【作者】 姜南；

【导师】 崔树森；

【作者基本信息】 吉林大学 ， 外科学， 2014， 博士

【摘要】 目的意义：随着社会的发展，周围神经损伤的患者日益增多。与中枢神经损伤不同，周围神经损伤后，其具有一定程度的自我再生能力。而这种自发再生的过程受到周围神经细胞多种分子基因水平的调控。早期人们更偏向于形态学研究，随着基因技术的发展，近阶段的研究发现了很多对周围神经损伤后具有再生调节能力的基因。而目前，尚缺乏对周围神经损伤后近、远端基因的数量及功能方面动态综合的研究，以及对比研究。本实验通过建立大鼠坐骨神经离断的模型，对其近远端基因所反映的生物学进程的变化趋势进行分析并加以对比。拟在已有研究的基础上，对周围神经损伤后再生的分子调控机制进行深入的研究。材料方法：成年雄性SD大鼠150只，根据坐骨神经离断时间的不同，随机分为6组，分别在第0天、4天、7天、14天、21天、28天处死大鼠，并在离断点近、远端分别取0.5厘米坐骨神经片段用于生物学分析。Trizol提取总RNA，制备基因表达芯片，每个时间点重复三次，运用相关软件对芯片数值进行标准化分析。并通过T检验对离断后不同时间点与空白对照组（0天）行统计学分析比较差异，选取p<0.05，fold change>2的基因作为统计学分析有差异显著性的基因进行生物学功能分析。我们应用DAVID在线软件的Gene Ontology (GO)的分析功能，对近远端差异基因所反映的刺激反应、炎症反应、免疫反应、细胞增殖、细胞迁移、细胞凋亡、轴突再生和导向、髓鞘生成、细胞外基质、信号转导和蛋白激酶活性共11个生物学进程进行生物学表达分析，观察相关差异基因在这11种生物学进程中的表达趋势变化，并对近远端的不同进行分析总结。为了验证本实验基因芯片的准确性，选取4个已知对周围神经有调控作用的基因，分别用实时荧光定量RT-PCR分析、Western Blot蛋白印迹分析以及组织化学免疫荧光染色从分子、蛋白、形态三个方面分别验证。研究结果：基因芯片分析结果显示，在坐骨神经近端片段，在7天时，上调基因表达最多，在4天后，下调基因表达稳定，在14天后两者基本维持同一水平，总体数量上上调基因表达占优势。在坐骨神经远端片段，在7天时，上调基因表达最多，而下调基因表达随着时间越来越多，两者总体水平基本相当。在生物学进程方面，刺激反应、炎症反应、免疫反应、细胞增殖、细胞迁移、细胞凋亡、轴突导向和再生、髓鞘生成、细胞外基质、信号转导和蛋白激酶活性这11个可概括周围神经再生性变化的生物学特性，在近远端每个时间点都有其各自特异性的变化，而这些都与我们已知的周围神经再生的过程基本一致。研究结论：1、在大鼠坐骨神经离断的模型中，在近端片段，上调基因总体占优势，而远端片段两者总体数量基本相当；无论是近端片段还是远端片段，都在同一时段（7天）具有最多的上调表达，而后两者表达趋势相反。2、在生物学进程方面，刺激反应、炎症反应、免疫反应、细胞增殖、细胞迁移、细胞凋亡、轴突导向和再生、髓鞘生成、细胞外基质、信号转导和蛋白激酶活性这11个生物学特性相关的差异基因最大值和正负向调控的最大差值大多数在第7天出现，它们在近远端每个时间点都有其各自特异性的变化，互有关联，组成了一个动态发展的调控网络。3、根据实时荧光定量RT-PCR分析、Western Blot蛋白印迹分析以及组织化学免疫荧光染色结果，基因芯片的准确性真实可靠。更多还原

