000003814 drug Substances 0.000 title claims abstract description 17
- 208000003167 cholangitis Diseases 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 241000713666 Lentivirus Species 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 2
- 210000000013 bile duct Anatomy 0.000 abstract description 42
- 210000002919 epithelial cell Anatomy 0.000 abstract description 37
- 230000037361 pathway Effects 0.000 abstract description 11
- 210000000440 neutrophil Anatomy 0.000 abstract description 10
- 230000008506 pathogenesis Effects 0.000 abstract description 5
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 238000012544 monitoring process Methods 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 230000004913 activation Effects 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 230000008859 change Effects 0.000 abstract description 2
- 230000006698 induction Effects 0.000 abstract description 2
- 210000000941 bile Anatomy 0.000 abstract 1
- 230000001225 therapeutic effect Effects 0.000 abstract 1
- 102100027152 Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial Human genes 0.000 description 43
- 101710205862 Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial Proteins 0.000 description 43
- 230000014509 gene expression Effects 0.000 description 24
- 101000964754 Homo sapiens Zinc finger protein 709 Proteins 0.000 description 16
- 102100040662 Zinc finger protein 709 Human genes 0.000 description 15
- 238000001262 western blot Methods 0.000 description 13
- 238000010586 diagram Methods 0.000 description 8
- 230000002018 overexpression Effects 0.000 description 8
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 5
- 210000005259 peripheral blood Anatomy 0.000 description 5
- 239000011886 peripheral blood Substances 0.000 description 5
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 4
- 102100034533 Histone H2AX Human genes 0.000 description 4
- 101001067891 Homo sapiens Histone H2AX Proteins 0.000 description 4
- 102000004890 Interleukin-8 Human genes 0.000 description 4
- 108090001007 Interleukin-8 Proteins 0.000 description 4
- 102100023974 Keratin, type II cytoskeletal 7 Human genes 0.000 description 4
- 101710194919 Keratin, type II cytoskeletal 7 Proteins 0.000 description 4
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 4
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 238000003197 gene knockdown Methods 0.000 description 4
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 4
- 238000003119 immunoblot Methods 0.000 description 4
- 210000005228 liver tissue Anatomy 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 4
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 3
- 108020004459 Small interfering RNA Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000004075 alteration Effects 0.000 description 3
