本文選題：急性重復(fù)缺氧 + 模型��； 參考：《第三軍醫(yī)大學(xué)》2008年博士論文

【摘要】： 缺氧時,機體會出現(xiàn)各種代償反應(yīng)以對抗缺氧的影響。機體這種與缺氧做斗爭的能力,即缺氧的耐受力顯然是有一定限度的。近年來的研究表明,機體的這種對缺氧的耐受性可以通過缺氧預(yù)適應(yīng)等措施得到加強。缺氧預(yù)適應(yīng)是指機體經(jīng)短暫時間或亞致死量的缺氧后,對后續(xù)的更長時間或更嚴(yán)重缺氧性損傷產(chǎn)生明顯耐受的能力。其機制除了與缺氧引發(fā)的由缺氧誘導(dǎo)因子-1等轉(zhuǎn)錄因子介導(dǎo)的各種適應(yīng)代償性反應(yīng)蛋白表達改變有關(guān)外,組織、細(xì)胞的能量代謝產(chǎn)物,特別是ATP的分解代謝產(chǎn)物等在缺氧預(yù)適應(yīng)中也起重要作用。 腦是對缺氧最敏感的器官,是影響機體缺氧耐受能力的主要限制因素。因此中樞神經(jīng)系統(tǒng)的機能或代謝變化可能是機體缺氧耐受形成的主要機制。研究表明,缺氧時腦組織的物質(zhì)變化不僅對于機體缺氧耐受能力的建立具有重要作用,而且缺氧預(yù)適應(yīng)小鼠的腦勻漿液也能提高動物甚至離體細(xì)胞對缺氧的耐受能力。積極尋找并研究急性重復(fù)缺氧預(yù)適應(yīng)小鼠腦勻漿液中的可提高機體缺氧耐受能力的生物活性物質(zhì)具有重要的理論和實際意義。本研究通過觀察不同實驗條件對急性重復(fù)缺氧預(yù)適應(yīng)小鼠模型的影響,建立穩(wěn)定可靠、效果顯著的小鼠缺氧預(yù)適應(yīng)模型。由于動物對缺氧的耐受性個體差異較大,影響因素較多,因此為便于控制實驗條件和探索增強缺氧耐受性的物質(zhì)特性,本研究采用離體細(xì)胞觀察急性重復(fù)缺氧預(yù)適應(yīng)小鼠腦勻漿液對離體細(xì)胞缺氧耐受性的影響。在確定急性重復(fù)缺氧預(yù)適應(yīng)小鼠腦勻漿液對不同離體培養(yǎng)細(xì)胞(未用NGF誘導(dǎo)的PC12細(xì)胞、HepG2細(xì)胞)缺氧耐受性具有雙相效應(yīng)的基礎(chǔ)上,以NGF誘導(dǎo)分化的PC12細(xì)胞作為觀察對象,探索急性重復(fù)缺氧預(yù)適應(yīng)小鼠腦勻漿液對分化PC12細(xì)胞只起保護作用、無損傷作用的最適濃度,并進一步將該提取液分成蛋白部分和非蛋白部分,探討其中存在的增強細(xì)胞缺氧耐受性的可能組分。實驗分三部分進行。 一、方法 第一部分急性重復(fù)缺氧預(yù)適應(yīng)小鼠模型的復(fù)制 (1).使小鼠一次性缺氧致死,以觀察小鼠缺氧致死前的變化規(guī)律。 (2).將小鼠連續(xù)4次重復(fù)缺氧,以觀察小鼠每次缺氧耐受時間的變化。 (3).將小鼠分成2組,均經(jīng)過連續(xù)4次缺氧,2組小鼠每次結(jié)束缺氧的標(biāo)準(zhǔn)分別是呼吸頻率約40次/min或出現(xiàn)第1次喘呼吸,以觀察每次不同時間缺氧對小鼠缺氧耐受性的影響。 (4).將小鼠分成2組,均經(jīng)過連續(xù)4次缺氧,2組小鼠每次結(jié)束缺氧的標(biāo)準(zhǔn)分別是第一次喘呼吸或末次喘呼吸,以觀察每次不同時間缺氧對小鼠缺氧耐受性的影響。 (5).將小鼠分成4組,均經(jīng)過連續(xù)4次缺氧,但4組小鼠連續(xù)4次缺氧的環(huán)境溫度分別是:9-11℃、13-15℃、17-19℃、21-23℃,以觀察不同環(huán)境溫度對小鼠缺氧耐受性的影響。 (6).將小鼠分成2組,均經(jīng)連續(xù)4次缺氧,2次缺氧間的換瓶操作時間分別是5-15s或20-30s,以觀察不同復(fù)氧時間對小鼠缺氧耐受性的影響。 (7).將經(jīng)過連續(xù)4次缺氧的小鼠恢復(fù)常氧, 30分鐘后再放回缺氧瓶缺氧1次,以觀察小鼠復(fù)氧30分鐘后缺氧耐受性的變化。 第二部分缺氧預(yù)適應(yīng)小鼠腦勻漿液對未用NGF誘導(dǎo)分化的PC12細(xì)胞、HepG2細(xì)胞以及NGF誘導(dǎo)分化的PC12細(xì)胞缺氧耐受性的影響 1.以不同濃度的小鼠腦勻漿液分別作用于未用NGF誘導(dǎo)的PC12細(xì)胞,觀察缺氧24h、48h、72h時PC12細(xì)胞的活力、LDH透出率、早期凋亡率、晚期凋亡率的變化規(guī)律,以確定小鼠腦勻漿液對細(xì)胞缺氧耐受性的影響。 