本文選題：海水淹溺 + ALI��； 參考：《第四軍醫(yī)大學(xué)》2014年博士論文

【摘要】：研究背景： 海水淹溺性急性肺損傷（Seawater aspiration-induced acute lung injury, Seawateraspiration-induced ALI）是海水淹溺后的嚴(yán)重并發(fā)癥。不可控性的炎癥反應(yīng)是海水淹溺性ALI的主要發(fā)病機(jī)制，同時(shí)海水有著其特殊的理化因素及細(xì)胞外液的高滲性。JAK/STAT1是炎癥反應(yīng)中的重要信號(hào)轉(zhuǎn)導(dǎo)通路，在誘導(dǎo)上皮細(xì)胞凋亡中也起到重要作用，同時(shí)JAK，STAT1又能夠被細(xì)胞外液的高滲環(huán)境激活，因此，我們考慮JAK/STAT1通路可能在海水淹溺性ALI炎癥反應(yīng)及上皮細(xì)胞凋亡中起到重要的作用。 表沒食子兒茶素沒食子酸酯（Epigallocatechin-3-gallate，EGCG）是綠茶中兒茶素的主要活性成分，是STAT1的抑制劑。近年來(lái)大量文獻(xiàn)報(bào)道EGCG具有抗腫瘤抑制腫瘤生長(zhǎng)及轉(zhuǎn)移的作用，除此以外在糖尿病，帕金森，老年癡呆等疾病中都有較好的療效。研究表明EGCG能夠通過抑制STAT1的活性，降低心肌細(xì)胞缺血再灌注損傷，而EGCG在肺損傷特別是海水淹溺性ALI中會(huì)起到什么樣的作用，有待我們進(jìn)一步研究。 研究目的： 1.觀察EGCG對(duì)海水淹溺性ALI炎癥反應(yīng)及上皮細(xì)胞凋亡的作用； 2.探討JAK/STAT1通路在海水淹溺性ALI發(fā)病中的作用； 3.探討EGCG保護(hù)海水淹溺性ALI的作用機(jī)制。 研究意義： 通過離體及活體實(shí)驗(yàn)，探討EGCG保護(hù)海水淹溺性ALI的作用機(jī)制，明確JAK/STAT1通路在海水淹溺性ALI發(fā)病中的作用機(jī)制，為海水淹溺性ALI的治療提供新的思路。 實(shí)驗(yàn)方法： 1．動(dòng)物實(shí)驗(yàn) （1）雄性SD大鼠（180-220g），暴露氣管并氣管內(nèi)滴注配方海水4ml/kg建立海水淹溺性ALI模型。隨機(jī)分為五組：正常組（n=8），海水損傷后1小時(shí)組（n=8），3小時(shí)組（n=8），6小時(shí)組（n=8），12小時(shí)組（n=8）。分別在致傷后1,3,6,12小時(shí)檢測(cè)動(dòng)脈血氧分壓，IL-1，IL-10，TNF-α炎癥因子含量，PCR檢測(cè)JAK，STAT家族各成員的mRNA，Western blot檢測(cè)STAT1，P-STAT1的蛋白含量。并進(jìn)行組織病理學(xué)檢測(cè)。 （2）雄性SD大鼠（180-220g）。隨機(jī)分為四組：①正常組：暴露氣管并術(shù)前半小時(shí)腹腔注射1ml生理鹽水（n=12）；②EGCG組：暴露氣管并術(shù)前半小時(shí)腹腔注射10mg/kg EGCG溶于1ml生理鹽水中（n=12）；③海水組：暴露氣管后氣管內(nèi)滴注配方海水4ml/kg并致傷前0.5小時(shí)腹腔注射1ml生理鹽水（n=12）；④海水+EGCG組：暴露氣管后氣管內(nèi)滴注配方海水4ml/kg并致傷前0.5小時(shí)腹腔注射10mg/kgEGCG溶于1ml生理鹽水中（n=12）。于致傷后0-3小時(shí)動(dòng)態(tài)觀察動(dòng)脈血氧分壓變化，致傷后6小時(shí)檢測(cè)肺干濕重比，肺泡灌洗液蛋白含量，IL-1，IL-10，TNF-α炎癥因子含量，PCR檢測(cè)STAT1mRNA含量，Western blot檢測(cè)STAT1，P-STAT1，caspase-3，P21的蛋白含量，熒光TUNEL，免疫組化，電鏡觀察肺組織超微結(jié)構(gòu)及組織病理學(xué)檢測(cè)。 2．細(xì)胞實(shí)驗(yàn) （1）培養(yǎng)NR8383細(xì)胞，指數(shù)生長(zhǎng)期的NR8383細(xì)胞無(wú)血清饑餓16小時(shí)后，分為四組：正常組，EGCG組（10цM EGCG干預(yù)細(xì)胞），海水組（30%海水干預(yù)細(xì)胞），海水+EGCG組（10цM EGCG干預(yù)細(xì)胞后0.5小時(shí)再給予30%海水干預(yù)細(xì)胞）。海水干預(yù)6小時(shí)后取細(xì)胞上清液檢測(cè)IL-1，IL-10，TNF-α炎癥因子含量，PCR檢測(cè)JAK1，JAK2，STAT1的mRNA含量，Western blot檢測(cè)JAK1，P-JAK1，JAK2，P-JAK2，STAT1，P-STAT1含量。 （2）培養(yǎng)A549細(xì)胞，指數(shù)生長(zhǎng)期的A549細(xì)胞無(wú)血清饑餓16小時(shí)后，分為兩步實(shí)驗(yàn)：第一步用不同濃度海水干預(yù)細(xì)胞，分為五組：正常組，10%海水組，20%海水組，30%海水組，40%海水組，干預(yù)細(xì)胞后6小時(shí)流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡程度；第二步用EGCG干預(yù)細(xì)胞，分為四組：正常組，EGCG組（10цM EGCG干預(yù)細(xì)胞），海水組（30%海水干預(yù)細(xì)胞），海水+EGCG組（10цM EGCG干預(yù)細(xì)胞后0.5小時(shí)再給予30%海水干預(yù)細(xì)胞），干預(yù)細(xì)胞后6小時(shí)流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率。 實(shí)驗(yàn)結(jié)果： 1．海水淹溺性ALI在6小時(shí)損傷最重，STAT1在損傷后1小時(shí)即升高6小時(shí)表達(dá)量達(dá)最高值。 