miRNAs调节的免疫炎性机制在氧醚复合吸入治疗脓毒症中的作用

发布时间：2018-04-25 15:39

本文选题：盲肠结扎穿孔 + 脓毒症　；参考：《第四军医大学》2014年硕士论文

【摘要】：脓毒症（sepsis）是感染引起的、以全身炎症反应综合征(systemic inflammatoryresponse syndrome, SIRS)为特征的、证实有细菌存在或有高度可疑感染灶的疾病，是外科重症监护室（Intensive Care Unit, ICU）的主要死亡原因。研究表明，ICU中约40%的患者患有脓毒症，病死率高达36%。近年来，脓毒症发病呈上升趋势，住院费用极昂贵，尚缺乏有效的救治手段。因此，寻找安全、有效、经济的救治策略具有十分重要的意义。高氧和麻醉浓度的异氟醚已经被证实对脓毒症动物有保护效应。氧气和异氟醚在临床上已被广泛应用，但高浓度氧长时间吸入会产生过量氧自由基，可能导致氧毒性的发生，而麻醉剂量的异氟醚会引起病人麻醉，重症病人不能耐受，因此，两者在临床应用会受到病人情况的限制。我们最近的研究表明，0.5最低肺泡有效浓度（minimum alveolar concentration, MAC）异氟醚复合60%氧吸入对酵母多糖所致小鼠脓毒症具有保护作用。因此，本课题采用临床公认的脓毒症模型---盲肠结扎穿孔（cecal ligation and puncture, CLP）法诱导小鼠脓毒症及脂多糖（lipopolysaccharide, LPS）诱导离体细胞脓毒症模型，证实0.5MAC异氟醚复合60%氧吸入对脓毒症具有保护作用，并通过芯片研究说明miRNA调控的免疫炎症机制可能在其中发挥重要作用。材料与方法根据随机表法把雄性昆明小鼠（25-30g，无病原）随机分为6组，“脓毒症+空气”组、“脓毒症+100%氧气”组、“脓毒症+0.5MAC异氟醚+60%氧气”组、“假手术+空气”组、“假手术+100%氧气”组、“假手术+0.5MAC异氟醚+60%氧气”组。给予动物CLP诱导脓毒症模型，动物单纯开腹关腹作为对照模型。氧醚治疗：脓毒症模型制作后1h、6h分别给动物100%氧吸入或0.5MAC异氟醚复合60%氧气吸入1h；对照组动物吸入空气。CLP后连续7天观察各组动物存活率。取全血分离血清检测血生化指标、血清C反应蛋白（C-reactive protein, CRP）、血乳酸、炎症因子等指标，取心、肺、肝和肾各脏器，计算肺湿干比、观察各脏器病理损伤并进行病理评分。进行肺泡灌洗，检测肺蛋白渗出。提白细胞，检测白细胞miRNA表达谱。 RAW264.7巨噬细胞系，随机分为6组，“脂多糖”组、“脂多糖+100%氧气”组、“脂多糖+0.5MAC异氟醚+60%氧气”组、“安慰剂”组、“安慰剂+100%氧气”组、“安慰剂+0.5MAC异氟醚+60%氧气”组。 LPS刺激诱导细胞脓毒症模型，，脂多糖溶剂DMED培养基作为阴性对照。氧醚治疗：CLP后即刻给细胞100%氧或0.5MAC异氟醚复合60%氧环境处理。处理结束后收集细胞培养上清液、抽提细胞核蛋白和（或）取爬片的细胞，检测炎症因子及核因子κB（nuclear factor-kappa B，NF-κB）的活化。结果 1．CLP后24h，脓毒症小鼠心、肺、肝和肾脏发生显著病理性改变，血乳酸和血生化水平显著高于对照组小鼠，血中炎症因子水平显著增高，7天存活率仅为40%。造模后1h和6h分别吸入1h的100%氧或0.5MAC异氟醚复合60%氧，可显著减轻CLP所致脓毒症小鼠各指标的异常变化。 2．造模后不同时间点，脓毒症小鼠血中炎症因子发生显著异常。造模后1h和6h分别吸入100%氧或0.5MAC异氟醚复合60%氧1h，可显著改变CLP后小鼠miRNA表达谱。 3．100ng/ml脂多糖刺激2h后，细胞炎症因子水平及NF-κB的活化显著高于对照组。100%氧或0.5MAC异氟醚复合60%氧处理可显著抑制炎症因子的异常变化及降低NF-κB的活化。结论综上所述，我们的实验结果证明，0.5MAC异氟醚复合60%氧气可能是通过miRNA调节的免疫炎性机制及抑制NF-κB的活化对脓毒症产生保护作用。
[Abstract]:Sepsis (sepsis) is an infection, characterized by systemic inflammatory response syndrome (systemic inflammatoryresponse syndrome, SIRS), which confirms the presence of bacteria or a highly suspected infection, which is the main cause of death in the surgical intensive care unit (Intensive Care Unit, ICU). The study showed that about 40% of the patients in ICU were Huan Younong. In recent years, the incidence of toxosis is up to 36%., the incidence of sepsis is rising, the cost of hospitalization is very expensive, and it is still lack of effective treatment. Therefore, it is very important to find a safe, effective and economical treatment strategy. It is widely used clinically, but high concentration of oxygen long time inhalation produces oxygen free radicals, which may lead to oxygen toxicity, and anesthetic isoflurane can cause patients to be anesthetized and severe patients are intolerant. Therefore, both in clinical application will be limited by the patient's condition. Our recent study shows that 0.5 of the lowest alveoli are effective. The concentration (minimum alveolar concentration, MAC) isoflurane combined with 60% oxygen inhalation can protect the sepsis caused by yeast polysaccharide in mice. Therefore, this subject uses a clinically recognized sepsis model - cecum ligation and perforation (cecal ligation and puncture, CLP) to induce sepsis and lipopolysaccharide (lipopolysaccharide, LPS) induction in mice The sepsis model of isolated cells confirmed the protective effect of 0.5MAC isoflurane combined with 60% oxygen inhalation on sepsis, and a chip study showed that the immune inflammatory mechanism regulated by miRNA may play an important role in it.
Materials and methods
The male Kunming mice (25-30g, without pathogens) were randomly divided into 6 groups, "sepsis + air" group, "sepsis +100% oxygen" group, "sepsis +0.5MAC isoflurane +60% oxygen" group, "sham operation + air" group, "sham operation +100% oxygen" group, "sham operation +0.5MAC isoflurane +60% oxygen" group. Animal CLP induction was given. The model of sepsis, animals simply open the abdominal cavity as the control model. Oxygen and ether treatment: after the sepsis model was made 1H, 6h was given to animals 100% oxygen inhalation or 0.5MAC isoflurane combined with 60% oxygen inhalation of 1H, and the control group of animals inhaled air.CLP for 7 days to observe the survival rate of each group. The blood biochemical indexes were detected by whole blood separated serum, serum C The C-reactive protein (CRP), blood lactic acid, and inflammatory factors were used to measure the lung wet dry ratio, the pathological injury of the organs and the pathological scores were observed. The alveolar lavage, the pulmonary protein exudation, the leukocyte and the miRNA expression of leukocyte were detected.
RAW264.7 macrophage system, randomly divided into 6 groups, "lipopolysaccharide" group, "lipopolysaccharide +100% oxygen" group, "lipopolysaccharide +0.5MAC isoflurane +60% oxygen" group, "placebo" group, "placebo +100% oxygen" group, "placebo +0.5MAC isoflurane +60% oxygen" group. LPS stimulation induced cell sepsis model, lipopolysaccharide solvent DMED medium For the negative control. Oxygen ether treatment: the cells were treated with 100% oxygen or 0.5MAC isoflurane immediately after CLP. After treatment, the cell culture supernatant was collected, the cell of nuclear protein and / or crawling tablet were extracted, and the activation of the inflammatory factors and nuclear factor kappa B (nuclear factor-kappa B, NF- kappa B) was detected.
Result
After 1.CLP 24h, there were significant pathological changes in the heart, lung, liver and kidney of sepsis mice. Blood lactic acid and blood biochemical levels were significantly higher than those in the control group. The levels of inflammatory factors in the blood were significantly higher. The 7 day survival rate was only 100% oxygen or 0.5MAC isoflurane combined with 60% oxygen after 1h and 6h, respectively, which could significantly reduce CLP induced sepsis. Abnormal changes in each index of the rat.
2. after modeling at different time points, the inflammatory factors in the blood of sepsis mice were significantly abnormal. After the model, 1H and 6h inhaled 100% oxygen or 0.5MAC isoflurane combined with 60% oxygen 1H, which could significantly change the miRNA expression profiles in CLP mice.
After 3.100ng/ml lipopolysaccharide stimulated 2h, the levels of inflammatory factors and the activation of NF- kappa B were significantly higher than those of the control group of.100% oxygen or 0.5MAC isoflurane combined with 60% oxygen treatment, which could significantly inhibit the abnormal changes of inflammatory factors and reduce the activation of NF- kappa B.
conclusion
In summary, our experimental results show that 0.5MAC isoflurane combined with 60% oxygen may be a protective effect of miRNA mediated immuno inflammatory mechanism and inhibition of activation of NF- kappa B on sepsis.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614

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