孕期炎症刺激致子代大鼠心室重构及机制的研究

发布时间：2018-03-20 18:34

  本文选题：孕期炎症　切入点：心室重构　出处：《第三军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：近年来，炎症在心血管疾病发病中重要作用的认识是对高血压的病因寻找中最重要的进展之一。本研究室将高血压的病因追溯至母体孕期，选取脂多糖(Lipopolysaccharide, LPS)作为炎症刺激剂，建立了孕期炎症刺激导致子代大鼠血压升高的动物模型；在此模型的基础上，选取了炎症通路上的关键靶点核转录因子kappa-B(Nuclear factor kappa-B, NF-κB)在母体孕期进行干预，可以有效阻止子代出生后高血压的发生，但关于其机制尚待深入阐明。心室重构是高血压、心力衰竭和心肌病等许多心血管疾病发生发展的共同病理生理过程，从病理生理学角度来说，一方面是心肌细胞发生代偿性肥厚、细胞凋亡、坏死和增生；另一方面是心肌细胞外基质胶原的沉积，胶原网的组成和含量的改变；最终引起心肌实质和间质比例的失衡，使心脏的结构改变和功能失偿。近年来研究认为，神经内分泌因子的过度激活在心室重构病程的发生、发展中极为重要，其中肾素-血管紧张素系统(renin angiotensin system, RAS)则发挥着尤为重要的作用。研究发现，RAS系统中除了ACE-AngⅡ-AT1R通路以外，还存在着与其作用相拮抗的另外一条通路即血管紧张素转化酶-血管紧张素(1-7)-Mas受体轴(Angiotensin converting enzyme2-Angiotensin(1-7)-Mas axis)，ACE2-Ang(1-7)-MasR轴，此条通路通过ACE2降解AngⅡ，其在高血压、心力衰竭的防治及改善心肌纤维化等方面均得到了证实。我室前期研究发现，与循环RAS系统变化不明显相比，肾小球数量减少和肾功能的下降的同时存在肾脏局部RAS系统的明显激活。在脂肪RAS系统中也得到了相同的结论，提示局部组织RAS系统的变化可能是高血压发病的另一重要原因之一。在我室研究中还发现孕期炎症刺激会引起子代大鼠心室肌发生凋亡和重构，且这种重构的发生早于高血压的发生，提示子代的心室重构是引起高血压发生的机制之一。但子代大鼠哪些因子的变化引起了心室重构的发生？以及孕期炎症刺激对子代大鼠心脏局部RAS系统是否产生影响？其规律和特征是什么等尚待进一步研究。 本课题拟通过观察心肌组织胶原的变化，胶原代谢酶MMP-2、TIMP-2及促纤维化因子TGFβ-1、TGFβ-2的变化来研究心肌发生纤维化及心肌重构。并通过观察子代大鼠心脏局部RAS系统上两条代谢通路即ACE-AngⅡ-AT1R通路和ACE2-Ang(1-7)-MasR通路的变化，进一步了解孕期炎症刺激后子代大鼠发生心肌纤维化及心肌间质重构的机制进行研究。 方法： 1.孕鼠分组及给药：将SD孕鼠采用完全随机法分为3组。正常对照组：在孕第8~14天每天腹腔注射无菌生理盐水0.5ml；LPS模型组：在孕第8、10、12天腹腔注射LPS0.79mg/kg，孕第9、11、13、14天给予生理盐水；LPS+PDTC组：在孕第8、10、12天腹腔注射LPS0.79mg/kg，孕第8~14天每天腹腔注射PDTC100mg/kg。 2.称重指标的测定：子大鼠每周进行体重(body weight,BW)的测定，三组实验动物在6周龄和16周龄时麻醉，打开腹腔，快速取出心脏，置于冰冻无菌生理盐水中洗净后，滤纸吸干水分称重，天平称量心脏重量(heart weight,HW)。 3.用天狼星红及Masson胶原染色观察心肌胶原形态学及心肌胶原容积分数的变化。 4.免疫组织荧光的方法检测6周龄和16周龄子代大鼠心肌组织AngⅡ及Mas受体蛋白水平； 5.免疫组织化学方法检测6周龄和16周龄心肌组织中AT1受体的蛋白表达。 6.采用Real-time PCR测定6周龄和16周龄子大鼠心脏组织中ACE、ACE2、AT1R、MasR、MMP-2、TIMP-2、TGFβ-1及TGFβ-2mRNA基因表达水平。 7.采用Western blotting方法测定6周龄和16周龄子代大鼠心肌组织ACE、ACE2、MMP-2、TIMP-2、TGFβ-1及TGFβ-2的蛋白含量； 结果 1.孕期炎症刺激对子代大鼠发生心室重构的影响 (1)孕期炎症刺激对子代大鼠心脏指数的影响 与对照组相比，子代大鼠6周龄时，LPS组心脏重量、心脏质量分数明显升高(P0.05)，PDTC干预后明显下降(P0.05)；子代大鼠16周龄时，LPS组较对照组心脏重量、心脏质量分数明显升高(P0.05)，PDTC干预后显著性降低(P0.01)，LPS组体重明显升高(P0.05)；差异有统计学意义。 (2)孕期炎症刺激对子代大鼠胶原蛋白的影响 天狼星红及Masson染色结果显示，与正常对照组相比，6w、16w，LPS组沉积在心肌间质的胶原表达明显增多；L+P组，红色胶原表达较LPS组减少。 (3)孕期炎症刺激对子代大鼠心肌组织MMP-2、TIMP-2、TGFβ-1及TGFβ-2mRNA水平的影响 子代大鼠6周龄时，与正常对照组相比，LPS组TGFβ-1mRNA表达显著性升高(P0.01)；TGFβ-2mRNA表达水平明显升高(P0.05)，PDTC干预后显著性降低(P0.01)；LPS组MMP-2mRNA表达明显降低(P0.05)；TIMP-2mRNA表达显著性升高(P0.01)。子代大鼠16周龄时，LPS组较正常对照组TGFβ-1mRNA表达明显升高(P0.05)，PDTC干预后明显降低(P0.05)；TGFβ-2mRNA表达明显升高(P0.05)；TIMP-2mRNA表达明显升高(P0.05)PDTC干预后显著性降低(P0.01)；MMP-2mRNA表达明显降低(P0.01)。 (4)孕期炎症刺激对子代大鼠心肌组织MMP-2和TIMP-2蛋白水平的影响 子代大鼠6w、16w，LPS模型组较正常对照组MMP-2蛋白水平显著性降低(P0.05)；TIMP-2蛋白水平显著性升高(P0.05)；LPS组TIMP2-/MMP-2的蛋白水平比值显著升高(P0.05)，PDTC干预后蛋白水平明显降低(P0.05)。 (5)孕期炎症刺激对子代大鼠心肌组织TGFβ-1和TGFβ-2蛋白水平的影响 子代大鼠6w、16w，LPS模型组较正常对照组TGFβ-1的蛋白水平明显升高(P0.05)，PDTC干预后明显降低(P0.05)；TGFβ-2的蛋白水平明显升高(P0.05)。 2.孕期炎症刺激对子代大鼠心脏局部RAS系统的影响 (1)免疫组织荧光测定子代大鼠心肌组织中AngⅡ：LPS组较对照组荧光强度及荧光点明显增强，L+P组较LPS组减少；MasR蛋白表达水平LPS组较对照组荧光强度明显减弱，荧光点明显减少，L+P组红色荧光区较LPS组增多。 (2)免疫组织化学测定子代大鼠心肌组织中AT1R蛋白水平：对照组，阳性细胞数(细胞核着色)少，LPS组，阳性细胞数(细胞核着色)明显增多，L+P组，，阳性细胞(细胞核着色)较LPS组减少。 (3)Real time PCR检测子代大鼠心肌组织ACE、ACE2、MasR及AT1R mRNA水平 子代大鼠6周龄时，与正常对照组比，LPS组ACE mRNA表达有升高趋势，PDTC干预后明显降低(P0.05)；AT1R mRNA表达明显升高(P0.05)； MasR mRNA表达显著性降低(P0.01)，PDTC干预后显著性升高(P0.01)。子代大鼠16周龄时，与正常对照组比，LPS组较ACE mRNA表达明显升高(P0.05)，PDTC干预后显著降低(P0.01)AT1R mRNA表达显著性升高(P0.01)，PDTC干预后明显降低(P0.05)；MasR、ACE2mRNA表达明显降低(P0.05)。 (4)Western blotting检测子代大鼠心肌组织ACE和ACE2蛋白水平 子代大鼠6w、16w，LPS模型组较正常对照组ACE蛋白水平显著性升高(P0.05)，PDTC干预后明显降低(P0.05)；LPS组ACE2蛋白水平显著性降低(P0.05)；LPS组ACE/ACE2的蛋白水平比值显著升高(P0.05)，PDTC干预后明显降低(P0.05)。 结论 1.孕期炎症刺激可导致子代大鼠发生心室重构，NF-κB的抑制剂PDTC能有效逆转心室重构。 2.孕期炎症刺激可引起子代大鼠发生基质金属蛋白酶及基质金属蛋白酶抑制酶表达失衡，转化生长因子β1、转化生长因子β2表达增加，胶原代谢异常，从而引起心肌纤维化，促进了心室重构的发生。 3.孕期炎症刺激一方面可导致子代大鼠心肌组织ACE表达增加，AngⅡ生成增多，激活ACE-AngⅡ-AT1R通路；另一方面使ACE2表达降低减少了AngⅡ的降解和Ang(1-7)的生成，降低了ACE2-Ang(1-7)-MasR通路的活性，造成心脏局部ACE-AngⅡ-AT1R和ACE2-Ang(1-7)-MasR两条通路失衡，引起心脏局部RAS系统的过度激活，而这可能是孕期炎症刺激致子代大鼠发生心室重构的重要分子基础。
[Abstract]:In recent years, understanding of the important role of inflammation in the pathogenesis of cardiovascular diseases is one of the most important progress in the search for the causes of hypertension. This study room will be traced to maternal hypertension, lipopolysaccharide (Lipopolysaccharide, LPS) were selected as inflammatory stimuli, established the animal model of blood pressure during pregnancy leads to inflammation of rat offspring increased; on the basis of this model, the key target inflammatory pathways on nuclear transcription factor kappa-B (Nuclear factor kappa-B, NF- K B) to intervene in the mother during pregnancy, can effectively prevent the occurrence of hypertension sub generation after birth, but the mechanism needs to be further elucidated. Ventricular remodeling is a common hypertension. The pathophysiology of heart failure and cardiomyopathy of many cardiovascular diseases, from the perspective of pathophysiology, one is myocardial cells have compensatory hypertrophy, apoptosis Death, necrosis and hyperplasia; on the other hand is the deposition of myocardial extracellular