本文關(guān)鍵詞：不同誘因急性肺損傷模型大鼠肺內(nèi)外炎癥動(dòng)態(tài)變化　出處：《河北醫(yī)科大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 急性肺損傷 急性呼吸窘迫綜合癥 大腸桿菌 脂多糖 腫瘤壞死因子α TNF-α

【摘要】：目的:急性肺損傷是一常見(jiàn)病和多發(fā)病,較高的病死率引起人們關(guān)注,它的發(fā)病機(jī)制到目前為止還不是十分清楚。本研究通過(guò)尾靜脈注射脂多糖和氣管內(nèi)滴入大腸桿菌來(lái)模擬肺內(nèi)外兩種模型,觀(guān)察這兩種模型中的炎癥反應(yīng)有何不同,并觀(guān)察小劑量糖皮質(zhì)激素對(duì)這兩種模型治療效果有何不同。從而更好的指導(dǎo)臨床中急性肺損傷的治療。 方法:清潔級(jí)雄性SD大鼠112只,按隨機(jī)數(shù)字表法分為14組:尾靜脈注射生理鹽水1 ml對(duì)照組、尾靜脈注射脂多糖( LPS 8 mg/kg ) 2h、4h、6h模型組、氣管內(nèi)滴入大腸桿菌標(biāo)準(zhǔn)菌株懸液( E.coli, 5.0×10~9 cfu/ml, 3 ml/kg ) 12h、24h、36h模型組,NS+Dex組及LPS 2h+Dex組、4h+Dex組、6h+Dex組、E.coli 12h+Dex組、24h+Dex組、36h+Dex干預(yù)組,每組8只動(dòng)物。干預(yù)組在造模結(jié)束后立即腹腔內(nèi)注射地塞米松( 2mg/kg )。 10%水合氯醛腹腔注射麻醉( 3ml/kg ),股動(dòng)脈放血收集血液,離心( 3000 rpm, 4℃, 10 min ),吸取上清,-80℃保存,檢測(cè)血清中TNF-α的含量。開(kāi)胸處死動(dòng)物后,取右肺中葉測(cè)定肺的濕干比;取右肺下葉,10%中性福爾馬林溶液固定,常規(guī)石蠟包埋,切片,HE染色,觀(guān)察肺組織病理形態(tài)學(xué)變化并評(píng)分,經(jīng)氣管插管行左肺支氣管肺泡灌洗,取支氣管肺泡灌洗液( Bronchoalveloar lavag fluid, BALF ),離心( 1200 rpm, 4℃, 10 min ),吸取上清,-80℃保存,檢測(cè)BALF中TNF-α、蛋白的含量。細(xì)胞沉淀懸浮于200μl生理鹽水中,進(jìn)行白細(xì)胞計(jì)數(shù)。 結(jié)果: 1肺組織病理形態(tài)學(xué):光鏡下觀(guān)察LPS2h后開(kāi)始出現(xiàn)肺血管充血,炎癥細(xì)胞的浸潤(rùn),肺間質(zhì)水腫, 4h出現(xiàn)典型ALI的表現(xiàn):透明膜形成,6h與4h病理半定量評(píng)分比較無(wú)統(tǒng)計(jì)學(xué)差異;在氣管內(nèi)滴入大腸桿菌12h后,發(fā)現(xiàn)大量炎癥細(xì)胞浸潤(rùn)、氣道上皮細(xì)胞的脫落,肺泡水腫,24h出現(xiàn)肺泡上皮細(xì)胞空泡化,壞死,細(xì)胞增殖,大量纖維蛋白的滲出,透明膜形成,部分肺實(shí)變。36h較24h病理半定量評(píng)分無(wú)統(tǒng)計(jì)學(xué)差異。給予激素干預(yù)后,LPS模型在2h、4h、6h可見(jiàn)浸潤(rùn)的炎癥細(xì)胞減少,蛋白滲出減少,病理半定量評(píng)分也相應(yīng)減少;E.coli模型在24h、36h出現(xiàn)炎癥細(xì)胞浸潤(rùn)及透明膜減少,但病理半定量評(píng)分無(wú)統(tǒng)計(jì)學(xué)差異。 2 BALF中細(xì)胞總數(shù)、蛋白含量和肺組織W/D的變化:在LPS模型中BALF中細(xì)胞計(jì)數(shù)隨著時(shí)間的延長(zhǎng),肺泡灌洗液中細(xì)胞計(jì)數(shù)明顯增多,給予激素后肺泡灌洗液中細(xì)胞計(jì)數(shù)明顯下降;肺泡灌洗液中蛋白4h達(dá)高峰,6h開(kāi)始下降,激素干預(yù)組同比相應(yīng)下降;濕干比,在LPS組2h較對(duì)照組明顯升高,但隨著時(shí)間的推移,未出現(xiàn)統(tǒng)計(jì)學(xué)意義的進(jìn)一步增加,激素干預(yù)組同比相應(yīng)下降;在Ecoli模型中肺泡灌洗液中細(xì)胞計(jì)數(shù),肺泡灌洗液中蛋白,濕干比,隨著時(shí)間的延長(zhǎng),未出現(xiàn)統(tǒng)計(jì)學(xué)意義的增加,12h、24h與36h比較無(wú)統(tǒng)計(jì)學(xué)意義,給予激素后24h、36h均明顯下降。 3血清中TNF-α含量的變化:血清中TNF-α的水平,在LPS模型2h、4h、6h與NS組比明顯升高,三組隨著時(shí)間的延長(zhǎng),出現(xiàn)升高→高峰→降低的變化趨勢(shì)。給予激素干預(yù)后,TNF-α的含量較模型組同比明顯下降(P0.01)。在Ecoli模型中12h、24h、36h與NS組比明顯升高,三組隨著時(shí)間的發(fā)展,同樣出現(xiàn)升高→高峰→降低的變化趨勢(shì)。給予激素后,TNF-α的含量較模型組無(wú)明顯變化。 4肺泡灌洗液( BALF )中TNF-α含量的變化:血清中TNF-α的水平,在LPS模型2h、4h、6h與NS組比明顯升高,三組隨著時(shí)間的延長(zhǎng),出現(xiàn)升:升高→高峰→降低的變化趨勢(shì)。給予激素干預(yù)后較模型組同比下降( P0.01 )。在Ecoli模型中12h、24h、36h與NS組比明顯升高,三組趨勢(shì)同LPS模型;給予激素干預(yù)后,較模型組無(wú)明顯改善。 結(jié)論: 1不同時(shí)間點(diǎn)脂多糖和大腸桿菌誘發(fā)肺內(nèi)外ALI模型均成功。 2 LPS模型在4h達(dá)到典型ALI病理表現(xiàn),大腸桿菌模型在24h同樣達(dá)到典型表現(xiàn),說(shuō)明這兩種模型ALI的發(fā)展過(guò)程的時(shí)間窗存在不同。 3糖皮質(zhì)激素的療效與導(dǎo)致肺損傷的誘因密切相關(guān)。兩種模型激素干預(yù)后,LPS主要表現(xiàn)為纖維蛋白滲出減少,肺水腫減輕。大腸桿菌主要表現(xiàn)為炎細(xì)胞浸潤(rùn)減少,纖維蛋白滲出也有所減輕,但總的病理評(píng)分并無(wú)明顯改善。借以說(shuō)明臨床治療中對(duì)肺內(nèi)和肺外ALI應(yīng)用激素的干預(yù)治療要區(qū)別對(duì)待。 4兩種模型血清和BALF中TNF-α隨時(shí)間改變出現(xiàn)相同的趨勢(shì)變化,激素干預(yù)后,LPS模型血清和BALF有明顯改善;大腸桿菌模型血清和BALF中基本無(wú)改善。可得出激素對(duì)內(nèi)毒素導(dǎo)致的肺外型肺損傷治療效果較理想,對(duì)重癥感染造成的肺內(nèi)型肺損傷治療效果欠佳。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R-332;R563.8

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