CN114250194A - 一种急性胰腺炎细胞模型的构建方法和用途 - Google Patents
一种急性胰腺炎细胞模型的构建方法和用途 Download PDFInfo
- Publication number
- CN114250194A CN114250194A CN202111446561.0A CN202111446561A CN114250194A CN 114250194 A CN114250194 A CN 114250194A CN 202111446561 A CN202111446561 A CN 202111446561A CN 114250194 A CN114250194 A CN 114250194A
- Authority
- CN
- China
- Prior art keywords
- acute pancreatitis
- cell model
- glucose
- cell
- time
- 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.)
- Granted
Links
- 206010033645 Pancreatitis Diseases 0.000 title claims abstract description 58
- 206010033647 Pancreatitis acute Diseases 0.000 title claims abstract description 56
- 201000003229 acute pancreatitis Diseases 0.000 title claims abstract description 56
- 238000010276 construction Methods 0.000 title claims abstract description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 50
- 239000008103 glucose Substances 0.000 claims abstract description 50
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 30
- 239000000194 fatty acid Substances 0.000 claims abstract description 30
- 229930195729 fatty acid Natural products 0.000 claims abstract description 30
- 150000004665 fatty acids Chemical class 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 25
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 9
- 239000003814 drug Substances 0.000 claims abstract description 7
- 229940079593 drug Drugs 0.000 claims abstract description 5
- 238000012216 screening Methods 0.000 claims abstract description 5
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 claims description 32
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 16
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 16
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims description 16
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 16
- 239000005642 Oleic acid Substances 0.000 claims description 16
- 235000021314 Palmitic acid Nutrition 0.000 claims description 16
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 16
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 claims description 16
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 16
- 238000011534 incubation Methods 0.000 claims description 8
- 241001465754 Metazoa Species 0.000 claims description 5
- 230000017074 necrotic cell death Effects 0.000 abstract description 31
- 238000011282 treatment Methods 0.000 abstract description 27
- 230000000694 effects Effects 0.000 abstract description 9
- 238000011161 development Methods 0.000 abstract description 5
