二氧化铈纳米颗粒对大鼠心肌缺血再灌注损伤的影响

发布时间：2018-05-25 11:08

本文选题：二氧化铈纳米颗粒 + 心肌缺血再灌注损伤　；参考：《郑州大学》2014年硕士论文

【摘要】：1研究背景和目的目前，随着我国经济发展和人民生活水平的不断提高，缺血性心脏疾病的发生率和由此引发的死亡率正不断上升。各种临床治疗手段的不断更新发展，为此类疾病提供了更多更有效的治疗方法，但同时心肌缺血再灌注损伤(Myocardium ischemic reperfusion injury，MIRI)在患者病情的恢复中起到的不利影响也逐渐突显了出来。心肌缺血再灌注损伤表现为当心肌细胞经过一段时间的缺血期，血流供应恢复之后，心肌细胞的损伤并没有减轻或者恢复，反而出现了较再灌注以前更加严重的损伤的现象。如何有效的防治缺血再灌注损伤对心肌细胞造成的损害成为有关医学研究的一个热点，相关的研究有如β-受体阻滞剂，钙离子拮抗剂，肾素血管紧张素抑制剂等，但效果却都不是十分的理想。因此，寻找到一种切实而有效的方法或药物来治疗或者改善缺血再灌注损伤成为临床上一个亟待解决的问题。心肌缺血再灌注损伤的发生机制十分复杂。细胞凋亡被认为是其重要的发生机制之一。凋亡又称为细胞的程序性死亡，再灌注后心肌梗死面积扩大即认为是细胞凋亡引起的。缺血再灌注过程中的细胞凋亡可能是由氧自由基(oxygenfree radicals，OFR)的大规模爆发性产生、细胞内的钙超载、线粒体损伤等几个原因引起的，其中氧自由基的大量产生被认为是最主要的原因之一。在心肌的缺血再灌注过程中，氧自由基起到了至关重要的作用。心肌缺血再灌注后出现的心律失常、心肌梗死面积扩大等现象均是由于大量爆发性产生的氧自由基引起的。缺血再灌注过程中，大量产生的氧自由基对细胞结构进行了破坏，其与细胞膜磷脂中含量较多的不饱和脂肪酸发生脂质过氧化反应，使细胞膜上的离子通道、酶等膜蛋白的活性降低，从而影响了细胞膜及细胞器膜的功能，使其完整性遭到破坏，流动性降低。我国的稀土资源丰富，二氧化铈作为一种稀土材料被广泛的应用于各个领域。纳米材料因其自身尺度的原因而具有特殊的物理化学性质，针对纳米材料的研究也日渐深入。二氧化铈纳米颗粒(CeO2nanoparticles)作为一种纳米材料，在近些年来被应用于工业、汽车制造业及人民日常生活中的多个方面。有研究证实，二氧化铈纳米颗粒具有能够清除氧自由基，减弱有机体的氧化应激反应等很强的生物活性。本研究旨在观察二氧化铈纳米颗粒对大鼠缺血再灌注损伤心肌组织中超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathioneperoxidase, GSH-Px)、丙二醛(malondialdehyde,MDA)和细胞凋亡(apoptosis)的影响，从而探讨其对大鼠缺血再灌注损伤心肌的保护作用，为对缺血再灌注损伤提供更多的防治手段。 2材料和方法 2.1二氧化铈纳米颗粒悬液的制备：分别将不同纳米粒径的二氧化铈纳米颗粒溶解在PBS缓冲液（PH7.4，0.01mM）中，并使其充分混匀，注射前，再将其放入超声细胞破碎仪中，进行超声震荡2min的操作，，使悬液再次充分混合均匀，完成后再用0.2μm的针头过滤器过滤方可使用。 2.2实验动物分组：健康雄性SD大鼠50只，体重在250~300g之间，随机分成5组，假手术(sham)组、模型(I/R)组、1-10nm粒径组、10-25nm粒径组、50nm粒径组，每组10只。适应性饲养一周后进行模型的制作。大鼠采用10%水合氯醛（300mg/kg）进行腹腔注射麻醉，仰卧位固定牢固，分离气管行气管插管，开胸时连接呼吸机。小动物呼吸机潮气量设定为3ml/100g，呼吸频率为70次/分，呼吸比2:1。在大鼠胸骨左缘心脏搏动最为明显处切开皮肤，自第4、5肋间进胸，撕破心包膜，充分暴露心脏。于肺动脉圆锥与左心耳之间距离主动脉根部约3~4mm处穿线，在结扎线和心脏表面垫一带有凹槽的乳胶管，待心律规整后结扎左冠状动脉前降支（left anterior decreased coronary artery,LAD），结扎线远端心肌颜色先变白后变为紫绀证明结扎成功。结扎45min后松解结扎线，再灌注120min，缺血部位心肌颜色恢复为模型制作成功。假手术组于术前24h尾静脉注射PBS缓冲液（0.2ml/100g），左冠状动脉前降支只穿线不结扎；模型组于术前24h经尾静脉注射PBS缓冲液（0.2ml/100g），左冠状动脉前降支结扎45min，松解120min，成功制作缺血再灌注模型；三种不同纳米粒径组均于术前24h经尾静脉注射各自粒径CeO2纳米悬液（0.2ml/100g），模型制作同模型组。 2.3心肌再灌注120min后处死大鼠，迅速取出心脏，冷生理盐水充分冲洗至无血液残留，取左心室缺血区心肌组织，分为两部分，一部分迅速用滤纸吸干水分后转移至-80℃冰箱中保存待用，另外一部分置于甲醛溶液中固定，用于组织病理学观察和细胞凋亡的检测。将-80℃冰箱中保存的新鲜心肌组织在低温环境下制备心肌组织匀浆，应用黄嘌呤氧化酶法测定SOD活性，比色法测定GSH-Px活性，硫代巴比妥酸法测定MDA含量。具体操作步骤严格按照试剂盒说明书进行。将甲醛溶液中固定的部分大鼠心肌组织制备常规石蜡切片，用于苏木素-伊红染色（HE染色）、细胞凋亡的检测。 