【作者】 查丁胜；

【导师】 陈建庭；

【作者基本信息】 南方医科大学 ， 骨外科学， 2011， 博士

【摘要】 研究背景骨质疏松症是中老年人群的常见病,以全身骨量减少,骨组织的微细结构破坏,骨强度降低和骨折危险性增高为特征。骨质疏松症最大的危害是骨质疏松性骨折,其中以髋部骨折危害最大,不仅髋部活动功能损伤明显,而且死亡率高。随着老龄化社会的到来,骨质疏松症防治的临床与基础研究已成为医学界研究的热点。运动是预防和治疗骨质疏松的重要因素。近年来,具有无创、副反应小、依从性好等特点的物理因子疗法防治骨质疏松症逐渐受到重视。全身振动运动是振动疗法中的一种,是指借助于振动仪器,使振动刺激通过下肢或躯干作用于全身,使机体整体振动,以达到防治疾病目的的方法。对去卵巢模型动物、低骨量人群及绝经后女性的研究显示,低于引起组织损伤的机械振动信号---低强度高频率振动(Low-magnitude, high-frequency vibration, LMHFV),且有较好的增加骨形成和抑制骨吸收作用。作为一种治疗骨质疏松症的新方法,LMHFV具有无创、副反应小、高依从性的特点,能降低骨质疏松性骨折的发生率,在骨质疏松症的预防和治疗领域越来越受到重视。但是,LMHFV增加骨形成、抑制骨吸收的具体机制仍不清楚。研究证明,振动应力可以影响成骨细胞的生物学特性,但振动应力影响成骨细胞生物学特性的具体机制尚不清楚。因此,研究振动应力影响成骨细胞生物学特性的分子机制,以其作为切入点进一步阐明LMHFV预防和治疗骨质疏松的机制,将为全身振动运动预防和治疗骨质疏松提供科学依据。本研究通过自行研制的细胞振动仪,对体外培养的成骨前体MC3T3-E1细胞施加低强度(0.3g)、不同高频率(0、30、45、60、90Hz)振动(LMHFV),结合分子生物学检测技术,观察LMHFV对成骨前体MC3T3-E1细胞生物学特性的影响及其机制的初步研究。研究内容如下：目的1.研究低强度高频率振动(LMHFV)对成骨细胞生物学特性的影响。2.研究环氧合酶-2(COX-2)在LMHFV影响成骨细胞生物学特性中的作甩。3。研究LMHFV诱导成骨细胞COX-2蛋白表达的信号转导机制。方法I。低强度、高频率振动(LMHFV)影响成骨细胞生物学特性的作用1. LMHFV影响成骨细胞OPG/RANKL比率的作用(1)LMHFV对成骨细胞OPG/RANKL比率的影响对MC3T3-E1细胞施加频率0、30、45、60、90Hz,强度0.3g,时间30min的振动,分别于振动后0、0.5、1.5、3、6h收集细胞培养上清液。用酶联免疫吸附法(ELISA)检测LMHFV加载后不同时间MC3T3-E1细胞分泌可溶性OPG、RANKL的情况。通过计算OPG/RANKL比率,将增加OPG/RANKL比率最高的振动频率做为本实验继续研究的频率。(2)LMHFV加载成骨细胞条件培养基(Conditioned medium, CM)对破骨细胞分化成熟的影响LMHFV (30Hz)加载MC3T3-E1细胞后含OPG/RANKL比率最高的条件培养基(Conditioned medium, CM30HZ)及LMHFV (OHz)加载后的条件培养基(Conditioned medium, CM0Hz),分别与含40ng/ml sRANKL、20ng/ml M-CSF的1640培养基按体积比1：1混合形成混合培养基。将破骨细胞前体RAW264.7细胞随机分为Control组(CM0Hz)、实验组(CM30Hz),混合培养基分别诱导破骨细胞前体RAW264.7细胞分化5、7天。①实时荧光定量聚合酶链式反应(Real-time quantitative PCR, qPCR)分别检测第5、7天各组抗酒石酸酸性磷酸酶(TRAP) mRNA的表达水平；②TRAP检测试剂盒检测第5、7天各组TRAP活性；③TRAP染色计算第7天各组多核破骨细胞形成数目。2. LMHFV影响成骨细胞分化的作用(1) LMHFV对成骨细胞碱性磷酸酶(ALP)、骨钙素(OCN) mRNA表达的影响：MC3T3-E1细胞随机分为Control组(0Hz)、LMHFV组(30Hz), Control组不予振动刺激,LMHFV组给予振动刺激(0.3g,30Hz,30min/day),分别加载4、8天后,收集细胞,提取细胞总RNA。实时荧光定量聚合酶链式反应(Real-time quantitative PCR, qPCR)检测ALP mRNA、OCN mRNA表达,以GAPDH(?)内对照,分析ALP mRNA、OCN mRNA相对表达的变化。(2) LMHFV对成骨细胞碱性磷酸酶(ALP)活性、骨钙素(OCN)表达的影响：MC3T3-E1细胞随机分为Control组(0Hz)、LMHFV组(30Hz), Control组不予振动刺激,LMHFV组给予振动刺激(0.3g,30Hz,30min/day),分别加载4、8天后,收集细胞,提取细胞总蛋白,ALP检测试剂盒检测ALP活性；OCN ELISA试剂盒检测OCN表达；ALP活性、OCN表达均以细胞总蛋白量标准化,分析ALP活性、OCN表达相对改变的变化。(3) LMHFV对成骨细胞矿化结节形成的影响：MC3T3-E1细胞随机分为Control组(0Hz)、LMHFV组(30Hz), Control组不予振动刺激,LMHFV组给予振动刺激(0.3g,30Hz,30min/day),培养14天后,茜素红染色观察矿化结节形成情况。Ⅱ.环氧合酶(COX-2)在LMHFV影响成骨细胞生物学特性中的作用1. LMHFV对成骨细胞COX-2 mRNA及蛋白表达的影响MC3T3-E1细胞随机分为Control组(OHz). LMHFV组(30Hz), Control组不予振动刺激,LMHFV组给予振动刺激(0.3g,30Hz,30min)。分别于LMHFV干预结束后0、0.5、1.5、3、6h-①提取细胞总RNA, qPCR法检测COX-2 mRNA表达,以GAPDH为内对照,分析COX-2 mRNA相对表达的变化；②提取细胞总蛋白,Western blot检测COX-2蛋白表达,以β-actin为内对照,分析COX-2蛋白相对表达的变化。2. LMHFV对成骨细胞表达PGE2活性的影响MC3T3-E1细胞随机分为Control组(OHz)、LMHFV组(30Hz), Control组不予振动刺激,LMHFV组给予振动刺激(0.3g,30Hz,30min)。分别于LMHFV加载结束后0、0.5、1.5、3、6h收集细胞培养上清、提取细胞总蛋白；在COX-2特异性抑制剂NS-398抑制实验中,MC3T3-E1细胞先与10μM NS-398孵育1h,再施加LMHFV刺激,在LMHFV干预结束后0.5h分别收集细胞培养上清、提取细胞总蛋白；ELISA法检测细胞培养上清液中PGE2活性,并以细胞总蛋白量标准化,分析PGE2活性相对表达的变化。3。COX-2特异性抑制剂NS-398对LMHFV增加成骨细胞OPG分泌的影响MC3T3-E1细胞随机分为Control (OHz)+VEHICLE组、Control (OHz) +NS-398组、LMHFV (30Hz)+VEHICLE组、LMHFV (30Hz)+NS-398组。VEHICLE为溶解NS-398的等量DMSO, MC3T3-E1细胞先与10μMNS-398孵育1h,再施加LMHFV (0.3g,30Hz,30min)刺激,在LMHFV干预结束后6h分别收集细胞培养上清、提取细胞总蛋白；ELISA法检测细胞培养上清液中可溶性OPG活性,并以细胞总蛋白量标准化,分析OPG分泌的变化。4。COX-2特异性抑制剂NS-398对LMHFV诱导成骨细胞分化作用的影响MC3T3-E1细胞随机分为Control (OHz)+VEHICLE组、Control (OHz)+NS-398组、LMHFV (30Hz)+VEHICLE组、LMHFV (30Hz)+NS-398组。干预方法为：对细胞施加8天的LMHFV (0.3g,30Hz,30min/day)刺激,VEHICLE为溶解NS-398的等量DMSO,细胞与10μM NS-398孵育8天,干预结束后,分别收集细胞、提取细胞总蛋白；按前述方法分别检测ALP活性、OCN表达,并以细胞总蛋白量标准化,分析ALP活性、OCN表达相对改变的变化。