- 230000004761 fibrosis Effects 0.000 description 3
- 238000003125 immunofluorescent labeling Methods 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 229960001661 ursodiol Drugs 0.000 description 3
- QCHQVPPBLFSODM-MZWCVTMLSA-N Ac-Asp-Lys-Ala-Thr-Ile-Gly-Phe-Glu-Val-Gln-Glu-Glu Chemical compound OC(=O)C[C@H](NC(C)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O)CC1=CC=CC=C1 QCHQVPPBLFSODM-MZWCVTMLSA-N 0.000 description 2
- 230000005778 DNA damage Effects 0.000 description 2
- 231100000277 DNA damage Toxicity 0.000 description 2
- 102000012199 E3 ubiquitin-protein ligase Mdm2 Human genes 0.000 description 2
- 108050002772 E3 ubiquitin-protein ligase Mdm2 Proteins 0.000 description 2
- 108010040476 FITC-annexin A5 Proteins 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- -1 N-cadherein Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000004700 fetal blood Anatomy 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 208000003343 Antiphospholipid Syndrome Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 description 1
- 206010008635 Cholestasis Diseases 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010072579 Granulomatosis with polyangiitis Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000012654 Primary biliary cholangitis Diseases 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 208000021386 Sjogren Syndrome Diseases 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007870 cholestasis Effects 0.000 description 1
- 231100000359 cholestasis Toxicity 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000009274 differential gene expression Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000008971 epithelial apoptosis Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 208000018191 liver inflammation Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002175 menstrual effect Effects 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 239000002644 phorbol ester Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000012764 semi-quantitative analysis Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Developmental Biology & Embryology (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Public Health (AREA)
- Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Toxicology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Epidemiology (AREA)
- Food Science & Technology (AREA)
- Rheumatology (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
Abstract
本发明涉及生物医学领域，具体涉及ZNF709基因在制备治疗PBC的药物中的应用。所述ZNF709基因的序列如SEQ ID NO.1所示，所述PBC为原发性胆汁性胆管炎。本发明的研究结果表明，原发性胆汁性胆管炎发病机理与中性粒细胞胞外诱捕网(NETs)的形成增加、诱导正常胆管上皮细胞发生PBC样改变密切相关。本发明还提供了PBC治疗新靶点，过表达ZNF709基因后，可以部分抵消NETs所导致的P53‑MDM2通路激活，并且能够部分缓解NETs诱导胆管上皮细胞的PBC样改变，该结果为进一步诊断和治疗PBC及监测PBC病情进展提供了重要的科学依据。
Description
技术领域
本发明涉及生物医学领域，具体涉及ZNF709基因在制备治疗PBC的药物中的应用。
背景技术