2.4统计各组数据，所有数据均以均数±标准差（x±s）表示，各组数据间比较采用单因素方差分析，两两比较采用LSD法，应用SPSS17.0统计分析软件进行分析，检验水准α取0.05，P0.05认为有统计学意义。 3结果 3.1模型组大鼠心肌组织中SOD、GSH-Px活性比假手术组明显降低，组间差异显著（P0.05），差异具有统计学意义；三组不同粒径二氧化铈纳米组心肌组织中SOD、GSH-Px活性比模型组明显升高（P0.05），差异具有统计学意义；三组不同粒径二氧化铈纳米颗粒组之间相比，10-25nm粒径组SOD、GSH-Px活性明显高于另外两组（P0.05），差异具有统计学意义，1-10nm粒径组与50nm粒径组比较，SOD、GSH-Px活性明显升高（P0.05），差异具有统计学意义。 3.2模型组大鼠心肌组织中MDA含量比假手术组明显升高（P0.05），差异具有统计学意义；三组不同粒径二氧化铈纳米颗粒组心肌组织中MDA含量比模型组明显降低（P0.05），差异具有统计学意义；三组不同粒径二氧化铈纳米颗粒组之间相比，10-25nm粒径组MDA含量明显低于另外两组（P0.05），差异具有统计学意义，1-10nm粒径组与50nm粒径组比较，MDA含量明显降低（P0.05），差异具有统计学意义。 3.3大鼠心肌组织病理学显示，假手术组较模型组心肌细胞结构完整，三种不同粒径二氧化铈纳米颗粒组和模型组比较细胞形态有明显的改善，但仍然不如假手术组，存在炎症细胞浸润等现象。 3.4心肌细胞凋亡检测结果显示，模型组心肌细胞凋亡指数（apoptoticindex, AI）较假手术组显著升高（P0.05），差异具有统计学意义；三种不同粒径二氧化铈纳米颗粒组AI均明显低于模型组（P0.05），差异具有统计学意义，其中10-25nm组AI明显低于另外两组（P0.05），差异具有统计学意义，1-10nm组、50nm组两组之间无显著差异（P0.05）。 4结论 4.1二氧化铈纳米颗粒可显著提高大鼠缺血再灌注损伤心肌组织中SOD、GSH-Px的活性，降低MDA的含量。 4.2二氧化铈纳米颗粒明显降低了心肌细胞凋亡的发生，改善了再灌注损伤引起的心肌细胞的形态改变。 4.3减轻了大鼠心肌细胞缺血再灌注损伤过程中的氧化应激，对大鼠心肌细胞起到了一定程度的保护作用。
[Abstract]:1 background and purpose of research
At present, with the development of China's economy and the continuous improvement of the people's living standards, the incidence of ischemic heart disease and the resulting mortality are increasing. The continuous renewal and development of various clinical treatments provide more effective treatment for this kind of disease, but the myocardial ischemia reperfusion injury (Myocardium ischem) The adverse effects of IC reperfusion injury, MIRI) in the recovery of the patient's condition are also gradually highlighted. Myocardial ischemia reperfusion injury is manifested by a period of ischemia period of the cardiac muscle cells, after the recovery of the blood supply, the damage of the cardiac myocytes is not mitigated or recovered, but is more severe than before the reperfusion. The phenomenon of heavy damage. How to effectively prevent and cure the damage caused by ischemia-reperfusion injury to myocardial cells has become a hot spot in medical research. Related research is such as beta blocker, calcium antagonist, renin angiotensin inhibitor and so on, but the effect is not very ideal. Therefore, to find a practical and effective method. Effective methods or drugs to treat or improve ischemia-reperfusion injury have become an urgent problem in clinical practice.
