冲击波对骨髓间充质干细胞中c-Fos、c-Jun蛋白表达的影响

背景:目前,冲击波疗法被广泛的用于治疗骨不连、骨折延迟愈合和股骨头坏死以及运动系统慢性劳损性疾病,取得了很好的疗效,但其作用机制并不明确.目的:观察体外冲击波作用于骨髓间充质干细胞后c-Fos、c-Jun蛋白表达的变化情况.设计、时间及地点:随机分组设计,对比观察,于2007-03/2008-03在吉林大学基础医学院病理学国家重点实验室完成.材料:骨髓来源于健康志愿者.方法:体外培养人骨髓间充质干细胞,取P4代细胞,加入2.5 g/L胰蛋白酶消化制成细胞悬液,用无血清低糖DMEM培养基调整细胞密度在1.0×10~9L~(-1)的水平,然后取6 mL细胞悬液,分装入6个1.5 mL.的Eerrendorf管内,1管为对照组,5管为诱导组.将诱导组Eerrendorf管置入体外液电冲击波碎石机中,采用最佳冲击波强度(工作电压8.5 kV,脉冲次数为120次)作用于细胞,分别在作用后5,15,30 min,1,2 h提取蛋白质.主要观察指标:倒置显微镜观察骨髓间充质干细胞形态变化:四甲基偶氮唑盐法测定细胞生长曲线;Western Blot检测细胞内c-Fos、c-Jun蛋白表达的变化.结果:①骨髓间充质干细胞接种于培养板,细胞呈圆形,24 h后,细胞开始缓慢分裂、增殖,半量换液后见贴壁细胞形态 呈梭形.第3天后细胞增殖加快,呈团簇集落样生长.10～14 d各个细胞克隆增大,直至铺满培养板底面达到融合状态.传代培养的骨髓间充质干细胞呈圆形,24 h内完全贴壁,形态与原代培养的细胞类似,1周左右达到完全融合,呈漩涡状排列.传至10代以后,细胞分裂增殖速度明显减慢,细胞逐渐老化.②四甲基偶氮唑盐结果显示:原代及P2、P3代的人骨髓间充质干细胞生长曲线呈S型,在第3-5天为增殖旺盛期.⑨体外冲击波诱导后,人骨髓间充质干细胞中c-Fos、c-Jun的磷酸化水平开始增高,分别于30,15 min后达高峰,分别为对照组的2.56倍,1.68倍(P<0.01),之后逐渐下降;c-Fos、c-Jun的总量未见明显变化(P>0.05).结论:体外冲击波作用于体外培养的人骨髓间充质干细胞后,呈现了细胞内c-Fos、c-Jun蛋白活性增高的变化.

Effects of shock wave on c-Fos and c-Jun protein expression in bone marrow mesenchymal stem cells

BACKGROUND: Now, Shock Wave Therapy is used to cure the ununion and delayed union of bone, the avascular necrosis of the femoral head and the chronic injury of locomotor system, what has got a good curative effect. But, the mechanism is not clear. OBJECTIVE: To investigate the expression of c-Fos, c-Jun protein in human bone marrow mesenchymal stem cells (BMSCs) Influenced by shock wave.DESIGN, TIME AND SETTING: Randomized group, the controlled study was performed at the State Key Laboratory of Pathology, College of Basic Medicine, Jilin University, from March 2007 to March 2008. MATERIALS: Bone marrow was obtained from healthy volunteers.METHODS: Human BMSCs were cultured in vitro. And the fourth generation cells were digested into cell suspension with 2.5 g/L trypsin and adjusted at a density of 1.0×10~9/L with DMEM-LG. Then, the cells were divided into 6 1.5-mL Eerrendorf tubes, one was control group and the other five were experimental groups. The experimental groups were treated with an optimal dose of shock wave (8.5 kV, 120 times) by liquid-electric shock wave lithotripsy. Then the protein was extracted at different time points (5, 15, 30 minutes, 1, 2 hours) after treatment.MAIN OUTCOME MEASURES: Inverted microscope was used to observe the morphologic feature of BMSCs. The growth curves were charted by MTT method. The change of activations of c-Fos, c-Jun were tested by western blot. RESULTS: ①BMSCs were seeded in culture plate. The cells began to divide and proliferate slowly 24 hours later, and became fusiform shape after adhering to the wall. 3 days later, the speed of proliferation quickened, and cells accumulated colony. At day 10-14, the number of hMSCs grew till they covered the bottom of the culture plate. The round passage hMSCs adhered to the wall completely in 24 hours, which were similar to primary cells. The cells connected together during one week, and showed vortex-like. The speed of proliferation became slower and the cells became older when hMSCs were passaged to the tenth generation. ②MTT method showed that the growth curve of original, P2, P3 hMSCs looked like S shape, and the third to fifth days were growing period. ③The phosphorylation level of c-Fos and c-Jun began to increase after induced by shock wave and reached a peak at 30 and 15 minutes, respectively. And their phosphorylation level were 2.56-fold and 1.68-fold (P < 0.01) compared with the average level in control groups, respectively. Then they began to decrease. There was no apparent change in the total dose (P > 0.05). CONCLUSION: Following the treatment of shock wave, the activations of c-Fos and c-Jun in BMSCs increased.