本文選題：海水 + 肺水腫��； 參考：《第四軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：研究背景： 海水淹溺是航海、海上作戰(zhàn)、作業(yè)時常發(fā)生的意外傷害事故，吸入海水可導(dǎo)致肺泡-毛細(xì)血管膜損傷并伴有通透性增高，富含蛋白的液體聚集于肺泡腔，引起海水淹溺型肺水腫(pulmonary edema induced by seawaterdrowning，PE-SWD)，是海水淹溺引起死亡的主要原因之一。PE-SWD的主要病理變化是肺通透性增高，肺水腫形成，引起低氧血癥和呼吸困難。如不及時救治和控制病情，，易發(fā)展為海水型呼吸窘迫綜合征(seawaterrespiratory distress syndrome, SW-RDS)。SW-RDS是急性呼吸窘迫綜合征(acute respiratory distress syndrome， ARDS)的一種。近年來關(guān)于PE-SWD/SW-RDS的研究逐漸增多，但是其發(fā)病機(jī)制仍不完全清楚，缺乏有效的治療手段，死亡率仍很高。PE-SWD/SW-RDS時，肺泡上皮屏障功能破壞，肺水腫液的清除功能障礙，加之吸入的高滲海水在肺泡內(nèi)聚集，驅(qū)使血管內(nèi)液體沿滲透壓力梯度流向肺泡內(nèi)，加重缺氧。 縫隙連接蛋白43(connexin43，Cx43)在肺泡上皮和毛細(xì)血管內(nèi)皮普遍存在，是細(xì)胞間縫隙連接的基本組成單位，不僅發(fā)揮細(xì)胞間的通訊功能，而且對細(xì)胞的生長、分化和凋亡甚至腫瘤形成都有重要作用。有文獻(xiàn)報道Cx43與血管通透性有關(guān)。近年來研究表明Cx43參與肺損傷的過程，但Cx43在PE-SWD/SW-RDS中的作用及其機(jī)制尚不清楚。本實驗通過復(fù)制大鼠海水淹溺型肺水腫的模型，觀察Cx43在海水淹溺型肺水腫中的作用，并進(jìn)一步研究其可能的作用機(jī)制。 實驗?zāi)康模?⑴觀察大鼠海水淹溺型肺水腫模型中Cx43的表達(dá)變化。 ⑵探討Cx43在海水淹溺型肺水腫中的作用及其機(jī)制。 實驗方法： 實驗一 體內(nèi)實驗 24只SD大鼠隨機(jī)分為4組，正常對照組(0h)、海水淹溺1h組(1h)、4h組(4h)和8h組(8h)。氣管內(nèi)滴注海水的方法復(fù)制大鼠海水淹溺型肺水腫的模型，通過肺組織濕干重比值(W/D)、支氣管肺泡灌洗液(BALF)中的蛋白含量和伊文思藍(lán)漏出指數(shù)(ELI)測定肺組織的通透性；RT-PCR和Western Blot等分子生物學(xué)實驗方法檢測肺組織Cx43的含量變化。 體外實驗 采用25%體積濃度的海水孵育A549細(xì)胞模擬海水淹溺的肺內(nèi)環(huán)境(作用時間為4小時)，觀察Cx43的表達(dá)變化。A549細(xì)胞隨機(jī)分為2組：空白對照組(Control)、海水組(SW)，通過RT-PCR和Western Blot方法檢測海水對Cx43的表達(dá)的影響。通過對FITC標(biāo)記的葡聚糖的相對通透性測定單層A549細(xì)胞的通透性，觀察海水對單層細(xì)胞通透性的影響。 實驗二 (一)A549細(xì)胞隨機(jī)分為空白對照組(Control)和海水組(SW)，WesternBlot方法檢測海水對MAPK信號通路的作用。 (二)分離純化大鼠肺泡Ⅱ型上皮細(xì)胞隨機(jī)分為5組：空白對照組(Control)、海水組(SW)、ERK1/2抑制劑組(PD)、JNK抑制劑組(SP)、p38抑制劑組(SB)，并設(shè)陰性對照組。通過FCM分析細(xì)胞膜Cx43的表達(dá)量。 (三)A549細(xì)胞隨機(jī)分為6組：空白對照組(Control)、海水組(SW)、ERK1/2抑制劑組(PD)、JNK抑制劑組(SP)、p38抑制劑組(SB)和反義寡核苷酸干預(yù)組(ASODN)。通過Western Blot方法檢測各組細(xì)胞Cx43的表達(dá)含量。培養(yǎng)單層細(xì)胞，通過對FITC標(biāo)記的葡聚糖的通透性的檢測觀察單層細(xì)胞的通透性，統(tǒng)計分析Cx43含量與單層細(xì)胞通透性的關(guān)系。 