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学科主题: 外科学
题名:
SMCX基因在周围神经创伤修复分子调控中的作用机制研究
作者: 韩端阳
答辩日期: 2016-05-10
导师: 姜保国
专业: 外科学
授予单位: 北京大学
授予地点: 北京大学第二临床医学院
学位: 博士
关键词: 高通量RNA sequencing测序 ; 周围神经损伤修复 ; schwann细胞 ; SMCX基因
其他题名: The regulatory mechanism of SMCX gene in peripheral nerve repair
分类号: R651.3
摘要:

【背景】周围神经损伤(Peripheral Nerve Injury)是临床常见的创伤类型,常造成 长期患者远侧肢体功能障碍,从而严重威胁患者生活质量,是现代社会越来越突 出并亟待攻克的临床难题。尽管近些年来治疗周围神经损伤的各种外科技术不断 改进, 然而现有各种方法都无法做到损伤神经远近端感觉与运动神经的精确对 接和快速愈合。周围神经损伤修复涉及细胞迁移、清除、凋亡、再生等一系列过 程,其中的分子调控机制尚未完全明确。我们的研究表明, SMCX 基因在周围神经 修复中起重要调节作用。尽管 SMCX 基因在中枢神经发育和修复过程中的作用 已经逐渐引起国际学界关注,但是 SMCX 基因周围神经修复过程中的作用及分 子调控机制尚未见报道。揭示探索其作用机制可使我们进一步了解周围神经创伤 修复过程的分子调控机制,并为进一步干预治疗􏰀供新靶点。

【目的】 1:通过高通量RNA-sequencing基因组测序,筛查与周围神经创伤修复相关的基

因谱; 2:研究SMCX基因对周围神经schwann细胞各项细胞功能的分子信号调控机制

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【方法】建立大鼠坐骨神经卡压伤动物模型,􏰀取总 RNA 用于高通量 RNA-sequencing 测序。采用 Illumina HiSeq2000 上机测序,所得大数据经过 Illumina CASAVA 1.8.2 系统分析检测;通过 DAVID 系统分析显著差异表达基因 谱;通过GO系统分析检测出周围神经损伤中差异表达的生物过程;通过IPA分 析检测出在周围神经损伤修复过程中表达活跃的分子信号通路和互相关联的网 状关联谱;通过 GSEA 基因富集分析方法得到周围神经损伤基因表达热图; Western blot 检测 PDCD1、IL10、IL18、IFN gamma 蛋白水平的表达;通过细胞 划痕实验及 Transwell 实验检测 SMCX 基因敲除对周围神经 schwann 细胞的迁移 能力的影响;通过细胞扩增实验检测 SMCX 基因敲除对 schwann 细胞增殖能力 的影响;通过软琼脂糖实验检测 SMCX 基因敲除对 schwann 细胞抗凋亡能力的 影响。

【结果】

第一部分:筛选周围神经损伤修复过程中相关基因谱的研究
通过高通量 RNA-sequencing 技术大规模测序分析了大鼠周围神经卡压损伤 修复过程中的整体基因谱表达变化情况,在损伤后第 7 天的时间点上,通过 DAVID 在线系统分析共检测出 2847 个差异表达基因位点。基因本体分析(GO analysis)检测发现上调的基因谱主要跟髓鞘化、轴突包裹、神经元包裹以及神 经电脉冲等 23 个生理过程相关;另一方面,下调的基因谱主要跟细胞凋亡调节、 自身免疫反应调节、先天免疫反应和创伤修复等 27 个生理过程相关。进一步的, 我们通过 Panther Pathway analysis 分析总结与周围神经损伤修复相关的分子信号 通路。结果显示,上调的基因谱主要集中在胆固醇生物合成通路、Oxytocin 受体 介导通路、Alpha 肾上腺素受体介导信号通路、五羟色胺受体介导信号通路以及 促甲状腺素释放激素受体介导信号通路等 17 条主要分子信号通路;另一方面, 下调的基因谱主要集中在炎性反应介导信号通路、gamma 干扰素介导信号通路、 Toll 样受体信号通路以及 JAK/STAT 信号通路这 4 条主要分子信号通路中。接下 来,我们通过 Ingenuity Pathway Analysis 系统综合分析发现,周围神经损伤修复 过程中最主要相关的 NFkB 信号通路、炎症反应与 TNFa 信号通路这三大分子信号调节通路。

