应用差异显示技术研究实验性脑脓肿的早期相关基因
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摘要
目的
研究脑组织对金黄色葡萄球菌的免疫反应及脑脓肿形成和发展过程中所涉及的分子机制。
方法
采用金黄色葡萄球菌大鼠右大脑半球皮层下接种的方法建立实验性脑脓肿模型；通过改进了的mRNA差异显示PCR技术对实验性脑脓肿早期的组织标本进行了检测；对差异表达基因的扩增片段进行克隆、测序、鉴定，再与Genbank数据库中的已知序列进行同源性匹配，确定脑脓肿相关基因或发现新基因，对新基因进行EST序列的注册；通过生物信息学技术对脑脓肿相关基因进行染色体定位，对部分脑脓肿相关EST片段进行电子延伸并用RT-PCR加以证实，并作开放阅读框架(ORF)及翻译蛋白功能的预测分析；应用5′末端快速扩增法(5′ RACE)对1个脑脓肿相关EST片段进行扩增序列全长的尝试。
结果
建立了稳定的实验性脑脓肿模型；经克隆、测序及鉴定获得了17个脑脓肿相关的差异表达基因，其中7个为功能明确的已知基因，3个为功能未明的已知基因，5个只与大鼠EST片段有高度的同源性，其余2个为新基因。2个新基因为片段G3-2和片段G3-3，进行EST序列的注册后得到EST数据库的登陆号18522605和21767476以及GenBank数据库的登陆号CD490310和CK826599。1个功能明确的已知基因分别为片段G2-1代表的异类核核蛋白F
(hnRNPF)、GZ一2代表的次要组织相容性杭原H 13、片段G 18一1代表的鸟
普二磷酸解离抑制因子3(GDI3)、片段G20一l代表的交集素(ITSN)、片段
G20一代表的x染色体连锁的核糖体蛋白s4(RPs4x)、片段A18一2代表的
SPT3相关因子42相似蛋白、以及片段CZ一1代表的Ras相关蛋白.对其余脑
脓肿相关基因进行了染色体定位;对3个脑脓肿相关EST片段成功地进行了
电子延伸并获得RT一PCR证实.应用5‘RACE进行扩增序列全长的尝试未
获成功.
结论
采用金黄色葡萄球菌大鼠右大脑半球皮层下接种的方法建立的实验性脑
脓肿模型完全符合脑脓肿的病理变化过程;脑组织对金萄菌的免疫反应及脑
脓肿形成和发展过程中涉及复杂的分子机制，G蛋白信号系统的激活，特别
是G二亚基效应因子信号传导通路的激活起着更为重要的作用.此外，机体
通过不同的转录前调控机制启动了大量的基因表达，而且在转录后通过对
mRNA前体进行不同方式的拼接合成新的蛋白对脑脓肿做出反应.H13杭原
过表达就是转录增强的结果，并可能由此引发了免疫反应的异常，即在脑脓
肿晚期可能存在炎性细月色付脑组织的自身免疫.
Objective
To search the related genes associated with early phase of experimental brain abscess and to explore the molecular mechanism of brain abscess.
Method
Brain abscesses were produced in the rat by direct intracerebral injection of stapbylococcus aureus. In early phase of experimental brain abscess, abscess tissue samples were compared with normal and control group by mRNA differential display PCR. Differentially expressed cDNA fragments were cloned into the pGEM-Teasy vector. Positive clones were sequenced and then confirmed by Northern blot or RT-PCR. All the sequences were put into Genebank database and analyzed by BLASTN software to search for their genetics origins or to find novel genes. Then novel genes were submitted to Genebank database. Chromosome location and electronic cloning were performed by bioinformatics. The sequences obtained from electronic cloning were confirmed by RT-PCR and predicted. An effort to obtain full-length cDNA was attempted by 5' rapid ampliation ofcDNA ends (5' RACE).
Result
The experimental brain abscess model was established successfully in the rat. 17 differentially expressed cDNA fragments were cloned and identified. Among these fragments, 7 fragments showed highly homologous to known sequences
with known function and 3 fragments showed known sequences with unknown function in Genebank nr database, 5 fragments showed highly homologous to EST in EST database, 2 fragments showed no homologous in nr or EST database. The sequence data of these 2 fragments, Frag G3-2 and Frag G3-3, were submitted to Genebank with accession No. CD490310 and CK.826599. 7 known sequences with known function includes heterogeneous nuclear ribonucleoprotein F (hnRNP F), minor histocompatibility antigen (mHA) HI3, guanosine diphosphate dissociation inhibitor 3(GDI3), intersectin 1(ITSN), ribosomal protein S4 X-linked(Rps4x), SPT3-associated factor 42 and Ras-associated protein(Rap1). All fragments were located in chromosomes. 3 fragments were prolongated by electronic cloning and confirmed by RT-PCR. The attempt to obtain full-length cDNA was unsuccessful.
