缝隙连接蛋白43在海水淹溺型肺水肿中的作用及其机制的研究

发布时间：2018-05-11 22:44

本文选题：海水 + 肺水肿　；参考：《第四军医大学》2012年硕士论文

【摘要】：研究背景：海水淹溺是航海、海上作战、作业时常发生的意外伤害事故，吸入海水可导致肺泡-毛细血管膜损伤并伴有通透性增高，富含蛋白的液体聚集于肺泡腔，引起海水淹溺型肺水肿(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的主要病理变化是肺通透性增高，肺水肿形成，引起低氧血症和呼吸困难。如不及时救治和控制病情，，易发展为海水型呼吸窘迫综合征(seawaterrespiratory distress syndrome, SW-RDS)。SW-RDS是急性呼吸窘迫综合征(acute respiratory distress syndrome， ARDS)的一种。近年来关于PE-SWD/SW-RDS的研究逐渐增多，但是其发病机制仍不完全清楚，缺乏有效的治疗手段，死亡率仍很高。PE-SWD/SW-RDS时，肺泡上皮屏障功能破坏，肺水肿液的清除功能障碍，加之吸入的高渗海水在肺泡内聚集，驱使血管内液体沿渗透压力梯度流向肺泡内，加重缺氧。缝隙连接蛋白43(connexin43，Cx43)在肺泡上皮和毛细血管内皮普遍存在，是细胞间缝隙连接的基本组成单位，不仅发挥细胞间的通讯功能，而且对细胞的生长、分化和凋亡甚至肿瘤形成都有重要作用。有文献报道Cx43与血管通透性有关。近年来研究表明Cx43参与肺损伤的过程，但Cx43在PE-SWD/SW-RDS中的作用及其机制尚不清楚。本实验通过复制大鼠海水淹溺型肺水肿的模型，观察Cx43在海水淹溺型肺水肿中的作用，并进一步研究其可能的作用机制。实验目的： ⑴观察大鼠海水淹溺型肺水肿模型中Cx43的表达变化。 ⑵探讨Cx43在海水淹溺型肺水肿中的作用及其机制。实验方法：实验一体内实验 24只SD大鼠随机分为4组，正常对照组(0h)、海水淹溺1h组(1h)、4h组(4h)和8h组(8h)。气管内滴注海水的方法复制大鼠海水淹溺型肺水肿的模型，通过肺组织湿干重比值(W/D)、支气管肺泡灌洗液(BALF)中的蛋白含量和伊文思蓝漏出指数(ELI)测定肺组织的通透性；RT-PCR和Western Blot等分子生物学实验方法检测肺组织Cx43的含量变化。体外实验采用25%体积浓度的海水孵育A549细胞模拟海水淹溺的肺内环境(作用时间为4小时)，观察Cx43的表达变化。A549细胞随机分为2组：空白对照组(Control)、海水组(SW)，通过RT-PCR和Western Blot方法检测海水对Cx43的表达的影响。通过对FITC标记的葡聚糖的相对通透性测定单层A549细胞的通透性，观察海水对单层细胞通透性的影响。实验二 (一)A549细胞随机分为空白对照组(Control)和海水组(SW)，WesternBlot方法检测海水对MAPK信号通路的作用。 (二)分离纯化大鼠肺泡Ⅱ型上皮细胞随机分为5组：空白对照组(Control)、海水组(SW)、ERK1/2抑制剂组(PD)、JNK抑制剂组(SP)、p38抑制剂组(SB)，并设阴性对照组。通过FCM分析细胞膜Cx43的表达量。 (三)A549细胞随机分为6组：空白对照组(Control)、海水组(SW)、ERK1/2抑制剂组(PD)、JNK抑制剂组(SP)、p38抑制剂组(SB)和反义寡核苷酸干预组(ASODN)。通过Western Blot方法检测各组细胞Cx43的表达含量。培养单层细胞，通过对FITC标记的葡聚糖的通透性的检测观察单层细胞的通透性，统计分析Cx43含量与单层细胞通透性的关系。实验结果：实验一体内实验大鼠吸入海水后，肺组织W/D比值增加，BALF中的蛋白含量和ELI也明显增加(P0.05)，说明吸入海水导致肺通透性增高。肺组织Cx43mRNA和蛋白含量明显增加，4h达最高值(P0.05)。体外实验 RT-PCR和Western Blot检测A549细胞Cx43表达，海水作用后，Cx43表达明显增多，单层细胞通透性增高(P0.05)。实验二 Western Blot结果表明海水导致A549细胞p-ERK1/2、p-JNK表达减少，p-p38增多。ERK1/2抑制剂、JNK抑制剂分别上调Cx43的表达，p38信号通路对Cx43的表达无明显作用，同时ERK1/2抑制剂、JNK抑制剂使单层A549细胞通透性增加(P0.05)。通过统计相关分析，Cx43表达量与单层细胞的通透性呈正相关关系，r=0.940, P0.05。结论： ⑴在海水淹溺型肺水肿模型中，肺组织Cx43表达增多，且Cx43表达量与肺泡-毛细血管通透性正相关。 ⑵海水可通过抑制ERK1/2和JNK信号通路引起Cx43表达量增加进而影响肺泡-毛细血管的通透性。
[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.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R649.3

【参考文献】