应用mRNA差异显示技术筛选大鼠小脑颗粒神经元凋亡模型中差异表达基因  

Screening differentially expressed genes involved in apoptosis of primary cultured rat cerebellar granule neurons by mRNA differential display RT-PCR

作　　者：银巍[1] 黄奕俊 皮荣标[3] 苏兴文[2] 赵灵芝[3] 邱鹏新[2] 颜光美[2] 

机构地区：[1]中山大学中山医学院生化教研室 [2]中山大学中山医学院药理教研室 [3]中山大学药学院,广东广州510089

出　　处：《中国病理生理杂志》2007年第11期2141-2146,共6页Chinese Journal of Pathophysiology

基　　金：国家杰出青年基金资助项目(No.39625022);广东省自然科学基金资助项目(No.05001739);广东省科技计划资助项目(No.2KM028091);广东省自然科学基金团队资助项目(No.039191);广东省关键领域重点突破资助项目(No.2003A10905)

摘　　要：目的:应用mRNA差异显示技术对大鼠小脑颗粒神经元凋亡模型中差异表达基因进行筛选,克隆以及鉴定。方法:大鼠小脑颗粒神经元的分离和原代培养;对大鼠小脑颗粒神经元凋亡逆转模型进行荧光mRNA差异显示逆转录PCR(FDDRT-PCR,fluorescent differential display RT-PCR);EST片段亚克隆入pGEM-T Ea-syTM,克隆后测序并进行序列同源性检索;反Northern杂交重鉴定与筛选。结果:通过DDRT-PCR获得164个在小脑颗粒神经元凋亡模型中差异表达的ESTs;对其中的17个ESTs进行了克隆并测序;通过反Northern杂交的初步筛选初步鉴定出5个阳性片段。结论:阳性差异表达EST的筛选、克隆以及鉴定为神经元凋亡与保护分子机制研究奠定基础。AIM： To Screen and identify differentially expressed genes that involved in apoptosis model in rat cerebellar granule neurons （CGNs）. METHODS ： The rat cerebellar granule neurons were isolated and primarily cultured. Fluorescent differential display RT- PCR （FDD RT- PCR） was performed to screen differentially expressed ESTs in the apoptosis model of primarily cultured rat CGNs. ESTs were subcloned into pGEM - T Easy^TM vector and then sequenced. Alignment assay in non - redunant database was applied for encoding information. Reverse Northern blotting was used to appraise the results from DDRT - PCR. RESULTS： 164 pieces of differentially expressed ESTs were obtained by FDDRT - PCR. 17 of them were subcloned and sequenced. 5 ESTs of 17 were confirmed to be positive results by reverse Northern blotting. CONCLUSION： DD- PCR is a rapid, simple -operation and sensitive method for screening differentially expressed genes, which would contribute to the molecular mechanisms of apoptosis/survive of CGNs.

关 键 词：细胞凋亡 差异表达基因 小脑 神经元 

分 类 号：R363[医药卫生—病理学]