【作者】 俞坚武；

【导师】 徐耕；

【作者基本信息】 浙江大学 ， 内科学(心血管病)， 2001， 硕士

【摘要】 目的：交感神经系统功能亢进、过高的儿茶酚胺水平对临床心血管疾病是一个不良的预后因素，但其影响心脏病变确切的细胞分子水平机制和途径并未完全明确。本实验通过观察在异丙肾上腺素引起的大鼠心肌损伤过程中心肌细胞凋亡的改变，以及凋亡相关基因Fas与Bcl-2是否参与调控由异丙肾上腺素引起的心肌损伤过程中心肌细胞凋亡的进程，并探讨在此过程中异丙肾上腺素对心肌细胞凋亡的促发和演变可能的影响，为进一步研究儿茶酚胺对心脏损伤的细胞分子水平机制及临床药物干预建立实验基础。 方法：采用大剂量异丙肾腺素（Isoproterenol，ISO）皮下注射造成大鼠心肌损伤模型；50只SD大鼠随机分成三组：（1）心肌损伤早期组20只 ISO15mg/kg/d一次皮下注射后6小时处死；（2）心肌损伤组20只 ISO15mg/kg/d间隔24小时连续三次皮下注射，首次注射后72小时处死；（3）对照组10只 予等量生理盐水代替ISO皮下注射，其余方法同前；分别以HE染色光镜下观察心肌组织病理学改变，原位末端标记法（TUNEL）及免疫组化法检测心肌凋亡细胞、心肌细胞Fas与Bcl-2基因的蛋白表达变化，资料统计采用t检验、χ~2检验、Ridit分析及Spearman等级相关分析。结果： 对照组：HE染色见心肌无病理损伤；TUNEL标记染色阴性；Fas蛋白、Bcl-2蛋白表达均较低，其中仅1例Fas蛋白弱阳性（+）表达，2例Bcl-2蛋白弱阳性（+）表达。 心肌损伤早期组：20例HE染色病理损伤分级归属为Ⅰ级；TUNEL染色5例呈阳性标记（5/20，25％）；细胞凋亡指数（AI，‰）为7.56±1.53；Bcl-2蛋白 兔疫组化染色检测结果2例呈弱阳性（＋）、l例呈阳性（－H－）表达；BCI－2基因的 蛋白表达等级经m山t分析，与对照组比较无明显统计差异（P＞0刀5）。Fas蛋白免 疫组化染色检测结果示6例弱阳性（＋），4例阳性（＋＋），l例强阳性（＋＋＋）；Fas 基因的蛋白表达等级经＊山t分析；与对照组比较有显著性差异（P＜0刀5）。 心肌损伤组：光镜下HE染色病理损伤分级结果为1级5例、11级2例、Ill 级 8例、IVS例；HE病理损伤级别经 Ridit分析，与损伤早期组比较有显著性差 异（P＜0．001）。TU’NEL标记 19例阳性 （19／2，95％）；细胞凋亡指数（AI，hi） 为14．82士4．IO，与损伤早期组比较有显著性差异（卜．83，P＜0刀01）。免疫组化染 色检测显示其中 Fas蛋白表达弱阳性（＋）2例、阳性（＋＋）13例、强阳性卜＋＋） 5例：B。1－2蛋白表达弱阳性（十）12例、阳性（十十）3例、强阳性（＋十＋）1例； Fas基因的蛋白表达等级经Ridit分析，与对照组及心肌损伤早期组比较有显著性 差异（P＜0刀of，P＜0刀of）；BCI－2基因的蛋白表达等级经Ridt分析，与对照组 及心肌损伤早期组比较亦有显著性差异（P＜0刀1，P＜0刀1）；而且在心肌损伤组 中，Fas基因的蛋白表达强度上调更为明显，经Ridit分析，与Bcl－2基因的蛋白 表达等级比较有显著性差异（P＜口刀of）。异丙肾上腺素致大鼠心肌损伤组中，Fas 基因的蛋白表达等级与心肌细胞凋亡指数之间有较高的正相关性，其相关系数 r＝0．776（P＜0刀of）；Bcl－2基因的蛋白表达等级与心肌细胞凋亡指数之间也有一定 的正相关性，r。0．482（P（刀5）。 结论：在大剂量异丙肾上腺素引起的大鼠心肌损伤过程中存在着心肌细胞凋 亡现象。大剂量异丙肾上腺素一次皮下注射后6小时，已经存在心肌细胞凋亡过 程的激活，Fas基因的蛋白表达水平上调。心肌细胞凋亡在异丙肾上腺素引起的 心肌损伤早期即己出现。大剂量异丙肾上腺素连续皮下注射引起的心肌损伤过程丑 中心肌细胞凋亡现象明显活跃。在异丙肾上腺素引起的心肌损伤过程中，心肌凋 亡阳性细胞及Ns、sd－2蛋白阳性表达心肌细胞主要位于心内膜下心肌变性的损 伤区以及在心肌凝固性坏死灶的周围区域。凋亡相关基因Fas。Bcl－2参与异丙肾 上腺素引起的心肌损伤过程中心肌细胞凋亡过程的调控，Fas基因的表达在异丙 肾上腺素引起的心肌损伤过程中，对心肌细胞凋亡的发生及进展过程起着重要调 控作用。更多还原

