深低温冷冻对大鼠髌腱肌腱干细胞干性及多向分化能力的影响

Effects of cryopreservation on stemness and multi-differentiation ability of tendon-derived stem cells in rat patellar tendon

目的:研究深低温冷冻对大鼠髌腱肌腱干细胞(tendon-derived stem cells,TDSCs)的干性以及成骨、成脂肪、成软骨和成肌腱分化能力的影响。方法:实验组大鼠髌腱组织用深低温冷冻处理2周,对照组未作特殊处理。从两组髌腱组织中分离培养出TDSCs,采用细胞免疫荧光检测TDSCs干性相关标志物Nanog、Oct4和Sox2的表达,成骨、成脂肪、成软骨诱导分化实验分别检测成骨、成脂肪、成软骨分化能力,细胞免疫荧光和实时定量逆转录聚合酶链反应(qRT-PCR)检测成肌腱能力。结果:两组TDSCs培养7 d(P0代)时均可见大的多边形和星形细胞形态,P3代时均可见均一的成纤维样细胞形态。两组TDSCs均表达干性相关标志物Nanog、Oct4和Sox2,均具有成骨、成脂肪、成软骨和成肌腱分化特性,且两组细胞的干性相关标志物Nanog、Oct4及Sox2的表达和多向分化能力之间差异无统计学意义(P>0.05)。结论:深低温冷冻处理的髌腱组织中检测到有活性的TDSCs,且TDSCs干性和多向分化能力无明显变化,这些发现可能为研究同种异体肌腱移植治疗肌腱缺损中关于内源性修复的存在提供依据。

Objective:To study the effects of cryopreservation on stemness and osteogenic,adipogenic,chondrogenic and tenogenic differentiation ability of tendon-derived stem cells(TDSCs)in rat patellar tendon.Methods:The patellar tendon tissue of the experimental group was treated with cryopreservation for two weeks,while the control group was not treated with such processing.TDSCs were isolated and cultured from patellar tendon tissue of the two groups.Cellular immunofluorescence was used to detect the expression of stemness-related markers Nanog,Oct4 and Sox2 of TDSCs.Osteogenic,adipogenic and chondrogenic induction differentiation assays were used to detect osteogenic,adipogenic and chondrogenic differentiation ability of TDSCs.Immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)were used to detect tenogenic ability of TDSCs.Results:TDSCs from both groups showed large polygonal and star-shaped morphology at P0,and uniform fibroblast-like cell morphology was observed at P3.TDSCs from both groups expressed stemness-related markers Nanog,Oct4 and Sox2,and they possessed osteogenic,adipogenic,chondrogenic and tenogenic differentiation ability,and the expression levels of stemness-related markers Nanog,Oct4 and Sox2 and multi-differentiation ability of TDSCs from the two groups were similar,and there was no statistical difference(P>0.05).Conclusion:Active TDSCs were observed in the patellar tendon tissue treated with cryopreservation,and the stemness and multi-differentiation ability of TDSCs do not alter after cryopreservation.These findings may provide a new theoretical basis for the existence of endogenous repair in the study of allogeneic tendon transplantation in the treatment of tendon defects.