CN114480255A - 于污染条件下分离肠道上皮细胞的方法 - Google Patents
于污染条件下分离肠道上皮细胞的方法 Download PDFInfo
- Publication number
- CN114480255A CN114480255A CN202210240520.4A CN202210240520A CN114480255A CN 114480255 A CN114480255 A CN 114480255A CN 202210240520 A CN202210240520 A CN 202210240520A CN 114480255 A CN114480255 A CN 114480255A
- Authority
- CN
- China
- Prior art keywords
- cell
- intestinal
- epithelial cells
- tissue
- supernatant
- 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.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 38
- 210000002490 intestinal epithelial cell Anatomy 0.000 title abstract description 22
- 210000004027 cell Anatomy 0.000 claims abstract description 80
- 210000002919 epithelial cell Anatomy 0.000 claims abstract description 29
- 230000000968 intestinal effect Effects 0.000 claims abstract description 19
- 210000001035 gastrointestinal tract Anatomy 0.000 claims abstract description 14
- 241000894006 Bacteria Species 0.000 claims abstract description 13
- 238000000746 purification Methods 0.000 claims abstract description 13
- 238000000432 density-gradient centrifugation Methods 0.000 claims abstract description 7
- 239000008055 phosphate buffer solution Substances 0.000 claims description 46
- 210000001519 tissue Anatomy 0.000 claims description 43
- 239000006228 supernatant Substances 0.000 claims description 40
- 230000008014 freezing Effects 0.000 claims description 18
- 238000007710 freezing Methods 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 18
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 239000003085 diluting agent Substances 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 14
- 238000000926 separation method Methods 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 10
- 238000007789 sealing Methods 0.000 claims description 10
- 210000002966 serum Anatomy 0.000 claims description 10
- 102100023972 Keratin, type II cytoskeletal 8 Human genes 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- 230000029087 digestion Effects 0.000 claims description 8
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 8
- 210000004379 membrane Anatomy 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 8
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 8
- 239000001103 potassium chloride Substances 0.000 claims description 8
- 235000011164 potassium chloride Nutrition 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 6