第二部分:SMCX 基因对周围神经 schwann 细胞功能的分子调控机制 首先我们通过包装 SMCX 慢病毒载体敲除人类来源的周围神经 schwann 细 胞。通过形态学观察,我们发现相比较于触手数目较多且长度较短的对照组人类 周围神经 schwann 细胞,SMCX 基因敲除组的人类 schwann 细胞的触手数目明显 减少,触手长度变得相对延长。进一步的,通过软琼脂糖抗凋亡实验,我们发现 相比较于正常的人类 Schwann 细胞组,SMCX 基因的敲除显著削弱周围神经 schwann 细胞的抗凋亡能力。再进一步的,我们通过 Transwell 实验检测 SMCX 基因对周围神经 schwann 细胞运动性的影响。结果显示,与正常的人类来源周围 神经schwann细胞活跃的细胞移动性相比较,SMCX基因敲除组的人类schwann 细胞的移动性显著下降,这一发现证明 SMCX 基因对 schwann 细胞移动性的调 控起到重要作用。为了进一步验证 SMCX 基因对 schwann 细胞移动性的影响, 我们进行了细胞划线实验。实验结果表明,在正常的人类来源周围神经 schwann 细胞中,划线两侧细胞在第二天时间点能够完全融合。然而在 SMCX 基因敲除

组的 schwann 细胞移动性明显受到影响,直到第二天时间点仍然存留相当大距离。 可见 SMCX 基因的存在对周围神经 schwann 细胞的移动性具有重要意义,起到 正性调节作用。同样的,我们在大鼠来源的 schwann 细胞上也进行了相同的实验, 同样证实了 SMCX 基因对大鼠周围神经 schwann 细胞的移动性具有重要的正性 调节的作用。进一步的,我们在大鼠来源的周围神经 schwann 细胞上进行了细胞 倍增计数实验。实验结果显示,相比较于正常对照组的 schwann 细胞的细胞增殖 速度,SMCX 基因敲除组的 schwann 细胞的增殖速度显著减缓,生长曲线低平, 在实验第五天的时间点上,细胞数量仅相当于对照组的大约三分之一。这个发现 证明,SMCX 对周围神经 schwann 细胞的细胞增殖性具有重要的促进作用,是 schwann 细胞正常增殖过程中不可或缺的调节基因位点。接下来,我们进一步对 SMCX 基因敲除 schwann 细胞的髓鞘化相关基因谱进行了针对性的表达强度检 测,分别对 IntA4、Cdh2、Ncam、Gap43、Sox2、Galc、Pmp22、P0、以及 Prx 等 9 个重要的髓鞘化相关基因进行了表达强度检测。结果显示,其中除了 Cdh2、 Ncam 与 P0 三个相关基因表达显著增强以外,其余 6 个相关基因全部表达下降。 另外,SMCX 敲除显著下调大鼠 schwann 细胞系中 Wnt 信号通路上 AXIN2、DKK1以及 CCND1 等三个主要靶基因的表达强度。从而强烈􏰀示 SMCX 对周围神经 schwann 细胞的细胞形态及功能的影响很可能是通过 Wnt 分子信号通路机制实现 的。

【结论】(1)本研究系统的􏰁绘了在周围神经损伤修复过程中的全基因谱表达 变化的宏观情况,发现了 NFkB 信号通路与炎症反应是周围神经损伤修复早起最 为重要活跃的分子信号调节通路;(2)SMCX 基因对周围神经 schwann 细胞的 细胞形态、细胞增殖性、细胞移动性以及髓鞘化能力均具有重要的调节作用。 SMCX 基因位点可能成为一个有重要意义的促进周围神经创伤修复的全新治疗 靶点。

 

英文摘要:

Background

Peripheral nerve injury is a common clinical event, which often cause extremity disability. Peripheral nerve injury patient pose both economical and sociological burden to their family. Though the surgical technique on treating peripheral nerve injury have developed, the efficacies of these techniques are still not satisfactory. Peripheral nerve repair engage a series of cytological process such as cell migration, myelin clearance, axon regeneration etc. The exact mechanism regulating all these processes is still not clear. Our preliminary research demonstrated that SMCX gene play a vital role in peripheral nerve repair. Although the SMCX gene function has been reported in the central nerve system, the knowledge of SMCX gene regulatory mechanism in peripheral nerve repair is still very limited. By revealing SMCX gene regulatory mechanism in PNS, it stands for a promising novel treatment target for peripheral nerve injury.

Objective

1: By utilizing RNA sequencing technique, profile the whole gene set involved in peripheral nerve repair process.

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2: Study regulatory mechanism of SMCX gene to peripheral nerve schwann cell.

Methods

Utilizing next generation sequencing technique to analyze total RNA extract from peripheral nerve crush model. Using Illumina HiSeq2000 system to finish all sequencing work and use Illumina CASAVA 1.8.2 system to analyze the data. Using DAVID online system analyze differential expressed genes. Using GO system analyze differential expressed biological process. Using IPA system analyzes active molecular signaling pathways in peripheral nerve repair and network analysis. Western blot analyze PDCD1、IL10、IL18、IFN gamma protein expression level. Using cell scratch test to evaluate impact of SMCX gene knockout to schwann cell migration function. Using cell proliferation test and soft agar assay to evaluate impact of SMCX gene knockout to schwann cell growth ability and cell-cluster formation.

