新生鼠窒息致心肌损伤及相关因素影响的实验研究

【作者】 何平；

【导师】 杨达宽； 杨绍军；

【作者基本信息】 昆明医学院 ， 外科学， 2010， 博士

【副题名】新生鼠窒息致心肌损伤与氧化应激及HO-1mRNA表达关系的实验研究

【摘要】 第一部分新生鼠窒息致心肌损伤模型及观测指标的建立目的建立近似人类新生儿窒息的病理过程的新生鼠窒息心肌损伤模型,并监测CK-MB,cTnI及心肌细胞的凋亡等与心肌损伤有关的指标。方法7d SD大鼠48只,雌雄不拘,随机分为对照组F组(n=8只),造模组(n=40只)。造模组分为A、B、C、D、E 5组,每组8只(分别为窒息后6h、24h、48h、72h、7d)。将实验动物分别放入55ml的磨口瓶中,瓶内放入0.005kg的钠石灰,待动物安静后,造模组塞紧瓶塞半小时,取出吸氧120min后,放回母鼠身边继续喂养,分别于窒息后6h、24h、48h、72h、7d后处死;对照组8只放入55ml的磨口瓶中半小时不塞瓶塞,取出120min后放回母鼠身边继续喂养,6h后全部处死。采用ELISA法测CK-MB、cTnI,TUNEL法测心肌纽胞凋亡并做HE染色的心肌病理切片,每组随机抽取一个标本做电镜切片。结果病理切片:1.造模组心肌病理切片有明显的心肌细胞坏死、炎性细胞浸润和出血;电镜切片下有心肌细胞线粒体普遍肿胀,细胞核肿胀,染色质边集,肌丝溶解,Z线不清,线粒体空泡,脊模糊稀疏;对照组光镜和电镜病理切片均正常;2.造模组的CK-MB、cTnI、心肌细胞凋亡均明显增高,与对照组比较,有统计学意义(P＜0.05);3 CK-MB 24h达最高,尔后逐渐下降;cTnI 72h达最高,持续到7d;心肌细胞凋亡随时间延长而增加。结论1.复制常压下新生鼠窒息模型近似于人类新生儿窒息的病理过程的致病机制,相对简单易得。2.模型具有窒息致心肌损伤的病理和临床特征,心肌细胞凋亡是窒息致心肌损伤的一种形式。3.模型存在光镜和电镜下的病理改变,心肌细胞凋亡明显增高,血中CK-MB、cTnI明显增高,为观察分析新生鼠窒息心肌损伤奠定了良好的基础。模型稳定易得,适用于窒息致心肌损伤或(和)窒息致多器官损伤及后遗症的发病机制和相关因素的研究。第二部分新生鼠窒息致心肌损伤与氧化应激及HO-1mRNA表达关系的实验研究目的:缺氧导致心肌损伤是窒息导致的多器官损伤的一个严重的并发症,除缺氧的因素外,还有哪些相关因素的参与,目前还没有完全清楚。利用新生鼠窒息模型,观测氧自由基,抗氧化酶和HO-1mRNA与心肌损伤的关系以及HO-1mRNA抑制剂ZnPP对各观测指标的影响,揭示HO-1mRNA,氧化反应激与窒息致心肌损伤以及心肌组织病理改变之间的内在联系。阐明与窒息心肌损伤密切相关的因素。为新生儿窒息致心肌损伤的预防和治疗提供可靠实验依据。方法:正常对照组8只,窒息组80只(分为窒息组40只,窒息+ZnPP40只。)又分为窒息后6h,24h,48h,72h,7d,5个不同的时间段采血和心肌样本,制作病理切片待检。1.光学显微镜和电镜观察心肌组织病理损伤;2.TUNEL法检测心肌细胞凋亡;3.原位杂交技术检测HO-1mRNA在心肌组织的表达;4.ELISA技术检测血中CK-MB,cTnI水平;5.黄嘌呤氧化酶法测心肌组织的匀浆SDD活性;二硫代二硝基苯甲酸法检测心肌组织匀浆GSH-PX活力;硫代巴比妥酸法检测心肌组织匀浆MDA含量;化学比色法测定心肌组织匀浆XOD活性。结果:1.窒息组心肌组织病理改变:①光学显微镜显示窒息后各时段心肌组织均有少量的炎性细胞浸润和少量出血;电镜下有线粒体肿胀,核固缩,肌浆网肿胀;②窒息+ZnPP组心肌病理改变:光学显微镜下有较多的炎性细胞浸润和出血;电镜显示:心肌细胞融合,肌丝溶解,线粒体肿胀,线粒体脊溶解。窒息+ZnPP组的病理损伤比窒息严重。2.①窒息组血中CK-BM,cTnI水平明显高于对照组,有极显著性差异(P＜0.01);②窒息+ZnPP组血中CK-MB,cTnI水平也明显高于对照组,有极显著性差异(P＜0.01);⑧窒息组血中CK-MB,cTnI水平明显低于窒息+ZnPP组,比较有显著性差异(P＜0.05)。3.①窒息组心肌细胞凋亡率明显高于对照组,有显著性差异(P＜0.01):②窒息+ZnPP组心肌细胞凋亡率明显高于对照组,有极显著性差异(P＜0.01);③窒息+ZnPP组明显高于窒息组,有显著性差异(P＜0.05)。4.①窒息组心肌组织匀浆的SOD活性,GSH-PX活力低于对照组,有极显著性差异(P＜0.01),MDA含量高于对照组,XOD活性高于对照组,有极显著性差异(P＜0.01);②窒息+ZnPP组的SOD活性,GSH-PX活力明显低于对照组,有极显著性差异(P＜0.01);MDA含量高于对照组,有极显著性差异,XOD活性明显高于对照组,有极显著性差异(P＜0.01):③窒息+ZnPP组与窒息组比较,SOD活性,GSH-PX活力明显低于窒息组,MDA含量明显高于窒息组,XOD活性明显高于窒息组,有极显著性差异(P＜0.01)。5.①窒息组心肌组织细胞HO-1mRNA表达高于对照组,有极显著性差异(P＜0.01);②窒息+ZnPP组心肌细胞HO-1mRNA表达高于对照组有显著性差异(P＜0.05);③窒息+ZnPP组与窒息组比较,HO-1mRNA表达明显降低,有极显著性差异(P＜0.01);6.窒息后心肌组织病理损伤和心肌细胞凋亡持续到窒息后7d开始减轻。结论:1、新生鼠窒息后心肌组织和细胞受到明显的损伤,CK-MB,cTnI在外周血中明显升高,心肌有出血,炎性细胞浸润,细胞坏死和凋亡率增高。2、外周血中CK-MB持续时间较短,cTnI在血中持续时间较长,约7d,心肌细胞坏死,凋亡率增加在窒息后7d仍存在,提示对心肌损伤的防治应持续至窒息后7d,以免加重心肌损伤和后遗症发生。3、HO-1mRNA表达与SOD、GSH-PX的活性呈正相关,与XOD的活性,MDA的含量呈负相关。4、窒息组,窒息+ZnPP组的SOD、GSH-PX,XOD,MDA及HO-1mRNA表达的差异均有统计学意义。窒息+ZnPP组氧化应激反应更为强烈,心肌损伤更为严重,证实HO-1mRNA可减轻氧化应激对心肌的损伤作用。5、窒息组窒息+ZnPP组的SOD、GSH-PX,XOD,MDA与对照组比较差异有统计学意义,证实氧自由基参与窒息对心肌损伤。6、该研究提示在窒息致心肌损伤的防治中,可适时使用HO-1mRNA诱导剂,减少氧化应激对心肌的损伤。7、窒息后新生鼠全身和心肌组织均存在氧化应激的改变,这种改变与心肌损伤密切相关,因此,抗氧化治疗可能是在窒息致心肌损伤中的一种重要的治疗方法。更多还原