- 101000975496 Homo sapiens Keratin, type II cytoskeletal 8 Proteins 0.000 claims description 6
- 108010019160 Pancreatin Proteins 0.000 claims description 6
- 210000002808 connective tissue Anatomy 0.000 claims description 6
- 230000001079 digestive effect Effects 0.000 claims description 6
- 239000012091 fetal bovine serum Substances 0.000 claims description 6
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 6
- 229940055695 pancreatin Drugs 0.000 claims description 6
- 239000008188 pellet Substances 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
- 239000013049 sediment Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000012894 fetal calf serum Substances 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 10
- 241001465754 Metazoa Species 0.000 abstract description 7
- 238000000605 extraction Methods 0.000 abstract description 5
- 241000894007 species Species 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 2
- 230000035790 physiological processes and functions Effects 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract description 2
- 210000000813 small intestine Anatomy 0.000 description 18
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 239000000834 fixative Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 241000283707 Capra Species 0.000 description 7
- 241000283973 Oryctolagus cuniculus Species 0.000 description 7
- 241000700159 Rattus Species 0.000 description 7
- 239000002244 precipitate Substances 0.000 description 5
- 241001494479 Pecora Species 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 239000002131 composite material Substances 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 210000003097 mucus Anatomy 0.000 description 4
- 239000012089 stop solution Substances 0.000 description 4
- 210000004876 tela submucosa Anatomy 0.000 description 4
- 108010070511 Keratin-8 Proteins 0.000 description 3
- 241000282887 Suidae Species 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 210000004347 intestinal mucosa Anatomy 0.000 description 3
- 210000000936 intestine Anatomy 0.000 description 3
- 238000003307 slaughter Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 241000272814 Anser sp. Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282836 Camelus dromedarius Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 206010061307 Neck deformity Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000025174 PANDAS Diseases 0.000 description 1