2.以不同濃度的小鼠腦勻漿液分別作用于HepG2細(xì)胞,觀察缺氧24h、48h、72h缺氧時HepG2細(xì)胞的活力、早期凋亡率、晚期凋亡率的變化規(guī)律,以了解小鼠腦勻漿液對非神經(jīng)元模型細(xì)胞的影響。 3.根據(jù)上述第二部分實驗的結(jié)果,僅以較低濃度范圍小鼠腦勻漿液分別作用于NGF誘導(dǎo)分化的PC12細(xì)胞,觀察缺氧24h、48h、72h時分化PC12細(xì)胞的活力、LDH透出率,晚期凋亡率的變化規(guī)律,以探索缺氧預(yù)適應(yīng)小鼠腦勻漿液對缺氧、NGF誘導(dǎo)分化的PC12細(xì)胞只起保護作用、沒有損傷作用的最適濃度。 第三部分:缺氧預(yù)適應(yīng)小鼠腦勻漿液不同組分對NGF誘導(dǎo)分化的PC12細(xì)胞缺氧耐受性的影響及其可能有效成分的初步探討 (1).以高氯酸除蛋白法制備缺氧預(yù)適應(yīng)小鼠腦勻漿去蛋白液,HPLC法測定其腺苷含量。用小鼠腦勻漿去蛋白液作用于NGF誘導(dǎo)分化PC12細(xì)胞,以確定去蛋白液對缺氧的分化PC12細(xì)胞有無保護作用;以小鼠腦勻漿去蛋白液合并腺苷A1或A2A受體阻斷劑作用于分化PC12細(xì)胞,以確定小鼠腦勻漿去蛋白液的作用是否與腺苷有關(guān)。 (2).透析法除去缺氧預(yù)適應(yīng)小鼠腦勻漿液中小分子物質(zhì),并觀察透析保留液對缺氧的分化PC12細(xì)胞有無保護作用;分別用RT-PCR、Western blot方法檢測缺氧預(yù)適應(yīng)小鼠腦組織中VEGF mRNA、VEGF的蛋白水平是否增加;用重組VEGF純品作用于分化PC12細(xì)胞,以確定透析保留液對分化PC12細(xì)胞的保護作用是否與VEGF有關(guān)。 二、結(jié)果與討論 通過上述3部分的工作獲得以下結(jié)果 (一)影響急性重復(fù)缺氧模型的因素 1.小鼠經(jīng)4次急性重復(fù)缺氧后,每次缺氧耐受時間顯著增加。這一結(jié)果提示,小鼠在每次缺氧后,均在體內(nèi)產(chǎn)生了增強缺氧耐受性的物質(zhì),并且各次缺氧后缺氧耐受的物質(zhì)有積累效應(yīng)。 2.在小鼠出現(xiàn)第一次喘呼吸時將小鼠換入下一個缺氧瓶與小鼠呼吸頻率在40次/min時換瓶比較,可顯著提高小鼠每次耐受時間。這是因為小鼠在呼吸頻率為40次/min時的缺氧時間比出現(xiàn)喘呼吸時的缺氧時間短,缺氧程度尚未達到缺氧耐受形成的程度,因此對缺氧的耐受性也不強,因為缺氧預(yù)適應(yīng)的形成需要足夠程度的缺氧。 3.在小鼠出現(xiàn)第一次喘呼吸時將小鼠換入下一個缺氧瓶與小鼠出現(xiàn)末次喘呼吸時換瓶比較,缺氧耐受時間無顯著差異。其原因是,不同小鼠喘呼吸次數(shù)不同,有的小鼠喘呼吸3-5次即死亡,有的小鼠喘呼吸卻可達30次以上,因此小鼠的個體差異較大,使組間無顯著差異;并且由于末次喘呼吸后小鼠隨即死亡,因此末次喘呼吸難以判斷,以其作為結(jié)束缺氧標(biāo)準(zhǔn)導(dǎo)致小鼠死亡率增加。 4.隨著環(huán)境溫度的增加,小鼠的缺氧耐受時間顯著降低。這可能是因為低溫可減少小鼠體內(nèi)有害物質(zhì)的產(chǎn)生、降低代謝,有利于缺氧預(yù)適應(yīng)的形成。 5.每次對小鼠換瓶操作時間長于15秒會顯著降低小鼠的缺氧耐受時間。其原因可能是復(fù)氧時間太長,因為復(fù)氧時間短暫有利于預(yù)適應(yīng)的形成。由于經(jīng)過重復(fù)4次缺氧的小鼠恢復(fù)常氧30分鐘后,其缺氧耐受性還可以部分保留,說明小鼠4次缺氧耐受性的完全消失需要至少30分鐘以上的時間,因此賦予預(yù)適應(yīng)小鼠缺氧耐受性的物質(zhì),可能部分對機體恢復(fù)常氧供應(yīng)較為敏感,但部分能維持較長時間。 (二)缺氧預(yù)適應(yīng)小鼠腦勻漿液對未用NGF誘導(dǎo)的PC12細(xì)胞、HepG2細(xì)胞、NGF誘導(dǎo)分化的PC12細(xì)胞缺氧耐受性的影響 1.缺氧預(yù)適應(yīng)小鼠腦勻漿液對缺氧、未用NGF誘導(dǎo)的PC12細(xì)胞、HepG2細(xì)胞缺氧耐受性的影響是:缺氧預(yù)適應(yīng)小鼠腦勻漿液可顯著提高細(xì)胞對缺氧的耐受性。