肉用杂交犊牛小肠上皮细胞的分离培养和鉴定
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摘要
本试验旨在建立肉用杂交犊牛小肠上皮细胞分离培养和鉴定的方法,为研究犊牛小肠上皮细胞营养吸收、免疫调控及肠道屏障功能提供原代细胞培养模型。选用新生未吮乳的肉用杂交犊牛的空肠组织,应用胶原酶Ⅰ和中性酶联合消化法对小肠上皮细胞进行分离,应用相差消化法和相差贴壁法对犊牛小肠上皮细胞进行纯化。通过细胞形态学观察、生长曲线、免疫荧光及染色体核型分析等方法来鉴定细胞。结果表明:1)应用胶原酶Ⅰ和中性酶联合消化法得到的犊牛小肠上皮细胞生长状况良好,经纯化后得到90%以上纯度的犊牛小肠上皮细胞,并稳定传代至10代以上; 2)犊牛小肠上皮细胞的生长曲线为"S"形,符合细胞增殖规律; 3)免疫荧光鉴定犊牛小肠上皮细胞特异性表达角蛋白13和绒毛蛋白; 4)透射电镜观察发现犊牛小肠上皮细胞边缘有微绒毛结构,细胞与细胞之间的紧密连接结构清晰可见; 5)染色体核型分析显示细胞内含有60条染色体,形态正常,呈二倍体核型。综上所述,应用胶原酶Ⅰ和中性酶联合消化及相差贴壁纯化成功得到犊牛小肠上皮细胞,为研究犊牛小肠上皮细胞营养物质吸收、免疫调控和肠道屏障功能提供了细胞素材。
This experiment was designed to establish a method for isolation,culture and identification of hybrid calf intestinal epithelial cells,in order to provide primary cell culture models for the study of intestinal nutrient absorption,immune regulation and intestinal barrier function. Jejunum tissue of newborn hybrid calf was cut down and digested with mixture of collagenase Ⅰ and dispase. The calf intestinal epithelial cells were purified by differential digestion and differential attachment methods. The methods of cell morphology observation,growth curve,immunofluorescence and karyotype analysis were used to identify the cells. The results showed as follows: 1) calf intestinal epithelial cells obtained using mixture of collagenase Ⅰ and dispase digestive method grown well. The calf intestinal epithelial cells could be purified to more than 90%,and could pass more than 10 generations stably. 2) The"S"shape of growth curve of calf intestinal epithelial cells was accordance with the pattern of cell proliferation. 3) The calf intestinal epithelial cells were stained positive for cytokeratin-13 and villin by immunofluorescence identification. 4) Morphological features such as microvilli structure and tight junctions between cells were observed under transmission electron microscope. 5) Karyotype analysis showed that the number of chromosomes with normal morphology and the karyotype was diploid. The above results show that calf intestinal epithelial cells has been successfully obtained by collagenase Ⅰ and dispase combined digestion and differential attachment purification,which can used for the study of intestinal nutrient absorption,immune regulation and the intestinal barrier function.[Chinese Journal of Animal Nutrition,2019,31( 5) : 2278-2286]
This experiment was designed to establish a method for isolation,culture and identification of hybrid calf intestinal epithelial cells,in order to provide primary cell culture models for the study of intestinal nutrient absorption,immune regulation and intestinal barrier function. Jejunum tissue of newborn hybrid calf was cut down and digested with mixture of collagenase Ⅰ and dispase. The calf intestinal epithelial cells were purified by differential digestion and differential attachment methods. The methods of cell morphology observation,growth curve,immunofluorescence and karyotype analysis were used to identify the cells. The results showed as follows: 1) calf intestinal epithelial cells obtained using mixture of collagenase Ⅰ and dispase digestive method grown well. The calf intestinal epithelial cells could be purified to more than 90%,and could pass more than 10 generations stably. 2) The"S"shape of growth curve of calf intestinal epithelial cells was accordance with the pattern of cell proliferation. 3) The calf intestinal epithelial cells were stained positive for cytokeratin-13 and villin by immunofluorescence identification. 4) Morphological features such as microvilli structure and tight junctions between cells were observed under transmission electron microscope. 5) Karyotype analysis showed that the number of chromosomes with normal morphology and the karyotype was diploid. The above results show that calf intestinal epithelial cells has been successfully obtained by collagenase Ⅰ and dispase combined digestion and differential attachment purification,which can used for the study of intestinal nutrient absorption,immune regulation and the intestinal barrier function.[Chinese Journal of Animal Nutrition,2019,31( 5) : 2278-2286]
引文
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[4]李艳,彭春燕,梁榕旺,等.鸡小肠上皮细胞的分离培养与鉴定[J].中国畜牧兽医,2011(2):68-72.
