动式吸入纳米级碳黑对大鼠肺组织的损伤作用及机制研究

发布时间：2018-04-19 23:01

本文选题：纳米级碳黑 + 动式吸入染毒　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:碳黑是一种无定形碳,由于其稳定性的特点,经常被用于阴性对照来解释一些纳米材料的毒性。纳米级碳黑具有较大的表面积,因而也具有较强的生物学作用。已有证据表明纳米级碳黑颗粒可诱导肺部炎症和组织病理损伤。国际癌症研究机构(IARC)将碳黑分类为人类可能致癌物。但其作用机制仍然不清。外源性异物进入机体后,在对机体造成氧化损伤作用的同时机体自身也出现抗氧化应激保护机制。而FOXO家族的转录因子牵涉细胞周期阻滞,细胞死亡和保护应激刺激多个过程,在抗氧化应激修复损伤中有着重要的作用。本研究通过动式吸入染毒的方法,建立碳黑染毒大鼠模型,观察碳黑对大鼠肺组织的影响,并以FOXO3a、FOXO1为切入点,对纳米级碳黑的毒作用机制进行深入研究,探讨其导致机体细胞毒作用靶点,寻找切实可行的预防控制方法。方法:1使用Tecnai G220透射电子显微镜测量纳米级碳黑颗粒的尺寸和形态;并通过扫描电子显微镜观察其表面形态。使用Brunauer-Emmett-Teller(BET)吸附等温式计算纳米级碳黑颗粒的比表面积。2 48只健康SD雄性大鼠,体重150-180g,随机分为两组,每组24只。建立大鼠动式吸入染毒模型,实验组每天吸入纳米级碳黑颗粒6h,浓度为30mg/m3,染毒14天28天和恢复14天后每组分别处死8只动物留取肝、肾、心、脑、肺组织于-80℃冰箱冻存。3在染毒过程中每隔3天记录一次大鼠的体重,比较实验组与对照组间大鼠体重变化差异,并观察大鼠一般体征。腹主动脉取血后处死动物,处死前称重,并称量各脏器重量计算脏器系数。4肺组织形态及病理学观察:碳黑吸入染毒不同时间段大鼠的肺组织的外观形态发生显著改变,制作肺组织病理切片,光镜下观察纳米级碳黑颗粒染毒后肺的病理变化。5碳黑在肺组织中的沉积率和清除率:根据计算公式Lung burden=(MV)×(T)×(CON)×(DF)=(7.3ml/min)(360min)(30mg/m3)(33.8%)(MV为大鼠通气量,T为每天暴露时间,CON为染毒罐内碳黑浓度,DF为可进入肺泡的碳黑量占总碳黑量的百分比)计算碳黑在肺内的沉积量。比较肺组织病理切片内纳米级碳黑颗粒的分布及不同时间点的含量计算清除率。6大鼠肺组织氧化应激水平的测定:利用试剂盒检测纳米级碳黑颗粒染毒后,大鼠肺组织中SOD、GSH-Px活力以及MDA含量的变化。将肺组织细胞制成单细胞悬液,使用2,7-二氢二氯荧光黄双乙酸钠(2,7-ddichlorodihydrf-luorescein diacetate,DCFH-DA)荧光探针孵育,流式细胞仪检测不同时间点实验组与对照组肺组织细胞活性氧(reactive oxygen species,ROS)含量。7大鼠肺组织细胞凋亡的测定:Tunel法检测细胞凋亡。Tunel法可使用石蜡包埋肺组织切片,对完整的单个凋亡细胞核或凋亡小体进行原位染色,检测纳米级碳黑颗粒染毒后,肺组织内凋亡细胞的数量。8大鼠肺组织DNA损伤的测定:运用彗星试验(SCGE),检测纳米级碳黑颗粒染毒后,大鼠肺组织细胞OTM值和Tail DNA%的变化。9纳米级碳黑颗粒对大鼠肺组织中FOXO蛋白表达的影响免疫组化:对FOXO3a、P-FOXO3a、FOXO1及P-FOXO1进行定位、定性及定量的研究,Western blot:检测FOXO3a、P-FOXO3a、FOXO1及P-FOXO1蛋白表达情况。结果:1纳米级碳黑颗粒的特征通过扫描电子显微镜(SEM)和透射电子显微镜(TEM)证实碳黑(CB)的尺寸为30到50nm的球形颗粒组成了数十到数百纳米的聚集体,但无中间体。BET结果显示CB颗粒比表面积约为74.85m2/g。2大鼠的一般状况在实验过程中,实验大鼠全部存活,一般情况良好;在染毒和恢复期间没有观察到纳米级碳黑颗粒的明显毒性作用。实验组大鼠进食及饮水正常,精神状态良好。3碳黑对大鼠体重的变化影响碳黑染毒28天的过程中,染毒组的体重增长速度略低于非暴露组,两组的体重差异无统计学意义(P0.05)。4大鼠的肺脏器系数变化脏器系数分析碳黑吸入染毒14天后,肺的绝对重量与肺的脏器系数同对照组相比无显著性差异(P0.05),在吸入染毒28天后,肺的绝对重量与对照组相比无显著性差异(P0.05),肺的脏器系数显著增加(P0.05),恢复14天后,肺的脏器系数仍显著高于对照组(P0.05)。