本文選題：納米級(jí)碳黑 + 動(dòng)式吸入染毒�。� 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:碳黑是一種無(wú)定形碳,由于其穩(wěn)定性的特點(diǎn),經(jīng)常被用于陰性對(duì)照來(lái)解釋一些納米材料的毒性。納米級(jí)碳黑具有較大的表面積,因而也具有較強(qiáng)的生物學(xué)作用。已有證據(jù)表明納米級(jí)碳黑顆�？烧T導(dǎo)肺部炎癥和組織病理?yè)p傷。國(guó)際癌癥研究機(jī)構(gòu)(IARC)將碳黑分類(lèi)為人類(lèi)可能致癌物。但其作用機(jī)制仍然不清。外源性異物進(jìn)入機(jī)體后,在對(duì)機(jī)體造成氧化損傷作用的同時(shí)機(jī)體自身也出現(xiàn)抗氧化應(yīng)激保護(hù)機(jī)制。而FOXO家族的轉(zhuǎn)錄因子牽涉細(xì)胞周期阻滯,細(xì)胞死亡和保護(hù)應(yīng)激刺激多個(gè)過(guò)程,在抗氧化應(yīng)激修復(fù)損傷中有著重要的作用。本研究通過(guò)動(dòng)式吸入染毒的方法,建立碳黑染毒大鼠模型,觀(guān)察碳黑對(duì)大鼠肺組織的影響,并以FOXO3a、FOXO1為切入點(diǎn),對(duì)納米級(jí)碳黑的毒作用機(jī)制進(jìn)行深入研究,探討其導(dǎo)致機(jī)體細(xì)胞毒作用靶點(diǎn),尋找切實(shí)可行的預(yù)防控制方法。方法:1使用Tecnai G220透射電子顯微鏡測(cè)量納米級(jí)碳黑顆粒的尺寸和形態(tài);并通過(guò)掃描電子顯微鏡觀(guān)察其表面形態(tài)。使用Brunauer-Emmett-Teller(BET)吸附等溫式計(jì)算納米級(jí)碳黑顆粒的比表面積。2 48只健康SD雄性大鼠,體重150-180g,隨機(jī)分為兩組,每組24只。建立大鼠動(dòng)式吸入染毒模型,實(shí)驗(yàn)組每天吸入納米級(jí)碳黑顆粒6h,濃度為30mg/m3,染毒14天28天和恢復(fù)14天后每組分別處死8只動(dòng)物留取肝、腎、心、腦、肺組織于-80℃冰箱凍存。3在染毒過(guò)程中每隔3天記錄一次大鼠的體重,比較實(shí)驗(yàn)組與對(duì)照組間大鼠體重變化差異,并觀(guān)察大鼠一般體征。腹主動(dòng)脈取血后處死動(dòng)物,處死前稱(chēng)重,并稱(chēng)量各臟器重量計(jì)算臟器系數(shù)。4肺組織形態(tài)及病理學(xué)觀(guān)察:碳黑吸入染毒不同時(shí)間段大鼠的肺組織的外觀(guān)形態(tài)發(fā)生顯著改變,制作肺組織病理切片,光鏡下觀(guān)察納米級(jí)碳黑顆粒染毒后肺的病理變化。5碳黑在肺組織中的沉積率和清除率:根據(jù)計(jì)算公式Lung burden=(MV)×(T)×(CON)×(DF)=(7.3ml/min)(360min)(30mg/m3)(33.8%)(MV為大鼠通氣量,T為每天暴露時(shí)間,CON為染毒罐內(nèi)碳黑濃度,DF為可進(jìn)入肺泡的碳黑量占總碳黑量的百分比)計(jì)算碳黑在肺內(nèi)的沉積量。比較肺組織病理切片內(nèi)納米級(jí)碳黑顆粒的分布及不同時(shí)間點(diǎn)的含量計(jì)算清除率。6大鼠肺組織氧化應(yīng)激水平的測(cè)定:利用試劑盒檢測(cè)納米級(jí)碳黑顆粒染毒后,大鼠肺組織中SOD、GSH-Px活力以及MDA含量的變化。將肺組織細(xì)胞制成單細(xì)胞懸液,使用2,7-二氫二氯熒光黃雙乙酸鈉(2,7-ddichlorodihydrf-luorescein diacetate,DCFH-DA)熒光探針?lè)跤?流式細(xì)胞儀檢測(cè)不同時(shí)間點(diǎn)實(shí)驗(yàn)組與對(duì)照組肺組織細(xì)胞活性氧(reactive oxygen species,ROS)含量。7大鼠肺組織細(xì)胞凋亡的測(cè)定:Tunel法檢測(cè)細(xì)胞凋亡。Tunel法可使用石蠟包埋肺組織切片,對(duì)完整的單個(gè)凋亡細(xì)胞核或凋亡小體進(jìn)行原位染色,檢測(cè)納米級(jí)碳黑顆粒染毒后,肺組織內(nèi)凋亡細(xì)胞的數(shù)量。8大鼠肺組織DNA損傷的測(cè)定:運(yùn)用彗星試驗(yàn)(SCGE),檢測(cè)納米級(jí)碳黑顆粒染毒后,大鼠肺組織細(xì)胞OTM值和Tail DNA%的變化。9納米級(jí)碳黑顆粒對(duì)大鼠肺組織中FOXO蛋白表達(dá)的影響免疫組化:對(duì)FOXO3a、P-FOXO3a、FOXO1及P-FOXO1進(jìn)行定位、定性及定量的研究,Western blot:檢測(cè)FOXO3a、P-FOXO3a、FOXO1及P-FOXO1蛋白表達(dá)情況。結(jié)果:1納米級(jí)碳黑顆粒的特征通過(guò)掃描電子顯微鏡(SEM)和透射電子顯微鏡(TEM)證實(shí)碳黑(CB)的尺寸為30到50nm的球形顆粒組成了數(shù)十到數(shù)百納米的聚集體,但無(wú)中間體。BET結(jié)果顯示CB顆粒比表面積約為74.85m2/g。2大鼠的一般狀況在實(shí)驗(yàn)過(guò)程中,實(shí)驗(yàn)大鼠全部存活,一般情況良好;在染毒和恢復(fù)期間沒(méi)有觀(guān)察到納米級(jí)碳黑顆粒的明顯毒性作用。實(shí)驗(yàn)組大鼠進(jìn)食及飲水正常,精神狀態(tài)良好。3碳黑對(duì)大鼠體重的變化影響碳黑染毒28天的過(guò)程中,染毒組的體重增長(zhǎng)速度略低于非暴露組,兩組的體重差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。4大鼠的肺臟器系數(shù)變化臟器系數(shù)分析碳黑吸入染毒14天后,肺的絕對(duì)重量與肺的臟器系數(shù)同對(duì)照組相比無(wú)顯著性差異(P0.05),在吸入染毒28天后,肺的絕對(duì)重量與對(duì)照組相比無(wú)顯著性差異(P0.05),肺的臟器系數(shù)顯著增加(P0.05),恢復(fù)14天后,肺的臟器系數(shù)仍顯著高于對(duì)照組(P0.05)。其他臟器的臟器系數(shù)均無(wú)明顯變化。5碳黑在肺組織中的沉積率和清除率碳黑染毒14天、28天后,每只大鼠肺部碳黑的理論沉積量為373.03mg、746.06mg。碳黑染毒28天后,碳黑顆粒大部分沉積于肺間質(zhì)內(nèi),少量存在于肺泡間,在恢復(fù)14天后碳黑可被清除為28天的82.5%,且與染毒28天相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。6大鼠肺組織的病理變化對(duì)照組,光鏡下觀(guān)察支氣管上皮排列整齊管腔規(guī)則,呼吸性細(xì)支氣管和周?chē)闻�、血管等組織結(jié)構(gòu),纖毛完整無(wú)脫落,管腔內(nèi)無(wú)滲出物。