持续与间歇低载荷振动对骨折愈合影响的细胞实验研究

发布时间：2018-02-24 23:11

  本文关键词： 骨折愈合 成骨细胞 持续与间歇低载荷振动　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的:随着全球经济的飞速发展,交通事故伤与高处坠落伤等高能量损伤病人逐年增多,此类损伤已成为和平时期创伤发生的最常见原因。在以上损伤中,四肢损伤,特别是四肢骨折的发生率极高,约有5%-10%患者会发生骨折延迟愈合或骨不连,严重影响了骨折的康复进程。因此,怎样加快骨折愈合,有效防治骨折延迟愈合或不愈合已经成为热门课题。近十年来的研究表明,低载荷(频率32-37Hz,加速度0.2-0.3g)振动(low-magnitude high-frequency vibration LMHFV)能够促进骨的生长、对抗骨质疏松、促进骨折愈合。然而绝大多数文献报导的振动对骨及骨细胞的影响均是采用持续的振动加载方式鲜有文献报导研究持续与间歇振动对骨及骨细胞是否会有差别。因此本实验拟通过对体外培养的MC3T3-E1细胞施加持续低载荷振动(sostenuto s LMHFV)与间歇低载荷振动(intermittent LMHFV i LMHFV)两种不同的低载荷振动加载模式,通过细胞层面的研究来探索持续与间歇低载荷振动对骨折愈合是否会产生不同的影响及其作用机理。研究方法:使用振动加载设备,对MC3T3-E1细胞施加振幅35Hz,强度0.25g的低载荷机械振动。分成空白对照组,持续振动0.5h组,振动15min间歇0.5h、1h、3h、6h后再振动15min组。选取振动加载的第1、3、5、7天进行MTT检测,来测定MC3T3-E1细胞的增殖活性。连续三天低载荷振动加载结束后运用ALP试剂盒检测胞内ALP活性,运用Western-blot、RT-q PCR技术来检测低载荷振动刺激对成骨细胞ALP、OCN、COLⅠ、OPG、RANKL等因子的表达水平,运用ELISA试剂盒检测MC3T3-E1细胞成骨细胞分泌RANKL及M-CSF两种破骨细胞刺激因子的情况。实验结果:不论在哪一天检测,持续与间歇低载荷振动组的细胞增殖活性与对照组无明显差别,持续低载荷振动组的细胞增殖活性与间歇低载荷振动组的细胞增殖活性也没有明显差别。所有振动组的MC3T3-E1细胞的ALP、OCN、COLⅠ的表达量及OPG/RANKL比值均明显优于对照组,并且间歇0.5h振动组较持续振动组效果更好。低载荷振动抑制了MC3T3-E1细胞分泌RANKL及M-CSF因子,并且间歇0.5h振动组、间歇1h振动组两组的RANKL及M-CSF的分泌量较持续振动组受到的更加明显的抑制。结论:低载荷振动(35Hz,0.25g)可以促进成骨细胞的分化,并且可抑制成骨细胞分泌破骨细胞刺激因子,进而抑制破骨细胞活性;间隔0.5h的低载荷振动促进成骨细胞分化的效果优于持续振动组;低载荷振动对成骨细胞的增殖无显著影响。
[Abstract]:Background and objective: with the rapid development of the global economy, the number of patients with high-energy injuries, such as traffic accidents and falling injuries, has increased year by year, and such injuries have become the most common cause of peacetime trauma. The incidence of limb injuries, especially limb fractures, is extremely high. About 5- 10 percent of the patients have delayed union or nonunion, which seriously affects the recovery process of fractures. Therefore, how to accelerate fracture healing, The effective prevention and treatment of delayed healing or nonunion of fracture has become a hot topic. Recent studies have shown that low-load vibration (frequency 32-37Hz, acceleration 0.2-0.3 g) can promote bone growth and combat osteoporosis. However, the effect of vibration on bone and bone cells is mostly reported in the literature. It is rarely reported that there is a difference between continuous vibration and intermittent vibration on bone and bone cells. In this study, two different modes of low load vibration loading were proposed for MC3T3-E1 cells: continuous low load vibration (Sostenuto s LMHFV) and intermittent low load vibration LMHFV I (LMHFV). Through cell-level studies to explore whether sustained and intermittent low-load vibration has different effects on fracture healing and its mechanism. The MC3T3-E1 cells were subjected to low load mechanical vibration with amplitude of 35 Hz and intensity of 0.25 g. The cells were divided into blank control group, continuous vibration group for 0.5 h, vibration interval for 15 minutes for 0.5 h and 1 h for 3 h and then 15 min group for 15 min. The MTT was detected on the 5th day of vibration loading. After three days of low load vibration loading, ALP kit was used to detect intracellular ALP activity, Western-blottRT-q PCR technique was used to detect the expression level of factors such as ALP OCNCOL 鈪，

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