经鼻给予不同浓度的NGF对SOD1-93A小鼠内源性神经干细胞增殖作用影响的研究

发布时间：2018-05-21 13:06

  本文选题：经鼻给药 + 运动神经元病　； 参考：《河北医科大学》2014年硕士论文

【摘要】：目的：运动神经元病（motor neuron disease，MND）是一组病因未明的选择性侵犯脊髓前角细胞、脑干运动神经元、皮层锥体细胞及锥体束的慢性进行性神经变性疾病。肌萎缩侧索硬化(amyotrophic lateral sclerosis，ALS)是运动神经元病中发病率最高的一种类型，是选择性累及脊髓前角细胞、脑干运动神经核以及大脑运动皮质的进行性致死性神经变性病，以同时累及上、下运动神经元为特点[1-2]。患者多在首次出现症状后的3～5年内因肌肉逐渐萎缩无力、呼吸衰竭而死亡。目前，临床上除利鲁唑可以有限的延缓患者病程外，尚无有效治疗手段[3]。干细胞移植治疗、基因治疗等新方法尚在研究中[4-6],家族性ALS占5～10％，其中20％与铜／锌超氧化物歧化酶(SODl)基因突变有关。 神经生长因子（nerve growth factor,NGF）由Levi Montalcini于20世纪50年代首次在小鼠肉瘤细胞中发现。NGF具有维持神经干细胞的存活，促进神经干细胞的增殖、分化及迁移，抑制细胞凋亡，营养神经元，保护和修复受损神经等效应[7]。NGF可促进体外培养的神经前体细胞分化成未成熟的神经元和星形胶质细胞。NGF有促进神经干细胞存活、增殖、分化及定向迁移等作用，有助于中枢神经系统损伤后神经的再生及修复,为MND治疗带来了希望。由于肝脏的首过效应和血脑屏障(Blood-brain barrierBBB)的阻挡，经典的静脉及皮下给药途径并未使药物最大程度的发挥效应。SOD1—G93A转基因小鼠是国际上公认、代表家族性ALS的动物模型。所以，本实验研究采用经鼻给予SOD1小鼠不同浓度的NGF观察其脑内海马齿状回的BrdU、Nestin的表达及神经元的数量的方法，，评价其对SOD1转基因小鼠内源性神经干细胞的增殖调节作用，旨在寻找一种可以使NGF绕过BBB并在中枢靶部位迅速达到最佳效应浓度的新型给药途径。 方法： 1实验模型及分组：100天雌性非转基因正常对照组小鼠6只，分为A组；国际公认的100天（症状期）SOD1-G93A雌性转基因小鼠36只(均由中国医学科学院实验动物研究中心提供)，随机分为:B组（空白对照组）6只；C组（经鼻给药组）30只。再按照经鼻给予NGF不同浓度，将经鼻给药组分为：C1组（15mg/kg浓度组）；C2组（20mg／kg浓度组）、C3组（25mg／kg浓度组）、C4组（30mg／kg浓度组）、C5组（35mg／kg浓度组），每组6只。 2给药途径及方法：A组及B组经鼻给予生理盐水，C组经鼻给予NGF，给药量每次给予5ul，间隔2min再次给药，根据各组分别给予总量A:15ul、B:15ul、C1:15ul、C2:20ul、C3:25ul、C4:30ul、C5:35ul。用同样给药方法连续给3天药。 3各组随机取一半标本通过免疫组织化学染色以显示内源性神经干细胞及脑内增殖细胞。进行图像分析并计数Nestin和Brdu阳性细胞数目，分析比较A组、B组、C组之间脑内神经细胞增殖及内源性神经干细胞增殖和分化的变化情况。 4另一半标本腹腔内注射戊巴比妥钠（40mg/Kg）对小鼠进行麻醉后，断头取脑，脑组织中分离出海马组织，采用Western-Blot法检测Nestin和Brdu蛋白表达水平。 结果： 1. H.E.及Masson染色结果显示：A组中，海马齿状回存在较少BrdU阳性细胞。B组皮质均有较多的Nestin和Brdu阳性细胞，同时双侧的海马齿状回区也有Nestin阳性细胞，但该区域Brdu阳性细胞数量相对较少；但C1-C5组Nestin和Brdu阳性细胞数目均有明显增多(P0．05)；当NGF浓度达到25mg／kg以后，随着浓度增加，Nestin和Brdu阳性细胞增殖数目较以前增殖不明显。 2.Western Blot相关蛋白基因变化：Western blot检测结果显示：正常对照组脑内有一定水平的Nestin和Brdu蛋白表达；B组小鼠相对于正常对照组小鼠海马区齿状回Nestin和Brdu蛋白表达较高。C组中随着经鼻给药剂量逐渐加大，Nestin和Brdu蛋白表达也随之逐渐增高，但C4、C5组蛋白表达较C3组有所减弱，差异有显著性意义(P0．01)。 结论： 1.症状期SOD1转基因小鼠脑内发现有内源性神经干细胞增殖，说明本实验操作效果明显。 2.NGF对SOD1转基因小鼠脑内作用与其给药量密切相关。低给药量时(15ul)，主要表现为对脑内某些神经元的保护作用，对内源性神经干细胞增殖作用较强，当给药量大于25ul以上时，对神经干细胞的增殖作用不明显，当给药量达到35ul时，小鼠可产生明显的不良反应症状。
[Abstract]:Objective: motor neuron disease (MND) is a group of unknown causes of selective invasion of the spinal cord anterior horn cells, brainstem motoneurons, cortical pyramidal cells and pyramidal bundles of chronic progressive neurodegenerative disease. Amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS) is the highest incidence of motor neuron disease. A type of progressive neurodegenerative neurodegenerative disease involving the spinal cord anterior horn cells, the brainstem motor nucleus, and the motor cortex of the brain, which is involved in the simultaneous involvement of the lower motor neurons in the [1-2]. patients in the 3~5 years after the first symptom of the onset of the atrophy of the muscles and the death of respiratory failure. In addition to the limited delay in the course of the patient's disease, there is no effective treatment for [3]. stem cell transplantation. The new method of gene therapy, such as gene therapy, is still in the study of [4-6], and familial ALS accounts for 5 to 10%, of which 20% are related to the mutation of copper / zinc superoxide dismutase (SODl) gene.
Nerve growth factor (NGF) was first found in mouse sarcoma cells by Levi Montalcini in 1950s that.NGF has the survival of neural stem cells, promoting the proliferation, differentiation and migration of neural stem cells, inhibiting apoptosis, nourishing neurons, protecting and repairing damaged nerve effects [7].NGF can promote in vitro. The differentiation of cultured neural precursor cells into immature neurons and astrocytes.NGF can promote the survival, proliferation, differentiation and directional migration of neural stem cells, which can help the regeneration and repair of the nerve after the central nervous system injury, and bring hope for the treatment of MND. The first effect of the liver and the blood brain barrier (Blood-brain Barr) IerBBB), the classical vein and subcutaneous administration pathway did not make the maximum effect of the drug.SOD1 G93A transgenic mice as an internationally recognized animal model representing familial ALS. Therefore, this experiment studied the BrdU of the dentate gyrus of the intracerebral sea, the expression of Nestin, and the NGF of different concentrations of the SOD1 mice given to SOD1 mice. The number of neurons is used to evaluate the proliferation regulation of endogenous neural stem cells in SOD1 transgenic mice, aiming to find a new way to make NGF bypass BBB and quickly reach the best effect concentration at the central target site.
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