【作者】 王岩；

【导师】 潘杰；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2010， 硕士

【摘要】 脑积水是以脑萎缩和脑脊液流动障碍为主要特点的神经系统疾病,多种因素参与该疾病的形成。转化生长因子β1(TGF-β1)是一种重要的生长信号分子,其主要通过TGF-β1/Smad信号通路发挥生物学效应,在细胞外基质的积聚中起重要作用。研究表明,TGF-β1过表达的小鼠能够自发产生脑积水,其机制可能同基质金属蛋白酶-9(MMP-9)和基质金属蛋白酶组织抑制剂-1(TIMP-1)的异常表达有关;在发生交通性脑积水小鼠的脑组织中,也发现TGF-β1表达量升高。我们在利用脂代谢关键基因(apoE/LDLR,AL以及apoE)敲除的动脉粥样硬化小鼠模型的研究中发现不同年龄和性别的个体会自发性地发生脑部膨隆,脑室被不同程度的积水填充。为了探究其发生机制,本实验利用AL双基因敲除小鼠,主要研究了TGF-β1/Smad信号通路相关因子以及MMP-9和TIMP-1在该小鼠脑积水发生中的作用。我们选用不同年龄、不同饲喂条件的脑积水AL小鼠及非脑积水对照小鼠,利用RT-PCR、qRT-PCR、Western免疫印记等方法,检测了小鼠脑组织中TGF-β1/Smad、MMP-9、TIMP-1等因子的表达变化特征,以期了解该脂代谢关键基因缺失小鼠发生脑组织发育异常以及脑积水发生的可能机制。此外,通过伊文思蓝法及脑电图分别测定了apoE-/-/LDLR-/-、apoE-/-脑积水小鼠及同年龄对照小鼠血脑屏障通透性变化及脑细胞电活动情况,以期了解脑积水小鼠血脑屏障通透性及脑电活动是否出现异常。通过实验得到如下结果:1、不同年龄及不同饲喂条件下的AL小鼠均出现脑发育异常的个体。经统计,病变个体约占小鼠总体数目的5%-10%。病变小鼠脑实质发生萎缩,脑室扩大,皮层变薄,脑室内被不同程度的积水填充,确定该病症为脑积水。2、通过对五组不同年龄、饲喂条件下脑积水小鼠及对照小鼠TGF-β1/Smad信号通路mRNA表达水平的测定,发现脑积水小鼠脑组织中该信号通路相关因子的表达量与正常小鼠相比有差异;但在这五组脑积水小鼠的不同组之间相比,该通路相关因子变化趋势并不一致。3、通过对五组不同年龄、饲喂条件下脑积水小鼠及对照小鼠脑组织中MMP-9/TIMP-1 mRNA表达水平的测定,我们发现,总体而言,脑积水小鼠脑组织中MMP-9 mRNA表达量较对照组小鼠降低,TIMP-1 mRNA表达量较对照组升高。4、通过对两组不同年龄脑积水小鼠及对照小鼠血脑屏障通透性的测定,发现脑积水小鼠同正常小鼠相比,血脑屏障通透性无明显变化。5、通过对脑积水小鼠及对照小鼠脑电图的测定,发现脑积水小鼠同正常小鼠相比,脑电图出现明显异常,脑电波幅值显著增大。上述实验结果提示,(1)aopE-/-/LDLR-/-小鼠(以及apoE-/-小鼠)以较高频率发生脑积水,具体病症表现为脑实质发生萎缩,脑室系统扩张,皮层变薄,脑室内被积水填充。(2)TGF-β1信号通路参与该小鼠脑积水的发生、发展过程,但其具体作用机制尚不明确,推测该信号通路可能在脑积水发生的不同阶段发挥不同的作用。(3)在脑积水小鼠脑组织中存在MMP-9/TIMP-1调节的失衡,MMP-9/TIMP-1参与细胞外基质的积聚,推测在脑积水小鼠中,该平衡的失调可能通过促进细胞外基质的积聚,参与该病的发生。(4)该小鼠脑积水的发生与血脑屏障通透性异常可能无直接关系。(5)小鼠发生脑积水时,脑电图出现异常变化,表明脑细胞的电活动发生异常。推测是由于脑脊液的积聚导致脑部特定区域出现缺血及继发性脑周白质萎缩,最终出现脑细胞电生理兴奋性增高现象。更多还原