造模成功后檢測(cè)組織病理學(xué)，動(dòng)脈血氧分壓，炎癥因子，蛋白表達(dá)量指標(biāo)，結(jié)果顯示組織病理學(xué)檢測(cè)肺組織明顯水腫，炎細(xì)胞浸潤(rùn)，隨著時(shí)間延長(zhǎng)肺損傷程度逐漸加重，在6小時(shí)點(diǎn)肺損傷程度最重。致傷后出現(xiàn)明顯低氧血癥。IL-1，TNF-α促炎因子明顯升高，IL-10抑炎因子明顯下降，差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。STAT1蛋白表達(dá)量在損傷后1小時(shí)開始升高6小時(shí)達(dá)最高，此后開始下降。 2．在體內(nèi)實(shí)驗(yàn)中EGCG預(yù)處理能夠減輕肺組織炎癥并抑制肺組織細(xì)胞凋亡。 通過體內(nèi)實(shí)驗(yàn)觀察組織病理學(xué)，動(dòng)脈血氧分壓，肺含水量，，炎癥因子，蛋白表達(dá)量，凋亡，超微結(jié)構(gòu)等指標(biāo)明確EGCG對(duì)海水淹溺性ALI的保護(hù)作用。結(jié)果顯示EGCG預(yù)處理組能夠減輕海水淹溺性ALI導(dǎo)致的低氧血癥，差異具有統(tǒng)計(jì)學(xué)意義（P0.05）；減輕肺水腫，降低肺泡灌洗液蛋白含量，差異具有統(tǒng)計(jì)學(xué)意義（P0.05）；降低IL-1，TNF-α促炎因子，升高IL-10抑炎因子，差異具有統(tǒng)計(jì)學(xué)意義（P0.05）；Western及免疫組化結(jié)果均顯示能夠降低STAT1，P-STAT1蛋白的表達(dá)量；Western結(jié)果顯示能夠降低STAT1下游P21及caspase-3的蛋白表達(dá)量；熒光TUNEL結(jié)果顯示能夠減少組織細(xì)胞凋亡；電鏡觀察肺組織超微結(jié)構(gòu)及組織病理學(xué)檢測(cè)均顯示能夠減輕肺水腫，炎細(xì)胞浸潤(rùn)程度。 3.在離體實(shí)驗(yàn)中EGCG預(yù)處理能夠減輕NR8383細(xì)胞炎癥反應(yīng)并抑制A549細(xì)胞凋亡。 通過離體實(shí)驗(yàn)觀察炎癥因子，蛋白表達(dá)量，流式檢測(cè)細(xì)胞凋亡率指標(biāo)明確EGCG在離體細(xì)胞中對(duì)炎癥反應(yīng)及細(xì)胞凋亡的影響。結(jié)果顯示EGCG預(yù)處理能夠減輕海水干預(yù)NR8383細(xì)胞的炎癥反應(yīng)，降低IL-1，TNF-α促炎因子，升高IL-10抑炎因子，差異具有統(tǒng)計(jì)學(xué)意義（P0.05），降低P-JAK1， P-JAK2，STAT1，P-STAT1的蛋白含量，對(duì)JAK1，JAK2總蛋白含量無(wú)明顯影響。用不同濃度海水干預(yù)A549細(xì)胞后顯示隨著海水濃度的升高A549細(xì)胞的凋亡率升高（P0.05），EGCG預(yù)處理后可以降低海水干預(yù)后引起A549細(xì)胞的凋亡率（P0.05）。 結(jié)論： 1.海水淹溺性ALI中STAT1表達(dá)明顯升高，且STAT1的激活與海水淹溺性ALI的損傷程度相關(guān)。 2. EGCG能夠減輕海水淹溺性ALI中肺組織炎癥反應(yīng)，減輕肺水腫及血管通透性，這與其負(fù)調(diào)節(jié)JAK/STAT1通路有關(guān)。 3. EGCG能夠減輕海水淹溺性ALI中肺泡上皮細(xì)胞的凋亡，這與其負(fù)調(diào)節(jié)JAK/STAT1通路及凋亡蛋白caspase-3，P21有關(guān)。
[Abstract]:Research background:
Seawater drowning acute lung injury (Seawater aspiration-induced acute lung injury, Seawateraspiration-induced ALI) is a serious complication after seawater drowning. The uncontrollable inflammatory reaction is the main pathogenesis of seawater drowning ALI, and the sea water has its special physical and chemical factors and the hypertonic.JAK/STAT1 of the extracellular fluid. The important signal transduction pathway in the inflammatory response also plays an important role in inducing apoptosis of epithelial cells. At the same time, JAK, STAT1 can be activated by the hypertonic environment of extracellular fluid. Therefore, we consider that the JAK/STAT1 pathway may play an important role in the ALI inflammatory response and epithelial cell apoptosis in seawater drowning.
