氧化应激诱导肩袖肌腱干细胞自噬的表达

Expression of autophagy in rotator cuff tendon stem cells induced by oxidative stress

背景:目前肩袖腱骨愈合机制尚未完全阐明。有研究表明氧化应激状态下可导致组织细胞损害,不利于创面愈合。临床发现肩袖损伤腱骨止点常常愈合效果不佳,推测这可能与创面在缺血缺氧应激状态下出现异常细胞凋亡有关。目的:以氧化应激刺激诱导肩袖肌腱干细胞自噬促进细胞生存为突破点,设想通过自噬来缓解细胞氧化应激性损害。方法:采用肩袖肌腱干细胞体外培养体系,分别用0,50,100,200,400μmol/L H_(2)O_(2)处理人肌腱干细胞24,48,72 h,CCK-8法检测细胞活力,然后选择合适的H_(2)O_(2)浓度用于建立肩袖肌腱干细胞体外氧化应激损伤模型。将第3代肩袖肌腱干细胞分为空白组、H_(2)O_(2)(50μmol/L)干预组、自噬抑制剂3-甲基腺嘌呤预处理(5 mmol/L,30 min)+H_(2)O_(2)组,干预48 h后,采用MDC、Lyso-Tracker Red染色检测细胞自噬现象,基因表达谱芯片检测自噬相关基因,DCFH-DA检测活性氧水平,Annexin V/PI检测细胞凋亡情况,Western blot检测各组细胞Beclin-1、p-mTOR、mTOR、LC3A/B和Caspase-3蛋白表达。结果与结论:①随着H_(2)O_(2)浓度的增加,肩袖肌腱干细胞增殖活性下降,差异有显著性意义(P<0.05);当H_(2)O_(2)浓度为50μmol/L时,肩袖肌腱干细胞增殖活性最强;②与空白组及3-甲基腺嘌呤+H_(2)O_(2)组比较,H_(2)O_(2)组细胞自噬水平明显增强,差异有显著性意义(P<0.05);③与空白组及3-甲基腺嘌呤+H_(2)O_(2)组比较,H_(2)O_(2)组细胞活性氧水平明显升高(P<0.05),自噬正调控因子Beclin-1蛋白表达上调(P<0.05),自噬负调控因子mTOR蛋白表达下降(P<0.05),自噬标志蛋白LC3A/B表达上调(P<0.05),凋亡相关Caspase-3蛋白表达上调(P<0.05);④结果表明,低浓度的H_(2)O_(2)可促进人肌腱干细胞增殖,但细胞凋亡和活性氧水平也一定范内小幅度升高,这是一种细胞高转化状态。细胞自噬可能发挥细胞保护作用,促进这种高转化状态,具体机制还需进一步研究。

BACKGROUND:The mechanism of rotator cuff tendon-bone healing has not yet been fully elucidated.Studies have shown that oxidative stress can lead to tissue cell damage,which is not conducive to wound healing.It is clinically found that the tendon-bone insertion point of rotator cuff injury often has a poor healing effect,and it is speculated that this may be related to the abnormal cell apoptosis in the wound under ischemia and hypoxia stress.OBJECTIVE:To induce autophagy of rotator cuff tendon stem cells to promote cell survival as a breakthrough point by oxidative stress stimulation,it is envisaged that autophagy can alleviate cellular oxidative stress damage.METHODS:The rotator cuff tendon stem cells were cultured in vitro and treated with 0,50,100,200,400μmol/L H_(2)O_(2) respectively for 24,48 and 72 hours,followed by CCK-8 assay to detect the cell viability.The oxidative stress injury model of rotator cuff tendon stem cells in vitro was established with an appropriate concentration of H_(2)O_(2).The cultured rotator cuff tendon stem cells at passage 3 were divided into three groups:blank group,H_(2)O_(2)(50μmol/L)intervention group,and 3-methyladenine pretreatment(5 mmol/L,30 minutes)+H_(2)O_(2) group.At 48 hours after intervention,autophagy was detected by MDC and Lyso-Tracker Red staining.Autophagy related genes were detected by gene expression microarray.Reactive oxygen species level was detected by DCFH-DA.Apoptosis was detected by Annexin V/PI.Protein expression levels of Beclin-1,p-mTOR,mTOR,LC3A/B and Caspase-3 were detected by western blot assay.RESULTS AND CONCLUSION:(1)With the increase of H_(2)O_(2) concentration,the proliferation activity of rotator cuff tendon stem cells significantly decreased(P<0.05).When the concentration of H_(2)O_(2) was 50μmol/L,the proliferation activity of rotator cuff tendon stem cells was the strongest.(2)Compared with the blank group and 3-methyladenine+H_(2)O_(2) group,the autophagy level of H_(2)O_(2) group was significantly increased(P<0.05).(3)Compared with the blank group and 3-methyladenine+H_(2)O_(2) group,the level of reactive oxygen species was significantly increased(P<0.05);the expression of Beclin-1 protein was up-regulated(P<0.05);the expression of mTOR protein was down-regulated(P<0.05);the expression of LC3A/B protein was up-regulated(P<0.05);and the expression of Caspase-3 protein was up-regulated(P<0.05)in the H_(2)O_(2) group.(4)These results indicate that the low concentration of H_(2)O_(2) can promote the proliferation of human tendon stem cells,and the degree of apoptosis and reactive oxygen species level also increase slightly in a certain range,which is a state of high cell transformation.Autophagy may play a cytoprotective role and promote this high transformation state,but the specific mechanism needs to be further studied.