NRG1-ErbB4信号在Fmr1基因敲除鼠脑组织中的改变及意义

发布时间：2018-04-17 05:14

本文选题：脆性X综合征 + 抑制性中间神经元　；参考：《广州医学院》2012年硕士论文

【摘要】：脆性X综合征（fragile X syndrome，FXS）是常见的遗传性智力低下性疾病之一，其发病率男性约为1/4000，，女性约为1/8000。其根本病因是脆性X智障基因（fragile X mental retardation1，FMR1）5，端非编码区（CGG）n三核苷酸重复序列不稳定扩增及其相邻部位CpG岛异常甲基化，使Fmr1基因失活，导致其编码的产物--脆性X智力低下相关蛋白（fragile X mental retardation protein，FMRP）表达减少或缺失。临床上主要表现为程度不等的智力障碍、注意力缺陷、孤独症及活动过度等，此外有报道20%-25%的FXS患者并发癫痫。脆性X综合征的模型小鼠Fmr1基因敲除（knockout，KO）小鼠的许多行为学表现与脆性X综合征患者非常相似，包括癫痫易感性。除听源性惊厥易感性增加外，我们课题组前期研究还发现Fmr1KO鼠较野生（wild type，WT）小鼠具有更高的热性惊厥易感性。有研究表明抑制性中间神经元功能降低可导致大脑皮层兴奋性增加，从而导致FXS癫痫的发生。我们课题组前期研究的结果也支持中间神经元的异常参与了FXS发病机制：与WT小鼠相比，KO小鼠脑组织中小白蛋白[parvalbumin, PV)及谷氨酸脱羧酶（glutamatedecarboxylase, GAD）神经元数量减少；海马抑制性中间神经元上Ⅰ型电压门控性钠通道（Sodium channel,voltage-gated,alpha1, Nav1.1）电流密度降低。我们课题组还发现2周龄Fmr1KO小鼠纹状皮质、颞听皮质、梨状皮质的PV细胞面积较同龄WT小鼠缩小。4周龄Fmr1KO与WT小鼠比较，相应的CA2、CA3、颞听皮质以及梨状皮质的PV细胞面积较同龄WT小鼠减小。而我们课题组也发现14-17天龄的Fmr1KO小鼠GABAARα5蛋白的的表达以及在mRNA水平均低于WT小鼠。目前大量研究表明,神经调节蛋白1（Neuregulin1）及其受体ErbB4在神经元发育、突触功能及可塑性、神经递质的传递、以及N-甲基D-天冬氨酸的调节，GABAA受体、乙酰胆碱受体亚基的表达以及在维持神经环路的平衡中起着至关重要的作用。因此本课题组提出假设：KO小鼠中NRG1-ErbB4信号的改变有可能是导致PV阳性中间神经元数量减少及结构改变的原因之一。本研究通过比较不同年龄组Fmr1KO鼠和WT鼠脑组织中NRG1和ErbB4蛋白表达量，以及NRG1和ErbB4阳性神经元数量的差异，并观察比较PV阳性中间神经元上ErbB4分布的变化，探讨NRG1-ErbB4信号在FXS中癫痫易感性增高中的作用。材料与方法： FVB品系小鼠在19～21℃自然光的条件下饲养，让它们自由获取食物和水分。实验前取其尾巴提取DNA，PCR技术鉴定小鼠基因型。随机选取出生后2周、4周雄性Fmr1基因敲除（knockout mouse，KO小鼠，KO~(2w)和KO~(4w)）用免疫组化法检测NRG1和ErbB4神经元数量（每组各取6只同窝小鼠）；蛋白印迹法检测NRG1和ErbB4蛋白含量（每组各取3只同窝小鼠）；免疫荧光双标记法检测ErbB4蛋白在PV阳性中间神经元上的表达（每组各取3只同窝小鼠）。上述所有实验都以同龄的雄性野生型（wild type，WT）小鼠作为对照组。结果： 1. KO与WT小鼠脑组织NRG1阳性神经元数量的比较 KO~(2w)和KO~(4w)小鼠大脑皮质、海马CA1及CA3区NRG1阳性神经元的数量均较同龄WT小鼠减少，差异有统计学意义；而在齿状回区，KO~(2w)和KO~(4w)小鼠NRG1阳性神经元均较同龄WT小鼠增加，差异有统计学意义。 2. KO与WT小鼠脑组织ErbB4阳性神经元数量的比较 KO~(2w)小鼠大脑皮质、海马CA1区、CA3区及齿状回区ErbB4阳性神经元的数量均较WT~(2w)小鼠减少，差异有统计学意义；KO~(4w)小鼠大脑皮质、海马CA3区ErbB4阳性神经元的数量均较WT~(4w)小鼠减少，差异有统计学意义，而海马CA1区及齿状回区与WT~(4w)小鼠相比差异无统计学意义。 3. KO与WT小鼠大脑皮质、海马组织中NRG1、ErbB4蛋白表达量的比较 KO~(2w)和KO~(4w)小鼠大脑皮质NRG1、ErbB4含量分别较WT~(2w)和WT~(4w)小鼠减少，差异有统计学意义；KO~(2w)和KO~(4w)小鼠海马中NRG1、ErbB4含量分别较WT~(2w)和WT~(4w)小鼠减少，差异有统计学意义。 4. KO与WT小鼠脑组织PV阳性神经元ErbB4表达量的比较 KO~(2w)和KO~(4w)小鼠皮质、海马CA1及CA3区PV与ErbB4共标记的神经元上，共定位阳性神经元均较WT~(2w)和WT~(4w)小鼠减少，差异有统计学意义；而海马齿状回区PV与ErbB4共表达的神经元与同龄小鼠相比差异无统计学意义。结论： KO~(2w)和KO~(4w)小鼠大脑皮质及海马NRG1、ErbB4阳性神经元数量、NRG1、ErbB4表达量以及PV阳性神经元ErbB4表达量均减少，提示NRG1-ErbB4信号的降低可能与KO小鼠PV阳性GABA能中间神经元的减少有关，可能在FXS癫痫易感性增高中发挥重要作用。
[Abstract]:Fragile X syndrome (fragile X, syndrome, FXS) is one of the most common inherited mental retardation disease, the incidence rate of male is about 1/4000, the female is about 1/8000. is the fundamental cause of fragile X mental retardation gene (fragile X mental retardation1 FMR1, 5), non encoding region (CGG) n trinucleotide repeat sequence is not stable amplification and adjacent parts of abnormal methylation of