- 230000007246 mechanism Effects 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 4
- 230000001939 inductive effect Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 93
- 206010028851 Necrosis Diseases 0.000 description 28
- 230000001965 increasing effect Effects 0.000 description 13
- 239000000243 solution Substances 0.000 description 10
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 239000007995 HEPES buffer Substances 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 238000010306 acid treatment Methods 0.000 description 6
- 210000004923 pancreatic tissue Anatomy 0.000 description 6
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 6
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 3
- 102000029816 Collagenase Human genes 0.000 description 3
- 108060005980 Collagenase Proteins 0.000 description 3
- 229930186217 Glycolipid Natural products 0.000 description 3
- 210000001015 abdomen Anatomy 0.000 description 3
- 229960002424 collagenase Drugs 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 241000269435 Rana <genus> Species 0.000 description 2
- TYWXNGXVSZRXNA-NVZSGMJQSA-N Ranunculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC[C@@H]1C=CC(=O)O1 TYWXNGXVSZRXNA-NVZSGMJQSA-N 0.000 description 2
- TYWXNGXVSZRXNA-UHFFFAOYSA-N Ranunculin Natural products OC1C(O)C(O)C(CO)OC1OCC1C=CC(=O)O1 TYWXNGXVSZRXNA-UHFFFAOYSA-N 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 102000038379 digestive enzymes Human genes 0.000 description 2
- 108091007734 digestive enzymes Proteins 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000001338 necrotic effect Effects 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 108010010737 Ceruletide Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 241001235578 Rana dybowskii Species 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000018690 Trypsinogen Human genes 0.000 description 1
- 108010027252 Trypsinogen Proteins 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 229930190815 caerulein Natural products 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- YRALAIOMGQZKOW-HYAOXDFASA-N ceruletide Chemical compound C([C@@H](C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)[C@@H](C)O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(OS(O)(=O)=O)C=C1 YRALAIOMGQZKOW-HYAOXDFASA-N 0.000 description 1
- 229960001706 ceruletide Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 238000013227 male C57BL/6J mice Methods 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000277 pancreatic duct Anatomy 0.000 description 1