Ⅲ. LMHFV诱导成骨细胞COX-2表达的信号转导机制1. LMHFV诱导成骨细胞COX-2表达的信号转导途径初探分别于MC3T3-E1细胞施加LMHFV (0.3g,30Hz,30min)刺激前1h加入各种信号通路抑制剂NS-398、Staurosporine、H-89、U0126、SP600125、SB203580,于LMHFV加载结束后3小时,提取细胞总蛋白。Western blot检测COX-2蛋白表达,以β-actin为内对照,分析COX-2蛋白相对表达的变化。2. cAMP/PKA信号通路对LMHFV诱导成骨细胞COX-2表达的影响(1) LMHFV对成骨细胞PKA信号通路的影响MC3T3-E1细胞随机分为Control组(OHz)、LMHFV组(30Hz), Control组不予振动刺激,LMHFV组给予振动刺激(0.3g,30Hz,30min),分别于LMHFV加载结束后0、0.5、1.5、3、6h收集细胞,提取细胞总蛋白。Western blot检测PKA总蛋白及磷酸化蛋白含量,以总蛋白为对照,分析PKA磷酸化蛋白含量相对值的变化。(2) LMHFV对成骨细胞内cAMP水平的影响MC3T3-E1细胞随机分为Control组(0Hz)、LMHFV组(30Hz), Control组不予振动刺激,LMHFV组给予振动刺激(0.3g,30Hz,30min),分别于LMHFV加载结束后0、0.5、1.5、3、6h收集细胞。cAMP ELISA试剂盒检测LMHFV对成骨细胞内cAMP水平的影响。(3) LMHFV加载成骨细胞含PGE2条件培养基(Conditioned medium,CMPEG2)对成骨细胞内cAMP水平的影响：LMHFV加载MC3T3-E1细胞结束后0.5h,分别收集0Hz、30Hz组含PGE2的条件培养基(CMPGE2),随机将MC3T3-E1细胞分为CM0Hz组、CM30Hz组,与相应的CMPGE2分别共孵育0、0.5、1.5、3、6h后收集细胞,cAMP ELISA检测试剂盒检测CMPGE2对成骨细胞内cAMP水平的影响。(4)腺苷酸环化酶激活剂(Forskolin, FSK)对成骨细胞内cAMP水平的影响：MC3T3-E1细胞与15μM FSK分别共孵育0、0.5、1.5、3、6h或分别与0、5、10、15、20μM FSK共孵育3h后,按cAMP ELISA检测试剂盒要求收集细胞,检测含FSK对成骨细胞内cAMP水平的影响。(5)PKA抑制剂对LMHFV、FSK、CMPGE2诱导成骨细胞COX-2蛋白表达的影响在MC3T3-E1细胞接受各种干预因素前加入30μM H-89孵育1h,分别于LMHFV (0.3g、30Hz、30min)加载细胞后3h、15μM FSK刺激细胞后3h、CMPGE2孵育细胞后3h,收集细胞,提取总蛋白。Western blot检测COX-2蛋白表达情况,β-actin为内对照,分析COX-2蛋白相对表达的变化。结果Ⅰ。低强度高频率振动(LMHFV)影响成骨细胞生物学特性的作用1. LMHFV影响成骨细胞OPG/RANKL比率的作用(1) LMHFV对成骨细胞表达OPG/RANKL比率的影响：LMHFV加载结束后6h,与0Hz相比,30Hz LMHFV能明显促进成骨细胞分泌OPG(P<0.001),但对RANKL的分泌无明显影响,30Hz LMHFV提高成骨细胞OPG/RANKL比率最显著(P<0.001)。(2) LMHFV加载成骨细胞条件培养基(Conditioned medium, CM)对破骨细胞分化成熟的影响LMHFV加载结束后6小时,与0Hz相比,30Hz形成的CM含OPG/RANKL比率最高。①与含CM0Hz的混合培养基组相比,在第5天,CM30Hz组TRAP mRNA及活性均较CM0Hz组低,差异无统计学意义(P=0.069,P=0.160)；在第7天,CM30Hz组TRAP mRNA及活性均较CM0Hz组低,差异有统计学意义(P<0.001,P=0.043)；②与含CM0Hz的混合培养基组相比,在第7天,CM30Hz组形成的多核(≥3个核)破骨细胞数量减少,其中10核以上破骨细胞数量减少的差异有统计学意义(P<0.001)。2. LMHFV影响成骨细胞分化的作用(1) LMHFV对成骨细胞碱性磷酸酶(ALP)、骨钙素(OCN) mRNA表达的影响：LMHFV加载MC3T3-E1细胞4天,与0Hz组相比,30Hz组增加MC3T3-E1细胞ALP mRNA水平,其差异有统计学意义(P=0.006); LMHFV加载MC3T3-E1细胞8天,与0Hz组相比,30Hz组增加MC3T3-E1细胞ALP mRNA及OCN mRNA水平,其差异均有统计学意义(P=0.003,P=0.001)。(2) LMHFV对成骨细胞ALP活性、OCN表达的影响：LMHFV加载MC3T3-E1细胞4天,与0Hz组相比,30Hz组增加MC3T3-E1细胞ALP活性,其差异有统计学意义(P=0.031)；LMHFV加载MC3T3-E1细胞8天,与0Hz组相比,30Hz组增加MC3T3-El细胞ALP活性及OCN表达,其差异均有统计学意义(P=0.003,P=0.001)。(3) LMHFV对成骨细胞矿化结节形成的影响：生长至80%-90%汇合的MC3T3-E1细胞,经OHz.30Hz LMHFV加载14天后,茜素红染色结果显示0Hz组、30Hz组LMHFV均可见橘红色钙化结节,但30Hz组钙化结节数目较0Hz组多,颜色较0Hz组深。Ⅱ.COX-2在LMHFV影响成骨细胞生物学特性中的作用1.LMHFV对成骨细胞COX-2 mRNA及蛋白表达的影响(1)与Control组相比,30Hz LMHFV加载成骨细胞后0、0.5、1.5、3、6h,振动组COX-2 mRNA表达均较Control组增加(all P<0.01),其中LMHFV加载后0.5h COX-2 mRNA表达水平最高(P<0.001)；(2)与Control组相比,30Hz LMHFV加载成骨细胞后0.5、1.5、3、6h,振动组COX-2蛋白表达均较Control组增加(all P<0.01),其中LMHFV加载后3h COX-2蛋白表达水平达到高峰(P<0.01)。2. LMHFV对成骨细胞PGE2活性表达的影响与Control组相比,30Hz LMHFV加载成骨细胞后0、0.5、1.5、3、6h,振动组PGE2活性表达均较Control组增加(all P<0.05),其中LMHFV加载后0.5h PGE2活性表达水平最高(P=0.032)。LMHFV加载后0.5h,COX-2特异性抑制剂NS-398对LMHFV诱导MC3T3-E1细胞PGE2活性增加具有明显的抑制作用(P<0.001)。3.COX-2特异性抑制剂对LMHFV增加成骨细胞OPG分泌的影响COX-2特异性抑制剂NS-398对30Hz LMHFV加载成骨细胞后6h OPG的分泌具有抑制作用(P=0.012),NS-398可抑制LMHFV诱导的OPG/RANKL比率升高。4.COX-2特异性抑制剂对LMHFV诱导成骨细胞分化作用的影响对30Hz LMHFV加载成骨细胞8天,COX-2特异性抑制剂NS-398可明显抑制LMHFV诱导的ALP活性、OCN表达增加(P=0.005,P=0.001)。Ⅲ. LMHFV诱导成骨细胞COX-2蛋白表达的信号转导机制1.LMHFV诱导成骨细胞COX-2蛋白表达的信号转导途径初探COX-2特异性抑制剂NS-398、PKA特异性抑制剂H-89、ERK1/2特异性抑制剂U0126、p38.特异性抑制剂SB203580均可抑制LMHFV诱导成骨细胞COX-2蛋白的表达(a11 P<0.05),而PKC特异性抑制剂Staurosporine和JNK特异性抑制剂SP600125对LMHFV诱导成骨细胞COX-2蛋白表达无明显影响。2. cAMP/PKA信号通路对LMHFV诱导成骨细胞COX-2蛋白表达的影响(1) LMHFV对成骨细胞PKA信号通路的影响与Control组相比,30Hz LMHFV加载成骨细胞后0、0.5、1.5、3h,LMHFV组PKA磷酸化活性均较Control组高,其差异均有统计学意义(allP<0.01),其中LMHFV加载后1.5h PKA磷酸化水平最高(P<0.001)。