乌司他丁对急性肺损伤大鼠肺保护作用的研究

发布时间：2018-02-14 11:28

本文关键词： 急性肺损伤肿瘤坏死因子白细胞介素乌司他丁脂多糖　出处：《大连医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：通过观察乌司他丁(ulinastatin，UTI)对急性肺损伤大鼠肺组织肿瘤坏死因子(TNF-α)、白细胞介素（IL-1β)、白介素1受体抗体（IL-1ra）mRNA表达的影响，探讨乌司他丁治疗急性肺损伤机制。方法：将72只雄性SD大鼠随机分为4组，即A组实验对照组，，B组内毒素组和C组乌司他丁低剂量组(UTI50U/kg），D组乌司他丁高剂量组(UTI100U/kg)，制模前20分钟给予C组和D组大鼠，分别经腹腔注射不同剂量的乌司他丁，BCD组均采用尾静脉注射内毒素磷酸脂多糖（15mg/kg）制作急性肺损伤模型，A组则经腹腔及尾静脉注射等量的生理盐水，各组于注射内毒素磷酸脂多糖（LPS）后2小时、6小时、24小时各取样6只；以1%戊巴比妥钠50mg/kg腹腔注射麻醉，大鼠仰卧位固定于操作台，开胸取肺，10%甲醛固定右下肺叶组织，待行病理检查。右下肺组织冻存于-80℃，应用HE染色，光镜下观察各组大鼠肺组织变化情况，应用引物设计软件primer primer5.0设计引物，应用逆转录聚合酶链反应（ReverseTranscription-Polymerase Chain Reaction，RTPCR）法扩增并测定大鼠肺组织细胞TNF-α mRNA、IL-1β mRNA、IL-1ra mRNA表达情况。检测不同剂量的乌司他丁干预下，内毒素（LPS）致急性肺损伤大鼠肺组织TNF-α、IL-1β、IL-1ra的mRNA表达情况差异。结果:大鼠右下肺组织经HE染色检查，光镜下观察显示对照组大鼠肺泡结构清晰完整，无破坏，间隔无增宽；内毒素脂多糖(LPS)组肺组织炎症表现明显，肺泡结构不同程度受损，肺组织间隔增厚变宽，炎细胞浸润明显；乌司他丁(ulinastatin)组则显示较内毒素组呈现一定的炎症抑制，表现为肺泡组织间隔变窄，炎细胞浸润减轻；对肺损伤大鼠肺组织内TNF-α mRNA、IL-1β mRNA、IL-1ra mRNA表达情况，应用RT-PCR法、凝胶电泳计算机灰度分析得出结果，经SPSS19软件统计分析，应用方差分析及t检验，分别比较同一时间点不同处理组的TNF-α表达情况的组间差异，结果提示乌司他丁干预组与内毒素组之间统计学差异显著(P0.001)，乌司他丁低剂量组与高剂量组相比较，差异也显著(P0.001)，再分别比较IL-1β mRNA、IL-1ra mRNA表达情况，应用乌司他丁组与内毒素组比较均有显著差异(P0.005)，乌司他丁低剂量组与高剂量组相比较，差异也显著(P0.001)。结论:乌司他丁对于脓毒症引起的急性肺损伤具有一定的治疗作用，其机制可能为降低以TNF-α IL-1β IL-1ra为代表的炎症因子表达，抑制肺部炎症反应的发生。
[Abstract]:Objective: To observe the effect of ulinastatin (Ulinastatin, UTI) on the expression of tumor necrosis factor (TNF- alpha), interleukin (IL-1 beta) and interleukin 1 receptor antibody (IL-1ra) mRNA in lung tissue of rats with acute lung injury, and to explore the mechanism of ulinastatin in the treatment of acute lung injury.
Methods: 72 male SD rats were randomly divided into 4 groups: A group, experimental control group, B group, LPS group and ulinastatin group C low dose group (UTI50U/kg), group D Ulinastatin high dose group (UTI100U/kg), mold 20 minutes before giving C group and D group rats respectively, by intraperitoneal injection of different doses of ulinastatin, group BCD by intravenous injection of endotoxin lipopolysaccharide (15mg/kg) phosphate production model of acute lung injury in A group with normal saline intraperitoneal and intravenous injection of the same amount of phosphate groups in the injection of endotoxin lipopolysaccharide (LPS) after 2 hours, 6 hours, 24 hours the sampling of 6; with 1% 50mg/kg intraperitoneal injection of pentobarbital sodium anesthesia, rats supine fixed on the operation table, lung, 10% formalin fixed to the lower lobe of the right lung tissue, lung tissue for pathological examination. The right under the freezing at -80 deg.c, using HE staining, observe the changes of lung tissue of rats under the light microscope, using primer Design software primer Primer5.0 primers by reverse transcriptase polymerase chain reaction (ReverseTranscription-Polymerase Chain Reaction, RTPCR) were amplified by TNF- and determination of mRNA cells in lung tissue of rats with IL-1 alpha, beta mRNA, IL-1ra mRNA expression. The effects of different doses of statin intervention detection, endotoxin (LPS) injury of TNF- alpha, lung tissue of rats with acute the expression of IL-1ra induced lung IL-1 beta, mRNA difference.
Results: the lung tissue of rats after right HE staining, light microscope observation showed that rat alveolar structure of control group was clear and complete, no damage, no interval widened; lipopolysaccharide (LPS) group showed obvious inflammation of lung tissue, alveolar structure damaged to varying degrees, lung septum thick and wide, infiltration of inflammatory cells obviously; ulinastatin (Ulinastatin) group showed endotoxin group showed a suppression of inflammation, tissue showed alveolar septum, infiltration of inflammatory cells to reduce; lung injury in rats TNF- mRNA IL-1 alpha, beta mRNA, IL-1ra mRNA expression, using RT-PCR method, gel electrophoresis and computer gray analysis results by SPSS19 software, statistical analysis, analysis of variance and t test, respectively, compared with the same time point of different groups of TNF- expression differences between the groups. The results suggest that the effects between the intervention group and endotoxin group he d statistics Significant differences (P0.001), ulinastatin low dose group and high dose group compared with significant difference (P0.001), and then were compared with IL-1 IL-1ra beta mRNA, mRNA expression and application of ulinastatin group and endotoxin group were significantly different (P0.005), ulinastatin low dose group and high dose compared to the group, a significant difference (P0.001).
Conclusion: Ulinastatin has certain therapeutic effect on acute lung injury caused by sepsis, and its mechanism may be to reduce the expression of inflammatory factors represented by TNF- alpha IL-1 beta IL-1ra and inhibit the occurrence of pulmonary inflammatory reaction.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.8

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