- 210000001819 pancreatic juice Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical group C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- YRALAIOMGQZKOW-UHFFFAOYSA-N sulfated caerulein Natural products C=1C=CC=CC=1CC(C(N)=O)NC(=O)C(CC(O)=O)NC(=O)C(CCSC)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)CNC(=O)C(C(C)O)NC(=O)C(NC(=O)C(CC(O)=O)NC(=O)C(CCC(N)=O)NC(=O)C1NC(=O)CC1)CC1=CC=C(OS(O)(=O)=O)C=C1 YRALAIOMGQZKOW-UHFFFAOYSA-N 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 230000008718 systemic inflammatory response Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Images
Classifications
-
- 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
- C12N5/0676—Pancreatic 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/507—Pancreatic 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/36—Lipids
-
- 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
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供了一种急性胰腺炎细胞模型的构建方法和用途,属于细胞模型领域。本发明首次发现葡萄糖处理能够显著增加胰腺腺泡细胞坏死数量,显著降低半最大效应时间,成功构建急性胰腺炎细胞模型。本发明首次发现葡萄糖联合脂肪酸处理能够进一步增加诱导胰腺腺泡细胞坏死程度,成功构建急性胰腺炎细胞模型。本发明采用的原料易得,价格便宜,降低了建模成本;本发明在较短的时间内就能成功构建急性胰腺炎细胞模型,缩短了建模时间。本发明的建模方法方便快捷,容易操作,成本较低,具有良好的可重复性。本发明构建的急性胰腺炎细胞模型不仅可以用于急性胰腺炎治疗药物的筛选,还能够为急性胰腺炎的发生发展机制提供良好的平台,应用前景广阔。
Description
技术领域
本发明属于细胞模型领域,具体涉及一种急性胰腺炎细胞模型的构建方法和用途。
背景技术
急性胰腺炎(acute pancreatitis,AP)是由多种病因导致胰腺组织自身消化所致的胰腺水肿、出血及坏死等炎性损伤。临床主要表现为急性、持续的中上腹疼痛,伴有恶心、呕吐等,血淀粉酶或脂肪酶升高超过正常值上限的3倍,同时伴有典型影像学改变,重症患者炎症波及全身,常常合并局部或全身并发症,预后较差。胰腺炎特征性的表现包括高淀粉酶血症,消化酶在腺泡内异常活化(如胰蛋白酶原变为胰蛋白酶),腺泡细胞中大空泡的积聚,促炎介质的释放(如关键转录因子NF-K B)导致炎症细胞侵入胰腺和全身炎症反应,进而引起凋亡和坏死,最终引起腺泡细胞的死亡。目前普遍认为急性胰腺炎开始于胰腺腺泡细胞,胰腺腺泡细胞坏死能够诱发强烈的炎症反应,进而导致急性胰腺炎。
急性胰腺炎是一种发病率和死亡率很高的可导致死亡的疾病,它的病理机制尚不明确,目前尚无特殊或有效的治疗方法。为了进一步研究急性胰腺炎的发展机制,并开发出有效治疗急性胰腺炎的药物,构建急性胰腺炎体外模型具有重要意义。
雨蛙素(Caerulein),又名雨蛙肽,是澳大利亚产的蛙HYlaCaerulea的皮肤提取物。它是一种胆囊收缩素类似物,作用于胰腺腺泡细胞可引起大量消化酶和胰液的分泌,从而导致急性水肿性胰腺炎。文献(中国现代医学杂志,2014年6月,第24卷第18期)报道了应用雨蛙肽刺激大鼠胰腺腺泡细胞AR42J细胞24小时来构建急性胰腺炎体外细胞模型的方法。但是,该方法采用的刺激物雨蛙素价格昂贵,难以获得,增加了构建急性胰腺炎细胞模型的成本;此外,该方法需要处理24小时,时间较长,增加了构建急性胰腺炎细胞模型的时间。因此,开发出一种成本更低、时间更短的构建急性胰腺炎细胞模型的新方法具有重要意义。
发明内容
本发明的目的在于提供一种急性胰腺炎细胞模型的构建方法和用途。
本发明提供了葡萄糖在构建急性胰腺炎细胞模型中的用途。
本发明还提供了葡萄糖与脂肪酸联用在构建急性胰腺炎细胞模型中的用途。
进一步地,所述脂肪酸为棕榈酸、油酸中的一种或两种。
本发明还提供了一种急性胰腺炎细胞模型的构建方法,所述构建方法包括以下步骤:将胰腺腺泡细胞与葡萄糖共同孵育,得到急性胰腺炎细胞模型。
本发明还提供了另一种急性胰腺炎细胞模型的构建方法,所述构建方法包括以下步骤:将胰腺腺泡细胞与葡萄糖和脂肪酸共同孵育,得到急性胰腺炎细胞模型。
进一步地,所述脂肪酸为棕榈酸、油酸中的一种或两种。
进一步地,所述脂肪酸为棕榈酸和油酸;其中,棕榈酸的浓度为150~250μM,优选为200μM,油酸的浓度为150~250μM,优选为200μM。
进一步地,所述共同孵育的温度为室温,时间为30min以上;
所述葡萄糖的浓度20mM以上;
所述胰腺腺泡细胞为动物胰腺腺泡细胞。
进一步地,所述共同孵育的时间为12小时以上;
所述葡萄糖的浓度25~100mM;
所述胰腺腺泡细胞为小鼠胰腺腺泡细胞。
