本文選題：六溴環(huán)十二烷 + SH-SY5Y細(xì)胞　； 參考：《上海大學(xué)》2016年博士論文

【摘要】：以急性毒性指標(biāo)可以將六溴環(huán)十二烷(HBCD)界定為低毒化合物,但研究表明HBCD具有內(nèi)分泌干擾毒性、免疫抑制毒性、生殖發(fā)育毒性、神經(jīng)毒性等效應(yīng)。其中,神經(jīng)發(fā)育毒性是HBCD一個較為敏感的毒性終點(diǎn),氧化應(yīng)激會引起線粒體功能障礙,從而導(dǎo)致的能量代謝失常和線粒體依賴性凋亡是HBCD誘發(fā)神經(jīng)發(fā)育毒性的機(jī)制之一。本研究使用β-NGF誘導(dǎo)神經(jīng)母細(xì)胞瘤SH-SY5Y分化模型,研究HBCD對分化細(xì)胞的毒性。應(yīng)用免疫熒光、流式細(xì)胞術(shù)、蛋白免疫印跡、高效液相色譜等技術(shù)檢測了β-NGF和HBCD單獨(dú)及聯(lián)合作用于SH-SY5Y細(xì)胞后突起的生長狀況、細(xì)胞凋亡、ROS的產(chǎn)生、線粒體膜電位的變化、細(xì)胞內(nèi)鈣離子的改變、細(xì)胞ATP含量,并用N-乙酰-L-半胱氨酸(NAC)消除ROS后再檢測相關(guān)指標(biāo)變化,結(jié)果表明:1.分化發(fā)育過程中的SH-SY5Y細(xì)胞比不分化的細(xì)胞更易受到損傷。在無凋亡誘導(dǎo)濃度下,分化細(xì)胞在HBCD作用下的凋亡率顯著升高,且具有時間效應(yīng)關(guān)系。同時,線粒體依賴性凋亡相關(guān)蛋白Cyt-c、Apaf-1和Caspase-9表達(dá)明顯上升,表明HBCD啟動了細(xì)胞的線粒體依賴性凋亡途徑。2.β-NGF誘導(dǎo)SH-SY5Y細(xì)胞分化時線粒體膜電位上升,ROS和胞內(nèi)鈣離子略升高,與多數(shù)研究發(fā)現(xiàn)神經(jīng)細(xì)胞分化過程中ROS水平和線粒體膜電位上升的結(jié)果相符。而HBCD作用于分化中的SH-SY5Y細(xì)胞時,線粒體膜電位顯著下降,伴隨ROS和細(xì)胞內(nèi)鈣濃度明顯升高;抗氧化劑NAC預(yù)處理可明顯抑制HBCD誘導(dǎo)的氧化應(yīng)激效應(yīng),細(xì)胞ROS水平與對照組相近,線粒體膜電位回升,胞內(nèi)鈣離子濃度回落,同時凋亡率有所下降,可見氧化應(yīng)激引起線粒體損傷誘導(dǎo)凋亡是HBCD神經(jīng)發(fā)育毒性的原因之一。3.HBCD可抑制β-NGF誘導(dǎo)的SH-SY5Y細(xì)胞突起的生長,神經(jīng)樹突標(biāo)志性蛋白MAP2表達(dá)下降,同時伴隨胞內(nèi)ATP水平降低,細(xì)胞能勢下降;NAC能部分減輕HBCD對神經(jīng)突起生長的抑制作用,提高ATP合成的水平和細(xì)胞能勢,但對MAP2表達(dá)沒有作用。以上結(jié)果表明HBCD對突起生長的抑制作用很可能通過HBCD刺激ROS累積,引起線粒體功能障礙繼而影響ATP合成,無法提供足夠供細(xì)胞分化的能量而引起,但ROS蓄積并不能解釋MAP2蛋白的表達(dá)被抑制的原因。4.繼續(xù)對能量敏感的蛋白AMPK和神經(jīng)分化密切相關(guān)的PI3K/AKT/m TOR通路進(jìn)行分析后發(fā)現(xiàn),在β-NGF作用下,AMPK、PI3K、AKT磷酸化水平均顯著上升,m TOR磷酸化低于對照組,并具有時間效應(yīng)關(guān)系。另外,AMPK的表達(dá)與細(xì)胞內(nèi)AMP/ATP比值水平呈正相關(guān),并促進(jìn)MAP2表達(dá)。HBCD作用后細(xì)胞內(nèi)m TOR磷酸化水平隨時間延長而顯著上升,AMPK磷酸化受抑制,用PI3K和AKT抑制劑(LY294002,MK-2006)處理細(xì)胞后,AMPK磷酸化顯著增加,MAP2表達(dá)水平回升。以上結(jié)果表明,在β-NGF誘導(dǎo)SH-SY5Y分化時m TOR受到AMPK的負(fù)調(diào)控,HBCD作用時m TOR磷酸化水平升高并下調(diào)了AMPK的磷酸化,阻礙了MAP2的表達(dá),HBCD的發(fā)育毒性作用與PI3K/AKT/m TOR通路對AMPK的負(fù)調(diào)控有關(guān)。HBCD的立體異構(gòu)體選擇性和對映異構(gòu)體選擇性代謝在不同物種,不同組織器官都有差異,沒有普適的規(guī)律可以進(jìn)行模式生物的研究并進(jìn)行異構(gòu)體單體的風(fēng)險評價。肝臟是HBCD的主要代謝器官,為了解HBCD異構(gòu)體在人體內(nèi)的代謝選擇性和毒性差異,本研究利用正常人肝細(xì)胞L-02和人肝癌細(xì)胞Hep G2進(jìn)行HBCD異構(gòu)體的代謝和毒性研究,分別以10-7,10-6,10-5 mol/L的α-、β-和γ-HBCD染毒L-02和Hep G2細(xì)胞1 d、2 d、4 d和6 d,采用LC-MS/MS對細(xì)胞內(nèi)三種異構(gòu)體的含量變化、生物異構(gòu)化情況和對映異構(gòu)體選擇性進(jìn)行分析,應(yīng)用CCK8、熒光探針法及彗星實(shí)驗(yàn)檢測HBCD對L-02和Hep G2細(xì)胞的生物學(xué)效應(yīng);Real-time PCR檢測代謝酶的表達(dá)變化;結(jié)果發(fā)現(xiàn):1.α-HBCD、β-HBCD和γ-HBCD在兩種肝細(xì)胞內(nèi)的半衰期為α-HBCDγ-HBCDβ-HBCD,α-HBCD和γ-HBCD的代謝速率到第6天時基本一致。同時,Hep G2對HBCD的代謝能力比L-02強(qiáng),同時間段Hep G2細(xì)胞內(nèi)的異構(gòu)體比L-02細(xì)胞內(nèi)同一種異構(gòu)體含量更低。2.在Hep G2細(xì)胞中,α-HBCD和β-HBCD能發(fā)生異構(gòu)化轉(zhuǎn)化成γ-HBCD,而γ-HBCD染毒組不能檢出α-HBCD或β-HBCD;在L-02細(xì)胞中,β-HBCD染毒組能檢出少量γ-HBCD,其它兩個異構(gòu)體沒有檢測到生物轉(zhuǎn)化產(chǎn)物。3.在L-02細(xì)胞中,α-,β-,γ-HBCD的手性富集值EF分別為0.54±0.02,0.80±0.02,0.31±0.01;在Hep G2細(xì)胞中α-,β-,γ-HBCD的EF分別為0.54±0.01,0.79±0.02,0.32±0.02,兩種細(xì)胞均表現(xiàn)為(+)-α-HBCD略高于(-)-α-HBCD,而(+)-β-HBCD和(-)-γ-HBCD為優(yōu)勢異構(gòu)體。4.