猪肝细胞的分离培养和AMPK活性调控的研究
摘要
本试验在简化细胞培养方法的基础上，研究激活剂5-氨基-4-咪唑羧基酰氨核苷(AICAR)和抑制剂8-溴-腺甘一磷酸(Br~8-AMP)对猪肝细胞AMPK活性的影响。猪肝细胞的分离采用二步胶原酶灌注法，细胞进行悬浮培养，简化之处是在培养过程不充氧气(95∶5)；AMPK活性研究采用单因素设计，设三个处理，分别为对照组、激活组和抑制组，在激活组和抑制组的培养液分别加入AICAR和Br~8-AMP，浓度均为0.2mmol／1。测定30min、60min、180min细胞培养液中的谷丙转氨酶(ALT)、乳酸脱氢酶(LDH)、尿素(BU)和细胞AMPK的活性。结果表明：
本试验采用的猪肝细胞分离方法，能分离得到较多的猪肝细胞，并保证细胞有较好的结构功能完整性，简化的培养操作能在180min内维持细胞较高的活性，可用于AMPK的研究；
在猪肝细胞培养液中分别加入0.2mmol／1的AICAR和Br~8-AMP对仔猪肝细胞AMPK的活性有着不同程度的影响。猪肝细胞培养30min、60min，AICAR使仔猪肝细胞AMPK活性升高分别为8.11％、46.20％；Br~8-AMP使AMPK的活性降低分别为9.13％、22.39％。
本试验表明：在体外研究中激活剂AICAR和抑制剂Br~8-AMP可以有效的调节猪肝细胞AMPK的活性。
The experiment was conducted to study the effects of activator 5-amino-4-imidozolecarboxamide riboside (AICAR) and inhibitor Br8-AMP on AMPK of porcine hepatocytes and explore a method of isolation and culture of pig liver cells. Hepatocytes were isolated by two-step perfusion collagenase and cultured in suspension without gasing with O2/CO2(95 : 5) .The harvested cells were divided three treatments: the control, the activation and inhibiton incubated with 0.2mmol/l AICAR and'Br -AMP in medium respectively. The ALT,LDH ,BU of medium and the activity AMPK of hepatocytes were measured. The result showed:
A number of hepatocytes with fine viability and specific function were harvested by the method of isolation and simplified culture in the experiment. The activity of AMPK were increased by 8.11% and 46.20% (p<0.01) respectively with AICAR at SOmin and 60min, Br8-AMP decreased the activity by 9.13%<, 22.39% respectively.The data indicate
AICAR and Br -AMP can regulate effectively the activity of AMPK in vitro.
本试验采用的猪肝细胞分离方法，能分离得到较多的猪肝细胞，并保证细胞有较好的结构功能完整性，简化的培养操作能在180min内维持细胞较高的活性，可用于AMPK的研究；
在猪肝细胞培养液中分别加入0.2mmol／1的AICAR和Br~8-AMP对仔猪肝细胞AMPK的活性有着不同程度的影响。猪肝细胞培养30min、60min，AICAR使仔猪肝细胞AMPK活性升高分别为8.11％、46.20％；Br~8-AMP使AMPK的活性降低分别为9.13％、22.39％。
本试验表明：在体外研究中激活剂AICAR和抑制剂Br~8-AMP可以有效的调节猪肝细胞AMPK的活性。
The experiment was conducted to study the effects of activator 5-amino-4-imidozolecarboxamide riboside (AICAR) and inhibitor Br8-AMP on AMPK of porcine hepatocytes and explore a method of isolation and culture of pig liver cells. Hepatocytes were isolated by two-step perfusion collagenase and cultured in suspension without gasing with O2/CO2(95 : 5) .The harvested cells were divided three treatments: the control, the activation and inhibiton incubated with 0.2mmol/l AICAR and'Br -AMP in medium respectively. The ALT,LDH ,BU of medium and the activity AMPK of hepatocytes were measured. The result showed:
A number of hepatocytes with fine viability and specific function were harvested by the method of isolation and simplified culture in the experiment. The activity of AMPK were increased by 8.11% and 46.20% (p<0.01) respectively with AICAR at SOmin and 60min, Br8-AMP decreased the activity by 9.13%<, 22.39% respectively.The data indicate
AICAR and Br -AMP can regulate effectively the activity of AMPK in vitro.
