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建鲤体内外急性肝损伤模型的建立
曹丽萍,贾睿,丁炜东,杜金梁,殷国俊
0
(中国水产科学研究院淡水渔业研究中心,农业部水生动物遗传育种和养殖生物学重点开放实验室,无锡 214081)
摘要:
以四氯化碳(CCl4)为肝毒剂,建立建鲤体内外急性肝损伤模型。体内,采用腹腔注射法造模,按0.1 mL/10 g一次性分别注射0、6.25%、12.5%和15% CCl4-橄榄油溶液(v/v),于造模后24~144 h取血,测定建鲤血清中谷丙转氨酶(GPT)、谷草转氨酶(GOT)、乳酸脱氢酶(LDH)和 丙二醛(MDA)水平;体外,0、2、4、8、16、32 mmol·L-1 CCl4分别作用于原代培养的建鲤肝细胞4 h后,用噻唑蓝(MTT)法检测细胞生存状况,同时收集细胞培养上清,测定其GPT、 GOT 、LDH、MDA、超氧化物歧化酶(SOD)和谷胱甘肽(GSH)活力。结果显示,12.5%、15% CCl4-橄榄油溶液腹腔注射建鲤24 h后即能引起血清中GOT、GPT和MDA显著升高,15% CCl4溶液造模组48 h能显著提高血清中LDH水平,4个指标均在CCl4作用96 h后恢复到接近正常水平; 8~32 mmol·L-1 CCl4作用体外培养肝细胞4 h时,细胞上清中GPT、 GOT 、LDH和MDA含量显著升高、SOD和GSH活力显著下降,肝细胞存活率显著下降,分别为空白对照组的66.29%、54%和37%,且有剂量依赖性。结果表明,分别采用15% CCl4-橄榄油腹腔注射建鲤72 h和8 mmol·L-1 CCl4作用体外培养肝细胞4 h可以构建体内外急性肝损伤模型。
关键词:  四氯化碳  建鲤  模型  MTT
DOI:
投稿时间:2012-03-14
基金项目:科技部国际科技合作项目(2009DFA32620), 无锡市科技计划(国际科技合作)(CZE00906)和中央级公益性科研院所基本科研业务费专项资金(中国水产科学研究院淡水渔业研究中心)项目(2011JBFC05)共同资助。
Establishment of the acute hepatic injury model induced by CCl4 in Cyprinus carpio var. jian in vivo and in vitro
CAO Li-ping,JIA Rui,DING Wei-dong,DU Jin-liang,YIN Guo-jun
(Key Open Laboratory for Genetic Breeding of Aquatic Animals and Aquaculture Biology, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Science, Wuxi 214081)
Abstract:
The present study aimed to develop acute liver injury models in vivo and in vitro using carbon tetrachloride (CCl4) as hepatotoxicant to lay a foundation for screening and evaluating liver-protective drugs. In vivo, Jian carps were injected intraperitoneally with CCl4 0.1 mL/10 g of body weight once and the blood samples were collected in 4 groups with concentrations (0, 6.25%, 12.5% and 15% CCl4 in olive oil, v/v) and in different periods (24, 48, 72, 96 and 144 h). Glutamate oxalate transaminase (GOT), glutamate pyruvate transaminase (GPT), lactate dehydrogenase (LDH) and malondialdehyde (MDA) of serum were detected. In vitro, the hepatocytes were exposed to 6 concentrations of CCl4 (0, 2, 4, 8, 16 and 32 mmol/L) for 4 h and the activities of GPT, GOT, LDH, MDA, superoxide dismutase (SOD) and glutathione (GSH) in cell culture supernatant and the survival rate of the injured hepatocytes were determined. Results revealed the groups injected with 12.5% and 15% (v/v) of CCl4 in olive oil at a volume of 0.1 mL/10 g of body weight for 24 h could significantly elevate the serum levels of GOT, GPT and MDA, and the group with 15% of CCl4 for 48 h produced a significant effect in the case of LDH activities, but all four hepatic function markers were recovered to normal level at 96 h. Hepatocytes treated with CCl4 at 8, 16 and 32 mmol·L-1 increased levels of GOT, GPT and MDA, suppressed SOD and GSH activities; meanwhile, significantly decreased cell viabilities (66.29%, 54% and 37%, respectively) compared to the control dose-dependently. Overall results proved the optimal damaged conditions of acute liver injury models induced by CCl4 in vivo and in vitro were 15% CCl4 0.1 mL/10 g.b.w for 72 h once intraperitoneally and 8 mmol·L-1 for 4 h.
Key words:  carbon tetrachloride  Cyprinus carpio var. jian  model  MTT

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