鈣穩(wěn)態(tài)在硒拮抗鎘致LMH細胞內(nèi)質(zhì)網(wǎng)應(yīng)激中的作用
發(fā)布時間：2019-04-27 06:11
【摘要】：鎘是自然環(huán)境和工業(yè)環(huán)境中毒性最強的污染物之一,其半衰期長、排泄率低、蓄積能力強,能夠由食物鏈不斷蓄積,進而增強其對人類和動物健康的危害。肝臟是鎘蓄積和毒性作用的主要靶器官之一,肝細胞是鎘主要侵害的靶細胞之一,肝臟又被稱為硒庫,飼料中硒能夠拮抗鎘的肝毒性,但其具體機制仍不清楚。鈣穩(wěn)態(tài)與細胞功能與存活密切相關(guān)。為了揭示硒拮抗鎘致肝細胞毒性效應(yīng)的機制,本研究以LMH細胞為試驗對象,在培養(yǎng)體系中加入硒與鎘,并加入2-APB抑制鈣離子通道,采用MTT、MDC、q RT-PCR、流式細胞術(shù)及western blot等方法,對細胞活性、內(nèi)質(zhì)網(wǎng)應(yīng)激、細胞自噬、鈣離子調(diào)控等相關(guān)指標進行了檢測。研究表明:(1)成功表達了雞CNX和CRT蛋白抗原多肽,制備了兔抗雞CNX和CRT多克隆抗體,建立了檢測肝細胞及組織中CNX和CRT蛋白表達水平的Western blot方法。(2)鎘與硒致LMH細胞的24 h半數(shù)抑制濃度分別為3.18μM和15.24μM,表明LMH細胞對鎘具有高度敏感性,在鎘硒比為2:1和1:1時,硒均能夠拮抗鎘致肝細胞毒性作用。(3)鎘可以干擾內(nèi)質(zhì)網(wǎng)應(yīng)激標志分子ATF4、ATF6、GRP78、GRP94 m RNA表達,即鎘能夠誘導(dǎo)LMH細胞內(nèi)質(zhì)網(wǎng)應(yīng)激,同時鎘也可以增加LMH細胞自噬泡的數(shù)量及自噬標志分子Beclin1、P62、ATG3、ATG5和ATG9基因表達,而硒能夠有效拮抗這些指標變化,表明鎘能夠誘發(fā)LMH細胞內(nèi)質(zhì)網(wǎng)應(yīng)激和自噬,這是鎘發(fā)揮毒性作用的重要機制之一,硒能夠通過有效抑制內(nèi)質(zhì)網(wǎng)應(yīng)激與自噬來發(fā)揮拮抗效應(yīng)。(4)鎘能夠干擾LMH細胞內(nèi)游離鈣離子含量、Ca M與Ca M-IV基因表達,增加CNX與CRT基因及蛋白表達水平,而硒能夠有效拮抗這些指標變化,表明鎘能夠誘發(fā)LMH細胞鈣穩(wěn)態(tài)調(diào)節(jié)紊亂,這是鎘發(fā)揮毒性作用的重要機制之一,硒能夠通過有效抑制鈣穩(wěn)態(tài)紊亂來發(fā)揮拮抗效應(yīng)。(5)應(yīng)用鈣池操縱鈣通道抑制劑2-APB建立鈣釋放阻斷細胞模型,染鎘后,細胞毒性、內(nèi)質(zhì)網(wǎng)應(yīng)激、自噬現(xiàn)象均有所減輕,表明鈣穩(wěn)態(tài)失衡是鎘發(fā)揮毒性作用的關(guān)鍵機制,同時硒的拮抗效應(yīng)也所減弱,揭示鈣穩(wěn)態(tài)在硒拮抗鎘致LMH細胞內(nèi)質(zhì)網(wǎng)應(yīng)激中發(fā)揮著關(guān)鍵作用。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S859.8
本文編號：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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S859.8
【參考文獻】
相關(guān)期刊論文 前1條
1 段耀奎;曹文華;周欣;李金國;趙曉光;王慶寶;;硒鎘對裸鼠人肝癌細胞的協(xié)同影響作用[J];泰山醫(yī)學(xué)院學(xué)報;2005年06期
，本文編號：2466752
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