【Abstract】 Objective To establish an animal model similar to the neonatal asphy- xia with injure of myocardium and to obsever CK-MB,cTnI in blood and apoptosis of myocardial cells.Methods Seven old SD ras(n=48) were radomly divided into Control group(n:8) and model group A、B、C、D、E 5groups(each group=8,after asphyxia6h、24h、48h、72h、7d).The model groups SD rats were put into separably 55ml contaner with CaO-NaOH0.005Kg,the mouth of bottl was sealed up a half of an hour,then being given oxygen 120 min and coming back with mother ras.After 6h、24h、48h、72h、7dWere deid.Blood CK-MB、cTnI was detected by ELISA Methods,apoptosis of myocardiumwas detected by TUNEL Methods and myocardium been made into pathological sections,pathological injure of myocardium tissue was observed through optical microscope and electron microscope.Result 1.The pathological injure of myocardium tissue was remarkable through optical microscope and electron microscope;2.Blood CK-MB、cTnI and apoptosis of myocardium the model groups was higher than control group(P＜0.05);3.Blood CK-MB was highest at 24h,than was lower gradually,Blood cTnI was highest at 72h,than was stable until 7d;the apoptosis of myocardium was more and more growed as time longer gradually.Conclusion1.Relatively convenient and easily got experimental normal pressure neonatal rat asphyxia modle which reproduced human neonatal asphyxia pathogenic mechanism and was successfully.2.Neonatal rat asphyxia modle had pathological injure of myocardium tissue and clinical fecture,apoptosis of myocardium cell was a styl of injure of myocardium after asphyxia.3.The pathological injure of myocardium tissue was remarkable through optical microscope and electron microscope,apoptosis of myocardium and Blood CK-MB、cTnI was higher remarkablein neonatal rat asphyxia modle.The modle was of long term stability and suitable for pathogenesy research of injure of myocardium produced by asphyxia and multiorgan damage which established a good foundation for observing After effect and reaseach of Relatively elements of asphyxia experim-ental. Objective:Myocardium damage was produced asphyxia which is important organof multiple organs disorder in asphyxia.Besides asphyxia,there were.other factors which take part in myocardium damage in asphyxia,it is unclear at present.Making neonatal rat asphyxia modle,revealing internal association between myocardium damage of asphyxia and oxyradical,antioxidase,expression of Heme oxygenase-1mRNA and heme oxygenase-1mRNA imhibition ZnPP.Observing affection of oxyradical,antioxidase,expression of heme oxygenase-1mRNA and heme oxygenase-1mRNA inhibition ZnPP to asphyxia myocardium damage.To illuminate intensive assocition factors with asphyxia myocardium damage.In