实验性脑脓肿组织中G蛋白信号系统相关基因的克隆

Preliminary study on G proteins signaling pathway in early phase of experimental brain abscess by mRNA differential display PCR

目的筛选和克隆实验性脑脓肿的早期相关基因,研究脑组织对病原菌的免疫反应过程中所涉及的分子机制。方法通过脑组织内直接注射金黄色葡萄球菌获得大鼠脑脓肿,利用mRNA荧光差异显示PCR技术比较脑脓肿组脑组织中mRNA的表达与正常对照组、手术对照组之间的差异,获得的差异表达cDNA片段经克隆、测序及BLAST软件进行同源性比较分析,并采用Northern杂交和RTPCR进行鉴定。结果共获得25条差异表达的cDNA条带,经Northern杂交和RTPCR鉴定其中17条为阳性(阳性率为68%)。克隆和测序结果显示其中3个差异表达片段与G蛋白相关的细胞信号传导系统有关:片段G181代表的鸟苷二磷酸解离抑制因子3(GDI3)、片段G201代表的交集素(ITSN)以及片段C21代表的Ras相关蛋白(Rap1)。结论在实验性脑脓肿早期GDI3、ITSN和Rap1差异表达,推导G蛋白信号系统的激活可能与脑脓肿的发病相关。

Objective To identify related genes associated with early phase of experimental brain abscess and to investigate the molecular mechanism of brain abscess. Methods Brain abscess was produced in the rat by direct intracerebral injection of Staphylococcus attreus. In early phase of experimental brain abscess, abscess tissue samples were compared with normal and control group by rnRNA differential display PCR. Differentially expressed cDNA fragments were cloned into the pGEM-T easy vector. Positive clones were sequenced and then confinned by Northern blot and RT-PCR. All the sequences were put into GenBank database and analyzed by BLAST software to search for their genetics origins. Results Twenty-five differentially expressed cDNA fragments were obtained, 17 of which were confirmed by Northern blot and RT-PCR. Analysis of the sequences by BLAST software showed that three fragments shared high homology with the genes in G proteins signaling pathway, guanosine diphosphate dissociation inhibitor 3 （GDI3）, intersectin 1 （ITSN） and Ras-aasociated protein（Rap1）. Conclusion In this study, GD13, ITSN and Rapl were confmned to be differentially expressed at the early stage of experimental brain abscess. The activation of G proteins signaling pathway might play an important role in the pathogenesis of brain abscess.