在L-02細(xì)胞中,β-HBCD和γ-HBCD對細(xì)胞增殖的抑制率高于α-HBCD,而β-HBCD對Hep G2細(xì)胞的抑制率高于α-HBCD和γ-HBCD;α-HBCD、β-HBCD和γ-HBCD均可誘導(dǎo)細(xì)胞ROS升高和線粒體膜電位降低,效應(yīng)強(qiáng)度為β-HBCDγ-HBCDα-HBCD,且Hep G2細(xì)胞比L-02細(xì)胞更加敏感,同一種異構(gòu)體在Hep G2細(xì)胞內(nèi)刺激ROS產(chǎn)生的效應(yīng)濃度要低于L-02細(xì)胞的效應(yīng)濃度;β-HBCD誘導(dǎo)DNA損傷的效應(yīng)最強(qiáng),在L-02細(xì)胞中,α-HBCD和γ-HBCD之間沒有差別,在Hep G2細(xì)胞中,α-HBCD較之γ-HBCD更易引起DNA損傷,兩種細(xì)胞L-02細(xì)胞比Hep G2細(xì)胞更易產(chǎn)生DNA損傷。5.β-HBCD可下調(diào)L-02細(xì)胞中P450酶CYP1A1的表達(dá),在Hep G2細(xì)胞中產(chǎn)生該效應(yīng)的是α-HBCD和γ-HBCD。β-HBCD和γ-HBCD在L-02細(xì)胞中誘導(dǎo)CYP2B6的表達(dá),且γ-HBCD還誘導(dǎo)了L-02細(xì)胞GST基因的表達(dá),在兩種細(xì)胞中,三種異構(gòu)體對代謝酶的表達(dá)有明顯差異,且L-02細(xì)胞比Hep G2細(xì)胞更易受到誘導(dǎo)。綜上所述,HBCD能抑制SH-SY5Y細(xì)胞分化,線粒體依賴性凋亡和能量代謝障礙引起的突起生長障礙是重要的原因之一,PI3K/AKT/m TOR通路在調(diào)控AMPK的磷酸化和MAP2的表達(dá)中有重要作用,該條通路的激活是HBCD神經(jīng)發(fā)育毒性的重要分子機(jī)制。HBCD在L-02和Hep G2兩種細(xì)胞的代謝有立體異構(gòu)體選擇性和對映異構(gòu)體選擇性;α-、β-和γ-HBCD三種異構(gòu)體的細(xì)胞毒性效應(yīng)在L-02中比HepG2弱,但在DNA損傷效應(yīng)強(qiáng)度上L-02細(xì)胞反應(yīng)比Hep G2強(qiáng)。三種異構(gòu)體在兩種細(xì)胞中對CYP和GST的誘導(dǎo)效應(yīng)有差異,推測這是L-02細(xì)胞對HBCD毒性反應(yīng)程度比HepG2較弱的原因之一,也可能是β-HBCD毒性較強(qiáng)的原因之一。
[Abstract]:Six bromo ring twelve alkanes (HBCD) can be defined as low toxic compounds with acute toxicity, but the study shows that HBCD has endocrine disrupting toxicity, immunosuppressive toxicity, reproductive development toxicity, neurotoxicity and other effects. Among them, neurodevelopmental toxicity is a more sensitive toxicity end of HBCD, and oxidative stress causes mitochondrial dysfunction, The resulting energy metabolic disorder and mitochondrial dependent apoptosis are one of the mechanisms of HBCD induced neurodevelopmental toxicity. This study used beta -NGF to induce the SH-SY5Y differentiation model of neuroblastoma and study the toxicity of HBCD to differentiated cells. Immunofluorescence, flow cytometry, protein immunization, high performance liquid chromatography and other techniques were used to detect beta -NG. The growth of F and HBCD alone and in combination with SH-SY5Y cells, apoptosis, ROS production, changes in mitochondrial membrane potential, intracellular calcium ion change, cell ATP content, and N- acetyl -L- cysteine (NAC) elimination of ROS after ROS and then change the correlation index, the results show that the ratio of SH-SY5Y cells in 1. differentiation and development process The undifferentiated cells are more susceptible to damage. The apoptosis rate of the differentiated cells under the action of HBCD is significantly increased and has a time effect relationship. At the same time, the expression of mitochondrial dependent apoptosis related protein Cyt-c, Apaf-1 and Caspase-9 increased obviously, indicating that HBCD activated the mitochondria dependent apoptosis pathway.2. beta -NGF of the cells. The mitochondrial membrane potential increased and the ROS and intracellular calcium ions increased slightly in the induction of SH-SY5Y cells differentiation. The results were consistent with the results of most studies, which were found to be consistent with the results of ROS and mitochondrial membrane potential in the process of neural differentiation. While HBCD acted on the differentiated SH-SY5Y cells, the mitochondrial membrane potential decreased significantly, with ROS and intracellular calcium concentration rising obviously. The