order to provid reliable laboratory proof for prevention and treatment of asphyxia myocardium damage.Methods:88 after born 7d SD rats,male or female were randomly divided into control group(n=8),asphyxia group(n=40),and asphyxia +ZnPP group(n=40).The asphyxia group was further divided into 6h(n=8),24h(n=8);48h(n=8);72h(n=8); 7d(n=8);and asphyxia +ZnPP group further divied 6h(n=8);24h(n=8);48h(n=8); 72h(n=8);7d(n=8) too.Then,blood and myocardium tissue were took at 6h,24h,48h, 72h,7d.Myocardium tissue been made into pathological sections.1.Pathological injury of myocardium tissue was observed through optical microscope and eleotron microscope;2.Myocardial cell apoptosis was observed through TUNEL method;3. Expression of heme oxygenase-1mRNA was observed through in stitu hybridization histochemistry;4.CK-MB,cTnI level in blood was observed through ELISA method; 5.The activity of SOD was determinated by xanthine oxidase method;the activity of GSH-Px was determinated by the method of dithiobisnitrobenzoing acid;the activity of XOD was determinated by chemical colorimetry method;the content of MDA was determinated by thiobarbituric method in myocardium tissue.Result:1.Myocardium tissue’s pathological changing;①asphyxia group:under optical microscop,myocardium cell swelling,there were a few inflammation cell and a little bleeding;under electon microscop,there were swelled mitochondrion, concentrated cell organs,condensed nucleu;②asphyxia+ZnPP:under optical microscop there was a lot of imflammation cell and bleed much,myocardial fibers arranged in disorder.Myocardial cell edema,becoming vacuole,under electron microscop,myocardial cell fusion,myocardial fibers melting,mitochondrion edema and pycnosis,mitochondrial crista melting,asphyxia+ZnPP group’s pathogenesis injury was serious more than asphyxia group’s.2.①The level of CK-MB,cTnI in blood in asphyxia group was higher much than that in control group(P＜0.01);②the level of CK-MB,cTnI in blood in asphyxia+ZnPP group was higher much than control group(P＜0.01);③the level of CK-MB,cTnI in blood in asphyxia+ZnPP group was higher than that in asphyxia group(P＜0.05).3.①Myocardial cell apoptosis in asphyxia group was higher much than that in control group(P＜0.01);②Myocardial cell apoptosis in asphyxia+ZnPPgroup was higher much than that in control group(P＜0.01);③the myocardial cell apoptosis in asphyxia+ZnPP group was higher than that in asphyxia group(P＜0.05).4.