其他脏器的脏器系数均无明显变化。5碳黑在肺组织中的沉积率和清除率碳黑染毒14天、28天后,每只大鼠肺部碳黑的理论沉积量为373.03mg、746.06mg。碳黑染毒28天后,碳黑颗粒大部分沉积于肺间质内,少量存在于肺泡间,在恢复14天后碳黑可被清除为28天的82.5%,且与染毒28天相比差异有统计学意义(P0.05)。6大鼠肺组织的病理变化对照组,光镜下观察支气管上皮排列整齐管腔规则,呼吸性细支气管和周围肺泡、血管等组织结构,纤毛完整无脱落,管腔内无渗出物。染毒14天和28天后,大鼠肺组织内碳黑颗粒增多,肺泡壁增厚。肺泡腔内可见较多的巨噬细胞,单核细胞增多,聚集成团,吞噬黑色碳黑颗粒增多。恢复期染毒组大鼠病变程度与14天染毒组相似,仍观察到肺泡间质内中有许多碳黑颗粒分布,但无大量炎性细胞。7大鼠肺组织细胞凋亡的变化Tunel法检测细胞凋亡结果显示,纳米级碳黑颗粒染毒14天与28天后,与对照组相比,染毒组细胞凋亡比例略有增加,差异有统计学意义(P0.05)。恢复14天后与染毒28天相比细胞凋亡比例略有降低,但差异无统计学意义(P0.05)。8纳米级碳黑颗粒对大鼠肺组织氧化应激水平的影响与对照组相比,纳米级碳黑染毒14天和28天后大鼠肺组织SOD、GSH-Px活力均显著降低(P0.05)。MDA含量显著升高(P0.05),恢复组SOD、GSH-Px活力均高于染毒组(P0.05),MDA含量有所降低(P0.05)。流式细胞仪检测ROS结果显示,纳米级碳黑染毒14天和28天后,大鼠肺组织ROS水平显著高于对照组,差异有统计学意义(P0.05),恢复组与28天染毒组相比有所降低,差异有统计学意义(P0.05),与对照组相比差异无统计学意义。9纳米级碳黑颗粒对大鼠肺组织DNA损伤的影响纳米级碳黑颗粒染毒14天后,大鼠肺组织中Tail DNA%同对照组相比无显著性差异(P0.05),OTM值与对照组相比无显著性差异(P0.05)。在吸入染毒28天后,大鼠肺组织中Tail DNA%与对照组相比均显著增加(P0.05);OTM值与对照组相比均显著增加(P0.05)。恢复14天后Tail DNA%与OTM值与28天染毒组相比均有明显降低,且差异有统计学意义(P0.05);与对照组相比差异无统计学意义(P0.05)。10、纳米级碳黑颗粒对大鼠肺组织中FOXO3a、P-FOXO3a、FOXO1及P-FOXO1蛋白表达的影响与对照组相比,纳米级碳黑染毒14天与28天后大鼠肺组织中FOXO3a与FOXO1蛋白表达量显著升高(P0.05),并且其主要在细胞核表达,且随着染毒时间的增加表达量增加。恢复组与28天染毒组相比FOXO3a与FOXO1蛋白表达量降低,差异有统计学意义(P0.05);恢复组与对照组相比,大鼠肺组织FOXO3a与FOXO1蛋白表达量的差异无统计学意义。染毒14天P-FOXO3a蛋白表达量与对照组相比差异无统计学意义,染毒28天P-FOXO3a蛋白表达量与对照组相比升高(P0.05),且在细胞质与细胞核内均有表达,恢复组与对照组相比P-FOXO3a蛋白表达量的差异无统计学意义(P0.05)。与对照组相比,染毒14天和28天后,大鼠肺组织P-FOXO1蛋白表达量显著升高(P0.05),细胞质与细胞核内均有表达,恢复14天与对照组相比P-FOXO1蛋白表达量的差异无统计学意义。结论:1建立大鼠动式吸入纳米级碳黑染毒模型,在28天动式吸入染毒过程中,实验组大鼠体重增长略低于对照组,差异无统计学意义。2大鼠吸入纳米级碳黑后,大部分沉积于肺间质内,少量存在于肺泡间,在停止染毒并恢复14天后肺间质内碳黑几乎不能被清除,表明碳黑在进入肺组织后短期内不能被清除。3纳米级碳黑颗粒经呼吸道染毒后可使大鼠肺组织出现不同程度的病理变化,且随着染毒时间的增加病变加重。并引起肺细胞的凋亡和坏死。4纳米级碳黑颗粒染毒后,可使大鼠肺组织细胞发生氧化损伤,抗氧化能力受损,14天后可缓解部分氧化应激水平。5纳米级碳黑颗粒经呼吸道染毒后,可使大鼠肺组织细胞发生轻微的DNA损伤,在恢复14天后机体自身修复可以恢复基因组完整性。6纳米级碳黑颗粒诱导的大鼠肺组织的氧化应激可能与FOXO3a和FOXO1的调节有关。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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