本文選題：脆性X綜合征 + 抑制性中間神經(jīng)元��； 參考：《廣州醫(yī)學(xué)院》2012年碩士論文

【摘要】：脆性X綜合征（fragile X syndrome，F(xiàn)XS）是常見的遺傳性智力低下性疾病之一，其發(fā)病率男性約為1/4000，，女性約為1/8000。其根本病因是脆性X智障基因（fragile X mental retardation1，F(xiàn)MR1）5，端非編碼區(qū)（CGG）n三核苷酸重復(fù)序列不穩(wěn)定擴增及其相鄰部位CpG島異常甲基化，使Fmr1基因失活，導(dǎo)致其編碼的產(chǎn)物--脆性X智力低下相關(guān)蛋白（fragile X mental retardation protein，F(xiàn)MRP）表達減少或缺失。臨床上主要表現(xiàn)為程度不等的智力障礙、注意力缺陷、孤獨癥及活動過度等，此外有報道20%-25%的FXS患者并發(fā)癲癇。脆性X綜合征的模型小鼠Fmr1基因敲除（knockout，KO）小鼠的許多行為學(xué)表現(xiàn)與脆性X綜合征患者非常相似，包括癲癇易感性。除聽源性驚厥易感性增加外，我們課題組前期研究還發(fā)現(xiàn)Fmr1KO鼠較野生（wild type，WT）小鼠具有更高的熱性驚厥易感性。有研究表明抑制性中間神經(jīng)元功能降低可導(dǎo)致大腦皮層興奮性增加，從而導(dǎo)致FXS癲癇的發(fā)生。我們課題組前期研究的結(jié)果也支持中間神經(jīng)元的異常參與了FXS發(fā)病機制：與WT小鼠相比，KO小鼠腦組織中小白蛋白[parvalbumin, PV)及谷氨酸脫羧酶（glutamatedecarboxylase, GAD）神經(jīng)元數(shù)量減少；海馬抑制性中間神經(jīng)元上Ⅰ型電壓門控性鈉通道（Sodium channel,voltage-gated,alpha1, Nav1.1）電流密度降低。我們課題組還發(fā)現(xiàn)2周齡Fmr1KO小鼠紋狀皮質(zhì)、顳聽皮質(zhì)、梨狀皮質(zhì)的PV細(xì)胞面積較同齡WT小鼠縮小。4周齡Fmr1KO與WT小鼠比較，相應(yīng)的CA2、CA3、顳聽皮質(zhì)以及梨狀皮質(zhì)的PV細(xì)胞面積較同齡WT小鼠減小。而我們課題組也發(fā)現(xiàn)14-17天齡的Fmr1KO小鼠GABAARα5蛋白的的表達以及在mRNA水平均低于WT小鼠。目前大量研究表明,神經(jīng)調(diào)節(jié)蛋白1（Neuregulin1）及其受體ErbB4在神經(jīng)元發(fā)育、突觸功能及可塑性、神經(jīng)遞質(zhì)的傳遞、以及N-甲基D-天冬氨酸的調(diào)節(jié)，GABAA受體、乙酰膽堿受體亞基的表達以及在維持神經(jīng)環(huán)路的平衡中起著至關(guān)重要的作用。因此本課題組提出假設(shè)：KO小鼠中NRG1-ErbB4信號的改變有可能是導(dǎo)致PV陽性中間神經(jīng)元數(shù)量減少及結(jié)構(gòu)改變的原因之一。本研究通過比較不同年齡組Fmr1KO鼠和WT鼠腦組織中NRG1和ErbB4蛋白表達量，以及NRG1和ErbB4陽性神經(jīng)元數(shù)量的差異，并觀察比較PV陽性中間神經(jīng)元上ErbB4分布的變化，探討NRG1-ErbB4信號在FXS中癲癇易感性增高中的作用。 材料與方法： FVB品系小鼠在19～21℃自然光的條件下飼養(yǎng)，讓它們自由獲取食物和水分。實驗前取其尾巴提取DNA，PCR技術(shù)鑒定小鼠基因型。