(2) LMHFV对成骨细胞内cAMP水平的影响与Control组相比,30Hz LMHFV加载成骨细胞后0、0.5、1.5h,振动组细胞内cAMP水平均较Control组增加(P<0.001,P=001,P=008),其中LMHFV加载后0.5h cAMP水平最高(P=008)。(3) LMHFV加载成骨细胞含PGE2条件培养基(Conditioned medium, CMPGE2)对成骨细胞内cAMP水平的影响：LMHFV加载成骨细胞后0.5h形成的CM含PGE2水平最高,与Control组相比,CMPGE2孵育细胞0.5、1.5、3h后,CMPGE2组细胞内cAMP水平均较Control组高(all P<0.05),其中在1.5h, CMPGE2组细胞内cAMP水平最高(P<0.05)。(4)腺苷酸环化酶激活剂(Forskolin, FSK)对成骨细胞内cAMP水平的影响：与Control组相比,15μM FSK与成骨细胞孵育0.5、1.5、3h后,FSK组细胞内cAMP水平较对照组明显升高(P=0.003,P<0.001,P<0.001),其中在3h, FSK组细胞内cAMP水平最高。(5)PKA抑制剂对LMHFV、FSK、CMPGE2诱导成骨细胞COX-2蛋白表达的影响与Control组相比,LMHFV, FSK及CMPGE2均能诱导成骨细胞COX-2蛋白表达(P<0.001,P=001,P=008),PKA特异性抑制剂H-89均能抑制LMHFV,FSK及CMPGE2诱导成骨细胞COX-2蛋白的表达(P=001,P=001,P<0.001)。结论1.低强度(0.3g),高频率(30Hz)振动(LMHFV)对成骨前体MC3T3-E1细胞生物学特性具有积极的影响：增加MC3T3-E1细胞OPG分泌,提高OPG/RANKL浓度比；促进MC3T3-E1细胞成骨分化。2.COX-2信号参与了LMHFV对成骨细胞生物学特性的影响。LMHFV诱导MC3T3-E1细胞COX-2蛋白表达可能通过PKA、ERK1/2及p38信号通路；PGE2的自分泌作用在LMHFV通过PGE2/cAMP/PKA信号通路诱导MC3T3-E1细胞COX-2蛋白表达中起重要作用。更多还原

【Abstract】 BackgroudOsteoporosis is a common disease in middle-aged and old people, characterized by inadequate bone mass and microarchitectural deterioration of bone tissue with a compromised bone strength and an increased risk of bone fragility fractures. The major consequence of osteoporosis was osteoporotic fracture, especially the fracture of hip, which had obviously decreased activity and a high mortality, was thought to the most dangerous consequence in osteoporosis. With the arrival of an aging society, the clinical and basic research of osteoporosis prevention and treatment has become a medical research focus.Exercise is an important factor in the prevention and treatment of osteoporosis.In recent years, physical factors characterized by a non-invasive method, less side effects, good compliance and other characteristics, have become an important way in the prevention and treatment of osteoporosis. As a physiotherapy method, whole-body vibration treatment produces a vibratory stimulation of the entire body for patients who stand on a vibrating platform through sinusoidal oscillations in a vertical or rotational mode. Recent studies have reported that low magnitude high frequency vibration (LMHFV) could increase bone formation and inhibit bone resorption in animal models and young adults and children with low bone mineral density or physical impairments, promote neuromuscular adaptation and increase muscle strength. As a new method for treating osteoporosis, whole body vibration exercise characterized by a non-invasive method, less side effects, good compliance, can effectively reduce the incidence of osteoporotic fractures, whole body vibration treatment increasingly gets more attention in the field of osteoporosis prevention and treatment.However, the mechanism of whole body vibration increases bone formation and inhibits bone resorption remains unclear.Studies have shown that vibration stress can affect the biological characteristics of osteoblasts, but the specific mechanisms of the vibration stress on the biological characteristics of osteoblasts remain unclear. Therefore, regarding the identification of the molecular mechanisms of vibration stress on the biological characteristics of osteoblasts as an entry point to further clarify the mechanism of whole body vibration in preventing and treating osteoporosis, which will provide a scientific basis for whole body vibration to prevent and cure osteoporosis.In