Epigallocatechin gallate (Epigallocatechin-3-gallate, EGCG) is the main active component of catechin in green tea and is an inhibitor of STAT1. In recent years, a large number of literatures have reported that EGCG has the effect of inhibiting tumor growth and metastasis. Besides, it has good curative effect in diabetes, Parkinson, Alzheimer's disease and so on. Studies have shown that EGCG can reduce the myocardial ischemia and reperfusion injury by inhibiting the activity of STAT1, and what role EGCG can play in lung injury, especially in seawater drowning ALI, is to be further studied.
The purpose of the study is:
1. to observe the effect of EGCG on ALI inflammatory response and epithelial cell apoptosis in seawater drowning.
2. to explore the role of JAK/STAT1 pathway in the pathogenesis of seawater drowning ALI.
3. to explore the mechanism of EGCG protecting ALI from seawater drowning.
Research significance:
In vitro and in vivo experiments, the mechanism of EGCG protection for seawater drowning ALI is discussed, and the mechanism of JAK/STAT1 pathway in the pathogenesis of seawater drowning ALI is clearly defined, which provides a new way of thinking for the treatment of seawater drowning ALI.
Experimental methods:
1. animal experiments
(1) the male SD rats (180-220g), exposed to the trachea and intratracheal infusion of seawater 4ml/kg to establish a seawater drowning ALI model, were randomly divided into five groups: normal group (n=8), 1 hour group (n=8) after seawater injury, 3 hour group (n=8), 6 hour group (n=8), 12 hour group (n =8). Levels of inflammatory factors, PCR were detected by JAK, mRNA and Western blot of STAT family members were tested for STAT1 and P-STAT1 protein content, and histopathological examination was performed.
(2) male SD rats (180-220g) were randomly divided into four groups: (1) normal group: exposed trachea and intraperitoneal injection of 1ml saline (n=12) half an hour before operation; (2) EGCG group: exposed trachea and intraperitoneal injection of 10mg/kg EGCG in 1ml physiological saline (n=12) for half an hour before operation; (3) seawater group: intratracheal intratracheal infusion of seawater 4ml/kg and caused by seawater 4ml/kg and cause Intraperitoneal injection of 1ml saline (n=12) 0.5 hours before injury; (4) seawater +EGCG group: intratracheal intratracheal infusion of seawater 4ml/kg and intraperitoneal injection of 10mg/kgEGCG in 1ml physiological saline (n=12) before injury. The changes of arterial blood oxygen pressure were observed at 0-3 hours after injury, and lung dry wet weight ratio and alveolar irrigation were detected at 6 hours after injury. The content of lotion protein, IL-1, IL-10, TNF- alpha inflammatory factor, PCR detection of STAT1mRNA content, Western blot to detect STAT1, P-STAT1, Caspase-3, P21 protein content, fluorescence TUNEL, immunohistochemistry, electron microscopy observation of ultrastructure of lung tissue and histopathological detection.