- 208000021155 Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection Diseases 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 241000282376 Panthera tigris Species 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003181 biological factor Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002183 duodenal effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 210000003405 ileum Anatomy 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000006467 substitution reaction Methods 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
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
-
- 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
- C12N5/0679—Cells of the gastro-intestinal tract
-
- 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
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- General Engineering & Computer Science (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Dermatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本申请公开了一种于污染条件下分离肠道上皮细胞的方法,污染条件包括肠道包含细菌数不低于105cfu/m3以及所述方法实施环境中浮游微生物不低于104cfu/L,该方法包括对肠道组织块进行消化、密度梯度离心得到纯化的上皮细胞步骤。本申请适用于不同年龄阶段和生理状态的不同种属动物(新生和已采食动物均可)肠道上皮细胞的分离纯化,且无需控制和使用无菌提取条件及细胞传代纯化技术,操作方法快速、简便、高效,所用仪器设备为实验常规仪器设备,成本低廉,在医学、动物学、营养学等领域的研究以及试剂盒的研发等具有重要的指导意义。
Description
技术领域
本申请涉及肠道上皮细胞分离技术领域,尤其涉及一种于污染条件下分离肠道上皮细胞的方法,适用于不同年龄阶段和生理状态的不同种属动物(新生和已采食动物均可)现场有菌条件下获取肠道上皮细胞。
背景技术
肠道上皮细胞是肠道主要功能性细胞,在肠道营养物质消化吸收、构成肠道免疫屏障抵御细菌等病原体的入侵以及应急反应中均发挥着重要的作用。由于肠道经常经受食物和药物等等异物的刺激,其微环境中存在着强大的抗感染生物因素。由于受这种特殊的生长条件限制,肠道上皮细胞原代培养较难。
现有技术对于肠道上皮细胞的分离纯化方法主要有酶消化法和组织块,这些方法虽然能提成和分离得到上皮细胞,但是这些操作过程较为繁琐,耗时较长,获取细胞纯度和活细胞数量较低,并且对于一些有污染的肠道或者已经产生肿瘤的肠道,其提取和分离得到的上皮细胞纯度不够,无法满足对于其继续传代培养的要求。
发明内容
有鉴于此,本申请的目的在于建立一种快速、简便、高效,且适用于污染条件下的肠道上皮细胞的分离纯化,在医学、动物学、营养学等领域的研究以及试剂盒的研发等具有重要的指导意义。
本发明的发明人深入研究比较了国内外不同种属动物肠道上皮细胞分离、纯化、以及原代培养等多种方法,通过大量的实验摸索,发明了适用于不同种属动物(不同年龄,不同生理阶段)肠道上皮细胞的分离纯化方法,适合现场有菌条件下提取,更能反映出肠道上皮细胞机能的实际情况,为动物肠道上皮细胞的结构、发育以及物种进化等研究奠定基础。此方法可应用于大鼠、猪、鸡、鸭、鹅、狗、骆驼、牛、马、羊、兔、猴、虎、猫、熊猫、人等肠道上皮的提取、分离及纯化。
为了达到上述目的,本发明具体通过以下技术方案实现:
本申请实施例公开了一种于污染条件下分离肠道上皮细胞的方法,所述污染条件包括肠道以活菌计数计包含细菌数不低于105cfu/m3、以及所述方法实施环境中采用GB/T16293-2010《医药工业洁净室(区)浮游菌的测试方法》检测浮游微生物不低于104cfu/L,所述方法包括以下步骤:
得到去除组织周围的结缔组织和脂肪的肠道组织块;
将所述肠道组织块用0.05~0.25%胰酶消化处理,并用完全培养液终止消化,以得到组织消化液;
将所述组织消化液依次通过100μm细胞滤器和70μm细胞过滤器,离心,得到第一细胞沉淀;
将所述第一细胞沉淀用样本稀释液重悬至浓度为2×108~1×109个/mL,进行密度梯度离心,即可得到第二细胞沉淀。
在本申请实施例中,在对所述肠道组织块进行消化的处理条件为温度-4~40℃,消化0-3h;在对所述组织消化液过滤后的离心条件为400~500g离心10~15min。
在本申请实施例中,所述样本稀释液包含8.0g/L氯化钠、0.2g/L氯化钾、41.44g/L磷酸氢二钠和0.24g/L磷酸二氢钾,所述样本稀释液的pH 7.2~7.5;所述完全培养基为包含10%胎牛血清、1%双抗的DMEM/F12培养基。
在本申请实施例中,所述方法还包括对第二细胞沉淀进行鉴定的步骤。
