本文選題：DNA甲基化　切入點：大鼠　出處：《天津醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：【目的】雪旺細胞(Schwann cells,SCs)是周圍神經(jīng)系統(tǒng)中最重要的細胞之一。成熟的SCs包裹神經(jīng)元的軸突形成髓鞘,此時的SCs稱為正常態(tài)雪旺細胞(Normal Schwann cells,NSCs)。周圍神經(jīng)損傷后,損傷遠端神經(jīng)纖維的軸突和髓鞘均被破壞,繼而完全崩解,這種現(xiàn)象被稱之為華勒變性。神經(jīng)損傷后SCs即表現(xiàn)出高生物活性,此時的SCs稱之為激活態(tài)雪旺細胞(Activated Schwann cells,ASCs),ASCs的高生物活性對軸突再生有良好的促進作用。然而,目前的研究絕大部分都集中在蛋白質(zhì)水平和miRNA水平,并且大量的研究已經(jīng)證實:ASCs和NSCs在蛋白質(zhì)水平和miRNA水平存在顯著差異,但是兩種狀態(tài)SCs存在差異的根本原因尚不清楚。近些年隨著基因工程技術(shù)的逐漸成熟,DNA甲基化的研究越來越受到研究者重視,并且研究發(fā)現(xiàn):周圍神經(jīng)系統(tǒng)損傷后,可使部分基因(如Shh和Olig1)發(fā)生甲基化狀態(tài)改變,這些基因的甲基化改變對軸突再生起到關(guān)鍵作用。然而,目前還沒有全基因組的比較ASCs和NSCs與軸突再生相關(guān)基因甲基化差異的實驗研究。本課題通過DNA甲基化免疫共沉淀測序(Methylated DNA immunoprecipitation sequencing,MeDIP-Seq)技術(shù)檢測Wistar大鼠ASCs和NSCs全基因組的甲基化差異,篩選軸突再生相關(guān)基因,并做功能分析�！痉椒ā拷】党赡闣istar大鼠(約150±5克)3只,坐骨神經(jīng)預(yù)損傷,臂叢神經(jīng)僅做分離。飼養(yǎng)7天后,脫頸處死Wistar大鼠,提取坐骨神經(jīng)損傷遠端和臂叢神經(jīng)各1.5厘米,采用低濃度胰蛋白酶消化法從坐骨神經(jīng)組織提取ASCs、從臂叢神經(jīng)提取NSCs。分別培養(yǎng)ASCs和NSCs至第3代,S-100抗體免疫熒光染色鑒定細胞,并計算細胞純度。CCK8法測定細胞增殖,并繪制ASCs和NSCs的增值曲線。收集第3代ASCs和NSCs,使用EDTA方法提取DNA。運用MeDIP-Seq技術(shù)篩選出ASCs和NSCs的差異性甲基化區(qū)域;使用KOBAS軟件對差異甲基化區(qū)域注釋和統(tǒng)計分析,篩選出軸突再生相關(guān)差異甲基化基因,使用InterProScan軟件分析差異基因的染色體分布情況、基因本體(GO)分析以及信號通路分析�！窘Y(jié)果】本研究獲得純度為95%以上的ASCs和NSCs,兩者S-100免疫熒光染色均表達陽性。在相同培養(yǎng)條件下,ASCs生長速度和貼壁速度更快。MeDIP-Seq共發(fā)現(xiàn)177176個差異性甲基化區(qū)域,其中位于啟動子(≤1 kb)內(nèi)1 097個、啟動子(1~2 kb)內(nèi)1 136個,CpG島內(nèi)567個�；蜃⑨尫诸惡�,共獲得214個與軸突再生相關(guān)的差異甲基化基因。這些基因分布于各個染色體上,以12號染色體上最多(22個),M染色體最少(2個)。GO分析結(jié)果顯示差異甲基化基因涉及軸突生長、軸突形成、軸突延伸和軸突導(dǎo)向等過程;并且與MAPK、細胞黏附分子和Ras等信號通路有關(guān)�！窘Y(jié)論】ASCs和NSCs的甲基化水平存在明顯的差異,214個甲基化差異基因與軸突再生有關(guān),分布于各個染色體上;涉及軸突生長、軸突形成、軸突延伸和軸突導(dǎo)向等過程,并且與MAPK、細胞黏附分子和Ras等主要信號通路有關(guān)。
[Abstract]:[objective] Schwann cells (Schwann cells) is one of the most important cells in the peripheral nervous system. The axons of mature SCs envelop neurons form myelin sheath. The SCs is called normal Schwann cells after peripheral nerve injury. The axons and myelin sheath of the injured distal nerve fibers are destroyed and then completely disintegrated. This phenomenon is called Walller degeneration. After nerve injury, SCs shows high biological activity. The high bioactivity of activated Schwann cells called activated Schwann cells at this time can promote axonal regeneration. However, most of the current studies have focused on protein and miRNA levels. And a large number of studies have confirmed that there are significant differences between NSCs and NSCs at protein and miRNA levels. However, the underlying reasons for the differences between the two states of SCs are not clear. In recent years, with the gradual development of genetic engineering technology, the study of DNA methylation has been paid more and more attention by researchers, and it has been found that: after the injury of the peripheral nervous system, Methylation changes in some genes, such as Shh and Olig1, play a key role in axon regeneration. There is no experimental study on the difference of methylation between ASCs and NSCs genes associated with axon regeneration. In this study, the methylation difference between ASCs and NSCs of Wistar rats was detected by DNA methylated DNA immunoprecipitation sequencing and MeDIP-Seq.Methylated DNA immunoprecipitation sequencing technique was used to detect the difference of methylation between ASCs and NSCs genomes in Wistar rats. Screening and functional analysis of genes related to axon regeneration. [methods] healthy adult Wistar rats (about 150 鹵5 g) were used to preinjure the sciatic nerve and to isolate the brachial plexus nerve. After feeding for 7 days, the Wistar rats were killed. Sciatic nerve injury and brachial plexus nerve were extracted from sciatic nerve by low concentration trypsin digestion method, and from brachial plexus nerve by ASCs and NSCs antibody immunofluorescence staining. Cell purity. CCK8 method was used to measure cell proliferation, and the increment curves of ASCs and NSCs were plotted. The third generation ASCs and NSCs were collected and extracted by EDTA method. The differential methylation regions of ASCs and NSCs were screened by MeDIP-Seq technique. The differential methylation genes associated with axon regeneration were screened by using KOBAS software to annotate the differential methylation regions and statistical analysis. The chromosomal distribution of differential methylation genes was analyzed by InterProScan software. [results] the purity of ASCs and NSCs were more than 95%, both of them were positive by S-100 immunofluorescence staining. Under the same culture conditions, the growth rate and adherent speed of ASCs were faster. MeDIP-Seq. A total of 177176 differentially methylated regions were found. Among them, 1 097 were located in promoter (鈮，

本文編號：1648163