本文選題：海水 + 肺損傷��； 參考：《第四軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：第一部分NFAT5在海水吸入型肺損傷中的作用及其機(jī)制的研究 研究背景： 吸入海水導(dǎo)致的急性肺損傷是海上生產(chǎn)作業(yè)和海上作戰(zhàn)等意外死亡的主要原因之一。海水吸入后大部分患者會(huì)因?yàn)楹眍^氣管反射性痙攣引發(fā)窒息而死亡，部分幸存患者可導(dǎo)致海水吸入型肺損傷(Seawater Inhalation Induced Acute Lung Injury,SWI-ALI)，甚至發(fā)展成為更為嚴(yán)重的海水吸入型呼吸窘迫綜合征(seawater inhalationinduced acute respiratory distress syndrome, SWI-ARDS)。目前國(guó)內(nèi)外對(duì)于海水吸入型肺損傷發(fā)生機(jī)制的研究報(bào)道非常有限，其理論基礎(chǔ)和分子機(jī)制尚有待闡明，而且未形成統(tǒng)一有效的臨床治療體系。因此，研究和闡明海水吸入型肺損傷特有的發(fā)病機(jī)制對(duì)于臨床治療方案的選擇和指導(dǎo)藥物的選用具有重要的意義。炎癥反應(yīng)是海水吸入型肺損傷的重要表現(xiàn)，與炎癥有關(guān)的炎癥因子和通路眾多，其中我們選擇了活化性T淋巴細(xì)胞核因子5(nuclear factor of the activated T cell5, NFAT5)和血管緊張素Ⅱ(angiotensinⅡ，AngⅡ)及其受體AT1兩個(gè)方面作為研究重點(diǎn)。 NFAT5是NF-κB/Rel家族成員之一，是哺乳動(dòng)物體內(nèi)唯一已知的關(guān)于滲透壓轉(zhuǎn)錄因子，可在多種組織中表達(dá)，參與維持細(xì)胞內(nèi)外滲透壓的平衡。此外，NFAT5還可誘導(dǎo)促炎因子的表達(dá)，促進(jìn)炎癥疾病的發(fā)生發(fā)展，如干眼病，炎性腸病(inflammatorybowel disease, IBD)等。因此，我們推測(cè)NFAT5可能在海水吸入型肺損傷的發(fā)生發(fā)展過(guò)程中起到非常重要的作用。 實(shí)驗(yàn)?zāi)康模?本實(shí)驗(yàn)通過(guò)復(fù)制大鼠海水吸入型肺損傷模型，研究了NFAT5的表達(dá)及其功能變化，以及NF-κB的變化，并在細(xì)胞水平上通過(guò)使用siRNA技術(shù)抑制NFAT5表達(dá)，觀察NF-κB及炎癥因子的變化，從而驗(yàn)證NFAT5在海水吸入型肺損傷中的重要作用。 實(shí)驗(yàn)方法： 體內(nèi)實(shí)驗(yàn)：健康雄性SD大鼠，體重190-210g，隨機(jī)分到對(duì)照組，海水吸入3h組，海水吸入6h組，海水吸入12h組四組中，每組6只。配制3%戊巴比妥鈉，按1.5ml/kg給予大鼠腹腔注射進(jìn)行麻醉，經(jīng)頸正中切口暴露氣管，將注射器插入氣管，按4ml/kg緩慢注入預(yù)先配置的海水，模擬海水吸入型肺損傷模型。待大鼠出現(xiàn)呼吸喘息氣急，耳鼻發(fā)紺，全肺滿布濕Up音，在鼻和口有粉紅色泡沫液體流出。在相應(yīng)時(shí)間點(diǎn)取0.5ml右側(cè)頸內(nèi)動(dòng)脈血進(jìn)行血?dú)夥治鰴z測(cè)，檢測(cè)動(dòng)脈血?dú)怙@示PaO250mmHg，SaO245%左右，及PaO2/FiO2（氧合指數(shù)）150mmHg提示海水吸入型肺損傷模型建立成功。然后處死大鼠，肺組織進(jìn)行石蠟包埋，蘇木精-伊紅染色法(hematoxylin-eosin staining, HE)染色，濕/干比(wet-to-dry ratios, W/D)檢測(cè)，勻漿肺組織通過(guò)反轉(zhuǎn)錄PCR (reverse transcription PCR, RT-PCR)和蛋白免疫印跡(Western blot)檢測(cè)肺組織內(nèi)NFAT5和NF-κB的表達(dá)，通過(guò)酶聯(lián)免疫吸附劑測(cè)定(enzyme-linkedimmunosorbentassay, ELISA)檢測(cè)肺組織內(nèi)的腫瘤壞死因子(tumornecrosis factor-α, TNF-α)、白介素1β(Interleukin-1β, IL-1β)和白介素8(Interleukin-8,IL-6)含量變化。 體外實(shí)驗(yàn)：采用大鼠肺泡巨噬細(xì)胞系NR8383細(xì)胞進(jìn)行孵育及相關(guān)處理。NR8383細(xì)胞生長(zhǎng)在含20%胎牛血清的F12培養(yǎng)液中，當(dāng)長(zhǎng)滿培養(yǎng)瓶底后，選用12.5%，25%，37.5%濃度的海水處理細(xì)胞，放入孵箱6h后，離心收集上清液及細(xì)胞進(jìn)行檢測(cè)，觀察細(xì)胞中NFAT5在蛋白及mRNA水平的表達(dá)和炎癥因子的變化情況。用NFAT5的小干擾RNA(siRNA)處理細(xì)胞，觀察NF-κB及炎癥因子變化情況。 實(shí)驗(yàn)結(jié)果： 體內(nèi)實(shí)驗(yàn)：大鼠氣管內(nèi)滴注海水后，PaO2和SaO2顯著降低，PaCO2逐漸升高。HE染色結(jié)果表明海水吸入6h后肺泡結(jié)構(gòu)紊亂，有大量的炎性細(xì)胞滲出，伴有局部肺不張，損傷嚴(yán)重。