本文選題：體溫過高　切入點(diǎn)：惡性　出處：《安徽醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景 新生兒缺氧-缺血性腦病(Hypoxic-ischemic encephalopathy，HIE)是窒息后的大腦表現(xiàn)[1]，是引起新生兒死亡和嚴(yán)重神經(jīng)系統(tǒng)功能障礙，包括腦性癱瘓、癲癇、精神發(fā)育遲滯、注意力不集中、記憶力減退以及學(xué)習(xí)能力喪失等的重要原因[2]。妊娠晚期胎兒宮內(nèi)窒息可由多種因素引起，包括短暫的臍血流中止、強(qiáng)烈的子宮收縮以及胎盤早剝等。急性胎兒窒息可發(fā)生在產(chǎn)前、產(chǎn)時(shí)及產(chǎn)后的任一時(shí)期，導(dǎo)致HIE的發(fā)生[3]。在發(fā)達(dá)國家足月新生兒中，HIE的發(fā)生率約為1-3‰[2]，在早產(chǎn)兒中接近60%[4]。HIE的預(yù)后與疾病的嚴(yán)重程度密切相關(guān)。輕度HIE患兒預(yù)后良好，中度HIE患兒經(jīng)過有效治療，約有30%-50%的患兒遺留嚴(yán)重的神經(jīng)系統(tǒng)功能障礙，而重度HIE患兒神經(jīng)系統(tǒng)功能障礙的發(fā)生率高達(dá)90%以上[5]，該病嚴(yán)重影響患兒的認(rèn)知功能及運(yùn)動發(fā)育，對個(gè)人、家庭及社會都造成沉重的負(fù)擔(dān)，且持續(xù)終身，嚴(yán)重影響了我國人民整體身心健康、國民經(jīng)濟(jì)發(fā)展和社會穩(wěn)定。 在很久之前人們已經(jīng)逐漸認(rèn)識到，HIE并非只是由于窒息所導(dǎo)致或加重，單純窒息僅僅能解釋HIE中的一小部分[6]，越來越多的其他危險(xiǎn)因素被人們所認(rèn)識。近年來流行病學(xué)調(diào)查顯示，臨床中一些非窒息的危險(xiǎn)因素能夠加重HIE的損傷程度，尤其是母親分娩過程中體溫高于38.5℃，是加重HIE的危險(xiǎn)因素之一，體溫增高對新生兒缺氧-缺血性腦損傷(Hypoxic-Ischemic brain damage，HIBD)有更加嚴(yán)重的影響[7,8]。探討體溫增高對未成熟大腦HIBD后腦損傷程度的影響，引起人們對高溫這一危險(xiǎn)因素的足夠重視，有利于HIBD損傷機(jī)制的研究以及臨床干預(yù)和預(yù)防，這是本項(xiàng)目的主要研究內(nèi)容之一。 到目前為止，對于HIE的管理策略都是支持性的，包括：維持組織通氣和血流灌注、維持血糖在正常較高水平、控制驚厥發(fā)作、高壓氧、營養(yǎng)腦細(xì)胞藥物、早期干預(yù)康復(fù)等，但以上治療措施都沒有定向于每個(gè)具體進(jìn)行性損傷的過程。因此，并不奇怪，盡管過去幾十年圍生醫(yī)學(xué)及康復(fù)醫(yī)學(xué)的快速發(fā)展， HIE導(dǎo)致腦癱的發(fā)生率基本沒有改變。最近，又有研究顯示全身降溫能夠減少HIE新生兒的死亡率或中、重度殘疾程度，但低體溫組HIE患兒仍有44%發(fā)生了死亡、中度、重度殘疾[9]。神經(jīng)元丟失是許多神經(jīng)系統(tǒng)疾病包括新生兒HIE的主要特征，不可逆的腦損傷和有限的神經(jīng)干細(xì)胞分化能力是HIE患兒死亡和殘疾的主要原因[10]。近幾十年，由于細(xì)胞療法的出現(xiàn)，為HIE患兒提供了新的希望。 干細(xì)胞是一類具有自我更新、多向分化潛能的祖細(xì)胞，能夠分化成為多種不同的組織。干細(xì)胞因其廣泛的分化潛能及低免疫原性成為近年來醫(yī)學(xué)及生物學(xué)領(lǐng)域最引人注目的熱點(diǎn)之一，從而為神經(jīng)系統(tǒng)疾病包括新生兒HIE、腦癱、腦卒中、脊髓損傷及神經(jīng)變性疾病等治療提供了光明的前景。現(xiàn)在關(guān)于干細(xì)胞的很多研究都來自骨髓，但是骨髓源性干細(xì)胞隨著年齡增長其細(xì)胞數(shù)量和擴(kuò)增、分化能力出現(xiàn)明顯下降趨勢，并存在病毒污染的高度危險(xiǎn)性。而且自病人抽吸骨髓是一個(gè)侵入性、疼痛的過程，自新生兒抽吸骨髓幾乎不可能。故尋找一種能夠替代骨髓，并可彌補(bǔ)其缺陷的干細(xì)胞來源是目前的緊急任務(wù)。而最近研究發(fā)現(xiàn)干細(xì)胞也能夠從臍帶血中分離。人臍帶血單核細(xì)胞（Human Cord Blood Mononuclear Cells，HCMNCs）內(nèi)含豐富的造血干細(xì)胞、間充質(zhì)干細(xì)胞（Mesenchymal Stem Cells，MSCs）等，擁有多重效用：移植的細(xì)胞擁有極好的遷移能力[11,12]，局部缺血性腦損傷增加了它們向損傷部位的遷移[13]；移植細(xì)胞遷移到損傷組織，可以分泌神經(jīng)保護(hù)因子，保護(hù)神經(jīng)細(xì)胞免受損傷和凋亡[14]；提高了內(nèi)源性神經(jīng)干細(xì)胞的增殖，同時(shí)抑制新產(chǎn)生細(xì)胞的死亡；部分可以分化進(jìn)入宿主腦組織中形成星形膠質(zhì)細(xì)胞樣細(xì)胞，這些星形膠質(zhì)細(xì)胞樣細(xì)胞可以支持神經(jīng)細(xì)胞的存活；在體外人類臍帶血衍生的MSCs能夠產(chǎn)生粒細(xì)胞集落刺激因子[15,16]。