[5]王静,张彦明,仝钢,等.新生仔猪小肠上皮细胞的分离培养和鉴定[J].畜牧兽医学报,2010,41(1):92-98.
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[7]李小芬,詹康,张响英,等.山羊小肠上皮细胞分离培养与鉴定[J].中国农业大学学报,2017,22(10):91-96.
[8]刘飞飞,车东升,穆成龙,等.兔小肠上皮细胞体外分离培养[J].吉林农业大学学报,2011,33(4):429-432.
[9] SCHIERACK P,NORDHOFF M,POLLMANN M,et al. Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in sw ine[J].Histochemistry and Cell Biology,2006,125(3):293-305.
[10] MIYAZAWA K,HONDO T,KANAYA T,et al.Characterization of new ly established bovine intestinal epithelial cell line[J]. Histochemistry and Cell Biology,2010,133(1):125-134.
[11] FOLLMANN W,WEBER S,BIRKNER S. Primary cell cultures of bovine colon epithelium:isolation and cell culture of colonocytes[J]. Toxicology in Vitro,2000,14(5):435-445.
[12]克莱尔·怀斯.上皮细胞培养指南[M].段恩奎,王莉,译.北京:科学出版社,2005:114.
[13] LI P,WILDE C J,FINCH L M,et al.Identification of cell types in the developing goat mammary gland[J].The Histochemical Journal,1999,31(6):379-393.
[14] LORET S,RUSU D,EL MOUALIJ B,et al. Preliminary characterization of jejunocyte and colonocyte cell lines isolated by enzymatic digestion from adult and young cattle[J]. Research in Veterinary Science,2009,87(1):123-132.
[15]詹康,左晓昕,陈银银,等.猪小肠上皮细胞分离培养与鉴定[J].动物营养学报,2015,27(5):1477-1484.
[16] XU Q B,WU Y M,LIU H Y,et al.Establishment and characterization of an omasal epithelial cell model derived from dairy calves for the study of small peptide absorption[J].PLoS One,2014,9(3):e88993.
[17]王治国,卜登攀,王加启.牛乳腺上皮细胞β酪蛋白的检测及核型分析[J].中国畜牧兽医,2007,34(2):8-11.
[18] XAVIER R J,PODOLSKY D K.Unravelling the pathogenesis of inflammatory bow el disease[J]. Nature,2007,448(26):427-434.
[19] RODA G,SARTINI A,ZAMBON E,et al. Intestinal epithelial cells in inflammatory bow el diseases[J].World Journal of Gastroenterology,2010,16(34):4264-4271.
[20] GLAYBURGH D R,SHEN L,TURNER J R. A porous defense:the leaky epithelial barrier in intestinal disease[J]. Laboratory Investigation,2004,84(3):282-291.
[21]王清静,张恒,龙民慧,等.小肠上皮细胞的培养及在动物营养上的研究进展[J].饲料工业,2012,33(4):53-56.
[22] MANICHANH C,REEDER J,GIBERT P,et al. Reshaping the gut microbiome w ith bacterial transplantation and antibiotic intake[J]. Genome Research,2010,20(10):1411-1419.
[23] WILLING B P,RUSSELL S L,FINLAY B B.Shifting the balance:antibiotic effects on host-microbiota mutualism[J].Nature Review s M icrobiology,2011,9(4):233-243.
[24] KUHLMANN I. The prophylactic use of antibiotics in cell culture[J].Cytotechnology,1996,19(2):95-105.
[25]李融,韩庆广,赵国琦.鹅小肠上皮细胞的分离培养研究[J].中国家禽,2009,31(16):22-25.
[26]孙秀梅,程帆,刘维,等.4种小鼠肠上皮细胞方法分离培养方法的比较[J].西北农林科技大学学报(自然科学版),2013,41(5):25-31.
[27]尹博文,陈玲霞,李振宇,等.树鼩小肠上皮细胞的体外分离培养与鉴定[J].实验动物科学,2017,34(2):11-15.
[28] GRAVES C L,HARDEN S W,LAPATO M,et al.A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function[J]. Journal of Immunological M ethods,2014,414:20-31.
[29]薛庆善.体外培养的原理与技术[M].北京:科学出版社,2001.
[30] KHALIL H A,LEI N Y,BRINKLEY G,et al.A novel culture system for adult porcine intestinal crypts[J].Cell and Tissue Research,2016,365(1):123-134.
[31] EHMANN U K,DEVRIES J T,CHEN M S C,et al.An in vitro model of epithelial cell grow th stimulation in the rodent mammary gland[J]. Cell Proliferation,2003,36(4):177-190.