Conclusions
The histopathologic features of the rat experimental abscesses produced by direct intracerebral injection of stapbylococcus aureus are similar to other animal models and to human abscess. The molecular mechanism of brain abscess is complicated. Activation of G proteins signaling pathway, especially activation of
G a effectors signaling pathway, play an important role. After stapbylococcus
aureus exposure, differential pre-transcriptional regulation mechanisms take place. Then large numbers of genes were expressed and new proteins were synthesized through the alternative splicing of pro-mRNA. As a result, over-expressed mHA H13 may cause autoimmunity of inflammatory cells versus brain tissue.
研究脑组织对金黄色葡萄球菌的免疫反应及脑脓肿形成和发展过程中所涉及的分子机制。
方法
采用金黄色葡萄球菌大鼠右大脑半球皮层下接种的方法建立实验性脑脓肿模型；通过改进了的mRNA差异显示PCR技术对实验性脑脓肿早期的组织标本进行了检测；对差异表达基因的扩增片段进行克隆、测序、鉴定，再与Genbank数据库中的已知序列进行同源性匹配，确定脑脓肿相关基因或发现新基因，对新基因进行EST序列的注册；通过生物信息学技术对脑脓肿相关基因进行染色体定位，对部分脑脓肿相关EST片段进行电子延伸并用RT-PCR加以证实，并作开放阅读框架(ORF)及翻译蛋白功能的预测分析；应用5′末端快速扩增法(5′ RACE)对1个脑脓肿相关EST片段进行扩增序列全长的尝试。
结果
建立了稳定的实验性脑脓肿模型；经克隆、测序及鉴定获得了17个脑脓肿相关的差异表达基因，其中7个为功能明确的已知基因，3个为功能未明的已知基因，5个只与大鼠EST片段有高度的同源性，其余2个为新基因。2个新基因为片段G3-2和片段G3-3，进行EST序列的注册后得到EST数据库的登陆号18522605和21767476以及GenBank数据库的登陆号CD490310和CK826599。1个功能明确的已知基因分别为片段G2-1代表的异类核核蛋白F
(hnRNPF)、GZ一2代表的次要组织相容性杭原H 13、片段G 18一1代表的鸟
普二磷酸解离抑制因子3(GDI3)、片段G20一l代表的交集素(ITSN)、片段
G20一代表的x染色体连锁的核糖体蛋白s4(RPs4x)、片段A18一2代表的
SPT3相关因子42相似蛋白、以及片段CZ一1代表的Ras相关蛋白.对其余脑
脓肿相关基因进行了染色体定位;对3个脑脓肿相关EST片段成功地进行了
电子延伸并获得RT一PCR证实.应用5‘RACE进行扩增序列全长的尝试未
获成功.
结论
采用金黄色葡萄球菌大鼠右大脑半球皮层下接种的方法建立的实验性脑
脓肿模型完全符合脑脓肿的病理变化过程;脑组织对金萄菌的免疫反应及脑
脓肿形成和发展过程中涉及复杂的分子机制，G蛋白信号系统的激活，特别
是G二亚基效应因子信号传导通路的激活起着更为重要的作用.此外，机体
通过不同的转录前调控机制启动了大量的基因表达，而且在转录后通过对
mRNA前体进行不同方式的拼接合成新的蛋白对脑脓肿做出反应.H13杭原
过表达就是转录增强的结果，并可能由此引发了免疫反应的异常，即在脑脓
肿晚期可能存在炎性细月色付脑组织的自身免疫.
Objective
To search the related genes associated with early phase of experimental brain abscess and to explore the molecular mechanism of brain abscess.
Method
Brain abscesses were produced in the rat by direct intracerebral injection of stapbylococcus aureus. In early phase of experimental brain abscess, abscess tissue samples were compared with normal and control group by mRNA differential display PCR. Differentially expressed cDNA fragments were cloned into the pGEM-Teasy vector. Positive clones were sequenced and then confirmed by Northern blot or RT-PCR. All the sequences were put into Genebank database and analyzed by BLASTN software to search for their genetics origins or to find novel genes. Then novel genes were submitted to Genebank database. Chromosome location and electronic cloning were performed by bioinformatics. The sequences obtained from electronic cloning were confirmed by RT-PCR and predicted. An effort to obtain full-length cDNA was attempted by 5' rapid ampliation ofcDNA ends (5' RACE).