染毒14天和28天后,大鼠肺組織內(nèi)碳黑顆粒增多,肺泡壁增厚。肺泡腔內(nèi)可見(jiàn)較多的巨噬細(xì)胞,單核細(xì)胞增多,聚集成團(tuán),吞噬黑色碳黑顆粒增多。恢復(fù)期染毒組大鼠病變程度與14天染毒組相似,仍觀(guān)察到肺泡間質(zhì)內(nèi)中有許多碳黑顆粒分布,但無(wú)大量炎性細(xì)胞。7大鼠肺組織細(xì)胞凋亡的變化Tunel法檢測(cè)細(xì)胞凋亡結(jié)果顯示,納米級(jí)碳黑顆粒染毒14天與28天后,與對(duì)照組相比,染毒組細(xì)胞凋亡比例略有增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)�；謴�(fù)14天后與染毒28天相比細(xì)胞凋亡比例略有降低,但差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。8納米級(jí)碳黑顆粒對(duì)大鼠肺組織氧化應(yīng)激水平的影響與對(duì)照組相比,納米級(jí)碳黑染毒14天和28天后大鼠肺組織SOD、GSH-Px活力均顯著降低(P0.05)。MDA含量顯著升高(P0.05),恢復(fù)組SOD、GSH-Px活力均高于染毒組(P0.05),MDA含量有所降低(P0.05)。流式細(xì)胞儀檢測(cè)ROS結(jié)果顯示,納米級(jí)碳黑染毒14天和28天后,大鼠肺組織ROS水平顯著高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05),恢復(fù)組與28天染毒組相比有所降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05),與對(duì)照組相比差異無(wú)統(tǒng)計(jì)學(xué)意義。9納米級(jí)碳黑顆粒對(duì)大鼠肺組織DNA損傷的影響納米級(jí)碳黑顆粒染毒14天后,大鼠肺組織中Tail DNA%同對(duì)照組相比無(wú)顯著性差異(P0.05),OTM值與對(duì)照組相比無(wú)顯著性差異(P0.05)。在吸入染毒28天后,大鼠肺組織中Tail DNA%與對(duì)照組相比均顯著增加(P0.05);OTM值與對(duì)照組相比均顯著增加(P0.05)�；謴�(fù)14天后Tail DNA%與OTM值與28天染毒組相比均有明顯降低,且差異有統(tǒng)計(jì)學(xué)意義(P0.05);與對(duì)照組相比差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。10、納米級(jí)碳黑顆粒對(duì)大鼠肺組織中FOXO3a、P-FOXO3a、FOXO1及P-FOXO1蛋白表達(dá)的影響與對(duì)照組相比,納米級(jí)碳黑染毒14天與28天后大鼠肺組織中FOXO3a與FOXO1蛋白表達(dá)量顯著升高(P0.05),并且其主要在細(xì)胞核表達(dá),且隨著染毒時(shí)間的增加表達(dá)量增加。恢復(fù)組與28天染毒組相比FOXO3a與FOXO1蛋白表達(dá)量降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05);恢復(fù)組與對(duì)照組相比,大鼠肺組織FOXO3a與FOXO1蛋白表達(dá)量的差異無(wú)統(tǒng)計(jì)學(xué)意義。染毒14天P-FOXO3a蛋白表達(dá)量與對(duì)照組相比差異無(wú)統(tǒng)計(jì)學(xué)意義,染毒28天P-FOXO3a蛋白表達(dá)量與對(duì)照組相比升高(P0.05),且在細(xì)胞質(zhì)與細(xì)胞核內(nèi)均有表達(dá),恢復(fù)組與對(duì)照組相比P-FOXO3a蛋白表達(dá)量的差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。與對(duì)照組相比,染毒14天和28天后,大鼠肺組織P-FOXO1蛋白表達(dá)量顯著升高(P0.05),細(xì)胞質(zhì)與細(xì)胞核內(nèi)均有表達(dá),恢復(fù)14天與對(duì)照組相比P-FOXO1蛋白表達(dá)量的差異無(wú)統(tǒng)計(jì)學(xué)意義。結(jié)論:1建立大鼠動(dòng)式吸入納米級(jí)碳黑染毒模型,在28天動(dòng)式吸入染毒過(guò)程中,實(shí)驗(yàn)組大鼠體重增長(zhǎng)略低于對(duì)照組,差異無(wú)統(tǒng)計(jì)學(xué)意義。2大鼠吸入納米級(jí)碳黑后,大部分沉積于肺間質(zhì)內(nèi),少量存在于肺泡間,在停止染毒并恢復(fù)14天后肺間質(zhì)內(nèi)碳黑幾乎不能被清除,表明碳黑在進(jìn)入肺組織后短期內(nèi)不能被清除。3納米級(jí)碳黑顆粒經(jīng)呼吸道染毒后可使大鼠肺組織出現(xiàn)不同程度的病理變化,且隨著染毒時(shí)間的增加病變加重。并引起肺細(xì)胞的凋亡和壞死。4納米級(jí)碳黑顆粒染毒后,可使大鼠肺組織細(xì)胞發(fā)生氧化損傷,抗氧化能力受損,14天后可緩解部分氧化應(yīng)激水平。5納米級(jí)碳黑顆粒經(jīng)呼吸道染毒后,可使大鼠肺組織細(xì)胞發(fā)生輕微的DNA損傷,在恢復(fù)14天后機(jī)體自身修復(fù)可以恢復(fù)基因組完整性。6納米級(jí)碳黑顆粒誘導(dǎo)的大鼠肺組織的氧化應(yīng)激可能與FOXO3a和FOXO1的調(diào)節(jié)有關(guān)。
[Abstract]:Objective: carbon black is an amorphous carbon. Due to its stability, it is often used as a negative control to explain the toxicity of some nanomaterials. Nanoscale carbon black has a larger surface area and therefore has a strong biological effect. The disease Research Institute (IARC) classifications of carbon black as a possible carcinogen of human beings. However, the mechanism of its action is still unclear. After exogenous foreign bodies enter the body, the body causes oxidative damage to the body and the body itself also has the protective mechanism