溴結(jié)構(gòu)域包含蛋白2在小鼠腦內(nèi)的表達及其對皮質(zhì)神經(jīng)元突起發(fā)育的影響
發(fā)布時間：2018-07-15 14:10
【摘要】：大腦皮質(zhì)神經(jīng)元在機體活動中發(fā)揮多種重要的生理功能,是大腦發(fā)揮調(diào)節(jié)作用的基本結(jié)構(gòu)和功能單位。大腦皮質(zhì)的發(fā)育形成是一個多步驟、復(fù)雜的漸進過程,在許多因素的嚴謹調(diào)控下,分步驟、有次序地進行。在皮質(zhì)的發(fā)育過程中,內(nèi)源性因素或外源性因素會通過影響神經(jīng)干細胞的增殖分化、神經(jīng)元的遷移定位以及神經(jīng)突起的數(shù)量、分支、形態(tài)、方向等對皮質(zhì)的發(fā)育造成影響,從而改變正常機體的生理功能。因此,對大腦皮質(zhì)發(fā)育各環(huán)節(jié)參與的分子機制研究尤為重要。溴結(jié)構(gòu)域包含蛋白2(Bromodomain containing protein 2,Brd2)是溴結(jié)構(gòu)域包含蛋白家族成員之一,其作為主要的表觀遺傳調(diào)節(jié)因子在一些臨床疾病,如癌癥、肥胖、2型糖尿病以及神經(jīng)精神性疾病中發(fā)揮重要作用。此外,Brd2亦通過調(diào)控多種靶蛋白的轉(zhuǎn)錄,對細胞增殖、分化、細胞周期甚至凋亡進行調(diào)控。既往研究表明Brd2在神經(jīng)系統(tǒng)胚胎的早期發(fā)育過程中具有豐富的表達,神經(jīng)元增殖后,Brd2能夠利用基因調(diào)控豐富新生神經(jīng)元的細胞種類;而如果缺失Brd2,將會引起神經(jīng)前體細胞的增殖變多,更嚴重的會導(dǎo)致神經(jīng)管延遲閉合。這些研究表明,Brd2在神經(jīng)系統(tǒng)的發(fā)育過程中作用明顯。然而,當(dāng)前關(guān)于Brd2在哺乳動物神經(jīng)系統(tǒng)正常發(fā)育中的分布及作用機制的研究還比較匱乏,亟需開展研究。因此本文通過綜合應(yīng)用發(fā)育神經(jīng)生物學(xué)研究方法和形態(tài)學(xué)方法,探討B(tài)rd2在中樞神經(jīng)系統(tǒng)中的定位分布以及其細胞化學(xué)特點,探究Brd2對皮質(zhì)神經(jīng)元發(fā)育的影響及其機制,為進一步闡明Brd2在大腦發(fā)育過程中的調(diào)控提供有力的研究證據(jù)。1.Brd2在成年小鼠大腦皮質(zhì)的定位分布及細胞化學(xué)特點運用熒光原位雜交技術(shù)觀察了成年小鼠腦內(nèi)Brd2基因的分布模式;借助熒光原位雜交技術(shù)聯(lián)合免疫熒光染色技術(shù),對Brd2陽性細胞的細胞化學(xué)特點進行了觀察。結(jié)果如下:1)熒光原位雜交組織化學(xué)結(jié)果顯示在成年小鼠腦內(nèi),Brd2 m RNA雜交信號在前腦主要表達于大腦皮質(zhì)、尾殼核、海馬、基底外側(cè)杏仁核,在韁核、丘腦以及下丘腦等區(qū)域也有部分表達;在中腦主要表達于導(dǎo)水管周圍灰質(zhì);在后腦及延髓主要表達于孤束核、小細胞網(wǎng)狀核以及小腦皮質(zhì)等。2)熒光原位雜交聯(lián)合免疫熒光標記技術(shù)結(jié)果顯示,在成年小鼠大腦皮質(zhì)中,brd2mrna陽性信號分布較廣泛,主要分布于大腦皮質(zhì)ii-iii層和v-vi層,在i層和iv層亦有少量分布。其中大多數(shù)陽性信號與神經(jīng)元標志物neun共存,而與星形膠質(zhì)細胞標志物gfap不共存。提示在成年大腦皮質(zhì)中brd2主要表達于神經(jīng)元中,可能對神經(jīng)元的發(fā)育調(diào)控發(fā)揮作用。3)借助brd2mrna熒光原位雜交聯(lián)合免疫熒光標記技術(shù),我們觀察了大腦皮質(zhì)中brd2陽性神經(jīng)元與gaba能中間神經(jīng)元及其亞類(pv,som,cb)的共存關(guān)系。結(jié)果顯示brd2/gaba雙標記神經(jīng)元約占brd2陽性神經(jīng)元的21%,約占gaba陽性神經(jīng)元的68%;brd2/pv雙標記神經(jīng)元約占brd2陽性神經(jīng)元的12%,約占pv陽性神經(jīng)元的27%;brd2/som雙標記神經(jīng)元約占brd2陽性神經(jīng)元的15%,約占som陽性神經(jīng)元的92%;brd2/som雙標記神經(jīng)元約占brd2陽性神經(jīng)元的23%,約占cb陽性神經(jīng)元的72%。2.