The mechanism of myocardial ischemia reperfusion injury is very complicated. Apoptosis is considered to be one of its important mechanisms. Apoptosis is also called programmed cell death. The enlargement of infarct size after reperfusion is considered to be caused by apoptosis. The apoptosis in the process of ischemia-reperfusion may be based on oxygen free radical (oxygenfree radi) CALS, OFR) produced in a large scale, calcium overload in cells, mitochondrial damage and other causes, among which oxygen free radicals are considered to be one of the most important causes. Oxygen free radicals play a vital role in myocardial ischemia and reperfusion. Cardiac arrhythmia, heart after myocardial ischemia and reperfusion, heart The enlargement of infarct area is caused by a large number of explosive oxygen free radicals. In the process of ischemia-reperfusion, a large number of oxygen free radicals are damaged by the oxygen free radicals, which have the lipid peroxidation with the more unsaturated fatty acids in the membrane phospholipids, and make the ion channels, enzymes and other membrane eggs on the membrane. The activity of white decreased, which affected the function of cell membrane and organelle membrane, and damaged its integrity and reduced its fluidity.
The rare earth resources of China are rich, two cerium oxide as a kind of rare earth material is widely used in various fields. Nano materials have special physical and chemical properties because of their own scale, and the research on nano materials is becoming more and more in-depth. Two cerium oxide nanoparticles (CeO2nanoparticles) as a kind of nanomaterial, in recent years, It has been applied to many aspects of industry, automobile manufacturing and people's daily life. Studies have proved that two cerium oxide nanoparticles have strong biological activity to remove oxygen free radicals and to weaken the oxidative stress reaction of organisms.
The purpose of this study was to observe the effects of two cerium oxide nanoparticles on superoxide dismutase (superoxide dismutase, SOD), glutathione peroxidase (glutathioneperoxidase, GSH-Px), malondialdehyde (malondialdehyde, MDA) and apoptosis (apoptosis) in the myocardial tissue of rats with ischemia-reperfusion injury, and to explore the effect of the ischemia reperfusion injury on rats. The protective effect of injecting damaged myocardium provides more prevention and treatment for ischemia-reperfusion injury.
2 materials and methods
2.1 the preparation of two cerium oxide nanoparticles suspension: dissolve two cerium oxide nanoparticles with different nanoparticle diameter in PBS buffer solution (PH7.4,0.01mM) and mix them well. Before injection, they are put into ultrasonic cell breakers, and the operation of ultrasonic oscillation 2min is carried out to make the suspension fully mix well again, and then then use 0.2 mu m. The filter filter of the needle filter can be used.
2.2 group of experimental animals: 50 healthy male SD rats and weight of 250~300g, randomly divided into 5 groups, the sham operation (sham) group, the model (I/R) group, the 1-10nm particle size group, the 10-25nm particle size group, the 50nm particle size group, and each group of 10 rats. The rats were fed with 10% chloral chloral (300mg/kg) for intraperitoneal injection, and supine with 10% chloral chloral aldehyde (300mg/kg). The position was firmly fixed, trachea cannula was separated from the trachea, and the ventilator was connected when opening the chest. The volume of the ventilator was set to 3ml/100g, the respiratory frequency was 70 times per cent. The respiration rate was more obvious than 2:1. in the left edge of the sternum of the rat. It was the most obvious incision in the left edge of the rat sternum, from the 4,5 intercostal chest, the pericardial membrane and the heart. The line between the ears and the root of the aorta was about 3~4mm. The ligation line and the heart surface were padded with a grooved latex tube. After the arrhythmia, the anterior descending branch of the left coronary artery was ligated (left anterior decreased coronary artery, LAD). The color of the distal myocardium in the ligation line changed to cyanosis to prove that the ligation was successful. After ligating 45min, the ligation line was loosened. The model group was injected with PBS buffer solution (0.2ml/100g) and the left anterior descending branch of the left coronary artery was not ligation, and the model group was injected with PBS buffer solution (0.2ml/100g) after 24h via the tail vein before operation, and the left anterior descending branch of the left coronary artery was ligated 45min and released 120min. The success was successful in the sham operation group. The PBS buffer solution (0.2ml/100g) was injected into the caudal vein of the left coronary artery before operation. The ischemia reperfusion model was made. The three different size groups were injected with CeO2 nanoscale suspension (0.2ml/100g) of each particle by injection of 24h through the tail vein before operation, and the model group was made in the same model.