這些多重效用促成了它們在神經(jīng)系統(tǒng)疾病中的治療潛能，因此臍帶血單核細(xì)胞最終有能力產(chǎn)生新生神經(jīng)細(xì)胞替代那些受損傷或疾病的神經(jīng)細(xì)胞。本研究的目的之二即是通過腹腔注射移植人HCMNCs，探討其對HIE后遠(yuǎn)期學(xué)習(xí)記憶功能的影響及突觸超微結(jié)構(gòu)的改變。為此，特開展以下研究： 第一部分體溫增高對缺氧缺血性腦損傷新生大鼠神經(jīng)功能的影響 目的建立新生大鼠缺氧缺血性腦損傷(Hypoxic-Ischemic brain damage，HIBD)動物模型，研究體溫增高對其神經(jīng)功能發(fā)育的影響。方法7日齡新生SD大鼠62只，隨機(jī)分成高溫HIBD組、常溫HIBD組、單純高溫組及常溫對照組。高溫HIBD組在42℃溫度下進(jìn)行缺氧缺血處理，常溫HIBD組在37℃溫度下進(jìn)行缺氧缺血處理，缺氧時(shí)間均為60min。單純高溫組幼鼠僅置于42℃水浴箱中60min，不進(jìn)行缺氧損傷。常溫對照組幼鼠不予以任何處理。應(yīng)用早期神經(jīng)反射及Morris水迷宮實(shí)驗(yàn)評估大鼠神經(jīng)行為學(xué)功能發(fā)育；后斷頭取腦，行普通HE染色光鏡下觀察，并利用透射電鏡觀察各組幼鼠大腦損傷部位超微結(jié)構(gòu)的改變。 結(jié)果高溫HIBD組與常溫HIBD組、常溫對照組相比，生長率降低[（54.57±10.27）比（83.05±20.42）、（80.81±16.84）]；生后14d早期神經(jīng)反射實(shí)驗(yàn)中，懸崖調(diào)轉(zhuǎn)反射[（14.60±2.83）比（12.64±1.93）、（9.69±2.76）]、陰性趨地性反射[（18.14±5.17）比（14.48±2.88）、（12.15±0.89）]、步態(tài)反射[（23.34±10.05）比（18.02±4.52）、（11.44±2.26）]均顯著減慢，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；Morris水迷宮實(shí)驗(yàn)中，從第3天開始逃避潛伏期延長[第3天：（65.20±11.46）比（58.24±6.01）、（47.55±8.87），第4天：（48.58±3.46）比（44.16±5.74）、（33.14±3.97）]，，穿臺次數(shù)減少[（2.12±1.05）比（3.06±1.16）、（3.82±1.25）]，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；腦組織形態(tài)學(xué)評分增加[（2.88±0.99）比（2.06±1.00）、（0.00±0.00）]；透射電鏡觀察突觸間隙增寬[（23.45±2.28）比（21.27±1.12）、（19.45±0.79）]，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；單純高溫組各項(xiàng)檢測指標(biāo)與常溫對照組比較差異均無統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論母鼠體溫增高加重了幼鼠HIBD的程度，嚴(yán)重影響神經(jīng)功能發(fā)育；單純母鼠體溫增高對大鼠遠(yuǎn)期學(xué)習(xí)記憶功能的影響不大。 第二部分人臍帶血單核細(xì)胞移植對新生大鼠缺氧缺血性腦損傷學(xué)習(xí)記憶功能的影響 目的探討人臍帶血單核細(xì)胞(HCMNCs)移植對新生大鼠缺氧缺血性腦損傷(HIBD)后遠(yuǎn)期學(xué)習(xí)記憶功能的影響及與突觸超微結(jié)構(gòu)改變的相關(guān)性。方法新生7日齡SD大鼠60只，隨機(jī)分為3組，采用Rice法建立HIBD動物模型，缺氧缺血后24h，HIBD+移植組大鼠腹腔內(nèi)注入1×107HCMNCs (在500ul的0.9%NaCl中)；HIBD+NaCl組腹腔內(nèi)注入等量的生理鹽水（NaCl）；另設(shè)空白對照組。生后10d及67d時(shí)行頭顱磁共振成像（MRI）檢查，生后35d時(shí)利用Morris水迷宮實(shí)驗(yàn)評估大鼠空間學(xué)習(xí)記憶能力，后斷頭取腦，進(jìn)行鼠腦大體形態(tài)學(xué)評分，并使用透射電鏡觀察各組鼠腦損傷部位神經(jīng)元突觸超微結(jié)構(gòu)的改變，與Morris水迷宮結(jié)果進(jìn)行相關(guān)性分析。 