antioxidant NAC pretreatment could obviously inhibit the oxidative stress induced by HBCD, the ROS level of the cells was similar to the control group, the mitochondrial membrane potential recovered, the intracellular calcium ion concentration fell, and the apoptosis rate decreased. The oxidative stress induced apoptosis induced by mitochondrial damage was one of the causes of HBCD neurotoxicity,.3.HBCD could inhibit beta. The growth of -NGF induced SH-SY5Y cell protuberance, the decrease of the expression of the nerve dendrite marker protein MAP2 and the decrease of the intracellular ATP level and the decrease of the cell energy potential; NAC can partially alleviate the inhibitory effect of HBCD on the growth of the neurite protuberance, improve the level of the ATP synthesis and the cell energy potential, but have no effect on the expression of MAP2. The above results indicate that HBCD is protruding. The inhibitory effect of growth is likely to stimulate the accumulation of ROS through HBCD, causing mitochondrial dysfunction and then affecting the synthesis of ATP, which can not provide enough energy for cell differentiation, but the accumulation of ROS does not explain the reason for the inhibition of the expression of MAP2 protein,.4. continues to be closely related to the energy sensitive protein AMPK and PI3K/AKT/m. After the analysis of TOR pathway, the phosphorylation level of AMPK, PI3K and AKT increased significantly under the action of beta -NGF, and the phosphorylation of M TOR was lower than that of the control group and had a time effect relationship. In addition, the expression of AMPK was positively correlated with the level of AMP/ATP ratio in the cells, and the phosphorylation level of the M cells in the cells increased with time after the MAP2 expression was.HBCD. The phosphorylation of AMPK was inhibited, and the phosphorylation of AMPK increased significantly with the treatment of PI3K and AKT inhibitor (LY294002, MK-2006), and the expression level of MAP2 increased. The above results showed that m TOR was regulated by AMPK in the induction of SH-SY5Y differentiation by beta -NGF. The developmental toxicity of HBCD is related to the negative regulation of the PI3K/AKT/m TOR pathway to the negative regulation of AMPK, the selectivity of the isomer of.HBCD and the selective metabolism of enantiomers in different species, different tissues and organs, and there is no universal law for the study of model organisms and the risk assessment of isomer monomers. The liver is H The main metabolic organs of BCD are to understand the metabolic selectivity and toxicity difference of HBCD isomers in the human body. The metabolism and toxicity of HBCD isomers in normal human hepatocyte L-02 and human hepatoma cell Hep G2 were studied in this study, respectively, with the alpha, beta and gamma -HBCD L-02 and Hep G2 cells of 10-7,10-6,10-5 mol/L, respectively, 2, 4 and 6. The changes in the content of three isomers, bioisomerization and enantiomer selectivity were analyzed by MS/MS. The biological effects of HBCD on L-02 and Hep G2 cells were detected by CCK8, fluorescence probe and comet