溴结构域包含蛋白2在小鼠脑内的表达及其对皮质神经元突起发育的影响
发布时间：2018-07-15 14:10
【摘要】：大脑皮质神经元在机体活动中发挥多种重要的生理功能,是大脑发挥调节作用的基本结构和功能单位。大脑皮质的发育形成是一个多步骤、复杂的渐进过程,在许多因素的严谨调控下,分步骤、有次序地进行。在皮质的发育过程中,内源性因素或外源性因素会通过影响神经干细胞的增殖分化、神经元的迁移定位以及神经突起的数量、分支、形态、方向等对皮质的发育造成影响,从而改变正常机体的生理功能。因此,对大脑皮质发育各环节参与的分子机制研究尤为重要。溴结构域包含蛋白2(Bromodomain containing protein 2,Brd2)是溴结构域包含蛋白家族成员之一,其作为主要的表观遗传调节因子在一些临床疾病,如癌症、肥胖、2型糖尿病以及神经精神性疾病中发挥重要作用。此外,Brd2亦通过调控多种靶蛋白的转录,对细胞增殖、分化、细胞周期甚至凋亡进行调控。既往研究表明Brd2在神经系统胚胎的早期发育过程中具有丰富的表达,神经元增殖后,Brd2能够利用基因调控丰富新生神经元的细胞种类;而如果缺失Brd2,将会引起神经前体细胞的增殖变多,更严重的会导致神经管延迟闭合。这些研究表明,Brd2在神经系统的发育过程中作用明显。然而,当前关于Brd2在哺乳动物神经系统正常发育中的分布及作用机制的研究还比较匮乏,亟需开展研究。因此本文通过综合应用发育神经生物学研究方法和形态学方法,探讨Brd2在中枢神经系统中的定位分布以及其细胞化学特点,探究Brd2对皮质神经元发育的影响及其机制,为进一步阐明Brd2在大脑发育过程中的调控提供有力的研究证据。1.Brd2在成年小鼠大脑皮质的定位分布及细胞化学特点运用荧光原位杂交技术观察了成年小鼠脑内Brd2基因的分布模式;借助荧光原位杂交技术联合免疫荧光染色技术,对Brd2阳性细胞的细胞化学特点进行了观察。结果如下:1)荧光原位杂交组织化学结果显示在成年小鼠脑内,Brd2 m RNA杂交信号在前脑主要表达于大脑皮质、尾壳核、海马、基底外侧杏仁核,在缰核、丘脑以及下丘脑等区域也有部分表达;在中脑主要表达于导水管周围灰质;在后脑及延髓主要表达于孤束核、小细胞网状核以及小脑皮质等。2)荧光原位杂交联合免疫荧光标记技术结果显示,在成年小鼠大脑皮质中,brd2mrna阳性信号分布较广泛,主要分布于大脑皮质ii-iii层和v-vi层,在i层和iv层亦有少量分布。其中大多数阳性信号与神经元标志物neun共存,而与星形胶质细胞标志物gfap不共存。提示在成年大脑皮质中brd2主要表达于神经元中,可能对神经元的发育调控发挥作用。3)借助brd2mrna荧光原位杂交联合免疫荧光标记技术,我们观察了大脑皮质中brd2阳性神经元与gaba能中间神经元及其亚类(pv,som,cb)的共存关系。结果显示brd2/gaba双标记神经元约占brd2阳性神经元的21%,约占gaba阳性神经元的68%;brd2/pv双标记神经元约占brd2阳性神经元的12%,约占pv阳性神经元的27%;brd2/som双标记神经元约占brd2阳性神经元的15%,约占som阳性神经元的92%;brd2/som双标记神经元约占brd2阳性神经元的23%,约占cb阳性神经元的72%。2.下调brd2对体外培养皮质神经元突起形态的影响从体外培养皮质神经元的转染率、神经元活性以及树突分支情况角度对非病毒基因转染技术进行了对比,表明脂质体转染优于电穿孔转染。之后借助优化的脂质体转染体系,观察下调brd2对体外培养皮质神经元形态的影响。结果如下:1)电穿孔转染对神经元的存活率影响较小,而神经元存活率随着用于转染脂质体的体积增加而减少。2)sholl分析结果显示树突分支的复杂性具有距离依赖性,在20-50μm为半径的圆轨迹内存在最复杂的树枝状分支;不同转染条件显著影响树突分支,在距胞体相同距离的情况下,神经元突起与同心圆交点个数最多的实验组为4μl脂质体转染组,最少的实验组为电穿孔转染组。此外还观察到不同组别的树突总长度有显著不同。实验组中4μl脂质体转染组神经元突起总长度最长,最短实验组为电穿孔转染组。3)借助3μl脂质体转染体系,针对原代培养皮质神经元给予brd2shrna下调质粒,pcr结果显示下调效率约为57%。免疫荧光染色结果显示下调brd2可以增加离体培养皮质神经元的突起总长度,提示brd2对神经元的突起生长具有抑制作用。3.体内电转下调brd2对皮质神经元突起形态的影响采用子宫内胚胎电转技术,借助brd2shrna重组质粒,在体观察下调brd2对皮质神经元突起形态的影响。结果如下:1)E15电转Brd2 shRNA重组质粒分别至胎鼠感觉皮质或前额叶皮质附近,出生后3周进行灌注取材,免疫荧光结果表明电转细胞在皮质中主要分布于V/VI层,在II/III层亦有分布。电转细胞在皮质各层的分布比例同对照组相比无统计学差异(P0.05)。提示E15下调Brd2对皮质神经元的迁移可能没有影响。2)E15电转乱序质粒至胎鼠感觉皮质或前额叶皮质附近,出生后3周进行灌注取材,免疫荧光结果可见V层中电转神经元胞体多为锥形,胞体大小、细胞形态等类似,且均发出长长的顶树突指向皮质表层。而电转Brd2 sh RNA重组质粒时可见大量迂曲样短突起。这就提示,E15下调Brd2可影响皮质神经元突起的发育。但是,Brd2具体是通过什么机制来调控皮质神经元突起的发育还有待进一步实验探明。综上所述,本文获得了以下主要结论:1)Brd2基因在成年小鼠脑内广泛表达,在成年大脑皮质中Brd2主要表达于神经元中,可能对神经元的发育调控发挥作用。2)对于体外培养皮质神经元突起形态的研究来说,脂质体转染优于电穿孔转染。下调Brd2可以增加离体培养皮质神经元的突起总长度。3)在体胚胎电转Brd2 sh RNA重组质粒对皮质神经元的迁移可能影响不大,但可影响皮质神经元突起的发育。
[Abstract]:Cerebral cortical neurons play a variety of important physiological functions in the activities of the body. It is the basic structure and functional unit of the brain to play a regulatory role. The development of cerebral cortex is a multi step, complicated and progressive process. Under the strict regulation of many factors, the formation of the cerebral cortex is carried out in sequence and step by step. Factors or exogenous factors can affect the development of the cortex by affecting the proliferation and differentiation of neural stem cells, the migration and localization of neurons and the number of neurites, branches, morphology, direction and so on, so as to change the physiological function of the normal body. Therefore, the study of the molecular mechanism involved in the various links of the cerebral cortex is particularly important. The domain containing protein 2 (Bromodomain containing protein 2, Brd2) is one of the members of the bromine domain containing protein family. It acts as a major epigenetic regulatory factor in some clinical diseases, such as cancer, obesity, type 2 diabetes and neuropsychiatric disorders. In addition, Brd2 also regulates the transcription of a variety of target proteins. Cell proliferation, differentiation, cell cycle and even apoptosis are regulated. Previous studies have shown that Brd2 has a rich expression during the early development of neural embryos. After the proliferation of neurons, Brd2 can use gene regulation to enrich the cell types of newborn neurons, and if Brd2 is missing, the proliferation of neural precursor cells will be increased. More serious may lead to