原发性胆汁性胆管炎(Primary biliary cholangitis，PBC)是一种慢性肝脏疾病，主要侵犯胆管上皮细胞，导致胆汁淤积、肝脏炎症和纤维化，PBC的确切发病机制仍不完全清楚，这导致了在治疗和管理方面的挑战。传统的PBC治疗方法主要是用熊去氧胆酸(ursodeoxycholic acid，UDCA)进行药物治疗，然而，由于患者对UDCA的反应性不同及UDCA疗效的有限性，有一部分PBC患者疾病进展快速，晚期可能需要肝移植。而中性粒细胞胞外诱捕网(Neutrophil extracellular traps，NETs)是一种由中性粒细胞释放的DNA-蛋白复合物，它们在类风湿关节炎、系统性红斑狼疮、抗磷脂抗体综合征、肉芽肿性多血管炎和干燥综合征等自身免疫性疾病中发挥着重要作用，但与PBC的关联尚不清楚。寻找NETs促进PBC发生及进展的关键基因，可为PBC的诊断、个体化治疗及疾病监测，提供重要的依据。
发明内容
针对现有技术存在的缺陷和不足，本发明旨在解析NETs与PBC之间的联系，发现NETs促进PBC发病的关键靶点和作用机制，有望发现一种全新的治疗靶点，为熊去氧胆酸治疗效果不佳的患者提供新的治疗选择，并为PBC患者诊断和疾病监测提供新靶点。
本发明的第一个方面在于提供ZNF709基因在制备治疗PBC的药物中的应用，所述ZNF709基因的序列如SEQ ID NO.1所示。
进一步的，所述药物中含有所述ZNF709基因或ZNF709蛋白。
更进一步的，所述药物还包括药学上可接受的载体。
更进一步的，所述原发性胆汁性胆管炎与中性粒细胞胞外诱捕网形成增加相关。
本发明的第二个方面在于提供包含所述ZNF709基因的重组载体。
本发明的第三个方面在于提供包含所述重组载体的重组慢病毒。
本发明的第四个方面在于提供转所述重组载体或所述重组慢病毒的干细胞。
所述的干细胞指造血干细胞或间充质干细胞，所述造血干细胞来源于骨髓、外周血或脐带血，所述间充质干细胞来源于骨髓、皮肤、脂肪、胎盘、脐带、脐带血或经血。
本发明的第五个方面在于提供所述重组载体、所述重组慢病毒或所述干细胞在制备治疗PBC的药物中的应用。
本发明的第六个方面在于提供一种治疗PBC的药物，所述药物包含所述ZNF709基因、所述重组载体、所述重组慢病毒或所述干细胞。
进一步的，所述药物还包括药学上可接受的载体，所述药学上可接受的载体包括常规的稀释剂，如注射用水。
本发明具有如下有益效果：
本发明的实验结果首次揭示了NETs在PBC发病机制中的关键作用，包括NETs通过调节ZNF709-P53-MDM2通路诱导胆管上皮细胞发生PBC样改变。本发明发现了PBC发病的新机制，ZNF709是治疗PBC的新靶点，该结果为进一步诊断和治疗PBC及监测PBC病情进展提供了重要的科学依据。
附图说明
图1为NETs在PBC发病机制中的作用，其中：
A为PBC患者外周血中性粒细胞NETs的代表性免疫荧光染色图；
B为PBC患者血清中游离DNA的相对定量分析散点图；
C为PBC患者肝脏NETs的代表性免疫荧光染色图；
D为PBC模型小鼠肝脏NETs的代表性免疫荧光染色图。
图2为NETs通过下调ZNF709表达，激活P53-MDM2通路，诱导胆管细胞发生PBC样改变，其中：
A为NETs刺激后胆管上皮细胞细胞的差异基因表达的火山图；
B为NETs刺激后胆管上皮细胞ZNF709-P53-MDM2通路蛋白表达水平的蛋白质免疫印迹图；
C为敲低ZNF709后，胆管上皮细胞P53-MDM2通路蛋白表达水平的蛋白质免疫印迹图；
D为敲低ZNF709后，胆管上皮细胞ZO-1、CK-7蛋白表达水平的蛋白质免疫印迹图；
E为敲低ZNF709后，胆管上皮细胞α-SMA、TGF-β、IL-8蛋白表达水平的蛋白质免疫印迹图；
F为敲低ZNF709后，胆管上皮细胞N-cadherin蛋白表达水平的蛋白质免疫印迹图；
G为敲低ZNF709后，胆管上皮细胞H2AX蛋白表达水平的蛋白质免疫印迹图；
H为敲低ZNF709后，胆管上皮细胞PDC-E2蛋白表达水平的蛋白质免疫印迹图；
I为敲低ZNF709后，流式细胞术检测胆管上皮细胞凋亡水平的散点图。
图3为过表达ZNF709能够部分缓解NETs诱导胆管上皮细胞发生的PBC样改变，其中：
A为过表达ZNF709后，胆管上皮细胞P53-MDM2通路蛋白表达水平的蛋白质免疫印迹图；
B为过表达ZNF709后，胆管上皮细胞ZO-1、CK-7蛋白表达水平的蛋白质免疫印迹图；
C为过表达ZNF709后，胆管上皮细胞α-SMA、TGF-β、IL-8蛋白表达水平的蛋白质免疫印迹图；
D为过表达ZNF709后，胆管上皮细胞N-cadherin蛋白表达水平的蛋白质免疫印迹图；
E为过表达ZNF709后，胆管上皮细胞H2AX蛋白表达水平的蛋白质免疫印迹图；
F为过表达ZNF709后，胆管上皮细胞PDC-E2蛋白表达水平的蛋白质免疫印迹图；
G为过表达ZNF709后，流式细胞术检测胆管上皮细胞凋亡水平的散点图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明下述实施例披露了ZNF709基因在制备治疗PBC的药物中的应用，所述ZNF709基因的序列如SEQ ID NO.1所示。
实施例1：NETs与PBC发病的关系
1实验方法
分别提取PBC患者及健康对照者外周血中性粒细胞，通过SytoxGreen和MPO-Cy3的免疫荧光共染色检测外周血中性粒细胞释放NETs的水平；对PBC患者及健康对照者肝脏组织进行冰冻切片，通过CitH3-FITC和MPO-Cy3的免疫荧光共染色观察肝脏组织中NETs的水平；对PBC患者及健康对照者血清中的游离DNA进行SytoxGreen荧光半定量分析。对PBC模型小鼠及野生C57/BL6小鼠的肝脏组织进行冰冻切片，通过CitH3-FITC和MPO-Cy3的免疫荧光共染色观察其肝脏组织中NETs的水平。
2实验结果
本实施例通过一系列实验和研究，首先发现了PBC患者(图1A-C)及2OA-BSA联合Poly I：C诱导的PBC动物模型(图1D)中显著有NETs发生。该结果表明，NETs与PBC的发病密切相关。
实施例2：ZNF709基因在胆管细胞中的表达
1实验方法
采用静脉穿刺法采集健康人外周血并分离出中性粒细胞。分离的中性粒细胞用RPMI-1640培养基重悬于24孔板(0.5×106/孔)中，用1nM佛波酯(PMA)在37℃、5％CO2条件下刺激4h生成NETs。4h后，用微孔板在500rpm下摇5分钟检测NETs。收集上清液，用0.45μm PES膜过滤器过滤细胞和细胞碎片，PBS洗涤3次后，获得NETs。
