【摘要】 [目的]研究在连续体外单层培养条件下不同接种密度对大鼠关节软骨细胞分化状态的影响,并探讨藻酸钠微球三维立体培养恢复去分化关节软骨细胞表型的调节机制。[方法]以正常密度(3×104/cm2)或低密度(3×102/cm2)分别连续体外单层传代培养大鼠膝关节软骨细胞使其去分化,RT-PCR检测I、II型胶原和聚集蛋白聚糖mRNA的表达以确定其分化状态。取正常密度培养时的去分化软骨细胞,藻酸钠微球包被4周以恢复其表型,在该立体培养过程中将特异的SIRT1抑制剂-EX-527加入到培养基中抑制其表达,甲苯胺兰染色微球中软骨细胞分泌的细胞外基质,W estern b lot检测各种条件下SIRT1和Sox-9的表达。[结果]当以正常密度连续体外单层培养时,软骨细胞在第4代发生去分化,低密度时于第1代即明显去分化,此时SIRT1和Sox9表达明显降低。藻酸钠微球三维立体培养能显著地增强去分化软骨细胞中II型胶原、聚集蛋白聚糖mRNA以及SIRT1、Sox9蛋白的表达,同时降低I型胶原mRNA的表达。EX-527不仅限制藻酸钠微球中的软骨细胞周围细胞外基质的大量生成而且还抑制了SIRT1和Sox9的表达。[结论]关节软骨细胞连续体外单层培养的去分化与细胞接种密度有关,低密度培养加速去分化过程。藻酸钠微球三维立体培养恢复去分化关节软骨细胞表型的作用可能是通过细胞-细胞、细胞-细胞外基质间的相互作用,从而激活SIRT1的表达,继而增强Sox9的转录活性来实现。更多还原

【Abstract】 [Objective]To investigate the influence of alginate suspension culture on the redifferentiation of dedifferentiated articular chondrocytes.[Methods]The rat knee dedifferentiated chondrocytes were obtained by serial monolayer culture using two seeding densities of 3×104/cm2 and 3×102/cm2.Dedifferentiated chondrocytes of seeding densities of 3×104/cm2 were encapsulated in alginate bead for four weeks and the specific SIRT1 inhibitor,EX-527,was introduced into the culture medium.The differentiation state was estimated by RT-PCR to detect collagen type I,II and aggrecan mRNA levels.Toluidine blue stain was used to visualize the extracellular matrix around the articular chondrocytes encapsulated in alginate bead and Western blot was used to estimate SIRT1 and Sox9 expression.[Results]When seeded with 3×104/cm2 chondrocyte dedifferentiation took place at passage 4 and seeded with 3×102/cm2 dedifferentiation took place at passage 1.The collagen type II,aggrecan mRNA and SIRT1,Sox9 protein expression significantly increased and the collagen type I expression significantly decreased by alginate suspension culture.EX-527 inhibited not only extracellular matrix accumulation around the chondrocytes when encapsulated in alginate bead but also the expression of SIRT1 and Sox9.[Conclusion]The process of chondrocytes dedifferentiation was accelerated when cells were seeded with lower density.Alginate suspension culture can recover the phenotype of dedifferentiated chondrocytes and this mechanism may be via cell-cell,cell-matrix interaction to upregulate SIRT1 and consequent Sox9 expression.更多还原