delayed closure of the nerve canal. These studies have shown that Brd2 plays a significant role in the development of the nervous system. However, the current research on the distribution and mechanism of Brd2 in the normal development of the mammalian nervous system is still scarce and needs to be studied urgently. This article is based on the comprehensive application of developmental neurobiology. To investigate the location and distribution of Brd2 in the central nervous system and its cytochemical characteristics, explore the effect and mechanism of Brd2 on the development of cortical neurons, to further clarify the evidence for the regulation of Brd2 in the development of the brain and the localization and distribution of.1.Brd2 in the cerebral cortex of adult mice. The cytochemical characteristics of the Brd2 gene in adult mice were observed by fluorescence in situ hybridization, and the cytochemical characteristics of Brd2 positive cells were observed by fluorescence in situ hybridization and immunofluorescence staining. The results were as follows: 1) the fluorescence in situ hybridization histochemical results showed that in the adult mouse brain, Brd2 The m RNA hybridization signal is mainly expressed in the cerebral cortex, the caudate putamen, the hippocampus and the basolateral amygdala, in the habenular nucleus, the thalamus and the hypothalamus. The middle brain is mainly expressed in the periaqueductal gray; the posterior brain and medulla are mainly expressed in the nucleus of the solitary tract, the small cell reticular nucleus, and the cerebellar cortex, and.2) in situ heterozygosity. The results of combined immunofluorescence staining showed that the brd2mrna positive signals were widely distributed in the cerebral cortex of adult mice, mainly distributed in the II-III and V-VI layers of the cerebral cortex, and in the I and IV layers. Most of the positive signals coexisted with the neuron marker NeuN, but did not coexist with the astrocyte marker GFAP. It is suggested that BRD2 is mainly expressed in the neurons in the adult cerebral cortex and may play a role in the regulation of the development and regulation of neurons. With the help of brd2mrna fluorescence in situ hybridization combined with immunofluorescence labeling technique, we observed the coexistence of BRD2 positive neurons in the cerebral cortex and the GABA energy intermediate neurons and their subclasses (PV, SOM, CB). The results showed that the relationship between the BRD2 positive neurons and the subclass of GABA (PV, SOM, CB) in the cerebral cortex was observed. Brd2/gaba double labeled neurons accounted for about 21% of BRD2 positive neurons, accounting for about 68% of GABA positive neurons, and brd2/pv double labeled neurons accounted for 12% of BRD2 positive neurons, accounting for 27% of PV positive neurons, and brd2/som double labeled neurons accounted for 15% of BRD2 positive neurons, accounting for 92% of the SOM positive neurons, and brd2/som double labeled neurons were approximately occupied. 23% of BRD2 positive neurons, which accounted for 72%.2. of CB positive neurons, reduced BRD2 to the morphology of cortical neurons in vitro. The ratio of the transfection rate of cortical neurons in vitro, the activity of neurons and the branch of dendrites were compared. The results showed that the transfection of liposome was superior to electroporation. The effect of down regulation of BRD2 on the morphology of cultured cortical neurons in vitro was observed with the optimized liposome transfection system. The results were as follows: 1) the effect of electroporation on the survival rate of neurons was less, and the survival rate of neurons decreased with the increase of the volume of transfected liposomes by.2) sholl analysis showed that the complexity of the dendritic branches was complex. In the distance dependence, the most complex branch like branch exists within the radius of 20-50 m radius, and the different transfection conditions significantly affect the branch of the dendrite. In the case of the same distance from the cell body, the experimental group with the largest