急性肺栓塞对脑钠肽及C型利钠肽受体影响的实验研究

  本文关键词：急性肺栓塞对脑钠肽及C型利钠肽受体影响的实验研究，由笔耕文化传播整理发布。

        一、研究背景与目的肺栓塞（pulmonary embolism，PE）是临床常见的一组疾病，由患者静脉系统血栓或粥样硬化斑块、组织碎片或其他途径进入静脉循环的肿瘤、脂肪、空气等异物，导致部份肺动脉及其分支阻塞而造成。依照病程发展又可分为急和慢性肺栓塞。患者会因呼吸困难而感到不适，可透过心电图、抽血或放射影像来诊断。而脑利钠肽（brainnatriuretic peptide，BNP）作为新的生物标志物，作为上述诊断的补充，也逐渐被用于PE的诊断及治疗中。BNP具有利钠、利尿、扩血管作用，在抑制心肌肥厚、纤维化和抑制心肌细胞凋亡的终末器官活性也十分明显。其释放受到心肌细胞张力增强的刺激，与HF导致的心室功能障碍密切相关。在APE患者中，其血浆浓度明显增加。然而一些研究表明，作为心血管系统的重要保护性激素之一，其高浓度却未带来与之相对应的靶器官效应，而表现出一种BNP缺乏或抵抗的一系列现象。C型利钠肽受体（C-type natriuretic peptide receptor，NPR-C）作为体内脑利钠肽激素的主要清除受体，近来受到越来越多的重视，在细胞信号转导及相应在心血管疾病领域的应用逐渐深入起来，虽然其表达水平亦被发现受诸多因素的影响，但急性肺栓塞对其产生的一系列相应影响目前尚不明确。结合BNP作为新的生物标志物在APE后其浓度及功能方面的改变来看，其很可能与NPR-C受体有关，此相关性尚未引起相应关注。本研究拟通过建立小鼠急性肺血栓栓塞模型，观察NPR-C在体内主要表达器官的水平变化，通过测定BNP的浓度改变，，检测两者之间的相关性，初步探讨其表达调控的主要可能机制，并试图进一步诠释体内脑利钠肽浓度升高的主要原因，为今后进一步研究肺血栓栓塞症的病理机制及筛选药物奠定了基础，并为临床诊断与治疗提供有益的思路。二、研究方法1、构建并且改进小鼠急性肺血栓栓塞症模型，通过与全血血栓模型比较，建立更为稳定的动物模型。通过肺组织石蜡切片HE及PTAH染色，BP及PAP检测，TNF-α测量，评估及比较模型优缺点。2、IHC检测NPR-C的表达分布情况；通过Western blotting及real-time PCR技术分别检测小鼠心、肺、肾的NPR-C蛋白及mRNA表达水平变化，心肌细胞BNP的mRNA表达水平变化；EIA法检测小鼠血浆BNP浓度变化。3、ELISA法检测肺栓塞小鼠内皮素（endothelin-1，ET-1）及肌钙蛋白I（cardiactroponin I，cTnI）的水平变化。三、研究结果1、HE及PTAH染色证明小鼠肺血栓栓塞确实、可靠，栓子大多位于肺动脉段或亚段水平。白色血栓栓塞组小鼠栓子24h、48h自溶率分别为5%、30%，全血血栓24h自溶率即已达100%。栓塞后小鼠血压明显下降，肺动脉压明显升高，呼吸急促，术后活动明显减少。2、IHC染色提示NPR-C于肺广泛表达，PE后其不同结构间表达的密度梯度明显改变。3、Western blotting显示NPR-C在心、肺含量明显降低，肾未见明显变化。4、RT-PCR证实BNP于肺、肾脏未见表达；Real-time PCR证实，mRNA水平NPR-C在心、肺表达明显下调，肾脏未见明显改变，心脏BNP mRNA表达明显增加。5、血浆BNP浓度明显，但增加幅度高于心脏BNP mRNA表达增加幅度。6、TNF-α与PE前后未见明显改变，仅见与手术操作相关的炎症反应；ET-1、cTnI升高明显，但cTnI增加幅度更为明显。四、研究结论1、小鼠肺白色血栓栓塞较以往模型栓溶明显减弱，可靠性高，未见明显由于异物栓子所引起的炎症反应，各项主要PE后表现符合实际，能更好的模拟APE这一病理生理过程，用于研究优越性明显。2、APE后小鼠NPR-C表达下调，主要表现与心、肺。且肺组织内NPR-C下调主要位于肺动脉。BNP浓度的增加不单是由于其合成的增加，还与NPR-C减少造成的其清除下降有关。3、NPR-C的表达调控可能与血管机械性张力有关。4、cTnI与BNP结合辅助肺栓塞的诊断在PE后短期内其价值较ET-1与BNP的联用价值更高，而后者在PE后较长时间的诊断中可能发挥一定的效果。

