不同诱因急性肺损伤模型大鼠肺内外炎症动态变化

发布时间：2018-01-06 07:37

本文关键词：不同诱因急性肺损伤模型大鼠肺内外炎症动态变化　出处：《河北医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:急性肺损伤是一常见病和多发病,较高的病死率引起人们关注,它的发病机制到目前为止还不是十分清楚。本研究通过尾静脉注射脂多糖和气管内滴入大肠杆菌来模拟肺内外两种模型,观察这两种模型中的炎症反应有何不同,并观察小剂量糖皮质激素对这两种模型治疗效果有何不同。从而更好的指导临床中急性肺损伤的治疗。方法:清洁级雄性SD大鼠112只,按随机数字表法分为14组:尾静脉注射生理盐水1 ml对照组、尾静脉注射脂多糖( LPS 8 mg/kg ) 2h、4h、6h模型组、气管内滴入大肠杆菌标准菌株悬液( E.coli, 5.0×10~9 cfu/ml, 3 ml/kg ) 12h、24h、36h模型组,NS+Dex组及LPS 2h+Dex组、4h+Dex组、6h+Dex组、E.coli 12h+Dex组、24h+Dex组、36h+Dex干预组,每组8只动物。干预组在造模结束后立即腹腔内注射地塞米松( 2mg/kg )。 10%水合氯醛腹腔注射麻醉( 3ml/kg ),股动脉放血收集血液,离心( 3000 rpm, 4℃, 10 min ),吸取上清,-80℃保存,检测血清中TNF-α的含量。开胸处死动物后,取右肺中叶测定肺的湿干比;取右肺下叶,10%中性福尔马林溶液固定,常规石蜡包埋,切片,HE染色,观察肺组织病理形态学变化并评分,经气管插管行左肺支气管肺泡灌洗,取支气管肺泡灌洗液( Bronchoalveloar lavag fluid, BALF ),离心( 1200 rpm, 4℃, 10 min ),吸取上清,-80℃保存,检测BALF中TNF-α、蛋白的含量。细胞沉淀悬浮于200μl生理盐水中,进行白细胞计数。结果: 1肺组织病理形态学:光镜下观察LPS2h后开始出现肺血管充血,炎症细胞的浸润,肺间质水肿, 4h出现典型ALI的表现:透明膜形成,6h与4h病理半定量评分比较无统计学差异;在气管内滴入大肠杆菌12h后,发现大量炎症细胞浸润、气道上皮细胞的脱落,肺泡水肿,24h出现肺泡上皮细胞空泡化,坏死,细胞增殖,大量纤维蛋白的渗出,透明膜形成,部分肺实变。36h较24h病理半定量评分无统计学差异。给予激素干预后,LPS模型在2h、4h、6h可见浸润的炎症细胞减少,蛋白渗出减少,病理半定量评分也相应减少;E.coli模型在24h、36h出现炎症细胞浸润及透明膜减少,但病理半定量评分无统计学差异。 2 BALF中细胞总数、蛋白含量和肺组织W/D的变化:在LPS模型中BALF中细胞计数随着时间的延长,肺泡灌洗液中细胞计数明显增多,给予激素后肺泡灌洗液中细胞计数明显下降;肺泡灌洗液中蛋白4h达高峰,6h开始下降,激素干预组同比相应下降;湿干比,在LPS组2h较对照组明显升高,但随着时间的推移,未出现统计学意义的进一步增加,激素干预组同比相应下降;在Ecoli模型中肺泡灌洗液中细胞计数,肺泡灌洗液中蛋白,湿干比,随着时间的延长,未出现统计学意义的增加,12h、24h与36h比较无统计学意义,给予激素后24h、36h均明显下降。 3血清中TNF-α含量的变化:血清中TNF-α的水平,在LPS模型2h、4h、6h与NS组比明显升高,三组随着时间的延长,出现升高→高峰→降低的变化趋势。给予激素干预后,TNF-α的含量较模型组同比明显下降(P0.01)。在Ecoli模型中12h、24h、36h与NS组比明显升高,三组随着时间的发展,同样出现升高→高峰→降低的变化趋势。给予激素后,TNF-α的含量较模型组无明显变化。 4肺泡灌洗液( BALF )中TNF-α含量的变化:血清中TNF-α的水平,在LPS模型2h、4h、6h与NS组比明显升高,三组随着时间的延长,出现升:升高→高峰→降低的变化趋势。给予激素干预后较模型组同比下降( P0.01 )。在Ecoli模型中12h、24h、36h与NS组比明显升高,三组趋势同LPS模型;给予激素干预后,较模型组无明显改善。结论: 1不同时间点脂多糖和大肠杆菌诱发肺内外ALI模型均成功。 2 LPS模型在4h达到典型ALI病理表现,大肠杆菌模型在24h同样达到典型表现,说明这两种模型ALI的发展过程的时间窗存在不同。 3糖皮质激素的疗效与导致肺损伤的诱因密切相关。两种模型激素干预后,LPS主要表现为纤维蛋白渗出减少,肺水肿减轻。大肠杆菌主要表现为炎细胞浸润减少,纤维蛋白渗出也有所减轻,但总的病理评分并无明显改善。借以说明临床治疗中对肺内和肺外ALI应用激素的干预治疗要区别对待。 4两种模型血清和BALF中TNF-α随时间改变出现相同的趋势变化,激素干预后,LPS模型血清和BALF有明显改善;大肠杆菌模型血清和BALF中基本无改善。可得出激素对内毒素导致的肺外型肺损伤治疗效果较理想,对重症感染造成的肺内型肺损伤治疗效果欠佳。
[Abstract]:Objective: acute lung injury is a common and frequently occurring disease, high mortality cause people attention, its pathogenesis is still not very clear so far. This study by intravenous injection of lipopolysaccharide and tracheal instillation of tail of Escherichia coli to simulate two kinds of inside and outside the lung model, to observe the inflammatory response from the two models in the observation, and low dose glucocorticoid treatment effect of the two models. The difference in treatment of acute lung injury in order to better guide clinical.
Methods: male SD 112 rats were randomly divided into 14 groups: intravenous injection of 1 ml normal saline control group, intravenous injection of lipopolysaccharide (LPS 8 mg/kg) 2h, 4h, 6h model group, intratracheal instillation of standard strain of Escherichia coli suspension (E.coli, 5 * 10~9 cfu/ml ml/kg, 3) 12h, 24h, 36h group, NS+Dex group and LPS 2h+Dex group, 4h+Dex group, 6h+Dex group, E.coli 12h+Dex group, 24h+Dex group, 36h+Dex group, 8 rats in each animal. Dexamethasone was immediately after the modeling injection intervention group (2mg/kg).
Anesthesia by intraperitoneal injection of 10% chloral hydrate (3ml/kg), femoral artery blood collection blood centrifugation (3000 rpm, 4 C, 10 min), supernatant was stored at -80, and the content of serum alpha TNF-. Open chest animal were sacrificed after taking the right middle lobe of the lung was wet and dry; and the right lung under the leaves, 10% formalin fixed, paraffin embedded sections, HE staining, observe pathological changes of lung tissue and score of endotracheal intubation and left lung bronchoalveolar lavage, and bronchoalveolar lavage fluid (Bronchoalveloar lavag, fluid, BALF), centrifugation (1200 rpm, 4 C, 10 min), draw the supernatant, -80 C preservation, BALF detection of TNF- alpha protein content. Cell pellet was suspended in 200 L saline, white blood cell count.
Result:
1 histology: under the light microscope after LPS2h began to appear in pulmonary vascular congestion, inflammatory cell infiltration, pulmonary interstitial edema, 4H showed typical ALI manifestations: hyaline membrane formation, there was no statistical difference between 6h and 4H were semi quantitative score; after intratracheal instillation of Escherichia coli 12h, found that a large number of the infiltration of inflammatory cells and airway epithelial cell shedding, alveolar edema, 24h vacuoles in alveolar epithelial cells, cell proliferation, necrosis, large amount of fibrin exudation, hyaline membrane formation, lung consolidation.36h compared with 24h pathological semi quantitative score showed no statistically difference. Given the hormone intervention of LPS model in 2H, 4H 6h, infiltrative inflammatory cells decreased, protein exudation reduced, pathological semi quantitative score is reduced; the E.coli model in 24h 36h, inflammatory cell infiltration and hyaline membrane decreased, but the semi quantitative pathological score was no significant difference.
2 of the total number of BALF cells, changes of protein content and lung tissue of W/D: BALF in the LPS model in cell counting with the extension of time, bronchoalveolar lavage fluid cell count increased significantly after given hormone cell count in bronchoalveolar lavage fluid were significantly decreased; 4H protein in bronchoalveolar lavage fluid reached the peak, 6H began to decline in hormone intervention an appropriate group decreased; wet dry ratio, LPS in group 2H was significantly increased compared with the control group, but with the passage of time, there was no statistical significance to further increase the hormone intervention group decreased compared to the same period; in the Ecoli model cell count in bronchoalveolar lavage fluid in bronchoalveolar lavage fluid in protein, wet and dry, with time prolong, increase, there was no statistical significance of 12h, 24h and 36h were not significant, after given hormone 24h, 36h decreased significantly.
The change of TNF- content in the serum of 3 alpha TNF- alpha levels in serum in LPS model of 2h, 4h, 6h and NS were much higher than the three group increased with the extension of time, to reduce the peak to variation. Given the hormone intervention, the content of TNF- alpha compared with the model group obviously decreased compared to the same period (P0.01). In the Ecoli model, 12h, 24h, 36h and NS were much higher than the three groups over time, also increased to variation of peak to reduce. After given hormone, the content of TNF- alpha compared with the model group had no obvious change.
4 in bronchoalveolar lavage fluid (BALF) changes in TNF- content: alpha TNF- alpha levels in serum in LPS model of 2h, 4h, 6h and NS were much higher than the three group with the extension of time, appear rise to variation to reduce the peak increased. Compared with the model group after intervention give hormone down (P0.01). In the Ecoli model, 12h, 24h, 36h and NS were much higher than the three group trend with LPS model; give hormone intervention, compared with the model group had no obvious improvement.
Conclusion:
1 at different time points, both lipopolysaccharide and Escherichia coli induced ALI models both inside and outside the lung.
The 2 LPS model reached typical ALI pathological findings in 4H. The Escherichia coli model also showed typical performance in 24h, indicating that the time windows of ALI development process of these two models were different.
3 Effect of glucocorticoids and cause lung injury are closely related. Two models of hormone intervention, LPS mainly reduce fibrin exudation, pulmonary edema. To reduce the infiltration of inflammatory cells mainly Escherichia coli, fibrin has been reduced, but the total pathological score was not significantly improved. In order to explain the intervention treatment of lung and extrapulmonary ALI hormone application in the clinical treatment should be treated differently.
4 two kinds of serum TNF- and BALF model in a change with time change in trend of the same hormone intervention, serum BALF and LPS model has obvious improvement; there is no improvement in serum and BALF in Escherichia coli model. The treatment of extrapulmonary lung injury caused by endotoxin effect of hormones on the ideal, for poor pulmonary lung the damage caused by the treatment of severe infection.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332;R563.8

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