高温对原代培养神经元损伤的研究

发布时间：2018-03-23 17:31

本文选题：神经元　切入点：原代培养　出处：《第二军医大学》2016年博士论文

【摘要】：中暑是严重威胁人类生命健康的疾病,发病率高,致死率高,致残率高。随着全球温度变暖,发病率逐年上升。中暑可以引起全身多器官和系统的损害,对神经系统的损害尤为明显和严重。中暑患者可以出现头痛、抽搐、意识丧失、躁狂、淡漠、认知功能下降等神经系统受累表现。有关中暑的研究中以人和动物为研究对象居多,但是面临两个问题就是:一、制造中暑的动物模型不统一,不同动物模型中动物加热采用的高温和时间各不相同[1-4],这就会对各种实验结果产生一定的影响。绕过动物模型,直接加热原代培养的大鼠皮层神经元,可以很好的解决上述矛盾。二、没有全面掌握不同高温情况对神经元本身受到的伤害表现和规律特点。我们通过设定不同高温和加热时间的组合,全面观察不同情况高温处理下神经元的病理变化,全面模拟高温损伤后神经元元损伤的病理变化规律,为进一步防治中暑奠定基础。我们通过大量实验研究,摸索建立了一种新的通过新生乳鼠皮层培养高纯度、高成活率、高产量神经元的方法。通过在不同温度和暴露时间加热神经元,观察不同高温设置下神经元的死亡率,探索高温加热神经元后神经元死亡的病理变化规律。同时我们还进一步研究了常见中暑情况下神经元坏死和凋亡的具体情况以及相关蛋白Cas-3、HSP70含量的变化,为进一步防治中暑做了初步的研究。第一部分构建改良的大鼠原代皮层神经元的培养模型研究目的:建立稳定的高纯度、低死亡率的大鼠皮层神经元培养方法。研究方法:参考Beaudoin等人[5]和Giordano G等人[6]、王旭辉等人[7]的实验方法,并经过适当摸索改进。采用新生12h内的Wistar大鼠,麻醉后低温下干净分离出双侧大脑皮层,剪碎组织后予胰酶消化,消化后轻柔吹打组织,然后过滤再离心,弃上清并添加适量接种液,轻轻吹匀后再次过滤,光镜下计数细胞密度,用接种液稀释至适当浓度接种。接种后第4h、45h、96h全量更换培养液。结果:神经元培养第5天,光镜下见神经元形态饱满透亮、背景干净,固缩的死亡细胞少。行神经元特异性标志物NSE免疫组化鉴定,NSE阳性率超过95%。行NMDAR1抗体染色,观察发现神经元细胞突起长、交联丰富充分。行台盼兰染色,阳性细胞非常少,神经元死亡率小于5%。结论:建立了一套简便、稳定、高纯度、低死亡率、高产量、低背景的皮层神经元培养方法。第二部分不同高温和暴露时间处理下神经元损伤情况的研究研究目的:探索不同高温和暴露时间处理下神经元的病理改变和死亡情况的变化规律。研究方法:神经元培养第5d,在37℃5%CO2培养箱中分别给予细胞39℃、41℃、43℃、45℃、47℃加热,加热45min或者1h。加热24h后,光镜下观察神经元形态改变;台盼兰染色后观察细胞形态并统计各个模式下细胞的死亡率;Annexin/PI染色后荧光显微镜下观察细胞在各种温度下坏死和凋亡情况。结果:加热45min情况下,神经元对热不敏感,最高可以耐受45℃的高温。加热1h的情况下,随着温度逐渐升高,神经元死亡率逐渐增高。43℃及以下的高温作用1h,神经元死亡以凋亡为主。45度及以上高温作用1h,神经元死亡以坏死为主。结论:不同高温和暴露时间是影响神经元死亡率的重要因素。神经元可以最高耐受45℃度的高温45min。43度以下的热打击主要造成神经元部分凋亡,43度以上的高温,神经元开始大面积坏死。第三部分模拟临床常见高温中暑情况下神经元损伤的进一步研究研究目的:探索临床常见致中暑温度下神经元坏死凋亡的具体情况以及相关蛋白的变化规律。研究方法:神经元培养第5d,在37℃5%CO2培养箱中分别给予细胞41℃、43℃加热1h。加热24h后,光镜观察神经元形态学变化;Annexin/PI荧光双标染色后流式细胞仪计数神经元坏死、凋亡比例并统计学分析;免疫细胞化学染色Cas-3、HSP70,Image-Pro Plus 6.0软件进行IOD值的扫描后统计分析。结果:神经元41℃、43℃加热1h后,神经元小部分死亡,以细胞凋亡为主,温度越高凋亡细胞越多。存活细胞形态无明显变化。Caspase-3表达强度:37度对照组41度1h组43度1h组。HSP70表达强度:43度1h组37度对照组41度1h组。结论:临床常见致中暑高温热打击情况下,神经元仅小部分死亡,主要以凋亡为主。41度1h较43度1h加热情况下凋亡细胞少,Cas-3表达低,HSP70表达高,这可能与HSP70高表达的抗凋亡保护作用有关。
[Abstract]:Heat stroke is a serious threat to human life and health disease, high morbidity, high mortality, high disability rate. With the global warming, the incidence increased year by year. Heat stroke can cause multiple organ and system damage, nervous system damage is obvious and serious. Heat stroke can occur in patients with headache, seizures, loss of consciousness. Mania, indifference, cognitive decline and nervous system involvement. Research on heatstroke in human and animal studies mostly, but faced with two problems: one is that the animal model of manufacturing the heat is not uniform, no animal animal model used in heating with high temperature and time vary [1-4], which will produce the effects of various experimental results. Around the animal model, the direct heating of primary cultured rat cortical neurons, can solve the above contradiction. Two, did not fully grasp the different situation of high temperature Damage performance and characteristics of the neurons themselves. We set through the combination of different high temperature and heating time, comprehensive observation of pathological changes of neurons under different high temperature treatment, comprehensive simulation of the pathological changes of nerve injury, after high temperature damage, lay the foundation for further prevention and treatment of heatstroke. Our experiments has been established a new through the cortex of neonatal rats cultured with high purity, high survival rate, high yield of neurons. In the different temperature and time of exposure to heat setting temperature observation of different neurons, neuronal mortality, explore the pathological changes of neuronal death after heating neurons. At the same time we also studied the specific circumstances of neuronal necrosis the common case of heatstroke and apoptosis and related protein Cas-3, HSP70 content changes, for further prevention and treatment in the summer to do A preliminary study on the construction of training. The first part of the modified rat primary cortical neurons model objective: to establish a stable method for high purity, low mortality cultured cortical neurons of rats. Methods: Beaudoin [5] and Giordano et al reference G et al [6], Wang Xuhui [7] experimental method, and through appropriate improvement exploration by Wistar of newborn rats within 12h after anesthesia, low temperature clean separation of bilateral cerebral cortex tissue after cut to trypsin digestion, digestion and soft tissue, then filtering and centrifugation, supernatant and adding appropriate amount of inoculum, gently blowing evenly filtered again, under the light microscope by counting the cell density and inoculation diluted to the appropriate concentration of inoculation. 