牦牛小肠上皮细胞分离培养及鉴定
|
摘要
为体外研究牦牛肠道粘膜免疫及肠道微生物与肠粘膜之间的互作关系提供细胞,本试验采用组织块培养法培养牦牛小肠上皮细胞(intestinal epithelial cell,IEC),差异消化法进行纯化,免疫细胞化学法进行鉴定,并用细胞计数法测定其生长曲线。组织块培养法结合差异消化法可分离纯化出牦牛小肠上皮细胞,免疫细胞化学法检测显示该细胞波形蛋白染色为阴性,角蛋白染色为阳性,阳性率达到90%;细胞生长曲线显示细胞接种1d后开始贴壁生长,3-6d进入对数生长期,从第7d开始细胞增殖基本处于停滞期。采用组织块培养法成功获得了牦牛小肠上皮细胞,证明该方法可为研究牦牛肠道免疫和肠道微生物与肠粘膜之间的互作提供牦牛小肠上皮细胞。
To provide cells for interaction between the in vitro study of yak intestinal mucosal immunity, intestinal microflora and intestinal mucosa in this experiment, intestinal epithelial cells of yak were cultured by explant culture method. The cells were purified by differential digestion, and identified by immunocytochemistry.The growth curves were determined by cell counting method. The results showed that the method of explant culture combined with differential digestion can purify intestinal epithelial cells of yak, immunohistochemistry showed that the cells were negative for vimentin staining and cytokeratin staining was positive, the positive rate reached 90%; The cell growth curve showed that the cells began to adhere to the wall after inoculation with 1 d, and 3-6 d entered the logarithmic growth phase. The cell proliferation began to stagnate at the beginning of 7 d. Yak small intestinal epithelial cells were successfully obtained by tissue mass culture, it is proved that this method can provide the yak intestinal epithelial cells for the study of yak intestinal immunity and the interaction between intestinal microorganism and intestinal mucosa.
To provide cells for interaction between the in vitro study of yak intestinal mucosal immunity, intestinal microflora and intestinal mucosa in this experiment, intestinal epithelial cells of yak were cultured by explant culture method. The cells were purified by differential digestion, and identified by immunocytochemistry.The growth curves were determined by cell counting method. The results showed that the method of explant culture combined with differential digestion can purify intestinal epithelial cells of yak, immunohistochemistry showed that the cells were negative for vimentin staining and cytokeratin staining was positive, the positive rate reached 90%; The cell growth curve showed that the cells began to adhere to the wall after inoculation with 1 d, and 3-6 d entered the logarithmic growth phase. The cell proliferation began to stagnate at the beginning of 7 d. Yak small intestinal epithelial cells were successfully obtained by tissue mass culture, it is proved that this method can provide the yak intestinal epithelial cells for the study of yak intestinal immunity and the interaction between intestinal microorganism and intestinal mucosa.
引文
[1]朱翠,师子彪,蒋宗勇,等.乳酸杆菌在调节肠道屏障功能中的作用[J].中国畜牧兽医,2012,39(09):118-122.
[2]尚沁沁,李雅丽,史艳云,等.益生菌对动物肠上皮细胞免疫功能的研究进展[J].中国畜牧杂志,2014,50(13):87-90.
[3]杨雪菲,漆艳娥,太珍珍,等.肠道菌群在机体免疫调节功能中的作用[J].中国微生态学杂志,2016,28(08):979-983.
[4]王静,何生虎,葛松,等.犊牛腹泻的发病原因及治疗的研究进展[J].农业科学研究,2013,34(03):59-62.
[5]付永凯.牛肠道疾病的发病原因及防治策略[J].畜牧兽医科学(电子版),2017,(04):37.
[6]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(01):92-98
[7]詹康,左晓昕,陈银银,等.猪小肠上皮细胞分离培养与鉴定[J/OL].动物营养学报,2015,27(05):1477-1484.
[8]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.
[9]Liu Z,Zhang P,Zhou Y,et al.Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3[J].Brazilian Journal of Medical and Biological Research,2010,43(05):451-459.
[10]黄鸿眉,程茜.双歧杆菌对肠上皮细胞生长和白介素-8分泌功能的影响[J].中国微生态学杂志,2008,20(02):137-139.
[11]古少鹏,王敏霞,赵素芬,等.鸡胚盲肠上皮细胞原代培养与鉴定[J].畜牧兽医学报,2009,40(11):1699-1704.
[12]王雪云,刘芳,蒋大伟,等.鸡肠上皮细胞原代培养与鉴定[J].黑龙江畜牧兽医,2015,(21):10-12+284.
[13]Yamada K,Sato K,Morishita S,et al.Establishment of a primary culture method for mouse intestinal epithelial cells by organ culture of fetal small intestine[J].Bioscience,Biotechnology,and Biochemistry,2009,73(8):1849-1855.
[14]韩庆广,徐晓燕,赵国琦,等.小鼠小肠上皮细胞的分离培养研究[J].家畜生态学报,2009,30(01):48-51.