the present study, MC3T3-E1 osteoblastic cells were subjected to low-magnitude (0.3g), various high-frequency (0,30,45,60,90 Hz) vibration (LMHFV) by using cell vibration stress loading system developed by ourselves and observed the effects of LMHFV on biological characteristics of osteoblasts in vitro. In addition, cyclooxygenase-2 protein synthesis and signal transduction pathways that mediated cellular responses to LMHFV were investigated using specific inhibitors and agonists. The research works are as follows:Objectives1. To explore the effects of low magnitude high frequency vibration (LMHFV) on the biological characteristics of osteoblasts in vitro.2. To investigate the roles played by cyclooxygenase-2 in effects of LMHFV on biological characteristics of osteoblasts in vitro.3. To investigate the preliminary mechanisms of LMHFV on expression of cyclooxygenase-2 protein in osteoblasts.MethodsI. Effects of low magnitude high frequency vibration (LMHFV) on the biological characteristics of osteoblasts in vitro.1. To investigate the effects of LMHFV on ratio of OPG/RANKL in MC3T3-E1 osteoblastic cells in vitro.(1) OPG/RANKL ratio assay:MC3T3-E1 cells were subjected to 0.3 g, and different high frequencies (0,30,45,60,90 Hz) LMHFV by using cell vibration stress loading system for 30 min, then soluble OPG, soluble RANKL levels in the cell medium collected at 0,0.5,1.5,3,6h post-LMHFV, respectively, were detected by ELISA Kit. By calculating the OPG/RANKL ratio, the frequency which showed a largest increase in OPG/RANKL ratio was selected as the one to used at subsequent experiments.(2) Effect of conditioned medium from the vibrated MC3T3-E1 cells on the regulation of osteoclasts:30Hz LMHFV-conditioned medium (CM) and OHz LMHFV-conditioned medium (CM) in MC3T3-E1 cells were collected at 6h post-LMHFV, respectively, then the CM (30Hz or OHz) was mixed (1:1,V/V) with the 1640 medium which contained 40ng/ml sRANKL,20ng/ml M-CSF.RAW264.7 cells were induced by 1640 medium which has been mixed with CM(30Hz) or CM(OHz) for 5,7 days,respectively. TRAP mRNA level of RAW264.7 cells in CM (30Hz) or CM (OHz) groups was analyzed by real-time quantitative PCR (qPCR) at 5 and 7 days, respectively. Samely, TRAP activity of RAW264.7 cells in CM (30Hz) or CM (OHz) groups was analyzed by TRAP Detection Kit at 5 and 7 days, respectively. At day 7, the RAW264.7 cells were stained by TRAP Staining Kit, the number of TRAP-positive multinuclear RAW-OCs in CM (30Hz) or CM (OHz) groups was recorded and analysed, respectively.2. To investigate the effect of LMHFV on osteogenic differentiation in MC3T3-E1 osteoblastic cells in vitro.(1) Effects of LMHFV on expression of alkaline phosphatase (ALP) mRNA and osteocalcin (OCN) mRNA in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were divided into two groups at random:Control group and LMHFV group, Control group was loaded with OHz,0.3g,30min/day vibration stress, LMHFV group was loaded with 30Hz,0,3g,30min/day vibration stress.Two groups were vibrated for 4,8 days, respectively. Cells were collected and total RNA was extracted using TaKaRa Kit according to the manufacturer’s instruction. The mRNA expression of ALP, OCN were analyzed by qPCR. Relative expression of ALP,OCN were calculated using GAPDH mRNA expression as an internal control.