①Activity of SOD、GSH-PxXin myocardium tissue in asphyxia group was lower much than that in control group,activity of XOD,MDA content in myocardium tissue in asphyxia group was highermuch than that in control group(P＜0.01);②Activity of SOD,GSH-Px in myocardium tissue in asphyxia+ZnPP group was lower much than that in control group,activity of XOD,content of MDA in myocardium tissum in asphyxia+ZnPP was higher much than incontrol grou(P＜0.01);③Activity of SOD, GSH-PX in myocardium tissue in asphyxia+ZnPP was lower much than that in asphyxia group,activity of XOD,content of MDA in myocardium tissue in asphyxia+ZnPP group was higher much than in asphyxia group(P＜0.01).5.①Expression of HO-1mRNA in myocardial cell in asphyxia group was highermuch than that in control group(P＜0.01;②expression of HO-1 mRNA in myocardial cell in asphyxia+ZnPP group was higher a little than that in control (P＜0.05);③expression of HO-1mRNA in myocardial cell in asphyxia group was higher much than that in asphyxia+ZnPP group(P＜0.01).6.Myocardium tissue pathological damage and apoptosis of myocardial cell contint until 7d after asphyxia.Conclusion:1.After asphyxia,myocardium tissue damage after asphyxia was obvious,apoptosis of myocardial cell was higher much,there was bleed,a few inflammation cells and CK-MB,cTnI level in blood is higher much.2.After asphyxia,CK-MB,cTnI level in blood is higher much.apoptosis of myocardial cell was higher much than that in control group,which indicated apparent apoptosis of myocardial cell was a style of myocardium tissue damage.3.After asphyxia,oxidative stress increased strong in myocardium,the lower activity of SOD、GSH-PX,the higher activity of XODand MDA content,the more serious in myocardium tissue was,which indicated apparent:Oxidative stress is a important factor in myocardium tissue after asphyxia.4.Expression of HO-1mRNA in myocardial cell in asphyxia group was higher much than that in control group and asphyxia+ZnPP group,expression of HO-1mRNA in myocardial cell in asphyxia group was higher little than that in control group,which means hypoxia inducted high expression of HO-1mRNA of myocardial cell.ZnPP can inhibition expression of HO-1mRNA under hypoxia.5.When hypoxia,there was colse relationship between myocardium tissue damage and expression of HO-1mRNA.The higher expression of HO-1mRNA in myocardial cell was,the more little damage of myocardium tissue was.6.Expression of HO-1mRNA was closed relationship with oxidative stress,the higher expression of HO-1mRNA was,the more activity of SOD,GSH-PX was,the lower activity of XOD and content of MDA was.So maybe using HO-1 induction to protect and treat damage of myocardium tissue after asphyxia is necessary.7.Oxidative stress plays a important role in myocardium tissue damage in asphyxia stress.Increasing expression of HO-1mRNA can inhibiton strong oxidative stress, which indicated apprent;HO-1mRNA and HO-1 can decreased damage of myocardium tisssue when hypoxia.So maybe using antioxidative stress drug to be important treating method to damage of myocardium tissue after asphyxia.更多还原