2. cell experiment
(1) NR8383 cells were cultured, and the NR8383 cells in the exponential growth period were divided into four groups after 16 hours without serum starvation: the normal group, the group EGCG (10 M EGCG intervention cells), the seawater group (30% seawater intervention cells), the seawater +EGCG group (10 0.5 hours after the intervention cells of M EGCG) and the 30% seawater intervention cells after 0.5 hours. The cell supernatant was tested for IL- after the seawater intervention for 6 hours. 1, IL-10, TNF- alpha inflammatory factor content, PCR detection of JAK1, JAK2, STAT1 mRNA content, Western blot detection JAK1, P-JAK1, blot, ",", "," content.
(2) to cultivate A549 cells, the A549 cells in the exponential growth period were divided into two steps after 16 hours without serum starvation: the first step was divided into five groups: normal group, 10% sea water group, 20% seawater group, 30% seawater group, 40% seawater group, and 6 small time flow cytometry to detect cell apoptosis after 6 small time flow cytometry; second steps were performed with EGCG dry. The precells were divided into four groups: normal group, group EGCG (10 M EGCG intervention cells), sea water group (30% seawater intervention cells), and seawater +EGCG group (30% seawater intervened cells after 0.5 hours after 10 M EGCG intervention cells), and 6 hour flow cytometry was used to detect the cell withering rate after 6 hours of flow cytometry.
Experimental results:
1. seawater drowning ALI was the most serious injury at 6 hours, and STAT1 increased 6 hours at 1 hours after injury.
After the success of the model, histopathology, arterial oxygen pressure, inflammatory factors and protein expression were detected. The results showed that the lung tissue was obviously edema, inflammatory cells infiltrated, and the lung injury was gradually increased with time, and the lung injury was the most severe at 6 hours. The obvious hypoxemia.IL-1, TNF- alpha proinflammatory cause appeared after the injury. The IL-10 anti inflammatory factor significantly decreased, and the difference was statistically significant (P0.05), the expression of.STAT1 protein increased at 1 hours after 6 hours, and then began to decline.
2. in vivo experiments, EGCG pretreatment can reduce lung tissue inflammation and inhibit apoptosis in lung tissue.
In vivo observation of histopathology, arterial oxygen pressure, lung water content, inflammatory factors, protein expression, apoptosis, ultrastructure and other indicators of the protective effect of EGCG on seawater drowning ALI. The results showed that the EGCG pretreatment group could reduce the hypoxemia caused by seawater drowning ALI, the difference was statistically significant (P0.05); Pulmonary edema and reducing the protein content of alveolar lavage fluid were statistically significant (P0.05); reducing IL-1, TNF- alpha, and increasing IL-10 anti inflammatory factors were statistically significant (P0.05); Western and immunohistochemical results showed that the expression of STAT1, P-STAT1 protein could be reduced, and Western results showed that the downstream P2 of STAT1 could be reduced. The protein expression of 1 and Caspase-3, and the results of fluorescence TUNEL showed that the apoptosis of the tissue cells could be reduced, and the ultrastructure of the lung tissue and the histopathological examination of the lung tissue showed that the pulmonary edema and the degree of infiltration of inflammatory cells could be reduced by electron microscopy.
3. in vitro experiments, EGCG pretreatment can reduce the inflammatory response of NR8383 cells and inhibit the apoptosis of A549 cells.
The effects of inflammatory factors, protein expression and flow cytometry on the inflammatory response and apoptosis in isolated cells were observed through the experiment in vitro. The results showed that EGCG pretreatment could reduce the inflammatory response of NR8383 cells in seawater intervention, reduce IL-1, TNF- alpha pro-inflammatory factors and increase IL-10 anti inflammatory factors, and the difference was found. Statistical significance (P0.05), reducing the protein content of P-JAK1, P-JAK2, STAT1 and P-STAT1 had no obvious effect on the total protein content of JAK1 and JAK2. The apoptosis rate of A549 cells increased with the increase of seawater concentration (P0.05) with the concentration of seawater in different concentrations (P0.05), and the apoptosis rate of A549 cells could be reduced after the pretreatment of sea water. .05).
Conclusion:
1. the expression of STAT1 in seawater drowning ALI increased significantly, and the activation of STAT1 was related to the degree of ALI damage in seawater.
2. EGCG can reduce the inflammatory reaction of lung tissue and reduce pulmonary edema and vascular permeability in seawater drowning ALI, which is related to the negative regulation of JAK/STAT1 pathway.
3. EGCG can reduce the apoptosis of alveolar epithelial cells in seawater drowning ALI, which is related to the negative regulation of JAK/STAT1 pathway and apoptotic protein caspase-3 and P21.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R649.3

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