低温论文：炎症因子在低温恶化心力衰竭中作用及EPO的保护机制.doc

蚇羁芀蚁薃羀莃蒃袂肀肂虿螈聿膅蒂蚄肈莇蚇蚀肇葿薀罿肆腿莃袅肅芁薈螁肄莃莁蚇膄肃薇薃膃膅荿袁膂芈薅螇膁蒀莈螃膀膀蚃虿腿节蒆羈膈莄蚁袄膈蒇蒄螀芇膆蚀蚆袃艿蒃薂袂莁蚈袀袂膀蒁袆袁芃螆螂袀莅蕿蚈衿蒇莂羇袈膇薇袃袇艿莀蝿羆莂薆蚅羆肁荿薁羅芄薄羀羄莆蒇袆羃蒈蚂螂羂膈蒅蚇羁芀蚁薃羀莃蒃袂肀肂虿螈聿膅蒂蚄肈莇蚇蚀肇葿薀罿肆腿莃袅肅芁薈螁肄莃莁蚇膄肃薇薃膃膅荿袁膂芈薅螇膁蒀莈螃膀膀蚃虿腿节蒆羈膈莄蚁袄膈蒇蒄螀芇膆蚀蚆袃艿蒃薂袂莁蚈袀袂膀蒁袆袁芃螆螂袀莅蕿蚈衿蒇莂羇袈膇薇袃袇艿莀蝿羆莂薆蚅羆肁荿薁羅芄薄羀羄莆蒇袆羃蒈蚂螂羂膈蒅蚇羁芀蚁薃羀莃蒃袂肀肂虿螈聿膅蒂蚄肈莇蚇蚀肇葿薀罿肆腿莃袅肅芁薈螁肄莃莁蚇膄肃薇薃膃膅荿袁膂芈薅螇膁蒀莈螃膀膀蚃虿腿节蒆羈膈莄蚁袄膈蒇蒄螀芇膆蚀蚆袃艿蒃薂袂莁蚈袀袂膀蒁袆袁芃螆螂袀莅蕿蚈衿蒇莂羇袈膇薇袃袇艿莀蝿羆莂薆蚅羆肁荿薁羅芄薄羀羄莆蒇袆羃蒈蚂螂羂膈蒅蚇羁芀蚁薃羀莃蒃袂肀肂虿螈聿膅蒂蚄肈莇蚇蚀肇葿薀罿肆腿莃袅肅芁薈螁肄莃莁蚇膄肃薇薃膃膅荿袁膂芈薅螇膁蒀莈螃膀膀蚃虿腿节蒆羈膈莄蚁袄膈蒇蒄螀芇膆蚀蚆袃艿蒃薂袂莁蚈袀袂膀蒁袆袁芃螆螂袀莅蕿蚈衿蒇莂羇袈膇薇袃袇艿莀蝿羆莂薆蚅羆肁荿薁羅芄 低温论文：炎症因子在低温恶化心力衰竭中作用及EPO的保护机制【中文摘要】探讨炎症因子在低温恶化心力衰竭中的作用及促红细胞生成素(hypothermia, EPO)的保护机制。方法：雄性SD大鼠120只,2-3月龄,体重240-260g,随机分2组,分别为对照组(A组)10只；阿霉素(adriamycin, ADR)组110只；ADR组均予腹腔注射ADR制作大鼠心衰模型(2.5mgkg-1d-1,2次/周,连续5周),A组予腹腔注射同体积的生理盐水(2次/周,连续5周)。后行经胸壁超声心动图(transthoracic echocardiography,TTE)测定心功能,判断心衰模型是否建立成功。再将ARD组中造模成功存活下的76只大鼠按照随机原则分为常温心衰组(B组)19只；低温心衰组(C组)19只；EPO常温心衰组(D组)19只；EPO低温心衰组(E组)19只。第6周,D、E组腹腔注射EPO(4000IUkg-1d-1,连续4天)：A、B、C组均予腹腔注射同体积的生理盐水(1次/天,连续4天)；第7周将C、E组大鼠放置于气候箱中(4,4小时/天,连续4天),A、B、D组温度与外界温度相同,整个过程重复4天。实验过程中定期观察大鼠的精神状态及死亡情况,实验第7周末(即将C、E组从气候箱中取出当天)行心脏彩超测定心功能及终止实验。心脏标本常规HE染色观察心室肌病理变化,免疫组化法观测心室肌组织中炎症因子IL-6、IL-1、TNF-a的表达水平、实时荧光定量PCR法测定心室肌组织中炎症因子IL-6、IL-1、TNF-a mRNA表达量。结果：在腹腔注射ADR诱导建立心衰模型过程中,大鼠出现阿霉素中毒的表现,且死亡34只；A组未见上述中毒症状及死亡情况；心衰造模成功后,C组在放置低温气候箱过程中死亡4只,E组死亡1只。(1)心功能水平：与A组相比,其余四组经ADR处理后,LVEDD、LVESD增加(p0.05), LVEF、FS降低(p0.05)；与B组相比,C组LVEDD、LVESD增加(p0.05), LVEF、FS值增加(p0.01),D组LVEF、FS值下降(p0.01),LVEDD、LVESD减小(p0.01)；与D组相比,E组LVESD增加(p0.01), LVEF、FS值下降(p0.01),上述结果显示低温能进一步使心功能恶化,EPO可改善心衰大鼠心功能。(2)HE染色：光镜下可见对照组大鼠心肌细胞形态结构基本正常,其余四组心肌组织内可见不同程度的少量炎性细胞浸润、间质充血水肿、心肌细胞玻璃样变性及坏死。与常温心衰组相比,低温心衰组玻璃样变性及坏死更明显；EPO常温心衰组上述改变较常温心衰组轻。(3)免疫组化：光镜下可见心肌细胞胞浆内棕黄或棕褐着色。与A组相比,其余四组心肌组织内IL-6、IL-1、TNF-a阳性细胞百分比增高,差异有统计学意义(P0.05)；与B组相比,C组心肌组织IL-6、IL-1、TNF-a阳性细胞百分率增高,D组心肌组织IL-6、IL-1、TNF-a阳性细胞百分率下降,差异均有统计学意义(P0.05)；与D组相比,E组心肌组织IL-6、IL-1、TNF-a阳性细胞百分率下降,差异有统计学意义(P0.05)。(4)基因表达：与A组相比,其余四组心衰大鼠心室肌组织IL-6、IL-1、TNF-a mRNA表达量上调,差异有显著意义(p0.01；与B组相比,C组心衰大鼠心室肌组织IL-6、IL-1、TNF-a mRNA表达上调,差异有显著意义(p0.01),D组IL-6、IL-1、TNF-a mRNA表达下调,差异有统计学意义(P0.05)；与D组相比,E组IL-6、IL-1、TNF-a mRNA表达上调,差异有统计学意义(p0.05)。结论：(1)低温促使心衰大鼠心功能进一步恶化,其机制之一可能为低温环境刺激心衰大鼠心肌组织炎症反应加重。(2)EPO调节IL-6、IL-1、TNF-a的转录水平,从而抑制炎症反应,达到改善及拮抗低温恶化心衰大鼠心功能的作用。