在缺氧初期(24h),較高濃度的保護作用顯著強于低濃度的保護作用,這提示缺氧預(yù)適應(yīng)小鼠腦組織中產(chǎn)生了某種或某些可以增強細(xì)胞缺氧耐受性的物質(zhì)或使腦組織中抗缺氧成分表達增加。隨著缺氧時間延長,保護作用下降,高濃度的下降更快。至缺氧72h,高濃度提取液的缺氧損傷作用顯著高于低濃度組,該結(jié)果提示,缺氧預(yù)適應(yīng)小鼠腦組織中可能產(chǎn)生了半衰期較短的抗缺氧物質(zhì)及在高濃度時才顯示作用的、作用時間持久的促缺氧細(xì)胞損傷的物質(zhì),即提示缺氧預(yù)適應(yīng)有著雙相效應(yīng),缺氧預(yù)適應(yīng)小鼠腦勻漿的作用存在量效關(guān)系和時間效應(yīng)。但是由于本研究的目的是為了尋找缺氧預(yù)適應(yīng)小鼠腦勻漿液中增強缺氧耐受性的物質(zhì),因此實驗中應(yīng)避免損傷性物質(zhì)的影響。為此,在后續(xù)缺氧預(yù)適應(yīng)小鼠腦勻漿液對分化PC12細(xì)胞的作用研究中,以蛋白濃度對提取液進行定量,并降低實驗中所用提取液的濃度范圍,以期找到提取液對NGF誘導(dǎo)分化的PC12細(xì)胞只起保護作用,無損傷作用(即只增強NGF誘導(dǎo)分化的PC12細(xì)胞缺氧耐受性,對分化PC12細(xì)胞無損傷作用)的最適濃度,為進一步研究缺氧預(yù)適應(yīng)小鼠腦勻漿液的保護效應(yīng)及保護性物質(zhì)奠定基礎(chǔ)。 2.經(jīng)較低濃度的缺氧預(yù)適應(yīng)小鼠腦勻漿液對NGF誘導(dǎo)分化的PC12細(xì)胞的作用實驗發(fā)現(xiàn),蛋白終濃度為100.0μg/mL的缺氧預(yù)適應(yīng)小鼠腦勻漿液與同濃度正常小鼠腦勻漿液比較,可顯著增強NGF誘導(dǎo)分化的PC12細(xì)胞的缺氧耐受性,并且此濃度的缺氧預(yù)適應(yīng)小鼠腦勻漿液對分化PC12細(xì)胞未顯示出損傷作用,為增強分化PC12細(xì)胞缺氧耐受性的適合濃度。 (三)缺氧預(yù)適應(yīng)小鼠腦勻漿液不同組分對NGF誘導(dǎo)分化的PC12細(xì)胞缺氧耐受性的影響及其可能有效成分的初步探討 1.向離體培養(yǎng)的NGF誘導(dǎo)分化的PC12細(xì)胞中加入不同濃度的腺苷,隨著腺苷濃度增加,細(xì)胞的缺氧耐受性增高,10.0μmol/L的腺苷對細(xì)胞的保護作用可維持24h。急性4次重復(fù)缺氧可使小鼠腦組織腺苷含量上升。缺氧預(yù)適應(yīng)小鼠腦勻漿去蛋白液只在缺氧的24h內(nèi)顯著增加分化PC12細(xì)胞的缺氧耐受性,腺苷A2A受體阻斷劑可阻斷其保護作用。正常小鼠腦勻漿去蛋白液對分化PC12細(xì)胞無保護作用。因此,缺氧預(yù)適應(yīng)小鼠腦勻漿液的非蛋白組分的抗缺氧作用時間較短,腺苷可能是其中一個重要的活性物質(zhì)。 2.在缺氧24h和48h,缺氧預(yù)適應(yīng)小鼠腦勻漿透析保留液與未經(jīng)透析的缺氧預(yù)適應(yīng)小鼠腦勻漿液對分化PC12細(xì)胞均有保護作用,且經(jīng)兩種孔徑(3.5kD、7.0kD)透析袋透析的缺氧預(yù)適應(yīng)小鼠腦勻漿透析保留液和未經(jīng)透析的缺氧預(yù)適應(yīng)小鼠腦勻漿液對缺氧細(xì)胞的保護作用無顯著差異,說明缺氧預(yù)適應(yīng)小鼠腦組織中起保護作用的主要是分子量大于7.0kD的蛋白成分。急性重復(fù)缺氧預(yù)適應(yīng)小鼠腦組織中VEGF表達雖增加,但應(yīng)用重組VEGF直接作用于分化的PC12細(xì)胞發(fā)現(xiàn),不同濃度的VEGF對NGF誘導(dǎo)分化的、不同缺氧時間的PC12細(xì)胞均無保護作用。這一結(jié)果提示:缺氧預(yù)適應(yīng)小鼠腦勻漿液的蛋白組分抗缺氧作用較為持久,其中VEGF表達量雖增高,但并不參與NGF誘導(dǎo)分化的PC12細(xì)胞的抗缺氧作用。由于缺氧預(yù)適應(yīng)小鼠腦勻漿液的蛋白組分抗缺氧作用時間持久,提示蛋白質(zhì)組學(xué)方法可能是研究其中抗缺氧成分的有效方法,因此本研究結(jié)果為今后進一步研究缺氧預(yù)適應(yīng)小鼠腦勻漿液中抗缺氧物質(zhì)提示了方向。 三、結(jié)論 1.賦予重復(fù)缺氧小鼠缺氧耐受性的物質(zhì),部分對機體恢復(fù)常氧供應(yīng)較為敏感,部分則能持續(xù)較長時間; 2.