【作者】 廖波；

【导师】 张克英； 陈代文；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2003， 硕士

【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。更多还原

【Abstract】 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 indicateAICAR and Br -AMP can regulate effectively the activity of AMPK in vitro.更多还原