of antioxidant stress. The transcription factors of the FOXO family involve cell cycle block, cell death and protection of stress. Stimulation of multiple processes plays an important role in the repair of oxidative stress. In this study, the effect of carbon black on the lung tissue of rats was observed by the method of dynamic inhalation, and the effect of carbon black on the lung tissue of rats was observed. The mechanism of the toxic effect of nano carbon black was studied with FOXO3a and FOXO1 as the breakthrough point. The target of cytotoxic effect is to find practical prevention and control methods. Method: 1 the size and morphology of nano carbon black particles were measured by Tecnai G220 transmission electron microscope, and the surface morphology was observed by scanning electron microscope. The specific surface area of nano carbon black particles was calculated by Brunauer-Emmett-Teller (BET) adsorption isotherm.2 48 Only healthy SD male rats and body weight 150-180g were randomly divided into two groups, each group was 24. The rat model of dynamic inhalation was established. The experimental group inhaled 6h of nanoscale carbon black particles every day, the concentration was 30mg/m3, 14 days 28 days and 14 days after the recovery. The kidney, heart, brain and lung tissues were exposed to.3 in -80 freezer. The body weight of rats was recorded every 3 days, and the body weight difference between the experimental group and the control group was compared and the general physical signs were observed. The animals were killed after the abdominal aorta was taken from the blood. The weight of the organs was weighed before death, and the lung tissue morphology and pathology of the organ coefficients of each organ were weighed to calculate the lung tissue and pathology of the organ coefficient.4: the lung of the rats in different time periods of carbon black inhalation. The appearance of the tissue was significantly changed, the lung tissue was made, the pathological sections of lung tissue were made, and the pathological changes of lung were observed under the light microscope. The deposition rate and clearance rate of.5 carbon black in lung tissues were observed: according to the formula Lung burden= (T) * (CON) * (DF) = (7.3ml/min) (360min) (30mg/m3) (30mg/m3) (30mg/m3) (30mg/m3) (30mg/m3) (MV), T was daily Exposure time, CON is the concentration of carbon black in the poisoned tank, DF is the percentage of carbon black that can enter the alveoli, the amount of carbon black in the lung is calculated. Comparison of the distribution of nanoscale black particles in the pathological sections of lung tissue and the content of different time points in the calculation of the oxidative stress level of lung tissue in.6 rats: using reagents The changes of SOD, GSH-Px activity and MDA content in lung tissues of rats were detected by the case of nanoscale carbon black particles. The lung tissue cells were made into single cell suspension, using 2,7- two hydrogen two chlorine fluorescent yellow diacetate (2,7-ddichlorodihydrf-luorescein