(2) Effects of LMHFV on ALP activity and OCN expression in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were divide into two groups at random:Control group and LMHFV group, Control group was loaded with OHz,0.3g,30min/day vibration stress, LMHFV group was loaded with 30Hz,0,3g,30min/day vibration stress.Two groups were vibrated for 4,8 days, respectively. Cells were collected and their total protein was then prepared with RIPA lysis buffer. ALP commercial reagents and osteocalcin ELISA Kit were used to study the effects of LMHFV (OHz or 30Hz) on ALP activity and expression of OCN, respectively.The results of ALP activity, expression of OCN were normalised using the amount of total protein.(3) Effect of LMHFV on the mineralized nodule formation in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were divide into two groups at random:Control group and LMHFV group, Control group was loaded with OHz,0.3g,30min/day vibration stress, LMHFV group was loaded with 30Hz,0,3g,30min/day vibration stress.Two groups were vibrated for 14 days, respectively. Mineralized nodule formation was examined by the alizarin red staining.Ⅱ. To investigate the roles played by cyclooxygenase-2 (COX-2) in effects of LMHFV on biological characteristics of osteoblasts in vitro.1. Effect of LMHFV on expression of COX-2 mRNA, COX-2 protein in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were subjected to 0.3g,0 or 30 Hz LMHFV by using cell vibration stress loading system for 30 min.(1) Cells were collected and total RNA was extracted using TaKaRa Kit according to the manufacturer’s instruction at 0,0.5,1.5,3,6h post-LMHFV, respectively. The mRNA expression of COX-2 was analyzed by qPCR. Relative expression of COX-2 mRNA were calculated using GAPDH mRNA expression as an internal control.(2) Cells were collected and their total protein was then prepared with RIPA lysis buffer at 0,0.5,1.5,3,6h post-LMHFV, respectively. The expression of COX-2 protein was analyzed by Western blot.2. Effect of LMHFV on prostaglandin E2 (PGE2) activity in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were subjected to 0.3g,0 or 30 Hz LMHFV by using cell vibration stress loading system for 30 min, then cell medium, cells were collected and their total protein was then prepared with RIPA lysis buffer at 0,0.5,1.5,3,6h post-LMHFV, respectively. Inhibition experiments were performed by 1h pretreatment with COX-2 inhibitor NS-398 before LMHFV loading. The PGE2 activity in cell medium was analyzed by PGE2 ELISA Kit, and the results of PGE2 activity were normalised using the amount of total protein.3. Effect of COX-2 inhibitor NS-398 on LMHFV-induced OPG secretion in MC3T3-E1 osteoblastic cells in vitro.MC3T3-E1 cells were subjected to 0.3g,0 or 30 Hz LMHFV by using cell vibration stress loading system for 30 min, then cell medium, cells were collected and their total protein was then prepared with RIPA lysis buffer at 6h post-LMHFV, respectively. Inhibition experiments were performed by 1h pretreatment with COX-2 inhibitor NS-398 before LMHFV loading. The OPG secretion in cell medium was analyzed by OPG ELISA Kit, and the results of OPG secretion were normalised using the amount of total protein.4. Effect of COX-2 inhibitor NS-398 on LMHFV-induced ALP activity and OCN expression in MC3T3-E1 osteoblastic cells in vitro.The MC3T3-E1 cells were exposed to 0.3g,0 or 30 Hz LMHFV for 8 days with or without COX-2 inhibitor NS-398, respectively. ALP commercial reagents and osteocalcin ELISA Kit were used to meastred ALP activity and expression of OCN, respectively.The results of ALP activity, expression of OCN were normalised using the amount of total protein.