CpG Island, the inactivation of the Fmr1 gene, leading products: fragile X mental retardation protein encoding (fragile X mental retardation protein, FMRP) reduced or absent expression. The main clinical performance for varying degrees of mental disorder, attention deficit hyperactivity and autism, etc. in addition, there are FXS patients with epilepsy 20%-25%. A mouse model of Fmr1 gene of fragile X syndrome (knockout, KO) knockout mice many behavior with fragile X syndrome were very similar, including seizure susceptibility. In addition to increase the susceptibility of source, preliminary studies of our group found that Fmr1KO rats compared with wild-type (wild type, WT) in mice with febrile seizure susceptibility higher. Studies have shown that inhibitory interneuron function can lead to decrease the excitability of the cerebral cortex increased, resulting in the occurrence of epilepsy. The results of our FXS project preliminary studies also support interneurons may be involved in the pathogenesis of FXS: compared with WT mice, KO mice brain tissue and albumin [parvalbumin, PV) and glutamate decarboxylase (glutamatedecarboxylase, GAD) to reduce the number of neurons; inhibitory interneurons of voltage-gated sodium channels in hippocampal Sodium (channel, voltage-gated, alpha1 Nav1.1), current density decreased. Our research group also found that 2 week old Fmr1KO mice striate cortex, temporal auditory cortex, piriform cortex cell surface PV product compared with age-matched WT mice reduced Small.4 week old Fmr1KO and WT mice, the corresponding CA2, CA3, temporal auditory cortex and piriform cortex of the PV cell area compared with age-matched WT mice decreased. And our research group also found that 14-17 day old Fmr1KO mice GABAAR alpha 5 protein expression and were lower than those of WT mice at mRNA level. At present, a large amount of research shows that neuregulin 1 (Neuregulin1) and its receptor ErbB4 in neuronal development, synaptic function and plasticity, neurotransmitter, and regulation of N- methyl D- aspartate receptor GABAA, plays a crucial role in the expression of nicotinic acetylcholine receptor subunits and balance in maintaining neural loop in this. The research group put forward a hypothesis: NRG1-ErbB4 signal in KO mice the change may be the cause of changes in structure and the number of PV positive interneurons decreased. This study compares the different age groups Fmr1KO and WT rats brain tissue of N The expression of RG1 and ErbB4, and the difference in the number of NRG1 and ErbB4 positive neurons, and to observe the change of ErbB4 distribution in PV positive interneurons, and to explore the role of NRG1-ErbB4 signal in the increase of epilepsy susceptibility in FXS.