【Abstract】 OBJECTIVE: The activation of sympathetic nerves system and elevation of the catecholamine correlates with the severity of ventricular dysfunction in the cardiovascular disease. But the mechanism of the cellular and molecular level is not well known. So the purpose of our study is to observe the phenomena of apoptosis in cardiomyocytes and to investigate the influence of the Isoproterenol (ISO) in the progression of cardiornyocytes apoptosis on the model of rat myocardium injury induced by the ISO, and also to assess the expression of apoptosis related Gene Fas and Bcl-2 during the rat cardiac injury followed by the ISO administration. We make experimental exploration in the mechanism of cardiac injury induced by the cathecholamine. METHODS: To establish the rat myocardium injury model by ISO subcutaneous injection. Fifty male Sprague-Dawley rats were divided randomly into three groups. (1) Early stage of cardiac injury group (n=20): The rats were sacrificed at 6 hours after single 15mg/kg ISO subcutaneous injection; (2) Cardiac injury group (n20): The rats received subcutaneous injections of ISO (1 5mg/kg/d for 3 days), and were sacrificed at 72 hours ?? followed the first injection; (3) Control group (n= 10): The rats were received subcutaneous injections with normal saline as substitution for ISO. Myocardial damage stained with hematoxylin and eosin was examined under the microscope in all S-D rats, Fas and Bcl-2 were identified by imrnunohistochemistry analysis, Apoptotic cells were stained in situ by terminal deoxynucleotidyl transferase mediated-dUTP nick end Labeling. The statistical data was analyzed by student t test, Chi square test, test of relative to an identified distribution unit and Spearman rank correlation analysis. Value of P<0.05 were considered to be significant. RESULTS: Control: No. myocardium damage stained with HE under microscope; In TUNEI analysis, myocardial tissue sections were negative; the expression of Fas and Bcl-2 were very low. Early stage of cardiac injury group: The myocardium damage stained with HE in 20 rats was grade I ; TU7NEL staining show positive in 5 myocardial tissue sections of rats, The apoptosis index was 7.56 ?1 53?No significant difference in the degree of the expression of Bcl-2 between the control and the early stage of cardiac injury group, but Ridit static analysis showed that the degree of the expression of Fas was significantly up-regulation (p<O.O5). Cardiac injury group: The myocardium damage stained with HE was obviously serious; The myocardial tissue sections in 19 rats appeared apoptotic by TUNEL analysis, The apoptosis index in the cardiac injury group was 14.82?.10 vs 7.56?1.53 in the early stage of cardiac injury group (P<0.00 1); The intensity of staining of Fas was apparently significantly increased respectively compared with the control and the early stage of cardiac injury group(P<0.00l, P<0.001), and the degree of ?.--- expression of Bcl-2 was also significantly increased respectively compared with the control and the early stage of cardiac injury group (P<0.O1,P<0.01), But the up-regulation in Fas expression was more higher than in Bcl-2 expression by Ridit analysis (P<0.00 l).The level of expression of ap更多还原