本文選題：二氧化鈰納米顆粒 + 心肌缺血再灌注損傷��； 參考：《鄭州大學(xué)》2014年碩士論文

【摘要】：1研究背景和目的 目前，隨著我國(guó)經(jīng)濟(jì)發(fā)展和人民生活水平的不斷提高，缺血性心臟疾病的發(fā)生率和由此引發(fā)的死亡率正不斷上升。各種臨床治療手段的不斷更新發(fā)展，為此類疾病提供了更多更有效的治療方法，但同時(shí)心肌缺血再灌注損傷(Myocardium ischemic reperfusion injury，MIRI)在患者病情的恢復(fù)中起到的不利影響也逐漸突顯了出來(lái)。心肌缺血再灌注損傷表現(xiàn)為當(dāng)心肌細(xì)胞經(jīng)過(guò)一段時(shí)間的缺血期，血流供應(yīng)恢復(fù)之后，心肌細(xì)胞的損傷并沒(méi)有減輕或者恢復(fù)，反而出現(xiàn)了較再灌注以前更加嚴(yán)重的損傷的現(xiàn)象。如何有效的防治缺血再灌注損傷對(duì)心肌細(xì)胞造成的損害成為有關(guān)醫(yī)學(xué)研究的一個(gè)熱點(diǎn)，相關(guān)的研究有如β-受體阻滯劑，鈣離子拮抗劑，腎素血管緊張素抑制劑等，但效果卻都不是十分的理想。因此，尋找到一種切實(shí)而有效的方法或藥物來(lái)治療或者改善缺血再灌注損傷成為臨床上一個(gè)亟待解決的問(wèn)題。 心肌缺血再灌注損傷的發(fā)生機(jī)制十分復(fù)雜。細(xì)胞凋亡被認(rèn)為是其重要的發(fā)生機(jī)制之一。凋亡又稱為細(xì)胞的程序性死亡，再灌注后心肌梗死面積擴(kuò)大即認(rèn)為是細(xì)胞凋亡引起的。缺血再灌注過(guò)程中的細(xì)胞凋亡可能是由氧自由基(oxygenfree radicals，OFR)的大規(guī)模爆發(fā)性產(chǎn)生、細(xì)胞內(nèi)的鈣超載、線粒體損傷等幾個(gè)原因引起的，其中氧自由基的大量產(chǎn)生被認(rèn)為是最主要的原因之一。在心肌的缺血再灌注過(guò)程中，氧自由基起到了至關(guān)重要的作用。心肌缺血再灌注后出現(xiàn)的心律失常、心肌梗死面積擴(kuò)大等現(xiàn)象均是由于大量爆發(fā)性產(chǎn)生的氧自由基引起的。缺血再灌注過(guò)程中，大量產(chǎn)生的氧自由基對(duì)細(xì)胞結(jié)構(gòu)進(jìn)行了破壞，其與細(xì)胞膜磷脂中含量較多的不飽和脂肪酸發(fā)生脂質(zhì)過(guò)氧化反應(yīng)，使細(xì)胞膜上的離子通道、酶等膜蛋白的活性降低，從而影響了細(xì)胞膜及細(xì)胞器膜的功能，使其完整性遭到破壞，流動(dòng)性降低。 我國(guó)的稀土資源豐富，二氧化鈰作為一種稀土材料被廣泛的應(yīng)用于各個(gè)領(lǐng)域。納米材料因其自身尺度的原因而具有特殊的物理化學(xué)性質(zhì)，針對(duì)納米材料的研究也日漸深入。二氧化鈰納米顆粒(CeO2nanoparticles)作為一種納米材料，在近些年來(lái)被應(yīng)用于工業(yè)、汽車制造業(yè)及人民日常生活中的多個(gè)方面。有研究證實(shí)，二氧化鈰納米顆粒具有能夠清除氧自由基，減弱有機(jī)體的氧化應(yīng)激反應(yīng)等很強(qiáng)的生物活性。 