發(fā)布時(shí)間：2018-09-11 14:53

【摘要】：水母是廣泛分布在全球各海域的無脊椎浮游動物。近年來,海洋水溫升高、海水富營養(yǎng)化等多種因素造成某些海域中特定種系水母暴發(fā)。多數(shù)種類水母無明顯毒性作用,少數(shù)種類水母的刺絲囊中攜帶有毒性很強(qiáng)的毒液,其刺絲囊因化學(xué)或機(jī)械刺激發(fā)射刺絲釋放毒素可以造成人員的嚴(yán)重蜇傷。全球每年被水母蜇傷的患者數(shù)以十萬計(jì),其中致死病例也并不罕見。不同種類水母蜇傷的癥狀和體征有所不同,有些水母蜇傷只造成一過性刺痛或輕微紅腫,而有些水母蜇傷較嚴(yán)重,患者出現(xiàn)持續(xù)性疼痛,蕁麻疹和水皰,表皮壞死,循環(huán)系統(tǒng)損傷,多器官功能障礙,甚至死亡。針對水母蜇傷預(yù)防和治療藥物的研究越來越受到關(guān)注,取得了一些進(jìn)展,但尚沒有達(dá)成救治措施方面的廣泛共識。水母毒素成分復(fù)雜,易粘附和失活,毒作用靶點(diǎn)多樣,毒素的這些理化特點(diǎn)增加了尋找特效抗毒藥物的難度。本文第一部分在細(xì)胞水平和整體動物水平分析了低分子右旋糖酐(Dextran-40)對致命性水母蜇傷的保護(hù)作用。第二部分著重分析了5種金屬陽離子對水母毒素溶血作用的干預(yù)效應(yīng)。實(shí)驗(yàn)方法第一部分:Dextran-40對致命性水母蜇傷的保護(hù)作用本部分研究首先應(yīng)用激光掃描共聚焦顯微鏡,在細(xì)胞水平研究Dextran-40對發(fā)形霞水母Cyanea capillata觸手提取物(tentacle extract,TE)造成的小鼠心室肌細(xì)胞和紅細(xì)胞損傷的保護(hù)作用。然后,應(yīng)用本課題組建立的致命性水母蜇傷綜合征(acute jellyfish envenomation syndrome,AJES和delayed jellyfish envenomation syndrome,DJES)模型,在整體動物水平分別研究Dextran-40對AJES小鼠模型生存率的影響、Dextran-40對AJES大鼠模型循環(huán)功能的影響以及Dextran-40對DJES大鼠模型血生化指標(biāo)的影響。第二部分:金屬陽離子對水母毒素溶血作用的干預(yù)效應(yīng)本部分研究首先應(yīng)用分光光度計(jì)法檢測TE的溶血效應(yīng),觀察5種據(jù)報(bào)道具有潛在抗溶血效應(yīng)的陽離子(La3+,Mn2+,Zn2+,Cu2+和Fe2+)對TE溶血作用的影響,然后,分別測定陽離子溶液的吸光度值、陽離子對紅細(xì)胞懸液的吸光度值影響、陽離子對血紅蛋白溶液的吸光度值影響,并且在顯微鏡下對紅細(xì)胞直接計(jì)數(shù)。綜合分析La3+,Mn2+,Zn2+,Cu2+和Fe2+對水母毒素溶血作用的干預(yù)效應(yīng)。實(shí)驗(yàn)結(jié)果第一部分:Dextran-40對致命性水母蜇傷的保護(hù)作用本部分實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),TE會引起心肌細(xì)胞和紅細(xì)胞胞內(nèi)Ca2+含量呈現(xiàn)劑量依賴性增加,心肌細(xì)胞和紅細(xì)胞存活數(shù)量呈現(xiàn)劑量依賴性減少。Dextran-40可以對TE引起的心肌細(xì)胞胞內(nèi)Ca2+含量升高和細(xì)胞存活率降低起到一定的保護(hù)作用;對TE引起的紅細(xì)胞形態(tài)損傷和溶血作用沒有明顯保護(hù)作用,但是顯著抑制TE引起的紅細(xì)胞胞內(nèi)鈣超載。應(yīng)用本課題組建立的致命性水母蜇傷綜合征(AJES和DJES)模型,在整體動物水平研究結(jié)果發(fā)現(xiàn),Dextran-40在0.8 g/kg時(shí)能明顯降低TE所致AJES小鼠死亡率。Dextran-40在0.6 g/kg時(shí)能明顯緩解TE所致AJES大鼠的血壓下降,明顯改善TE所致DJES大鼠的肝功能指標(biāo)(ALT,AST,A/G)、心肌損傷相關(guān)酶指標(biāo)(CK,LDH)。第二部分:金屬陽離子對水母毒素溶血作用的干預(yù)效應(yīng)本部分實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),有輕微顏色的Zn2+,Cu2+和Fe2+離子溶液導(dǎo)致紅細(xì)胞懸液的吸光度值降低,而Mn2+溶液增加了紅細(xì)胞懸液吸光度值,透明的La3+溶液對紅細(xì)胞懸液的吸光度值幾乎沒有影響,這表明陽離子自身顏色可能干擾紅細(xì)胞懸液中血紅蛋白的吸收光譜。我們測試了5種陽離子對紅細(xì)胞懸浮液釋放的血紅蛋白溶液的影響,表明結(jié)果透明La3+溶液不對血紅蛋白溶液的吸光度值產(chǎn)生明顯影響,Mn2+溶液導(dǎo)致血紅蛋白溶液吸光度值輕微降低,有輕微顏色的Zn2+,Cu2+和Fe2+溶液則顯著降低血紅蛋白溶液的吸光度值。經(jīng)校正分析,除La3+顯示完全抑制TE的溶血作用之外,Zn2+,Cu2+,Fe2+和Mn2+使TE溶血曲線右移,即其實(shí)際抑制效應(yīng)比之前報(bào)道的抑制效果偏弱。實(shí)驗(yàn)結(jié)論第一部分:Dextran-40對致命性水母蜇傷的保護(hù)作用我們推測Dextran-40一方面通過封堵水母毒素在細(xì)胞膜上形成的孔道,對細(xì)胞的鈣超載起到了抑制作用,從而保護(hù)心肌細(xì)胞;另一方面可能通過提高血漿膠體滲透壓,吸收血管外水分而增加血容量,穩(wěn)定血壓,以拮抗水母毒素引起冠脈痙攣導(dǎo)致的機(jī)體低血壓癥及其他心臟病變。第二部分:金屬陽離子對水母毒素溶血作用的干預(yù)效應(yīng)研究結(jié)果表明除La3+之外,其他4種金屬陽離子(Mn2+,Zn2+,Cu2+和Fe2+)會干擾血紅蛋白的吸光度,所以測試含金屬離子類試劑對水母毒素溶血作用的影響時(shí),應(yīng)該進(jìn)行校正分析。