缺氧預(yù)適應(yīng)小鼠腦勻漿液在濃度較高時對離體缺氧培養(yǎng)細(xì)胞的作用表現(xiàn)為雙相性--缺氧早期,腦勻漿液對缺氧細(xì)胞具有保護效應(yīng),延長缺氧時間,其損傷作用逐漸顯現(xiàn),這種雙相作用存在量效關(guān)系和時間效應(yīng);其只起保護作用的濃度是在較低濃度; 3.缺氧預(yù)適應(yīng)小鼠腦勻漿液可顯著提高不同來源離體培養(yǎng)細(xì)胞的缺氧耐受性,提示對缺氧細(xì)胞具有普遍的保護作用; 4.缺氧預(yù)適應(yīng)小鼠腦勻漿液的蛋白組分抗缺氧作用較為持久,其中VEGF表達量雖增高,但并不參與NGF誘導(dǎo)分化的PC12細(xì)胞的抗缺氧作用; 5.缺氧預(yù)適應(yīng)小鼠腦勻漿液的非蛋白組分的抗缺氧作用時間較短,腺苷可能是其中一個重要的活性物質(zhì)。
[Abstract]:In the case of hypoxia, the body has a variety of compensatory responses to the effect of anoxia. The body's ability to combat hypoxia, the tolerance of hypoxia, is obviously limited. In recent years, the body's tolerance to hypoxia can be strengthened through hypoxia preconditioning. Hypoxic preconditioning refers to the body. After a short or sublethal dose of hypoxia, the ability to tolerate further longer or more severe anoxic damage is associated with a variety of adaptable reactive protein expressions mediated by hypoxia inducible factor -1 and other adaptable reactive protein expressions, tissue, and cell energy metabolites, especially A TP catabolic products also play an important role in hypoxic preconditioning.
Brain is the most sensitive organ to hypoxia, which is the main limiting factor of the body's tolerance to hypoxia. Therefore, the changes in the function or metabolism of the central nervous system may be the main mechanism for the formation of the body's hypoxia tolerance. The hypoxic preconditioning of the brain homogenate of mice can also improve the tolerance to hypoxia in animals and even isolated cells. It is of great theoretical and practical significance to actively seek and study bioactive substances in the brain homogenate of acute repeated hypoxia preconditioning mice, which can improve the anoxic tolerance of the body. A stable, reliable and effective model of hypoxic preconditioning in mice was established with a stable and reliable model of hypoxic preconditioning in mice. In order to facilitate the control of the experimental