本发明还提供了上述方法构建的急性胰腺炎细胞模型在筛选预防和/或治疗急性胰腺炎的药物中的用途。
本发明首次发现葡萄糖处理能够显著增加胰腺腺泡细胞坏死数量,显著降低半最大效应时间,成功构建急性胰腺炎细胞模型。本发明首次发现葡萄糖联合脂肪酸处理能够进一步增加诱导胰腺腺泡细胞坏死程度,成功构建急性胰腺炎细胞模型。
本发明的建模方法采用的原料易得,价格便宜,降低了建模成本;本发明的建模方法在较短的时间内就能成功构建急性胰腺炎细胞模型,缩短了建模时间。本发明的建模方法方便快捷,容易操作,成本较低,具有良好的可重复性。
本发明构建的急性胰腺炎细胞模型不仅可以用于急性胰腺炎治疗药物的筛选,还能够为急性胰腺炎的发生发展机制提供良好的平台。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1葡萄糖处理胰腺腺泡细胞后的多功能荧光酶标仪测试结果:
(A)25mM葡萄糖处理荧光时间变化图;(B)50mM葡萄糖处理荧光时间变化图;(C)100mM葡萄糖处理荧光时间变化图;(D)25、50、100mM葡萄糖处理荧光时间变化图;(E)半最大效应(half-maximal response,HMR)时间统计结果。图中,*表示P<0.05,**表示P<0.01,***表示P<0.001。图2葡萄糖联合脂肪酸处理胰腺腺泡细胞后的多功能荧光酶标仪测试结果:(A)荧光时间变化图;(B)半最大效应(half-maximal response,HMR)时间统计结果。图中,**表示P<0.01,***表示P<0.001。
图3葡萄糖联合脂肪酸处理胰腺腺泡细胞后的荧光显微镜法测试结果:
(A)细胞荧光染色代表图;(B)细胞坏死比率结果。图中,***表示P<0.001。
具体实施方式
本发明所用原料与设备均为已知产品,通过购买市售产品所得。
实施例1:构建糖脂代谢紊乱病相关急性胰腺炎细胞模型的方法
一、实验方法
1、实验动物
SPF级雄性C57BL/6J小鼠(7-8周)购于北京华阜康生物科技股份有限公司,许可证:SCXK(京)2020-0004。于四川大学华西医院实验动物中心动物房分笼饲养,每5只一笼,恒温(25±2℃)且照明控制(12h白天/黑夜循环),自由摄食、饮水,适应性喂养1周后正式开始实验。本实验由四川大学华西医院实验动物中心伦理委员会审核通过,所有的动物实验及相关操作均按照学校和国家标准执行。
2、新鲜分离小鼠胰腺腺泡细胞
提前配制4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)溶液和胶原酶溶液。将小鼠颈椎脱臼处死后,消毒腹部,开腹迅速取出胰腺组织。将37℃预热好的胶原酶溶液沿主胰管注入胰腺组织至各小叶充盈透亮。将注射充盈的胰腺组织及胶原酶一起37℃水浴消化17min。消化结束后,吸出胰腺组织至适量HEPES溶液,进行吹打。吹打后静置沉淀,将上清不断转移至70μM细胞筛过滤。重复以上吹打、静置、过滤步骤至胰腺组织基本消化,无法吹下细胞为止。将所得细胞悬液260g室温离心2min后,倒去上清;重复一次再加入4-6mL HEPES溶液重悬混匀后即得小鼠胰腺腺泡细胞。取少量小鼠胰腺腺泡细胞重悬液于玻片上,在4×、10×视野下分别观察细胞形态(圆润透亮为细胞状态好;胰腺腺泡细胞易成团,3-4个细胞成团时细胞密度适宜)。
3、细胞处理
3.1葡萄糖处理:用不同浓度葡萄糖(HG,25、50、100mM)的HEPES溶液在室温下孵育新鲜分离胰腺腺泡细胞(细胞密度为5-10×106个/mL),以相同浓度甘露醇(25、50、100mM)的HEPES溶液为渗透压对照组。使用多功能荧光酶标仪法实时检测孵育12小时内的细胞坏死情况。
3.2葡萄糖联合脂肪酸处理:用不同浓度葡萄糖(25、50mM)联合脂肪酸(棕榈酸:PA,200μM;油酸:OA,200μM)在室温下孵育新鲜分离胰腺腺泡细胞(细胞密度为5-10×106个/mL),以不添加任何刺激物作为空白组。使用多功能荧光酶标仪法实时检测孵育12小时内的细胞坏死情况,使用荧光显微镜法检测孵育30min后的细胞坏死情况。
其中,使用多功能荧光酶标仪法实时检测细胞坏死情况的方法如下:
采用坏死细胞荧光探针碘化丙啶(propidium iodide,PI)标记细胞并使用多功能酶标仪检测其荧光随时间的变化。简单来说,向分离新鲜小鼠胰腺腺泡细胞中加入6mL含PI探针的HEPES溶液,PI终浓度为1.5μmol/L,注意避光。20min后,将细胞悬液转移至黑色透明平底96孔板,采用多功能荧光酶标仪测定荧光变化,设置激发光为535nm,发射光为617nm,读数间隔为120s每次。首先记录20min以内的荧光值(即前10个点),记为基线;再按照预先计算好的浓度加入刺激物,记录各时间点的荧光值;记基线荧光值的平均值为F0,各时间点荧光值为F,时间荧光曲线图以F/F0表示。每次实验各组均有4个复孔,相同的实验均独立重复3次以上,计算多次实验的平均值和标准误绘制时间荧光曲线图。计算半最大效应(half-maximal response,HMR)时间,HMR=最大死亡效应时间/2。
使用荧光显微镜法检测细胞坏死情况的方法如下:
将新鲜分离好的腺泡细胞平均分为空白组(Ctrl)、模型组(HG+PA+OA),每组1mL反应体系。模型组给予葡萄糖(25mM)联合脂肪酸(棕榈酸,PA,200μM;油酸,OA,200μM)室温孵育30min。孵育结束后,260g室温离心2min,加入0.5mL HEPES溶液重悬,再分别加入PI(终浓度为0.25μmol/L)和Hoechest33342核酸染色剂(终浓度为50μg/mL)充分混匀。取20μL细胞溶液置于载玻片上,盖上盖玻片静止后,于正置荧光显微镜下拍照。采用Hoechest33342染色(蓝色荧光)计数总细胞,PI染色(红色荧光)计数坏死细胞,用坏死细胞数量除以细胞总数再取百分数,即为坏死比率。每次实验独立重复三次。
二、实验结果
1、葡萄糖处理对胰腺腺泡细胞坏死的影响