實驗結(jié)果： 實驗一 體內(nèi)實驗 大鼠吸入海水后，肺組織W/D比值增加，BALF中的蛋白含量和ELI也明顯增加(P0.05)，說明吸入海水導(dǎo)致肺通透性增高。肺組織Cx43mRNA和蛋白含量明顯增加，4h達(dá)最高值(P0.05)。 體外實驗 RT-PCR和Western Blot檢測A549細(xì)胞Cx43表達(dá)，海水作用后，Cx43表達(dá)明顯增多，單層細(xì)胞通透性增高(P0.05)。 實驗二 Western Blot結(jié)果表明海水導(dǎo)致A549細(xì)胞p-ERK1/2、p-JNK表達(dá)減少，p-p38增多。ERK1/2抑制劑、JNK抑制劑分別上調(diào)Cx43的表達(dá)，p38信號通路對Cx43的表達(dá)無明顯作用，同時ERK1/2抑制劑、JNK抑制劑使單層A549細(xì)胞通透性增加(P0.05)。通過統(tǒng)計相關(guān)分析，Cx43表達(dá)量與單層細(xì)胞的通透性呈正相關(guān)關(guān)系，r=0.940, P0.05。 結(jié)論： ⑴在海水淹溺型肺水腫模型中，肺組織Cx43表達(dá)增多，且Cx43表達(dá)量與肺泡-毛細(xì)血管通透性正相關(guān)。 ⑵海水可通過抑制ERK1/2和JNK信號通路引起Cx43表達(dá)量增加進(jìn)而影響肺泡-毛細(xì)血管的通透性。
[Abstract]:Research background:
Sea water drowning is an accident that often occurs in navigation, maritime operations and operations. Inhalation of seawater can lead to alveolar capillary membrane damage and increased permeability, and protein rich liquid is gathered in the alveoli of the lung (pulmonary edema induced by seawaterdrowning, PE-SWD), which causes death in seawater drowning. One of the main causes of.PE-SWD is increased pulmonary permeability, pulmonary edema, hypoxemia and dyspnea. If not timely treatment and control of the condition, it is easy to develop into seawaterrespiratory distress syndrome (SW-RDS).SW-RDS as acute respiratory distress syndrome (acute respiratory). Distress syndrome, ARDS). In recent years, the study of PE-SWD/SW-RDS has gradually increased, but its pathogenesis is still not completely clear, the lack of effective treatment, the death rate is still high.PE-SWD/SW-RDS, the destruction of the alveolar epithelial barrier function, the clearance of pulmonary edema fluid, and the inhalation of hypertonic seawater in the alveoli. It drives blood vessels to flow into alveoli along the osmotic pressure gradient and aggravates hypoxia.
