大鼠颈脊髓不同传导束损伤后差异性基因表达的研究
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摘要
目的：比较大鼠红核脊髓束和皮质脊髓背侧束损伤后24h的差异性基因表达,并对其行基因本体论和信号通路的分析,从基因层面探索脊髓损伤后的修复是否遵循进化论的原则,为将来以进化论为指导治疗脊髓损伤奠定基础。
方法：使用SAM软件对前期实验中得到的基因表达谱芯片结果进行分析,筛选和比较两种传导束损伤后基因的差异性表达；并通过MAS、GO、KEGG和GenMapp对生物过程和信号通路进行分析和比较。
选取8个感兴趣的基因：Fos, Ntrk1, Gadd45α, Myc, Sox7, Tnfrsf12a,Slc5a3和Rnf6,设计引物行RT-PCR验证。实验选取雌性SD大鼠12只,体重200-230g,平均220g,随机分为4组：双侧皮质脊髓背侧束+薄束楔束损伤组3只,双侧薄束楔束损伤组3只,双侧红核脊髓束损伤组3只,假手术组3只。建立动物模型后24h后处死动物,以损伤处为中心取5mm脊髓组织,将每组中的标本混合提取总RNA行RT-PCR检验,并将结果与基因芯片结果进行比较。
结果：两种传导束损伤后24h基因表达变化存在差异,红核脊髓束损伤后表达发生变化的基因有153个,上调的有136个,下调的有17个。皮质脊髓背侧束损伤后表达发生变化的基因有26个,上调的有22个,下调的有4个。红核脊髓束损伤后发生显著性变化(P<0.05)的生物过程条目为164条,25条涉及到3个及以上的基因变化；发生显著性变化(P<0.05)的信号通路为25条,17条涉及到3个及以上的基因变化。皮质脊髓背侧束损伤后发生显著性变化(P<0.05)的生物过程条目为7条,发生显著性变化(P<0.05)的信号通路为1条,涉及到的基因均少于3个。这些基因主要都涉及到炎症、免疫、抗原处理及呈递等急性期反应,少数与神经发育和轴突再生相关。
RT-PCR验证结果显示两种传导束损伤后Myc、Fos、Tnfrsfl 2a和Gadd45a的变化和基因芯片结果完全一致,Sox7和Rnf6在红核脊髓束损伤后表达无显著性变化,Ntrkl和Slc5a3基因在红核脊髓束损伤后表达和基因芯片结果相反。
结论：红核脊髓束损伤后24h基因表达变化多于皮质脊髓背侧束损伤,该变化与损伤面积大小无关,是否与进化程度有关还需进一步验证。红核脊髓束损伤后利于神经再生的基因如Fos、TrkA表达上调高于皮质脊髓背侧束损伤组,利于轴突再生的信号通路如MAPK也被激活,在基因水平上支持红核脊髓束的修复能力强于皮质脊髓背侧束,但还需要蛋白质层面和组织学层面的进一步验证。红核脊髓束损伤后其他显著性上调的基因如Tnfrsfl2a和Gadd45a与神经再生有无直接关系值得进一步的研究。
Objective:To compare the different gene expressional changes and difference in gene ontology and pathway between RST and dCST after transection at 24h; try to explain different regeneration abilities of different evolved spinal cord tracts using evolutionism at the gene level; and provide a robust framework in the treatment of SCI in the light of evolutionism.
Methods:Gene expressional profiles obtained in former experiments were analyzed, and the differentially expressed genes were screened and compared by SAM. The biological processes and pathways between RST and dCST were analyzed by MAS, GO, Kegg, and GenMAPP.
To corroborate and validate microarray results, Fos, Ntrkl, Gadd45a, Myc, Sox7, Tnfrsfl2a, Slc5a3 and Rnf6 were selected for RT-PCR test.12 female Sprague-Dawley rats, which weighed 200-230g (average 220g), were divided randomly into 4 groups:bilateral dCST+FCFG transection group (n=3), bilateral FCFG transection group (n=3), bilateral RST transection group (n=3) and sham operation group (n=3). Rats were sacrificed24h after injury,5mm length spinal cord specimens centered injury site were procured. Total RNA were extracted and purified. RT-PCR was performed and the results were compared with results of microarray analysis.
Results:Differences of gene expressional profiles exist between RST and dCST after injury. The expression of 153 genes changed after transection of RST, 136 were up-regulated and 17 were down-regulated. The expression of 26 genes changed after transection of dCST,22 were up-regulated and 4 were down-regulated. The number of biological process terms significantly changed (P < 0.05) after RST transection was 164,25 terms involved with no less than 3 gene counts; the number of pathways significantly changed (P< 0.05) was 25,17 terms involved with no less than 3 gene counts. The number of biological process terms significantly changed (P< 0.05) after dCST transection was 7, the number of pathways significantly changed (P< 0.05) was 1, all involved with less than 3 gene counts. Most of the functions were related to acute phase response such as immune response, antigen processing and presentation, inflammatory response. Few genes were related to neural regeneration.
The expressional changes of Myc, Fos, Tnfrsfl2a, and Gadd45a tested by RT-PCR were in accordance with the results of microarray analysis. The expressional changes of Sox7 and Rnf6 showed no significant changes after RST transection. The expressional changes of Ntrkl and Slc5a3 after RST transection were contrast to the results of microarray analysis.
Conclusion:The different expressional changes of genes have nothing to do with injured area. Further experiments should be performed to testify if it is related to different evolution. Genes related to neural regeneration such as Fos and Ntrkl were more up-regulated after RST transection, pathways related to axon regeneration such as MAPK pathway was activated after RST transection, which suggest RST has stronger regeneration ability at the gene level. However, this should be testified at the protein and histology level. Other genes significantly up-regulated after RST injury, such as Tnfrsfl2a and Gadd45a may have relationship with axon regeneration, and deserves further research.
