Study On Mechanism Of SUMO Modification In Ossification Of Human Ligamentum Flavum

Objectives: To investigate the small ubiquitin-like modifier(SUMO)modification of ossification of ligament flavum(OLF)and to establish an OLF fibroblast ossification model by inducing bone marrow stromal cells(BMSCs)to differentiate into osteoblasts,to clarify the SUMO expression and cell differentiation after BMSCs were induced to differentiate into osteoblasts,and study the effect of SUMO1 on the osteogenic differentiation of BMSCs.After induced to differentiate into osteoblasts,The hypoxic stress ability of BMSCs was observed when the expression of SUMO1 was changed by applying hypoxic stress.Methods: The ligamentum flavum tissues of OLF patients and patients with normal ligamentum flavum were obtained by clinical operation.Fibroblasts were isolated and cultured by collagenase-1 digestion and tissue culture.The cell morphology was observed.The expression of SUMO1 and SUMO1 modification of OLF fibroblasts were detected by Western Blot method.BMSCs in femoral bone marrow of mice was isolated and cultured.Using ossification induction kit to induce ossification,established OLF cell model,detected the expression of ossification markers ALP(alkaline phosphatase),COL-I(collagen1),OCN(osteocalcin),Runx2(runt-related transcription factor 2),analysis the effect of osteogenic differentiation.The expression and localization of SUMO1 and SUMO2/3 in BMSCs before and after osteogenic differentiation were studied by immunofluorescence assay.The expression of SUMO in the process of osteogenic differentiation were detected by Western Blot method.Up/down-regulated SUMO1 by lentivirus transfection to investigate the effect of SUMO1 on osteogenic differentiation of BMSCs.By applying hypoxic stress to BMSCs induced to differentiate into osteoblasts,apoptosis was observed by TUNEL method,and the hypoxic stress ability of BMSCs induced to differentiate into osteoblasts was observed when the expression level of SUMO1 changed.Results:1.OLF ligamentum flavum fibroblasts can be successfully isolated and cultured by the combination of collagenase digestion and tissue culture.2.Western Blot assay showed that the expression of SUMO1 protein in OLF-derived fibroblasts was lower than that in normal tissue-derived fibroblasts,and the level of SUMO1 modification was down-regulated.3.BMSCs were selected as the basic cell model for predicting OLF.After induction of osteogenic differentiation,the expression levels of ALP,COL-I,OCN and Runx2 in BMSCs were significantly increased and the induction was effective.4.Cell immunofluorescence assay showed that SUMO1 was mainly expressed in nucleus and SUMO2/3 was expressed in both nucleus and cytoplasm before ossification induced BMSCs.After BMSCs were induced by ossification,the expression of SUMO1 in nucleus,SUMO2/3 in cytoplasm and SUMO2/3 in nucleus decreased significantly.5.When BMSCs were induced to differentiate into osteoblasts,the expression of bound SUMO1 decreased linearly after inducing BMSCs to differentiate into osteoblasts,while the expression of bound SUMO2/3 remained unchanged after transient overexpression.6.Increasing the expression of SUMO1 can decrease the expression of ALP,inhibit the osteogenic differentiation of BMSCs after induction,and inhibit the expression of SUMO1 can increase the expression of ALP and promote the osteogenic differentiation of BMSCs after induction.7.Imitating the hypoxic stress in the process of ossification of OLF fibroblasts,enhancing the expression of SUMO1 can reduce the apoptotic rate of BMSCs in the process of osteogenic differentiation,improve the ability of cells to hypoxic stress,and low expression of SUMO1 can increase the apoptotic rate of BMSCs in the process of osteogenic differentiation and reduce the ability of cells to hypoxic stress.Conclutions: 1.The combination of collagenase-1 digestion method and tissue culture method is more conducive to the migration of cells within the tissue block,and can improve the success rate of isolation and culture of ligamentum flavum fibroblasts.2.The level of SUMO1 modification of OLF fibroblasts was down-regulated.3.Mouse BMSCs can be induced to differentiate into osteoblasts,and BMSCs can be used as a basic cell model for predicting OLF.4.The expression level of bound SUMO1 protein decreased during osteogenic differentiation of BMSCs.It mainly acts in the nucleus.5.Enhanced expression of SUMO1 can inhibit osteogenic differentiation of BMSCs induced by ossification.It is speculated that the enhanced expression of SUMO1 in OLF fibroblasts may also up-regulate SUMO1 modification and inhibit osteogenic differentiation.6.Upregulation of SUMO1 expression enhances hypoxic stress and reduces apoptosis of BMSCs after osteogenic differentiation.Therefore,it is speculated that in the initial stage of OLF,human enhanced expression of SUMO1 in OLF fibroblasts can enhance the response of fibroblasts to hypoxic stress and reduce apoptosis.Stem cell transplantation through SUMO1 gene transfection may be a potential way to treat OLF.