    1. Background and PurposePulmonary embolism is a kind of relatively common cardiovascular emergency whichresults from the blocade of the pulmonary arteries and their branches by venousthrombi or atherosclerotic plaques, tissue fragments or other matters entering into thevenous circulation such as the tumor, fat, air and other foreign bodies. In accordancewith the progression of the disease, it is defined as aucte or chronic pulmonaryembolism which is accompanied by dyspnea or other discomforts. The diagnosis canbe made through electrocardiogram, blood biomarkers or radiological imagingtechnologies. Meanwhile, brain natriuretic peptide, as a new candidate of biomarkers,has also provided a supplementary of above diagnostic methods and been graduallyused to evaluate PE clinic outcome and treatment.Brain natriuretic peptide is mainly produced by the cardiac myocytes where theirrelease can be enhanced by increased vascular tension and closely related toventricular dysfunction. Together with atrial natriuretic peptide (ANP) and C typenatriuretic peptide they play an important role in cardiorenal homeostasis: Theycirculate as hormones to act in various tissues in the body inducing not onlyvasodilation, natriuresis, and diuresis but also the inhibition of cardiac hypertrophy,fibrosis and apoptosis in myocardial cells where they show an end-organ activity.Although among acute pulmonary embolism patients its concerntration signifcantlyincreased, its high level don’t produce more corresponding target organ effects.Recent studies support the notion that HF patients actually manifest a state of BNPinsuffciency or resist to its effect where they actually play an important cardiovascularprotective hormone.C-type natriuretic peptide receptor, as the main clearance receptor in our bodies,has been paid more and more attentions, whose applications in the cardiovascularand cellular signal transduction fields gradually increased. Although its expression level was also found to be affected by many factors, a series of corresponding effectsfollowing APE are still not clear. Combined with the fact that BNP acts as a newbiomarker for APE and the changes of its concentration and function, it might berelevant with NPR-C receptor, which has not caused corresponding attention.We has established a mouse APTE model and observed the changes of NPR-Clevels in vivo. By comparing with the alternations of BNP concerntraion, we aimed tofind their correlation, and to explore the possible mechanisms which regulate theirexpressions. This study lay the foundation for our further research on pathologicalmechanism of pulmonary thromboembolism, screening of drugs, and provided auseful train of thought for furture clinical diagnosis and treatment.2. Methods1. By comparing with the whole blood induced pulmonary embolism models, tryto modify and establish a more steady mouse APTE model. Through HE staining andPTAH staining of sections of lungs, monitoring BP and PAP, testing TNF-α level, wehave evaluated the advantages and disadvantages of these models.2. By DAB staining, distributions of NPR-C in lung were shown; By westernblotting and PCR technology, changes of protein level and mRNA level of NPR-Cwere tested; EIA kit and PCR were also used in the tests of BNP plasmaconcerntration and mRNA levels.3. Elisa kit was used to test the levels of endothelin-1(ET-1) and cardiac troponinI (cTnI) following induced mouse APTE.3. Results1. HE and PTAH staining have proved its reliable embolism whose most embolilay in the level of segments or subsegments of pulmonary arteries. White-autologousthrombi pulmonary embolism model showed no obvious fibrinolysis of the thrombi (5%and30%separately in24h and48h), while whole blood induced PE model showed a100%fibrinolysis of the thrombi. Mice following pulmonary showed significantdyspnea and decrease of BP, obvious increased PAP, and significantly reducedpostoperative activity.2. IHC staining suggested that NPR-C expressed widely in pulmonary tissues,and its distribution density gradient between different tissues chaned significantlyfollowing PE. 3. Western blotting showed that NPR-C proteins decreased significantly afterAPTE in heart and lung, while there were no differences in kidney.4. RT-PCR proved that BNP was not expressed in lung and kidney; BNP mRNAlevels have significantly been down-regulated after APTE in heart and lung, but nodifferences were seen in kindney.5. BNP concerntration in plasma increased significantly, but the degree of itsincrease is greater than the degree of mRNA increase.6. No significant relations between PE and TNF-α were seen except for thestimulation resulted from the surgery operation; ET-1and cTnI increased significantly,and the latter one showed more greater changes.4. Conclusions1. The obvious fibrinolysis of the thrombi in white-autologous thrombi pulmonaryembolism models was largely improved than in the whole blood group; No obviousinflammation was seen resulted from the in vitro operated thrombi, and the mainsymptoms after PE was in line with the actual situations. In all, our model can bettersimulate the pathophysiological processes of APE which showed an obviousadvantages in scientific researches.2. NPR-C was down-regulated in mice following APE, which mainly existed inheart and lung, and the changes in lung mainly exited in pulmonary arteries. Theincreased plasma BNP resulted not only from the increased secretion but from thedecreased clearance.3. The mechanisms of NPR-C regulation were probably associated with thevascular mechanical tension.4. BNP combined with cTnI probably shows greater value in the diagnosis ofshort period after PE, while combined with ET-1in longer period after PE.

        急性肺栓塞对脑钠肽及C型利钠肽受体影响的实验研究

英文缩写词一览表4-6Abstract6-8摘要9-11前言11-13第一部分 实验性急性肺自体血栓栓塞模型的建立13-23    前言13-14    材料与方法14-17        材料14-16        方法16-17    结果17-20    讨论20-23第二部分 急性肺栓塞后NPR-C的改变及意义23-43    前言23    材料与方法23-34        材料23-27        方法27-34    结果34-40    讨论40-43参考文献43-48全文结论48-49致谢49-50文献综述 利钠肽及利钠肽受体研究进展50-64    参考文献58-64在读期间发表论文64

  本文关键词：急性肺栓塞对脑钠肽及C型利钠肽受体影响的实验研究，由笔耕文化传播整理发布。