本文關(guān)鍵詞：急性肺栓塞對(duì)腦鈉肽及C型利鈉肽受體影響的實(shí)驗(yàn)研究，由筆耕文化傳播整理發(fā)布。

        一、研究背景與目的肺栓塞（pulmonary embolism，PE）是臨床常見(jiàn)的一組疾病，由患者靜脈系統(tǒng)血栓或粥樣硬化斑塊、組織碎片或其他途徑進(jìn)入靜脈循環(huán)的腫瘤、脂肪、空氣等異物，導(dǎo)致部份肺動(dòng)脈及其分支阻塞而造成。依照病程發(fā)展又可分為急和慢性肺栓塞�；颊邥�(huì)因呼吸困難而感到不適，可透過(guò)心電圖、抽血或放射影像來(lái)診斷。而腦利鈉肽（brainnatriuretic peptide，BNP）作為新的生物標(biāo)志物，作為上述診斷的補(bǔ)充，也逐漸被用于PE的診斷及治療中。BNP具有利鈉、利尿、擴(kuò)血管作用，在抑制心肌肥厚、纖維化和抑制心肌細(xì)胞凋亡的終末器官活性也十分明顯。其釋放受到心肌細(xì)胞張力增強(qiáng)的刺激，與HF導(dǎo)致的心室功能障礙密切相關(guān)。在APE患者中，其血漿濃度明顯增加。然而一些研究表明，作為心血管系統(tǒng)的重要保護(hù)性激素之一，其高濃度卻未帶來(lái)與之相對(duì)應(yīng)的靶器官效應(yīng)，而表現(xiàn)出一種BNP缺乏或抵抗的一系列現(xiàn)象。C型利鈉肽受體（C-type natriuretic peptide receptor，NPR-C）作為體內(nèi)腦利鈉肽激素的主要清除受體，近來(lái)受到越來(lái)越多的重視，在細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)及相應(yīng)在心血管疾病領(lǐng)域的應(yīng)用逐漸深入起來(lái)，雖然其表達(dá)水平亦被發(fā)現(xiàn)受諸多因素的影響，但急性肺栓塞對(duì)其產(chǎn)生的一系列相應(yīng)影響目前尚不明確。結(jié)合BNP作為新的生物標(biāo)志物在APE后其濃度及功能方面的改變來(lái)看，其很可能與NPR-C受體有關(guān)，此相關(guān)性尚未引起相應(yīng)關(guān)注。本研究擬通過(guò)建立小鼠急性肺血栓栓塞模型，觀察NPR-C在體內(nèi)主要表達(dá)器官的水平變化，通過(guò)測(cè)定BNP的濃度改變，，檢測(cè)兩者之間的相關(guān)性，初步探討其表達(dá)調(diào)控的主要可能機(jī)制，并試圖進(jìn)一步詮釋體內(nèi)腦利鈉肽濃度升高的主要原因，為今后進(jìn)一步研究肺血栓栓塞癥的病理機(jī)制及篩選藥物奠定了基礎(chǔ)，并為臨床診斷與治療提供有益的思路。二、研究方法1、構(gòu)建并且改進(jìn)小鼠急性肺血栓栓塞癥模型，通過(guò)與全血血栓模型比較，建立更為穩(wěn)定的動(dòng)物模型。通過(guò)肺組織石蠟切片HE及PTAH染色，BP及PAP檢測(cè)，TNF-α測(cè)量，評(píng)估及比較模型優(yōu)缺點(diǎn)。2、IHC檢測(cè)NPR-C的表達(dá)分布情況；通過(guò)Western blotting及real-time PCR技術(shù)分別檢測(cè)小鼠心、肺、腎的NPR-C蛋白及mRNA表達(dá)水平變化，心肌細(xì)胞BNP的mRNA表達(dá)水平變化；EIA法檢測(cè)小鼠血漿BNP濃度變化。3、ELISA法檢測(cè)肺栓塞小鼠內(nèi)皮素（endothelin-1，ET-1）及肌鈣蛋白I（cardiactroponin I，cTnI）的水平變化。三、研究結(jié)果1、HE及PTAH染色證明小鼠肺血栓栓塞確實(shí)、可靠，栓子大多位于肺動(dòng)脈段或亞段水平。白色血栓栓塞組小鼠栓子24h、48h自溶率分別為5%、30%，全血血栓24h自溶率即已達(dá)100%。栓塞后小鼠血壓明顯下降，肺動(dòng)脈壓明顯升高，呼吸急促，術(shù)后活動(dòng)明顯減少。2、IHC染色提示NPR-C于肺廣泛表達(dá)，PE后其不同結(jié)構(gòu)間表達(dá)的密度梯度明顯改變。3、Western blotting顯示NPR-C在心、肺含量明顯降低，腎未見(jiàn)明顯變化。4、RT-PCR證實(shí)BNP于肺、腎臟未見(jiàn)表達(dá)；Real-time PCR證實(shí)，mRNA水平NPR-C在心、肺表達(dá)明顯下調(diào)，腎臟未見(jiàn)明顯改變，心臟BNP mRNA表達(dá)明顯增加。5、血漿BNP濃度明顯，但增加幅度高于心臟BNP mRNA表達(dá)增加幅度。6、TNF-α與PE前后未見(jiàn)明顯改變，僅見(jiàn)與手術(shù)操作相關(guān)的炎癥反應(yīng)；ET-1、cTnI升高明顯，但cTnI增加幅度更為明顯。四、研究結(jié)論1、小鼠肺白色血栓栓塞較以往模型栓溶明顯減弱，可靠性高，未見(jiàn)明顯由于異物栓子所引起的炎癥反應(yīng)，各項(xiàng)主要PE后表現(xiàn)符合實(shí)際，能更好的模擬APE這一病理生理過(guò)程，用于研究?jī)?yōu)越性明顯。2、APE后小鼠NPR-C表達(dá)下調(diào)，主要表現(xiàn)與心、肺。且肺組織內(nèi)NPR-C下調(diào)主要位于肺動(dòng)脈。BNP濃度的增加不單是由于其合成的增加，還與NPR-C減少造成的其清除下降有關(guān)。3、NPR-C的表達(dá)調(diào)控可能與血管機(jī)械性張力有關(guān)。4、cTnI與BNP結(jié)合輔助肺栓塞的診斷在PE后短期內(nèi)其價(jià)值較ET-1與BNP的聯(lián)用價(jià)值更高，而后者在PE后較長(zhǎng)時(shí)間的診斷中可能發(fā)揮一定的效果。

    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.

        急性肺栓塞對(duì)腦鈉肽及C型利鈉肽受體影響的實(shí)驗(yàn)研究

英文縮寫(xiě)詞一覽表4-6Abstract6-8摘要9-11前言11-13第一部分 實(shí)驗(yàn)性急性肺自體血栓栓塞模型的建立13-23    前言13-14    材料與方法14-17        材料14-16        方法16-17    結(jié)果17-20    討論20-23第二部分 急性肺栓塞后NPR-C的改變及意義23-43    前言23    材料與方法23-34        材料23-27        方法27-34    結(jié)果34-40    討論40-43參考文獻(xiàn)43-48全文結(jié)論48-49致謝49-50文獻(xiàn)綜述 利鈉肽及利鈉肽受體研究進(jìn)展50-64    參考文獻(xiàn)58-64在讀期間發(fā)表論文64

  本文關(guān)鍵詞：急性肺栓塞對(duì)腦鈉肽及C型利鈉肽受體影響的實(shí)驗(yàn)研究，由筆耕文化傳播整理發(fā)布。