将胆管上皮细胞接种于6孔板(2.5×106/孔)中，用上述方法诱导的NETs与胆管上皮细胞共培养48h，收集胆管上皮细胞总RNA，使用RNA测序分析NETs对正常人胆管上皮细胞转录组的影响。
2实验结果
ZNF709在NETs作用后的胆管细胞中下调(图2A)。
实施例3：ZNF709-P53-MDM2通路在NETs诱导的PBC发展中的作用
1实验方法
通过Western blot检测NETs刺激胆管上皮细胞后ZNF709-P53-MDM2通路中各蛋白的变化，GAPDH作为内参。
2实验结果
在NETs作用下，胆管上皮细胞中ZNF709表达下调，P53、MDM2表达上调，P53-MDM2通路被激活(图2B)。
实施例4：敲低ZNF709基因对胆管上皮细胞的影响
1实验方法
(1)siRNA(序列见表1)转染胆管上皮细胞，敲低ZNF709表达水平，待细胞生长至80-90％，用RIPA提取总蛋白，通过Western blot检测胆管上皮细胞P53及MDM2的表达水平，GAPDH作为内参。
表1siRNA序列
(2)siRNA转染胆管上皮细胞，敲低ZNF709表达水平，待细胞生长至80-90％，用RIPA提取总蛋白，通过Western blot检测检测胆管上皮细胞的ZO-1、CK-7、α-SMA、TGF-β、IL-8、N-cadherin、H2AX、PDC-E2蛋白表达水平，GAPDH作为内参。
(3)siRNA转染胆管上皮细胞，敲低ZNF709表达水平，待细胞生长至80-90％，用流式细胞术检测细胞凋亡(Annexin V-FITC，PI)水平。
2实验结果
(1)敲低ZNF709后，P53-MDM2通路被激活(图2C)。
(2)敲低ZNF709后，胆管上皮细胞发生上皮改变(图2D)、炎症及纤维化改变(图2E)、EMT改变(图2F)、DNA损伤(图2G)、胆管上皮细胞PBC特异抗原PDC-E2过度表达(图2H)。
(3)敲低ZNF709后，胆管上皮细胞发生凋亡(图2I)。
实施例5：过表达ZNF709基因对胆管上皮细胞的影响
1实验方法
慢病毒构建：将SEQ ID NO.1所示ZNF709基因连接至pCDH-CMV-MCS-EF1-Puro载体(艾基生物)的BamH I/EcoR I位点之间，将上述载体转染至HEK293T细胞，获取病毒液。用慢病毒转染胆管上皮细胞，过表达ZNF709基因，NETs与胆管上皮细胞共培养48h，在NETs的刺激下，待细胞生长至80-90％，用RIPA提取总蛋白，通过Western blot检测胆管上皮细胞P53-MDM2通路蛋白的表达水平，ZO-1、CK-7、α-SMA、TGF-β、IL-8、N-cadherin、H2AX、PDC-E2蛋白表达水平，GAPDH作为内参。并通过流式细胞术检测细胞凋亡(Annexin V-FITC，PI)水平。
2实验结果
过表达ZNF709基因后，可以部分抵消NETs所导致的P53-MDM2通路激活(图3A)，并且能够部分缓解NETs诱导胆管上皮细胞的PBC样改变，如上皮改变(图3B)、炎症及纤维化改变(图3C)、EMT改变(图3D)、DNA损伤(图3E)、胆管上皮细胞PBC特异抗原PDC-E2过度表达(图3F)及细胞凋亡(图3G)。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (4)
1.ZNF709基因在制备治疗PBC药物中的应用，其特征在于，所述ZNF709基因的序列如SEQ ID NO.1所示，所述PBC为原发性胆汁性胆管炎。
2.根据权利要求1所述的应用，其特征在于，所述药物中含有所述ZNF709基因或ZNF709基因编码的蛋白。
3.根据权利要求2所述的应用，其特征在于，所述药物还包括药学上可接受的载体。
4.包含权利要求1中所述ZNF709基因的重组载体或包含所述重组载体的重组慢病毒在制备治疗PBC的药物中的应用，其特征在于，所述ZNF709基因的序列如SEQ ID NO.1所示，所述PBC为原发性胆汁性胆管炎。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311455183.1A CN117487816B (zh) | 2023-11-03 | 2023-11-03 | Znf709基因在制备治疗pbc的药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311455183.1A CN117487816B (zh) | 2023-11-03 | 2023-11-03 | Znf709基因在制备治疗pbc的药物中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117487816A CN117487816A (zh) | 2024-02-02 |
| CN117487816B true CN117487816B (zh) | 2024-06-04 |
Family
ID=89675815
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311455183.1A Active CN117487816B (zh) | 2023-11-03 | 2023-11-03 | Znf709基因在制备治疗pbc的药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117487816B (zh) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1307009A (zh) * | 2000-01-21 | 2001-08-08 | 上海博道基因技术有限公司 | 一种新的多肽——人锌指蛋白53和编码这种多肽的多核苷酸 |
| CN1338472A (zh) * | 2000-08-21 | 2002-03-06 | 上海博德基因开发有限公司 | 一种新的多肽——人锌指蛋白75.68和编码这种多肽的多核苷酸 |
| CN107271675A (zh) * | 2017-03-24 | 2017-10-20 | 郑猛 | 抗人adrb3单克隆抗体及其在疾病诊断和治疗中的应用 |
| CN107441089A (zh) * | 2017-08-14 | 2017-12-08 | 合肥工业大学 | 一种列酮类药物的新用途 |
