氟化钠、亚砷酸钠对大鼠肝细胞BRL-3A的联合毒性作用

发布时间：2018-05-29 15:15

本文选题：大鼠肝细胞 + 氟化钠　；参考：《南华大学》2012年硕士论文

【摘要】：目的：用不同浓度的氟化钠、亚砷酸钠对体外培养的大鼠肝细胞进行染毒，探讨氟和砷对大鼠肝细胞的损伤及其联合毒作用，阐明其致肝细胞损伤的机制，为氟砷联合作用对肝脏的综合毒性评定提供理论依据。方法：将体外培养至80%左右融合的大鼠BRL-3A细胞按下列方法处理：正常对照换以不含毒物的无血清DMEM培养基培养；单独亚砷酸钠染毒：分别用亚砷酸钠浓度为1.0×10~(-3)mol/L、1.0×10~(-4)mol/L、1.0×10~(-5)mol/L、1.0×10~(-6)mol/L、1.0×10~(-7)mol/L的无血清DMEM培养基培养；单独氟化钠染毒：分别用氟化钠浓度为1.0×10~(-1)mol/L、1.0×10~(-2)mol/L、1.0×10~(-3)mol/L、1.0×10~(-4)mol/L、1.0×10~(-5)mol/L的无血清DMEM培养基培养；氟砷联合染毒：用同时加入氟化钠和亚砷酸钠的无血清DMEM培养，培养基中氟化钠浓度为1.0×10-3mol/L，，亚砷酸钠浓度分别为1.0×10~(-4)mol/L、1.0×10~(-5)mol/L、1.0×10~(-6)mol/L。染毒24小时后HE染色法观察细胞形态结构变化，MTT法检测细胞增殖情况，同时检测培养液中谷丙转氨酶（ALT）、谷草转氨酶（AST）的活性；再分别用二硫对硝基苯甲酸（DTNB）法、黄嘌呤氧化酶法、硫代巴比妥酸法检测培养液中谷胱甘肽过氧化物酶(GSH—Px)活性、超氧化物歧化酶(SOD)活性、丙二醛（MDA）含量，并通过定量实时聚合酶链式反应（Q_PCR）测定大鼠BRL-3A细胞Bcl-2mRNA表达水平。结果： 1）氟化钠、亚砷酸钠染毒24小时后大鼠BRL-3A细胞收缩、变细、体积变小；细胞数量明显减少，双核细胞减少，细胞胞核固缩，有的细胞内出现空泡；较多细胞脱落，细胞间隙变宽，形成一片片的细胞脱失区。 2）氟化钠、亚砷酸钠均对大鼠肝细胞的增殖产生了抑制作用（P＜0.05），且随着浓度的增加，增殖抑制率增加；氟化钠与亚砷酸钠对BRL-3A细胞增殖抑制的交互作用有统计学意义（P＜0.01），交互作用呈拮抗作用。 3）氟化钠、亚砷酸钠增加了BRL-3A细胞培养物中ALT、AST活性（P＜0.05）；氟化钠与亚砷酸钠对ALT、AST活性影响的交互作用具有统计学意义（P＜0.05），交互作用呈拮抗作用。 4）氟化钠、亚砷酸钠降低了细胞GSH－Px、SOD活性（P＜0.05）、提高了培养液中MDA的浓度（P＜0.05）；氟化钠与亚砷酸钠对GSH－Px、SOD、MDA的交互作用具有统计学意义（P＜0.01），其交互作用呈拮抗作用。 5）氟化钠、亚砷酸钠均抑制了BRL-3A细胞中的Bcl-2mRNA表达（P＜0.05），氟化钠与亚砷酸钠对BRL-3A细胞中的Bcl-2mRNA表达影响的交互作用具有统计学意义（P＜0.01），交互作用呈拮抗作用。结论： 1）氟、砷能引起BRL-3A细胞形态改变，可抑制大鼠BRL-3A细胞的增殖。 2）氟、砷染毒可影响BRL-3A细胞的氧化应激系统，使GSH－Px、SOD活性降低，MDA浓度增高。 3）氟、砷染毒引起可导致BRL-3A细胞内的Bcl-2基因表达下降。 4）氟和砷对BRL-3A细胞的损伤存在交互作用，其联合作用方式表现为拮抗作用。
[Abstract]:Objective: Different concentrations of sodium fluoride and sodium arsenite were used to treat rat hepatocytes cultured in vitro. The damage of fluoride and arsenic to rat hepatocytes and its combined toxicity were studied, and the mechanism of the damage induced by fluoride and arsenic was elucidated. To provide a theoretical basis for the comprehensive toxicity assessment of the combined action of fluoride and arsenic on the liver. Methods: Rat BRL-3A cells cultured to about 80% in vitro were treated as follows: normal control was cultured on serum-free DMEM medium containing no poison, sodium arsenite alone was cultured on a serum-free DMEM medium of 1.0 脳 10 ~ (-3) mol / L ~ (-1) of sodium arsenite at the concentration of 1.0 脳 10 ~ (-3) mol / L ~ (-1) and 1.0 脳 10 ~ (-5) mol 路L ~ (-1) ~ 1 脳 10 ~ (-5) mol / L ~ (-1) of 1.0 脳 10 ~ (-1) 10~(-7)mol/L. Sodium fluoride alone: a serum-free DMEM medium containing 1.0 脳 10 ~ (-1) mol / L ~ (-1) sodium fluoride at a concentration of 1.0 脳 10 ~ (-1) mol / L ~ (-1) O ~ (-1) mol / L ~ (-1) ~ (-1) mol / L ~ (10) ~ (-1) mol 路L ~ (-1) (1.0 脳 10 ~ (10) mol 路L ~ (-1); a serum-free DMEM medium of 1.0 脳 10 ~ (-1) mol / L ~ (-1) (1.0 脳 10 ~ (10) mol 路L ~ (-1); a combination of fluoride and arsenic poisoning: serum-free DMEM was cultured with sodium fluoride and sodium arsenite. The concentration of sodium fluoride and sodium arsenite were 1.0 脳 10 ~ (-3) mol / L and 1.0 脳 10 ~ (-3) mol / L, respectively. After 24 hours of exposure, the morphological and structural changes of cells were observed by HE staining and the proliferation of cells was detected by MTT method, and the activity of alanine aminotransferase (alt) and glutamic oxaloacetic transaminase (AST) in the culture medium were also detected. The activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA) in culture medium were determined by xanthine oxidase method and thiobarbituric acid method. The expression of Bcl-2mRNA in rat BRL-3A cells was measured by quantitative real time polymerase chain reaction (QPCR). Results: 1) after exposure to sodium fluoride and sodium arsenite for 24 hours, the BRL-3A cells in rats contracted, became thinner and smaller in volume, the number of cells decreased significantly, the number of binucleated cells decreased, the nuclei of cells shrank, some cells appeared vacuoles, and more cells fell off. The intercellular gap widens, forming a strip of cell loss. 2) Sodium fluoride and sodium arsenite inhibited the proliferation of rat hepatocytes (P < 0.05), and the inhibition rate increased with the increase of concentration. The interaction between sodium fluoride and sodium arsenite on the proliferation inhibition of BRL-3A cells was statistically significant (P < 0.01), and the interaction was antagonistic. 3) Sodium fluoride and sodium arsenite increased the activity of alt in BRL-3A cell culture (P < 0.05), and the interaction between sodium fluoride and sodium arsenite on the activity of BRL-3A was statistically significant (P < 0.05), and the interaction was antagonistic. (4) Sodium fluoride and sodium arsenite decreased the activity of GSH-PxC and increased the concentration of MDA in the culture medium (P < 0.05), and the interaction between sodium fluoride and sodium arsenite on the activity of GSH-PxX / SOD was statistically significant (P < 0.01), and the interaction between sodium fluoride and sodium arsenite was antagonistic. 5) both sodium fluoride and sodium arsenite inhibited the expression of Bcl-2mRNA in BRL-3A cells (P < 0.05). The interaction between sodium fluoride and sodium arsenite on the expression of Bcl-2mRNA in BRL-3A cells was statistically significant (P < 0.01), and the interaction was antagonistic. Conclusion: 1) fluoride and arsenic can induce morphological changes of BRL-3A cells and inhibit the proliferation of BRL-3A cells in rats. 2) fluoride and arsenic exposure could affect the oxidative stress system of BRL-3A cells and increase the activity of GSH-PxCX SOD. 3) fluoride and arsenic could induce the decrease of Bcl-2 gene expression in BRL-3A cells. 4) there was interaction between fluoride and arsenic on BRL-3A cells, and the combined action was antagonistic.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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