大鼠视觉可塑性相关基因cDNA消减文库的构建及筛选
发布时间：2018-11-16 10:44
【摘要】： 目的:采用抑制性消减杂交技术构建大鼠视觉可塑性相关基因的cDNA消减文库,筛选与视觉可塑性关键期终止相关的基因及与视觉可塑性相关的基因,探讨视觉可塑性及其关键期终止的分子机制。 方法:本研究主要分为四个部分,各部分的具体研究方法如下:1、自行设计并制作专用大鼠暗饲养箱,建立暗饲养动物模型及暗饲养后予光照刺激动物模型。2、采用暗饲养动物模型(D67)及暗饲养后予光照刺激动物模型(D60+L7),应用抑制性消减杂交技术构建大鼠视皮质与视觉可塑性关键期终止相关基因的cDNA消减文库。并通过PCR筛选、反向Northern杂交、测序及同源性检索对差异表达基因进行分析,筛选视觉可塑性关键期终止相关基因。3、应用抑制性消减杂交技术构建幼年(P28)和成年(P60)大鼠视皮质差异表达基因的cDNA消减文库。并通过PCR筛选、反向Northern杂交、测序及同源性检索对差异表达基因进行分析,筛选视觉可塑性相关基因。4、采用半定量RT-PCR技术检测部分筛选到的基因在不同组大鼠视皮质中表达情况,进一步验证应用抑制性消减杂交技术筛选到的差异表达基因的真实性。 结果:1、自行设计并成功制作了能满足实验要求的专用大鼠暗饲养箱,成功建立了暗饲养动物模型及暗饲养后予光照刺激动物模型。2、成功构建了大鼠视皮质与视觉可塑性关键期终止相关基因的cDNA消减文库,经筛选,得到14个在D60+L7大鼠视皮质中上调表达基因的片段。其中13个为已知基因,一个为新基因片断。发现锌指蛋白9、烯醇酶-1、热休克蛋白8、脂肪细胞补体相关蛋白、肽基脯氨酸异构酶A、非神经元烯醇酶及ftp-3基因参与了视觉可塑性关键期的终止,同时,也筛选到既往有文献报道其功能与可塑性相关的β-微管蛋白基因、髓磷脂碱蛋白基因、亲环素基因参与了视觉可塑性关键期的终止。3、成功构建了幼年和成年大鼠视皮质差异表达基因的cDNA消减文库,经过筛选,最后确定了11个基因在视觉可塑性关键期内的大鼠(P28)视皮质中上调表达,4个基因在视觉可塑性关键期终止后的大鼠(P60)视皮质中上调表达。发现硬脂酰基-辅酶A去饱和酶2基因、α血红蛋白基因、谷氨酸-脯氨酸二肽重复蛋白基因、mDj4、不均一核糖核酸核蛋白C1/C2基因、strain BN/SsNHsdMCW
[Abstract]:Aim: to construct the cDNA subtractive library of rat visual plasticity related genes by suppression subtractive hybridization, and to screen the genes associated with the termination of visual plasticity and those related to visual plasticity. To explore the molecular mechanism of visual plasticity and the termination of critical period. Methods: this study was mainly divided into four parts. The specific research methods of each part were as follows: 1. The special rat dark feeding box was designed and made by ourselves, and the dark feeding animal model and the animal model of light stimulation after dark feeding were established. The dark feeding animal model (D67) and the dark fed animal model (D60L7) were used to construct the cDNA subtractive library of the related genes in the visual cortex and the critical period of visual plasticity by suppression subtractive hybridization. The differentially expressed genes were analyzed by PCR screening, reverse Northern hybridization, sequencing and homology retrieval. CDNA subtractive libraries of differentially expressed genes in the visual cortex of juvenile (P28) and adult (P60) rats were constructed by suppression subtractive hybridization. The differentially expressed genes were analyzed by PCR screening, reverse Northern hybridization, sequencing and homology retrieval. Semi-quantitative RT-PCR technique was used to detect the expression of partially screened genes in the visual cortex of different groups of rats to further verify the authenticity of differentially expressed genes screened by suppression subtractive hybridization. Results: 1. A special rat dark feeding box was designed and successfully made to meet the requirements of the experiment. The dark feeding animal model and the light stimulated animal model were established successfully. 2. The cDNA subtractive library was successfully constructed for the critical phase termination of visual plasticity in rat visual cortex. After screening, 14 up-regulated gene fragments were obtained in the visual cortex of D60L7 rats. Of these, 13 were known genes and one was a new gene fragment. It was found that zinc finger protein 9, enolase 1, heat shock protein 8, aliphatic complement associated protein, peptide proline isomerase A, non-neuronal enolase and ftp-3 genes were involved in the termination of the critical period of visual plasticity. The 尾 -tubulin gene, myelin protein gene and cyclin gene involved in the termination of the critical period of visual plasticity were also screened. The cDNA subtractive library of differentially expressed genes in the visual cortex of young and adult rats was successfully constructed. After screening, 11 genes were identified to be up-regulated in the visual cortex of rats (P28) during the critical period of visual plasticity. Four genes were up-regulated in the visual cortex of rats (P60) after the critical phase of visual plasticity ended. Stearyl CoA desaturase 2 gene, 伪 hemoglobin gene, glutamate proline dipeptide repeat protein gene, mDj4, heterogeneous ribonucleic acid protein C1/C2 gene, strain BN/SsNHsdMCW
