镉诱导鲫肝细胞内Ca2+-ATP酶与金属硫蛋白的表达

Ca2+-ATPase and metallothionein expressing induced by cadmium in the primary cultured hepatocytes of crucian carp(Carassius auratus)

  • 摘要: 研究了镉诱发鲫肝细胞相关的胞内游离钙离子变化,以及Ca2+-ATP酶及金属硫蛋白表达量的变化。试验分为对照组、5、10、15、20 μmol/L CdCl2 5个组。Ca2+用Fura-2/AM方法检测,试验后24 h用荧光倒置显微镜观察细胞内游离钙离子变化;分光光度法检测Ca2+-ATP酶;石墨炉—原子分光光度法检测了细胞内镉离子浓度;免疫酶联法(ELISA)检测了金属硫蛋白(MT)含量。结果显示,镉可导致细胞存活率下降,具有一定的毒性。镉离子引起胞内Ca2+荧光强度和Ca2+-ATP酶活性增加(P<0.01)。随镉浓度升高,处理组Ca2+--ATP酶浓度活性分别是对照组的4.52、6.73、6.68、7.19、6.18倍;暴露24 h后各组细胞内镉离子均有上升,其中5 μmol/L组最高,达(2.045±0.322) μmol/L;各处理组金属硫蛋白(MT)含量增高(P<0.01),且5 μmol/L低浓度组MT增幅最大,达17.15%。结果提示,镉诱导下细胞内Ca2+升高,MT表达量上升,且MT可螯合进入细胞内的镉离子,这种螯合可能是降低镉毒理作用的机制之一。

     

    Abstract: Cadmium is one of environment heavy metal pollutants which is ranked the eighth in the top toxic substances harmful to human health by the Agency for Toxic Substances and Disease Registry(ATSDR).Cadmium is easy to be absorbed by aquatic organisms and to be deposited in the body.We studied the performance of the Ca2+-ATPase and metallothionein in primary cultured hepatocytes of Carassius auratus.Hepatocytes were incubated with cadmium chloride at 5,10,15 and 20 μmol/L for 24 h.Fluo-2/AM fluorescent labeling is used to show the Ca2+ level.The activity of Ca2+-ATPase was measured by spectrophotometry graphite,cadmium by atomic adsorption spectroscopy and metallothionein(MT)by ELISA.Fluorescence intensity of Ca2+ was higher in the treatment groups than in control group; the activity of Ca2+-ATPase was significantly increased(P<0.01).Ca2+-ATPase activity in treatment group were 4.52,6.73,6.68,7.19,6.18 times higher than that in the control group respectively.MT expression level was significantly increased in each treatment group(P<0.01),and MT content of 5 μmol/L group increased by 17.15 %(P<0.01).The results indicate that cadmium increases levels of Ca2+ and MT in hepatocytes of Carassius auratus,and MT chelated cadmium in hepatocytes,which may be one of the mechanisms that reduce the cadmium toxicological influence.

     

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