肺組織W/D，，炎癥因子TNF-α，IL-1β和IL-8表達(dá)量明顯增多，NFAT5在mRNA和蛋白水平表達(dá)明顯增加。 體外實(shí)驗(yàn)：不同濃度的海水處理NR8383細(xì)胞，結(jié)果顯示NFAT5和p-NF-κB在海水濃度25%時(shí)表達(dá)量最多。使用NFAT5siRNA抑制NFAT5的表達(dá)后，觀測(cè)到p-NF-κB和炎癥因子含量的顯著下降。 實(shí)驗(yàn)結(jié)論： 以上結(jié)果證明了NFAT5參與了海水吸入型肺損傷的形成和發(fā)展過(guò)程，其機(jī)制可能是促進(jìn)NF-κB的激活。抑制NFAT5的表達(dá)后，肺損傷和炎癥因子表達(dá)量減輕。 第二部分：Ang-Ⅱ及其受體AT1在海水吸入型肺損傷中的作用機(jī)制及氯沙坦對(duì)海水吸入型肺損傷的保護(hù)作用的研究 研究背景： 研究表明，腎素-血管緊張素系統(tǒng)(renin-angiotensin system, RAS)與呼吸系統(tǒng)的生理和病理過(guò)程密切相關(guān)。肺臟及其血管內(nèi)存有單獨(dú)的、不依賴于全身RAS的局部RAS系統(tǒng)。在ALI初期，肺內(nèi)的局部RAS系統(tǒng)即被激活并在ALI的發(fā)生發(fā)展中起到了重要的作用。AngⅡ是RAS系統(tǒng)的關(guān)鍵效應(yīng)物。 AngII主要在肺血管內(nèi)皮細(xì)胞產(chǎn)生，通過(guò)與特定AngI受體(AT1)和AngII受體(AT2)兩種亞型結(jié)合而發(fā)揮作用。當(dāng)前已知的AngⅡ的作用大多數(shù)是通過(guò)AT1受體介導(dǎo)的。AngⅡ與受體AT1結(jié)合后激活交感神經(jīng)系統(tǒng)增加神經(jīng)遞質(zhì)的釋放，增加血管張力，維持體液和電解質(zhì)的平衡等參與血液循環(huán)的調(diào)節(jié)；AngⅡ還可以通過(guò)旁分泌、自分泌和細(xì)胞內(nèi)的結(jié)合等參與呼吸系統(tǒng)疾病的病理生理過(guò)程。最近的研究表明，AngⅡ本身可能是一種促炎肽。許多促炎癥基因的表達(dá)，主要通過(guò)AngⅡ及其受體AT1調(diào)節(jié)的信號(hào)通路完成的。此外，AT1受體的選擇性抑制劑氯沙坦(losartan)，已經(jīng)顯示在脂多糖(lipopolysaccharides, LPS)，敗血癥及機(jī)械通氣導(dǎo)致的肺損傷中通過(guò)多種機(jī)制起到保護(hù)作用。然而，AngⅡ及其受體AT1在海水吸入型肺損傷中的作用尚未見報(bào)道。因此，在本研究的目的在于觀察海水吸入導(dǎo)致的急性肺損傷中，AngⅡ及其受體AT1的作用及其機(jī)制，以及AT1受體拮抗劑氯沙坦是否具有保護(hù)作用。 實(shí)驗(yàn)?zāi)康模?本部分實(shí)驗(yàn)通過(guò)復(fù)制大鼠海水吸入型肺損傷模型，觀察AngⅡ及其受體AT1的表達(dá)變化，同時(shí)觀察不同劑量受體AT1的抑制劑氯沙坦對(duì)海水吸入型肺損傷的保護(hù)作用及其機(jī)制。 實(shí)驗(yàn)方法： 體內(nèi)實(shí)驗(yàn)：將健康SD雄性大鼠隨機(jī)分成5組，分別是對(duì)照組，海水處理組(3h，6h，12h)，5mg/kg氯沙坦+海水處理6h組，15mg/kg氯沙坦+海水處理6h組，30mg/kg氯沙坦+海水處理6h組。在海水處理之前30min，對(duì)照組和海水處理組通過(guò)腹腔注射給予生理鹽水，氯沙坦處理組則通過(guò)腹腔注射給予不同劑量的氯沙坦，海水處理同實(shí)驗(yàn)一。待大鼠出現(xiàn)呼吸喘息氣急，耳鼻發(fā)紺，全肺滿布濕Up音，在鼻和口有粉紅色泡沫液體流出。在相應(yīng)時(shí)間點(diǎn)取0.5ml右側(cè)頸內(nèi)動(dòng)脈血進(jìn)行血?dú)夥治鰴z測(cè)，檢測(cè)動(dòng)脈血?dú)怙@示PaO250mmHg，SaO245%左右，及PaO2/FiO（2氧合指數(shù)）150mmHg提示海水吸入型肺損傷模型建立成功。取左下肺進(jìn)行HE染色，觀察大鼠肺損傷病理變化情況，取右下肺進(jìn)行稱重烘干，分析大鼠肺水腫情況，其余肺組織進(jìn)行實(shí)時(shí)熒光定量PCR (real-time quantitative polymerase chain reaction, Real-Time PCR)，Westernblot，髓過(guò)氧化物酶(myeloperoxidase, MPO)和炎癥因子的檢測(cè)。 實(shí)驗(yàn)結(jié)果： 大鼠吸入海水后，迅速出現(xiàn)缺氧情況， PaO2降低和PaCO2升高。同時(shí)AngⅡ含量及其受體AT1表達(dá)量顯著增加，NF-κB磷酸化增多，ELISA檢測(cè)炎癥因子和MPO含量明顯增多，氯沙坦預(yù)處理后可以顯著改善肺損傷情況。 實(shí)驗(yàn)結(jié)論： 海水吸入導(dǎo)致大鼠急性肺損傷，并使AngII及其受體AT1的表達(dá)升高。氯沙坦預(yù)處理可以起到保護(hù)肺組織的作用，可能的機(jī)制是顯著抑制NF-κB磷酸化和炎癥因子的分泌，從而減輕肺損傷癥狀。
[Abstract]:Role of the first part NFAT5 in seawater - induced lung injury and its mechanism

