用mRNA差异显示法筛选和鉴定反复高G_Z暴露大鼠脑损伤相关基因

Screening and identification of genes related to the rat brain damage induced by high repeated +G_Z exposures with differential display polymerase chain reaction

目的 筛选和鉴定反复高 GZ暴露大鼠脑损伤相关基因。 方法 16只 SD大鼠随机分成对照组、+GZ重复暴露后 30 min、6 h和 2 4h4组 ,每组 4只。对照组大鼠 G值为 +1GZ,实验组大鼠在动物离心机上经历了 3次 +14GZ/ 45 s(两次间间隔 30 m in)作用。分别于暴露后 30 m in、6 h和2 4h处死大鼠取脑 ,提取总 RNA。取对照组和 +GZ暴露后 6 h组大鼠脑总 RNA,用 m RNA差异显示( DDPCR)的方法 ,筛选 +GZ暴露大鼠脑差异表达基因。用 3组锚定引物和 6组随机引物作不同组合进行 PCR扩增。用 Slot blot方法分析差异表达基因在对照组和 +GZ暴露后 30 m in、6 h和 2 4h组大鼠脑中表达情况。 结果 获得的差异表达片段 ,经斑点杂交进一步筛选 ,克隆了 3个强阳性片段 ,并进行序列分析 ,与 Genebank等数据库进行同源比较 ,没有同源基因。筛选的 3个差异表达基因在+GZ暴露后 6 h和 2 4h组大鼠脑中呈高表达。 结论 用 DDPCR筛选到 3个可能与反复高 GZ暴露大鼠脑损伤相关基因 ,它们在脑损伤的病理过程中可能起一定作用。

Objective To screen and identify the genes related to the rat brain damage induced by high repeated +G Z exposures. Methods Sixteen conscious SD rats were randomly divided into 4 groups: 1 control group, and 3 experimental groups. Using an animal centrifuge, control rats (n=4) were exposed to +1 G Z and experimental rats (n=12) were exposed to +14 G Z 3 times, each for 45 s with 30 min interval in between. The brains of 3 experimental groups were taken 30 min, 6 h and 24 h after the last centrifuge run respectively and total RNA was isolated. Differential display polymerase chain reaction (DDPCR) was used to compare gene expressions in brain of controls and those of experimentals 6 h after repeated +G Z exposures. Three sets of anchor primers and 6 sets of arbitrary ones were used for DDPCR amplification. The expression levels of differential display genes in the rat brains of controls and experimentals taken 30 min, 6 h and 24 h after repeated +G Z exposures were analyzed by Slot blot. Results The obtained differential display fragments were further selected by Dot blot, and 3 fragments with strong positive signals were cloned and sequenced. Homology searching in Genebank showed that the 3 gene fragments may be new genes. The results from Slot blot confirmed that the expression levels of the 3 differential display genes in rat brains taken 30 min, 6 h and 24 h after repeated +G Z exposures were significantly higher than those of control group. Conclusions The 3 new genes related to the rat brain damage induced by repeated +G Z exposures were found with DDPCR, and they might play an important role in the pathogenesis of rat brain damage induced by repeated +G Z exposures.