conditions and to explore the material characteristics of enhancing the hypoxia tolerance, this study adopted the view of isolated cells. The effects of acute repeated hypoxia preconditioning on hypoxia tolerance of isolated cells in mice were investigated. On the basis of determining the biphasic effect of acute hypoxia preconditioning mouse brain homogenate on the hypoxia tolerance of different cultured cells (without NGF induced PC12 cells, HepG2 cells), the differentiation of PC12 cells induced by NGF was used as a view. To explore the protective effect of acute hypoxic preconditioning on the differentiated PC12 cells, the optimal concentration of no damage was found, and the extract was further divided into protein and non protein parts, and the possible components of the enhanced cell hypoxia tolerance were discussed. The experiment was divided into three parts.
First, method
Part one replication of a mouse model of acute repeated hypoxia preconditioning
(1) let the mice die in a single dose of hypoxia to observe the change rule of mice before hypoxia.
(2) to observe the change of hypoxia tolerance time in mice by repeated 4 times of anoxia.
(3). The mice were divided into 2 groups, all after 4 consecutive anoxia. The standard of the 2 mice at each end of the hypoxia was about 40 times /min or first breath, in order to observe the effect of hypoxia on the hypoxia tolerance of mice at each time.
(4) the mice were divided into 2 groups, all after 4 consecutive anoxia. The standard of the 2 mice at the end of the hypoxia was the first breath or the last breath, to observe the effect of anoxia on the hypoxia tolerance of mice at each time.
(5) the mice were divided into 4 groups after 4 consecutive anoxia, but the ambient temperature of the 4 groups of mice for 4 consecutive anoxia were 9-11, 13-15, 17-19, 21-23, respectively, to observe the effects of different ambient temperatures on the hypoxia tolerance of mice.