在本申请实施例中,对第二细胞沉淀进行鉴定的步骤具体包括:
将所述第二细胞沉淀用样本稀释液重悬后,400~500g离心10~15min,弃上清;
加入0.5~1.0mL 4℃预冷的固定剂,室温或37℃孵育5~30min;
400~500g离心10~15min,弃上清;
加入预冷PBS洗涤;
加入1.0~3.0mL破膜剂室温或37℃孵育5~15min,400~500g离心10~15min,弃上清;
加入0.1~1.0mL重悬液后,完成细胞计数;
加1~10μL浓缩型血清,37℃孵育5~30min,洗涤;
加入KRT8抗体室温或37℃孵育30~120min,400~500g离心10~15min,弃上清,加1mL PBS重悬清洗2次;
加入荧光二抗,37℃避光孵育30~60min,400~500g离心10~15min,弃上清,加1mL PBS清洗2次,弃上清,加0.1mL PBS,混匀,最后上机检测。
在本申请实施例中,所述方法还包括对所述肠道组织块进行冻存与复苏的步骤。
在本申请实施例中,对所述肠道组织块进行冻存的步骤具体包括:
将所述肠道组织块装入冻存管中,加入冻存液,封口,置于4℃冰箱30min,随后放入-20℃冰箱1.5h,最后放入-80冰箱保存;
对所述肠道组织块进行复苏的步骤具体包括:将细胞冻存管从-80℃冰箱取出后迅速至于37℃水浴锅中,并摇动冻存管,使之迅速融化,取出组织块,用PBS缓冲液清洗干净,用于后续上皮细胞的分离、纯化及鉴定。
其中,所述PBS缓冲溶液包含8.0g/L氯化钠、0.2g/L氯化钾、41.44g/L磷酸氢二钠和0.24g/L磷酸二氢钾,所述PBS缓冲液的pH为7.2~7.5。
其中,所述细胞冻存液为无血清细胞冻存液或含有10%二甲基亚砜、90%胎牛血清的混合溶液。
与现有技术相比,本申请至少具有以下有益效果:
本申请实施例公开的方法可对污染条件的肠道上皮细胞进行有效的分离,得到的上皮细胞纯度高;并且本申请实施例提供的方法无需控制无菌提取条件和使用细胞传代纯化技术,操作方法快速、简便、高效,所用仪器设备为实验常规仪器设备,成本低廉。
附图说明
图1为本申请实施例提供的大鼠空肠上皮细胞鉴定图。
图2为本申请实施例提供的猪空肠上皮细胞鉴定图。
图3为本申请实施例提供的羊十二指空肠上皮细胞鉴定图。
图4为本申请实施例提供的兔回肠上皮细胞鉴定图。
具体实施方式
为了使本申请的目的、技术方案及优点更加清楚明白,以下结合实施例对本申请进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本申请,并不用于限定本申请。本申请中未详细单独说明的试剂均为常规试剂,均可从商业途径获得;未详细特别说明的方法均为常规实验方法,可从现有技术中获知。
实施例1大鼠肠道上皮细胞的提取与分离
1、材料
8周龄SD大鼠6只,购自南方医科大学实验动物中心。选取SD大鼠6只,短颈处死取出小肠肠段,采用活菌计数法检测其肠道肠段所含污染的微生物含量不低于105cfu/m3。
实施环境条件:采用GB/T16293-2010《医药工业洁净室(区)浮游菌的测试方法》检测浮游微生物不低于104cfu/L。
2、方法
(1)提取细胞
取SD大鼠肠道段,去除组织周围的结缔组织和脂肪,用预冷的PBS冲洗干净,纵向剖开肠段后,用预冷的PBS冲洗干净。将约0.6g肠道组织块剪碎并转入六孔板中,加入1.0mL0.25%胰酶,将六孔板用封口膜封好并转移到水浴锅里,温度37℃,消化30min,用完全培养液终止消化。将消化后的细胞先后通过100μm细胞滤器和70μm细胞过滤器,过滤去除粘液和粘膜下层。500g离心10min,弃上清。
其中,所用PBS缓冲溶液中各组分及含量如下:配方按1L配制,氯化钠8.0g,氯化钾0.2g,磷酸氢二钠41.44g,磷酸二氢钾0.24g,pH7.2~7.5。
所用的完全培养基终止液配方为DMEM/F12培养基(Gibco)、10%胎牛血清,1%双抗(Gibco)。
(2)细胞分离
用样本稀释液重悬组织细胞,将细胞悬液细胞浓度调整为2×108~1×109个/mL,大约1.8g分离的细胞,样本稀释液可以加到2mL。试剂和实验环境均需在20℃左右环境的条件下进行。采用细胞密度梯度离心的原理对上皮细胞进行纯化,具体操作详见试剂说明书,所得沉淀即为肠道上皮细胞。
其中,所用到的纯化试剂盒为大鼠组织上皮细胞分离液试剂盒(EP2012RATK,天津灏洋)。
(3)纯化细胞的鉴定
通过(1)和(2)获取的纯化上皮细胞,500g离心10min,弃上清。加入0.5mL 4℃预冷的固定剂,室温孵育10min。500g离心10min,弃上清。加入预冷PBS洗涤。加入1.5mL破膜剂室温孵育10min,500g离心10min,弃上清。加入0.1mL重悬液后,完成细胞计数。
加5μL浓缩型血清,37℃孵育10min。洗涤。加入KRT8抗体37℃孵育45min。500g离心10min,弃上清。加1mL PBS重悬清洗2次。荧光二抗,37℃避光孵育40min。500g离心10min,弃上清。加1mL PBS清洗2次,弃上清。加0.1mL PBS,混匀。最后上机检测。
其中,所用到的固定剂(上海懋康,MX1502),破膜剂为(上海懋康,MX1503),浓缩型正常山羊血清(AR1009上海懋康生物技术),一抗为Anti-KRT8 Antibody(onoclonal,3G9)(MO1421-3博士德生物工程有限公司)稀释比例为1~3μg/1×106个细胞,荧光二抗为Goatanti-Mouse IgG(H+L)Highly Cross-Adsorbed Secondary Antibo(A-11029invitrogen赛默飞)稀释比例10μg/mL。
3、结果
如图1所示,通过检测结果发现所得上皮细胞纯度可达到95.64%。
