本文選題：大鼠肝細(xì)胞 + 氟化鈉　； 參考：《南華大學(xué)》2012年碩士論文

【摘要】：目的： 用不同濃度的氟化鈉、亞砷酸鈉對(duì)體外培養(yǎng)的大鼠肝細(xì)胞進(jìn)行染毒，探討氟和砷對(duì)大鼠肝細(xì)胞的損傷及其聯(lián)合毒作用，闡明其致肝細(xì)胞損傷的機(jī)制，為氟砷聯(lián)合作用對(duì)肝臟的綜合毒性評(píng)定提供理論依據(jù)。 方法： 將體外培養(yǎng)至80%左右融合的大鼠BRL-3A細(xì)胞按下列方法處理：正常對(duì)照換以不含毒物的無(wú)血清DMEM培養(yǎng)基培養(yǎng)；單獨(dú)亞砷酸鈉染毒：分別用亞砷酸鈉濃度為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的無(wú)血清DMEM培養(yǎng)基培養(yǎng)；單獨(dú)氟化鈉染毒：分別用氟化鈉濃度為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的無(wú)血清DMEM培養(yǎng)基培養(yǎng)；氟砷聯(lián)合染毒：用同時(shí)加入氟化鈉和亞砷酸鈉的無(wú)血清DMEM培養(yǎng)，培養(yǎng)基中氟化鈉濃度為1.0×10-3mol/L，，亞砷酸鈉濃度分別為1.0×10~(-4)mol/L、1.0×10~(-5)mol/L、1.0×10~(-6)mol/L。染毒24小時(shí)后HE染色法觀察細(xì)胞形態(tài)結(jié)構(gòu)變化，MTT法檢測(cè)細(xì)胞增殖情況，同時(shí)檢測(cè)培養(yǎng)液中谷丙轉(zhuǎn)氨酶（ALT）、谷草轉(zhuǎn)氨酶（AST）的活性；再分別用二硫?qū)ο趸郊姿幔―TNB）法、黃嘌呤氧化酶法、硫代巴比妥酸法檢測(cè)培養(yǎng)液中谷胱甘肽過(guò)氧化物酶(GSH—Px)活性、超氧化物歧化酶(SOD)活性、丙二醛（MDA）含量，并通過(guò)定量實(shí)時(shí)聚合酶鏈?zhǔn)椒磻?yīng)（Q_PCR）測(cè)定大鼠BRL-3A細(xì)胞Bcl-2mRNA表達(dá)水平。 結(jié)果： 1）氟化鈉、亞砷酸鈉染毒24小時(shí)后大鼠BRL-3A細(xì)胞收縮、變細(xì)、體積變�。患�(xì)胞數(shù)量明顯減少，雙核細(xì)胞減少，細(xì)胞胞核固縮，有的細(xì)胞內(nèi)出現(xiàn)空泡；較多細(xì)胞脫落，細(xì)胞間隙變寬，形成一片片的細(xì)胞脫失區(qū)。 2）氟化鈉、亞砷酸鈉均對(duì)大鼠肝細(xì)胞的增殖產(chǎn)生了抑制作用（P＜0.05），且隨著濃度的增加，增殖抑制率增加；氟化鈉與亞砷酸鈉對(duì)BRL-3A細(xì)胞增殖抑制的交互作用有統(tǒng)計(jì)學(xué)意義（P＜0.01），交互作用呈拮抗作用。 3）氟化鈉、亞砷酸鈉增加了BRL-3A細(xì)胞培養(yǎng)物中ALT、AST活性（P＜0.05）；氟化鈉與亞砷酸鈉對(duì)ALT、AST活性影響的交互作用具有統(tǒng)計(jì)學(xué)意義（P＜0.05），交互作用呈拮抗作用。 4）氟化鈉、亞砷酸鈉降低了細(xì)胞GSH－Px、SOD活性（P＜0.05）、提高了培養(yǎng)液中MDA的濃度（P＜0.05）；氟化鈉與亞砷酸鈉對(duì)GSH－Px、SOD、MDA的交互作用具有統(tǒng)計(jì)學(xué)意義（P＜0.01），其交互作用呈拮抗作用。 5）氟化鈉、亞砷酸鈉均抑制了BRL-3A細(xì)胞中的Bcl-2mRNA表達(dá)（P＜0.05），氟化鈉與亞砷酸鈉對(duì)BRL-3A細(xì)胞中的Bcl-2mRNA表達(dá)影響的交互作用具有統(tǒng)計(jì)學(xué)意義（P＜0.01），交互作用呈拮抗作用。 結(jié)論： 1）氟、砷能引起B(yǎng)RL-3A細(xì)胞形態(tài)改變，可抑制大鼠BRL-3A細(xì)胞的增殖。 2）氟、砷染毒可影響B(tài)RL-3A細(xì)胞的氧化應(yīng)激系統(tǒng)，使GSH－Px、SOD活性降低，MDA濃度增高。 3）氟、砷染毒引起可導(dǎo)致BRL-3A細(xì)胞內(nèi)的Bcl-2基因表達(dá)下降。 4）氟和砷對(duì)BRL-3A細(xì)胞的損傷存在交互作用，其聯(lián)合作用方式表現(xiàn)為拮抗作用。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114

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