引文
程宝鸾主编．2000．动物细胞培养技术．华南理工大学出版社
鄂征主编．1995．组织培养和分子细胞学技术．北京出版社
D.L.斯佩克特 R.D.戈得曼 L.A.莱因万德著．黄培堂等译．2001．细胞实验指南．科学出版社
陈柯 高毅 潘玉先 杨继震．1999．有效原代猪肝细胞培养体系的研制．世界华人消化杂志．7(3)：206-209
陈代文 张克英．2002．AMPK对动物营养代谢的调节作用．动物营养学报．14(3)：1-6
丁义涛 陈钟 张鹤云．2001．肝细胞大规模培养的一种简便方法．江苏医药杂志．27(7)：492-494
施玮 朱惠刚 晏晓蓉 周至俊．2001．微囊藻提取物对大鼠原代培养的肝细胞酶学和形态学影响研究．环境与健康杂志．18(5)：259-261
陈凯耀 王宇明．2002．生物人工肝研究的若干新进展．世界华人消化杂志．10(1)：69-72
韩聚强．2002．体外肝细胞培养技术新进展．河北医科大学学报．23(3)：184-186
鹿晓晶 张华东 沈杏生．2002，肝细胞体外培养技术及其应用．防化研究．1：64-65
田京珠．2000．血清丙氨酸转氨酶增高的结果分析．实用医技．7(6)：419
杨红 骆抗生 王崇国．1996．慢性肝炎临床诊断和病理分级分期诊断的病理分析．临床肝胆杂志．12(4)：213
于聪慧 冷希圣 魏玉华 刘继超 杜如昱．2000．胶原酶及灌流时间对大鼠肝细胞分离效果的影响．中华实验外科杂志．17(6)：541-542
张阳德 范立侨 陈伟．2002．肝细胞体外培养研究进展．中国现代医学杂志．12(15)：1-4
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Bergeron.R.R.R.Russell.L.H.Young. J-M.Ren. M.Marcucci.A.Lee. G.I.Shulman. 1999.Effect of AMPKactivated on muscle glucose metabolism in conscious rats,American Journal Physiology. E938-E944
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Blair. E.C.Redwood.H.Ashrafian. M.Oliveira. J.Broxhholme. B.Kerr. A.Sslmon. I.O.Smith and H.Watkins.2001.Mutations in the γ_2 subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy:evidence for the central role of energy compromis in diseae pathogensie,Human Molecular Genetics. 10(11):1215-220
Bolster.D.R.S.J. Crozier. S.R. Kimball. and L.S. Jefferson,2002. AMP-activated protein kinase suppresses Protein Synthesis in Rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) Signaling,Journal Biological Chemistry.277(27):23977-23980
Brody. T. 1999,Nutritional Biochemistry(2nd. Ed)(M),Academic Press.P1006
Carling. D.D.G.Hardie. 1989.The substrate and sequence specificity of the AMP-activated protein kinase,Phosphorylation of glycogen synthase and phosphorylase kinase. Biochimica et Biophysica Acta. 1012:81-86
Carling.D.K.Aguan.A.Woods.A.J.M.Verhoeven.R.K.Beri.C.H.Brennan.C.Sidebottom.M.D.Davision.J.S cott,1994.Mammalian AMP-activated protein kinase is homologous to yeast and plant protein kinases involved in the regulation of carbon metabolism. The Journal of Biological Chemistry.269(115): 11442-11448
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Carlson.C.L.W.W.Winder. 1999． Liver AMP-activated protein kinase and acetyl-CoA carboxylase during and after exercise,Journal Apply Physiology 86(2) :669-674
Cheung.P.C.F.I.P.Salt.S.P.Davies.D.G.Hardie.D.Carling.2000． Characterization of AMP-activated protein kinaseγ-subnit isoforms and their role in AMP binding.Biochemical Journa.,346:659-669;
Daniel.T.D.Carling.2002． Functional analysis of mutations in the γ2 subunit of AMP-activated protein kinase associated with cardiac hypertrophy and wolff-parkinson-white syndrome.The Journal of Biological Chemistry.277(52) :51017-51024