assay, and Real-time PCR detected the changes in the expression of metabolic enzymes. The results showed that 1. alpha -HBCD, beta -HBCD and gamma -HBCD were in two kinds of liver. The half-life of the cell is alpha -HBCD gamma -HBCD beta -HBCD, the metabolic rate of alpha -HBCD and gamma -HBCD is basically the same as the sixth day. At the same time, the metabolic ability of Hep G2 to HBCD is stronger than that of L-02. The isomers in the Hep G2 cells of the same time period are lower than the same isomers in the L-02 cells. In gamma -HBCD, alpha -HBCD or beta -HBCD can not be detected by gamma -HBCD; in L-02 cells, a small amount of gamma -HBCD can be detected in the beta -HBCD group, and the other two isomers are not detected in the biotransformation product.3. in L-02 cells. The chiral enrichment value of alpha, beta, and gamma -HBCD is 0.54 + + + 0.01, respectively. The EF of D was 0.54 + 0.01,0.79 + 0.02,0.32 + 0.02 respectively. The two cells were (+) - alpha -HBCD was slightly higher than (-) - alpha -HBCD, while (+) - beta -HBCD and (-) - gamma -HBCD were dominant isomers in L-02 cells, and the inhibition rate of beta -HBCD and gamma -HBCD on cell proliferation was higher than that of alpha. Both -HBCD and gamma -HBCD can induce the increase of cell ROS and the decrease of mitochondrial membrane potential, the effect intensity is beta -HBCD gamma -HBCD alpha -HBCD, and Hep G2 cells are more sensitive than L-02 cells. The effect concentration of the same isomer in Hep G2 cells is lower than the effect concentration of the cell. In the cell, there is no difference between alpha -HBCD and gamma -HBCD. In Hep G2 cells, the alpha -HBCD is more likely to cause DNA damage than that of gamma -HBCD. The two cell L-02 cells are more likely to produce DNA damage.5. beta than Hep G2 cells. The expression of CYP2B6 was induced in the cells, and the expression of GST gene in L-02 cells was induced by gamma -HBCD. In two cells, the expression of metabolic enzymes was significantly different from three isomers, and L-02 cells were more easily induced than Hep G2 cells. To sum up, HBCD could inhibit the differentiation of SH-SY5Y cells, mitochondrial dependent apoptosis and energy metabolism disorder. The PI3K/AKT/m TOR pathway plays an important role in the regulation of the phosphorylation of AMPK and the expression of MAP2. The activation of this pathway is an important molecular mechanism of HBCD neurodevelopmental toxicity,.HBCD, in the metabolism of two cells of L-02 and Hep G2, which have stereoselective selectivity and enantiomer selectivity; alpha, beta - - The cytotoxic effects of three isomers of -HBCD and gamma are weaker than HepG2 in L-02, but L-02 cell reaction is stronger than Hep G2 in the intensity of DNA damage effect. The induction effect of CYP and GST in the three isomers is different in two cells. It is presumed that this is one of the reasons that L-02 cells are less toxic to HBCD than HepG2, and may also be beta toxicity. One of the stronger reasons.

【學(xué)位授予單位】：上海大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：X171.5

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