北京大学医学部机构知识库
Advanced  
IR@PKUHSC  > 北京大学第三临床医学院  > 学位论文
学科主题: 外科学
题名:
应力刺激下胸椎黄韧带骨化成骨潜能的差异及相关转录组测序研究
作者: 宁尚龙
答辩日期: 2016-05-10
导师: 陈仲强
专业: 外科学
授予单位: 北京大学
授予地点: 北京大学第三临床医学院
学位: 博士
关键词: 胸椎黄韧带骨化 ; 应力 ; 成骨潜能 ; 基因差异 ; 高通量测序 ; 差异表达基因 ; 生物信息学 ; 转录组
其他题名: Genetic differences in osteogenic differentiation potency in thoracic ossification of ligamentum flavum under cyclic mechanical stress and relevant Tranome high-throughput sequencing and bioinformatics analysis
分类号: R686.5
摘要:

第一部分  机械应力刺激下不同类型胸椎黄韧带骨化成骨分化潜能差异的研究

背景:

    胸椎黄韧带骨化(TOLF)是脊柱韧带异位骨化性疾病的一种,其发病机制尚不明确。研究证实机械应力是TOLF发生发展的重要因素,可以诱导TOLF韧带细胞向成骨方向分化。但同时TOLF病变范围有的局限,有的广泛,可以累及单节段或者多个节段。所以除应力外,遗传易感因素等也应在TOLF发病中发挥着重要作用。不同类型的TOLF发病机制可能存在差异,局限型中可能局部因素起主要作用,多节段范围广泛的TOLF可能受遗传易感因素影响更加明显,其成骨分化的潜能可能更强。迄今为止尚无研究针对单节段和多节段TOLF之间的成骨分化潜能以及基因差异进行对比分析。

目的:

研究机械应力刺激下不同类型TOLF成骨分化潜能的基因差异

方法:

在临床上纳入非TOLF、单节段和多节段TOLF三组各8例患者,培养原代细胞后,应用Flexcell FX-4000进行牵拉诱导成骨0、6、12和24小时。使用real-timePCR检测6个指标mRNA表达水平:Osteopontin、Osterix、BMP-2、Osteocalcin、RUNX2和ALP。使用western-blot检测Osteopontin、Osterix、BMP-2和ALP表达水平。而且也使用ALP活性检测和染色来评价各组的成骨分化情况。

结果:

镜下可见三组细胞形态基本类似,均为成纤维细胞样梭形细胞。我们分别对TOLF细胞及非TOLF细胞施加不同时间不同程度的牵张应力,得到15%是诱导TOLF韧带细胞成骨分化的最适宜应力条件。ALP活性检测及染色结果显示多节段TOLF组显著高于单节段组,而非TOLF组随时间增加无明显变化。real-time PCR结果显示多节段组的6个指标表达水平都高于单节段组,其中Osteopontin、Osterix、BMP-2、ALP有统计学差异,而多节段组除RUNX2外的指标都显著高于非TOLF组。在蛋白水平检测osterix及ALP可见,多节段TOLF组表达高于另两组并随诱导时间增加,而BMP-2和OPN在两组之间没有明显差异。

结论:

机械应力在TOLF的发病过程中起至关重要的作用,黄韧带细胞对一定范围内的应力诱导的成骨分化更加敏感。因而也提示在临床上OLF多发生于胸椎,颈椎腰椎很少见,可能是由于有这样潜在的因素发生影响。

机械应力刺激下不同类型TOLF成骨分化潜能存在基因差异,它们的骨化程度反应是不一致的,多节段TOLF的成骨潜能明显高于单节段TOLF。这提示除应力外,遗传差异及易感基因等重要的基础因素可能对骨化也发生重要影响。

 

第二部分 机械应力刺激前后胸椎黄韧带骨化转录组高通量测序研究

                            ——基因差异表达及相关信号通路

背景:

TOLF是指胸椎黄韧带的纤维组织发生异位骨化,文献中报道了各种各样的致病因素,迄今为止TOLF发病的分子机制并不清楚,仅有学者分别对一些相关的基因表达及信号通路进行了初步验证。另外有研究证实机械应力是TOLF发生发展的重要因素,可以诱导TOLF韧带细胞向成骨方向分化,也越来越多被用为体外诱导TOLF韧带细胞成骨的方式。而目前尚无关于OLF的mRNA转录组基因谱研究,并无研究从整体上系统地探索OLF发病的分子机制。

目的:

通过高通量测序的方法对比TOLF组和非TOLF组的韧带细胞,寻找出两者差异基因表达情况。

通过生物信息学方法分析获取和TOLF相关的基因及信号通路,为进一步在基因水平探讨TOLF的发病机制奠定基础。

通过高通量测序的方法对比分析机械应力成骨诱导前后的TOLF组韧带细胞,获得差异基因表达情况。

通过生物信息学方法分析获取TOLF中和应力诱导刺激相关的基因和信号通路,为进一步在基因水平探讨TOLF在应力作用下的发病机制奠定基础。

方法:

获取6例TOLF(单节段、多节段各3例)和3例非TOLF患者胸椎黄韧带标本,进行原代细胞培养,使用Flexcell FX-4000行牵拉诱导成骨0和24小时,提取mRNA,使用Illumina HiSeqTM 2500 测序平台测序。

筛查TOLF组和非TOLF组之间以及TOLF组机械应力诱导前后的差异表达基因,并进行基因本体(gene ontology,GO)功能分析及京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)信号通路分析,通过生物信息学分析、查阅文献并对与成骨相关的基因使用实时定量PCR进行验证。

结果:

同非TOLF组相比,TOLF组出现671个表达上调的基因和314个表达下调的基因。TOLF组机械应力诱导前后相比,出现341个表达上调的基因和502个表达下调的基因。

GO分析提示:

TOLF组和非TOLF组相比,排名前三位的细胞成分都与细胞外基质成分相关,差异表达基因主要与分布在细胞膜和细胞外基质的糖蛋白分子及受体相结合,基因功能主要是完成对刺激的应答及发育过程等。另外在得到的GO terms中可发现22个上调基因富集到的与成骨相关的GO功能分析。

TOLF组机械应力诱导前后相比,差异表达基因上调的生物学行为主要表现为细胞周期过程等,大多与细胞核内的染色体相关,并与微管结合相关;下调的GO terms分析中包括细胞外基质成分和细胞粘连过程等。

KEGG分析提示:

TOLF组和非TOLF组相比,差异表达基因富集到22个信号通路,排在前两位的是Cell cycle和ECM-receptor interaction。另外TGF-β、p53、FoxO和PI3K-Akt信号通路也具有较小的统计学矫正P值。

TOLF组机械应力诱导前后相比,差异表达基因富集到21个信号通路,Cell cycle和ECM-receptor interaction两个信号通路差异性仍比较显著,p53、Focal adhension、Wnt和PI3K-Akt等信号通路也具有较小的统计学P值。

对部分典型的差异表达基因行实时定量PCR验证结果同测序结果一致

结论:

TOLF组和非TOLF组及TOLF组机械应力诱导前后的转录组基因表达谱具有明显差异。

TOLF组韧带细胞与对照组相比表达显著的成骨相关基因,原代细胞有明显的成骨细胞表型。

L1RL1、PTHLH、DKK1、BMP6、SPP1和FGF1等差异表达基因可能与TOLF发病密切相关。

Cell cycle和ECM-receptor interaction等信号通路及所构成的网络可能分别在TOLF发病及感受机械应力诱导成骨中具有重要作用。

 

英文摘要:

Part one  Genetic differences in osteogenic differentiation potency between single- and multiple-level thoracic ossification of ligamentum flavum under cyclic mechanical stress

Background

Ossification of ligament flavm (OLF) of the spine is acterized by ectopic bone formation in flavum ligament with a most frequently prevalent in East Asians, especially in Japan. Epidemiology has shown that high incidence rate of OLF occurs in thoracic spine. Thoracic ossification of ligament flavum (TOLF) progresses insidiously over a long period and eventually cause devastating spinal cord compromise that always leads to serious myelopathy. Most promising studies have reported that OLF is influenced by multiple contributing factors, including the genetic background, dietary habits, metabolic abnormalities, and some local factors including mechanical stress.

