牛肝脏细胞体外培养和传代细胞的初步鉴定
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摘要
该文采用灌注胶原酶消化法分离牛肝细胞,通过DMEM培养基(含10%胎牛血清)体外连续进行原代和传代培养,观察细胞增殖变化及细胞培养过程中的形态消涨变化情况。该文还对第12代培养细胞的染色体情况及细胞生长情况进行分析,同时采用PAS染色法进行糖原含量鉴定。结果显示,分离的肝细胞群体倍增时间为62.8 h,染色体为60条, PAS染色观察发现,培养细胞质中有大量糖原颗粒。
Collagenase perfusion method was used to isolate the cells from bovine hepatocytes,in DMEM medium supplemented with 10% fetal bovine serum(FBS),continuous primary and subculture in vitro and proliferation and morphological changes during cell culture was observed.The chromosome and cell growth curve of the 12th passage of the cells were analyzed,at the same time,Glycogen was tested by periodic acid schiff(PAS).The results showed that the population doubling time for the 12th passage of the cell line was 62.8 h.There were 60 chromosomes and a large number of glycogen granules were observed in the cytoplasm PAS staining.
Collagenase perfusion method was used to isolate the cells from bovine hepatocytes,in DMEM medium supplemented with 10% fetal bovine serum(FBS),continuous primary and subculture in vitro and proliferation and morphological changes during cell culture was observed.The chromosome and cell growth curve of the 12th passage of the cells were analyzed,at the same time,Glycogen was tested by periodic acid schiff(PAS).The results showed that the population doubling time for the 12th passage of the cell line was 62.8 h.There were 60 chromosomes and a large number of glycogen granules were observed in the cytoplasm PAS staining.
引文
1徐雅玲,黄巨恩,刘华刚.体外原代肝细胞分离培养方法的比较.广西医科大学学报(Xu Yaling,Huang Juen,Liu Huagang.Comparison of isolation and culture methods of primary hepatocytes in vitro.Journal of Guangxi Medical University)2011;28(1):158-60.
2 Elgendy R,Giantin M,Dacasto M.Transcriptomic characterization of bovine primary cultured hepatocytes;a crosscomparison with a bovine liver and the Madin-Darby bovine kidney cells.Res Vet Sci 2017;113:40-9.
3刘丽,季辉,彭麟,阮祥春,吉利伟,江善祥,等.鸡肝原代细胞药物代谢模型的建立与优化.南京农业大学学报(Liu Li,Ji Hui,Peng Lin,Ruan Xiangchun,Ji Liwei,Jiang Shanxiang,et al.The establish mentand optimization of the drug metabolic model of adult chicken primary hepatocytes.Journal of Nanjing Agriculture University)2015;38(1):127-33.
4李瑞国,苗朝华,安晓荣,陈永福.牛乳腺上皮细胞体外分离、培养和鉴定的研究.中国农业科技(Li Ruiguo,Miao Chaohua,An Xiaorong,Chen Yongfu.Studies of in vitro isolation culture and identification of bovine mammary gland epithelial cells.Journal of Agricultural Science and Technology)2010;12(4):68-72.
5令世鑫,徐水林,马伟.兰州黑白花奶牛肾组织细胞系的建立,甘肃畜牧兽医(Ling Shixin,Xu Shuilin,Ma Wei.Establishment of kidney cell line in Lanzhou black and white flower cow.Gansu Animal Husbandry and Veterinary)2016;46(5):86-9.
6 Robinson J,Gospodarowicz D.Glycosaminoglycans synthesized by cultured bovine corneal endothelial cells.J Cell Physiol 1983;117(3):368-76.
7吴显实,卫程武,黄克和.原代牛肝细胞分离和培养方法的建立.中国兽医学报(Wu Xianshi,Wei Chengwu,Huang Kehe.Development of the technique for isolation and primary culture of bovine hepatocytes)2009;29(2):203-6.
8庄鹏,李志莹,范紫香,江元森,姚集鲁.改良原代大鼠肝细胞的体外分离培养和功能研究.热带医学杂志(Zhuang Peng,Li Zhiying,Fang Zixiang,Jiang Yuansen,Yao Jilu.Research on the function and morphometrics of cultured primary rat hepatocytes.Journal of Tropical Medicine)2008;8(10):1024-8.
9 Sahara K,Kawamura N,Iizuka T.Analysis of sex chromosome pairings in tetraploid females of the silkworm by using the sexlinked recessive gene.J Seric Sci Jpn 1990;59:196-201.
10张高峰,郭彤,魏华,马珂.采用Percoll法分离、纯化鲫肝细胞.中国水产科学(Zhang Gaofeng,Guo Tong,Wei Hua,Ma Ke.Isolation and purification of carassius auratus hepatocytes by the method of Percoll.Journal of Fishery Sciences of China)2007;14(2):208-15.
11刘学忠,李慧敏,王富民,顾建红,刘宗平.大鼠肝细胞分离和原代培养的简易方法.江苏农业学报(Liu Xuezhong,Li Huimin,Wang Fumin,Gu Jianhong,Liu Zhongping.Asimplified method for separation and primary culture of rat hepatocyte.Jiangsu Journal of Agricultural Sciences)2009;25(1):222-4.
12 Morsiani E,Pazzi P,Puviani AC,Brogli M,Valieri L,Gorini P,et al.Early experiences with a porcine hepatocyte-based bioartificial liver in acute hepatic failure patients.Int J Aritif Organs 2002;25(3):192-202.
13张才,王利民,刘国文,苏蔚,黄建龙,谢光洪,等.犊牛肝细胞的分离与原代培养.细胞生物学杂志(Zhang Cai,Wang Limin,Liu Guoliang,Su Wei,Huang Jianlong,Xie Guanghong,et al.The isolation and primary culture of calf hepatocytes.Chinese Journal of Cell Biology)2007;29:880-4.
