冲击波对骨髓间充质干细胞中c-Fos、c-Jun蛋白表达的影响

发布时间：2018-03-21 17:51

本文选题：冲击波　切入点：间充质干细胞　出处：《中国组织工程研究与临床康复》2009年49期 　论文类型：期刊论文

【摘要】：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2.5g/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1.0×109L-1的水平,然后取6mL细胞悬液,分装入6个1.5mL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8.5kV,脉冲次数为120次)作用于细胞,分别在作用后5,15,30min,1,2h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,24h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。10～14d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,24h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。③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30,15min后达高峰,分别为对照组的2.56倍,1.68倍(P0.01),之后逐渐下降;c-Fos、c-Jun的总量未见明显变化(P0.05)。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。
[Abstract]:Background: at present, shock wave therapy has been widely used in the treatment of nonunion, delayed union of fracture and necrosis of femoral head, and chronic strain disease of motor system. Objective: to observe the changes of c-Fosn c-Jun protein expression in bone marrow mesenchymal stem cells treated with extracorporeal shock wave. Completed in the State key Laboratory of Pathology, College of basic Medicine, Jilin University, 2007-03 / 2008-03. Materials: bone marrow derived from healthy volunteers. Methods: human bone marrow mesenchymal stem cells were cultured in vitro and P4 passage cells were taken. The cell suspension was digested with 2.5g/L trypsin. The cell density was adjusted to 1. 0 脳 109L-1 with serum free low glucose DMEM medium. Then the 6mL cell suspension was obtained. Six 1.5 mL Eerrendorf tubes were divided into 5 tubes of the control group. The Eerrendorf tubes of the induction group were placed in the extracorporeal hydroelectric shock wave lithotriptor. The optimal shock wave intensity (working voltage 8.5 kV, pulse number 120 times) was applied to the cells. Main outcome measures: morphological changes of bone marrow mesenchymal stem cells were observed by inverted microscope. The expression of c-Fossin-c-Jun protein was detected by Western Blot. Results the bone marrow mesenchymal stem cells (BMSCs) were inoculated into the culture plate for 24 hours, and the cells began to divide slowly and proliferate. The morphology of adherent cells was fusiform after half fluid exchange, and the proliferation of adherent cells accelerated after 3 days, and each cell clone increased after 10 ~ 14 days of cluster growth. The cultured bone marrow mesenchymal stem cells were completely adhered to the wall within 24 hours, and the morphology was similar to that of the primary cultured cells for about 1 week. The mesenchymal stem cells were arranged in a whirlpool shape. After 10 passage, the mesenchymal stem cells were arranged in a whirlpool shape. The cell division and proliferation slowed down obviously. The results showed that the growth curve of primary bone marrow mesenchymal stem cells (BMSCs) of primary generation and P2P3-generation was S-shaped, and the proliferation exuberant phase was 3. 3 in vitro shock wave induction on the 3rd day. The phosphorylation level of c-Fosn c-Jun in human bone marrow mesenchymal stem cells began to increase and reached its peak after 30 minutes. It was 2.56 times as much as 1.68 times that of the control group (P0.01), and then decreased gradually. The total amount of c-Fossin-c-Jun did not change significantly. Conclusion: the activity of c-Fosn c-Jun protein increased after cultured human bone marrow mesenchymal stem cells were exposed to shock wave in vitro.
【作者单位】：吉林大学第一医院脊柱外科;吉林大学第一医院骨关节二科;
【分类号】：R329

【参考文献】