2.3 after 120min reperfusion, the rats were killed and the heart was removed quickly. The cold physiological saline was fully washed to no blood residue, and the myocardial tissue of the left ventricular ischemic area was divided into two parts. A part of the left ventricular ischemic area was quickly absorbed by the moisture absorption of filter paper to the refrigerator of -80 C, and the other part was fixed in Formaldehyde Solution for histopathology. Detection and detection of cell apoptosis. The fresh myocardium preserved in the refrigerator at -80 C was prepared in the myocardial tissue homogenate under the low temperature environment. The activity of SOD was measured by xanthine oxidase method, GSH-Px activity was measured by colorimetric method, and the content of MDA was determined by thiobarbituric acid method. The specific operation procedure was carried out in the kit instructions strictly. The Formaldehyde Solution was carried out in the kit. Routine paraffin sections were made from the fixed part of rat cardiac muscle and used for hematoxylin eosin staining (HE staining) and apoptosis detection.
2.4 statistics of each group of data, all data are mean average number of standard deviation (x + s), each group of data is compared with single factor analysis of variance, 22 comparison using LSD method, the application of SPSS17.0 statistical analysis software analysis, test level alpha 0.05, P0.05 think there is statistical meaning.
3 Results
The activity of SOD and GSH-Px in the myocardium of the 3.1 model group was significantly lower than that in the sham group. The difference between the groups was significant (P0.05), and the difference was statistically significant. The activity of SOD in the myocardial tissue of the two cerium oxide nanometers of the three groups was significantly higher than that in the model group (P0.05), and the difference was statistically significant; the three groups of different particle sizes were two ceria. The activity of SOD and GSH-Px in the 10-25nm particle group was significantly higher than that of the other two groups (P0.05), and the difference was statistically significant. Compared with the 50nm particle size group, the 1-10nm particle size group was significantly higher in SOD, GSH-Px activity (P0.05), and the difference was statistically significant.
The content of MDA in the myocardial tissue of the 3.2 model group was significantly higher than that of the sham group (P0.05), and the difference was statistically significant. The content of MDA in the myocardial tissue of the two cerium oxide nanoparticles group of the three groups was significantly lower than that of the model group (P0.05), and the difference was statistically significant; the three groups of different particle sizes were compared with the two cerium oxide nanoparticles group, 10- The content of MDA in 25nm particle size group was significantly lower than that of the other two groups (P0.05), and the difference was statistically significant. Compared with the 50nm particle size group, the content of MDA was significantly decreased (P0.05), and the difference was statistically significant.
The myocardial histopathology of the 3.3 rats showed that the myocardial cell structure of the sham operation group was more complete than that of the model group. The morphology of the three different particle size two cerium oxide nanoparticles group and the model group were obviously improved, but it was still not as good as the sham operation group and the infiltration of inflammatory cells.
3.4 myocardial apoptosis detection results showed that the apoptosis index (apoptoticindex, AI) of the model group was significantly higher than that of the sham operation group (P0.05), and the difference was statistically significant. The three different particle size two cerium oxide nanoparticles group AI were significantly lower than the model group (P0.05), the difference was statistically significant, and the 10-25nm group AI was significantly lower than that of the other group. The difference between the two groups (P0.05) was statistically significant, but there was no significant difference between group 1-10nm and group 50nm (P0.05). There was no significant difference between the two groups.
4 Conclusion
4.1 two cerium oxide nanoparticles can significantly increase the activity of SOD and GSH-Px and reduce the content of MDA in rats with ischemia-reperfusion injury.
4.2 two cerium oxide nanoparticles significantly reduced the occurrence of cardiomyocyte apoptosis and improved the morphological changes of myocardial cells induced by reperfusion injury.
4.3 alleviated oxidative stress during myocardial ischemia-reperfusion injury in rats and played a protective role in rat cardiomyocytes.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

【参考文献】