(6). The mice were divided into 2 groups, all after 4 consecutive anoxia, and the operation time of the 2 anoxia was 5-15s or 20-30s, respectively, to observe the effect of different reoxygenation time on the hypoxia tolerance of mice.
(7) restore the normoxic mice after 4 consecutive anoxia, and then return to anoxic bottle for 1 times after 30 minutes to observe the change of hypoxia tolerance after 30 minutes of reoxygenation.
Second part of the hypoxic preconditioned mouse brain homogenate effect on the hypoxia tolerance of PC12 cells, HepG2 cells and NGF induced PC12 cells without NGF induced differentiation
1. the mouse brain homogenate of different concentrations was used to act on PC12 cells which were not induced by NGF, and the activity of PC12 cells, LDH permeability, early apoptosis rate and late apoptosis rate were observed at 24h, 48h and 72h, in order to determine the effect of mouse brain homogenate on cell hypoxia tolerance.
2. HepG2 cells were used in different concentrations of mouse brain homogenate to observe the activity of HepG2 cells, the early apoptosis rate and the late apoptosis rate of the hypoxia 24h, 48h and 72h, in order to understand the effect of the mouse brain homogenate on the non neuron model cells.
3. according to the results of the second parts of the experiment above, only the mouse brain homogenate in the lower concentration range was used to induce the differentiation of PC12 cells in NGF, and the activity of PC12 cells, LDH permeability and late apoptosis rate were observed at 24h, 48h and 72h, in order to explore the hypoxia preconditioning mouse brain homogenate solution to hypoxia and NGF induced differentiation of PC12. The cells only play a protective role, without the optimum concentration of injury.
The third part: the effect of different components of the hypoxic preconditioned mouse brain homogenate on the hypoxia tolerance induced by NGF induced PC12 cells and the preliminary study of its possible effective components
(1) the content of adenosine was determined by HPLC method with hypoxic preconditioning in the brain homogenate of mice with perchloric acid deproteinization. The mouse brain homogenate deprotein was used to induce the differentiation of PC12 cells by NGF to determine the protective effect of deproteinized solution on the anoxic differentiated PC12 cells, and the inhibition of adenosine A1 or A2A receptor blocker in the rat brain homogenate deproteinization solution. Disrupting agent acts on differentiated PC12 cells to determine whether the effect of mouse brain homogenate deproteinization is related to adenosine.
(2). The dialysis method was used to remove the small molecular substances in the mouse brain homogenate, and to observe the protective effect of the dialysis retention solution on the anoxic differentiated PC12 cells. RT-PCR and Western blot were used to detect VEGF mRNA in the brain tissue of the hypoxic preconditioned mice, and the protein level of VEGF increased, and the recombinant VEGF was used to differentiate PC12 fine. To determine whether the protective effect of dialysis fluid on differentiated PC12 cells is related to VEGF.
Two, results and discussion
The following results are obtained through the work of the above 3 parts
(1) factors affecting the model of acute anoxia
In 1. mice, after 4 acute anoxia, the tolerance time of hypoxia increased significantly each time. This result suggested that the mice produced an anoxic tolerance substance in the body after each anoxic, and the anoxic tolerance substance had accumulated effect after each anoxic.
2. during the first breath of mice, the mice were changed into the next hypoxic bottle and the breathing frequency of the mice at the 40 /min, which could significantly improve the mice tolerance time. This is because the hypoxia time of the mice at the respiratory rate of 40 times /min is shorter than that of the asthmatic respiration, and the degree of hypoxia has not reached the anoxic tolerance. The degree of formation, therefore, is also not strong enough to tolerate hypoxia, because the formation of hypoxic preconditioning requires adequate hypoxia.
3. during the first breath of mice, the mice were changed into the next hypoxic bottle and the last breath of the mice. There was no significant difference in the time of hypoxia tolerance. The reason was that the number of asthma in different mice was different, some of the mice were breathing 3-5 times or more than 30 times. There was no significant difference between the groups, and the mice died immediately after the last wheezing breathing, so the last wheezing respiration was difficult to judge, and the death rate of mice was increased as a standard to end the hypoxia.
4. with the increase of ambient temperature, the hypoxia tolerance time of mice decreased significantly. This may be because the low temperature can reduce the production of harmful substances in the mice, reduce metabolism, and be beneficial to the formation of hypoxic preconditioning.