| WO2019018440A1 (en) * | 2017-07-17 | 2019-01-24 | The Broad Institute, Inc. | HUMAN COLON CELL ATLAS IN GOOD HEALTH AND WITH HEMORRHAGIC RECTO-COLITIS |
| CN110261611A (zh) * | 2019-06-14 | 2019-09-20 | 上海四核生物科技有限公司 | Znf709蛋白作为胃癌血清生物标志物的应用及其试剂盒 |
| CN111304314A (zh) * | 2020-02-25 | 2020-06-19 | 四川省人民医院 | Znf124基因在视网膜色素变性疾病早期筛查或辅助诊断中的应用 |
| WO2022040299A2 (en) * | 2020-08-18 | 2022-02-24 | La Jolla Institute For Immunology | Methods and compositions for diagnosing and treating cancer and disease |
| CN115103670A (zh) * | 2019-12-24 | 2022-09-23 | 达纳-法伯癌症研究所股份有限公司 | 转录增强相关结构域(tead)转录因子抑制剂及其用途 |
| WO2022221413A2 (en) * | 2021-04-13 | 2022-10-20 | President And Fellows Of Harvard College | E3 ligase binders and uses thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2668691A1 (en) * | 2006-11-17 | 2008-06-05 | Genizon Biosciences Inc. | Genemap of the human genes associated with crohn's disease |
| WO2019089592A1 (en) * | 2017-10-31 | 2019-05-09 | The General Hospital Corporation | Molecular switch-mediated control of engineered cells |
-
2023
- 2023-11-03 CN CN202311455183.1A patent/CN117487816B/zh active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1307009A (zh) * | 2000-01-21 | 2001-08-08 | 上海博道基因技术有限公司 | 一种新的多肽——人锌指蛋白53和编码这种多肽的多核苷酸 |
| CN1338472A (zh) * | 2000-08-21 | 2002-03-06 | 上海博德基因开发有限公司 | 一种新的多肽——人锌指蛋白75.68和编码这种多肽的多核苷酸 |
| CN107271675A (zh) * | 2017-03-24 | 2017-10-20 | 郑猛 | 抗人adrb3单克隆抗体及其在疾病诊断和治疗中的应用 |
| WO2019018440A1 (en) * | 2017-07-17 | 2019-01-24 | The Broad Institute, Inc. | HUMAN COLON CELL ATLAS IN GOOD HEALTH AND WITH HEMORRHAGIC RECTO-COLITIS |
| CN107441089A (zh) * | 2017-08-14 | 2017-12-08 | 合肥工业大学 | 一种列酮类药物的新用途 |
| CN110261611A (zh) * | 2019-06-14 | 2019-09-20 | 上海四核生物科技有限公司 | Znf709蛋白作为胃癌血清生物标志物的应用及其试剂盒 |
| CN115103670A (zh) * | 2019-12-24 | 2022-09-23 | 达纳-法伯癌症研究所股份有限公司 | 转录增强相关结构域(tead)转录因子抑制剂及其用途 |
| CN111304314A (zh) * | 2020-02-25 | 2020-06-19 | 四川省人民医院 | Znf124基因在视网膜色素变性疾病早期筛查或辅助诊断中的应用 |
| WO2022040299A2 (en) * | 2020-08-18 | 2022-02-24 | La Jolla Institute For Immunology | Methods and compositions for diagnosing and treating cancer and disease |
| WO2022221413A2 (en) * | 2021-04-13 | 2022-10-20 | President And Fellows Of Harvard College | E3 ligase binders and uses thereof |
Non-Patent Citations (7)
| Title |
|---|
| Homo sapiens zinc finger protein 709 (ZNF709), mRNA;Strausberg RL等;Genbank Database;20221224;Accession NO.NM_152601 * |
| Mice deficient in poly(C)-binding protein 4 are susceptible to spontaneous tumors through increased expression of ZFP871 that targets p53 for degradation;Wensheng Yan等;GENES & DEVELOPMENT;20160225;第30卷;第522页摘要 * |
| Neutrophils and neutrophil extracellular traps in the liver and gastrointestinal system;Masaki Honda等;Nature Reviews Gastroenterology & Hepatology;20180131;第15卷;206-221 * |