[Abstract]:Jellyfish are invertebrate zooplankton widely distributed in various sea areas all over the world. In recent years, sea water temperature rises, eutrophication and other factors lead to the outbreak of certain species of jellyfish in certain sea areas. Most species of jellyfish have no obvious toxic effect. A few species of jellyfish carry highly toxic venom in their spiny sacs due to chemical factors. There are hundreds of thousands of jellyfish stings worldwide each year, and deaths are not uncommon. The symptoms and signs of different kinds of jellyfish stings vary. Some jellyfish stings cause only temporary stings or slight redness, while some jellyfish stings are more severe. Seriously, patients suffer from persistent pain, urticaria and blisters, epidermal necrosis, circulatory system damage, multiple organ dysfunction, and even death. Drug research for the prevention and treatment of jellyfish stings has attracted more and more attention, and some progress has been made, but there is no broad consensus on treatment measures. These physical and chemical characteristics of toxins make it difficult to find specific antitoxic drugs. In the first part of this paper, the protective effects of low molecular dextran (Dextran-40) on lethal jellyfish stings are analyzed at cellular and animal levels. In the second part, the solubility of five metal cations to jellyfish toxins is emphasized. Interventional effects of Dextran-40 on blood. Methods Part 1: Protective effects of Dextran-40 on lethal jellyfish stings. This part first studied the effects of Dextran-40 on ventricular myocytes and red and fine cells of mice induced by tentacle extract (TE) of Cyanea capillata at the cellular level by laser scanning confocal microscopy. Then, the effects of Dextran-40 on the survival rate of AJES mice model and the effects of Dextran-40 on AJES rat model were studied by using the acute jellyfish envenomation syndrome (AJES) and delayed jellyfish envenomation syndrome (DJES) models. The effect of Dextran-40 on blood biochemical parameters in DJES rats. Part 2: Intervention effect of metal cations on hemolysis of jellyfish toxin Part 2: The hemolysis effect of TE was detected by spectrophotometer, and five kinds of cations (La3+, Mn2+, Zn2+, Cu2+) with potential Anti-hemolysis effect were observed. Then, the absorbance of cationic solution, the absorbance of cationic solution to erythrocyte suspension, the absorbance of cationic solution to erythrocyte suspension, and the absorbance of cationic solution to erythrocyte were determined respectively. The erythrocyte count was also carried out under microscope. The hemolysis of jellyfish toxin by La3+, Mn2+, Zn2+, Cu2+ and Fe2+ was analyzed comprehensively. Intervention effect. Part I: Protective effect of Dextran-40 on lethal jellyfish