本文選題：神經(jīng)橫斷 + 近端片段��； 參考：《吉林大學》2014年博士論文

【摘要】：目的意義： 隨著社會的發(fā)展，周圍神經(jīng)損傷的患者日益增多。與中樞神經(jīng)損傷不同，周圍神經(jīng)損傷后，其具有一定程度的自我再生能力。而這種自發(fā)再生的過程受到周圍神經(jīng)細胞多種分子基因水平的調控。早期人們更偏向于形態(tài)學研究，隨著基因技術的發(fā)展，近階段的研究發(fā)現(xiàn)了很多對周圍神經(jīng)損傷后具有再生調節(jié)能力的基因。而目前，尚缺乏對周圍神經(jīng)損傷后近、遠端基因的數(shù)量及功能方面動態(tài)綜合的研究，以及對比研究。 本實驗通過建立大鼠坐骨神經(jīng)離斷的模型，對其近遠端基因所反映的生物學進程的變化趨勢進行分析并加以對比。擬在已有研究的基礎上，對周圍神經(jīng)損傷后再生的分子調控機制進行深入的研究。 材料方法： 成年雄性SD大鼠150只，根據(jù)坐骨神經(jīng)離斷時間的不同，隨機分為6組，分別在第0天、4天、7天、14天、21天、28天處死大鼠，并在離斷點近、遠端分別取0.5厘米坐骨神經(jīng)片段用于生物學分析。Trizol提取總RNA，制備基因表達芯片，每個時間點重復三次，運用相關軟件對芯片數(shù)值進行標準化分析。并通過T檢驗對離斷后不同時間點與空白對照組（0天）行統(tǒng)計學分析比較差異，選取p0.05，fold change2的基因作為統(tǒng)計學分析有差異顯著性的基因進行生物學功能分析。 我們應用DAVID在線軟件的Gene Ontology (GO)的分析功能，對近遠端差異基因所反映的刺激反應、炎癥反應、免疫反應、細胞增殖、細胞遷移、細胞凋亡、軸突再生和導向、髓鞘生成、細胞外基質、信號轉導和蛋白激酶活性共11個生物學進程進行生物學表達分析，觀察相關差異基因在這11種生物學進程中的表達趨勢變化，并對近遠端的不同進行分析總結。 為了驗證本實驗基因芯片的準確性，選取4個已知對周圍神經(jīng)有調控作用的基因，分別用實時熒光定量RT-PCR分析、Western Blot蛋白印跡分析以及組織化學免疫熒光染色從分子、蛋白、形態(tài)三個方面分別驗證。 研究結果： 基因芯片分析結果顯示，在坐骨神經(jīng)近端片段，在7天時，上調基因表達最多，在4天后，下調基因表達穩(wěn)定，在14天后兩者基本維持同一水平，總體數(shù)量上上調基因表達占優(yōu)勢。在坐骨神經(jīng)遠端片段，在7天時，上調基因表達最多，而下調基因表達隨著時間越來越多，兩者總體水平基本相當。 在生物學進程方面，刺激反應、炎癥反應、免疫反應、細胞增殖、細胞遷移、細胞凋亡、軸突導向和再生、髓鞘生成、細胞外基質、信號轉導和蛋白激酶活性這11個可概括周圍神經(jīng)再生性變化的生物學特性，在近遠端每個時間點都有其各自特異性的變化，而這些都與我們已知的周圍神經(jīng)再生的過程基本一致。 研究結論： 1、在大鼠坐骨神經(jīng)離斷的模型中，在近端片段，上調基因總體占優(yōu)勢，而遠端片段兩者總體數(shù)量基本相當；無論是近端片段還是遠端片段，都在同一時段（7天）具有最多的上調表達，，而后兩者表達趨勢相反。 2、在生物學進程方面，刺激反應、炎癥反應、免疫反應、細胞增殖、細胞遷移、細胞凋亡、軸突導向和再生、髓鞘生成、細胞外基質、信號轉導和蛋白激酶活性這11個生物學特性相關的差異基因最大值和正負向調控的最大差值大多數(shù)在第7天出現(xiàn)，它們在近遠端每個時間點都有其各自特異性的變化，互有關聯(lián)，組成了一個動態(tài)發(fā)展的調控網(wǎng)絡。 3、根據(jù)實時熒光定量RT-PCR分析、Western Blot蛋白印跡分析以及組織化學免疫熒光染色結果，基因芯片的準確性真實可靠。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R745

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