[32] KAEFFER B,BOTTREAU E,VELGE P,et al.Epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar(d/d haplotype)and immortalized by SV40 plasmid[J]. European Journal of Cell Biology,1993,62(1):152-162.
[33] RUSU D,LORET S,PEULEN O,et al.Immunochemical,biomolecular and biochemical characterization of bovine epithelial intestinal primocultures[J]. BM C Cell Biology,2005,6:42
[34] RUNEMBERT I,QUEFFEULOU G,FEDERICI P,et al. Vimentin affects localization and activity of sodiumglucose cotransporter SGLT1 in membrane rafts[J].Journal of Cell Science,2002,115(4):713-724.
[35] SNIDER N T,OMARY M B.Post-translational modifications of intermediate filament proteins:mechanisms and functions[J]. Nature Review s M olecular Cell Biology,2014,15(3):163-177.
[36] LELOUARD H,REGGIO H,MANGEAT P,et al.M ucin-related epitopes distinguish M cells and enterocytes in rabbit appendix and Peyer’s patches[J]. Infection and Immunity,1999,67(1):357-367.
[37] ONORI P,FRANCHITTO A,SFERRA R,et al. Peyer’s patches epithelium in the rat:a morphological,immunohistochemical,and morphometrical study[J].Digestive Diseases and Sciences,2001,46(5):1095-1104.
[38] BEYAZ F,ERGUN E,BAYRAKTAROGLU A G,et al.The identification of intestinal M cells in the sacculus rotundus and appendix of the Angora rabbit[J].Veterinary Research Communications,2010,34(3):255-265.
[39] IWATSUKI H,OGAWA C,SUDA M.Vimentin-positive cells in the villus epithelium of the rabbit small intestine[J]. Histochemistry and Cell Biology,2002,117(4):363-370.
[40]周传丽,刘铮铸,俞英,等.仔猪小肠黏膜上皮细胞体外分离培养及鉴定[J].中国农业科学,2011,44(21):4516-4524.
[2] LIEVIN-LE MOAL V,SERVIN A L.The front line of enteric host defense against unw elcome intrusion of harmful microorganisms:mucins,antimicrobial peptides,and microbiota[J]. Clinical M icrobiology Review s,2006,19(2):315-337.
[3] KAEFFER B. Mammalian intestinal epithelial cells in primary culture:a mini-review[J]. In Vitro Cellular&Developmental Biology-Animal,2002,38(3):123-134.
[4]李艳,彭春燕,梁榕旺,等.鸡小肠上皮细胞的分离培养与鉴定[J].中国畜牧兽医,2011(2):68-72.
[5]王静,张彦明,仝钢,等.新生仔猪小肠上皮细胞的分离培养和鉴定[J].畜牧兽医学报,2010,41(1):92-98.
[6] EVANS G S,FLINT N,SOMERS A S,et al.The development of a method for the preparation of rat intestinal epithelial cell primary cultures[J].Journal of Cell Science,101:219-231.
[7]李小芬,詹康,张响英,等.山羊小肠上皮细胞分离培养与鉴定[J].中国农业大学学报,2017,22(10):91-96.
[8]刘飞飞,车东升,穆成龙,等.兔小肠上皮细胞体外分离培养[J].吉林农业大学学报,2011,33(4):429-432.
[9] SCHIERACK P,NORDHOFF M,POLLMANN M,et al. Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in sw ine[J].Histochemistry and Cell Biology,2006,125(3):293-305.
[10] MIYAZAWA K,HONDO T,KANAYA T,et al.Characterization of new ly established bovine intestinal epithelial cell line[J]. Histochemistry and Cell Biology,2010,133(1):125-134.
[11] FOLLMANN W,WEBER S,BIRKNER S. Primary cell cultures of bovine colon epithelium:isolation and cell culture of colonocytes[J]. Toxicology in Vitro,2000,14(5):435-445.
[12]克莱尔·怀斯.上皮细胞培养指南[M].段恩奎,王莉,译.北京:科学出版社,2005:114.
[13] LI P,WILDE C J,FINCH L M,et al.Identification of cell types in the developing goat mammary gland[J].The Histochemical Journal,1999,31(6):379-393.
[14] LORET S,RUSU D,EL MOUALIJ B,et al. Preliminary characterization of jejunocyte and colonocyte cell lines isolated by enzymatic digestion from adult and young cattle[J]. Research in Veterinary Science,2009,87(1):123-132.
[15]詹康,左晓昕,陈银银,等.猪小肠上皮细胞分离培养与鉴定[J].动物营养学报,2015,27(5):1477-1484.
