【作者】 李晨；

【导师】 朱东；

【作者基本信息】 吉林大学 ， 临床医学硕士（专业学位）， 2019， 硕士

【摘要】 研究背景和目的:随着我国骨折的发病率逐年增高,除了传统的手术治疗方法外,我们急需探索一种新的非侵入性的治疗方法。近年来的研究表明,低载荷机械振动作为一种非药物疗法,通过促进成骨细胞的增殖,在骨质疏松症及骨折的治疗中取得了良好的疗效。当细胞接受力学信号的刺激时,细胞骨架发挥了重要的作用,其中MAPK(mitogen-activated protein kinase,MAPK)信号通路与细胞骨架的形成和功能密切相关。本项研究初步探讨低载荷机械振动下细胞骨架及MAPK信号通路对如何对成骨细胞增殖产生影响。研究方法:将小鼠胚胎成骨细胞前体细胞(MC3T3-E1)分为对照组和实验组,实验组给与每天0.5小时,持续一周的低载荷机械振动,加载参数为振动频率35Hz,振动强度0.25g。一周后,用CCK-8试剂盒检测对照组与实验组的细胞增殖情况。并采用激光共聚焦显微镜观察细胞骨架是否在低载荷机械振动下受到了结构性的损伤。然后,对于对照组和实验组进行microRNA(微小RNA,miRNA)基因芯片的检测,观察差异基因的表达情况,并采用荧光实时定量PCR(real-time quantitative PCR,RT-qPCR)验证基因芯片结果。实验结果:经过一周的低载荷机械振动,实验组的细胞增殖活性得到了加强。激光共聚焦显微镜显示在低载荷机械振动下的细胞骨架与对照组相比未发生明显变化。miRNA基因芯片结果显示实验组与对照组相比有明显的差异基因的表达,其中miR-182-5p(degree=17)、miR-150-5p(degree=13)、miR-125b-2-3p(degree=11)等差异基因是通过激活MAPK信号通路和肌动蛋白调节通路来影响成骨细胞的增殖的。RT-qPCR结果显示,实验组与对照组相比:miR-182-5p(***p<0.001)、miR-125b-2-3p(****p<0.0001)表达下降,miR-150-5p 表达上升(****p<0.0001)。实验结论:35Hz,0.25g的低载荷机械振动可以促进成骨细胞的增殖;低载荷机械振动可以通过上调miR-150-5p,下调miR-182-5p、miR-125b-2-3p的表达激活MAPK信号通路和肌动蛋白调节通路促进成骨细胞的增殖,而未对细胞骨架结构产生明显损害。更多还原

【Abstract】 Background and purpose:With the increasing incidence of fracture in China,in addition to the traditional surgical treatment,we urgently need to explore a new non-invasive treatment.Recent studies have shown that low load mechanical vibration,as a non-drug therapy,has achieved good results in the treatment of osteoporosis and fracture by promoting the proliferation of osteoblasts.When cells are stimulated by mechanical signals.cytoskeleton plays an important role.in which the mitogen-activated protein kinase(MAPK)signaling pathway is closely related to the formation and function of cytoskeleton.The aim of this study was to investigate the effects of cytoskeleton and MAPK signaling pathway on osteoblast proliferation under low load mechanical vibration.Methods:The mouse embryonic osteoblast precursor cells(MC3T3-E1)were divided into two groups:control group and experimental group.The experimental group was given low load mechanical vibration for one week.The loading parameters were vibration frequency of 35 Hz and vibration intensity of 0.25 g.One week later,the proliferation of the cells in the control group and the experimental group was detected by cck-8 kit.Laser confocal microscopy was used to observe whether the cytoskeleton suffered structural damage under low load mechanical vibration.Then,microRNA microarray was used to detect the expression of differentially expressed genes in the control group and the experimental group,and real-time quantitative PCR was used to verify the results of the microarray.Results:After a week of low load mechanical vibration,the cell proliferation activity of the experimental group was enhanced.Laser confocal microscopy showed no significant chances in cytoskeleton under low load mechanical vibration compared with the control group.MiRNA gene chip results showed that there was a significant difference in gene expression between the experimental group and the control group.Mir-182-5p(degree=17),mir-150-5p(degree=13),mir-125b-2-3p(degree=11)and other differentially expressed genes affect the proliferation of osteoblasts by activating mapk signaling pathway and actin regulation pathway.Compared with the control group,the expression of miR-182-5p<0.01),miR-125b-2-3p(****p<0.0001)decreased and the expression of miR-150-5p increased(****p<0.0001)in the experimental group.The results were consistent with the results of gene chip.Conclusion:At 35 Hz,0.25g low load mechanical vibration could promote the proliferation of osteoblasts.Low load mechanical vibration can promote the proliferation of osteoblasts by up-regulating miR-150-5p,down-regulating miR-182-5p,miR-125b-2-3p expression and activating MAPK signaling pathway and actin regulation pathway.There was no obvious damage to cytoskeleton structure.更多还原