大鼠坐骨神经横断后近远端神经基因表达和生物学进程分析的对比研究

发布时间：2018-06-04 10:29

本文选题：神经横断 + 近端片段　；参考：《吉林大学》2014年博士论文

【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p0.05，fold change2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。
[Abstract]:Objective significance:
With the development of society, the patients with peripheral nerve injury are increasing. Different from the central nerve injury, the peripheral nerve has a certain degree of self regenerative ability. This spontaneous regeneration process is regulated by a variety of molecular gene levels in the peripheral nerve cells. In recent years, many genes have been found to have the ability to regenerate and regulate the peripheral nerve injury. At present, there is still a lack of dynamic comprehensive research on the number and function of the peripheral nerve, the number and function of the distal genes, and the comparative study.
In this experiment, the model of the rat sciatic nerve disconnection was established to analyze and compare the trend of biological processes reflected by the proximal and distal genes. On the basis of the existing research, the molecular regulation mechanism of regeneration after peripheral nerve injury was studied.
Material methods:
150 adult male SD rats were randomly divided into 6 groups according to the time of the sciatic nerve break. They were randomly divided into 6 groups, zeroth days, 4 days, 7 days, 14 days, 21 days, and 28 days, and the distal part of the sciatic nerve was used for the biological analysis of the total RNA, and the gene expression chip was repeated three times at each time point. The correlation software was used to standardize the chip numeric analysis, and the difference was compared with the blank control group (0 days) by T test. The gene of P0.05 and fold change2 was selected as the statistical analysis of the biological function analysis with different significant genes.
We use the analysis function of Gene Ontology (GO) of DAVID online software to carry out 11 biological processes, such as stimulus response, inflammatory response, immune response, cell proliferation, cell migration, cell apoptosis, axon regeneration and guidance, myelin formation, extracellular matrix, signal transduction and protein kinase activity. To analyze the expression trends of the differential genes in these 11 biological processes, and analyze the differences between the proximal and distal regions.
In order to verify the accuracy of the experimental gene chip, 4 genes that have known regulating effects on the peripheral nerve were selected, respectively by real-time fluorescence quantitative RT-PCR analysis, Western Blot blot analysis and histochemical immunofluorescence staining, respectively, from three aspects of molecular, protein and morphology.
The results of the study:
The gene chip analysis showed that the up-regulated gene expression was most up-regulated at the 7 day of the proximal sciatic nerve, and the down regulated gene expression was stable at the end of the 4 days. In the 14 day, the two were basically maintained at the same level, and the overall number of genes was up-regulated. In the distal segment of the sciatic nerve, the up regulation of gene expression at the end of the 7 day was the most, and down regulated gene table. As time goes on, the overall level of the two is basically the same.
The biological characteristics of the biological processes, such as stimulus response, inflammatory response, immune response, cell proliferation, cell migration, cell apoptosis, axon guidance and regeneration, myelin formation, extracellular matrix, signal transduction, and protein kinase activity, 11 generalizes the biological characteristics of the regeneration of peripheral nerve, and have their respective specificity at each time point near the distal end. These changes are basically consistent with the process of peripheral nerve regeneration as we know it.
The conclusions are as follows:
1, in the model of the rat sciatic nerve disconnection, the up-regulated gene was dominant in the proximal segment, while the total number of the distal segments was basically the same; both the proximal and distal segments had the most up-regulated expression at the same time period (7 days), and the latter two expressed the opposite trend.
2, in biological processes, most of the maximum differences in the 11 biological characteristics related to the biological characteristics of the stimulus response, inflammatory response, immune response, cell proliferation, cell migration, cell apoptosis, axon guidance and regeneration, myelin formation, extracellular matrix, signal transduction, and protein kinase activity, most of the maximum differences in the positive and negative regulation are in seventh days. Now, they have their own specific changes at near and far end, and they are interrelated, forming a dynamic regulatory network.
3, according to real-time fluorescence quantitative RT-PCR analysis, Western Blot Western blot analysis and histochemical immunofluorescence staining results, the accuracy of gene chip is real and reliable.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R745

【参考文献】