多功能荧光酶标仪测试结果如图1所示,可以看出,与对照组相比,葡萄糖处理后的胰腺腺泡细胞坏死数量显著增加,HMR时间显著降低。
此外,胰腺腺泡细胞坏死数量随葡萄糖浓度的增加而增加,HMR时间随葡萄糖浓度的增加而降低,表明胰腺腺泡细胞的坏死程度随葡萄糖浓度的增加而增加。
上述实验结果表明,本发明用葡萄糖处理的方法能够诱导胰腺腺泡细胞坏死,成功构建糖脂代谢紊乱病相关急性胰腺炎细胞模型。
2、葡萄糖联合脂肪酸处理对胰腺腺泡细胞坏死的影响
多功能荧光酶标仪检测结果如图2所示,可以看出,与空白照组相比,葡萄糖联合脂肪酸处理30min后的胰腺腺泡细胞坏死数量就显著增加,并且随着处理时间的延长,葡萄糖联合脂肪酸处理组的胰腺腺泡细胞坏死数量越来越多。与空白照组相比,葡萄糖联合脂肪酸处理后的胰腺腺泡细胞坏死数量显著增加,HMR时间显著降低。
此外,葡萄糖联合脂肪酸处理能够诱导胰腺腺泡细胞坏死,并且,与葡萄糖(25mM)联合脂肪酸(棕榈酸,200μM;油酸,200μM)组相比,葡萄糖(50mM)联合脂肪酸(棕榈酸,200μM;油酸,200μM)组诱导胰腺腺泡细胞坏死的作用更佳;与葡萄糖(25mM)联合脂肪酸(棕榈酸,200μM;油酸,200μM)组相比,葡萄糖(50mM)联合脂肪酸(棕榈酸,200μM;油酸,200μM)组的HMR时间显著降低,表明胰腺腺泡细胞的坏死程度随联合处理药物中葡萄糖浓度的增加而增加。
结合图1和图2的数据还可以看出,与单独采用葡萄糖处理相比,葡萄糖联合脂肪酸处理能够进一步降低HMR时间,提高胰腺腺泡细胞的坏死程度。
荧光显微镜法检测结果如图3所示,可以看出,与空白组相比,葡萄糖(25mM)联合脂肪酸(棕榈酸,200μM;油酸,200μM)处理30min后能够显著提高胰腺腺泡细胞坏死比率,与多功能荧光酶标仪检测结果一致。
上述实验结果表明,本发明葡萄糖联合脂肪酸(棕榈酸和油酸)处理的方法能够诱导诱导胰腺腺泡细胞坏死,成功构建糖脂代谢紊乱病相关急性胰腺炎细胞模型。
综上,本发明提供了一种急性胰腺炎细胞模型的构建方法和用途。本发明首次发现葡萄糖处理能够显著增加胰腺腺泡细胞坏死数量,显著降低半最大效应时间,成功构建急性胰腺炎细胞模型。本发明首次发现葡萄糖联合脂肪酸处理能够进一步增加诱导胰腺腺泡细胞坏死程度,成功构建急性胰腺炎细胞模型。本发明采用的原料易得,价格便宜,降低了建模成本;本发明在较短的时间内就能成功构建急性胰腺炎细胞模型,缩短了建模时间。本发明的建模方法方便快捷,容易操作,成本较低,具有良好的可重复性。本发明构建的急性胰腺炎细胞模型不仅可以用于急性胰腺炎治疗药物的筛选,还能够为急性胰腺炎的发生发展机制提供良好的平台,应用前景广阔。
Claims (10)
1.葡萄糖在构建急性胰腺炎细胞模型中的用途。
2.葡萄糖与脂肪酸联用在构建急性胰腺炎细胞模型中的用途。
3.根据权利要求2所述的用途,其特征在于:所述脂肪酸为棕榈酸、油酸中的一种或两种。
4.一种急性胰腺炎细胞模型的构建方法,其特征在于:所述构建方法包括以下步骤:将胰腺腺泡细胞与葡萄糖共同孵育,得到急性胰腺炎细胞模型。
5.一种急性胰腺炎细胞模型的构建方法,其特征在于:所述构建方法包括以下步骤:将胰腺腺泡细胞与葡萄糖和脂肪酸共同孵育,得到急性胰腺炎细胞模型。
6.根据权利要求5所述的构建方法,其特征在于:所述脂肪酸为棕榈酸、油酸中的一种或两种。
7.根据权利要求6所述的构建方法,其特征在于:所述脂肪酸为棕榈酸和油酸;其中,棕榈酸的浓度为150~250μM,优选为200μM,油酸的浓度为150~250μM,优选为200μM。
8.根据权利要求5~7任一项所述的构建方法,其特征在于:所述共同孵育的温度为室温,时间为30min以上;
所述葡萄糖的浓度20mM以上;
所述胰腺腺泡细胞为动物胰腺腺泡细胞。
9.根据权利要求8所述的构建方法,其特征在于:所述共同孵育的时间为12小时以上;
所述葡萄糖的浓度25~100mM;
所述胰腺腺泡细胞为小鼠胰腺腺泡细胞。
10.权利要求4~9任一项所述方法构建的急性胰腺炎细胞模型在筛选预防和/或治疗急性胰腺炎的药物中的用途。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111446561.0A CN114250194B (zh) | 2021-11-30 | 2021-11-30 | 一种急性胰腺炎细胞模型的构建方法和用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111446561.0A CN114250194B (zh) | 2021-11-30 | 2021-11-30 | 一种急性胰腺炎细胞模型的构建方法和用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114250194A true CN114250194A (zh) | 2022-03-29 |
CN114250194B CN114250194B (zh) | 2023-04-11 |
Family
ID=80791465
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111446561.0A Active CN114250194B (zh) | 2021-11-30 | 2021-11-30 | 一种急性胰腺炎细胞模型的构建方法和用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114250194B (zh) |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040127406A1 (en) * | 2002-05-28 | 2004-07-01 | Presnell Sharon C. | Methods for in vitro expansion and transdifferentiation of human pancreatic acinar cells into insulin-producing cells |
