【作者】 王俊伟；

【导师】 段树民；

【作者基本信息】 浙江大学 ， 神经生物学， 2012， 硕士

【摘要】 神经系统是人体结构最精细功能最复杂的系统,由数以亿万计的高度相互联系的神经细胞组成,由于神经系统发育不良及后天的病变导致的各种神经性疾病已经成为危害人类的主要疾病之一,如神经系统先天性疾病引起脑积水,软骨发育不全与颅骨畸形,脑穿通畸形等。因此研究中枢神经系统发育机制有利于我们解决神经系统疾病。ADAM10是整合素金属蛋白酶家族的一员,具有金属蛋白酶结构域,整合素结合域胞内段信号域以及粘附活性。AD AM10具有蛋白水解酶活性,如Notch,淀粉样沉淀前体分子APP,跨膜生长因子TGFa,可与整合素结合。另外ADAM10在神经系统发育中起到调节细胞增殖、迁移、分化维持细胞的生存,以及参与轴突生长和髓鞘化。Adam10常规基因敲除小鼠死于胚胎发育早期,阻碍了进一步研究Adam10基因在后续器官及细胞发育中的功能。为了解决这一问题,我们采用GFAP-Cre基因小鼠与Adam10LoxP基因小鼠进行杂交得GFAP阳性细胞条件性敲除Adam10基因的小鼠,用来完成我们的实验目的：探究小鼠ADAM10在中枢神经系统中调节作用的研究。实验方法：利用小鼠杂交的方法在GFAP阳性细胞中特异性敲除Adam10基因的小鼠模型；PCR鉴定小鼠基因型；Western blot检测ADAM10蛋白表达量；HE染色法观察基因敲除后大脑皮层发育；免疫荧光染色法标记出放射状胶质细胞、星形胶质细胞、NG2细胞、神经元,观察特异性敲除Adam10基因对这几种细胞发育的影响。实验结果：1.PCR基因鉴定小鼠基因型,Western blot检测ADAM10蛋白在实验组(基因敲除型)小鼠表达下降,实验组小鼠表型：共济失调,生长缓慢,个体瘦小、掉毛等。2.HE染色发现实验组小鼠大脑皮层结构破坏,大脑皮层较薄且细胞分层紊乱,甚至大脑缺失；侧脑室腔增大；海马受损或者缺失。3.GFAP免疫荧光组织化学结果显示对照组大脑皮层GFAP阳性细胞形态呈“脚手架”式,长而无分支的丝状突起规则分布,实验组GFAP阳性细胞没有丝状突起结构出现；Western blot结果显示实验组相比对照组GFAP蛋白表达增多。BLBP的Western blot结果显示与对照组相比实验组BLBP蛋白表达量明显减少。4.NG2免疫荧光组织化学结果显示实验组与对照组相比较细胞形态、数目都没有明显差异；Western blot结果显示NG2蛋白表达量在对照组和实验组也没有明显差异。5.NeuN免疫荧光组织化学和Western blot结果显示实验组NeuN阳性细胞减少。结论：1Adam10基GFAP阳性细胞中条件性敲除造成个体生长迟缓、瘦小、掉毛,共济失调。大脑皮层有出血点,大脑偏小或者残缺甚至缺失。2放射状胶质细胞ADAM10功能正常是大脑皮层、海马生长发育正常进行的必要条件。Adam10基因在GFAP阳性细胞中特异性敲除,造成大脑皮层结构破坏,皮层较薄且细胞分层紊乱,侧脑室腔扩大；海马受损或者缺失。3Adam10基因GFAP阳性细胞中条件性敲除,放射状胶质细胞形态发生改变；星形胶质细胞GFAP蛋白表达增多；NG2细胞的生长发育影响不明显；大脑皮层神经元的生长发育受到抑制。更多还原

【Abstract】 The central nervous system, possessing highly sophisticated structure in mammals, plays complicated roles in preventing and curing diseases. In order to understand better about nervous system related deaeases, it is pivotal to study on the mechanism of the central nervous system development. ADAM10is a member of integrin metalloproteinase family and have metalloprotease, integrin-binding, intracellular signaling and cell adhesion activities. AD AM10are important for a secretase activity of proteins such as Notch, amyloid precursor protein (APP) and transforming growth factor a (TGFa), and can bind integrins. What’s more AD AM10has been shown to play important roles in the development of the nervous system, where it regulates proliferation, migration, differentiation and survival of various cells, as well as axonal growth and myelination.Central nervous system is mainly made up of two types of cells:neurons and glia cells. And glial cells are classified into several different types:astrocyte, oligodendrocyte, radial glial cell, ependymal cell and microglial cell. In quantity, the number of glial cells in brain is about10to50times as the number of neurons and glial cells account for about half the size of the brain, in which, neurons and glial cells wrap together closely, interval width20nm and they interact with each other closely. Glial cells have many functions, such as support and nutrition neurons, repair and regeneration of the nervous system, immune response, material metabolism and center, keeping stable on nerve cells extracellular K+concentration, enhancement the synapse formation, synaptic plasticity and improvement of synaptic transmission.In this study, we established the mouse model that Adam10gene was knocked out in GFAP-positive cells to complete the experimental purpose:exploring the role of ADAM10in regulating the nerve cell development in central nervous system.Methods:To build mouse model of Adam10gene was specific knocked out in GFAP-positive cells, Cre/LoxP system was applied. Mice genotype was identified by PCR. Western blot was used to test the expression of ADAM10. HE staining was observed to compare the test group with control group. Immunofluorescence staining was used to mark astrocytes, NG2cells, neurons to see whether knockout of Adam10influence the nerve cells development.Results:1. Western blot test showed ADAM10expression decreased in the knockout group. And PCR detected genotype of the mice. The phenotype of knockout group showed ataxia, thin body, hair loss et al.2. HE staining showed that the cerebral cortex structure was damaged in knockout mice and the cell layer disorder, larger ventricular cavity; hippocampus damage or loss.3. The expression of NeuN deceased in the brain where ADAM10is knocked out in GFAP-positive cells.4. GFAP immunofluorescence staining and Western blot showed that the GFAP expression increased in knockout group comparing with the control group.5. NG2immunofluorescence staining showed that there was no significance difference of NG2cells number in knockout mice and the control group. Western blot showed NG2expression didn’t change in knockout group.Conclusion:Adam10gene is knocked out in GFAP-positive cells, seriously affecting brain development, resulting in structure of cerebral cortex damage, cortical thinning and the cell layer disorder, lateral cavity expandation, even the missing of brain, damage or loss of hippocampus. That Adam10gene is knocked out in GFAP-positive cells reduced the number of cortical neurons.更多还原