实施例2猪肠道上皮细胞的分离
1、材料
13月龄长白猪6头,购自某屠宰场,屠宰后取出小肠肠段,采用活菌计数法检测其肠道肠段所含污染的微生物含量不低于105cfu/m3。
实施环境条件:采用GB/T16293-2010《医药工业洁净室(区)浮游菌的测试方法》检测浮游微生物不低于104cfu/L。
2、方法
(1)细胞分离
取长白猪小肠肠段,去除组织周围的结缔组织和脂肪,用预冷的PBS冲洗干净,纵向剖开肠段后,用预冷的PBS冲洗干净。将约0.6g肠道组织块剪碎并转入六孔板中,加入1.0mL 0.25%胰酶,将六孔板用封口膜封好并转移到水浴锅里,温度37℃,消化30min,用完全培养液终止消化。将消化后的细胞先后通过100μm细胞滤器和70μm细胞过滤器,过滤去除粘液和粘膜下层。500g离心10min,弃上清。
其中,所用PBS缓冲溶液中各组分及含量如下:配方按1L配制,氯化钠8.0g,氯化钾0.2g,磷酸氢二钠41.44g,磷酸二氢钾0.24g,pH7.2~7.5。
所用的完全培养基终止液配方为DMEM/F12培养基(Gibco)、10%胎牛血清,1%双抗(Gibco)。
(2)细胞纯化
用样本稀释液重悬组织细胞,将细胞悬液细胞浓度调整为2×108~1×109个/mL,大约1.8g分离的细胞,样本稀释液可以加到2mL。试剂和实验环境均需在20℃左右环境的条件下进行。采用细胞密度梯度离心的原理对上皮细胞进行纯化,具体操作详见试剂说明书,最终所得沉淀即为肠道上皮细胞。
其中,所用到的纯化试剂盒为猪组织上皮细胞分离液试剂盒(EPHY2012PK,天津灏洋)。
(3)纯化细胞的鉴定
通过(1)和(2)获取的纯化上皮细胞,500g离心10min,弃上清。加入0.5mL 4℃预冷的固定剂,室温孵育10min。500g离心10min,弃上清。加入预冷PBS洗涤。加入1.5mL破膜剂室温孵育10min,500g离心10min,弃上清。加入0.1mL重悬液后,完成细胞计数。
加5μL浓缩型血清,37℃孵育10min。洗涤。加入KRT8抗体37℃孵育45min。500g离心10min,弃上清。加1mL PBS重悬清洗2次。荧光二抗,37℃避光孵育40min。500g离心10min,弃上清。加1mL PBS清洗2次,弃上清。加0.1mL PBS,混匀。最后上机检测。
其中,所用到的固定剂(上海懋康,MX1502),破膜剂为(上海懋康,MX1503),浓缩型正常山羊血清(AR1009上海懋康生物技术),一抗为Cytokeratin8抗体[C-43](FITC)(GTX22530-06,GeneTex),荧光二抗为Goat anti-Mouse IgG(H+L)Highly Cross-AdsorbedSecondary Antibo(A-11029invitrogen赛默飞)稀释比例10μg/mL。
如图2所示,通过检测结果发现所得上皮细胞纯度为96.16%。实施例3羊肠道上皮 细胞的分离
1、材料
13月龄长白猪6头,购自某屠宰场,屠宰后取出小肠肠段,采用活菌计数法检测其肠道肠段所含污染的微生物含量不低于105cfu/m3。
实施环境条件:采用GB/T16293-2010《医药工业洁净室(区)浮游菌的测试方法》检测浮游微生物不低于104cfu/L。
2、方法
(1)细胞分离
取羊小肠肠段,去除组织周围的结缔组织和脂肪,用预冷的PBS冲洗干净,纵向剖开肠段后,用预冷的PBS冲洗干净。将约0.6g肠道组织块剪碎并转入六孔板中,加入1.0mL0.25%胰酶,将六孔板用封口膜封好并转移到水浴锅里,温度37℃,消化30min,用完全培养液终止消化。将消化后的细胞先后通过100μm细胞滤器和70μm细胞过滤器,过滤去除粘液和粘膜下层。500g离心10min,弃上清。
其中,所用PBS缓冲溶液中各组分及含量如下:配方按1L配制,氯化钠8.0g,氯化钾0.2g,磷酸氢二钠41.44g,磷酸二氢钾0.24g,pH7.2~7.5。所用的完全培养基终止液配方为DMEM/F12培养基(Gibco)、10%胎牛血清,1%双抗(Gibco)。
(2)细胞纯化
用样本稀释液重悬组织细胞,将细胞悬液细胞浓度调整为2×108~1×109个/mL,大约1.8g分离的细胞,样本稀释液可以加到2mL。试剂和实验环境均需在20℃左右环境的条件下进行。采用细胞密度梯度离心的原理对上皮细胞进行纯化,具体操作详见试剂说明书,最终所得沉淀即为肠道上皮细胞。
其中,所用到的纯化试剂盒为羊组织上皮细胞分离液试剂盒(EP2012GOX,天津灏洋)。
(3)纯化细胞的鉴定
通过(1)和(2)获取的纯化上皮细胞,500g离心10min,弃上清。加入0.5mL 4℃预冷的固定剂,室温孵育10min。500g离心10min,弃上清。加入预冷PBS洗涤。加入1.5mL破膜剂室温孵育10min,500g离心10min,弃上清。加入0.1mL重悬液后,完成细胞计数。
加5μL浓缩型血清,37℃孵育10min。洗涤。加入KRT8抗体37℃孵育45min。500g离心10min,弃上清。加1mL PBS重悬清洗2次。荧光二抗,37℃避光孵育40min。500g离心10min,弃上清。加1mL PBS清洗2次,弃上清。加0.1mL PBS,混匀。最后上机检测。
其中,所用到的固定剂(上海懋康,MX1502),破膜剂为(上海懋康,MX1503),浓缩型正常山羊血清(AR1009上海懋康生物技术),一抗为Cytokeratin8抗体[C-43](FITC)(GTX22530-06,GeneTex),荧光二抗为Goat anti-Mouse IgG(H+L)Highly Cross-AdsorbedSecondary Antibo(A-11029invitrogen赛默飞)稀释比例10μg/mL。