Gap connexin 43 (connexin43, Cx43) is common in the alveolar epithelium and capillary endothelium. It is the basic unit of intercellular gap junction. It not only plays an important role in cell communication, but also plays an important role in cell growth, differentiation, apoptosis and even tumor formation. It is reported that Cx43 is related to vascular permeability in recent years. The study shows that Cx43 is involved in the process of lung injury, but the role and mechanism of Cx43 in PE-SWD/SW-RDS is not clear. By replicating the model of seawater drowning induced pulmonary edema in rats, the role of Cx43 in seawater drowning induced pulmonary edema was observed and the possible mechanism of its action was further studied.
Objective:
(1) to observe the expression of Cx43 in rats with pulmonary edema induced by seawater drowning.
(2) to explore the role and mechanism of Cx43 in pulmonary edema induced by seawater drowning.
Experimental methods:
Experiment 1
In vivo experiment
24 SD rats were randomly divided into 4 groups, the normal control group (0h), the seawater drowning 1H group (1H), the 4H group (4h) and the 8h group (8h). The model of the rat seawater drowning pulmonary edema was replicated by intratracheal infusion of seawater. The protein content in the lung tissue wet dry weight (W/D), the protein content in the bronchoalveolar lavage fluid (BALF) and the Evans blue leakage index (ELI) were used to determine the lung group. The permeability of the lung tissue was detected by RT-PCR and Western Blot, and the Cx43 content in lung tissue was detected by molecular biology.
In vitro experiment
A549 cells were incubated with 25% volume concentration of sea water to simulate the intrapulmonary environment of seawater drowning (time of action of 4 hours). The expression of Cx43 was observed to be divided into 2 groups randomly: blank control group (Control), sea water group (SW), and RT-PCR and Western Blot methods to detect the effect of seawater on the expression of Cx43. The relative permeability of the sugar was measured and the permeability of monolayer A549 cells was measured to observe the effect of seawater on the permeability of monolayer cells.
Experiment two
(1) A549 cells were randomly divided into blank control group (Control) and seawater group (SW). WesternBlot method was used to detect the effect of seawater on MAPK signaling pathway.
(two) the isolated and purified rat alveolar type II epithelial cells were randomly divided into 5 groups: blank control group (Control), sea water group (SW), ERK1/2 inhibitor group (PD), JNK inhibitor group (SP), p38 inhibitor group (SB), and negative control group. The expression of cell membrane Cx43 was analyzed by FCM.
(three) A549 cells were randomly divided into 6 groups: blank control group (Control), seawater group (SW), ERK1/2 inhibitor group (PD), JNK inhibitor group (SP), p38 inhibitor group (SB) and antisense oligodeoxynucleotide intervention group (ASODN). The permeability of monolayer cells was observed and the relationship between Cx43 content and permeability of monolayer cells was statistically analyzed.
Experimental results:
Experiment 1
In vivo experiment
After inhalation of seawater, the W/D ratio of lung tissue increased, and the protein content and ELI in BALF increased significantly (P0.05), indicating that the inhaled seawater resulted in increased pulmonary permeability. The content of Cx43mRNA and protein in lung tissue increased significantly, and the highest value of 4H was (P0.05).
In vitro experiment
RT-PCR and Western Blot detected the expression of Cx43 in A549 cells. After seawater treatment, the expression of Cx43 increased significantly and the permeability of monolayer cells increased (P0.05).
Experiment two
Western Blot results showed that seawater resulted in A549 cells p-ERK1/2, p-JNK expression decreased, p-p38 increased.ERK1/2 inhibitors, JNK inhibitors increased the expression of Cx43, respectively, and p38 signaling pathway had no obvious effect on Cx43 expression. There was a positive correlation between the amount and the permeability of monolayer cells, r=0.940, P0.05.
Conclusion:
(1) in the model of pulmonary edema induced by seawater drowning, the expression of Cx43 increased in lung tissue, and the expression of Cx43 was positively correlated with alveolar capillary permeability.
(2) seawater can increase the expression of Cx43 through inhibiting the ERK1/2 and JNK signaling pathways, thereby affecting the permeability of alveolar capillaries.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R649.3

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