Slit—Robo GTPase激活蛋白在坐骨神经切断后脊神经节的表达变化

目的：观察Slit—Robo GTP酶激活蛋白（srGAP）在成年哺乳类动物脊神经节（DRG）的表达及其变化，了解srGAP在神经再生中的作用。方法：应用RT-PCR、免疫印迹法、免疫组织化学方法，检测成年SD大鼠正常和坐骨神经切断后1、3、7d的DRGsrGAP1、2、3mRNA及其蛋白的表达。结果：srGAP1、3mRNA在正常DRG均有弱表达，而srGAP2不表达；坐骨神经切断后3、7d，srGAP1、3mRNA表达增强，以术后7d最高；免疫组织化学显示，srGAP1在神经元胞体、突起均有表达，在DRG内胶质细胞亦有表达，坐骨神经切断后3、7d表达增强。结论：本研究结果说明Slit-Robo信号通路存在于大鼠周围感觉神经元内，周围神经损伤可激活此信号通路。

srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons by inactivating RAC1

Objective:To explore effect of srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons.Methods:In this study,expression of Slit1 was observed predominantly in the glia.while expression of Robo2 and srCAP3 was detected in sensory neurons of postnatal rat cultured dorsal root ganglion(DRG).Furthermore,upregulation of srGAP3 following sciatic nerve transection was detected by immunohistochemistry and Western blotting.Results:It was observed that inhibition of nenrite outgrowth in cultured adult DRG neurons following treatment with anti-srGAP3 or anti-Robo2 was more effectively(1.5-fold higher) than that following treatment with an anti-BDNF positive control antibody.It demonstrated that srGAP3 interacted with Robo2 and Slit1 protein to decrease Rac1-CTP activity in cultured adult rat DRG neurons and the opposite effect on Rac1-GTP activity was detected by co-immunoprecipitation and immunoblotting analyses following treatment with anti-Robo2 or anti-srGAP3.These data demonstrated a role for srGAP3 in nenrite outgrowth ol DRG sensory neurons.Conclusions:Our observations suggest that srGAP3 promotes neurile outgrowth and filopodial growth cones by interacting with Robo2 to inactivate Rac1 in mammalian DRG neurons.

靶向srGAP家族的串联短发夹RNA表达

为了解析不同srGAP蛋白在神经元发育中的补偿效应,构建了一种同时靶向srGAP家族成员srGAP1～3的串联短发夹RNA表达系统.通过设计,合成多个寡核苷酸,利用微小RNA表达载体构建相应srGAP基因的短发夹RNA表达质粒,并利用蛋白免疫印迹法筛选敲减作用明显的靶向不同srGAP基因的短发夹RNA,然后,将其串联到同一载体而构建成可以同时靶向srGAP家族的短发夹RNA串联重组质粒.结果表明,筛选得到了分别针对srGAP1～3的短发夹RNA J17、J24和J33,并构建出能够同时靶向srGAP1～3基因的串联短发夹RNA J1+2+3.

Differential expression of microRNAs in dorsal root ganglia after sciatic nerve injury

This study investigated the possible involvement of microRNAs in the regulation of genes that participate in peripheral neural regeneration.A microRNA microarray analysis was conducted and 23 microRNAs were identified whose expression was significantly changed in rat dorsal root ganglia after sciatic nerve transection.The expression of one of the downregulated microRNAs,microRNA-214,was validated using quantitative reverse transcriptase-PCR.MicroRNA-214 was predicted to target the 3′-untranslated region of Slit-Robo GTPase-activating protein 3.In situ hybridization verified that microRNA-214 was located in the cytoplasm of dorsal root ganglia primary neurons and was downregulated following sciatic nerve transection.Moreover,a combination of in situ hybridization and immunohistochemistry revealed that microRNA-214 and Slit-Robo GTPase-activating protein 3 were co-localized in dorsal root ganglion primary neurons.Western blot analysis suggested that Slit-Robo GTPase-activating protein 3 was upregulated in dorsal root ganglion neurons after sciatic nerve transection.These data demonstrate that microRNA-214 is located and differentially expressed in dorsal root ganglion primary neurons and may participate in regulating the gene expression of Slit-Robo GTPase-activating protein 3 after sciatic nerve transection.

人SRGAP1蛋白结构和功能预测

目的:利用生物信息学对人Slit-Robo GTP酶激活蛋白1(SRGAP1)蛋白质的理化性质,信号肽,亲水性/疏水性,跨膜区域,蛋白质二级结构、三级结构,蛋白质间的相互作用及GO注释进行预测分析。方法:使用多种分析软件对SRGAP1蛋白进行在线预测分析。结果:SRGAP1理化性质分析结果显示,人SRGAP1蛋白有1 085个氨基酸,等电点为6.36;生物信息学预测分析结果显示,SRGAP1是无信号肽、无跨膜结构的亲水不稳定蛋白质。二级结构存在19个α螺旋和11个β折叠,预测到10个与SRGAP1存在相互作用的蛋白。SRGAP1蛋白在调控GTP酶活性,细胞迁移,信号通路中有重要的作用。结论:系统预测分析了等电点为6.36的人SRGAP1蛋白理化性质、结构和功能,为将来探索SRGAP1具体功能和分子机制提供了一定的思路和理论基础。

难治性癫痫患儿脑组织srGAP2γ-氨基丁酸及MCP-1表达的意义

目的研究srGAP2(Slit-Robo GTPase activating Protein 2)、γ-氨基丁酸以及单核细胞趋化蛋白-1(MCP-1)在难治性癫痫患儿脑组织中的表达情况及意义。方法选取11例患儿的颞叶脑组织和同期11例正常标本的颞叶脑组织,使用免疫组化和免疫印迹等方法检测srGAP2、γ-氨基丁酸以及MCP-1的表达情况。结果难治性癫痫患儿脑组织中,srGAP2的阳性表达率显著高于正常脑组织。难治性癫痫患儿脑组织和正常脑组织可以看到染成棕褐色的神经元,但难治性癫痫患儿脑组织γ-氨基丁酸能神经元表达量明显低于正常脑组织。正常脑组织中MCP-1的表达较弱,而难治性癫痫患儿脑组织中MCP-1的表达较强。结论难治性癫痫患儿脑组织srGAP2和MCP-1表达上升而γ-氨基丁酸表达下降,srGAP2、γ-氨基丁酸及MCP-1与难治性癫痫发病密切相关,可能作为潜在治疗靶标。