Ⅲ. To investigate the preliminary mechanisms of LMHFV on expression of cyclooxygenase-2 protein in osteoblasts. The expression of COX-2 protein was analyzed by Western blot.1. Preliminary investigation of signal transduction pathway that mediated LMHFV-induced expression of COX-2 protein in MC3T3-E1 cells.Inhibitors including NS-398,Staurosporine,H-89,U0126,SP600125 and SB203580 were used.After the MC3T3-E1 cells had been pre-incubated in the presence of each inhibitor for 60 min to permit these compounds to penetrate the cells and block their respective pathways, LMHFV at 0.3g, OHz or 30Hz was applied in culture for 30-min, and then cells were collected and their total protein was then prepared with RIPA lysis buffer at 6h post-LMHFV. The expression of COX-2 protein was analyzed by Western blot.2. Effect of cAMP/PKA signal pathway on LMHFV-induced expression of COX-2 protein in MC3T3-El cells.(1) Effect of LMHFV on the phosphorylation of PKA in MC3T3-E1 cells.MC3T3-E1 cells were subjected to 0.3g, OHz or 30 Hz LMHFV by using cell vibration stress loading system for 30 min, then cells were collected and their total protein was then prepared with RIPA lysis buffer at 0,0.5,1.5,3,6h post-LMHFV, respectively. The total protein and phosphorylated protein of PKA were analyzed by Western blot.(2) Effect of LMHFV on intracellular cAMP level in MC3T3-E1 cells.MC3T3-E1 cells were subjected to 0.3g, OHz or 30 Hz LMHFV by using cell vibration stress loading system for 30 min, then cells were collected at 0,0.5,1.5,3, 6h post-LMHFV, respectively and cAMP ELISA Kit was used to measure intracellular cAMP level in MC3T3-E1 cells according to the manufacturer’s instruction.(3) Effect of LMHFV-conditioned medium (LMHFV-CM) on intracellular cAMP level in MC3T3-El cells.MC3T3-E1 cells were subjected to LMHFV-conditioned medium (LMHFV-CM) for 0,0.5,1.5,3 and 6h, CM from the 30Hz LMHFV loaded cells was regarded as Experiment group and CM from the OHz LMHFV loaded cells was regarded as Control group.Cells were collected at various time point, respectively and cAMP ELISA Kit was used to measure intracellular cAMP level in MC3T3-E1 cells according to the manufacturer’s instruction.(4) Effect of adenylate cyclase activator forskolin (FSK) on intracellular cAMP level in MC3T3-E1 cells.The MC3T3-E1 cells were exposed to 15μM FSK for 0,0.5,1.5,3,6h or 0,5, 10,15,20μM FSK for 3h, respectively. Cells were collected at various time point, respectively and cAMP ELISA Kit was used to measure intracellular cAMP level in MC3T3-E1 cells according to the manufacturer’s instruction.(5) Effect of PKA inhibitor H-89 on the stimulation of COX-2 protein in MC3T3-E1 cells by the LMHFV, FSK, LMHFV-conditioned medium (LMHFV-CM).After the MC3T3-E1 cells had been pre-incubated in the presence of 30μM H-89 for 60 min to permit H-89 to penetrate the cells and block its respective pathway, MC3T3-E1 cells were exposed to LMHFV for 30min and followed 3h post-LMHFV incubation, as well as exposed to 15μM FSK and LMHFV-CM for 3h, respectively. Cells were collected and their total protein was then prepared with RIPA lysis buffer at the corresponding detection time. The expression of COX-2 protein was analyzed by Western blot.ResultsI. Effects of low magnitude high frequency vibration (LMHFV) on the biological characteristics of osteoblasts in vitro.1. To investigate the effects of LMHFV on ratio of OPG/RANKL in MC3T3-E1 osteoblastic cells in vitro.