結(jié)果HIBD+NaCl組與HIBD+移植組、空白對照組相比，Morris水迷宮實(shí)驗(yàn)中，第2天開始逃避潛伏期延長[第2天：（61.17±6.22）比（53.09±7.41）、（53.53±5.28），第3天：（57.26±14.98）比（49.76±9.40）、（47.11±7.36），第4天：（52.10±6.17）比（44.16±5.74）、（41.96±8.97）]，穿臺次數(shù)減少[（2.12±1.05）比（3.06±1.16）、（3.70±1.17）]，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；腦組織形態(tài)學(xué)評分增加[（2.82±1.02）比（1.61±0.92）、（0.00±0.00）]；透射電鏡觀察突觸間隙增寬[（26.43±2.33）比（23.19±2.11）、（21.66±4.80）]，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；生后67d頭顱MRI檢查示殘存腦容量減少[（61.17±6.22）比（78.11±4.01）、（100±0.00）]，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。而HIBD+移植組與空白對照組比較，上述各項(xiàng)指標(biāo)間差異均無統(tǒng)計(jì)學(xué)意義（P0.05）；在第4天的Morris水迷宮實(shí)驗(yàn)中，各組大鼠的逃避潛伏期與透射電鏡觀察突觸間隙寬度之間具有高度相關(guān)性，相關(guān)系數(shù)r為0.857（P0.05）。 結(jié)論HCMNCs腹腔內(nèi)移植可促進(jìn)新生大鼠HIBD后損傷腦細(xì)胞的恢復(fù)及學(xué)習(xí)記憶功能的改善；Morris水迷宮實(shí)驗(yàn)中逃避潛伏期與透射電鏡觀察突觸間隙寬度具有高度相關(guān)性。
[Abstract]:Research background
Neonatal hypoxic ischemic encephalopathy (Hypoxic-ischemic encephalopathy HIE) is asphyxia brain [1], is the cause of neonatal death and severe neurological dysfunction, including cerebral palsy, epilepsy, mental retardation, inattention, memory loss and [2]. loss of the ability to learn an important reason of late pregnancy fetal asphyxia can be caused by a variety of factors, including short umbilical blood flow stops, strong uterine contractions and placental abruption. Acute fetal distress can occur in any period of prenatal, intrapartum and postpartum, leading to the occurrence of HIE [3]. in the developed countries in the HIE of newborn, the incidence rate is about 1-3 per thousand [2], closely related to the severity and prognosis of disease in preterm infants close to 60%[4].HIE. The mild HIE had good prognosis, moderate HIE patients after effective treatment, about 30%-50% of patients from severe nerve System dysfunction, and the severe HIE children with nervous system dysfunction rate is as high as 90% [5], the disease seriously affect cognitive function and motor development of children, for individuals, families and society have caused a heavy burden, and lifelong, seriously affected the Chinese people's overall physical and mental health, national economic development and social stability.
