腺病毒重组血管生成素1对光气引起的大鼠急性肺损伤的保护作用

发布时间：2018-04-17 07:52

本文选题：血管生成素1 + 基因治疗　；参考：《复旦大学》2013年硕士论文

【摘要】：研究的背景及目的：光气是一种重要的化学物质被广泛的运用于现代工业。作为一种高毒性的化学制品,可以导致急性肺损伤,肺水肿,甚至于死亡。目前基因治疗已经成为一种新型的治疗急性肺损伤的方法。血管生成素1(Ang1)是拯救内皮及维持内皮稳定性方面的决定性因子,其可能的原理是减少内皮的渗出和削弱内皮的炎症。本课题中,我们观察光气吸入引起的急性肺损伤的病理改变,假设Ang1基因治疗能逐渐降低炎症因子导致的肺血管的渗出。方法： 1.选取SPF级雄性SD大鼠(180-220g)60只随机分为6组,光气组暴露于浓度为8.33g/m3光气5min造成急性肺损伤模型,空气组则放入相同体积的容器内,吸入5min空气；设空气对照组三组,分别为：空气组,空气+Ad组,空气+Ad/Ang1组；,光气组三组,分别为：光气组,光气+Ad组,光气+Ad/Ang1组。腺病毒在吸入相应气体后1小时,由尾静脉注射干预。 2.气体(空气,光气)暴露5min,36小时后,取右肺下叶行湿／干比检测,取部分右肺上叶石蜡固定,HE染色行病理学检查,部分右肺上叶OCT包埋,新鲜冰冻切片观察免疫荧光,取右肺中叶行Western blot检测肺组中Ang1蛋白含量,RT-PCR法检测肺组中Ang1,IL-1β,IL-17和VEGF mRNA含量。 3.左肺行肺泡灌洗,取部分肺泡灌洗液行粒细胞计数。ELISA法检测肺水中Ang1, IL-1β, IL-17和VEGF的浓度。 4.腹主动脉采血,离心后取血清,ELISA法检测血清中Ang1,IL-1β,IL-17和VEGF的浓度。结果： 1.光气暴露组肺组织内及血清中的Ang1是呈下降趋势的。 2.通过腺病毒尾静脉注射法可以成功转染动物,且病毒本身对动物无致病性。 3.腺病毒所携带的目的基因可以在动物体内得到表达,肺组织中Ang1蛋白水平, mRNA水平以及血清中Angl水平均高于空白腺病毒对照组。 4.外源性的Ang1可以一定程度上缓解光气引起的急性肺损伤,如减少了肺泡灌洗液中的粒细胞数,肺湿／干比等。 5.Angl可以下调某些炎症因子,如IL-1β,IL-17。结论： 1.腺病毒携带Angl目的基因转染动物模型制造成功,且腺病毒本身对动物无明显影响。 2.Angl肺内高表达,发挥其抗炎和稳定血管减轻渗漏的作用,腺病毒介导的Angl基因治疗不失为一种可行性的干预光气引起的急性肺损伤的方法。
[Abstract]:Background and objectives of the study:Phosgene is an important chemical substance which is widely used in modern industry.As a highly toxic chemical, it can lead to acute lung injury, pulmonary edema, and even death.Gene therapy has become a new therapy for acute lung injury.Angiopoietin 1Ang1) is a decisive factor in saving endothelium and maintaining endothelial stability. Its possible mechanism is to reduce endothelial exudation and weaken endothelial inflammation.In this study, we observed the pathological changes of acute lung injury induced by phosgene inhalation, assuming that Ang1 gene therapy could gradually reduce pulmonary vascular exudation induced by inflammatory factors.Methods:1.60 male SD rats of SPF grade were randomly divided into 6 groups. The phosgene group was exposed to 8.33g/m3 phosgene 5min to induce acute lung injury model, the air group was put into the same volume container and inhaled the 5min air, and the air control group was divided into three groups.They were air group, air Ad group, air Ad/Ang1 group and phosgene group. They were phosgene group, phosgene Ad group and phosgene Ad/Ang1 group.Adenovirus was injected into the tail vein for 1 hour after inhalation of the corresponding gas.2.After exposure to air (air, phosgene) for 36 hours, the lower lobe of the right lung was taken for wet / dry ratio detection, the paraffin fixed part of the upper lobe of the right lung was stained with HE for pathological examination, the OCT was embedded in the upper lobe of the right lung, and the immunofluorescence was observed by fresh frozen sections.The content of Ang1 protein in lung group was detected by Western blot. The contents of IL-17 and VEGF mRNA in lung group were detected by reverse transcription-polymerase chain reaction (RT-PCR).3.The concentration of Ang1, IL-1 尾, IL-17 and VEGF in lung water was detected by Elisa and granulocyte count in partial alveolar lavage fluid.4.Blood samples were collected from abdominal aorta and serum levels of IL-17 and VEGF were determined by Elisa after centrifugation.Results:1.In phosgene exposure group, Ang1 in lung tissue and serum showed a decreasing trend.2.Adenovirus tail vein injection can successfully transfect animals, and the virus itself has no pathogenicity to animals.3.The target gene carried by adenovirus could be expressed in animals. The levels of Ang1 protein, mRNA and serum Angl in lung tissue were higher than those in control group.4.Exogenous Ang1 can alleviate acute lung injury induced by phosgene to some extent, such as reducing the number of granulocytes in alveolar lavage fluid and lung wet / dry ratio.5.Angl can down-regulate some inflammatory factors, such as IL-1 尾 -IL-17.Conclusion:1.Adenovirus carrying Angl gene was successfully transfected into animal model, and adenovirus itself had no obvious effect on animal.Angl gene therapy mediated by adenovirus is a feasible method to interfere with phosgene induced acute lung injury.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.8

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