TOLF most frequently affects single or dual levels and presents mainly in the lower thoracic spine (T10-T12), where is a mobile transition region between thoracic and lumbar spine that may be more prone to degeneration due to the high tensile forces present in the posterior column. The facilitating role of mechanical axial overload and subsequent increased repetitive tensile strain on surgically resected tissues are contributed to the ossification process of thoracic ligamentum flavum. Our previous study also found that mechanical stress induced the osteogenic differentiation of TOLF cells. Therefore, the locally abnormal mechanical stress is believed to play an important role in the progression of TOLF. However, there are also a large amount cases of multiple-level TOLF in immobile segments as well as mobile segments, and the disease progression and clinical outcomes of patients with multiple-level lesion are significantly different from single-level lesion. Actually, the multiple-level TOLF presents a poor prognosis and a possibly continuous development after the surgical resection, which indicated that its osteogenic differentiation potential may be intrinsically stronger than single-level TOLF.

Objectives

This study aimed to investigate he genetic differences and osteogenic differentiation potency between single- and multiple-level TOLF under cyclic mechanical stress.

Methods

Clinically, the patients with non-TOLF, single-level TOLF and multiple-level TOLF (n=8 in each group) were included in this study. The primary ligament cells that derived from the three groups were separately cultured and induced osteogenesis with different strength of cyclic mechanical stress (0%, 5%, 10%, 15% and 20%) for 12h and 24h to using a device called Flexcell FX-4000. The optimal stress was ed and applied in latter experiments. The ALP activity was determined to evaluate the osteogenesis using quantitative analysis and ALP staining assay. Real-time PCR was used to detect the mRNA expression of osteogenic-related genes including ALP, BMP-2, Runx2, Osterix, Osteopontin and Osteocalcin. Western blot was applied to determine the expression of these indicator at protein level.

Results

The morphology of the cells that derived from three groups was basically similar, all presented an elongate spindle-shape. After the osteogenic induction with different strength of cyclic mechanical stress on non-TOLF and TOLF cells for 12h and 24h, we determined the change of ALP activity and observed that 15% stress was the optimal condition in the induction of TOLF cells in this study. To evaluate the ostogenesis, ALP activity assays including quantitative and staining assays were performed. The results showed that either quantitative analysis or ALP staining in the multiple-level TOLF group was significantly higher than single-level group, but no significant change was found in non-TOLF. The results of real-time PCR indicated that the expression of ostegenic markers above in the multiple-level TOLF group was more than single-level group. Therein, the significant difference was found in ALP, BMP-2, osteopontin and osterix. However, in single-level TOLF, all indicators in the single-level group were significantly higher than non-TOLF group, except the Runx2. At the protein level, the expression of osterix and ALP in the multiple-level TOLF group was higher than single-level and non-TOLF groups and gradually increased with the increase of induction time. No significant differences were observed in BMP-2 and OPN.

Conclusions

There are obviously genetic differences in osteogenic differentiation potency between single- and multiple-level TOLF under cyclic mechanical stress. The osteogenic differentiation of multiple-level TOLF is stronger than single-level TOLF

Part two  Tranome high-throughput sequencing and bioinformatics analysis of the differentially expressed genes for thoracic ossification of ligamentum flavum before and after mechanical stress

 

Background

Thoracic ossification of ligament flavum (TOLF) of the spine is acterized by ectopic bone formation in the flavum ligament. Many factors contribute to OLF, including genetic background, dietary habits, metabolic abnormalities, and mechanical stress.Up to now, the molecular mechanism of is not clear yet, and only some genes or signaling pathways were verified preliminarily in several researches. Many studies have demonstrated the critical role of mechanical stress in the development of TOLF, and it was reported that mechanical stress induced osteogenic differentiation of ligament cells derived from TOLF. But there is no correlated study on the tranome analysis of TOLF to explore the molecular pathogenesis of TOLF systematically and roundly.

Objectives

The purpose of this study was to detect the differentially expressed genes between TOLF and non-TOLF and between TOLF before and after mechanical stress. In addition, we sought to identify a few candidate genes and pathways by using bioinformatics analysis.

