【作者】 罗羽慧；

【导师】 李法琦；

【作者基本信息】 重庆医科大学 ， 内科学， 2003， 硕士

【摘要】 背景、目的 细胞凋亡的概念于1972年由Kerr提出，它参与许多生理和病理改变，并已成为生物学和医学研究领域中的一个热门话题。近年来，随着人们对心衰发病机制的认识不断深入，提出了细胞凋亡作为一种不同于坏死的细胞损伤机制，可能参与充血性心力衰竭（CHF）的病理生理过程，是心力衰竭发生、发展的重要机制；细胞凋亡与某些凋亡相关基因的调节与表达有关，凋亡刺激基因Fas是调控凋亡信号途径的重要基因，最近的研究证明Fas/Fas配体（FasL）系统参与多种心血管疾病的发生和发展，Fas/FasL系统可能参与CHF的病理生理过程。检测心肌组织Fas/FasL基因的蛋白或mRNA表达水平可以了解凋亡的程度与心力衰竭的严重程度，这对于我们进一步理解CHF的发病机制及治疗有着重要的启示。血管紧张素转换酶抑制剂（angiotensin converting enzyme inhibitor, ACEI）可通过抑制神经内分泌过度激活，减轻心脏负荷，预防和逆转心肌重塑等机制，有效地预防和治疗心力衰竭。福辛普利是一种长效ACEI，在临床工作中发现使用它能有效地缓解心力衰竭患者的症状，使扩大的心脏恢复正常。本实验通过缩窄大鼠腹主动脉构建慢性CHF模型，探讨福辛普利干预对心肌细胞凋亡及Fas/FasL基因表达的影响。方法 以腹主动脉缩窄法建立大鼠慢性CHF模型。30只Wistar大鼠随机分成假手术组、手术-左室代偿性肥厚组（简称肥厚组）及手术-心衰组。采用原位末端脱氧核糖核苷酸转移酶介导的dUTP缺口末端标记法（terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, TUNEL）观察发生心衰的实验性高血压大鼠和同期仅左室肥厚而未发生心衰的实验性高血压大鼠的心肌细胞凋亡情况，同时以免疫组化ABC显色法分别检测Fas与FasL蛋白水平变化，以逆转录聚合酶链反应法检测Fas基因mRNA的表达改变，从而探讨心<WP=7>肌组织中Fas基因的蛋白与mRNA表达水平与心肌细胞凋亡的关系以及二者在心衰发展过程中的作用。此外，我们以福辛普利对心衰组大鼠进行干预，对照观察血流动力学、心肌细胞凋亡、心肌组织Fas和FasL蛋白及其mRNA表达的变化。结果 假手术组心肌中仅有少许心肌细胞凋亡，实施手术的代偿性肥厚组与心衰组大鼠均有心肌细胞凋亡发生，但心衰组心肌细胞凋亡的数目明显高于对照组（P<0.05）。大鼠经腹主动脉缩窄术后4周，左室肥厚而未发生心衰的大鼠其心肌组织Fas蛋白阳性染色指数明显高于假手术组（P<0.05），但FasL蛋白阳性染色指数与假手术组间无显著性差异（P>0.05）；而发生心衰的大鼠其心肌组织Fas及FasL蛋白阳性染色指数明显高于肥厚组，差异有显著性意义（P<0.05）。与假手术组和肥厚组相比较，心衰组心肌组织Fas基因的mRNA表达也上调（P<0.05）。应用福辛普利干预心衰组大鼠后，与假手术组相比，心衰组LVEDP显著升高（P<0.01），＋dp/dtmax和－dp/dtmax显著降低（P<0.01），福辛普利组LVEDP显著低于心衰组（P<0.01），＋dp/dtmax和－dp/dtmax显著高于心衰组（P<0.01）。心衰组心肌细胞凋亡指数（AI）、心肌组织Fas和FasL蛋白及其mRNA表达水平显著高于对照组（P<0.05），福辛普利组AI、心肌组织Fas和FasL蛋白及其mRNA表达水平显著低于心衰组（P<0.05）。结论 实验性CHF大鼠心肌细胞凋亡、Fas基因的蛋白与mRNA表达水平及FasL蛋白表达均增加，心肌细胞出现凋亡可能是心脏由代偿性心肌肥厚向心衰转变过程中的重要机制，心肌组织Fas水平的升高可能与这一改变有关，并可能是其促进因素；心肌细胞凋亡与Fas/FasL系统参与了慢性CHF的发生、发展过程。福辛普利长期干预慢性CHF，能够降低细胞凋亡及Fas和FasL基因的表达，可能与其减少Fas/FasL系统介导的细胞凋亡的发生，改善心肌功能有关。更多还原