如图3所示,通过检测结果发现所得上皮细胞纯度为91.11%。实施例4兔肠道上皮 细胞的分离
1、材料
13月龄长白猪6头,购自某屠宰场,屠宰后取出小肠肠段,采用活菌计数法检测其肠道肠段所含污染的微生物含量不低于105cfu/m3。
实施环境条件:采用GB/T16293-2010《医药工业洁净室(区)浮游菌的测试方法》检测浮游微生物不低于104cfu/L。
2、方法
(1)细胞分离
取兔小肠肠段,去除组织周围的结缔组织和脂肪,用预冷的PBS冲洗干净,纵向剖开肠段后,用预冷的PBS冲洗干净。将约0.6g肠道组织块剪碎并转入六孔板中,加入1.0mL0.25%胰酶,将六孔板用封口膜封好并转移到水浴锅里,温度37℃,消化30min,用完全培养液终止消化。将消化后的细胞先后通过100μm细胞滤器和70μm细胞过滤器,过滤去除粘液和粘膜下层。500g离心10min,弃上清。
其中,所用PBS缓冲溶液中各组分及含量如下:配方按1L配制,氯化钠8.0g,氯化钾0.2g,磷酸氢二钠41.44g,磷酸二氢钾0.24g,pH7.2~7.5。所用的完全培养基终止液配方为DMEM/F12培养基(Gibco)、10%胎牛血清,1%双抗(Gibco)。
(2)细胞纯化
用样本稀释液重悬组织细胞,将细胞悬液细胞浓度调整为2×108~1×109个/mL,大约1.8g分离的细胞,样本稀释液可以加到2mL。试剂和实验环境均需在20℃左右环境的条件下进行。采用细胞密度梯度离心的原理对上皮细胞进行纯化,具体操作详见试剂说明书,最终所得沉淀即为肠道上皮细胞。
其中,所用到的纯化试剂盒为兔组织上皮细胞分离液试剂盒(EPH2012PK,天津灏洋)。
(3)纯化细胞的鉴定
通过(1)和(2)获取的纯化上皮细胞,500g离心10min,弃上清。加入0.5mL 4℃预冷的固定剂,室温孵育10min。500g离心10min,弃上清。加入预冷PBS洗涤。加入1.5mL破膜剂室温孵育10min,500g离心10min,弃上清。加入0.1mL重悬液后,完成细胞计数。
加5μL浓缩型血清,37℃孵育10min。洗涤。加入KRT8抗体37℃孵育45min。500g离心10min,弃上清。加1mL PBS重悬清洗2次。荧光二抗,37℃避光孵育40min。500g离心10min,弃上清。加1mL PBS清洗2次,弃上清。加0.1mL PBS,混匀。最后上机检测。
其中,所用到的固定剂(上海懋康,MX1502),破膜剂为(上海懋康,MX1503),浓缩型正常山羊血清(AR1009上海懋康生物技术),一抗为Cytokeratin8抗体[C-43](FITC)(GTX22530-06,GeneTex),荧光二抗为Goat anti-Mouse IgG(H+L)Highly Cross-AdsorbedSecondary Antibo(A-11029invitrogen赛默飞)稀释比例10μg/mL。
如图4所示,通过检测结果发现所得上皮细胞纯度为91.49%。
以上所述,仅为本申请较佳的具体实施方式,但本申请的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本申请揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本申请的保护范围之内。
Claims (7)
1.一种于污染条件下分离肠道上皮细胞的方法,所述污染条件包括肠道以活菌计数计包含细菌数不低于105cfu/m3、以及所述方法实施环境中采用GB/T16293-2010《医药工业洁净室(区)浮游菌的测试方法》检测浮游微生物不低于104cfu/L,所述方法包括以下步骤:
得到去除组织周围的结缔组织和脂肪的肠道组织块;
将所述肠道组织块用0.05~0.25%胰酶消化处理,并用完全培养液终止消化,以得到组织消化液;
将所述组织消化液依次通过100μm细胞滤器和70μm细胞过滤器,离心,第一细胞沉淀;
将所述第一细胞沉淀用样本稀释液重悬至浓度为2×108~1×109个/mL,进行密度梯度离心,即可得到第二细胞沉淀。
2.根据权利要求1所述的方法,其特征在于,在对所述肠道组织块进行消化的处理条件为温度-4~40℃,消化0-3h;在对所述组织消化液过滤后的离心条件为400~500g离心10~15min。
3.根据权利要求1所述的方法,其特征在于,所述样本稀释液包含8.0g/L氯化钠、0.2g/L氯化钾、41.44g/L磷酸氢二钠和0.24g/L磷酸二氢钾,所述样本稀释液的pH 7.2~7.5;
所述完全培养基为包含10%胎牛血清、1%双抗的DMEM/F12培养基。
4.根据权利要求1所述的方法,其特征在于,所述方法还包括对第二细胞沉淀进行鉴定的步骤。
5.根据权利要求4所述的方法,其特征在于,对第二细胞沉淀进行鉴定的步骤具体包括:
将所述第二细胞沉淀用样本稀释液重悬后,400~500g离心10~15min,弃上清;
加入0.5~1.0mL 4℃预冷的固定剂,室温或37℃孵育5~30min;
400~500g离心10~15min,弃上清;
加入预冷PBS洗涤;
加入1.0~3.0mL破膜剂室温或37℃孵育5~15min,400~500g离心10~15min,弃上清;
加入0.1~1.0mL重悬液后,完成细胞计数;
加1~10μL浓缩型血清,37℃孵育5~30min,洗涤;
加入KRT8抗体室温或37℃孵育30~120min,400~500g离心10~15min,弃上清,加1mLPBS重悬清洗2次;
加入荧光二抗,37℃避光孵育30~60min,400~500g离心10~15min,弃上清,加1mLPBS清洗2次,弃上清,加0.1mL PBS,混匀,最后上机检测。
6.根据权利要求5所述的方法,其特征在于,所述方法还包括对所述肠道组织块进行冻存与复苏的步骤。
