钙稳态在硒拮抗镉致LMH细胞内质网应激中的作用
发布时间：2019-04-27 06:11
【摘要】：镉是自然环境和工业环境中毒性最强的污染物之一,其半衰期长、排泄率低、蓄积能力强,能够由食物链不断蓄积,进而增强其对人类和动物健康的危害。肝脏是镉蓄积和毒性作用的主要靶器官之一,肝细胞是镉主要侵害的靶细胞之一,肝脏又被称为硒库,饲料中硒能够拮抗镉的肝毒性,但其具体机制仍不清楚。钙稳态与细胞功能与存活密切相关。为了揭示硒拮抗镉致肝细胞毒性效应的机制,本研究以LMH细胞为试验对象,在培养体系中加入硒与镉,并加入2-APB抑制钙离子通道,采用MTT、MDC、q RT-PCR、流式细胞术及western blot等方法,对细胞活性、内质网应激、细胞自噬、钙离子调控等相关指标进行了检测。研究表明:(1)成功表达了鸡CNX和CRT蛋白抗原多肽,制备了兔抗鸡CNX和CRT多克隆抗体,建立了检测肝细胞及组织中CNX和CRT蛋白表达水平的Western blot方法。(2)镉与硒致LMH细胞的24 h半数抑制浓度分别为3.18μM和15.24μM,表明LMH细胞对镉具有高度敏感性,在镉硒比为2:1和1:1时,硒均能够拮抗镉致肝细胞毒性作用。(3)镉可以干扰内质网应激标志分子ATF4、ATF6、GRP78、GRP94 m RNA表达,即镉能够诱导LMH细胞内质网应激,同时镉也可以增加LMH细胞自噬泡的数量及自噬标志分子Beclin1、P62、ATG3、ATG5和ATG9基因表达,而硒能够有效拮抗这些指标变化,表明镉能够诱发LMH细胞内质网应激和自噬,这是镉发挥毒性作用的重要机制之一,硒能够通过有效抑制内质网应激与自噬来发挥拮抗效应。(4)镉能够干扰LMH细胞内游离钙离子含量、Ca M与Ca M-IV基因表达,增加CNX与CRT基因及蛋白表达水平,而硒能够有效拮抗这些指标变化,表明镉能够诱发LMH细胞钙稳态调节紊乱,这是镉发挥毒性作用的重要机制之一,硒能够通过有效抑制钙稳态紊乱来发挥拮抗效应。(5)应用钙池操纵钙通道抑制剂2-APB建立钙释放阻断细胞模型,染镉后,细胞毒性、内质网应激、自噬现象均有所减轻,表明钙稳态失衡是镉发挥毒性作用的关键机制,同时硒的拮抗效应也所减弱,揭示钙稳态在硒拮抗镉致LMH细胞内质网应激中发挥着关键作用。
[Abstract]:Cadmium is one of the most toxic pollutants in natural environment and industrial environment. Its half-life is long, its excretion rate is low, and its accumulation ability is strong. Cadmium can accumulate continuously from the food chain, and then enhance its harm to human and animal health. Liver is one of the main target organs of accumulation and toxicity of cadmium. Hepatocyte is one of the target cells which are mainly damaged by cadmium. The liver is also called selenium pool. Selenium in feed can antagonize the hepatotoxicity of cadmium, but the specific mechanism is still unclear. Calcium homeostasis is closely related to cell function and survival. In order to reveal the mechanism of selenium antagonizing the cytotoxicity of cadmium induced hepatocytes, LMH cells were added selenium and cadmium, and 2-APB was added to the culture system to inhibit calcium channels. MTT,MDC,q RT-PCR, was used in this study. Cell viability, endoplasmic reticulum stress, autophagy and calcium regulation were measured by flow cytometry and western blot. The results showed that: (1) Rabbit anti-chicken CNX and CRT polyclonal antibodies were successfully expressed and the polyclonal antibodies against chicken CNX and CRT were prepared. A Western blot method was established to detect the expression of CNX and CRT protein in hepatocytes and tissues. (2) the 24-hour inhibitory concentrations of cadmium and selenium in LMH cells were 3.18 渭 M and 15.24 渭 M, respectively, indicating that LMH cells were highly sensitive to cadmium. When the ratio of cadmium to selenium was 2:1 and 1: 1, selenium could antagonize the cytotoxicity induced by cadmium. (3) cadmium could interfere with the expression of endoplasmic reticulum stress marker molecule ATF4,ATF6,GRP78,GRP94 m RNA, that is, cadmium could induce endoplasmic reticulum stress in LMH cells. Cadmium can also increase the number of autophagy vesicles and the expression of autophagy marker molecules Beclin1,P62,ATG3,ATG5 and ATG9 in LMH cells. Selenium can effectively antagonize the changes of these indexes, indicating that cadmium can induce endoplasmic reticulum stress and autophagy in LMH cells. This is one of the important mechanisms of cadmium toxicity, selenium can effectively inhibit endoplasmic reticulum stress and autophagy to play an antagonistic effect. (4) cadmium can interfere with the intracellular free calcium content, Ca M and Ca M-IV gene expression in LMH cells. Se could effectively antagonize the changes of CNX and CRT gene and protein expression, which indicated that cadmium could induce disturbance of calcium homeostasis in LMH cells, which was one of the important mechanisms of cadmium toxicity. Selenium could exert its antagonistic effect by effectively inhibiting calcium homeostasis disorder. (5) calcium release blocking cell model was established by using calcium pool to manipulate calcium channel inhibitor 2-APB. After cadmium exposure, the cytotoxicity, endoplasmic reticulum stress and autophagy were alleviated. It is suggested that calcium homeostasis imbalance is the key mechanism of cadmium toxicity, and the antagonistic effect of selenium is also weakened. It is suggested that calcium homeostasis plays a key role in the antagonism of cadmium induced endoplasmic reticulum stress in LMH cells by selenium.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.8
