单轴拉力刺激对人股薄肌腱干细胞炎性因子表达的影响

Expression changes of inflammatory cytokines of tendon-derived stem cells in response to uniaxial stretching

目的 观察单轴拉力刺激对肌腱干细胞（TDSCs）炎性因子表达的影响.方法 通过胶原酶法和低密度接种法,从人的股薄肌腱中分离TDSCs;流式细胞术检测分离出的TDSCs的表面标记因子;体外诱导检测其多向分化能力.将细胞接种在体外细胞力学加载仪上,分别施加4％、8％和10％的单轴拉力.显微镜观察拉力刺激后的排列情况.荧光定量PCR和Western blot 观察拉力后TDSCs环氧合酶-2（COX-2）和基质金属蛋白酶-1（MMP-1）的基因和蛋白表达情况.结果 从人股薄肌腱中分离出TDSCs,表达CD29、CD44和CD105阳性,CD45和CD14阴性.拉力刺激4h,TDSCs未发生重新排列.4％拉力强度下MMP-1基因表达为0.090±0.007,与0％的0.247±0.032比较显著降低（P ＜0.05）;COX-2基因表达为0.005±0.001,与0％的0.005±0.001比较差异无统计学意义（P＞0.05）.与0％拉力强度比较,8％拉力强度下MMP-1和COX-2基因表达差异均无统计学意义（8％分别为0.168±0.040和0.007±0.001,而0％分别为0.134±0.075和0.006±0.003）（P＞0.05）.10％拉力强度下MMP-1和COX-2基因表达较0％拉力强度比较差异均显著升高（10％分别为0.047±0.003和0.496±0.036,而0％分别为0.011±0.003和0.005±0.003）（P＜0.05）.蛋白表达与基因表达相一致,与0％拉力强度比较,4％拉力强度下COX-2和MMP-1蛋白表达均降低,8％拉力强度下COX-2和MMP-1蛋白表达均无明显变化,10％拉力强度下COX-2蛋白表达升高,MMP-1有微弱升高.结论 不同强度单轴拉力引起炎性因子的表达不同,低强度拉力降低炎性因子表达,高强度拉力升高炎性因子表达.

Objective To observe the expression changes of inflammatory cytokines of human tendon-derived stem cells （TDSCs） under uniaxial stretching.Methods TDSCs were isolated from human gracilis tendon by collagenase digestion at very low density.Cells were detected for surface markers by flow cytometry,and tested for multi-differentiation potential in vitro.Repetitive uniaxial stretching was applied on the cells at 0%,4%,8% or 10% strain.Under the microscopy,cell alignment was observed after mechanical stretching.Expressions of inflammation factors COX-2 and MMP-1 were detected by qPCR and western blotting.Results TDSCs were successfully isolated from human gracilis tendon.Inflammatory cytokines CD29,CD44 and CD105 were positive,but CD45 and CD14 were negative.TDSCs could differentiate into osteocytes,adipocytes and chondrocytes in vitro.Cells were not realigned4 hours after mechanical stretching.MMP-1 mRNA expression was significantly down-regulated at 4% strain （0.090 ± 0.007） compared to that at 0% strain （0.247 ± 0.032,P 〈 0.05）.No significant difference was found in COX-2 mRNA expression at 4% and 8% strain （both was 0.005 ±0.001,P 〉0.05）.MMP-1 and COX-2 mRNA expressions at 8% strain （0.168 ± 0.040 and 0.007 ± 0.001）revealed no significant differences from those at 0% strain （0.134 ±0.075 and 0.006 ±0.003） （P 〉0.05）,whereas at 10% strain MMP-1 and COX-2 mRNA expressions were significantly up-regulated （0.047 ± 0.003 and 0.496 ± 0.036） compared to those at 0% strain （0.011 ± 0.003 and 0.005 ± 0.003）（P 〈 0.05）.Changes in MMP-1 and COX-2 protein expressions revealed similar trend as their mRNA expressions.In contrast to the setting of 0% strain,4% strain induced down-regulated MMP-1 and COX-2 proteins,8% strain induced no significant changes in MMP-1 and COX-2 proteins,and 10% strain induced up-regulated COX-2 protein despite minor increase in MMP-1 protein.Conclusions Mechanical stretching can affect the secretion of inflammatory cytokines.Low tensile stretch is associated with decreased expression of inflammatory cytokines while high tensile stretch promotes secretion of inflammatory cytokines.