【Abstract】 Hydrocephalus is a central nervous system disease characterized by brain atrophy and abnormal cerebrospinal fluid flow. Many factors are related with the development of hydrocephalus, such as abnormality, infection and subarachnoid hemorrhage, etc.Transforming growth factorβ1 is an important growth factor involved in individual development. Previous studies indicate that TGF-β1 overexpressing mice can develop hydrocephalus spontaneously, by regulating the expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in the brain. Besides, the expression of TGF-β1 is also found to be up-regulated in the brain of hydrocephalic mice.During our research of the artherosclerosis mouse model--apoE/LDLR double knockout mice, we find that approximately 5%-10% of them manifest abnormal brain development at different age or of different sex. The lateral ventricle were hydrocephalic. In order to investigate the mechnism of this disease, we use this double knockout mice to study the role of TGF-β1/Smad pathway as well as MMP-9 and TIMP-1 during hydrocephalus. RT-PCR, qRT-PCR, Western blot had been used to detect the expression characters of TGF-β1/Smad signaling pathway as well as MMP-9 and TIMP-1. We aim at investigating the mechnism of abnormal brain development and hydrocephalus of the key lipid metabolism gene knockout mice. Moreover, for the detection of blood brain barrier permeability of the hydrocephalic mice, peritoneal injection of Evans blue was practiced and the the content of Evans blue in the brain tissue was determined. To find whether these hydrocephalic mice differ electrophysiologically from non-hydrocephalic mice, electrocorticographics of the hydrocephalic as well as the control mice were recorded.Our research revealed that:1. AL mice of different age and diets are inclined to develop abnormal brain and the ratio is approximately 5%-10%. There are brain atrophy, enlarged brain lateral, thinned cortex in the diseased mice, and the disease is classified as hydrocephalus due to the hydrocephalic brain.2. Assays of mRNA expression levels of TGF-β1/Smad signal pathway revealed that the expression level of hydrocephalic mice was different from that of control mice, while the tendency was different between different groups.3. Assays of mRNA expression levels of MMP-9 and TIMP-1 revealed that the MMP-9 expression was down-regulated in the hydrocephalic mice on the whole, while the expression of TIMP-1 was up-regulated in the hydrocephalic mice.4. Assays of blood-brain barrier permeability of two different groups revealed that no significant difference of permeability existed between the hydrocephalic mice and the control mice.5. Electroencephalography assays revealed that abnormal electroencephalography signs appeared in the mice with hydrocephalus. The EEG wave amplitude of the hydrocephalic mice was significantly increased.The results implied that,(1) AL as well as apoE-/- mice are inclined to develop hydrocephalus frequently, and there are brain atrophy, enlarged brain lateral, thinned cortex in the diseased mice. (2) TGF-β1/Smad signal pathway took part in the development of hydrocephalus, while the mechnism by which this occurs remains obscure. It is estimated that the pathway exerts different roles during different stage of hydrocephalus. (3) The imbalance of MMP-9/TIMP-1 regulation of the hydrocephalic mice might participate in the develoment of this disease by promoting the accumulation of extracellular matrix. (4) The development of hydrocephalus is not directly related to abnormality of blood-brain-barrier permeability. (5) The electroencephalography signs in the hydrocephalic mice revealed abnormal electric activity of the brain cells. It is probably that the accumulation of cerebrospinal fluid induced ischemia in certain area of brain and insecondary atrophy of pericerebral white matter, which lead to the increased electric excitability of the brain cells.更多还原