胸椎黄韧带骨化患者韧带细胞成骨潜能的评估及相关高通量测序分析  

The evaluation of osteogenic potency of ligament cells in thoracic ossification of the ligamentum flavum and relevant transcriptome high-throughput sequencing analysis

作　　者：宁尚龙[1,2] 陈仲强[2] 马信龙[1] 范东伟[2] 孙垂国[2] 苗军[1] 曾岩[2] 李危石[2] 

机构地区：[1]天津市天津医院脊柱外科,300211 [2]北京市北京大学第三医院骨科,100091

出　　处：《中华骨科杂志》2017年第20期1300-1309,共10页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金（81572101,81272031）;天津市天津医院科技基金（TJYY1505）

摘　　要：目的评估胸椎黄韧带骨化患者韧带细胞的成骨潜能，并采用转录组高通量测序进一步分析差异基因表达。方法选择2015年10月至2016年4月入院的10例胸椎黄韧带骨化患者（骨化组）与10例不伴胸椎黄韧带骨化的胸椎疾病患者（非骨化组），术中收集黄韧带标本行细胞培养，采用Flexeell FX-4000细胞应力加载系统对细胞施加周期性牵张应力来诱导成骨并检测两组细胞成骨指标的表达，再各纳入3例样本进行基因测序分析。结果碱性磷酸酶（alkaline phosphatase，ALP）活性染色镜下观察染色细胞数和面积半定量分析，以及ALP活性定量检测结果显示骨化组表达在0h明显高于非骨化组（t=5．757，5．141，2．358，P〈0．05），且随诱导成骨而增加，其中定量检测及染色面积的差异有统计学意义（F=6．464，P=0．003；F=17．35，P=0.000）。Read-time PCR结果显示骨化组ALP、骨形成蛋白2（bone morphogenetic protein-2，BMP-2）及骨钙素（Osteoealein，OCN）的表达水平在0h、12h和24h几乎均高于非骨化组，且随诱导成骨而增加，除BMP-2和OCN在12h外差异均有统计学意义。Western Blot结果显示各指标在两组的表达与Real-time PCR结果类似。而在各项检测中非骨化组各指标的表达均无明显变化。高通量测序结果显示骨化组出现671个上调基因和314个下调基因。在得到的基因本体功能分析（gene ontology terms，GO terms）中可发现22个上调基因富集到的与成骨相关的GOterms。结论胸椎黄韧带骨化韧带细胞具有较高的成骨潜能，并明显表达成骨相关基因。差异表达基因LIRL1、PTHLH、DKK1、BMP6、SPP1和FGF1等可能与TOLF的成骨紧密相关。Objective To investigate the osteogenic differentiation potency of ligament cells in thoracic ossification of the ligamentum flavum （TOLF） and analyze further by using transcriptome high-throughput sequencing. Methods Clinically, the patients with non-TOLF and TOLF （n=10 in each group） who underwent surgery in our hospital from October 2015 to April 2016 were included in this study. The primary ligament cells that derived from the two groups were separately cultured and induced osteogenesis with 15% strength of cyclic mechanical stress for 12h and 24h using a device called Flexcell FX-4000. The ALP activity was determined to evaluate the osteogenesis using quantitative analysis and ALP staining assay. Real-time PCR and western-blotting were used to detect the mRNA and protein expression of osteogenic-related genes including ALP, BMP-2 and Osteocalcin. Then, three patients in each group were included in the study of transcriptome high-throughput sequencing and bioinformatics analysis using Illumina HiSeqTM 2500 sequencing platform to compare further. Results The morphology of the cells that derived from two groups was basically similar, all presented an elongate spindle-shape. To evaluate the ostogenesis, ALP activity assays including quantitative and staining assays were performed. Under microscope, the ALP staining in the TOLF group was higher than non-TOLF group and increased with the longer duration of stress induction. The result of semi-quantitative analysis showed the stained area and positive cells in TOLF group were more than non-TOLF group significantly at 0 h, and were increased with the induction. The results of quantitative analysis showed ALP activity in the TOLF group was significantly higher than non-TOLF group and were increased with the induction significantly all the time. But no significant change in ALP staining or quantitative analysis was found in non-TOLF. The results of real-time PCR indicated that the expression of ostegenic markers above in the TOLF group was more than non-TOLF g

关 键 词：胸椎 黄韧带 骨化 异位性 序列分析 

分 类 号：R681.5[医药卫生—骨科学]