体温增高对缺氧缺血性脑损伤新生大鼠神经功能的影响及人脐带血单核细胞移植的效果研究

发布时间：2018-03-18 01:05

  本文选题：体温过高　切入点：恶性　出处：《安徽医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景 新生儿缺氧-缺血性脑病(Hypoxic-ischemic encephalopathy，HIE)是窒息后的大脑表现[1]，是引起新生儿死亡和严重神经系统功能障碍，包括脑性瘫痪、癫痫、精神发育迟滞、注意力不集中、记忆力减退以及学习能力丧失等的重要原因[2]。妊娠晚期胎儿宫内窒息可由多种因素引起，包括短暂的脐血流中止、强烈的子宫收缩以及胎盘早剥等。急性胎儿窒息可发生在产前、产时及产后的任一时期，导致HIE的发生[3]。在发达国家足月新生儿中，HIE的发生率约为1-3‰[2]，在早产儿中接近60%[4]。HIE的预后与疾病的严重程度密切相关。轻度HIE患儿预后良好，中度HIE患儿经过有效治疗，约有30%-50%的患儿遗留严重的神经系统功能障碍，而重度HIE患儿神经系统功能障碍的发生率高达90%以上[5]，该病严重影响患儿的认知功能及运动发育，对个人、家庭及社会都造成沉重的负担，且持续终身，严重影响了我国人民整体身心健康、国民经济发展和社会稳定。 在很久之前人们已经逐渐认识到，HIE并非只是由于窒息所导致或加重，单纯窒息仅仅能解释HIE中的一小部分[6]，越来越多的其他危险因素被人们所认识。近年来流行病学调查显示，临床中一些非窒息的危险因素能够加重HIE的损伤程度，尤其是母亲分娩过程中体温高于38.5℃，是加重HIE的危险因素之一，体温增高对新生儿缺氧-缺血性脑损伤(Hypoxic-Ischemic brain damage，HIBD)有更加严重的影响[7,8]。探讨体温增高对未成熟大脑HIBD后脑损伤程度的影响，引起人们对高温这一危险因素的足够重视，有利于HIBD损伤机制的研究以及临床干预和预防，这是本项目的主要研究内容之一。 到目前为止，对于HIE的管理策略都是支持性的，包括：维持组织通气和血流灌注、维持血糖在正常较高水平、控制惊厥发作、高压氧、营养脑细胞药物、早期干预康复等，但以上治疗措施都没有定向于每个具体进行性损伤的过程。因此，并不奇怪，尽管过去几十年围生医学及康复医学的快速发展， HIE导致脑瘫的发生率基本没有改变。最近，又有研究显示全身降温能够减少HIE新生儿的死亡率或中、重度残疾程度，但低体温组HIE患儿仍有44%发生了死亡、中度、重度残疾[9]。神经元丢失是许多神经系统疾病包括新生儿HIE的主要特征，不可逆的脑损伤和有限的神经干细胞分化能力是HIE患儿死亡和残疾的主要原因[10]。近几十年，由于细胞疗法的出现，为HIE患儿提供了新的希望。 干细胞是一类具有自我更新、多向分化潜能的祖细胞，能够分化成为多种不同的组织。干细胞因其广泛的分化潜能及低免疫原性成为近年来医学及生物学领域最引人注目的热点之一，从而为神经系统疾病包括新生儿HIE、脑瘫、脑卒中、脊髓损伤及神经变性疾病等治疗提供了光明的前景。现在关于干细胞的很多研究都来自骨髓，但是骨髓源性干细胞随着年龄增长其细胞数量和扩增、分化能力出现明显下降趋势，并存在病毒污染的高度危险性。而且自病人抽吸骨髓是一个侵入性、疼痛的过程，自新生儿抽吸骨髓几乎不可能。故寻找一种能够替代骨髓，并可弥补其缺陷的干细胞来源是目前的紧急任务。而最近研究发现干细胞也能够从脐带血中分离。人脐带血单核细胞（Human Cord Blood Mononuclear Cells，HCMNCs）内含丰富的造血干细胞、间充质干细胞（Mesenchymal Stem Cells，MSCs）等，拥有多重效用：移植的细胞拥有极好的迁移能力[11,12]，局部缺血性脑损伤增加了它们向损伤部位的迁移[13]；移植细胞迁移到损伤组织，可以分泌神经保护因子，保护神经细胞免受损伤和凋亡[14]；提高了内源性神经干细胞的增殖，同时抑制新产生细胞的死亡；部分可以分化进入宿主脑组织中形成星形胶质细胞样细胞，这些星形胶质细胞样细胞可以支持神经细胞的存活；在体外人类脐带血衍生的MSCs能够产生粒细胞集落刺激因子[15,16]。这些多重效用促成了它们在神经系统疾病中的治疗潜能，因此脐带血单核细胞最终有能力产生新生神经细胞替代那些受损伤或疾病的神经细胞。本研究的目的之二即是通过腹腔注射移植人HCMNCs，探讨其对HIE后远期学习记忆功能的影响及突触超微结构的改变。