本研究旨在觀察二氧化鈰納米顆粒對(duì)大鼠缺血再灌注損傷心肌組織中超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽過(guò)氧化物酶(glutathioneperoxidase, GSH-Px)、丙二醛(malondialdehyde,MDA)和細(xì)胞凋亡(apoptosis)的影響，從而探討其對(duì)大鼠缺血再灌注損傷心肌的保護(hù)作用，為對(duì)缺血再灌注損傷提供更多的防治手段。 2材料和方法 2.1二氧化鈰納米顆粒懸液的制備：分別將不同納米粒徑的二氧化鈰納米顆粒溶解在PBS緩沖液（PH7.4，0.01mM）中，并使其充分混勻，注射前，再將其放入超聲細(xì)胞破碎儀中，進(jìn)行超聲震蕩2min的操作，，使懸液再次充分混合均勻，完成后再用0.2μm的針頭過(guò)濾器過(guò)濾方可使用。 2.2實(shí)驗(yàn)動(dòng)物分組：健康雄性SD大鼠50只，體重在250~300g之間，隨機(jī)分成5組，假手術(shù)(sham)組、模型(I/R)組、1-10nm粒徑組、10-25nm粒徑組、50nm粒徑組，每組10只。適應(yīng)性飼養(yǎng)一周后進(jìn)行模型的制作。大鼠采用10%水合氯醛（300mg/kg）進(jìn)行腹腔注射麻醉，仰臥位固定牢固，分離氣管行氣管插管，開胸時(shí)連接呼吸機(jī)。小動(dòng)物呼吸機(jī)潮氣量設(shè)定為3ml/100g，呼吸頻率為70次/分，呼吸比2:1。在大鼠胸骨左緣心臟搏動(dòng)最為明顯處切開皮膚，自第4、5肋間進(jìn)胸，撕破心包膜，充分暴露心臟。于肺動(dòng)脈圓錐與左心耳之間距離主動(dòng)脈根部約3~4mm處穿線，在結(jié)扎線和心臟表面墊一帶有凹槽的乳膠管，待心律規(guī)整后結(jié)扎左冠狀動(dòng)脈前降支（left anterior decreased coronary artery,LAD），結(jié)扎線遠(yuǎn)端心肌顏色先變白后變?yōu)樽辖C證明結(jié)扎成功。結(jié)扎45min后松解結(jié)扎線，再灌注120min，缺血部位心肌顏色恢復(fù)為模型制作成功。假手術(shù)組于術(shù)前24h尾靜脈注射PBS緩沖液（0.2ml/100g），左冠狀動(dòng)脈前降支只穿線不結(jié)扎；模型組于術(shù)前24h經(jīng)尾靜脈注射PBS緩沖液（0.2ml/100g），左冠狀動(dòng)脈前降支結(jié)扎45min，松解120min，成功制作缺血再灌注模型；三種不同納米粒徑組均于術(shù)前24h經(jīng)尾靜脈注射各自粒徑CeO2納米懸液（0.2ml/100g），模型制作同模型組。 2.3心肌再灌注120min后處死大鼠，迅速取出心臟，冷生理鹽水充分沖洗至無(wú)血液殘留，取左心室缺血區(qū)心肌組織，分為兩部分，一部分迅速用濾紙吸干水分后轉(zhuǎn)移至-80℃冰箱中保存待用，另外一部分置于甲醛溶液中固定，用于組織病理學(xué)觀察和細(xì)胞凋亡的檢測(cè)。將-80℃冰箱中保存的新鮮心肌組織在低溫環(huán)境下制備心肌組織勻漿，應(yīng)用黃嘌呤氧化酶法測(cè)定SOD活性，比色法測(cè)定GSH-Px活性，硫代巴比妥酸法測(cè)定MDA含量。具體操作步驟嚴(yán)格按照試劑盒說(shuō)明書進(jìn)行。將甲醛溶液中固定的部分大鼠心肌組織制備常規(guī)石蠟切片，用于蘇木素-伊紅染色（HE染色）、細(xì)胞凋亡的檢測(cè)。 