Long before people have come to realize that HIE is not only due to cause or aggravate the asphyxia, simple asphyxia can only explain a small part of the HIE [6] and other risk factors are increasingly recognized. Epidemiological survey in recent years, the damage degree of some non clinical factors in danger of suffocation can aggravate HIE. Especially the mother during childbirth temperature higher than 38.5 DEG C, is one of the risk factors for exacerbation of HIE, increased body temperature on neonatal hypoxic ischemic brain damage (Hypoxic-Ischemic brain damage, HIBD) have a more serious impact on [7,8]. high temperature effect on the degree of brain injury in immature brain HIBD, cause people to pay enough attention to the danger of high temperature factors, can be helpful to research the mechanism of HIBD damage and clinical prevention and intervention, which is one of the main research contents of this project.
So far, support, for the HIE management strategies include: maintenance of tissue perfusion and ventilation, maintain a high level of blood glucose in the normal control, seizure, hyperbaric oxygen, medical nutrition brain cells, early intervention rehabilitation, but the above treatment measures are not directed to each specific process of injury. It is not surprising that, despite the rapid development in the past few decades of perinatal medicine and rehabilitation medicine, HIE lead to the incidence of cerebral palsy is basically not changed. Recently, studies have shown that the body temperature and to reduce the death rate of newborn HIE or in severe disability, but the low temperature HIE patients were still 44% deaths have occurred. Moderate and severe disability [9]. neuronal loss is the main feature of many neurological diseases including neonatal HIE, irreversible brain damage and the differentiation of neural stem cells is HIE with the death and disability The main cause of the disease, [10]., has provided new hope for children with HIE in recent decades, due to the emergence of cell therapy.
Stem cells are self-renewal and multi-directional differentiation of progenitor cells can differentiate into different tissues. Stem cells because of its wide differentiation potential and low immunogenicity has become one of the hot spots in the field of medicine and biology in recent years, which include neonatal HIE, for nervous system disease of cerebral palsy, stroke. Provides a bright prospect for the treatment of spinal cord injury and neurodegenerative disease. Now a lot of research on stem cells from bone marrow, but bone marrow derived stem cells with age and the number of cells amplified, differentiation capacity decline significantly, and there is high risk of viral contamination. And since the patient is a bone marrow aspiration invasive, painful procedures from neonatal aspiration of bone marrow is almost impossible. So looking for a substitute for bone marrow, and can remedy the defects of stem cells is present An urgent task. And recent studies found that stem cells can be isolated from umbilical cord blood. Human umbilical cord blood mononuclear cell (Human Cord Blood Mononuclear Cells, HCMNCs) contains abundant hematopoietic stem cells, mesenchymal stem cells (Mesenchymal Stem Cells, MSCs), has the multiple effect: the transplanted cells have excellent the migration ability of [11,12], ischemic brain injury increased their migration to [13] injury; transplantation cells migrate to the damaged tissue, can secrete neurotrophic factor, protect nerve cells from damage and apoptosis of [14]; improve the stem cell proliferation of endogenous neural cells, and inhibition of the new part can form astrocyte death; cell differentiation into the host brain tissue, the survival of these astrocyte like cells can support nerve cells derived from human umbilical cord blood in vitro; the MSCs can produce Students of granulocyte colony-stimulating factor [15,16]. of these multiple factors contributing to their utility in nervous system disease therapeutic potential, therefore umbilical cord blood mononuclear cells are able to generate new nerve cells to replace those affected by the disease or injury of nerve cells. The purpose of this study is two by intraperitoneal injection of transplanted human HCMNCs, and explore its influence on learning and memory function of HIE after long-term and synaptic ultrastructure changes. Therefore, to carry out the following research:
The effect of body temperature increase on the nerve function of neonatal rats with hypoxic ischemic brain injury
Objective to establish neonatal rats with hypoxic-ischemic brain damage (Hypoxic-Ischemic brain, damage, HIBD) animal model of increased body temperature effect on the growth and development of the neurological function. Methods 7 day old SD rats were randomly divided into 62 groups of HIBD, high temperature, room temperature HIBD group, hyperthermia group and normal control group. HIBD group of hypoxia at high temperature ischemia treatment at 42 DEG C, normal temperature group HIBD hypoxia ischemia treatment at 37 DEG C, hypoxia time were 60min 60min. hyperthermia group were only in 42 DEG C water bath, not hypoxia injury. Normal control group were not given any treatment. Early application of neural reflex and Morris water maze test to evaluate nerve the development of learning behavior in rats; after the rats were observed under light microscope for HE staining, and observed the brain injury ultrastructure by transmission electron microscopy.
緇撴灉楂樻俯HIBD緇勪笌甯告俯HIBD緇

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