为此，特开展以下研究： 第一部分体温增高对缺氧缺血性脑损伤新生大鼠神经功能的影响 目的建立新生大鼠缺氧缺血性脑损伤(Hypoxic-Ischemic brain damage，HIBD)动物模型，研究体温增高对其神经功能发育的影响。方法7日龄新生SD大鼠62只，随机分成高温HIBD组、常温HIBD组、单纯高温组及常温对照组。高温HIBD组在42℃温度下进行缺氧缺血处理，常温HIBD组在37℃温度下进行缺氧缺血处理，缺氧时间均为60min。单纯高温组幼鼠仅置于42℃水浴箱中60min，不进行缺氧损伤。常温对照组幼鼠不予以任何处理。应用早期神经反射及Morris水迷宫实验评估大鼠神经行为学功能发育；后断头取脑，行普通HE染色光镜下观察，并利用透射电镜观察各组幼鼠大脑损伤部位超微结构的改变。 结果高温HIBD组与常温HIBD组、常温对照组相比，生长率降低[（54.57±10.27）比（83.05±20.42）、（80.81±16.84）]；生后14d早期神经反射实验中，悬崖调转反射[（14.60±2.83）比（12.64±1.93）、（9.69±2.76）]、阴性趋地性反射[（18.14±5.17）比（14.48±2.88）、（12.15±0.89）]、步态反射[（23.34±10.05）比（18.02±4.52）、（11.44±2.26）]均显著减慢，差异有统计学意义（P0.05）；Morris水迷宫实验中，从第3天开始逃避潜伏期延长[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）]，，穿台次数减少[（2.12±1.05）比（3.06±1.16）、（3.82±1.25）]，差异有统计学意义（P0.05）；脑组织形态学评分增加[（2.88±0.99）比（2.06±1.00）、（0.00±0.00）]；透射电镜观察突触间隙增宽[（23.45±2.28）比（21.27±1.12）、（19.45±0.79）]，差异有统计学意义（P0.05）；单纯高温组各项检测指标与常温对照组比较差异均无统计学意义（P0.05）。 结论母鼠体温增高加重了幼鼠HIBD的程度，严重影响神经功能发育；单纯母鼠体温增高对大鼠远期学习记忆功能的影响不大。 第二部分人脐带血单核细胞移植对新生大鼠缺氧缺血性脑损伤学习记忆功能的影响 目的探讨人脐带血单核细胞(HCMNCs)移植对新生大鼠缺氧缺血性脑损伤(HIBD)后远期学习记忆功能的影响及与突触超微结构改变的相关性。方法新生7日龄SD大鼠60只，随机分为3组，采用Rice法建立HIBD动物模型，缺氧缺血后24h，HIBD+移植组大鼠腹腔内注入1×107HCMNCs (在500ul的0.9%NaCl中)；HIBD+NaCl组腹腔内注入等量的生理盐水（NaCl）；另设空白对照组。生后10d及67d时行头颅磁共振成像（MRI）检查，生后35d时利用Morris水迷宫实验评估大鼠空间学习记忆能力，后断头取脑，进行鼠脑大体形态学评分，并使用透射电镜观察各组鼠脑损伤部位神经元突触超微结构的改变，与Morris水迷宫结果进行相关性分析。 结果HIBD+NaCl组与HIBD+移植组、空白对照组相比，Morris水迷宫实验中，第2天开始逃避潜伏期延长[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）]，穿台次数减少[（2.12±1.05）比（3.06±1.16）、（3.70±1.17）]，差异有统计学意义（P0.05）；脑组织形态学评分增加[（2.82±1.02）比（1.61±0.92）、（0.00±0.00）]；透射电镜观察突触间隙增宽[（26.43±2.33）比（23.19±2.11）、（21.66±4.80）]，差异有统计学意义（P0.05）；生后67d头颅MRI检查示残存脑容量减少[（61.17±6.22）比（78.11±4.01）、（100±0.00）]，差异有统计学意义（P0.05）。而HIBD+移植组与空白对照组比较，上述各项指标间差异均无统计学意义（P0.05）；在第4天的Morris水迷宫实验中，各组大鼠的逃避潜伏期与透射电镜观察突触间隙宽度之间具有高度相关性，相关系数r为0.857（P0.05）。 结论HCMNCs腹腔内移植可促进新生大鼠HIBD后损伤脑细胞的恢复及学习记忆功能的改善；Morris水迷宫实验中逃避潜伏期与透射电镜观察突触间隙宽度具有高度相关性。
[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.
缁撴灉楂樻俯HIBD缁勪笌甯告俯HIBD缁

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