大鼠視覺可塑性相關基因cDNA消減文庫的構建及篩選
發(fā)布時間：2018-11-16 10:44
【摘要】： 目的:采用抑制性消減雜交技術構建大鼠視覺可塑性相關基因的cDNA消減文庫,篩選與視覺可塑性關鍵期終止相關的基因及與視覺可塑性相關的基因,探討視覺可塑性及其關鍵期終止的分子機制。 方法:本研究主要分為四個部分,各部分的具體研究方法如下:1、自行設計并制作專用大鼠暗飼養(yǎng)箱,建立暗飼養(yǎng)動物模型及暗飼養(yǎng)后予光照刺激動物模型。2、采用暗飼養(yǎng)動物模型(D67)及暗飼養(yǎng)后予光照刺激動物模型(D60+L7),應用抑制性消減雜交技術構建大鼠視皮質與視覺可塑性關鍵期終止相關基因的cDNA消減文庫。并通過PCR篩選、反向Northern雜交、測序及同源性檢索對差異表達基因進行分析,篩選視覺可塑性關鍵期終止相關基因。3、應用抑制性消減雜交技術構建幼年(P28)和成年(P60)大鼠視皮質差異表達基因的cDNA消減文庫。并通過PCR篩選、反向Northern雜交、測序及同源性檢索對差異表達基因進行分析,篩選視覺可塑性相關基因。4、采用半定量RT-PCR技術檢測部分篩選到的基因在不同組大鼠視皮質中表達情況,進一步驗證應用抑制性消減雜交技術篩選到的差異表達基因的真實性。 結果:1、自行設計并成功制作了能滿足實驗要求的專用大鼠暗飼養(yǎng)箱,成功建立了暗飼養(yǎng)動物模型及暗飼養(yǎng)后予光照刺激動物模型。2、成功構建了大鼠視皮質與視覺可塑性關鍵期終止相關基因的cDNA消減文庫,經篩選,得到14個在D60+L7大鼠視皮質中上調表達基因的片段。其中13個為已知基因,一個為新基因片斷。發(fā)現鋅指蛋白9、烯醇酶-1、熱休克蛋白8、脂肪細胞補體相關蛋白、肽基脯氨酸異構酶A、非神經元烯醇酶及ftp-3基因參與了視覺可塑性關鍵期的終止,同時,也篩選到既往有文獻報道其功能與可塑性相關的β-微管蛋白基因、髓磷脂堿蛋白基因、親環(huán)素基因參與了視覺可塑性關鍵期的終止。3、成功構建了幼年和成年大鼠視皮質差異表達基因的cDNA消減文庫,經過篩選,最后確定了11個基因在視覺可塑性關鍵期內的大鼠(P28)視皮質中上調表達,4個基因在視覺可塑性關鍵期終止后的大鼠(P60)視皮質中上調表達。發(fā)現硬脂�；�-輔酶A去飽和酶2基因、α血紅蛋白基因、谷氨酸-脯氨酸二肽重復蛋白基因、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, 偽 hemoglobin gene, glutamate proline dipeptide repeat protein gene, mDj4, heterogeneous ribonucleic acid protein C1/C2 gene, strain BN/SsNHsdMCW
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R346
本文編號：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, 偽 hemoglobin gene, glutamate proline dipeptide repeat protein gene, mDj4, heterogeneous ribonucleic acid protein C1/C2 gene, strain BN/SsNHsdMCW
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R346
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