diacetate, DCFH-DA) fluorescence probe to incubate, and the flow cytometry was used to detect the experimental group at different time points. The determination of apoptosis in lung tissue of.7 rats with the content of reactive oxygen species (ROS) in the lung tissue of the control group: Tunel assay of apoptosis.Tunel method can use paraffin embedded lung tissue section to dye the intact single apoptotic cell nucleus or apoptotic body in situ, and detect the lung tissue after the nano-sized carbon black particles are infected. Determination of DNA damage in lung tissue of.8 rats: using comet test (SCGE), detection of nano carbon black particles, OTM value of lung tissue and the changes of Tail DNA% in rat lung,.9 nanoscale carbon black particles on the expression of FOXO protein in lung tissue of rats: the localization of FOXO3a, P-FOXO3a, FOXO1 and P-FOXO1. Western blot: was used to detect the expression of FOXO3a, P-FOXO3a, FOXO1 and P-FOXO1 protein. Results: the characteristics of 1 nanoscale carbon black particles were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), which confirmed that the spheroidal particles with the size of 30 to 50nm of the carbon black (CB) formed tens to hundreds of nanoscale aggregates, but there was no middle The results of body.BET showed that the specific surface area of CB particles was about 74.85m2/g.2 rats. During the experimental process, the experimental rats were all alive and well, and the obvious toxic effects of nanoscale carbon black particles were not observed during the period of exposure and recovery. The rats in the experimental group had normal eating and drinking water and good mental state of.3 carbon black on the rat body. The weight growth rate of the 28 days was slightly lower than that of the non exposure group. There was no significant difference in weight difference between the two groups (P0.05) the lung organ coefficient of the two groups was not significantly different, and the coefficient of lung organ coefficient changes of the organ coefficient was analyzed for 14 days after carbon black inhalation, the absolute weight of the lung and the lung visceral coefficient were not significantly different from those of the control group. P0.05), after 28 days of inhalation, the absolute weight of lung was not significantly different from that of the control group (P0.05), and the lung visceral coefficient increased significantly (P0.05). After 14 days, the lung visceral coefficient was still significantly higher than that of the control group (P0.05). There was no obvious change in the deposition rate and scavenging rate of.5 carbon black in the lung tissues by other visceral coefficients of other organs. After 14 days of poison, 28 days later, the theoretical deposition of carbon black in each rat's lungs was 373.03mg. After 28 days of 746.06mg. carbon black exposure, most of the carbon black particles were deposited in the pulmonary interstitium, and a small amount existed in the alveoli. After 14 days of recovery, carbon black could be cleared to 82.5% of the 28 days, and