14 Giantin M,Gallina G,Pegolo S,Lopparelli RM,Sandron C,Zancanella V.Primary hepatocytes as an useful bioassay to characterize metabolism and bioactivity of illicit steroids in cattle.Toxicol In vitro 2012;26(7):1224-32.
15张莉萍,康格非.原代肝细胞培养的研究现状.国外医学临床生物化学与检验学分册(Zhang Liping,Kang Gefei.Research status of primary hepatocyte culture.Sect Clin Biochem&Lab Med Foreign Med Sci)2004;25(3):193-4.
16梁岳,马广智,方展强.剑尾鱼肝细胞原代培养.中国比较医学杂志[Liang Yue,Ma Guangzhi,Fang Zhanqiang.Primary culture of Swordtail fish(Xiphophorus helleri)hepatocytes.Chinese Journal of Comparative Medicine]2006;16(3):185-8.
2 Elgendy R,Giantin M,Dacasto M.Transcriptomic characterization of bovine primary cultured hepatocytes;a crosscomparison with a bovine liver and the Madin-Darby bovine kidney cells.Res Vet Sci 2017;113:40-9.
3刘丽,季辉,彭麟,阮祥春,吉利伟,江善祥,等.鸡肝原代细胞药物代谢模型的建立与优化.南京农业大学学报(Liu Li,Ji Hui,Peng Lin,Ruan Xiangchun,Ji Liwei,Jiang Shanxiang,et al.The establish mentand optimization of the drug metabolic model of adult chicken primary hepatocytes.Journal of Nanjing Agriculture University)2015;38(1):127-33.
4李瑞国,苗朝华,安晓荣,陈永福.牛乳腺上皮细胞体外分离、培养和鉴定的研究.中国农业科技(Li Ruiguo,Miao Chaohua,An Xiaorong,Chen Yongfu.Studies of in vitro isolation culture and identification of bovine mammary gland epithelial cells.Journal of Agricultural Science and Technology)2010;12(4):68-72.
5令世鑫,徐水林,马伟.兰州黑白花奶牛肾组织细胞系的建立,甘肃畜牧兽医(Ling Shixin,Xu Shuilin,Ma Wei.Establishment of kidney cell line in Lanzhou black and white flower cow.Gansu Animal Husbandry and Veterinary)2016;46(5):86-9.
6 Robinson J,Gospodarowicz D.Glycosaminoglycans synthesized by cultured bovine corneal endothelial cells.J Cell Physiol 1983;117(3):368-76.
7吴显实,卫程武,黄克和.原代牛肝细胞分离和培养方法的建立.中国兽医学报(Wu Xianshi,Wei Chengwu,Huang Kehe.Development of the technique for isolation and primary culture of bovine hepatocytes)2009;29(2):203-6.
8庄鹏,李志莹,范紫香,江元森,姚集鲁.改良原代大鼠肝细胞的体外分离培养和功能研究.热带医学杂志(Zhuang Peng,Li Zhiying,Fang Zixiang,Jiang Yuansen,Yao Jilu.Research on the function and morphometrics of cultured primary rat hepatocytes.Journal of Tropical Medicine)2008;8(10):1024-8.
9 Sahara K,Kawamura N,Iizuka T.Analysis of sex chromosome pairings in tetraploid females of the silkworm by using the sexlinked recessive gene.J Seric Sci Jpn 1990;59:196-201.
10张高峰,郭彤,魏华,马珂.采用Percoll法分离、纯化鲫肝细胞.中国水产科学(Zhang Gaofeng,Guo Tong,Wei Hua,Ma Ke.Isolation and purification of carassius auratus hepatocytes by the method of Percoll.Journal of Fishery Sciences of China)2007;14(2):208-15.
11刘学忠,李慧敏,王富民,顾建红,刘宗平.大鼠肝细胞分离和原代培养的简易方法.江苏农业学报(Liu Xuezhong,Li Huimin,Wang Fumin,Gu Jianhong,Liu Zhongping.Asimplified method for separation and primary culture of rat hepatocyte.Jiangsu Journal of Agricultural Sciences)2009;25(1):222-4.
12 Morsiani E,Pazzi P,Puviani AC,Brogli M,Valieri L,Gorini P,et al.Early experiences with a porcine hepatocyte-based bioartificial liver in acute hepatic failure patients.Int J Aritif Organs 2002;25(3):192-202.
13张才,王利民,刘国文,苏蔚,黄建龙,谢光洪,等.犊牛肝细胞的分离与原代培养.细胞生物学杂志(Zhang Cai,Wang Limin,Liu Guoliang,Su Wei,Huang Jianlong,Xie Guanghong,et al.The isolation and primary culture of calf hepatocytes.Chinese Journal of Cell Biology)2007;29:880-4.
14 Giantin M,Gallina G,Pegolo S,Lopparelli RM,Sandron C,Zancanella V.Primary hepatocytes as an useful bioassay to characterize metabolism and bioactivity of illicit steroids in cattle.Toxicol In vitro 2012;26(7):1224-32.
15张莉萍,康格非.原代肝细胞培养的研究现状.国外医学临床生物化学与检验学分册(Zhang Liping,Kang Gefei.Research status of primary hepatocyte culture.Sect Clin Biochem&Lab Med Foreign Med Sci)2004;25(3):193-4.
16梁岳,马广智,方展强.剑尾鱼肝细胞原代培养.中国比较医学杂志[Liang Yue,Ma Guangzhi,Fang Zhanqiang.Primary culture of Swordtail fish(Xiphophorus helleri)hepatocytes.Chinese Journal of Comparative Medicine]2006;16(3):185-8.