【作者】 孙建刚；

【导师】 凌树才；

【作者基本信息】 浙江大学 ， 神经生物学， 2006， 硕士

【摘要】 中枢神经系统的发育是一个复杂而又精密的过程,各种基因在目前尚不完全清楚的机制诱导下按照一定的时序表达,它们的产物又有序地调控神经细胞的迁移、增殖、生长、分化与消亡。大脑皮质细胞的产生按照自内向外的原则,神经元定位在大脑皮质的哪一层,受到内源性和外源性信号调节,主导作用的是内源性信号。 RAGs的编码产物RAG-1和RAG-2的复合物能特异性识别Ig胚系基因中V、DH、J基因片段两侧的重组信号序列,切断DNA双链,引发V(D)J基因片段重排。RAGs是在淋巴细胞发育过程中产生抗体多样性的基础。哺乳动物中枢神经系统也可能存在位点特异性的重组。 本研究通过免疫组织化学和RT-PCR,旨在观察RAG-1在小鼠脑组织正常发育期间出现的表达变化,并对其进行组织定位,以探讨它在中枢发育过程所起到的作用,并为下一步科研工作提供理论依据。 [方法] 取E11(胚胎11天)、E13、E15、E17、E19、新生的和成年的ICR品系小鼠脑组织,抽提脑组织总RNA进行RT-PCR,RT-PCR产物用凝胶成像观察。引物:RAG-1 widetype:sense:5’ACTCAATTCTGACTCAACG 3’Antisense:5’AACAGATGTCACAGGACG 3’。脑组织经多聚甲醛固定,冠状切面,隔四取一,制作连续冰冻切片,行酶标免疫组织化学和尼氏染色。免疫组化结果用显微镜成像并做灰度统计分析。 [结果] 在E11的组织切片上可见RAG-1免疫阳性细胞首先出现于室管膜层;到E13在杏仁更多还原

【Abstract】 The development of central nervous system is a complicated and precise procedure. All kinds of genes express temporally under the control of unknowing mechanism, their products also control the migration、proliferation、differentiation、 growth and extinction of nerve cells. The development of cerebral cortex cells is according to the rule from interior to exterior, which layer was localized in cerebral cortex, was controlled by endogenic and ectogenic signals, but the endogenic signals are in the most.The compound of RAG-1 and RAG-2 can identify specifically recombination signal sequence which lie two sides of the V、 DH、J gene segment in Ig germline gene, then break DNA and induce the recombination of V(D)J gene segment. RAGs are the basis of antibody diversity during the lymphocyte development. Central nervous system maybe exist the site specific recombination.The changes of expression level of recombination activating gene 1 (RAG-1) during brain developmental were investigated and tissue localizated by RT-PCR and immunohistochemistry. Then we try to find the RAG-l’s action during the development of central nervous system and offer theory basis for next research.[Methods]The brain tissues of embryonic day (E)11、E13、E15、E17、E19、 new-born and adult mice were taken out, the total RNA were extracted and the changes of RAG-1 expression were investigated with the method of RT-PCR. The primer: RAG-1 wide type: sense: 5’ ACTCAATTCTGACTCAACG 3’ Antisense: 5’ AACAGATGTCACAGGACG 3’. Afterwards, each group brain tissues were fixated by formaldehydum polymerisatum, the frozen coronalsections of each group brain tissues were stained with immunohistochemistry method and Nissl’s staining, to localize the expression of RAG-1. the results of immunohistochemistry were statistical analysised.[Results]We observed the expression of RAG-1 in Ell localized ventricular zone (VZ);the expression of RAG-1 in El3 localize VZ> IZ> nucleus amygdalae and hypothalamus, the RAG-1 positive neurons are mostly immature and few apophysis;the expression of RAG-1 in El5 is decrease in VZ, RAG-1 mostly expresses in corticle plate (CP) , there are many positive nerve fibers in nucleus amygdalae > hypothalamus and thalamus;with the development of the cerebral cortex, the RAG-1 positive neurons and nerve fibers also express subventricular zone (SVZ) and CP in E17;the expression of RAG-1 in E19 is the same to that in El7, but the expression is decrease in VZ and SVZ and increase in CP and subcorticle plate(SP);the expression of RAG-1 localize mostly in CP and Hippocampus in new-born mouse, the form of RAG-1 positive neurons is the same to that in adult mouse;RAG-1 express hippocampus, granular cell in cerebellum, several in the cortex of frontal lobe and temporal lobe in adult mouse.The result showed that the expression is increase from Ell to El9 and decrease in the new-born mouse brain, but the expression level is also higher than that in the adult mouse. The result of RT-PCR is coincided with the result of immunohistchemisty, but the expression peak occured in El 7.I Conclusion!RAG-1 express in mouse embryonic brain tissue and the expression is related with the neuron development;the expression level of RAG-1 in embryonic brain tissue is higher than that in the adult mouse.更多还原