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346
本文编号：2335295
[Abstract]:Aim: to construct the cDNA subtractive library of rat visual plasticity related genes by suppression subtractive hybridization, and to screen the genes associated with the termination of visual plasticity and those related to visual plasticity. To explore the molecular mechanism of visual plasticity and the termination of critical period. Methods: this study was mainly divided into four parts. The specific research methods of each part were as follows: 1. The special rat dark feeding box was designed and made by ourselves, and the dark feeding animal model and the animal model of light stimulation after dark feeding were established. The dark feeding animal model (D67) and the dark fed animal model (D60L7) were used to construct the cDNA subtractive library of the related genes in the visual cortex and the critical period of visual plasticity by suppression subtractive hybridization. The differentially expressed genes were analyzed by PCR screening, reverse Northern hybridization, sequencing and homology retrieval. CDNA subtractive libraries of differentially expressed genes in the visual cortex of juvenile (P28) and adult (P60) rats were constructed by suppression subtractive hybridization. The differentially expressed genes were analyzed by PCR screening, reverse Northern hybridization, sequencing and homology retrieval. Semi-quantitative RT-PCR technique was used to detect the expression of partially screened genes in the visual cortex of different groups of rats to further verify the authenticity of differentially expressed genes screened by suppression subtractive hybridization. Results: 1. A special rat dark feeding box was designed and successfully made to meet the requirements of the experiment. The dark feeding animal model and the light stimulated animal model were established successfully. 2. The cDNA subtractive library was successfully constructed for the critical phase termination of visual plasticity in rat visual cortex. After screening, 14 up-regulated gene fragments were obtained in the visual cortex of D60L7 rats. Of these, 13 were known genes and one was a new gene fragment. It was found that zinc finger protein 9, enolase 1, heat shock protein 8, aliphatic complement associated protein, peptide proline isomerase A, non-neuronal enolase and ftp-3 genes were involved in the termination of the critical period of visual plasticity. The 尾 -tubulin gene, myelin protein gene and cyclin gene involved in the termination of the critical period of visual plasticity were also screened. The cDNA subtractive library of differentially expressed genes in the visual cortex of young and adult rats was successfully constructed. After screening, 11 genes were identified to be up-regulated in the visual cortex of rats (P28) during the critical period of visual plasticity. Four genes were up-regulated in the visual cortex of rats (P60) after the critical phase of visual plasticity ended. Stearyl CoA desaturase 2 gene, 伪 hemoglobin gene, glutamate proline dipeptide repeat protein gene, mDj4, heterogeneous ribonucleic acid protein C1/C2 gene, strain BN/SsNHsdMCW
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346
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