【英文摘要】:To investigate the effect of inflammatory factors and the protective mechanism of EPO on hypothermia heart failure.Methods:120 male SD rats were randomly divided into two groups.Control group (group A,n=10),Adriamycin group (ARD group,n=110).To construct heart failured model,all rats in ARD group were intraperitoneally injected with adriamycin (2.5mgkg-1d-1,twice every week,general 5 weeks).76 rats successfully modeled in ARD group were randomly divided into 4 groups.Homoeothermy heart failure group(group B,n=19),Hypothermia heart failure group(group C,n=19),Homoeothermy heart failure group interved by EPO(group D,n=19),Hypothermia heart failure group interved by EPO(group E,n=19). Both group D and E received intraperitoneal injection with EPO(4000Ukg-1d-1,continuously 4 days),group A,B and C received intraperitoneal injection with the same volume of 0.9% saline(once a day,continuously 4 days). During the seventh week, rats in group C and E were put into the climate chamber once a day(4,4 hours each time, continuously 4 days),while rats in group A,B and D remained in the homoeothermy condition. In the eighth week, all rats heart function was measured by transthoracic echocardiography(TTE).The pathological changes of myocardial tissue were observed by HE sataining, the protein expression of IL-6,IL-1and TNF-a were observed by immunohistochemistry and the mRNA expression of IL-6,IL-1and TNF-a were measured by qRT-PCR.Result:During the process of modeling,34 rats died for ADR poisoning,All rats in group A lived normally without poisoning or death. And then,4 rats died in group C and one rat died in group E during the experiment of low temperature.(1)Cardiac function:Compared with group A, LVEDD and LVESD of the other four groups increased (p0.05), LVEF and FS decreased (p0.05). Compared with group B, the LVEDD and LVESD of group C and D increased (p0.05,P0.01 respectely). LVEF、FS of group C decreased (p0.05, P0.01 respectely). Compared with group D, LVEDD and LVESD values of group E increased (p0.01),LVEF and FS decreased (p0.01).The results showed hypothermia could lead to further deterioration while EPO could improve cardiac function of heart failure rats.