Background of Study :

Acute lung injury caused by inhalation of seawater is one of the main causes of accidental death such as marine production operations and marine operations . Most of the patients after seawater intake may die due to tracheal reflex spasm , and some surviving patients may lead to seawater - induced acute lung injury ( SWI - ALI ) , and even develop into a more serious seawater intake - induced acute respiratory distress syndrome ( SWI - ARDS ) . Therefore , it is important to study and clarify the pathogenesis of seawater inhalation type lung injury . Therefore , it is important to study and clarify the pathogenesis of seawater inhalation type lung injury . Therefore , it is important to study and clarify the pathogenesis of seawater inhalation type lung injury .

NFAT5 is one of the members of NF - 魏B / Rel family . It is the only known osmotic transcription factor in mammals . It can be expressed in various tissues and can be involved in maintaining the balance of osmotic pressure inside and outside the cell . In addition , NFAT5 can induce the expression of pro - inflammatory factors and promote the development of inflammatory diseases , such as dry eye disease , inflammatory bowel disease ( IBD ) , etc . Therefore , we hypothesized that NFAT5 may play a very important role in the development of seawater - induced lung injury .

Purpose of the experiment :

The expression of NFAT5 and the changes of NF - 魏B and the changes of NF - 魏B and the changes of NF - 魏B and inflammatory factors were studied by using siRNA technique to inhibit NFAT5 expression .

Test Method :

In vivo experiments were performed in healthy male SD rats weighing 190 - 210 g , randomly divided into control group , seawater intake 3h group , seawater inhalation 6h group , seawater inhalation 12h group four groups , 6 rats in each group . After the rats were sacrificed , lung tissue was injected into the trachea , and the lung tissue was injected into the trachea , and the contents of TNF - 偽 , IL - 1尾 and IL - 6 were measured by enzyme - linked immunosorbent assay ( ELISA ) . The levels of TNF - 偽 , IL - 1尾 and IL - 6 were measured by enzyme - linked immunosorbent assay ( ELISA ) .

NR8383 cells were cultured in F12 medium containing 20 % fetal bovine serum .

Experimental results :

The results of HE staining showed that there was a significant increase in pulmonary tissue W / D , inflammatory cytokines TNF - 偽 , IL - 1尾 and IL - 8 expression , and the expression of NFAT5 increased significantly at mRNA and protein levels .

In vitro experiment : NR8383 cells were treated with different concentrations of seawater . The results showed that NFAT5 and p - NF - 魏B were the most expressed in seawater concentration of 25 % . After the expression of NFAT5 was inhibited by NFAT5 siRNA , there was a significant decrease in the content of p - NF - 魏B and inflammatory factors .

Experimental Conclusion :

These results demonstrate that NFAT5 is involved in the formation and development of seawater - induced lung injury . Its mechanism may be to promote the activation of NF - 魏B . After inhibition of NFAT5 expression , lung injury and inflammatory factor expression are reduced .

The second part : the mechanism of Ang - 鈪

本文編號(hào)：1734518