Materials and methods:
FVB mice at 19~21 DEG C under the natural light condition feeding, give them free access to food and water before the experiment. The tail extraction DNA, PCR technology to identify mouse genotypes. Randomly selected 2 weeks after birth, 4 week male Fmr1 knockout mice (knockout mouse, KO, KO~ and KO~ (2W) (4W)) were detected by immunohistochemistry and NRG1 ErbB4 neurons (n = 6 littermate mice); Western blot was used to detect NRG1 and ErbB4 protein content (n = 3 littermate mice); expression of ErbB4 protein was detected by immunofluorescence double staining in PV positive interneurons (n = on the 3 littermate mice). Male wild type all of the above experiments with age (wild type, WT) mice as the control group.
Result:
Comparison of the number of NRG1 positive neurons in the brain tissue of 1. KO and WT mice
The number of NRG1 positive neurons in cerebral cortex, hippocampus CA1 and CA3 region of KO~ (2W) and KO~ (4W) mice was decreased compared with that of the same age WT mice, but the difference was statistically significant in the hippocampus, but in the dentate gyrus, the positive neurons in KO~ (2W) and KO~ (KO~) mice increased compared with the same age mice.
Comparison of the number of ErbB4 positive neurons in the brain tissue of 2. KO and WT mice
KO~ (2W) in cerebral cortex, hippocampus CA1 region, CA3 region and dentate gyrus of the number of ErbB4 positive neurons was significantly lower than that of WT~ (2W) were decreased, the difference was statistically significant; KO~ (4W) in the cerebral cortex, the number of CA3 positive neurons in hippocampal ErbB4 region were lower than WT~ (4W) mice have reduced statistically significant difference, while the hippocampus CA1 and dentate gyrus (4W and WT~) had no significant difference compared to mice.
Comparison of the expression of NRG1 and ErbB4 protein in the cerebral cortex and hippocampus of 3. KO and WT mice
The contents of NRG1 and ErbB4 in cerebral cortex of KO~ (2W) and KO~ (4W) mice were decreased compared with those of WT~ (2W) and WT~ (4W) mice, and the difference was statistically significant.
Comparison of the ErbB4 expression of PV positive neurons in the brain tissue of 4. KO and WT mice
KO~ (2W) and KO~ (4W) PV and ErbB4 mice cortex, CA1 and CA3 in hippocampus were labeled neurons, CO localization of positive neurons was significantly lower than that of WT~ (2W) and WT~ (4W) were decreased, the difference was statistically significant; and dentate gyrus neurons PV and ErbB4 co expression difference with the age of mice was not statistically significant.
Conclusion:
KO~ (2W) and KO~ (4W) in mouse cerebral cortex and hippocampus of NRG1, number of ErbB4 positive neurons in NRG1, ErbB4 expression and PV expression of ErbB4 positive neurons decreased, suggesting that NRG1-ErbB4 may reduce the signal with KO mice PV positive GABA can reduce intermediate neurons, may play an important role in FXS increased seizure susceptibility.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.94

【共引文献】