7.根据权利要求6所述的方法,其特征在于,对所述肠道组织块进行冻存的步骤具体包括:
将所述肠道组织块装入冻存管中,加入冻存液,封口,置于4℃冰箱30min,随后放入-20℃冰箱1.5h,最后放入-80冰箱保存;
对所述肠道组织块进行复苏的步骤具体包括:将细胞冻存管从-80℃冰箱取出后迅速至于37℃水浴锅中,并摇动冻存管,使之迅速融化,取出组织块,用PBS缓冲液清洗干净,用于后续上皮细胞的分离、纯化及鉴定。
其中,所述PBS缓冲溶液包含8.0g/L氯化钠、0.2g/L氯化钾、41.44g/L磷酸氢二钠和0.24g/L磷酸二氢钾,所述PBS缓冲液的pH为7.2~7.5。
其中,所述细胞冻存液为无血清细胞冻存液或含有10%二甲基亚砜、90%胎牛血清的混合溶液。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210240520.4A CN114480255A (zh) | 2022-03-10 | 2022-03-10 | 于污染条件下分离肠道上皮细胞的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210240520.4A CN114480255A (zh) | 2022-03-10 | 2022-03-10 | 于污染条件下分离肠道上皮细胞的方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114480255A true CN114480255A (zh) | 2022-05-13 |
Family
ID=81485652
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210240520.4A Pending CN114480255A (zh) | 2022-03-10 | 2022-03-10 | 于污染条件下分离肠道上皮细胞的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114480255A (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5891651A (en) * | 1996-03-29 | 1999-04-06 | Mayo Foundation For Medical Education And Research | Methods of recovering colorectal epithelial cells or fragments thereof from stool |
CN103060266A (zh) * | 2012-12-14 | 2013-04-24 | 广州呼吸疾病研究所 | 人肺泡ii型上皮细胞的分离培养方法 |
CN104818238A (zh) * | 2015-03-26 | 2015-08-05 | 四川农业大学 | 一种鸡肠上皮细胞分离培养方法 |
CN109294972A (zh) * | 2018-10-17 | 2019-02-01 | 南京农业大学 | 一种羔羊小肠上皮细胞体外培养方法 |
US20190293634A1 (en) * | 2018-02-28 | 2019-09-26 | Albert Li | Isolated intestinal mucosa and uses thereof |
-
2022
- 2022-03-10 CN CN202210240520.4A patent/CN114480255A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5891651A (en) * | 1996-03-29 | 1999-04-06 | Mayo Foundation For Medical Education And Research | Methods of recovering colorectal epithelial cells or fragments thereof from stool |
CN103060266A (zh) * | 2012-12-14 | 2013-04-24 | 广州呼吸疾病研究所 | 人肺泡ii型上皮细胞的分离培养方法 |
CN104818238A (zh) * | 2015-03-26 | 2015-08-05 | 四川农业大学 | 一种鸡肠上皮细胞分离培养方法 |
US20190293634A1 (en) * | 2018-02-28 | 2019-09-26 | Albert Li | Isolated intestinal mucosa and uses thereof |
CN109294972A (zh) * | 2018-10-17 | 2019-02-01 | 南京农业大学 | 一种羔羊小肠上皮细胞体外培养方法 |
Non-Patent Citations (4)
Title |
---|
GHISELLI F等: "Isolation, culture, and characterization of chicken intestinal epithelial cells", 《BMC MOL CELL BIOL》, vol. 22, no. 01, pages 1 - 14 * |
刘倚帆等: "肉用杂交犊牛小肠上皮细胞的分离培养和鉴定", 《动物营养学报》, vol. 31, no. 05, pages 2278 - 2286 * |
张均田等: "《医学实验技术的理论与应用》", vol. 2, 中国协和医科大学出版社, pages: 303 - 305 * |