方法：使用SAM软件对前期实验中得到的基因表达谱芯片结果进行分析,筛选和比较两种传导束损伤后基因的差异性表达；并通过MAS、GO、KEGG和GenMapp对生物过程和信号通路进行分析和比较。
选取8个感兴趣的基因：Fos, Ntrk1, Gadd45α, Myc, Sox7, Tnfrsf12a,Slc5a3和Rnf6,设计引物行RT-PCR验证。实验选取雌性SD大鼠12只,体重200-230g,平均220g,随机分为4组：双侧皮质脊髓背侧束+薄束楔束损伤组3只,双侧薄束楔束损伤组3只,双侧红核脊髓束损伤组3只,假手术组3只。建立动物模型后24h后处死动物,以损伤处为中心取5mm脊髓组织,将每组中的标本混合提取总RNA行RT-PCR检验,并将结果与基因芯片结果进行比较。
结果：两种传导束损伤后24h基因表达变化存在差异,红核脊髓束损伤后表达发生变化的基因有153个,上调的有136个,下调的有17个。皮质脊髓背侧束损伤后表达发生变化的基因有26个,上调的有22个,下调的有4个。红核脊髓束损伤后发生显著性变化(P<0.05)的生物过程条目为164条,25条涉及到3个及以上的基因变化；发生显著性变化(P<0.05)的信号通路为25条,17条涉及到3个及以上的基因变化。皮质脊髓背侧束损伤后发生显著性变化(P<0.05)的生物过程条目为7条,发生显著性变化(P<0.05)的信号通路为1条,涉及到的基因均少于3个。这些基因主要都涉及到炎症、免疫、抗原处理及呈递等急性期反应,少数与神经发育和轴突再生相关。
RT-PCR验证结果显示两种传导束损伤后Myc、Fos、Tnfrsfl 2a和Gadd45a的变化和基因芯片结果完全一致,Sox7和Rnf6在红核脊髓束损伤后表达无显著性变化,Ntrkl和Slc5a3基因在红核脊髓束损伤后表达和基因芯片结果相反。
结论：红核脊髓束损伤后24h基因表达变化多于皮质脊髓背侧束损伤,该变化与损伤面积大小无关,是否与进化程度有关还需进一步验证。红核脊髓束损伤后利于神经再生的基因如Fos、TrkA表达上调高于皮质脊髓背侧束损伤组,利于轴突再生的信号通路如MAPK也被激活,在基因水平上支持红核脊髓束的修复能力强于皮质脊髓背侧束,但还需要蛋白质层面和组织学层面的进一步验证。红核脊髓束损伤后其他显著性上调的基因如Tnfrsfl2a和Gadd45a与神经再生有无直接关系值得进一步的研究。
Objective:To compare the different gene expressional changes and difference in gene ontology and pathway between RST and dCST after transection at 24h; try to explain different regeneration abilities of different evolved spinal cord tracts using evolutionism at the gene level; and provide a robust framework in the treatment of SCI in the light of evolutionism.
Methods:Gene expressional profiles obtained in former experiments were analyzed, and the differentially expressed genes were screened and compared by SAM. The biological processes and pathways between RST and dCST were analyzed by MAS, GO, Kegg, and GenMAPP.
To corroborate and validate microarray results, Fos, Ntrkl, Gadd45a, Myc, Sox7, Tnfrsfl2a, Slc5a3 and Rnf6 were selected for RT-PCR test.12 female Sprague-Dawley rats, which weighed 200-230g (average 220g), were divided randomly into 4 groups:bilateral dCST+FCFG transection group (n=3), bilateral FCFG transection group (n=3), bilateral RST transection group (n=3) and sham operation group (n=3). Rats were sacrificed24h after injury,5mm length spinal cord specimens centered injury site were procured. Total RNA were extracted and purified. RT-PCR was performed and the results were compared with results of microarray analysis.
Results:Differences of gene expressional profiles exist between RST and dCST after injury. The expression of 153 genes changed after transection of RST, 136 were up-regulated and 17 were down-regulated. The expression of 26 genes changed after transection of dCST,22 were up-regulated and 4 were down-regulated. The number of biological process terms significantly changed (P < 0.05) after RST transection was 164,25 terms involved with no less than 3 gene counts; the number of pathways significantly changed (P< 0.05) was 25,17 terms involved with no less than 3 gene counts. The number of biological process terms significantly changed (P< 0.05) after dCST transection was 7, the number of pathways significantly changed (P< 0.05) was 1, all involved with less than 3 gene counts. Most of the functions were related to acute phase response such as immune response, antigen processing and presentation, inflammatory response. Few genes were related to neural regeneration.
The expressional changes of Myc, Fos, Tnfrsfl2a, and Gadd45a tested by RT-PCR were in accordance with the results of microarray analysis. The expressional changes of Sox7 and Rnf6 showed no significant changes after RST transection. The expressional changes of Ntrkl and Slc5a3 after RST transection were contrast to the results of microarray analysis.
Conclusion:The different expressional changes of genes have nothing to do with injured area. Further experiments should be performed to testify if it is related to different evolution. Genes related to neural regeneration such as Fos and Ntrkl were more up-regulated after RST transection, pathways related to axon regeneration such as MAPK pathway was activated after RST transection, which suggest RST has stronger regeneration ability at the gene level. However, this should be testified at the protein and histology level. Other genes significantly up-regulated after RST injury, such as Tnfrsfl2a and Gadd45a may have relationship with axon regeneration, and deserves further research.
引文
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