【Abstract】 Background and Purpose The transition from compensated ventricular hypertrophy to heart failure is associated with a marked reduction in myocyte mass and reduction in contractile force. The reduction in myocyte mass and the functional loss may be ascribed to the increased cell death by apoptosis, while the increased expression of Fas gene may accelerate the process. In this experiment, we investigated the changes of myocardial apoptosis and the expression of Fas and Fas ligand (FasL) genes in myocardium of rats with congestive heart failure, and studied the effects of fosinopril (angiotensin converting enzyme inhibitor, ACEI) on myocardial apoptosis and the expression of Fas and FasL genes of cardiomyocyte in experimental congestive heart failure rats. Methods Rat models were experimental hypertensive rats. Thirty Wistar-Kyoto rats were divided randomly into sham-operated group, operated group-left ventricular compensated hypertrophy group and operated group-congestive heart failure group. Terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) and ABC immunohistochemical staining were used respectively to detect the changes of apoptosis and protein expression of Fas and FasL genes, and mRNA expression of Fas gene was evaluated by RT-PCR analysis. After the model of chronic congestive heart failure was established by gradually constricting the abdominal aorta of Wistar-Kyoto rats, the animals were divided into heart failure group and fosinopril treated group. Another 10 rats were employed as control with sham-operated group. Hemodynamic parameters were determined, and changes of apoptosis and the expression of Fas and FasL genes were detected as well.<WP=9> Results The data showed that few apoptotic myocardial cells were found in sham-operated group, while apoptotic myocardial cells were found both in congestive heart failure group and compensated hypertrophy group. The number of apoptotic myocardial cells in congestive heart failure group increased significantly in comparison with that in compensated hypertrophy group (P<0.05). There was significant difference (P<0.05) in positive index (PI) of Fas protein, but no significant difference (P>0.05) in PI of FasL protein between sham-operated group and compensated hypertrophy group. When the congestive heart failure occurred, positive index (PI) of Fas and FasL protein in myocardial cells were increased very significantly (P<0.05) than those in myocardium of Wistar-Kyoto rats with compensated hypertrophy. The expression of Fas gene in myocardium of rats with congestive heart failure was significantly higher than that in sham-operated group and compensated hypertrophy group (P<0.05). Compared with sham-operated group, left ventricular end-diastolic pressure (LVEDP) in congestive heart failure group was increased (P<0.01), while ＋dp/dtmax and －dp/dtmax were decreased significantly (P<0.01). The LVEDP was lower but ＋dp/dtmax and －dp/dtmax were significantly higher in fosinopril treated group (P<0.01). The apoptosis index (AI) and the expression of Fas and FasL genes in fosinopril group were significantly lower than those in congestive heart failure group (P<0.01).Conclusions Increased myocardial apoptosis may be the important mechanism during the transition from compensated hypertrophy to heart failure in experimental congestive heart failure rats. Changes of myocardial apoptosis and the expression of Fas and FasL genes suggest that apoptosis and Fas/FasL system are involved in the experimental congestive heart failure rats. Fosinopril administration for a long time in case of chronic congestive heart failure may decrease the expression of Fas and FasL genes, and this can contribute to its role of reducing myocardial apoptosis and improve the myocardial function.更多还原