下調(diào)brd2對體外培養(yǎng)皮質(zhì)神經(jīng)元突起形態(tài)的影響從體外培養(yǎng)皮質(zhì)神經(jīng)元的轉(zhuǎn)染率、神經(jīng)元活性以及樹突分支情況角度對非病毒基因轉(zhuǎn)染技術(shù)進行了對比,表明脂質(zhì)體轉(zhuǎn)染優(yōu)于電穿孔轉(zhuǎn)染。之后借助優(yōu)化的脂質(zhì)體轉(zhuǎn)染體系,觀察下調(diào)brd2對體外培養(yǎng)皮質(zhì)神經(jīng)元形態(tài)的影響。結(jié)果如下:1)電穿孔轉(zhuǎn)染對神經(jīng)元的存活率影響較小,而神經(jīng)元存活率隨著用于轉(zhuǎn)染脂質(zhì)體的體積增加而減少。2)sholl分析結(jié)果顯示樹突分支的復(fù)雜性具有距離依賴性,在20-50μm為半徑的圓軌跡內(nèi)存在最復(fù)雜的樹枝狀分支;不同轉(zhuǎn)染條件顯著影響樹突分支,在距胞體相同距離的情況下,神經(jīng)元突起與同心圓交點個數(shù)最多的實驗組為4μl脂質(zhì)體轉(zhuǎn)染組,最少的實驗組為電穿孔轉(zhuǎn)染組。此外還觀察到不同組別的樹突總長度有顯著不同。實驗組中4μl脂質(zhì)體轉(zhuǎn)染組神經(jīng)元突起總長度最長,最短實驗組為電穿孔轉(zhuǎn)染組。3)借助3μl脂質(zhì)體轉(zhuǎn)染體系,針對原代培養(yǎng)皮質(zhì)神經(jīng)元給予brd2shrna下調(diào)質(zhì)粒,pcr結(jié)果顯示下調(diào)效率約為57%。免疫熒光染色結(jié)果顯示下調(diào)brd2可以增加離體培養(yǎng)皮質(zhì)神經(jīng)元的突起總長度,提示brd2對神經(jīng)元的突起生長具有抑制作用。3.體內(nèi)電轉(zhuǎn)下調(diào)brd2對皮質(zhì)神經(jīng)元突起形態(tài)的影響采用子宮內(nèi)胚胎電轉(zhuǎn)技術(shù),借助brd2shrna重組質(zhì)粒,在體觀察下調(diào)brd2對皮質(zhì)神經(jīng)元突起形態(tài)的影響。結(jié)果如下:1)E15電轉(zhuǎn)Brd2 shRNA重組質(zhì)粒分別至胎鼠感覺皮質(zhì)或前額葉皮質(zhì)附近,出生后3周進行灌注取材,免疫熒光結(jié)果表明電轉(zhuǎn)細胞在皮質(zhì)中主要分布于V/VI層,在II/III層亦有分布。電轉(zhuǎn)細胞在皮質(zhì)各層的分布比例同對照組相比無統(tǒng)計學(xué)差異(P0.05)。提示E15下調(diào)Brd2對皮質(zhì)神經(jīng)元的遷移可能沒有影響。2)E15電轉(zhuǎn)亂序質(zhì)粒至胎鼠感覺皮質(zhì)或前額葉皮質(zhì)附近,出生后3周進行灌注取材,免疫熒光結(jié)果可見V層中電轉(zhuǎn)神經(jīng)元胞體多為錐形,胞體大小、細胞形態(tài)等類似,且均發(fā)出長長的頂樹突指向皮質(zhì)表層。而電轉(zhuǎn)Brd2 sh RNA重組質(zhì)粒時可見大量迂曲樣短突起。這就提示,E15下調(diào)Brd2可影響皮質(zhì)神經(jīng)元突起的發(fā)育。但是,Brd2具體是通過什么機制來調(diào)控皮質(zhì)神經(jīng)元突起的發(fā)育還有待進一步實驗探明。綜上所述,本文獲得了以下主要結(jié)論:1)Brd2基因在成年小鼠腦內(nèi)廣泛表達,在成年大腦皮質(zhì)中Brd2主要表達于神經(jīng)元中,可能對神經(jīng)元的發(fā)育調(diào)控發(fā)揮作用。2)對于體外培養(yǎng)皮質(zhì)神經(jīng)元突起形態(tài)的研究來說,脂質(zhì)體轉(zhuǎn)染優(yōu)于電穿孔轉(zhuǎn)染。下調(diào)Brd2可以增加離體培養(yǎng)皮質(zhì)神經(jīng)元的突起總長度。3)在體胚胎電轉(zhuǎn)Brd2 sh RNA重組質(zhì)粒對皮質(zhì)神經(jīng)元的遷移可能影響不大,但可影響皮質(zhì)神經(jīng)元突起的發(fā)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R338
本文編號：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R338
【參考文獻】
相關(guān)期刊論文 前1條
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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