4h after inoculation, 45h, 96h of the total replacement medium. Results: the neurons cultured for fifth days, see neurons full of bright light, clean background, pyknotic cell death row. Neuron specific markers of NSE were detected by immunohistochemistry, the positive rate of NSE is more than 95%. for NMDAR1 antibody staining, observed neuronal cell processes, crosslinking fully enriched. Trypan blue staining, positive cells of neurons is very small, the rate is lower than 5%.. Conclusion: to establish a set of simple, stable, high purity, low mortality rate. High yield cultivation method, low background cortical neurons. Objective to study on the second different high temperature and exposure time under the treatment of the injury of the neurons: explore the variation of high temperature and different exposure time under the treatment of pathological changes of neurons and death. Methods: Cultured 5D neurons, at 37 DEG 5%CO2 incubator were given cell 39 C, 41 C, 43 C, 45 C, 47 C heating, heating or heating 45min 1h. 24h after the change of neuronal morphology under light microscope; trypan blue staining to observe cell morphology and statistics of each Under the mode of cell death; cell necrosis was observed at various temperatures and apoptosis after Annexin/PI staining under fluorescence microscope. Results: heating 45min, neuron is not sensitive to heat, the highest temperature can tolerance 45 DEG C. Heating 1H, with the temperature gradually increased, neuronal mortality increased high temperature 1H.43 C and below, neuronal death through apoptosis of.45 degrees and above high temperature 1H, neuronal death by necrosis. Conclusion: the different temperature and exposure time is an important factor affecting neuronal mortality. Neurons to thermal impact of high temperature 45min.43 degrees C the maximum tolerated 45 degrees below the main cause neurons apoptosis, the high temperature of 43 degrees above, a large area of necrosis of neurons. The third part is the purpose of the simulation research of clinical common heat stroke under neuronal injury: a search for clinical Changes of common concrete conditions caused by temperature of heat stroke neuron apoptosis and related protein. Methods: cultured neurons in 5D at 37 DEG 5%CO2 in the cell culture box were given 41 C, 43 C 1h. 24h after heating heating, to observe the morphological changes of the neurons in the light microscope; flow cytometry staining after Annexin/PI neuron necrosis double immunofluorescence staining, the apoptosis rate and statistical analysis; immunocytochemical staining of Cas-3, HSP70, statistical analysis of IOD value scanning Image-Pro Plus 6 software. Results: the neurons of 41 DEG, 43 DEG 1h after heating, small part of neuron death, cell apoptosis, the higher the temperature the more apoptotic cells. Cell survival form significant changes in the expression of.Caspase-3 in the control group: 37 degrees 41 degrees 43 degrees 1H group 1H group.HSP70 group 1H expression intensity: 43 degrees 37 degrees and 41 in the control group 1H group. Conclusion: the clinical pathogenic heat stroke attack Only a small part of neurons died, mainly in apoptosis..41 degree 1H was less than that of 43 degree 1H heating, and Cas-3 expression was low, and HSP70 expression was high, which may be related to the anti apoptotic protective effect of HSP70.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R594.12

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