the difference was statistically significant (P0.05).6 rat lung tissue disease compared to the day 28 days. In the control group, the rules of the bronchial epithelium arranged neatly, the respiratory bronchioles and the surrounding alveoli, blood vessels and other tissue structures, the cilia were intact and no exudate was found in the lumen. The pulmonary carbon black particles in the lung tissue increased and the alveolar wall thickened in the lung tissue for 14 and 28 days. More macrophages and mononuclear cells in the alveolar cavity were observed. Cell proliferation, aggregation and black carbon black particles increased. The degree of pathological changes of rats in the recovery stage was similar to that of the 14 day exposure group. There were many carbon black particles in the alveolar interstitium, but there was no change of apoptosis in the lung tissue of.7 rats with inflammatory cells. Tunel method was used to detect the apoptosis. Compared with the control group, the proportion of apoptotic cells increased slightly compared with the control group, the difference was statistically significant (P0.05). Compared with the 28 days after the recovery, the percentage of apoptosis decreased slightly, but the difference was not statistically significant (P0.05) the effect of.8 nano carbon black particles on the oxidative stress level of lung tissue in rats was compared with the control group. SOD, GSH-Px activity of lung tissues of rats exposed to nano carbon black for 14 and 28 days was significantly decreased (P0.05).MDA content was significantly increased (P0.05), SOD in the recovery group, GSH-Px activity was higher than that of the infected group (P0.05), MDA content decreased (P0.05). The flow cytometry showed that the lung tissues of rats were exposed to nanoscale carbon black for 14 and 28 days. Significantly higher than the control group, the difference was statistically significant (P0.05), the recovery group was lower than the 28 day exposure group, the difference was statistically significant (P0.05). There was no significant difference between the control group and the control group (P0.05). The effect of.9 nanoscale carbon black particles on the DNA damage in the lung tissue of the rats was found to be Tail DNA% in the lung tissue of the rats after exposure to nanoscale carbon black particles. There was no significant difference in the OTM value compared with the control group (P0.05). The Tail DNA% in the lung tissue of the rats increased significantly after 28 days of inhalation (P0.05), and the OTM value increased significantly compared with the control group (P0.05). The Tail DNA% and OTM were compared with those of the 28 day exposure group after 14 days of recovery. The difference was statistically significant (P0.05), and there was no significant difference between the control group and the control group (P0.05).10. The effects of nano carbon black particles on the expression of FOXO3a, P-FOXO3a, FOXO1 and P-FOXO1 in the lung tissue of rats were compared with those of the control group. The expression of FOXO3a