持续牵张应力诱导肌腱干细胞向成骨细胞分化的作用机制研究

Study on the Mechanism of Continuous Stretch Stress Inducing The Differentiation of Tendon Stem Cells Into Osteoblasts

目的本研究通过构建不同牵张力处理的肌腱干细胞(tendon stem cells,TSCs)模型,探讨其对TSCs向成骨细胞定向分化的作用及机制。方法利用不同程度的牵张力处理TSCs 48 h,观察细胞形态,采用细胞计数试剂(cell counting kit-8,CCK-8)法检测细胞相对活力;应用逆转录聚合酶链式反应(reverse transcriotion-polymerase chain reaction,RT-PCR)及Western-blot分别检测其分化情况、干细胞维持性相关转录因子SOX2、OCT4基因及其蛋白表达水平、成骨细胞Runt相关转录因子2(Runt related transcription factor 2,RUNX2)、DLX5及COL1a的相对表达量及RUNX2的蛋白表达量。结果细胞活力检测结果显示,各牵张力处理组均出现细胞相对活性呈上升的趋势,但仅在牵张力为6%Strain时的升高差异有统计学意义(P<0.05);当牵张力达到4%Strain后,SOX2基因及蛋白表达出现明显下降(P<0.05)。对于OCT4基因,在6%Strain牵张力处理后其表达出现明显下降(P<0.05);成骨基因RUNX2仅在2%Strain组和6%Strain组升高,差异有统计学意义(P<0.05)。而COL1a仅在6%Strain组升高,差异有统计学意义(P<0.05)。对RUNX2蛋白检测发现仅在6%Strain组显著升高,差异有统计学意义(P<0.05)。进一步通过碱性磷酸酶检测后发现4%Strain和6%Strain能够有效提高成骨分化的进程。结论适度温和的牵张力有助于提高TSCs细胞的存活率,适度的牵张力也可以通过调节干细胞维持性相关转录因子SOX2、OCT4基因及其蛋白表达水平、成骨细胞RUNX2、DLX5及COL1a的相对表达量及RUNX2的蛋白表达量来加速诱导TSCs的分化,并具有加速其向成骨细胞分化的能力。

Objective To explore the mechanism of continuous stretch stress inducing differentiation of tendon stem cells(TSCs)into osteoblasts.Methods TSCs were treated with different degrees of Strain for 48 h to observe the cell morphology,and the cell relative viability rate was detected by CCK-8 method.RT-PCR and Western-blot were used to detect the differentiation,stem cell maintenance-related transcription factors SOX2,OCT4 gene and protein expression,osteoblast RUNX2(Runt related transcription factor 2),DLX5 and COL1a relative expression and RUNX2 protein expression.Results The cell relative viability by CCK-8 method showed that the cell relative viability rate increased in each Strain treatment group,but the increase was only statistically significant when the Strain was 6%(P<0.05).Compared with the control group,when the Strain reached 4%,the expression of SOX2 gene and protein decreased significantly(P<0.05).For OCT4 gene,expression was significantly decreased after 6%Strain treatment(P<0.05).The osteogenic gene RUNX2 increased only in the 2%Strain group and the 6%Strain group(P<0.05).However,COL1a only increased in the 6%Strain group(P<0.05).RUNX2 protein was detected only in the 6%Strain group,and the difference was statistically significant(P<0.05).After further testing by alkaline phosphatase,it was found that 4%Strain and 6%Strain can effectively improve the process of osteogenic differentiation.Conclusion Moderately mild tension helps to improve the survival rate of TSCs cells,and moderate tension can also accelerate the differentiation of TSCs and accelerate its ability to differentiate into osteoblasts by regulating the expression level SOX2、OCT4 genes and their proteins,the relative expression of osteoblast RUNX2、DLX5 and COL1a,and the protein expression of RUNX2.