小脑颗粒神经元凋亡差异表达基因的分离克隆研究

目的筛选分离低钾诱导的小脑颗粒细胞凋亡的差异表达基因。方法采用低钾处理的小脑颗粒细胞,抽提细胞总RNA,用荧光法mRNA差异显示技术分离差异表达基因,并对差异表达基因片段进行回收、克隆,经反向RNA印迹及RT鄄PCR技术验证。结果发现51条差异片段,成功克隆8个片段,其中2个未知功能的基因片段证实在低钾诱导的小脑颗粒细胞凋亡中表达增加。结论mRNA差异显示法是分离差异表达基因的一个良好的工具,分离出的差异基因与神经细胞凋亡有关。

Screening and identification of differentially expressed genes during the apoptosis of cerebellar granule neurons

Objective To isolate and identify differentially expressed genes involved in neurons apoptosis. Methods The low-potassium-induced apoptosis model in cerebellar granule cells(CGCs) was established and total cellular mRNA isolation ensued. Fluorescence differential display technique was applied to screen possible differentially expressed genes. Those apparent different cDNA fragments were reclaimed from the DD-PCR products,and then cloned into vectors for better sequence analysis. The sequences were compared with GenBank database. Reverse Northern blot and RT-PCR techniques were applied to identify the desired clones. Results Fifty-one differentially expressed genes were found by DD-PCR. Eight cDNA fragments were successfully cloned, two of which were confirmed to be new differentially expressed genes during apoptosis in potassium-deprived CGCs. Conclusion The isolated gene fragments, named 5# and 7#, are associated with low-potassium-induced apoptosis in CGCs.