Methods

Clinically, the patients with non-TOLF, single-level TOLF and multiple-level TOLF (n=3 in each group) were included in this study. The primary ligament cells that derived from the three groups were separately cultured and induced osteogenesis with cyclic mechanical stress for 24h using a device called Flexcell FX-4000. Purified mRNA and cDNA extracted from the samples was subjected to sequencing. NOISeq method was used to statistically identify the differentially expressed genes (DEGs) between the two groups.

Deep analysis using bioinformatics tools based on DEGs was performed using Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction network analysis.

The Classic DEGs were verified using real-time quantitative polymerase chain reaction (real-time qPCR).

Results

671 genes of TOLF group were up-regulated and 314 genes were found to be down-regulated as compared to the control group, And 341 genes of TOLF after stress induction group were up-regulated and 502 genes were found to be down-regulated as compared to the group without stress stimulus.

In the comparision between TOLF group and non-TOLF group,the top three cellular components in GO ontologies analysis were extra cellular matrix components. GO functions were mainly related to the glycoprotein in the cell membrane and extra cellular matrix (ECM). GO process were related to completing response to stimulus or developmental process.In addition,22 significant GO terms associated with upregulated genes were found to be closely related to ossification.And in the comparision between TOLF-SI group and TOLF control group,the top GO terms associated with up-regulated genes included cell cycle,and so on,and they are related to nuclear omosome and tubulin binding.Then,extracellular matrix and cell adhension could be found in the top down-regulated GO terms.

In the comparision between TOLF group and non-TOLF group,22 significant KEGG enrichment pathways were found(corrected P<0.05),and The top 2 pathways were Cell cycle and ECM-receptor interaction,in addition, TGF-β、p53、FoxO and PI3K-Akt pathway also had a very significant difference. And in the comparision between TOLF-SI group and TOLF control group,21 significant KEGG enrichment pathways were found(corrected P<0.05),and Cell cycle and ECM-receptor interaction pathway were also on the top,in addition, p53、Focal adhension、Wnt and PI3K-Akt pathway also had a very significant difference.

Some differentially expressed genes were verified with realtime PCR.

Conclusion

The gene expression profiling of TOLF group and non-TOLF group revealed differential gene expression. The similar result was achieved in TOLF-SI group and TOLF control group.

Global tranome analysis revealed TOLF cells expressed more ossification related genes than non-TOLF group,and TOLF ligament cells exhibita osteoblast phenotype.

Some genes including L1RL1,PTHLH,DKK1,BMP6,SPP1 and FGF1 may be closely related to TOLF,and so are some signaling pathway ,for example Cell cycle and ECM-receptor interaction,and the network they constituted.Wnt pathway abnormality and local inflammation may be related to disc ossification.

 


 

 

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/400002259/124736
Appears in Collections:北京大学第三临床医学院_学位论文

Files in This Item:

There are no files associated with this item.


作者单位: 北京大学第三临床医学院

Recommended Citation:
宁尚龙. 应力刺激下胸椎黄韧带骨化成骨潜能的差异及相关转录组测序研究[D]. 北京大学第三临床医学院. 北京大学. 2016.
Service
Recommend this item
Sava as my favorate item
Show this item's statistics
Export Endnote File
Google Scholar
Similar articles in Google Scholar
[宁尚龙]'s Articles
CSDL cross search
Similar articles in CSDL Cross Search
[宁尚龙]‘s Articles
Related Copyright Policies
Null
Social Bookmarking
Add to CiteULike Add to Connotea Add to Del.icio.us Add to Digg Add to Reddit
所有评论 (0)
暂无评论
 
评注功能仅针对注册用户开放,请您登录
您对该条目有什么异议,请填写以下表单,管理员会尽快联系您。
内 容:
Email:  *
单位:
验证码:   刷新
您在IR的使用过程中有什么好的想法或者建议可以反馈给我们。
标 题:
 *
内 容:
Email:  *
验证码:   刷新

Items in IR are protected by copyright, with all rights reserved, unless otherwise indicated.

 

 

Valid XHTML 1.0!
Copyright © 2007-2017  北京大学医学部 - Feedback
Powered by CSpace