Davies.S.P.D.Carling.D.G.Hardie.1989． Tissue distribution of the AMP-activated protein kinase.and lack of activation by cyclic-AMP-dependent protein kinase,studied using a specific and sensitive peptide assay.European Journal Biochemistry. 186:123-128
Dubey.R.K.D.G.Gillespie.E.K.Jackson. 1998． Cyclic AMP-adenosine pathway induces nitric oxide synthesis in aortic smooth muscle cell.Hypertension.31(2) :296-302
Fluckiger-Isler.R.E.Kux.P.Walter. 1996． 5-Iodotubercidin and proglycosyn:a comparision of two glycogenic compounds in hepatocytes from fasted rats,Biochimica et Biophysica Acta. 1311:77-84
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鄂征主编．1995．组织培养和分子细胞学技术．北京出版社
D.L.斯佩克特 R.D.戈得曼 L.A.莱因万德著．黄培堂等译．2001．细胞实验指南．科学出版社
陈柯 高毅 潘玉先 杨继震．1999．有效原代猪肝细胞培养体系的研制．世界华人消化杂志．7(3)：206-209
陈代文 张克英．2002．AMPK对动物营养代谢的调节作用．动物营养学报．14(3)：1-6
丁义涛 陈钟 张鹤云．2001．肝细胞大规模培养的一种简便方法．江苏医药杂志．27(7)：492-494
施玮 朱惠刚 晏晓蓉 周至俊．2001．微囊藻提取物对大鼠原代培养的肝细胞酶学和形态学影响研究．环境与健康杂志．18(5)：259-261
陈凯耀 王宇明．2002．生物人工肝研究的若干新进展．世界华人消化杂志．10(1)：69-72
韩聚强．2002．体外肝细胞培养技术新进展．河北医科大学学报．23(3)：184-186
鹿晓晶 张华东 沈杏生．2002，肝细胞体外培养技术及其应用．防化研究．1：64-65
田京珠．2000．血清丙氨酸转氨酶增高的结果分析．实用医技．7(6)：419
杨红 骆抗生 王崇国．1996．慢性肝炎临床诊断和病理分级分期诊断的病理分析．临床肝胆杂志．12(4)：213
于聪慧 冷希圣 魏玉华 刘继超 杜如昱．2000．胶原酶及灌流时间对大鼠肝细胞分离效果的影响．中华实验外科杂志．17(6)：541-542
张阳德 范立侨 陈伟．2002．肝细胞体外培养研究进展．中国现代医学杂志．12(15)：1-4
Beg. Z.H.J.A.gtonik.H.B.Brewer. JR.1979.Characterization and regulation of reductase kinase that modulates the enzymic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase,Proc. Natl. A cad. Sci. USA.76:4375-4379
Bergeron.R.R.R.Russell.L.H.Young. J-M.Ren. M.Marcucci.A.Lee. G.I.Shulman. 1999.Effect of AMPKactivated on muscle glucose metabolism in conscious rats,American Journal Physiology. E938-E944
Beri.R.K.A.E.Marley. C.G.See. W.F. Sopwith.K.Aguan.D.Carling. J.Scott.F. Carey. 1994.Molecular cloning,expression and chomsomal localization of human AMP-activated protein kinase,FEBS Letters.356:117-121
Blair. E.C.Redwood.H.Ashrafian. M.Oliveira. J.Broxhholme. B.Kerr. A.Sslmon. I.O.Smith and H.Watkins.2001.Mutations in the γ_2 subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy:evidence for the central role of energy compromis in diseae pathogensie,Human Molecular Genetics. 10(11):1215-220
Bolster.D.R.S.J. Crozier. S.R. Kimball. and L.S. Jefferson,2002. AMP-activated protein kinase suppresses Protein Synthesis in Rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) Signaling,Journal Biological Chemistry.277(27):23977-23980
Brody. T. 1999,Nutritional Biochemistry(2nd. Ed)(M),Academic Press.P1006
