间歇性循环张力对人间充质干细胞成软骨分化的影响

Impact of intermittent cyclic mechanical tension on chondrogenic differentiation in human mesenchymal stem cells

目的:研究间歇性循环张力(ICMT)对人间充质干细胞(h MSCs)成软骨分化的影响。方法:取人间充质干细胞建立体外软骨分化的模型,将人间充质干细胞以8×10~4/cm^2的密度接种于被Ⅰ型胶原覆盖处理过的Bio Flex TM六孔加力板中,置于37℃、5%CO_2培养箱中培养,待细胞贴壁后加入成软骨诱导液培养。根据间歇性循环张力的影响分为加力组和对照组,加力组:细胞通过Flexcell FX-5000TM应变加载系统在培养箱中(37℃、5%CO_2)给予力学刺激(10%伸长率、0. 5 Hz、8 h/d)处理;对照组:不加以力学刺激放在相同环境下一起培养。两组细胞分别在加成软骨诱导液后1、3、6、8、10 d用CCK-8法检测细胞增殖。第14天用番红O染色观察细胞成软骨变化,用流式细胞术检测细胞凋亡情况,用Real-time PCR的方法检测细胞成软骨基因ACAN、COLⅡ、SOX9的表达变化。结果:间歇性循环张力可以抑制间充质干细胞的增殖。第14天的加力组细胞明显凋亡,番红O染色比对照组浅,软骨基因的表达被抑制。结论:间歇性循环张力抑制人间充质干细胞成软骨分化。

Objective: To investigate the impact of intermittent cyclic mechanical tension (ICMT) on chondrogenic differentiation in human mesenchymal stem cells (hMSCs).Methods: Human mesenchymal stem cells were obtained to develop the chondrogenic differentiation model using in vitro technique.hMSCs were planted in density of 8×10 4/cm 2 in a BioFlexTM six-well multi-directional force plate coated with type I collagen,and cultured in 5% CO 2 incubator at 37 ℃.Upon cell adhesion,chondrogenic inducer was added in vitro .Then hMSCs were divided into afterburning group and control group according to the influence of intermittent cyclic tension.Cells in the afterburning group were treated by mechanical stimulation in a 5% CO 2 incubator(10%,0.5 Hz,8 h/d,at 37 ℃) using Flexcell FX-5000TM cell strain loading system,and hMSCs in the control group were cultured in the same environment without mechanical stimulation.Cell proliferation was detected by CCK-8 method at day 1,3,6,8 and 10 after addition of chondrogenic inducing agent.Cartilage changes were observed at the 14 th day using Safranin O staining kit.Flow cytometry was performed to examine the cell apoptosis,and expression changes of the cartilage genes ACAN,COLII and SOX9 were detected by RT-PCR.Results: ICMT inhibited the proliferation of mesenchymal stem cells,and cells in the afterburning group were found to be obviously apoptotic at the 14 th day,with lighter Safranin O staining.Inhibited chondrogenic gene expression was also found in the afterburning group.Conclusion: ICMT is able to inhibit the differentiation of human mesenchymal stem cells into cartilage.