number of neuron projection and concentric circle is 4 Mu liposome transfection group, and the least experimental group is electroporation transfection group. The total length of the dendrites of different groups was significantly different. In the experimental group, the 4 L liposome transfected group had the longest neurite length, the shortest experimental group was electroporation group.3) with the aid of 3 mu l liposome transfection system, and the primary cultured cortical neurons were given brd2shrna. The PCR results showed that the down regulation efficiency was about 57%. immunofluorescence. The results of light staining showed that down regulation of BRD2 could increase the total protuberance length of cultured cortical neurons in vitro, suggesting that BRD2 had inhibitory effect on the growth of neurons in the neurons. The effect of BRD2 on the morphology of cortical neurons in.3. was controlled by the technique of intrauterine embryo transfer, and the brd2shrna recombinant plasmid was used to observe the regulation of BRD2 against the skin in vivo. The results were as follows: 1) the results were as follows: 1) E15 Brd2 shRNA recombinant plasmids were transferred to the sensory cortex or prefrontal cortex of fetal rats, respectively, and were harvested for 3 weeks after birth. The immunofluorescence results showed that the electric transfer cells were mainly distributed in the V/VI layer in the cortex and distributed in the II/III layer. The distribution of electrotransfer cells in the cortex of the cortex was also distributed. Compared with the control group, there was no statistical difference (P0.05). It was suggested that the migration of Brd2 to cortical neurons could not affect the migration of cortical neurons to.2).2) E15 electrical disorder plasmids to the sensory cortex or prefrontal cortex of fetal rats. 3 weeks after birth, perfusion was carried out. The immunofluorescence results showed that the cell bodies of the electrotransfer neurons in the V layer were mostly conical, the size of the cell body, and the cell size. Morphology is similar, and a long apical dendrite is sent to the surface of the cortex. A large number of circuitous short protrusions can be seen when the Brd2 sh RNA recombinant plasmid is transferred. This suggests that the downregulation of Brd2 by E15 can affect the development of cortical neurites. However, the mechanism of Brd2 to regulate the development of the cortical neuron protuberance is still to be further tested. To sum up, in summary, the following main conclusions are obtained: 1) Brd2 gene is widely expressed in the brain of adult mice. In adult cerebral cortex, Brd2 is mainly expressed in neurons and may play a role in the development and regulation of neurons..2) is superior to electroporation in the study of the morphology of cortical neurons in vitro. The down-regulation of Brd2 can increase the total length of.3 in the cultured cortical neurons in vitro.) the transfer of Brd2 sh RNA plasmid to the cortical neurons may not affect the migration of cortical neurons, but it can affect the development of cortical neurons.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R338
本文编号：2124335
[Abstract]:Cerebral cortical neurons play a variety of important physiological functions in the activities of the body. It is the basic structure and functional unit of the brain to play a regulatory role. The development of cerebral cortex is a multi step, complicated and progressive process. Under the strict regulation of many factors, the formation of the cerebral cortex is carried out in sequence and step by step. Factors or exogenous factors can affect the development of the cortex by affecting the proliferation and differentiation of neural stem cells, the migration and localization of neurons and the number of neurites, branches, morphology, direction and so on, so as to change the physiological function of the normal body. Therefore, the study of the molecular mechanism involved in the various links of the cerebral cortex is particularly important. The domain containing protein 2 (Bromodomain containing protein 2, Brd2) is