脑源性微粒的提

发布时间：2018-04-23 19:40

本文选题：脑源性微粒 + 消化法　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:微粒(microparticle)是一种细胞激活或凋亡时所分泌的直径在0.1-1um之间的细胞囊泡。近年来,研究发现微粒可以介导某些疾病的发生,也可以作为感染、外伤的生物学标记物,并且还具有潜在的治疗价值,更为有意义的是,已有报道证实微粒作为物质运输载体,发挥着细胞间m RNAs、micro RNAs、脂类与蛋白质的传递作用。由此得到了基础与临床医学研究越来越多的关注。本实验通过对BDMP的两种提取方法进行比较,探索流式细胞仪对微粒浓度的检测,并且探索冻存条件对BDMP的影响以及收集临床TBI患者血浆标本对其脑源性微粒(BDMP)含量进行检测,试图:(1)比较BDMP不同提取方法的特点,为BDMP分离、纯化工作奠定基础;(2)建立起一套关于微粒含量行之有效的检测鉴定方法,为以后大规模开展BDMP的研究提供保障;(3)明确不同温度环境下BDMP的变化规律,为BDMP的保存提供参考;(4)在目前已有研究证实鼠源性BDMP存在的基础上,证实颅脑创伤(TBI)后患者血中也有BDMP的存在,为TBI后临床患者凝血功能紊乱的发生机制提供新思路。方法:(1)通过应用流式细胞技术/RNA定量提取技术分别对酶解消化法与冻融研磨法所产生的BDMP进行检测,观察两种不同方法所产生的BDMP结构的不同;(2)应用流式细胞技术分别对荧光标准品进行检测并计算检测结果的日内差、日间差,建立TBI小鼠模型,应用流式细胞技术检测脑源性微粒的含量;(3)将提取的BDMP分别放入4℃、-20℃、-80℃以及-196℃环境中保存一段时间,在不同时间点取样应用流式细胞技术检测;(4)收集TBI临床患者血样标本,分别应用流式细胞技术、Western blot与透射电镜技术对样本中BDMP进行检测。结果:1)通过对比BDMP的两种提取方法—研磨法和消化法发现:研磨法提取的BDMP的浓度大于消化法,研磨法提取的GFAP单阳性BDMP含量大于消化法,研磨法提取的AnnexinⅤ单阳性BDMP含量小于消化法;两种方法提取AnnexinⅤ与GFAP双阳性的BDMP两种方法提取AnnexinⅤ与GFAP双阳性的BDMP没有差异。利用微电泳系统,通过检测荧光度,对样本核酸进行精确分析。结果显示,BDMP中含有遗传物质RNA,并且研磨法提取的BDMP中所含RNA的浓度与含量大于消化法;2)流式细胞测定方法学研究结果显示,其日内差和日间差分别为5.8%和4.8%。应用流式细胞测定方法进一步检测小鼠脑组织BDMP含量,发现脑创伤小鼠脑组织中BDMP的含量要明显高于正常小鼠脑组织,具有显著性差异(P0.05);3)应用流式细胞技术在不同温度下不同时间段测BDMP的含量,结果显示:3日/3周内在不同温度下BDMP性质均会发生改变,并且不同温度对于BDMP的影响未见差异;4)根据流式细胞技术、western blot以及电镜实验发现,脑创伤病人外周血中存在BDMP,且脑创伤病人外周血中的GFAP和/或AnnexinV阳性BDMP含量多于健康人的。其BDMP具有独特的结构与功能,直径大小在0.1~1μm之间,其磷脂双分子膜上富含磷脂酰丝氨酸和神经细胞特异性标记物。结论:1)应用消化法能够有效的提取BDMP,通过对比BDMP的两种提取方法—研磨法和消化法发现,两种方法产生的微粒的浓度、种类并不一致;且证明BDMP中含有遗传物质RNA,研磨法提取的BDMP中所含RNA的浓度与含量大于消化法;2)流式细胞技术检测BDMP的含量具有可靠性好、重复性高及操作简便等优点,可用于BDMP的常规检测;遭受颅脑创伤小鼠脑组织细胞释放了更多的BDMP;3)BDMP实验应该取新鲜BDMP标本,冷冻保存后的BDMP的性质会发生改变,从而造成实验结果不稳定;4)脑创伤病人与健康人外周血中存在具有独特的结构与功能的BDMP,且脑创伤病人外周血中的GFAP和/或AnnexinV阳性BDMP含量多于健康人的。因此,有理由推测机体在遭受颅脑创伤后,血脑屏障受到了破坏,神经元与神经胶质细胞激活产生BDMP,会通过受损的血脑屏障释放到外周血中。目前,脑创伤病人外周血中BDMP的含量与患者的体质、性别、年龄、病情严重程度以及病程的关系还有待于进一步的研究。
[Abstract]:Objective: microparticle is a cell vesicle that is secreted by a cell between 0.1-1um and cell activation or apoptosis. In recent years, it has been found that particles can mediate the occurrence of certain diseases and can also be used as biological markers for infection and trauma, and also have potential therapeutic value. As a carrier of material transport, solid particles play the role of intercellular m RNAs, micro RNAs, lipid and protein transfer. Thus, more and more attention has been paid to basic and clinical medical research. In this experiment, two methods of extraction of BDMP were compared to explore the detection of particle concentration by flow cytometry, and to explore the condition of cryopreservation to BD The effects of MP and the collection of plasma samples of TBI patients on the content of brain derived particles (BDMP) were collected, trying to: (1) compare the characteristics of different BDMP extraction methods and lay the foundation for BDMP separation and purification; (2) establish a set of effective detection and identification methods on the content of particles to provide insurance for large-scale development of BDMP in the future. (3) to clarify the changing rules of BDMP under different temperature conditions to provide reference for the preservation of BDMP; (4) on the basis of existing research confirmed the existence of mouse derived BDMP, the presence of BDMP in the blood of patients with traumatic brain injury (TBI) has been confirmed, which provides a new idea for the mechanism of coagulation dysfunction after TBI. Method: (1) through the application flow /RNA quantitative extraction technology was used to detect the BDMP produced by enzymatic digestion and freezing thawing grinding, and the differences of BDMP structure produced by two different methods were observed. (2) the daily difference of fluorescent standard products was detected by flow cytometry and the difference between day and day was calculated. The difference between day and day was used to establish a model of TBI mice. Flow cytometry was used to detect the content of brain derived particles; (3) the extracted BDMP was