and FOXO1 protein in lung tissues of rats exposed to nanoscale carbon black for 14 and 28 days was significant. The expression of FOXO3a and FOXO1 protein in the recovery group decreased with the increase of the expression of the nucleus, and the expression of FOXO3a and FOXO1 protein decreased with the increase of the time of exposure. The difference between the recovery group and the control group was statistically significant (P0.05), and there was no significant difference in the expression of FOXO3a and FOXO1 protein in the lung group of the recovery group compared with the control group. There was no significant difference in the expression of P-FOXO3a protein for 14 days compared with the control group. The expression of P-FOXO3a protein increased in 28 days compared with the control group (P0.05), and expressed in both cytoplasm and nucleus. There was no significant difference in the expression of P-FOXO3a protein in the recovery group compared with the control group (P0.05). Compared with the control group, the difference was 14. After day and 28 days, the expression of P-FOXO1 protein in the lung tissue of rats increased significantly (P0.05), the cytoplasm and the nucleus were expressed in the nucleus. The difference of the expression of P-FOXO1 protein was not statistically significant compared with the control group for 14 days. Conclusion: 1 the model of dynamic inhalation nanoscale carbon black in rats was established, and the experimental group was in the process of dynamic inhalation and exposure to 28 days. The weight growth was slightly lower than that of the control group. The difference was not statistically significant in.2 rats after inhaling nanoscale carbon black, most of them were deposited in the pulmonary interstitium, and a small amount existed in the alveoli. The carbon black in the pulmonary interstitial was almost impossible to be removed after 14 days of cessation and recovery, indicating that carbon black could not be removed from the.3 nanoscale carbon black particles in the short term after entering the lung group. Respiratory tract infection can cause pathological changes in the lung tissue of rats in different degrees, and with the increase of exposure time, and the apoptosis and necrosis of the lung cells and necrosis of.4 nanoscale carbon black particles can cause oxidative damage in the lung tissue cells of the rat, the antioxidant capacity is damaged, and the oxidative stress level of.5 can be relieved in 14 days. After the infection of the respiratory tract, the lung tissue cells of the rats may have a slight DNA damage. The oxidative stress in the lung tissue of the rats induced by the recovery of the body's self restoration after 14 days after the recovery of the body's self repair can be related to the regulation of FOXO3a and FOXO1.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R114

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王燕;康現(xiàn)江;丁士文;穆淑梅;王宇;曹輝彩;;納米二氧化鈦對(duì)小鼠肝腎的影響[J];環(huán)境與健康雜志;2008年02期

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本文編號(hào)：1775105