本文编号：2466752
[Abstract]:Cadmium is one of the most toxic pollutants in natural environment and industrial environment. Its half-life is long, its excretion rate is low, and its accumulation ability is strong. Cadmium can accumulate continuously from the food chain, and then enhance its harm to human and animal health. Liver is one of the main target organs of accumulation and toxicity of cadmium. Hepatocyte is one of the target cells which are mainly damaged by cadmium. The liver is also called selenium pool. Selenium in feed can antagonize the hepatotoxicity of cadmium, but the specific mechanism is still unclear. Calcium homeostasis is closely related to cell function and survival. In order to reveal the mechanism of selenium antagonizing the cytotoxicity of cadmium induced hepatocytes, LMH cells were added selenium and cadmium, and 2-APB was added to the culture system to inhibit calcium channels. MTT,MDC,q RT-PCR, was used in this study. Cell viability, endoplasmic reticulum stress, autophagy and calcium regulation were measured by flow cytometry and western blot. The results showed that: (1) Rabbit anti-chicken CNX and CRT polyclonal antibodies were successfully expressed and the polyclonal antibodies against chicken CNX and CRT were prepared. A Western blot method was established to detect the expression of CNX and CRT protein in hepatocytes and tissues. (2) the 24-hour inhibitory concentrations of cadmium and selenium in LMH cells were 3.18 渭 M and 15.24 渭 M, respectively, indicating that LMH cells were highly sensitive to cadmium. When the ratio of cadmium to selenium was 2:1 and 1: 1, selenium could antagonize the cytotoxicity induced by cadmium. (3) cadmium could interfere with the expression of endoplasmic reticulum stress marker molecule ATF4,ATF6,GRP78,GRP94 m RNA, that is, cadmium could induce endoplasmic reticulum stress in LMH cells. Cadmium can also increase the number of autophagy vesicles and the expression of autophagy marker molecules Beclin1,P62,ATG3,ATG5 and ATG9 in LMH cells. Selenium can effectively antagonize the changes of these indexes, indicating that cadmium can induce endoplasmic reticulum stress and autophagy in LMH cells. This is one of the important mechanisms of cadmium toxicity, selenium can effectively inhibit endoplasmic reticulum stress and autophagy to play an antagonistic effect. (4) cadmium can interfere with the intracellular free calcium content, Ca M and Ca M-IV gene expression in LMH cells. Se could effectively antagonize the changes of CNX and CRT gene and protein expression, which indicated that cadmium could induce disturbance of calcium homeostasis in LMH cells, which was one of the important mechanisms of cadmium toxicity. Selenium could exert its antagonistic effect by effectively inhibiting calcium homeostasis disorder. (5) calcium release blocking cell model was established by using calcium pool to manipulate calcium channel inhibitor 2-APB. After cadmium exposure, the cytotoxicity, endoplasmic reticulum stress and autophagy were alleviated. It is suggested that calcium homeostasis imbalance is the key mechanism of cadmium toxicity, and the antagonistic effect of selenium is also weakened. It is suggested that calcium homeostasis plays a key role in the antagonism of cadmium induced endoplasmic reticulum stress in LMH cells by selenium.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.8
【参考文献】
相关期刊论文 前1条
1 段耀奎;曹文华;周欣;李金国;赵晓光;王庆宝;;硒镉对裸鼠人肝癌细胞的协同影响作用[J];泰山医学院学报;2005年06期
，本文编号：2466752
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