天麻素对海人酸干预的神经干细胞分化的影响

发布时间：2018-01-09 12:37

本文关键词：天麻素对海人酸干预的神经干细胞分化的影响　出处：《郑州大学》2009年硕士论文　论文类型：学位论文

【摘要】：目的和背景自90年代科学家们分离、培养出神经干细胞(neural stem cells,NSCs)以来,人们相继从各种动物及人的中枢神经系统(central neural system,CNS)内分离、培养出了神经干细胞,因其具有很强的分裂、增殖及自我更新能力,从而打破了传统上认为神经细胞不能再生的观念。神经干细胞的发现对神经损伤、神经退行性疾病等的治疗以及深入研究动物的生长发育和分化具有重要的意义,为中枢神经系统的结构和功能重建提供了新的手段,具有广阔的应用前景,因而神经干细胞的研究也就成了当今生命科学的研究热点。从神经干细胞的分离、体外培养及其生物学特性的研究,到移植治疗神经系统疾病,都取得了可喜的成绩。但中枢神经系统的再生是一个十分复杂的课题,神经干细胞在体内如何分化也是个难题,尚存在大量的未知问题需要进一步深入研究。缺氧缺血性脑病是临床常见病、多发病,如脑血栓、脑梗塞、脑血管痉挛等,其致死和致残率很高。缺氧缺血性脑病也常发生于围产期的胎儿和新生儿,不仅可引起患儿死亡,而且是造成脑瘫、癫痫及智力发育迟缓或智力低下的重要原因。据统计,国内每年约有75万新生儿发生窒息,约有25万婴儿因此成为伤残儿或低智儿童。近年来的研究表明:缺血再灌注可导致脑内兴奋性氨基酸(EAA)的大量释放,并对神经细胞产生兴奋性毒性作用,兴奋性氨基酸的毒性作用可能是通过以下两条途径产生:一是主要由氨基-3-羟基-5-甲基-4-异恶唑丙酸受体(AMPA-R)和海人藻酸受体(KA-R)过度兴奋所介导的神经细胞急性渗透性肿胀,以Na~+内流为特征,可在数小时内发生;二是主要由N-甲基-D-天(门)冬氨酸受体(NMDA-R)过度兴奋所介导的神经细胞延迟性损伤,以Ca~(2+)内流为特征,可在数小时至数日后发生,细胞内钙离子超载可能是兴奋性氨基酸引起细胞损伤或死亡的共同病理学机制。兴奋性氨基酸受体过度兴奋可导致神经细胞急性渗透性肿胀或延迟性损伤,可能导致神经功能缺损、智能落后、姿势异常等后遗症。因此,本研究旨在观察天麻素在脑缺血后兴奋性氨基酸增多时的作用,研究其在神经干细胞分化中的影响,是否可诱导神经干细胞向神经元方向分化,改善脑缺氧后由于神经干细胞向胶质细胞分化导致的认知或肢体活动障碍,这对于研究如何诱导神经干细胞在缺血缺氧后向神经元分化,研究其在缺血性脑血管病、神经系统变性疾病、癫痫、神经损伤等疾病中的应用具有重要意义。材料与方法以新生24h内的Wistar大鼠为实验动物,体外分离并培养神经干细胞,传代培养,并进行细胞单克隆实验。在神经干细胞分化时加海人酸和天麻素干预,海人酸浓度分别为100μmol/l、500μmol/l、1mmol/l、2mmol/l、5mmol/l,天麻素浓度为0.5mg/l。免疫荧光法和免疫组化SP法检测加入海人酸和天麻素干预后神经干细胞分化后细胞比例的变化,AO/EB荧光双染检测加入海人酸后部分神经干细胞的凋亡,MTT比色法测定海人酸对神经干细胞分化速度的影响。结果 1.神经干细胞可以分化为神经元和胶质细胞,其比例为23.07%±1.51%和76.93%±1.51%。 2.KA干预组神经元和胶质阳性细胞比例分别为13.99%±2.07%和86.01%±2.07%。不同浓度的海人酸干预的组之间神经元和胶质细胞比例无明显差异(F=1.43,p＞0.05),正常分化组与KA干预组所分化细胞比例有显著统计学差异(F=368.21,p＜0.001),海人酸可以诱导神经干细胞向胶质细胞分化。 3.海人酸和天麻素干预组神经元和胶质阳性细胞比例分别约为17.16%±2.14%和82.84%±2.14%,与只加海人酸组相比差异有统计学意义(F=115.6,p＜0.01),天麻素可可在一定程度上减少海人酸兴奋性的影响,诱导神经干细胞向神经元分化。 4.MTT检测加入海人酸和天麻素后早期部分神经干细胞死亡,但可促进存活的神经干细胞分化。结论 1.本实验所分离细胞的可分化为神经元、少突胶质细胞和星形胶质细胞,是神经干细胞。 2.海人酸可以诱导神经干细胞向胶质细胞分化。 3.天麻素可在一定程度上减少海人酸兴奋性的影响,诱导神经干细胞向神经元分化。
[Abstract]:Purpose and background
From 90s scientists, culture of neural stem cells (neural stem cells, NSCs), have been from the central nervous system of various animal and human (central neural, system, CNS) in isolation, cultured from neural stem cells, because of its strong division, proliferation and self-renewal capacity, thus breaking the the traditional idea that neural cells cannot regenerate. The discovery of neural stem cells on neural injury, plays an important role in the treatment of neurodegenerative diseases such as growth and differentiation of animal research, provides a new method for central nervous system structure and function reconstruction, so it has broad application prospects. Neural stem cell research has become the research hotspot of life science. From neural stem cell separation, and Study on the biological characteristics of cultured in vitro, to transplantation for the treatment of diseases of the nervous system, are taken Gratifying achievements have been made. However, the regeneration of central nervous system is a very complex subject. How to differentiate neural stem cells in vivo is also a difficult problem. There are still a lot of unknown problems that need further study.
Hypoxic ischemic encephalopathy is a common clinical disease, frequently occurring disease, such as cerebral thrombosis, cerebral infarction, cerebral vasospasm, the mortality and disability rate is very high. Hypoxic ischemic encephalopathy often happen to perinatal fetuses and newborns, can not only cause of death in children, but also is an important reason causing cerebral palsy, epilepsy and mental retardation or low intelligence. According to statistics, about 750 thousand of neonatal asphyxia at home every year, about 250 thousand babies become disabled children or children with low intelligence. Recent studies show that ischemic reperfusion can lead to brain excitatory amino acid (EAA) release, and produce excitatory toxic effect on nerve cells, toxicity excitatory amino acids may be produced through the following