5. the hypoxia tolerance time of mice was significantly reduced each time the operation time of the mice was longer than 15 seconds. The reason may be that the reoxygenation time was too long, because the time of reoxygenation was short for the formation of preconditioning. The hypoxia tolerance of mice after repeated 4 times of hypoxia was partly retained after 30 minutes, indicating that the mice were deficient in 4 times. The complete disappearance of oxygen tolerance requires at least 30 minutes or more, so the substance that endows the hypoxic tolerance of the preconditioned mice may be partly sensitive to the recovery of the oxygen supply in the body, but partly for a long time.
(two) hypoxic preconditioning mice brain homogenate had no effect on the hypoxia tolerance of PC12 cells, HepG2 cells and NGF induced PC12 cells induced by NGF.
1. hypoxic preconditioning mice brain homogenate on hypoxia, without NGF induced PC12 cells, the effect of hypoxia tolerance in HepG2 cells is that hypoxia preconditioning mouse brain homogenate can significantly improve the tolerance to hypoxia. At the early stage of hypoxia (24h), the higher concentration of protective effect is stronger than low concentration of protective effect, which suggests hypoxic preconditioning. In the brain tissue of mice, some or some substances that can enhance the anoxic tolerance of the cells or increase the expression of anti anoxic components in the brain tissue. With the prolonged anoxia time, the protective effect drops and the high concentration decreases faster. To hypoxia 72h, the hypoxia injury in high concentration extract is significantly higher than the low concentration group, which suggests hypoxia. Preconditioning mice may produce a short half-life of anti hypoxia substance and its effect at high concentration. The substance that has a lasting effect on hypoxia cell injury suggests that anoxic preconditioning has a biphasic effect. The effect of hypoxia preconditioning on the brain homogenate of the mice remains in the dose effect relationship and time effect. The aim of the study is to find the substance of hypoxia tolerance in the hypoxic preconditioning mouse's brain homogenate, so we should avoid the effect of damaging substances in the experiment. Therefore, in the study of the effect of subsequent hypoxia preconditioning on the differentiation of PC12 cells from the mouse brain homogenate, the concentration of protein was used to quantify the extract, and the experiment was reduced. The concentration range of the solution is used to find the protective effect of the extract on the PC12 cells induced by NGF, and the optimum concentration of no damage (that is, only enhancing the NGF induced PC12 cells' anoxic tolerance, and the no damage effect on the differentiated PC12 cells), is to further study the protective effects and protective effects of the hypoxic preconditioned mouse brain homogenate. Quality lays the foundation.
2. the effect of hypoxic preconditioning on NGF induced PC12 cells induced by hypoxic hypoxia in mice showed that the hypoxia preconditioned mouse brain homogenate with the final concentration of 100 g/mL and the same concentration of normal mice could significantly enhance the hypoxia tolerance of PC12 cells induced by NGF, and the deficiency of this concentration was deficient. Oxygen preconditioning mice brain homogenate did not show any damage to differentiated PC12 cells, and was suitable for enhancing the tolerance of differentiated PC12 cells to hypoxia.
(three) the effects of different components of hypoxic preconditioning mice brain homogenate on hypoxia tolerance of NGF induced PC12 cells and possible effective components
1. different concentrations of adenosine was added to the differentiated PC12 cells induced by NGF in vitro. With the increase of adenosine concentration, the hypoxia tolerance of the cells increased. The protective effect of adenosine at 10 mu mol/L could maintain 24h. acute 4 repeated hypoxia to increase the content of adenosine in the brain tissue of mice. Hypoxia preconditioning in the brain homogenate deproteinized solution only The hypoxia tolerance significantly increased the anoxic tolerance of differentiated PC12 cells, and adenosine A2A receptor blocker blocked its protective effect. The normal mouse brain homogenate deprotein solution had no protective effect on the differentiated PC12 cells. Therefore, the anti oxygen deficiency time of the non protein components in the brain homogenate of the hypoxic mice was shorter, and adenosine might be one of them. An important active substance.
2. in hypoxic preconditioning mice, the hypoxic preconditioning of dialysis fluid and non dialysis was performed in hypoxia 24h and 48h.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R363

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