stored at 4, -20, -80, and -196 for a period of time, and the flow cytometry was sampled at different time points. (4) the samples of blood samples from TBI clinical patients were collected, the flow cytometry, Western blot and transmission electron microscopy were used. In the sample, BDMP was detected. Results: 1) by comparing two kinds of extraction methods of BDMP: grinding method and digestion method, the concentration of BDMP extracted by grinding method was greater than digestion method, and the single positive BDMP content of GFAP extracted by lapping method was larger than digestion method, and the single positive BDMP content of Annexin V extracted by grinding method was less than digestion method; two methods extracted Annexin v. GFAP double positive BDMP two methods have no difference between Annexin V and GFAP double positive BDMP. Using the microelectrophoresis system, the sample nucleic acid is accurately analyzed by detecting the fluorescence degree. The results show that the BDMP contains genetic material RNA, and the concentration and content of RNA in BDMP extracted by the grinding method is greater than the digestion method; 2) flow cytometry The experimental results showed that the difference between day difference and daytime difference was 5.8% and 4.8%. applied flow cytometry to further detect the BDMP content in the brain tissue of mice. It was found that the content of BDMP in brain tissue of brain trauma mice was significantly higher than that of normal mice (P0.05); 3) the application of flow cytometry at different temperatures. The content of BDMP in different time periods was measured. The results showed that the BDMP properties at different temperatures were changed at 3 days /3 weeks, and there was no difference in the influence of temperature on BDMP. 4) according to flow cytometry, Western blot and electron microscopy, there was BDMP in peripheral blood of patients with brain trauma, and GFAP in peripheral blood of patients with brain trauma. And / or AnnexinV positive BDMP content more than healthy people. Its BDMP has unique structure and function, the diameter is between 0.1~1 and m, and its phosphatidic bimolecular membrane is rich in phosphatidyl serine and nerve cell specific markers. Conclusion: 1) the application of digestion method can effectively extract BDMP, by comparing the two extraction methods of BDMP - grinding method And the digestion method found that the concentration of the particles produced by the two methods is not consistent, and it is proved that the BDMP contains the genetic material RNA, the concentration and content of RNA in BDMP extracted by the grinding method is greater than the digestion method; 2) the content of BDMP by flow cytometry has the advantages of good reliability, high repeatability and easy operation, which can be used in the routine of BDMP. The brain tissue cells of the traumatic brain injury mice released more BDMP; 3) the BDMP experiment should take the fresh BDMP specimens, the properties of the BDMP after cryopreservation would change, and the result of the experimental results were unstable; 4) there was a unique structure and function of BDMP in the peripheral blood of the brain trauma patients and the healthy people, and the peripheral blood of the patients with brain trauma. The content of GFAP and / or AnnexinV positive BDMP in blood is more than that of healthy people. Therefore, it is reasonable to speculate that the blood brain barrier is damaged after the traumatic brain injury, the neurons and glial cells are activated to produce BDMP, which will be released to the peripheral blood through the damaged blood brain barrier. The content of BDMP in the peripheral blood of the patients with brain trauma and the patient are in the patients before the eyes. The relationship between physical fitness, sex, age, severity and duration of disease remains to be further studied.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15

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