Result
The experimental brain abscess model was established successfully in the rat. 17 differentially expressed cDNA fragments were cloned and identified. Among these fragments, 7 fragments showed highly homologous to known sequences
with known function and 3 fragments showed known sequences with unknown function in Genebank nr database, 5 fragments showed highly homologous to EST in EST database, 2 fragments showed no homologous in nr or EST database. The sequence data of these 2 fragments, Frag G3-2 and Frag G3-3, were submitted to Genebank with accession No. CD490310 and CK.826599. 7 known sequences with known function includes heterogeneous nuclear ribonucleoprotein F (hnRNP F), minor histocompatibility antigen (mHA) HI3, guanosine diphosphate dissociation inhibitor 3(GDI3), intersectin 1(ITSN), ribosomal protein S4 X-linked(Rps4x), SPT3-associated factor 42 and Ras-associated protein(Rap1). All fragments were located in chromosomes. 3 fragments were prolongated by electronic cloning and confirmed by RT-PCR. The attempt to obtain full-length cDNA was unsuccessful.
Conclusions
The histopathologic features of the rat experimental abscesses produced by direct intracerebral injection of stapbylococcus aureus are similar to other animal models and to human abscess. The molecular mechanism of brain abscess is complicated. Activation of G proteins signaling pathway, especially activation of
G a effectors signaling pathway, play an important role. After stapbylococcus
aureus exposure, differential pre-transcriptional regulation mechanisms take place. Then large numbers of genes were expressed and new proteins were synthesized through the alternative splicing of pro-mRNA. As a result, over-expressed mHA H13 may cause autoimmunity of inflammatory cells versus brain tissue.
引文
1. Wispelwey B, Dacey RG, Scheld WM. Brain abscess. Infections of the central nervous system. Edited by Scheld WM, Whitely RJ, Durack DT. New York, Raven Press, 1991: 457-86
2. Svantenson B, Nordstrom CH, Rausing A. Nontraumatic brain abscess. Epidemiology, clinical symptoms and therapeutic results. Acta Neurochir(Wien) 1988, 94:57-65
3. Enzmann DR, et al. Experimental brain abscesses evolution: Computed tomographic and neuropathologic correlation. Radiology 1979, 133:113-22
4. Flaris NA and Hickey WF. Development and characterization of an experimental model of brain abscesses in the rat. Am J Pathol 1992, 141:1299-307
5. Kielian T and Hickey WF. Proinflammatory cytokine, chemokine, and cellular adhesion molecule expression during the acute phase of experimental brain abscess development. Am J Pathol 2000, 157:647-58
6. Kielian T, Barry B and Hickey WF. CXC Chemokine Receptor-2 Ligands Are Required for Neutrophil-Mediated Host Defense in Experimental Brain Abscesses. J Immunol 2001, 166:4634-43
7. Shuyuan Y and Chunsheng Z. Review of 140 patients with brain abscesses. Surg Neurol 1993, 39:290-6
8. Shuyuan Y. Brain abscess associated with congenital heart disease. Surg Neurol 1989, 31 : 129-32
9. Shuyuan Y. Brain abscess: a review of 400 cases. J Neurosurgery 1981, 55:794-9
10．杨树源，赵春生．实验性脑脓肿模型的建立．中华神经外科杂志 1992，9：134-5
11．杨树源，张建宁，赵春生等．实验性脑脓肿影响学改变的病理基础研究．中华神经外科杂志 1998，14：367-70
12. Liang P and Pardee AB. Differentail display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 1992, 257:967-971
2. Svantenson B, Nordstrom CH, Rausing A. Nontraumatic brain abscess. Epidemiology, clinical symptoms and therapeutic results. Acta Neurochir(Wien) 1988, 94:57-65
3. Enzmann DR, et al. Experimental brain abscesses evolution: Computed tomographic and neuropathologic correlation. Radiology 1979, 133:113-22
4. Flaris NA and Hickey WF. Development and characterization of an experimental model of brain abscesses in the rat. Am J Pathol 1992, 141:1299-307
5. Kielian T and Hickey WF. Proinflammatory cytokine, chemokine, and cellular adhesion molecule expression during the acute phase of experimental brain abscess development. Am J Pathol 2000, 157:647-58
6. Kielian T, Barry B and Hickey WF. CXC Chemokine Receptor-2 Ligands Are Required for Neutrophil-Mediated Host Defense in Experimental Brain Abscesses. J Immunol 2001, 166:4634-43
7. Shuyuan Y and Chunsheng Z. Review of 140 patients with brain abscesses. Surg Neurol 1993, 39:290-6
8. Shuyuan Y. Brain abscess associated with congenital heart disease. Surg Neurol 1989, 31 : 129-32
9. Shuyuan Y. Brain abscess: a review of 400 cases. J Neurosurgery 1981, 55:794-9
10．杨树源，赵春生．实验性脑脓肿模型的建立．中华神经外科杂志 1992，9：134-5
11．杨树源，张建宁，赵春生等．实验性脑脓肿影响学改变的病理基础研究．中华神经外科杂志 1998，14：367-70
12. Liang P and Pardee AB. Differentail display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 1992, 257:967-971