matrix collagen, composition and content of collagen network changes; causing imbalance myocardial stroma ratio, the structural change and functional cardiac decompensation. Recent studies suggest that, the excessive activation of neuroendocrine factors in ventricular remodeling course happen, is very important in the development, of which the renin-angiotensin system (renin angiotensin system, RAS) plays a particularly important role. The study found that the RAS system in addition to the ACE-Ang II -AT1R pathway also exists and its role in antagonizing a pathway that angiotensin-converting enzyme - vascular (1-7) angiotensin -Mas receptor axis (Angiotensin converting enzyme2-Angiotensin -Mas axis (1-7)), ACE2-Ang (1-7) -MasR axis, this pathway by ACE2 degradation in Ang II, the prevention and improvement of hypertension, heart failure Myocardial fibrosis were confirmed. Our previous studies showed that, compared with the circulating RAS system did not change significantly, significantly reduce the number of glomeruli activated simultaneously and renal function in the presence of reduced renal local RAS system. Also obtained the same conclusion in fat RAS system, suggests that the changes in local tissue RAS system may be the incidence of hypertension is another important factor. In our study also found that during pregnancy will cause inflammation of offspring rat ventricular myocytes apoptosis and remodeling, and this remodeling occurs earlier than the occurrence of hypertension, suggesting that ventricular remodeling offspring is one of the mechanism of hypertension. But the change of rat offspring which the factor caused the occurrence of ventricular remodeling and inflammation during pregnancy? Offspring rats cardiac RAS system impact? The rules and characteristics of what is yet to be further research.
This paper through the changes of collagen of myocardial tissue was observed and collagen metabolism enzymes MMP-2, TIMP-2 and profibrotic factor TGF beta -1 and TGF beta -2 to study the changes of myocardial fibrosis and myocardial remodeling. And through the observation of offspring rat cardiac RAS system on the two metabolic pathways: -AT1R pathway and ACE2-Ang ACE-Ang II (1-7) changes of -MasR pathway, further understanding of prenatal inflammation after stimulation of rat offspring occurrence of myocardial fibrosis and myocardial interstitial remodeling mechanism were studied.
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