| Strausberg RL等.Homo sapiens zinc finger protein 709 (ZNF709), mRNA.Genbank Database.2022,Accession NO.NM_152601. * |
| 中性粒细胞胞外诱捕网在原发性胆汁性胆管炎患者中的作用初步探析;偶绎颜等;临床肝胆病杂志;20220414;第38卷(第4期);810-814 * |
| 原发性胆汁性胆管炎中胆管上皮细胞损伤的机制研究进展;唐映梅等;世界华人消化杂志;20191231;第27卷(第1期);36-42 * |
| 基于单细胞转录组的肝癌微环境异质性和胃组织新冠易感性研究;冯超;中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑;20220315;E072-613 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117487816A (zh) | 2024-02-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jäger et al. | Th1, Th17, and Th9 effector cells induce experimental autoimmune encephalomyelitis with different pathological phenotypes | |
| Li et al. | Triptolide ameliorates IL-10-deficient mice colitis by mechanisms involving suppression of IL-6/STAT3 signaling pathway and down-regulation of IL-17 | |
| Lee et al. | Digoxin ameliorates autoimmune arthritis via suppression of Th17 differentiation | |
| Chen et al. | The effect of intra-articular injection of different concentrations of ozone on the level of TNF-α, TNF-R1, and TNF-R2 in rats with rheumatoid arthritis | |
| CN105535022A (zh) | 外泌体在制备治疗急性肝衰竭的药物中的用途和药物组合物 | |
| CN102076351A (zh) | 损伤组织的功能性再生促进药物 | |
| Zhang et al. | Cordycepin (3′-deoxyadenosine) down-regulates the proinflammatory cytokines in inflammation-induced osteoporosis model | |
| Kanai et al. | IL-35 suppresses lipopolysaccharide-induced airway eosinophilia in EBI3-deficient mice | |
| KR20150102957A (ko) | Hmgb1 단편을 이용한 척수 손상에 대한 신규 치료법 | |
| CN113018439A (zh) | 一种缓解重症哮喘的药物、应用及动物模型构建方法 | |
| Luo et al. | Treatment with recombinant Hsp72 suppresses collagen-induced arthritis in mice | |
| CN114990206A (zh) | Commonγ-chain受体作为药物靶点在制备治疗狼疮性肾炎药物中的应用 | |
| CN117487816B (zh) | Znf709基因在制备治疗pbc的药物中的应用 | |
| CN102552935B (zh) | 肝细胞核因子1α治疗慢性肝病的用途 | |
| Bai et al. | Cellular mechanisms of hepatoprotection mediated by M2-like macrophages | |
| Zhou et al. | THH relieves CIA inflammation by reducing inflammatory-related cytokines | |
| CN115120615B (zh) | miR-146a-5p过表达工程干细胞外泌体在制备治疗芥子气致肺损伤药物中的应用 | |
| CN102901826A (zh) | 人类潜在新细胞因子tmem98及其用途 | |
| CN114099495A (zh) | 珊瑚菜内酯及sirt1的上调剂在制备促进炎症疾病创面愈合的药物中的应用 | |
| WO2017066605A1 (en) | Sequential application of macrophages for wound healing | |
| CN113151357A (zh) | 一种Bmi-1-RING1B过表达的血清9型重组腺相关病毒的构建方法及其应用 | |
| CN112125969A (zh) | 生物因子rimbp2在维持内耳毛细胞特性上的应用 | |
| RU2615463C1 (ru) | Способ лечения аллергической бронхиальной астмы, основанный на подавлении экспрессии генов цитокинов IL-4 и IL-13 с использованием молекул миРНК | |
| Chen et al. | WTAP/YTHDF1-mediated m6A modification amplifies IFN-γ-induced immunosuppressive properties of human MSCs | |
| Bi et al. | Effects of leukotriene B4 on interleukin-32, interferon-γ and chemokines in rats with rheumatoid arthritis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |