FTY720与人脐带间充质干细胞协同治疗内毒素所致小鼠急性肺损伤的实验研究

发布时间：2018-02-04 22:55

本文关键词： 急性肺损伤内毒素 1-磷酸鞘氨醇人脐带间充质干细胞　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:急性肺损伤和急性呼吸窘迫综合征(acute lung injury/acute respiratory distress syndrome,ALI/ARDS)是临床危重症患者高发病率和死亡率的主要原因,因缺乏有效的治疗药物,所以亟待寻找新的治疗手段。近年研究发现人脐带间充质干细胞(Human Umbilical Cord-Mesenchymal Stem Cells,h UC-MSCs)可以有效缓解急性肺损伤和急性呼吸窘迫综合征,而1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)对增强血管内皮细胞屏障功能具有重要作用,可抵抗肺脏的炎症反应,减轻肺损伤。FTY720为其类似物,是修饰后化学合成物,也是S1P1激动剂,具有免疫抑制作用,有研究结果表明,小剂量的S1P1激动剂对于小鼠急性肺损伤有保护作用。因此我们根据其各自功能及特点,推测h UC-MSCs与FTY720之间在治疗ALI/ARDS中有协同性。本实验研究的目的是探讨h UC-MSCs与FTY720治疗内毒素所致急性肺损伤的协同作用,以期增加治疗效果,并减少各自的治疗缺憾,同时对其治疗机理进行初步研究。方法:将8-10周龄雌性C57BL/6小鼠随机分为5组,分别是正常组、LPS组、h UC-MSCs组、FTY720组、h UC-MSCs与FTY720联合给药组。正常组给予气管内滴注NS,其余各组滴注10 mg/kg内毒素制备小鼠急性肺损伤模型,模型建立后24 h,分别给h UC-MSCs组每只小鼠尾静脉注射2×105人脐带间充质干细胞,给FTY720组每只小鼠腹腔内注射剂量为0.1 mg/kg的FTY720,联合给药组给予尾静脉注射2×105人脐带间充质干细胞和腹腔内注射剂量为0.1 mg/kg的FTY720。分别于造模后48 h及7 d处死小鼠,观察各组肺组织病理变化(HE染色)和病理损伤评分、肺组织湿干重比值、肺Micro-CT(活体)、BALF总蛋白和炎症因子(IL-12p70、IL-10、IL-6、MCP-1、IFN-γ、TNF-α)含量在给药后48小时和7天分别测得,48小时后分析各组小鼠生存率。提取肺总蛋白,进行双向电泳测定,对获取的蛋白图谱进行分析,寻找差异表达的蛋白质,以观察用内毒素损伤后肺部蛋白质表达的差异。结果:1.小鼠肺组织损伤评分和肺CT结果表明我们成功建立了内毒素诱导的小鼠急性肺损伤模型。2.研究发现h UC-MSCs组、FTY720组、h UC-MSCs与FTY720联合给药组的小鼠给药后生存率都有所提高、肺病理损伤评分降低、湿干重比值降低、灌洗液中总蛋白含量减少以及灌洗液中炎症因子IL-6、TNF-α、MCP-1减少。联合给药组与单独使用h UC-MSCs或FTY720组相比,联合给药的治疗效果最好,但其中生存率和肺湿干重比值的降低并没有统计学意义。3.蛋白质双向电泳结果表明急性肺损伤小鼠的肺内蛋白量是超过正常小鼠的。蛋白质点数为(100±2),其中有21个表达的蛋白差异点,通过蛋白质谱鉴定和生物信息检索结果显示蛋白质过氧化物酶-6在损伤肺和正常肺中有明显差异。结论:气管内滴注内毒素建立急性肺损伤小鼠模型较稳定;使用h UC-MSCs或FTY720的单独给药组可以减少肺内炎症,h UC-MSCs和FTY720联合给药组与单独给药相比也可以通过彼此之间的相互作用有效缓解急性肺损伤;蛋白质双向电泳结果表明急性肺损伤小鼠肺组织中蛋白质过氧化物酶-6表达的增加,机制还待进一步研究。h UC-MSCs和FTY720联合给药与分别单独给药相比也许是更优的治疗策略,其机制还有待于进一步探究。
[Abstract]:Objective: acute lung injury and acute respiratory distress syndrome (acute lung injury/acute respiratory distress syndrome, ALI/ARDS) is a major cause of morbidity and mortality in patients with critical illness, due to lack of effective treatment, so it is urgent to find new treatments. Recent studies have found that human umbilical cord mesenchymal stem cells (Human Umbilical Cord-Mesenchymal Stem Cells, H UC-MSCs) can effectively alleviate the acute lung injury and acute respiratory distress syndrome, and 1- 1-phosphate (sphingosine-1-phosphate, S1P) plays an important role in enhancing vascular endothelial barrier function, inflammation resistance lung, reduce lung injury in.FTY720 for its analogues, is modified by chemical synthesis, is S1P1 agonists have immunosuppressive effects, the results show that small doses of the S1P1 agonist for the mouse acute lung injury protection So we use. According to their respective functions and features, that between H and FTY720 treatment in UC-MSCs ALI/ARDS in collaboration. The purpose of this study is to investigate the synergistic effect of H UC-MSCs and FTY720 in the treatment of acute lung injury induced by endotoxin, in order to increase the treatment effect, and reduce the treatment of their shortcomings, and make a preliminary study on the mechanism of treatment. Methods: 8-10 week old female C57BL/6 mice were randomly divided into 5 groups, namely the normal group, LPS group, H UC-MSCs group, FTY720 group, H UC-MSCs and FTY720 combined group. Normal group received intratracheal instillation of NS, other groups were infusion of 10 mg/kg endotoxin preparation model of acute lung injury of mice model after 24 h, respectively, to the H UC-MSCs group each mouse tail vein injection of 2 x 105 human umbilical cord mesenchymal stem cells, FTY720 group injected dose per mice intraperitoneally for 0.1 mg/kg FTY720, combined treatment groups were given tail vein 2 x 105 intravenous injection of human umbilical cord mesenchymal stem cells and intraperitoneal injection of 0.1 mg/kg FTY720. respectively at 48 h and 7 d mice were sacrificed to observe the pathological changes of lung tissue in each group (HE staining) and the pathological damage score, lung wet dry weight ratio, lung Micro-CT (in vivo), BALF total protein and inflammatory factors (IL-12p70, IL-10, IL-6, MCP-1, IFN- y, TNF- a) content in 48 hours after administration and 7 days were measured, analyzed the survival rate of mice after 48 hours. The total lung protein extraction, two-dimensional electrophoresis determination of protein profiles obtained were analyzed for different expression the protein, in order to observe the differences in protein expression of lung injury after endotoxin. Results: 1. mice lung injury score and lung CT results showed that we successfully established endotoxin induced acute lung injury in mice model of.2. h was found in UC-MSCs group, FTY720 group, H UC-MSCs and FTY720 Combined drug group mice survival rate increased, lung pathology score decreased, wet to dry weight ratio decreased, the total protein content in the lavage fluid and reduce inflammatory factors in the lavage fluid IL-6, TNF- alpha, MCP-1 reduced. The combined dose group and H alone or combined with UC-MSCs FTY720 group compared to drug treatment effect is best, but the lower the survival rate and the lung wet / dry weight ratio was not statistically significant.3. protein two-dimensional electrophoresis results showed that the lung protein in mice with acute lung injury is more than normal mice. The protein points (100 + 2), 21 proteins differentially expressed in the point, through the search the results of protein mass spectrometry and bioinformatics indicated that the protein peroxidase -6 in normal lung and lung injury is significant difference. Conclusion: intratracheal instillation of LPS induced acute lung injury in mice model is stable; the use of H UC-MSCs or FTY720 Single dose group can reduce the inflammation in the lungs, H UC-MSCs and FTY720 combination group and monotherapy compared can effectively alleviate the acute lung injury induced by the interaction between each other; two-dimensional electrophoresis results showed that the increase in protein peroxidase in mouse lung tissue -6 expression in acute lung injury, the mechanism also needs further study of UC-MSCs and.H FTY720 combined with medication and were administered alone may be better than treatment strategies, the mechanism remains to be further explored.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8

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