AU2005301152A1 (en) * | 2004-10-29 | 2006-05-11 | Genentech, Inc. | Disruptions of genes encoding secreted proteins, compositions and methods relating thereto |
US20120171168A1 (en) * | 2005-06-17 | 2012-07-05 | Song Sun Uk | Method for treating pancreatitis with mesenchymal stem cells |
CN102827253A (zh) * | 2011-06-17 | 2012-12-19 | 上海市第一人民医院 | 一种抑制炎症反应的小分子多肽及其应用 |
CN106727666A (zh) * | 2016-11-21 | 2017-05-31 | 江南大学 | 低酯果胶在防治急性胰腺炎症方面的用途 |
CN110170046A (zh) * | 2019-05-21 | 2019-08-27 | 温州医科大学 | 成纤维细胞生长因子21在制备治疗急性胰腺炎药物中的应用 |
WO2019236528A1 (en) * | 2018-06-05 | 2019-12-12 | Anji Pharma (Us) Llc | Compositions and methods for treating pancreatitis |
CN113424795A (zh) * | 2021-05-24 | 2021-09-24 | 四川大学华西医院 | 一种急性胰腺炎动物模型的构建方法和用途 |
-
2021
- 2021-11-30 CN CN202111446561.0A patent/CN114250194B/zh active Active
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040127406A1 (en) * | 2002-05-28 | 2004-07-01 | Presnell Sharon C. | Methods for in vitro expansion and transdifferentiation of human pancreatic acinar cells into insulin-producing cells |
CN1819838A (zh) * | 2002-05-28 | 2006-08-16 | 贝克顿·迪金森公司 | 人类胰腺腺泡细胞体外扩增并转分化为胰岛素产生细胞的方法 |
AU2005301152A1 (en) * | 2004-10-29 | 2006-05-11 | Genentech, Inc. | Disruptions of genes encoding secreted proteins, compositions and methods relating thereto |
US20120171168A1 (en) * | 2005-06-17 | 2012-07-05 | Song Sun Uk | Method for treating pancreatitis with mesenchymal stem cells |
CN102827253A (zh) * | 2011-06-17 | 2012-12-19 | 上海市第一人民医院 | 一种抑制炎症反应的小分子多肽及其应用 |
CN106727666A (zh) * | 2016-11-21 | 2017-05-31 | 江南大学 | 低酯果胶在防治急性胰腺炎症方面的用途 |
WO2019236528A1 (en) * | 2018-06-05 | 2019-12-12 | Anji Pharma (Us) Llc | Compositions and methods for treating pancreatitis |
CN110170046A (zh) * | 2019-05-21 | 2019-08-27 | 温州医科大学 | 成纤维细胞生长因子21在制备治疗急性胰腺炎药物中的应用 |
CN113424795A (zh) * | 2021-05-24 | 2021-09-24 | 四川大学华西医院 | 一种急性胰腺炎动物模型的构建方法和用途 |
Non-Patent Citations (8)
Title |
---|
SEO JEONGYEON 等: ""Protective effect of lycopene on oxidative stress-induced cell death of pancreatic acinar cells"" * |
SONG JY 等: ""Oxidative stress induces nuclear loss of DNA repair proteins Ku70 and Ku80 and apoptosis in pancreatic acinar AR42J cells"" * |
YANG XM 等: ""Stress Hyperglycemia Is Independently Associated with Persistent Organ Failure in Acute Pancreatitis"" * |
于金宁 等: "\"中性粒细胞对大鼠急性重症胰腺炎胰腺腺泡凋亡的作用\"" * |
曾悦 等: ""内质网应激在高脂血症相关性大鼠急性胰腺炎发病中的作用"" * |
朱斌: ""DNaseⅠ与2型糖尿病胰腺损伤关系探讨:从Bcl-2/Caspase-3通路探讨高血糖环境中DNaseⅠ增高与胰腺损伤的关系"" * |
