【摘要】：目的本研究通過(guò)建立鉛暴露大鼠模型,檢測(cè)鉛暴露大鼠中樞神經(jīng)系統(tǒng)海馬、皮質(zhì)、脈絡(luò)叢以及血液和腦脊液中銅含量變化;鉛暴露對(duì)血腦脊液屏障銅清除能力的影響,進(jìn)而從脈絡(luò)叢銅轉(zhuǎn)運(yùn)蛋白(CTR1、ATP7A)及伴侶蛋白(COX17)角度初步探討鉛對(duì)中樞神經(jīng)系統(tǒng)銅蓄積的機(jī)制。本研究結(jié)果將進(jìn)一步豐富鉛神經(jīng)毒性的機(jī)制,同時(shí)也為鉛神經(jīng)毒性的標(biāo)志物研究提供重要信息及相關(guān)靶點(diǎn)。方法45只健康雄性SD大鼠隨機(jī)分為對(duì)照組、300ppm鉛暴露組、600ppm鉛暴露組。適應(yīng)性飼養(yǎng)7天后,通過(guò)自由飲水的染毒方式給予鉛暴露組大鼠300ppm、600ppm的醋酸鉛飲用水,對(duì)照組大鼠給予600ppm的醋酸鈉飲用水,染毒時(shí)間為24周。每日觀察記錄大鼠精神狀態(tài)、活動(dòng)量、飲食及飲水等情況,每周檢測(cè)大鼠體重變化。染毒24周結(jié)束后,應(yīng)用Morris水迷宮和曠場(chǎng)實(shí)驗(yàn)檢測(cè)大鼠的神經(jīng)功能;應(yīng)用ICP-MS檢測(cè)大鼠全血、皮質(zhì)、海馬、脈絡(luò)叢及腦脊液中鉛、銅含量;利用側(cè)腦室灌注方法檢測(cè)鉛暴露對(duì)大鼠血腦脊液屏障銅清除能力的影響;應(yīng)用激光掃描共聚焦顯微鏡觀察脈絡(luò)叢上皮細(xì)胞銅相關(guān)蛋白(ATP7A、CTR1、COX17)表達(dá)變化及細(xì)胞中ROS水平;應(yīng)用real-time PCR檢測(cè)鉛暴露對(duì)大鼠脈絡(luò)叢atp7a、ctr1、cox17的mRNA表達(dá)變化的影響;應(yīng)用試劑盒檢測(cè)鉛暴露對(duì)大鼠皮質(zhì)氧化損傷的影響;應(yīng)用透射電子顯微鏡觀察鉛暴露對(duì)大鼠脈絡(luò)叢上皮細(xì)胞超微結(jié)構(gòu)的損傷。結(jié)果1鉛暴露24周后,與對(duì)照組比較,300ppm、600ppm鉛暴露組大鼠總運(yùn)動(dòng)距離、總穿格數(shù)、中央?yún)^(qū)域運(yùn)動(dòng)距離以及中央?yún)^(qū)域停留時(shí)間均降低(P0.05);鉛暴露組大鼠第3、4天的逃避潛伏期均延長(zhǎng)(P0.05),且600ppm鉛暴露組第3、4天逃避潛伏期最長(zhǎng)(P0.05);鉛暴露組大鼠穿越平臺(tái)次數(shù)均下降。尤其是600ppm鉛暴露組大鼠穿臺(tái)次數(shù)相比對(duì)照組減少了2.93次(P0.05)。2側(cè)腦室灌注實(shí)驗(yàn)結(jié)果顯示:腦脊液與灌注液的Cu64比值在20min達(dá)到平臺(tái)期,達(dá)到平臺(tái)期后鉛暴露組大鼠收集液中Cu64比值為0.769±0.026,高于對(duì)照組的0.687±0.018(P0.05);鉛暴露組大鼠BCB銅清除率低于對(duì)照組(P0.05);未見鉛暴露組和對(duì)照組腦脊液與灌注液的C14比值有差異(P0.05)。3與對(duì)照組比較,鉛暴露組大鼠全血、海馬、皮質(zhì)、脈絡(luò)叢、腦脊液中鉛、銅含量增加(P0.05);600ppm鉛暴露組大鼠全血、海馬、皮質(zhì)、脈絡(luò)叢、腦脊液中鉛含量以及全血、海馬、皮質(zhì)的銅含量高于300ppm鉛暴露組(P0.05)。4與對(duì)照組比較,鉛暴露組大鼠皮質(zhì)中AGEs含量增加,抑制羥自由基能力下降,GSH-ST活性下降,GSH含量降低(P0.05)。未見鉛暴露后皮質(zhì)中H2O2含量發(fā)生改變。5與對(duì)照組比較,鉛暴露組大鼠脈絡(luò)叢上皮細(xì)胞線粒體出現(xiàn)腫脹現(xiàn)象,部分線粒體Qg脊熔斷,細(xì)胞間緊密連接缺失,部分細(xì)胞核發(fā)生變形。6鉛暴露后,脈絡(luò)叢上皮細(xì)胞中CTR1、ATP7A、COX17的熒光強(qiáng)度均高于對(duì)照組。7鉛暴露組大鼠脈絡(luò)叢上皮細(xì)胞中ROS水平與對(duì)照組比較,明顯增加。8與對(duì)照組比較,鉛暴露組大鼠脈絡(luò)叢中ctr1、atp7a、cox17 mRNA表達(dá)水平均增加(P0.05)。結(jié)論鉛暴露導(dǎo)致中樞神經(jīng)系統(tǒng)銅穩(wěn)態(tài)失調(diào),氧化損傷增加,這可能與鉛暴露后脈絡(luò)叢上皮細(xì)胞超微結(jié)構(gòu)損傷,銅轉(zhuǎn)運(yùn)蛋白和伴侶蛋白表達(dá)改變,進(jìn)而導(dǎo)致血腦脊液屏障對(duì)銅清除能力下降有關(guān)。
[Abstract]:Objective to investigate the changes in copper content in the hippocampus, cortex, choroid plexus, blood and cerebrospinal fluid in the central nervous system of lead exposed rats, and the effect of lead exposure on the copper removal ability of the blood cerebrospinal fluid barrier (CSF), and the preliminary study of lead from the angle of CTR1 (ATP7A) and chaperone (COX17). The mechanism of copper accumulation in the central nervous system. The results of this study will further enrich the mechanism of lead neurotoxicity, and provide important information and targets for the study of biomarkers of lead neurotoxicity. Methods 45 healthy male SD rats were randomly divided into control group, 300ppm lead exposure group and 600ppm lead exposure group. After 7 days of adaptation, the results were passed. The free drinking water was given to lead exposed rats 300ppm, 600ppm and lead acetate drinking water. The control group was given 600ppm sodium acetate drinking water for 24 weeks. The rats' mental state, activity, diet and drinking water were observed daily, and the weight changes of rats were measured every week. After the end of 24 weeks, the Morris water fans were applied. The effects of lead and copper content in the whole blood, cortex, hippocampus, choroid plexus and cerebrospinal fluid were detected by ICP-MS, and the effects of lead exposure on the copper scavenging ability of the blood cerebrospinal fluid barrier in rats were detected by the lateral ventricle perfusion method, and the copper related eggs