本文選題：神經(jīng)元　切入點(diǎn)：原代培養(yǎng)　出處：《第二軍醫(yī)大學(xué)》2016年博士論文

【摘要】：中暑是嚴(yán)重威脅人類生命健康的疾病,發(fā)病率高,致死率高,致殘率高。隨著全球溫度變暖,發(fā)病率逐年上升。中暑可以引起全身多器官和系統(tǒng)的損害,對神經(jīng)系統(tǒng)的損害尤為明顯和嚴(yán)重。中暑患者可以出現(xiàn)頭痛、抽搐、意識(shí)喪失、躁狂、淡漠、認(rèn)知功能下降等神經(jīng)系統(tǒng)受累表現(xiàn)。有關(guān)中暑的研究中以人和動(dòng)物為研究對象居多,但是面臨兩個(gè)問題就是:一、制造中暑的動(dòng)物模型不統(tǒng)一,不同動(dòng)物模型中動(dòng)物加熱采用的高溫和時(shí)間各不相同[1-4],這就會(huì)對各種實(shí)驗(yàn)結(jié)果產(chǎn)生一定的影響。繞過動(dòng)物模型,直接加熱原代培養(yǎng)的大鼠皮層神經(jīng)元,可以很好的解決上述矛盾。二、沒有全面掌握不同高溫情況對神經(jīng)元本身受到的傷害表現(xiàn)和規(guī)律特點(diǎn)。我們通過設(shè)定不同高溫和加熱時(shí)間的組合,全面觀察不同情況高溫處理下神經(jīng)元的病理變化,全面模擬高溫?fù)p傷后神經(jīng)元元損傷的病理變化規(guī)律,為進(jìn)一步防治中暑奠定基礎(chǔ)。我們通過大量實(shí)驗(yàn)研究,摸索建立了一種新的通過新生乳鼠皮層培養(yǎng)高純度、高成活率、高產(chǎn)量神經(jīng)元的方法。通過在不同溫度和暴露時(shí)間加熱神經(jīng)元,觀察不同高溫設(shè)置下神經(jīng)元的死亡率,探索高溫加熱神經(jīng)元后神經(jīng)元死亡的病理變化規(guī)律。同時(shí)我們還進(jìn)一步研究了常見中暑情況下神經(jīng)元壞死和凋亡的具體情況以及相關(guān)蛋白Cas-3、HSP70含量的變化,為進(jìn)一步防治中暑做了初步的研究。第一部分構(gòu)建改良的大鼠原代皮層神經(jīng)元的培養(yǎng)模型研究目的:建立穩(wěn)定的高純度、低死亡率的大鼠皮層神經(jīng)元培養(yǎng)方法。研究方法:參考Beaudoin等人[5]和Giordano G等人[6]、王旭輝等人[7]的實(shí)驗(yàn)方法,并經(jīng)過適當(dāng)摸索改進(jìn)。采用新生12h內(nèi)的Wistar大鼠,麻醉后低溫下干凈分離出雙側(cè)大腦皮層,剪碎組織后予胰酶消化,消化后輕柔吹打組織,然后過濾再離心,棄上清并添加適量接種液,輕輕吹勻后再次過濾,光鏡下計(jì)數(shù)細(xì)胞密度,用接種液稀釋至適當(dāng)濃度接種。接種后第4h、45h、96h全量更換培養(yǎng)液。結(jié)果:神經(jīng)元培養(yǎng)第5天,光鏡下見神經(jīng)元形態(tài)飽滿透亮、背景干凈,固縮的死亡細(xì)胞少。行神經(jīng)元特異性標(biāo)志物NSE免疫組化鑒定,NSE陽性率超過95%。行NMDAR1抗體染色,觀察發(fā)現(xiàn)神經(jīng)元細(xì)胞突起長、交聯(lián)豐富充分。行臺(tái)盼蘭染色,陽性細(xì)胞非常少,神經(jīng)元死亡率小于5%。結(jié)論:建立了一套簡便、穩(wěn)定、高純度、低死亡率、高產(chǎn)量、低背景的皮層神經(jīng)元培養(yǎng)方法。第二部分不同高溫和暴露時(shí)間處理下神經(jīng)元損傷情況的研究研究目的:探索不同高溫和暴露時(shí)間處理下神經(jīng)元的病理改變和死亡情況的變化規(guī)律。研究方法:神經(jīng)元培養(yǎng)第5d,在37℃5%CO2培養(yǎng)箱中分別給予細(xì)胞39℃、41℃、43℃、45℃、47℃加熱,加熱45min或者1h。加熱24h后,光鏡下觀察神經(jīng)元形態(tài)改變;臺(tái)盼蘭染色后觀察細(xì)胞形態(tài)并統(tǒng)計(jì)各個(gè)模式下細(xì)胞的死亡率;Annexin/PI染色后熒光顯微鏡下觀察細(xì)胞在各種溫度下壞死和凋亡情況。結(jié)果:加熱45min情況下,神經(jīng)元對熱不敏感,最高可以耐受45℃的高溫。加熱1h的情況下,隨著溫度逐漸升高,神經(jīng)元死亡率逐漸增高。43℃及以下的高溫作用1h,神經(jīng)元死亡以凋亡為主。45度及以上高溫作用1h,神經(jīng)元死亡以壞死為主。結(jié)論:不同高溫和暴露時(shí)間是影響神經(jīng)元死亡率的重要因素。神經(jīng)元可以最高耐受45℃度的高溫45min。43度以下的熱打擊主要造成神經(jīng)元部分凋亡,43度以上的高溫,神經(jīng)元開始大面積壞死。第三部分模擬臨床常見高溫中暑情況下神經(jīng)元損傷的進(jìn)一步研究研究目的:探索臨床常見致中暑溫度下神經(jīng)元壞死凋亡的具體情況以及相關(guān)蛋白的變化規(guī)律。研究方法:神經(jīng)元培養(yǎng)第5d,在37℃5%CO2培養(yǎng)箱中分別給予細(xì)胞41℃、43℃加熱1h。加熱24h后,光鏡觀察神經(jīng)元形態(tài)學(xué)變化;Annexin/PI熒光雙標(biāo)染色后流式細(xì)胞儀計(jì)數(shù)神經(jīng)元壞死、凋亡比例并統(tǒng)計(jì)學(xué)分析;免疫細(xì)胞化學(xué)染色Cas-3、HSP70,Image-Pro Plus 6.0軟件進(jìn)行IOD值的掃描后統(tǒng)計(jì)分析。結(jié)果:神經(jīng)元41℃、43℃加熱1h后,神經(jīng)元小部分死亡,以細(xì)胞凋亡為主,溫度越高凋亡細(xì)胞越多。存活細(xì)胞形態(tài)無明顯變化。Caspase-3表達(dá)強(qiáng)度:37度對照組41度1h組43度1h組。HSP70表達(dá)強(qiáng)度:43度1h組37度對照組41度1h組。結(jié)論:臨床常見致中暑高溫?zé)岽驌羟闆r下,神經(jīng)元僅小部分死亡,主要以凋亡為主。41度1h較43度1h加熱情況下凋亡細(xì)胞少,Cas-3表達(dá)低,HSP70表達(dá)高,這可能與HSP70高表達(dá)的抗凋亡保護(hù)作用有關(guān)。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R594.12

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本文編號(hào)：1654465