大容量肺灌洗对家兔肺泡上皮claudin-4和E-cadherin表达的影响
发布时间：2018-01-11 11:20
本文关键词：大容量肺灌洗对家兔肺泡上皮claudin-4和E-cadherin表达的影响 出处：《西南医科大学》2016年硕士论文 论文类型：学位论文
更多相关文章： 大容量肺灌洗 肺损伤 肺泡上皮屏障 紧密连接蛋白-4 E-钙粘蛋白
【摘要】：目的:观察大容量肺灌洗(whole lung lavage,WLL)对家兔肺泡上皮紧密连接蛋白(claudin-4)和E-钙粘蛋白(E-cadherin)表达的影响,探讨WLL相关性肺损伤的修复机制。方法:将15只家兔随机分为对照组、大容量肺灌洗后2小时组(肺灌洗2h组)、大容量肺灌洗后72小时组(肺灌洗72h组),每组5只。行WLL前各组家兔均禁食12h,禁饮6h。肺灌洗2h组和肺灌洗72h组家兔耳缘静脉注入3%戊巴比妥(1mL/kg),待进入全麻状态后行气管切开,插入气管插管导管,确保左、右肺分隔良好,左肺给予氧气吸入,氧流量控制在160~200mL/min,生理盐水(温度约37℃)反复灌洗右肺,灌洗总量按25~30mL/kg计算,每次灌入约10mL,生理盐水沿灌洗导管注入(速度约5mL/min),每次注入完毕后将家兔处于头低足高位,让灌洗液自然引流干净,重复上述操作,灌洗液回收率均在90%以上,后拔出气管插管,逐层缝合,术后常规处理,肺灌洗72h组家兔清醒后可逐渐恢复正常饮食。对照组家兔进入全麻状态后行气管切开,插入气管插管导管,确保左、右肺分隔良好,左肺给予氧气吸入,氧流量控制在160~200mL/min,右肺不予以灌洗。对照组家兔经以上操作后2小时行肺部CT检查,后股动脉放血处死家兔;肺灌洗2h组和肺灌洗72h组分别于设定时间点前15min再次麻醉家兔,行肺部CT检查后股动脉放血处死家兔。测量右肺湿/干重比,取部份右肺组织行石蜡切片,he染色观察肺组织的病理改变并进行评分;电镜观察肺泡上皮的超微结构;免疫组化检测肺组织claudin-4及e-cadherin的表达,其相对表达量用平均光密度值(od值)表示。结果:(1)家兔表现:肺灌洗2h组及肺灌洗72h组家兔大容量肺灌洗时出现呼吸急促,唇鼻部皮肤发绀明显,偶有呛咳等不适反应,术毕后上述症状逐渐消失,肺灌洗72h组术后清醒后饮食、活动无明显异常;对照组家兔无上述反应。(2)家兔肺部ct改变:肺灌洗2h组家兔左侧肺组织未见明显异常,但右肺组织见不均匀磨玻璃样浸润影;对照组和肺灌洗72h组家兔双肺组织肺部ct检查未见明显异常改变。(3)肺组织的大体表现:肺灌洗2h组家兔右肺组织体积增大,弹性减退,部份色泽暗红,表面可见散在出血点,切面可见少量泡沫状液体流出,左侧肺组织未见明显异常;肺灌洗72h组及对照组两侧肺组织表面光滑,弹性好,无明显出血点及水肿。(4)肺湿/干重比(w/d):与对照组肺w/d值(4.34±0.06)比较,肺灌洗2h组的肺w/d值(4.72±0.09)明显增高,差异有统计学意义(p0.05);与肺灌洗2h组比较,肺灌洗72h组的肺w/d值(4.36±0.07)明显降低,差异有统计学意义(p0.05);肺灌洗72h组w/d值较对照组高,但差异无统计学意义(p0.05)。(5)he染色:肺灌洗2h组家兔肺泡壁毛细血管扩张充血,可见少量炎症细胞浸润,肺泡间隔增厚、局灶性肺水肿,肺泡腔内可见淡红色液体,但肺组织结构完整;肺灌洗72h组家兔肺组织病理改变较肺灌洗2h组减轻,肺泡结构完整,肺泡腔内无渗液,但仍有少许炎症细胞浸润,对照组家兔he染色未见明显异常改变。肺灌洗2h组的病理评分(5.0±0.7)明显高于对照组(0.2±0.4)和肺灌洗72h组(1.2±0.4),差异有统计学意义(P均0.05);肺灌洗72h组病理评分较对照组高,差异有统计学意义(P0.05)。(6)电镜显示:对照组肺泡上皮细胞间紧密连接结构完整;肺灌洗2h组细胞间紧密连接有破坏,细胞间隙增宽,部分I型肺泡上皮细胞断裂;肺灌洗72h组上述改变减轻。(7)免疫组织化学:对照组家兔claudin-4及E-cadherin主要在肺泡上皮细胞包膜呈连续的阳性表达,包浆有少许表达;肺灌洗2h组肺泡上皮细胞claudin-4及E-cadherin的表达均明显低于对照组,为不连续表达;肺灌洗72h组claudin-4及E-cadherin表达较肺灌洗2h组增强,但仍低于对照组。肺灌洗2h组claudin-4及E-cadherin表达的OD值分别为(0.93±0.12)、(1.12±0.09),较对照组claudin-4及E-cadherin表达的OD值(1.44±0.05)、(1.74±0.05)及肺灌洗72h组claudin-4及E-cadherin表达的OD值(1.32±0.04)、(1.62±0.07)明显降低,差异均有统计学意义(P0.05);与对照组比较,肺灌洗72h组claudin-4及E-cadherin表达的OD值仍有降低,差异有统计学意义(P0.05)。结论:(1)大容量肺灌洗可导致家兔肺泡上皮屏障一定程度的损伤。(2)大容量肺灌洗后家兔肺泡上皮claudin-4及E-cadherin表达降低。(3)大容量肺灌洗相关性肺损伤可在一定时间内自行修复。(4)Claudin-4及E-cadherin可能参与了肺灌洗液的清除及肺损伤的修复。
[Abstract]:Objective: To observe the effects of large volume lung lavage (whole lung, lavage, WLL) on rabbit alveolar epithelial tight junction protein (claudin-4) and E- cadherin (E-cadherin) expression, explore the repair mechanism of WLL induced lung injury. Methods: 15 rabbits were randomly divided into control group, large volume lung lavage group (2 hours after lung lavage group 2H), large volume lung lavage group (72 hours