of choroid plexus epithelial cells were observed by laser scanning confocal microscope. The expression of white (ATP7A, CTR1, COX17) expression and the level of ROS in cells; the effect of real-time PCR on the expression of ATP7A, Ctr1 and COX17 in the choroid plexus of rats by real-time PCR; the effect of lead exposure on the oxidative damage in the cortex of rats was detected by the application of the kit; the ultrastructural observation of lead exposure on the ultrastructure of the rat choroid plexus epithelial cells was observed by transmission electron microscopy Results after 1 lead exposure for 24 weeks, compared with the control group, the total movement distance, total puncture number, central regional movement distance and central area residence time in the 300ppm and 600ppm lead exposed rats were all decreased (P0.05); the escape latency of the lead exposed rats on 3,4 days was prolonged (P0.05), and the 600ppm lead exposed group 3,4 days escape latency. The longest (P0.05) in the lead exposure group decreased the number of cross platform, especially in the 600ppm lead exposure group, the number of rats in the exposure group decreased by 2.93 times compared with the control group (P0.05).2 lateral ventricle perfusion test, which showed that the Cu64 ratio of cerebrospinal fluid and perfusion solution at 20min reached the platform stage, and the Cu64 ratio in the rat collection solution after the platform stage was 0. .769 + 0.026, higher than the control group 0.687 + 0.018 (P0.05), lead exposure group BCB copper clearance rate was lower than the control group (P0.05), no lead exposure group and the control group of cerebrospinal fluid and perfusion C14 ratio is different (P0.05).3 compared with the control group, lead exposed group of rats whole blood, hippocampus, cortex, choroid plexus, cerebrospinal fluid lead, copper content increase (P0.05); 600pp (P0.05); 600pp (P0.05); 600pp The lead content in the whole blood, hippocampus, cortex, choroid plexus, cerebrospinal fluid and the content of copper in the whole blood, hippocampus, and cortex of M lead exposed rats were higher than that of 300ppm lead exposure group (P0.05).4 and the control group. The content of AGEs in the cortex of lead exposed rats increased, the ability to inhibit the hydroxyl radical decreased, the GSH-ST activity decreased, and the GSH content decreased (P0.05). No lead exposed skin was found. Compared with the control group, the content of H2O2 in the cytoplasm was compared with the control group. The mitochondria of the choroid plexus epithelial cells in the lead exposed rats were swollen, some of the mitochondrial Qg ridges were broken, the close connections between the cells were missing, and the.6 lead exposure in some nuclei of the nuclei of the nuclei, and the fluorescence intensity of CTR1, ATP7A and COX17 in the choroid plexus epithelial cells was higher than that of the control group of.7 lead exposure. Compared with the control group, the level of ROS in the choroid plexus epithelial cells of the rats was significantly increased by.8. The expression level of Ctr1, ATP7A and COX17 mRNA in the choroid plexus of the lead exposed rats increased (P0.05). Conclusion lead exposure leads to the imbalance of copper in the central nervous system and the increase of oxidative damage, which may be associated with the ultrastructure of the choroid plexus epithelial cells after lead exposure. Structural damage, altered expression of copper transporters and chaperone proteins, leading to a decrease in blood cerebrospinal fluid barrier to copper clearance.
【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

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