|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|
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。
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。
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。
Part one Genetic differences in osteogenic differentiation potency between single- and multiple-level thoracic ossification of ligamentum flavum under cyclic mechanical stress
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.
This study aimed to investigate he genetic differences and osteogenic differentiation potency between single- and multiple-level TOLF under cyclic mechanical stress.
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.
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.
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
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.
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.
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).
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.
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.
|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.|