隨機選取出生后2周、4周雄性Fmr1基因敲除（knockout mouse，KO小鼠，KO~(2w)和KO~(4w)）用免疫組化法檢測NRG1和ErbB4神經(jīng)元數(shù)量（每組各取6只同窩小鼠）；蛋白印跡法檢測NRG1和ErbB4蛋白含量（每組各取3只同窩小鼠）；免疫熒光雙標(biāo)記法檢測ErbB4蛋白在PV陽性中間神經(jīng)元上的表達（每組各取3只同窩小鼠）。上述所有實驗都以同齡的雄性野生型（wild type，WT）小鼠作為對照組。 結(jié)果： 1. KO與WT小鼠腦組織NRG1陽性神經(jīng)元數(shù)量的比較 KO~(2w)和KO~(4w)小鼠大腦皮質(zhì)、海馬CA1及CA3區(qū)NRG1陽性神經(jīng)元的數(shù)量均較同齡WT小鼠減少，差異有統(tǒng)計學(xué)意義；而在齒狀回區(qū)，KO~(2w)和KO~(4w)小鼠NRG1陽性神經(jīng)元均較同齡WT小鼠增加，差異有統(tǒng)計學(xué)意義。 2. KO與WT小鼠腦組織ErbB4陽性神經(jīng)元數(shù)量的比較 KO~(2w)小鼠大腦皮質(zhì)、海馬CA1區(qū)、CA3區(qū)及齒狀回區(qū)ErbB4陽性神經(jīng)元的數(shù)量均較WT~(2w)小鼠減少，差異有統(tǒng)計學(xué)意義；KO~(4w)小鼠大腦皮質(zhì)、海馬CA3區(qū)ErbB4陽性神經(jīng)元的數(shù)量均較WT~(4w)小鼠減少，差異有統(tǒng)計學(xué)意義，而海馬CA1區(qū)及齒狀回區(qū)與WT~(4w)小鼠相比差異無統(tǒng)計學(xué)意義。 3. KO與WT小鼠大腦皮質(zhì)、海馬組織中NRG1、ErbB4蛋白表達量的比較 KO~(2w)和KO~(4w)小鼠大腦皮質(zhì)NRG1、ErbB4含量分別較WT~(2w)和WT~(4w)小鼠減少，差異有統(tǒng)計學(xué)意義；KO~(2w)和KO~(4w)小鼠海馬中NRG1、ErbB4含量分別較WT~(2w)和WT~(4w)小鼠減少，差異有統(tǒng)計學(xué)意義。 4. KO與WT小鼠腦組織PV陽性神經(jīng)元ErbB4表達量的比較 KO~(2w)和KO~(4w)小鼠皮質(zhì)、海馬CA1及CA3區(qū)PV與ErbB4共標(biāo)記的神經(jīng)元上，共定位陽性神經(jīng)元均較WT~(2w)和WT~(4w)小鼠減少，差異有統(tǒng)計學(xué)意義；而海馬齒狀回區(qū)PV與ErbB4共表達的神經(jīng)元與同齡小鼠相比差異無統(tǒng)計學(xué)意義。 結(jié)論： KO~(2w)和KO~(4w)小鼠大腦皮質(zhì)及海馬NRG1、ErbB4陽性神經(jīng)元數(shù)量、NRG1、ErbB4表達量以及PV陽性神經(jīng)元ErbB4表達量均減少，提示NRG1-ErbB4信號的降低可能與KO小鼠PV陽性GABA能中間神經(jīng)元的減少有關(guān)，可能在FXS癲癇易感性增高中發(fā)揮重要作用。
[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.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R749.94

【共引文獻】

相關(guān)碩士學(xué)位論文 前1條

1 吳華平;FMRP在智力低下兒童的表達缺陷及其臨床研究[D];南昌大學(xué);2006年

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