after lung lavage 72h group), 5 rats in each group. All rabbits were fasted before WLL 12h, 6h. 2H drink ban lung lavage group and lung lavage group 72h rabbit ear vein injection of 3% pentobarbital (1mL/kg), to enter the state of general anesthesia endotracheal open, insert the endotracheal tube, to ensure that the left and right lung left lung separated well received oxygen inhalation and oxygen flow control in 160~200mL/min, normal saline (temperature of about 37 DEG C) repeated lavage right lung lavage volume calculated according to 25~30mL/kg, every time pour about 10mL, saline lavage catheter implantation along (speed of about 5mL/min), after each injection in rabbits in the Trendelenburg position, let the natural drainage lavage fluid clean, repeat the above operation, lavage fluid recovery was above 90% after extubation, sutured, routine postoperative treatment, lung lavage in 72h group awake after gradually returned to normal diet. The rabbits in control group were in the state of general anesthesia after tracheotomy, endotracheal intubation catheter insertion, ensure that the left and right lung left lung separated well received oxygen inhalation and oxygen flow control in 160~200mL/min, not to be the right lung lavage. Control group rabbits after more than 2 hours after operation for lung CT examination after femoral artery bleeding of rabbits; lung lavage in 2H group and 72h group respectively in lung lavage set time before 15min again for lung CT anesthetized rabbits, rabbits were killed by exsanguination after measuring. The right lung wet / dry weight ratio, lung tissue of right part of paraffin section, he staining The pathological changes of lung tissues and score the ultrastructure of alveolar epithelium; electron microscope; immunohistochemistry the expression of claudin-4 and E-cadherin in lung tissue were determined, the relative expression for the average optical density (OD). Results: (1) in rabbits: shortness of breath appeared lung lavage in 2H group and 72h group lung lavage rabbit mwll, lip nasal skin cyanosis, occasional cough and other symptoms, the symptoms disappeared after surgery, lung lavage diet group 72h after awake, no obvious abnormal activity; control group rabbits without the above reaction. (2) the rabbit lung CT change: lung lavage in 2H group the left lung tissue had no obvious abnormalities, but the right lung tissue is not homogeneous ground glass opacity; control group and lung lavage in 72h group lung tissue of lung CT examination showed no obvious abnormal change. (3) the gross appearance of lung: lung lavage group 2H rabbits right lung tissue 绉澶，
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