sting. This part of the experiment found that TE could induce a dose-dependent increase in Ca2+ content in cardiomyocytes and erythrocytes, and a dose-dependent decrease in cardiomyocytes and erythrocytes survival. The increase of intracellular Ca2+ content and the decrease of cell survival rate play a certain protective role; they have no obvious protective effect on erythrocyte morphological damage and hemolysis induced by TE, but significantly inhibit intracellular calcium overload of erythrocyte induced by TE. Dextran-40 significantly reduced TE-induced mortality in AJES mice at 0.8 g/kg. Dextran-40 significantly alleviated TE-induced hypotension in AJES rats, significantly improved TE-induced liver function (ALT, AST, A/G) and myocardial injury-related enzymes (CK, LDH). Intervention effect of jellyfish toxin on hemolysis This part of the experimental results showed that slightly colored Zn2+, Cu2+ and Fe2+ ion solutions decreased the absorbance of erythrocyte suspension, while Mn2+ solution increased the absorbance of erythrocyte suspension, transparent La3+ solution had little effect on the absorbance of erythrocyte suspension, indicating that the cations were self-contained. Body color may interfere with the absorption spectrum of hemoglobin in erythrocyte suspensions. We tested the effects of five cations on the release of hemoglobin from erythrocyte suspensions. The results showed that transparent La3+ solution had no significant effect on the absorbance of hemoglobin solution. Mn2+ solution caused a slight decrease in the absorbance of hemoglobin solution. In addition to the complete inhibition of TE hemolysis by La3 +, Zn2 +, Cu2 +, Fe2 + and Mn2 +, the hemolysis curves of TE were shifted to the right by Zn2 +, Cu2 + and Mn2 +, i.e. the actual inhibition effect was weaker than previously reported. The protective effect of Dextran-40 on lethal jellyfish stings is hypothesized to protect cardiomyocytes by blocking the channels formed by jellyfish toxins on the cell membrane, inhibiting calcium overload, and on the other hand, increasing blood volume and stabilizing blood pressure by increasing plasma colloid osmotic pressure and absorbing extravascular water. Antagonizing hypotension and other heart diseases caused by jellyfish toxin-induced coronary spasm. Part II: Intervention effects of metal cations on the hemolysis of jellyfish toxin. The results showed that four other metal cations (Mn2+, Zn2+, Cu2+, and Fe2+) interfered with the absorbance of hemoglobin except La3+, so the metal ions were tested. The effect of reagents on hemolytic effects of jellyfish toxins should be corrected.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R646

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本文編號：2237013