本文選題：腦源性微粒 + 消化法��； 參考：《天津醫(yī)科大學》2017年碩士論文

【摘要】：目的:微粒(microparticle)是一種細胞激活或凋亡時所分泌的直徑在0.1-1um之間的細胞囊泡。近年來,研究發(fā)現(xiàn)微�？梢越閷承┘膊〉陌l(fā)生,也可以作為感染、外傷的生物學標記物,并且還具有潛在的治療價值,更為有意義的是,已有報道證實微粒作為物質運輸載體,發(fā)揮著細胞間m RNAs、micro RNAs、脂類與蛋白質的傳遞作用。由此得到了基礎與臨床醫(yī)學研究越來越多的關注。本實驗通過對BDMP的兩種提取方法進行比較,探索流式細胞儀對微粒濃度的檢測,并且探索凍存條件對BDMP的影響以及收集臨床TBI患者血漿標本對其腦源性微粒(BDMP)含量進行檢測,試圖:(1)比較BDMP不同提取方法的特點,為BDMP分離、純化工作奠定基礎;(2)建立起一套關于微粒含量行之有效的檢測鑒定方法,為以后大規(guī)模開展BDMP的研究提供保障;(3)明確不同溫度環(huán)境下BDMP的變化規(guī)律,為BDMP的保存提供參考;(4)在目前已有研究證實鼠源性BDMP存在的基礎上,證實顱腦創(chuàng)傷(TBI)后患者血中也有BDMP的存在,為TBI后臨床患者凝血功能紊亂的發(fā)生機制提供新思路。方法:(1)通過應用流式細胞技術/RNA定量提取技術分別對酶解消化法與凍融研磨法所產生的BDMP進行檢測,觀察兩種不同方法所產生的BDMP結構的不同;(2)應用流式細胞技術分別對熒光標準品進行檢測并計算檢測結果的日內差、日間差,建立TBI小鼠模型,應用流式細胞技術檢測腦源性微粒的含量;(3)將提取的BDMP分別放入4℃、-20℃、-80℃以及-196℃環(huán)境中保存一段時間,在不同時間點取樣應用流式細胞技術檢測;(4)收集TBI臨床患者血樣標本,分別應用流式細胞技術、Western blot與透射電鏡技術對樣本中BDMP進行檢測。結果:1)通過對比BDMP的兩種提取方法—研磨法和消化法發(fā)現(xiàn):研磨法提取的BDMP的濃度大于消化法,研磨法提取的GFAP單陽性BDMP含量大于消化法,研磨法提取的AnnexinⅤ單陽性BDMP含量小于消化法;兩種方法提取AnnexinⅤ與GFAP雙陽性的BDMP兩種方法提取AnnexinⅤ與GFAP雙陽性的BDMP沒有差異。利用微電泳系統(tǒng),通過檢測熒光度,對樣本核酸進行精確分析。結果顯示,BDMP中含有遺傳物質RNA,并且研磨法提取的BDMP中所含RNA的濃度與含量大于消化法;2)流式細胞測定方法學研究結果顯示,其日內差和日間差分別為5.8%和4.8%。應用流式細胞測定方法進一步檢測小鼠腦組織BDMP含量,發(fā)現(xiàn)腦創(chuàng)傷小鼠腦組織中BDMP的含量要明顯高于正常小鼠腦組織,具有顯著性差異(P0.05);3)應用流式細胞技術在不同溫度下不同時間段測BDMP的含量,結果顯示:3日/3周內在不同溫度下BDMP性質均會發(fā)生改變,并且不同溫度對于BDMP的影響未見差異;4)根據(jù)流式細胞技術、western blot以及電鏡實驗發(fā)現(xiàn),腦創(chuàng)傷病人外周血中存在BDMP,且腦創(chuàng)傷病人外周血中的GFAP和/或AnnexinV陽性BDMP含量多于健康人的。其BDMP具有獨特的結構與功能,直徑大小在0.1~1μm之間,其磷脂雙分子膜上富含磷脂酰絲氨酸和神經細胞特異性標記物。結論:1)應用消化法能夠有效的提取BDMP,通過對比BDMP的兩種提取方法—研磨法和消化法發(fā)現(xiàn),兩種方法產生的微粒的濃度、種類并不一致;且證明BDMP中含有遺傳物質RNA,研磨法提取的BDMP中所含RNA的濃度與含量大于消化法;2)流式細胞技術檢測BDMP的含量具有可靠性好、重復性高及操作簡便等優(yōu)點,可用于BDMP的常規(guī)檢測;遭受顱腦創(chuàng)傷小鼠腦組織細胞釋放了更多的BDMP;3)BDMP實驗應該取新鮮BDMP標本,冷凍保存后的BDMP的性質會發(fā)生改變,從而造成實驗結果不穩(wěn)定;4)腦創(chuàng)傷病人與健康人外周血中存在具有獨特的結構與功能的BDMP,且腦創(chuàng)傷病人外周血中的GFAP和/或AnnexinV陽性BDMP含量多于健康人的。因此,有理由推測機體在遭受顱腦創(chuàng)傷后,血腦屏障受到了破壞,神經元與神經膠質細胞激活產生BDMP,會通過受損的血腦屏障釋放到外周血中。目前,腦創(chuàng)傷病人外周血中BDMP的含量與患者的體質、性別、年齡、病情嚴重程度以及病程的關系還有待于進一步的研究。
[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.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R651.15

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