Results

Part 1. Profiling of related gene spectrum changes in peripheral nerve repair

Using high throughput RNA-sequencing sequencing technology to analyze the overall gene spectrum expression changes of rat peripheral nerve in the healing process. On the seventh day after the injury time, through the analysis of the DAVID online system detected a total of 2847 differentially expressed genes. Gene ontology analysis (GO analysis) detected up-regulated genes related with 23 biological processes such as axon myelination, radial sorting, neuron ensheathment and nerve impulses. On the other hand, down regulated genes are related with 27 biological processes such as apoptosis regulation, autoimmune regulation, regulating innate immune response and wound healing. Furthermore, we use Panther Pathway analysis to analyze molecular signaling pathway in peripheral nerve repair. Of note, up regulated genes were related with 17 pathways, while down-regulated genes were only related with 4 pathways. Especially Among these pathways, all related genes in 8 signaling pathways namely axon guidance mediated by semaphorins, thyrotropin-releasing hormone receptor 6

pathway, alpha adrenergic receptor signaling, 5HT2 type receptor mediated signaling, oxytocin receptor mediated signaling, heterotrimeric G-protein signaling pathway, cholesterol biosynthesis and inflammation mediated by chemokine were either up or down regulated genes in each of these signaling pathways showing a significant active involvement of these signaling pathways in peripheral nerve repair. Next, we performed Ingenuity pathway analysis (IPA). Data showed that the identified genes were primarily involved in NFkB signaling pathways and inflammatory response. The majority target genes of NFkB pathway were up regulated, which indicated that the NFkB pathway plays an indispensable role in peripheral nerve repair process.

Part 2. Regulatory mechanism of SMCX gene in peripheral nerve schwann cell

First we through the packaging the lentiviral vector of SMCX knockdown Schwann cells from human peripheral nerve. By morphological observation, we found that compared to control group of human peripheral nerve Schwann cells, SMCX gene knockout group human Schwann cells significantly reduced cellular processes and process length becomes relatively prolonged. Furthermore, through the soft agar assay, we found that compared to normal human Schwann cells, SMCX gene knockout significantly reduced Schwann cells cluster formation ability. Furthermore, we used the transwell assay to evaluate impact of SMCX gene on Schwann cells migration ability. The results showed that compared with normal human Schwann, SMCX gene knockout group of human Schwann cell mobility decreased significantly, this finding suggests that the SMCX gene plays an important role on Schwann cell mobility. In order to verify the effect of SMCX gene on Schwann cell mobility, we conducted cell scratch test. The experimental results show that in normal human Schwann cells, cell fusion can be completely crossed on both sides in second days. However, in the SMCX gene knockout Schwann cells mobility was obviously affected, until second day, cell scratch line remained a considerable distance. There we can see that the SMCX gene plays an important role in peripheral nerve Schwann cell mobility, whichplay a positive regulatory role. Similarly, we also carried out the same experiment in rat derived Schwann cells, also confirmed that the SMCX gene has important positive regulatory role on rat Schwann cells mobility. Furthermore, we carried out cell proliferation experiments in Schwann cells. The experimental results show that compared with normal control group, the cell proliferation rate of SMCX gene knockout group significantly slowed down and the growth curve is flat, on the fifth day time point, the number of cells is equivalent to only about 1/3 of the control group. This discovery proves that SMCX gene plays an important role in promoting Schwann cell proliferation. Next, we evaluated the effect of SMCX gene knockout on myelin related gene expression. We detected 9 important myelination related genes expression intensity namely IntA4, Cdh2, Ncam, Gap43, Sox2, Galc, Pmp22, P0 and Prx. The results showed that only the expression of Cdh2, Ncam and P0 gene expression was significantly enhanced, all the other 6 genes decreased. In addition, SMCX knockdown significantly decreased AXIN2, DKK1 and CCND1 expression. These results strongly suggest that SMCX gene regulatory effect on schwann cell morphology and function is likely to be realized by Wnt molecular signaling mechanism.

Conclusion

(1)  In the current study, we profile the whole gene spectrum change in peripheral nerve repair. We reported that NFkB signaling pathway and inflammation response play the most important role in the early phase following peripheral nerve injury.

(2)  The SMCX gene plays an important role in the regulation of Schwann cell morphology, proliferation, mobility and myelination ability. SMCX gene stands for an important new therapeutic target for promoting peripheral nerve injury.

 

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/124610
Appears in Collections:北京大学第二临床医学院_学位论文

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作者单位: 北京大学第二临床医学院

Recommended Citation:
韩端阳. SMCX基因在周围神经创伤修复分子调控中的作用机制研究[D]. 北京大学第二临床医学院. 北京大学. 2016.
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