two ways: one is mainly composed of -3- amino hydroxy -5- methyl -4- isoxazole propionate receptor (AMPA-R) and kainate receptor (KA-R) mediated by nerve hyperexcitability fine Acute cellular osmotic swelling, with Na~+ internal flow characteristics, can occur within hours; the two is mainly composed of N- methyl -D- aspartate receptor (door) (NMDA-R) delayed injury hyperexcitability mediated by nerve cells in Ca~ (2+) internal flow characteristics, can occur in several the number of hours to days after, intracellular calcium overload may be the common pathological excitatory amino acids induced cell injury or death. The mechanism of excitatory amino acid receptors can lead to neuronal hyperexcitability of acute osmotic swelling or delayed injury may lead to neurological impairment, intelligent backward, abnormal posture and other sequelae.
Therefore, the purpose of this study was to observe the gastrodin in excitatory amino acids after cerebral ischemia increased the role of the research in the effect of neural stem cell differentiation, induce differentiation of neural stem cells into neurons, improve the brain after hypoxia for neural stem cell differentiation cognitive or limb movement disorder leads to glial cells, the for the study of how to induce cell differentiation to neurons in hypoxia ischemia after neural stem on the ischemic cerebrovascular disease, epilepsy, neurodegenerative diseases, nervous system diseases, has important significance in the application of nerve injury.
Materials and methods
In the neonatal Wistar rats within 24h as experimental animal, isolate and culture neural stem cells, cultured, and cell clone experiments. Plus kainic acid and gastrodin in the differentiation of neural stem cells when hormone intervention, kainic acid concentrations were 100 mol/l, 500 mol/l, 1mmol/l, 2mmol/l, 5mmol/l. The change of gastrodin concentration of 0.5mg/l. by immunofluorescence and immunohistochemistry SP method with kainic acid and gastrodin percentage of stem cells after neural stem cell differentiation and prognosis, AO/EB fluorescent staining with cell apoptosis after kainic acid part of neural stem, to determine the effects of kainic acid on the differentiation of neural stem cells speed MTT colorimetric method.
Result
1. neural stem cells can differentiate into neurons and glial cells, the proportion of which is 23.07% + 1.51% and 76.93% + 1.51%.
No significant difference between the proportion of neurons and glial cells 2.KA positive cells proportion of intervention group neurons and glia were 13.99% + 2.07% and 86.01% + 2.07%. different concentrations of kainic acid intervention group (F=1.43, P > 0.05), normal group and KA intervention group the differentiation proportion of differentiated cells with significant difference (F=368.21, P < 0.001), kainic acid can induce neural stem cells to differentiate into glial cells.
3. kainic acid and gastrodin intervention group positive cells were neurons and glia were approximately 17.16% + 2.14% and 82.84% + 2.14%, compared with only kainic acid group had significant difference (F=115.6, P < 0.01), gastrodin kainic acid reduced cocoa excitatory effects induced in a certain extent. Differentiation of neural stem cells into neurons.
4.MTT was used to detect the early death of some neural stem cells after the addition of sea human acid and gastrodin, but it could promote the differentiation of surviving neural stem cells.
conclusion
1. the cells isolated from the experiment can differentiate into neurons, oligodendrocytes and astrocytes, which are neural stem cells.
2. sea human acids can induce neural stem cells to differentiate into glial cells.
3. gastrodin can reduce the effect of the excitability of the sea human acid to some extent and induce the neural stem cells to differentiate into neurons.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329;R285

【参考文献】