杨鑫敏 等: ""高三酰甘油血症性急性胰腺炎的基因学研究进展"" * |
舒梅铃 等: ""游离脂肪酸在高脂血症性胰腺炎中的作用"" * |
Also Published As
Publication number | Publication date |
---|---|
CN114250194B (zh) | 2023-04-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2446892B1 (de) | Isolierte adulte pluripotente Stammzellen | |
Pan et al. | Viability and differentiation of neural precursors on hyaluronic acid hydrogel scaffold | |
Bjerkvig et al. | Glioma cell interactions with fetal rat brain aggregates in vitro and with brain tissue in vivo | |
US20030161818A1 (en) | Cultures, products and methods using stem cells | |
JP2021518331A (ja) | 細胞加齢の反転のための一過性細胞リプログラミング | |
JPS61108386A (ja) | インビトロ細胞培養系 | |
CN108451979B (zh) | 一种具有辅助治疗前列腺癌的番茄红素复方制剂及其应用 | |
Zhao et al. | The three‐dimensional nanofiber scaffold culture condition improves viability and function of islets | |
CN110475856A (zh) | 使用纳米纤维的细胞培养 | |
Silva | The onset of phagocytosis and identity in the embryo of Lytechinus variegatus | |
Wang et al. | Preparation of collagen/chitosan microspheres for 3D macrophage proliferation in vitro | |
CN109219661A (zh) | 蛋白质生产方法 | |
Lin et al. | Enhanced cell survival of melanocyte spheroids in serum starvation condition | |
Rattner et al. | Chromatin organization during meiotic prophase of Bombyx mori | |
Kanda et al. | A simple technique for in vivo observation of SCE in mouse ascites tumor and spermatogonial cells | |
CN114250194B (zh) | 一种急性胰腺炎细胞模型的构建方法和用途 | |
Sun et al. | Isolation of ready-made rat microvessels and its applications in effective in vivo vascularization and in angiogenic studies in vitro | |
JPWO2019178296A5 (zh) | ||
Yazdekhasti et al. | Improved isolation, proliferation, and differentiation capacity of mouse ovarian putative stem cells | |
Scalzone et al. | A cytokine-induced spheroid-based in vitro model for studying osteoarthritis pathogenesis | |
Sun et al. | Growth of miniature pig parotid cells on biomaterials in vitro | |
CN114350593A (zh) | 一种非酒精性脂肪性肝病肝脂肪变性模型的构建方法及其应用 | |
Hu et al. | Effects of different concentrations of type-I collagen hydrogel on the growth and differentiation of chondrocytes | |
Stiller et al. | Morphogenesis of intracytoplasmic dense (inclusion) bodies in a recurring digital fibrous tumor of childhood: Light-and electron-microscopic investigations | |
Moghaddam et al. | Human olfactory epithelium-derived stem cells ameliorate histopathological deficits and improve behavioral functions in a rat model of cerebellar ataxia |
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 |