赵倩明等: "奶牛小肠上皮细胞的原代培养和鉴定", 《中国农业大学学报》, vol. 22, no. 06, pages 84 - 90 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Blaser et al. | Campylobacter enteritis associated with unpasteurized milk | |
Miller et al. | Observations on abortions in cattle: a comparison of pathological, microbiological and immunological findings in aborted foetuses and foetuses collected at abattoirs. | |
CN111961627A (zh) | 一种胞内劳森菌的分离与培养方法 | |
Berrie et al. | Differential recoveries from faecal cultures of larvae of some gastro-intestinal nematodes of cattle | |
US10584370B2 (en) | Screening for L-form bacteria | |
CN114480255A (zh) | 于污染条件下分离肠道上皮细胞的方法 | |
CN108866045A (zh) | 一种人类粪便dna提取方法 | |
JPS58183627A (ja) | 免疫生物学的調製物 | |
CN109370986A (zh) | 一种犬脂肪干细胞的提取方法及其制剂和应用 | |
CN107267467B (zh) | 一种猪繁殖与呼吸综合征病毒的分离方法 | |
CN100392067C (zh) | 胞内劳森氏菌在McCoy细胞中的培养 | |
Elezebeth et al. | The occurrence of Listeria species and antibodies against listeriolysin-O in naturally infected goats | |
Ullmann | Chapter X Methods in Campylobacter | |
Porter et al. | In vivo photolabeling of cells in the colon to assess migratory potential of hematopoietic cells in neonatal mice | |
RU2567845C1 (ru) | СПОСОБ ВЫДЕЛЕНИЯ ЛИЧИНОК T.canis МЕТОДОМ ПЕРЕВАРИВАНИЯ ПАРЕНХИМЫ ЛЕГКИХ И ПЕЧЕНИ ПЛОТОЯДНЫХ ЖИВОТНЫХ | |
CN109633183B (zh) | 一种溶酪大球菌间接血凝抗体检测试剂盒及其制备和应用 | |
Bjöersdorff et al. | Microbiological characterization of porcine fetal islet‐like cell clusters for intended clinical xenografting | |
RU2625031C1 (ru) | Иммуноферментная тест-система для серологической диагностики анаэробной энтеротоксемии животных и контроля напряженности поствакцинального иммунитета | |
Kirkbride | Leptospiral abortion | |
Jacquelot et al. | A protocol to isolate bone marrow innate lymphoid cells for alymphoid mouse reconstitution | |
Hyatt et al. | Usefulness of a commercially available enzyme immunoassay for Shiga-like toxins I and II as a presumptive test for the detection of Escherichia coli O157: H7 in cattle feces | |
CN107236714B (zh) | 一种猪细小病毒的分离方法 | |
CN108060139B (zh) | 一种猪流行性腹泻病毒的分离培养方法 | |
CN118620960A (zh) | 一种犬回肠上皮永生化细胞系的构建方法 | |
Dima et al. | Small animal brucellosis: associated risk factors, seroprevalence and Characterization of Brucella isolates in two districts of South Omo Zone, Ethiopia |
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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220513 |
|
RJ01 | Rejection of invention patent application after publication |