Carling. D.D.G.Hardie. 1989.The substrate and sequence specificity of the AMP-activated protein kinase,Phosphorylation of glycogen synthase and phosphorylase kinase. Biochimica et Biophysica Acta. 1012:81-86
Carling.D.K.Aguan.A.Woods.A.J.M.Verhoeven.R.K.Beri.C.H.Brennan.C.Sidebottom.M.D.Davision.J.S cott,1994.Mammalian AMP-activated protein kinase is homologous to yeast and plant protein kinases involved in the regulation of carbon metabolism. The Journal of Biological Chemistry.269(115): 11442-11448
Carling. D.P.R.Clarke.V.A.Zammit. D.G.Hardie. 1989.Purification and characterization of the
AMP-activated protein kinase.European Journal of Biochemistry.186:129-136
Carlson.C.L.W.W.Winder. 1999． Liver AMP-activated protein kinase and acetyl-CoA carboxylase during and after exercise,Journal Apply Physiology 86(2) :669-674
Cheung.P.C.F.I.P.Salt.S.P.Davies.D.G.Hardie.D.Carling.2000． Characterization of AMP-activated protein kinaseγ-subnit isoforms and their role in AMP binding.Biochemical Journa.,346:659-669;
Daniel.T.D.Carling.2002． Functional analysis of mutations in the γ2 subunit of AMP-activated protein kinase associated with cardiac hypertrophy and wolff-parkinson-white syndrome.The Journal of Biological Chemistry.277(52) :51017-51024
Davies.S.P.D.Carling.D.G.Hardie.1989． Tissue distribution of the AMP-activated protein kinase.and lack of activation by cyclic-AMP-dependent protein kinase,studied using a specific and sensitive peptide assay.European Journal Biochemistry. 186:123-128
Dubey.R.K.D.G.Gillespie.E.K.Jackson. 1998． Cyclic AMP-adenosine pathway induces nitric oxide synthesis in aortic smooth muscle cell.Hypertension.31(2) :296-302
Fluckiger-Isler.R.E.Kux.P.Walter. 1996． 5-Iodotubercidin and proglycosyn:a comparision of two glycogenic compounds in hepatocytes from fasted rats,Biochimica et Biophysica Acta. 1311:77-84
Fluckiger-Isler.R.P.Walter. 1993． Stimulation of rat liver glycogen synthesis by the adenosine kinase inhibitor 5-iodotubercidin.Biochemical Journal.292:85-91
Freshney.R.I.2000． Culture of animal cells:a manual of basic techniques.Published simultaneously in Canada
Garton.A.J.D.G.Campbell.D.Carling.D.G.Hardie.R.J.Colbran.S.J.Yeaman.1989． Phosphorylation of bovine hormone-sensitive lipase by the AMP-activated protein kinase.The Biochemical Journal. 179:249-254
Hamilton.S.R.D.Stapleton.J.B.O'Donnell.Jr.J.T.Kung.S.R.Dalal.B.E.Kemp.L.A.witters.2001． An activating mutation in the γ1 subunit of the AMP-activated protein kinase.FEBS Letters.500:163-168
Hardie.D.G.D.Carling.1997． The AMP-activate protein kinase fuel gauge of the mammalian cell?European Journal Biochemistry.246:259-273
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