[15]范斌,余冰,田刚,等.新生家兔空肠上皮细胞的体外分离培养与鉴定[J].动物营养学报,2014,26(6):1570-1578.
[16]Lowiese MB Desmarets,Sebastiaan Theuns,Dominique AJOlyslaegers,et al.Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses[J].Veterinary Research,2013,44(01):71.
[17]刘烨,赵桂华,尹昆,等.猫肠上皮细胞的原代培养及c DNA文库的构建[J].中国血吸虫病防治杂志,2017,29(04):464-467+474.
[18]Rusu Dorina,Loret Suzanne,Peulen Olivier,et al.Immunochemical,biomolecular and biochemical characterization of bovine epithelial intestinal primocultures[J].BMC Cell Biology,2005,6:42.
[19]赵倩明,左晓昕,詹康,等.奶牛小肠上皮细胞的原代培养和鉴定[J].中国农业大学学报,2017,22(06):84-90.
[20]王海霞,匡伟,赵国琦.小肠上皮细胞分离纯化方法的研究进展[J].中国畜牧杂志,2008,44(01):54-57.
[21]李小芬,詹康,张响英,等.山羊小肠上皮分离培养与鉴定[J].中国农业大学学报,2017,22(10):91-96.
[22]Miyazawa,Kohtaro,Hondo,et al.Characterization of newly established bovine intestinal epithelial cell line[J].Histochemistry and Cell Biology,2010,133(01),125-34.
[2]尚沁沁,李雅丽,史艳云,等.益生菌对动物肠上皮细胞免疫功能的研究进展[J].中国畜牧杂志,2014,50(13):87-90.
[3]杨雪菲,漆艳娥,太珍珍,等.肠道菌群在机体免疫调节功能中的作用[J].中国微生态学杂志,2016,28(08):979-983.
[4]王静,何生虎,葛松,等.犊牛腹泻的发病原因及治疗的研究进展[J].农业科学研究,2013,34(03):59-62.
[5]付永凯.牛肠道疾病的发病原因及防治策略[J].畜牧兽医科学(电子版),2017,(04):37.
[6]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(01):92-98
[7]詹康,左晓昕,陈银银,等.猪小肠上皮细胞分离培养与鉴定[J/OL].动物营养学报,2015,27(05):1477-1484.
[8]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.
[9]Liu Z,Zhang P,Zhou Y,et al.Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3[J].Brazilian Journal of Medical and Biological Research,2010,43(05):451-459.
[10]黄鸿眉,程茜.双歧杆菌对肠上皮细胞生长和白介素-8分泌功能的影响[J].中国微生态学杂志,2008,20(02):137-139.
[11]古少鹏,王敏霞,赵素芬,等.鸡胚盲肠上皮细胞原代培养与鉴定[J].畜牧兽医学报,2009,40(11):1699-1704.
[12]王雪云,刘芳,蒋大伟,等.鸡肠上皮细胞原代培养与鉴定[J].黑龙江畜牧兽医,2015,(21):10-12+284.
[13]Yamada K,Sato K,Morishita S,et al.Establishment of a primary culture method for mouse intestinal epithelial cells by organ culture of fetal small intestine[J].Bioscience,Biotechnology,and Biochemistry,2009,73(8):1849-1855.
[14]韩庆广,徐晓燕,赵国琦,等.小鼠小肠上皮细胞的分离培养研究[J].家畜生态学报,2009,30(01):48-51.
[15]范斌,余冰,田刚,等.新生家兔空肠上皮细胞的体外分离培养与鉴定[J].动物营养学报,2014,26(6):1570-1578.
[16]Lowiese MB Desmarets,Sebastiaan Theuns,Dominique AJOlyslaegers,et al.Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses[J].Veterinary Research,2013,44(01):71.
[17]刘烨,赵桂华,尹昆,等.猫肠上皮细胞的原代培养及c DNA文库的构建[J].中国血吸虫病防治杂志,2017,29(04):464-467+474.
[18]Rusu Dorina,Loret Suzanne,Peulen Olivier,et al.Immunochemical,biomolecular and biochemical characterization of bovine epithelial intestinal primocultures[J].BMC Cell Biology,2005,6:42.
[19]赵倩明,左晓昕,詹康,等.奶牛小肠上皮细胞的原代培养和鉴定[J].中国农业大学学报,2017,22(06):84-90.
[20]王海霞,匡伟,赵国琦.小肠上皮细胞分离纯化方法的研究进展[J].中国畜牧杂志,2008,44(01):54-57.
[21]李小芬,詹康,张响英,等.山羊小肠上皮分离培养与鉴定[J].中国农业大学学报,2017,22(10):91-96.
[22]Miyazawa,Kohtaro,Hondo,et al.Characterization of newly established bovine intestinal epithelial cell line[J].Histochemistry and Cell Biology,2010,133(01),125-34.