【Abstract】 Objectives:In modern society, there are many peripheral nerve injuries in accident andmanufacturing works. Not as the central nervous system which could not regenerate,the injured peripheral nerves have the ability to regenerate by their own miraculously.And this kind of spontaneous regeneration process is controlled by various moleculargenetic levels of peripheral nerve cells. In earlier studies, people prefer to the researchof morphology. A lot of genes which have the adjustment ability of regeneration afterperipheral nerve injury were found in nearly study stage with the development of genetechnology. System knowledge and comparative study of gene expression changesand the related biological processes during peripheral nerve injury and regeneration,however, is still lacking.This study aimed to obtain system knowledge of gene expression changesfollowing the model of rat sciatic nerve transection injury and discover the possiblebiological processes via the contrast of differentiation of the proximal and distalsegments. We wish to have a deeply integrated insight into the molecular mechanismsregulating peripheral nerve regeneration from much broader perspectives on the basisof early research.Materials and Methods:150adult male Sprague-Dawley (SD) rats were randomly divided into6groupsaccording to the different time of sciatic nerve transection. The rats were executed at0day,4day,7day,14day,21day,28day respectively, and the proximal and distalsegments of0.5cm sciatic nerve were taken for biological analysis. The total RNAwere extracted by Trizol, the gene chips for expression were prepared, repeated threetimes at each time points, using the related software to chip numerical standardized analysis. We compared the data of different time points with blank control group (0day) for a statistical analysis through the T test, and choosed genes which were p <0.05, the fold change>2as have significant differences in statistical analysis to abiological function analysis.We applied the functin of Gene Ontology (GO) analysis of DAVID softwareonline, made the analysis of total11biology expression of biological processes whichare response to stimulus, inflammantory response, immune response, cell proliferation,cell migration, cell apoptosis, axon guidance and axonogenesis, myelination,extracellular matrix, signal transduction and protein kinase activity, were reflected bythe genes of proximal and distal segments. We observed expression trends inbiological process of the total of11biological processes about the variations of genes,analyzed and summarized the distinct of proximal and distal segments.In order to verify the experimental accuracy of the gene chips, we selected fourknown genes have a regulating effects on peripheral nerve, respectively, by used realtime fluorescence quantitative analysis, Western Blot protein analysis andhistochemistry immunofluorescence staining from three aspects of the molecules,protein, and morphology respectively.Research Results:The gene chips analysis showed that the most genes had the expression trend ofpositive regulation at7day, and the genes had a stable expression trend of negativeregulation at4day later, the two types maintained the balance after14day, the genesof positive regulation were dominant in the overall number in the proximal segmentsof sciatic nerve post transection. The genes had the top expression trend of positiveregulation at7day, and the numbers of genes with expression trend of negativeregulation were increased with time points, the two types maintained the balance intotal numbers in the distal segments of sciatic nerve post transection.In biological process, the11biological characteristics: inflammantory response,immune response, cell proliferation, cell migration, cell apoptosis, axon guidance andaxonogenesis, myelination, extracellular matrix, signal transduction and protein kinase activity which were associated with peripheral nerve regeneration, had thespecific changes in the proximal and distal segments in every time points. All thesewere had the accordance to peripheral nerve regeneration process we had knoun.Research Conclusions:1、In the model of rat with sciatic nerve transection, the genes of positive regulationwere dominant in the proximal segments, and the genes numbers of positive andnegative regulation were equally in the distal segments. There had the mostpositive expression in the same time point(7day), but a opposite expression trendafter the point in both proximal and distal segments however.2、The11biological characteristics which were inflammantory response, immuneresponse, cell proliferation, cell migration, cell apoptosis, axon guidance andaxonogenesis, myelination, extracellular matrix, signal transduction and proteinkinase activity which were related to peripheral nerve regeneration had thespecific changes in the proximal and distal segments in every time points inbiological process. The maximum of different genes and the biggest difference ofpositive and negative regulation appeared mostly in7day. They were connectedto each other and consisted a regulating and controlling networks that haddynamic development.3、The results of gene chips were true and correct according to the comfirmation ofreal-time fluorescence quantitative analysis, Western Blot protein analysis andhistochemistry immunofluorescence staining.更多还原