2.4統(tǒng)計(jì)各組數(shù)據(jù)，所有數(shù)據(jù)均以均數(shù)±標(biāo)準(zhǔn)差（x±s）表示，各組數(shù)據(jù)間比較采用單因素方差分析，兩兩比較采用LSD法，應(yīng)用SPSS17.0統(tǒng)計(jì)分析軟件進(jìn)行分析，檢驗(yàn)水準(zhǔn)α取0.05，P0.05認(rèn)為有統(tǒng)計(jì)學(xué)意義。 3結(jié)果 3.1模型組大鼠心肌組織中SOD、GSH-Px活性比假手術(shù)組明顯降低，組間差異顯著（P0.05），差異具有統(tǒng)計(jì)學(xué)意義；三組不同粒徑二氧化鈰納米組心肌組織中SOD、GSH-Px活性比模型組明顯升高（P0.05），差異具有統(tǒng)計(jì)學(xué)意義；三組不同粒徑二氧化鈰納米顆粒組之間相比，10-25nm粒徑組SOD、GSH-Px活性明顯高于另外兩組（P0.05），差異具有統(tǒng)計(jì)學(xué)意義，1-10nm粒徑組與50nm粒徑組比較，SOD、GSH-Px活性明顯升高（P0.05），差異具有統(tǒng)計(jì)學(xué)意義。 3.2模型組大鼠心肌組織中MDA含量比假手術(shù)組明顯升高（P0.05），差異具有統(tǒng)計(jì)學(xué)意義；三組不同粒徑二氧化鈰納米顆粒組心肌組織中MDA含量比模型組明顯降低（P0.05），差異具有統(tǒng)計(jì)學(xué)意義；三組不同粒徑二氧化鈰納米顆粒組之間相比，10-25nm粒徑組MDA含量明顯低于另外兩組（P0.05），差異具有統(tǒng)計(jì)學(xué)意義，1-10nm粒徑組與50nm粒徑組比較，MDA含量明顯降低（P0.05），差異具有統(tǒng)計(jì)學(xué)意義。 3.3大鼠心肌組織病理學(xué)顯示，假手術(shù)組較模型組心肌細(xì)胞結(jié)構(gòu)完整，三種不同粒徑二氧化鈰納米顆粒組和模型組比較細(xì)胞形態(tài)有明顯的改善，但仍然不如假手術(shù)組，存在炎癥細(xì)胞浸潤(rùn)等現(xiàn)象。 3.4心肌細(xì)胞凋亡檢測(cè)結(jié)果顯示，模型組心肌細(xì)胞凋亡指數(shù)（apoptoticindex, AI）較假手術(shù)組顯著升高（P0.05），差異具有統(tǒng)計(jì)學(xué)意義；三種不同粒徑二氧化鈰納米顆粒組AI均明顯低于模型組（P0.05），差異具有統(tǒng)計(jì)學(xué)意義，其中10-25nm組AI明顯低于另外兩組（P0.05），差異具有統(tǒng)計(jì)學(xué)意義，1-10nm組、50nm組兩組之間無(wú)顯著差異（P0.05）。 4結(jié)論 4.1二氧化鈰納米顆粒可顯著提高大鼠缺血再灌注損傷心肌組織中SOD、GSH-Px的活性，降低MDA的含量。 4.2二氧化鈰納米顆粒明顯降低了心肌細(xì)胞凋亡的發(fā)生，改善了再灌注損傷引起的心肌細(xì)胞的形態(tài)改變。 4.3減輕了大鼠心肌細(xì)胞缺血再灌注損傷過(guò)程中的氧化應(yīng)激，對(duì)大鼠心肌細(xì)胞起到了一定程度的保護(hù)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

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相關(guān)期刊論文 前1條

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