one of the members of the bromine domain containing protein family. It acts as a major epigenetic regulatory factor in some clinical diseases, such as cancer, obesity, type 2 diabetes and neuropsychiatric disorders. In addition, Brd2 also regulates the transcription of a variety of target proteins. Cell proliferation, differentiation, cell cycle and even apoptosis are regulated. Previous studies have shown that Brd2 has a rich expression during the early development of neural embryos. After the proliferation of neurons, Brd2 can use gene regulation to enrich the cell types of newborn neurons, and if Brd2 is missing, the proliferation of neural precursor cells will be increased. More serious may lead to delayed closure of the nerve canal. These studies have shown that Brd2 plays a significant role in the development of the nervous system. However, the current research on the distribution and mechanism of Brd2 in the normal development of the mammalian nervous system is still scarce and needs to be studied urgently. This article is based on the comprehensive application of developmental neurobiology. To investigate the location and distribution of Brd2 in the central nervous system and its cytochemical characteristics, explore the effect and mechanism of Brd2 on the development of cortical neurons, to further clarify the evidence for the regulation of Brd2 in the development of the brain and the localization and distribution of.1.Brd2 in the cerebral cortex of adult mice. The cytochemical characteristics of the Brd2 gene in adult mice were observed by fluorescence in situ hybridization, and the cytochemical characteristics of Brd2 positive cells were observed by fluorescence in situ hybridization and immunofluorescence staining. The results were as follows: 1) the fluorescence in situ hybridization histochemical results showed that in the adult mouse brain, Brd2 The m RNA hybridization signal is mainly expressed in the cerebral cortex, the caudate putamen, the hippocampus and the basolateral amygdala, in the habenular nucleus, the thalamus and the hypothalamus. The middle brain is mainly expressed in the periaqueductal gray; the posterior brain and medulla are mainly expressed in the nucleus of the solitary tract, the small cell reticular nucleus, and the cerebellar cortex, and.2) in situ heterozygosity. The results of combined immunofluorescence staining showed that the brd2mrna positive signals were widely distributed in the cerebral cortex of adult mice, mainly distributed in the II-III and V-VI layers of the cerebral cortex, and in the I and IV layers. Most of the positive signals coexisted with the neuron marker NeuN, but did not coexist with the astrocyte marker GFAP. It is suggested that BRD2 is mainly expressed in the neurons in the adult cerebral cortex and may play a role in the regulation of the development and regulation of neurons. With the help of brd2mrna fluorescence in situ hybridization combined with immunofluorescence labeling technique, we observed the coexistence of BRD2 positive neurons in the cerebral cortex and the GABA energy intermediate neurons and their subclasses (PV, SOM, CB). The results showed that the relationship between the BRD2 positive neurons and the subclass of GABA (PV, SOM, CB) in the cerebral cortex was observed. Brd2/gaba double labeled neurons accounted for about 21% of BRD2 positive neurons, accounting for about 68% of GABA positive neurons, and brd2/pv double labeled neurons accounted for 12% of BRD2 positive neurons, accounting for 27% of PV positive neurons, and brd2/som double labeled neurons accounted for 15% of BRD2 positive neurons, accounting for 92% of the SOM positive neurons, and brd2/som double labeled neurons were approximately occupied. 