The evaluation of osteogenic potency of ligament cells in thoracic ossification of the ligamentum flavum and relevant transcriptome high-throughput sequencing analysis / 中华骨科杂志

ABSTRACT

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 westernblotting 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 group significantly except the expression of OCN at 0 h.The expression of the three ostegenic markers in TOLF group was increased with the stress induction for 12 h and 24 h significantly except the expression of BMP-2 and OCN at 12 h.The results of western-blotting indicated that the expression of the three ostegenic markers above in the TOLF group was more than non-TOLF group significantly except the expression of ALP at 0 h.The expression of the three ostegenic markers in TOLF group was increased with the stress induction for 12 h and 24 h,but only the expression of ALP at 24 h was significant.And no significant change in the expression of mRNA and protein was found in non-TOLF group.In the transcriptome analysis,671 genes of TOLF group were up-regulated and 314 genes were found to be down-regulated compared to the control group.In addition,22 significant GO terms associated with upregulated genes were found to be closely related to ossification.Conclusion TOLF ligament cells have high osteogenic differentiation potency,which could express obvious osteogenesis-related gene spectrum,and differentially expressed genes including L1RL1 、PTHLH、DKK1 、BMP6、SPP1 and FGF1 may be related with the osteogenic potency of ligament cells in thoracic ossification.