[16] XU Q B,WU Y M,LIU H Y,et al.Establishment and characterization of an omasal epithelial cell model derived from dairy calves for the study of small peptide absorption[J].PLoS One,2014,9(3):e88993.
[17]王治国,卜登攀,王加启.牛乳腺上皮细胞β酪蛋白的检测及核型分析[J].中国畜牧兽医,2007,34(2):8-11.
[18] XAVIER R J,PODOLSKY D K.Unravelling the pathogenesis of inflammatory bow el disease[J]. Nature,2007,448(26):427-434.
[19] RODA G,SARTINI A,ZAMBON E,et al. Intestinal epithelial cells in inflammatory bow el diseases[J].World Journal of Gastroenterology,2010,16(34):4264-4271.
[20] GLAYBURGH D R,SHEN L,TURNER J R. A porous defense:the leaky epithelial barrier in intestinal disease[J]. Laboratory Investigation,2004,84(3):282-291.
[21]王清静,张恒,龙民慧,等.小肠上皮细胞的培养及在动物营养上的研究进展[J].饲料工业,2012,33(4):53-56.
[22] MANICHANH C,REEDER J,GIBERT P,et al. Reshaping the gut microbiome w ith bacterial transplantation and antibiotic intake[J]. Genome Research,2010,20(10):1411-1419.
[23] WILLING B P,RUSSELL S L,FINLAY B B.Shifting the balance:antibiotic effects on host-microbiota mutualism[J].Nature Review s M icrobiology,2011,9(4):233-243.
[24] KUHLMANN I. The prophylactic use of antibiotics in cell culture[J].Cytotechnology,1996,19(2):95-105.
[25]李融,韩庆广,赵国琦.鹅小肠上皮细胞的分离培养研究[J].中国家禽,2009,31(16):22-25.
[26]孙秀梅,程帆,刘维,等.4种小鼠肠上皮细胞方法分离培养方法的比较[J].西北农林科技大学学报(自然科学版),2013,41(5):25-31.
[27]尹博文,陈玲霞,李振宇,等.树鼩小肠上皮细胞的体外分离培养与鉴定[J].实验动物科学,2017,34(2):11-15.
[28] GRAVES C L,HARDEN S W,LAPATO M,et al.A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function[J]. Journal of Immunological M ethods,2014,414:20-31.
[29]薛庆善.体外培养的原理与技术[M].北京:科学出版社,2001.
[30] KHALIL H A,LEI N Y,BRINKLEY G,et al.A novel culture system for adult porcine intestinal crypts[J].Cell and Tissue Research,2016,365(1):123-134.
[31] EHMANN U K,DEVRIES J T,CHEN M S C,et al.An in vitro model of epithelial cell grow th stimulation in the rodent mammary gland[J]. Cell Proliferation,2003,36(4):177-190.
[32] KAEFFER B,BOTTREAU E,VELGE P,et al.Epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar(d/d haplotype)and immortalized by SV40 plasmid[J]. European Journal of Cell Biology,1993,62(1):152-162.
[33] RUSU D,LORET S,PEULEN O,et al.Immunochemical,biomolecular and biochemical characterization of bovine epithelial intestinal primocultures[J]. BM C Cell Biology,2005,6:42
[34] RUNEMBERT I,QUEFFEULOU G,FEDERICI P,et al. Vimentin affects localization and activity of sodiumglucose cotransporter SGLT1 in membrane rafts[J].Journal of Cell Science,2002,115(4):713-724.
[35] SNIDER N T,OMARY M B.Post-translational modifications of intermediate filament proteins:mechanisms and functions[J]. Nature Review s M olecular Cell Biology,2014,15(3):163-177.
[36] LELOUARD H,REGGIO H,MANGEAT P,et al.M ucin-related epitopes distinguish M cells and enterocytes in rabbit appendix and Peyer’s patches[J]. Infection and Immunity,1999,67(1):357-367.
[37] ONORI P,FRANCHITTO A,SFERRA R,et al. Peyer’s patches epithelium in the rat:a morphological,immunohistochemical,and morphometrical study[J].Digestive Diseases and Sciences,2001,46(5):1095-1104.
[38] BEYAZ F,ERGUN E,BAYRAKTAROGLU A G,et al.The identification of intestinal M cells in the sacculus rotundus and appendix of the Angora rabbit[J].Veterinary Research Communications,2010,34(3):255-265.
[39] IWATSUKI H,OGAWA C,SUDA M.Vimentin-positive cells in the villus epithelium of the rabbit small intestine[J]. Histochemistry and Cell Biology,2002,117(4):363-370.
[40]周传丽,刘铮铸,俞英,等.仔猪小肠黏膜上皮细胞体外分离培养及鉴定[J].中国农业科学,2011,44(21):4516-4524.