(2) Cardiac histopathological changes:The morphology of cardiac cells of group A was normal.while cardiac cells of the other four groups shows different degree of glassy degeneration or necrosis in myocardial cells, hyperemia and oedema in cardiac stroma.Compared with groupB, the histopathological changes of group C were worse, glassy degeneration or necrosis in myocardial cells and moderate hyperemia,oedema in cardiac stroma were more serious.The histopathological changes of group D were better than in group B.(3)Immunohistochemistry:The cytoplasm of the cardiomyocytes in myocardial tissue was buffy and brown stainned in the light microscope.Compared with group A, the percentage of cells expressed of IL-6,IL-1and TNF-a protein in myocardial tissue of the other four groups increased significantly (p0.05). Compared with group B, the percentage of positive cells of group C increased significantly (p0.05),and the percentage of positive cells of group D increased significantly (p0.05). Compared with group D, the percentage of positive cells of group E increased significantly (p0.05)(4) The expression of gene:Compared with group A,the expression of IL-6,IL-1and TNF-a mRNA in myocardial tissue of the other four groups increased significantly (p0.01). Compared with group B, the expression of IL-6,IL-1 (3 and TNF-a mRNA of group C increased significantly (p0.01), and the expression of IL-6,IL-1(3 and TNF-a mRNA of group D increased significantly(p0.05). Compared with group D,the expression of 1L-6.IL-1and TNF-a mRNA of group E increased significantly (p0.05)Conclusion:1.Hypothermia could lead to further cardiac function deterioration,The mechanism maybe that the hypothermia could aggravate the inflammation of myocardial tissue in rats of heart failure. 2. By adjusting the transcription expression of IL-6,IL-1and TNF-a,EPO could inhibit the inflammatory response, so as to improve cardiac function of hypothermia heart failure rats.【关键词】低温 促红细胞生成素 心力衰竭 心功能 炎症因子 阿霉素【英文关键词】hypothermia erythropoietin heart failure heart function Inflammatory factor adriamycin【目录】炎症因子在低温恶化心力衰竭中作用及EPO的保护机制摘要3-5ABSTRACT5-7第1章 前言11-13第2章 材料与方法13-212.1 实验材料13-142.1.1 实验动物132.1.2 主要仪器132.1.3 主要试剂13-142.1.4 主要溶液的配制142.2 实验方法14-212.2.1 动物模型的建立及分组14-152.2.2 观察项目及检测方法15-202.2