23% of BRD2 positive neurons, which accounted for 72%.2. of CB positive neurons, reduced BRD2 to the morphology of cortical neurons in vitro. The ratio of the transfection rate of cortical neurons in vitro, the activity of neurons and the branch of dendrites were compared. The results showed that the transfection of liposome was superior to electroporation. The effect of down regulation of BRD2 on the morphology of cultured cortical neurons in vitro was observed with the optimized liposome transfection system. The results were as follows: 1) the effect of electroporation on the survival rate of neurons was less, and the survival rate of neurons decreased with the increase of the volume of transfected liposomes by.2) sholl analysis showed that the complexity of the dendritic branches was complex. In the distance dependence, the most complex branch like branch exists within the radius of 20-50 m radius, and the different transfection conditions significantly affect the branch of the dendrite. In the case of the same distance from the cell body, the experimental group with the largest number of neuron projection and concentric circle is 4 Mu liposome transfection group, and the least experimental group is electroporation transfection group. The total length of the dendrites of different groups was significantly different. In the experimental group, the 4 L liposome transfected group had the longest neurite length, the shortest experimental group was electroporation group.3) with the aid of 3 mu l liposome transfection system, and the primary cultured cortical neurons were given brd2shrna. The PCR results showed that the down regulation efficiency was about 57%. immunofluorescence. The results of light staining showed that down regulation of BRD2 could increase the total protuberance length of cultured cortical neurons in vitro, suggesting that BRD2 had inhibitory effect on the growth of neurons in the neurons. The effect of BRD2 on the morphology of cortical neurons in.3. was controlled by the technique of intrauterine embryo transfer, and the brd2shrna recombinant plasmid was used to observe the regulation of BRD2 against the skin in vivo. The results were as follows: 1) the results were as follows: 1) E15 Brd2 shRNA recombinant plasmids were transferred to the sensory cortex or prefrontal cortex of fetal rats, respectively, and were harvested for 3 weeks after birth. The immunofluorescence results showed that the electric transfer cells were mainly distributed in the V/VI layer in the cortex and distributed in the II/III layer. The distribution of electrotransfer cells in the cortex of the cortex was also distributed. Compared with the control group, there was no statistical difference (P0.05). It was suggested that the migration of Brd2 to cortical neurons could not affect the migration of cortical neurons to.2).2) E15 electrical disorder plasmids to the sensory cortex or prefrontal cortex of fetal rats. 3 weeks after birth, perfusion was carried out. The immunofluorescence results showed that the cell bodies of the electrotransfer neurons in the V layer were mostly conical, the size of the cell body, and the cell size. Morphology is similar, and a long apical dendrite is sent to the surface of the cortex. A large number of circuitous short protrusions can be seen when the Brd2 sh RNA recombinant plasmid is transferred. This suggests that the downregulation of Brd2 by E15 can affect the development of cortical neurites. However, the mechanism of Brd2 to regulate the development of the cortical neuron protuberance is still to be further tested. To sum up, in summary, the following main conclusions are obtained: 1) Brd2 gene is widely expressed in the brain of adult mice. In adult cerebral cortex, Brd2 is mainly expressed in neurons and may play a role in the development and regulation of neurons..2) is superior to electroporation in the study of the morphology of cortical neurons in vitro. The down-regulation of Brd2 can increase the total length of.3 in the cultured cortical neurons in vitro.) the transfer of Brd2 sh RNA plasmid to the cortical neurons may not affect the migration of cortical neurons, but it can affect the development of cortical neurons.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R338
【参考文献】
相关期刊论文 前1条
1 ;Changes of learning, memory and levels of CaMKII, CaM mRNA, CREB mRNA in the hippocampus of chronic multiple-stressed rats[J];Chinese Medical Journal;2006年02期
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