【作者】 江文珊；

【导师】 阴正勤；

【作者基本信息】 第三军医大学 ， 眼科学， 2006， 博士

【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更多还原

【Abstract】 Objective:To construct subtractive cDNA libraries which associated with visual cortex plasticity in the rat by using suppression subtractive hybridization technique and screening the related genes which associated with termination of the critical period of the visual cortex plasticity or associated with visual cortex plasticity.And to investigate the molecular mechanism involved.Methods:1、Designed and made the dark rearing box for rats and established animal models of dark rearing rats and dark-reared for 60 days postnatal and light-exposed for 7 days rats.2、A subtracted cDNA library was constructed with the visual cortex of rats that were dark-reared for 67 days postnatal(D67) and of those that were dark-reared for 60 days postnatal and light-exposed for 7 days(D60+L7) by using suppression subtractive hybridization technique.Differentially expressed genes were screened by polymerase chain reaction(PCR),reverse Northern hybridization,sequencing and homology analysis to screening genes which related to termination the critical period.3、To construct subtractive cDNA libraries using juvenile(P28) and adult(P60) rats’ visual cortex with suppression subtractive hybridization technique.Using PCR technique,reverse Northern hybridization, sequencing and homology search to analysis the differential expression genes fragments to screening genes which associated with plasticity.4、Used semiquantitative RT-PCR to measure the expression level of several genes which screened by SSH in the visual cortex of different groups to further verify the reliability of the SSH.Results:1、The dark rearing box was made successfully and established animal models of dark rearing rats and light-exposed after dark-reared rats.2、The subtracted cDNA library associated with termination of the critical period of visual cortex plasticity in rats was constructed successfully.After screening,14 sequences which were up-regulated in the visual cortex of D60+L7 rats were obtained.There were 13 known genes fragments and a novel one.Of the known genes,we found Znf9、Eno-1、Hsp8、30 kDa adipocyte complement-related protein、Ppia、Non-Neuronal Enolase and ftp-3 gene participation the termination course of the critical period of visual cortex plasticity.And we also found those genes which encodingβ-Tubulin,myelin basic protein,and Cyclophilin participation the termination of the critical period which’s function had been reported to be associated with visual cortex plasticity.3、Juvenile and adult rats’ visual cortex differential expression genes’ subtractive cDNA libraries were set up successfully.11 genes were obtained by sequencing and homology search which were up-regulation expressed in the visual cortex of P28 and 4 genes were up-regulation expressed in P60.We found Scd2、Hba-al、Glu-Pro dipeptide repeat protein、mDj4、hnRNP C1/ hnRNP C2 and strain BN/SsNHsdMCW mitochondrion gene which were up-regulation expressed in the visual cortex of P28 rats, which participate the critical period of plasticity and were the genes which related to plasticity,Hsp8、strain F344 x BN F1 mitochondrion、BHE/Cdb tRNA-Lys gene、complete mitochondrial genome participate the termination of the critical period of visual cortex plasticity of adult rats.And we also found those genes encoding calmodulin 2,cytochrome b and proteolipid protein were up-regulation expressed in the visual cortex of P28 rats, whose function had been reported to be associated with visual cortex plasticity.4、The results of semiquantitative RT-PCR indicating that the level of Hsp8 mRNA expression was significant higher in the visual cortex of P60 rats than that of P28 rats,the level of Plp mRNA expression was significant higher in the visual cortex of P28 rats than that of P60 rats,the level of Mbp mRNA expression was significant higher in the visual cortex of D60+L7 rats than that of D67 rats,which were in agreement with the SSH results and further verifies the reliability of the subtractive cDNA library we constructed.The results confirmed that Hsp8 gene and Mbp gene were the genes which termination the critical period,and Plp gene was the plasticity gene.Conclusions:The successfully construction of the subtracted cDNA libraries laying an important basis for elucidating the molecular mechanism of the critical period of visual cortex plasticity.A set of candidate genes association with termination of the critical period and association with visual cortex plasticity were screened from the subtracted cDNA libraries.Further screening and verification these genes maybe found the most important genes which determine whether or not termination of the critical period.It will make it possible to use gene therapy to regulation the critical period of visual cortex plastitity and更多还原