正常态和激活态雪旺细胞与轴突再生相关基因甲基化差异的实验研究

发布时间：2018-03-22 10:02

  本文选题：DNA甲基化　切入点：大鼠　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：【目的】雪旺细胞(Schwann cells,SCs)是周围神经系统中最重要的细胞之一。成熟的SCs包裹神经元的轴突形成髓鞘,此时的SCs称为正常态雪旺细胞(Normal Schwann cells,NSCs)。周围神经损伤后,损伤远端神经纤维的轴突和髓鞘均被破坏,继而完全崩解,这种现象被称之为华勒变性。神经损伤后SCs即表现出高生物活性,此时的SCs称之为激活态雪旺细胞(Activated Schwann cells,ASCs),ASCs的高生物活性对轴突再生有良好的促进作用。然而,目前的研究绝大部分都集中在蛋白质水平和miRNA水平,并且大量的研究已经证实:ASCs和NSCs在蛋白质水平和miRNA水平存在显著差异,但是两种状态SCs存在差异的根本原因尚不清楚。近些年随着基因工程技术的逐渐成熟,DNA甲基化的研究越来越受到研究者重视,并且研究发现:周围神经系统损伤后,可使部分基因(如Shh和Olig1)发生甲基化状态改变,这些基因的甲基化改变对轴突再生起到关键作用。然而,目前还没有全基因组的比较ASCs和NSCs与轴突再生相关基因甲基化差异的实验研究。本课题通过DNA甲基化免疫共沉淀测序(Methylated DNA immunoprecipitation sequencing,MeDIP-Seq)技术检测Wistar大鼠ASCs和NSCs全基因组的甲基化差异,筛选轴突再生相关基因,并做功能分析。【方法】健康成年Wistar大鼠(约150±5克)3只,坐骨神经预损伤,臂丛神经仅做分离。饲养7天后,脱颈处死Wistar大鼠,提取坐骨神经损伤远端和臂丛神经各1.5厘米,采用低浓度胰蛋白酶消化法从坐骨神经组织提取ASCs、从臂丛神经提取NSCs。分别培养ASCs和NSCs至第3代,S-100抗体免疫荧光染色鉴定细胞,并计算细胞纯度。CCK8法测定细胞增殖,并绘制ASCs和NSCs的增值曲线。收集第3代ASCs和NSCs,使用EDTA方法提取DNA。运用MeDIP-Seq技术筛选出ASCs和NSCs的差异性甲基化区域;使用KOBAS软件对差异甲基化区域注释和统计分析,筛选出轴突再生相关差异甲基化基因,使用InterProScan软件分析差异基因的染色体分布情况、基因本体(GO)分析以及信号通路分析。【结果】本研究获得纯度为95%以上的ASCs和NSCs,两者S-100免疫荧光染色均表达阳性。在相同培养条件下,ASCs生长速度和贴壁速度更快。MeDIP-Seq共发现177176个差异性甲基化区域,其中位于启动子(≤1 kb)内1 097个、启动子(1~2 kb)内1 136个,CpG岛内567个。基因注释分类后,共获得214个与轴突再生相关的差异甲基化基因。这些基因分布于各个染色体上,以12号染色体上最多(22个),M染色体最少(2个)。GO分析结果显示差异甲基化基因涉及轴突生长、轴突形成、轴突延伸和轴突导向等过程;并且与MAPK、细胞黏附分子和Ras等信号通路有关。【结论】ASCs和NSCs的甲基化水平存在明显的差异,214个甲基化差异基因与轴突再生有关,分布于各个染色体上;涉及轴突生长、轴突形成、轴突延伸和轴突导向等过程,并且与MAPK、细胞黏附分子和Ras等主要信号通路有关。
[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 卤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，

本文编号：1648163