(1) OPG/RANKL ratio assay:Compared to OHz-LMHFV loading, the secretion of OPG in MC3T3-E1 cells significantly increased at 6h after 30Hz-LMHFV loading (P<0.001), however, the secretion of RANKL in MC3T3-E1 cells showed little change at 6h after 30Hz-LMHFV loading.Correspondingly, the ratio of OPG/RANKL in MC3T3-E1 cells was highest at 6h after 30Hz-LMHFV loading (P<0.001).(2) Effect of conditioned medium from the vibrated MC3T3-E1 cells on the regulation of osteoclasts: ①After 5 days in culture, compared to 0Hz-LMHFV group; 30Hz-LMHFV group showed an insignificant increase in TRAP mRNA and TRAP activity, respectively (P=0.069, P=0.160).②After 7 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed an significant increase in TRAP mRNA and TRAP activity, respectively (P<0.001,P=0.043).③After 7 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed a light but in the number of RAW-OCs containing three to nine nuclei per cell, however, the population of RAW-OCs containing 10 or greater number of nuclei was found to be significantly lower in the cultures containing CM from the 30Hz-LMHFV loaded MC3T3-E1 cells (P<0.001).2. To investigate the effect of LMHFV on osteogenic differentiation in MC3T3-E1 osteoblastic cells in vitro.(1) Effects of LMHFV on expression of alkaline phosphatase (ALP) mRNA and osteocalcin (OCN) mRNA in MC3T3-E1 osteoblastic cells in vitro.After 4 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed a significant increase in ALP mRNA level (P=0.006). After 8 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed a significant increase in ALP mRNA level and OCN mRNA level, respectively (P=0.003, P=0.001)。(2) Effects of LMHFV on ALP activity and OCN expression in MC3T3-E1 osteoblastic cells in vitro.After 4 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed a significant increase in ALP activity (P=0.031). After 8 days in culture, compared to 0Hz-LMHFV group,30Hz-LMHFV group showed a significant increase in ALP activity and OCN expression, respectively (P=0.003, P=0.001)。(3) Effect of LMHFV on the mineralized nodule formation in MC3T3-E1 osteoblastic cells in vitro.After 14 days in culture, alizarin red staining showed that the mineralized nodule formation could be seen in OHz-LMHFV group and 30Hz-LMHFV group, however 30Hz-LMHFV group showed greater number and deeper color.Ⅱ. To investigate the roles played by cyclooxygenase-2 (COX-2) in effects of LMHFV on biological characteristics of osteoblasts in vitro.1. Effect of LMHFV on expression of COX-2 mRNA, COX-2 protein in MC3T3-E1 osteoblastic cells in vitro.(1) COX-2 mRNA level assay:Compared to OHz-LMHFV group, qPCR showed that 30Hz-LMHFV stimulation resulted in greater COX-2 mRNA level at 0,0.5,1.5, 3,6h post-LMHFV (allP<0.01), especially at 0.5 h post-LMHFV, 30Hz-LMHFV stimulation showed greatest COX-2 mRNA level (P<0.001).(2) COX-2 protein expression assay:Compared to OHz-LMHFV group, Western blot showed that 30Hz-LMHFV stimulation resulted in greater COX-2 protein expression at 0.5,1.5,3,6h post-LMHFV (allP<0.01), especially at 3 h post-LMHFV,30Hz-LMHFV stimulation showed greatest COX-2 protein expression (P<0.001).2. Effect of LMHFV on prostaglandin E2 (PGE2) activity in MC3T3-E1 osteoblastic cells in vitro.Compared to OHz-LMHFV group,30Hz-LMHFV stimulation resulted in greater PGE2 activity at0,0.5,1.5,3,6h post-LMHFV (all P<0.05), especially at 0.5h post-LMHFV,30Hz-LMHFV stimulation showed greatest PGE2 activity (P=0.032).Pretreatment of COX-2 inhibitor NS-398 could inhibit PGE2 activity induced by LMHFV at 3h post-LMHFV(P<0.001).3. Effect of COX-2 inhibitor NS-398 on LMHFV-induced OPG secretion in MC3T3-E1 osteoblastic cells in vitro. Pretreatment of COX-2 inhibitor NS-398 could inhibit the OPG secretion induced by LMHFV at 6h post-LMHFV(t=4.396,P=0.012) and NS-398 showed an inhibition effect on the increase of LMHFV-induced OPG secretion.4. Effect of COX-2 inhibitor NS-398 on LMHFV-induced ALP activity and OCN expression in MC3T3-E1 osteoblastic cells in vitro.After 8 days in culture, COX-2 inhibitor NS-398 could inhibit the increasing ALP activity and OCN expression induced by LMHFV, respectively (t=5.478,P=0.005;t=8.060,P=0.001)。Ⅲ. To investigate the preliminary mechanisms of LMHFV on expression of cyclooxygenase-2 protein in osteoblasts. The expression of COX-2 protein was analyzed by Western blot.1. Preliminary investigation of signal transduction pathway that mediated LMHFV-induced expression of COX-2 protein in MC3T3-E1 cells.The induction of COX-2 protein expression by 30Hz-LMHFV load was inhibited by COX-2 inhibitor NS-398, PKA inhibitor H-89, ERK1/2 inhibitor U0126, p38 inhibitor SB203580, respectively (all P<0.05).2. Effect of cAMP/PKA signal pathway on LMHFV-induced expression of COX-2 protein in MC3T3-E1 cells.(1) Effect of LMHFV on the phosphorylation of PKA in MC3T3-E1 cells.Compared to OHz-LMHFV group, Western blot showed that 30Hz-LMHFV stimulation resulted in increasing phosphorylation level of PKA at 0,0.5,1.5,3h post-LMHFV (all P<0.01), especially at 1.5 h post-LMHFV,30Hz-LMHFV stimulation showed greatest phosphorylation level of PKA (P< 0.001).(2) Effect of LMHFV on intracellular cAMP level in MC3T3-E1 cells.Compared to OHz-LMHFV group,30Hz-LMHFV stimulation resulted in greater intracellular cAMP level at 0,0.5,1.5h post-LMHFV (t=-10.85, P<0.001; t=-9.637,P=001;t=-4.941,P=008), especially at 0.5 h post-LMHFV, 30Hz-LMHFV stimulation showed greatest intracellular cAMP level (t=-4.941,P=008).(3) Effect of LMHFV-conditioned medium (LMHFV-CM) on intracellular cAMP level in MC3T3-E1 cells.Compared to 0Hz-LMHFV-CM group,30Hz-LMHFV-CM stimulation resulted in greater intracellular cAMP level at 0.5,1.5,3h (all P<0.05), especially at 1.5 h, 30Hz-LMHFV stimulation showed greatest intracellular cAMP level (P<0.05).(4) Effect of adenylate cyclase activator forskolin (FSK) on intracellular cAMP level in MC3T3-E1 cells.Compared to 0μM FSK,15μM FSK stimulation resulted in greater intracellular cAMP level at 0.5,1.5,3h (t=-6.175,P=0.003;t=-14.877, P<0.001;t=-20.895, P<0.001), especially at 3 h,15μM FSK stimulation showed greatest intracellular cAMP level.(5) Effect of PKA inhibitor H-89 on the stimulation of COX-2 protein in MC3T3-E1 cells by the LMHFV, FSK, LMHFV-conditioned medium (LMHFV-CM).Compared to the blank control group, Western blot analyses showed that the phosphorylation of PKA could be induced by 30Hz-LMHFV load,15μM FSK and 30Hz-LMHFV-CM, respectively (t=-13.294,P=000;t=-13.382,P=001; t=-13.275,P=008). Pretreatment of PKA inhibitor H-89 could inhibit the phosphorylation of PKA induced by 30Hz-LMHFV load,15μM FSK and 30Hz-LMHFV-CM, respectively (t=9.026,P=001;t=8.518,P=001; t=14.216,P=000).Conclusions1. Low-magnitude (0.3g), high-frequency (30Hz) vibration (LMHFV) showed positive influences on the biological characteristics of MC3T3-E1 cells:increasing the secretion of OPG and raising the concentration ratio of OPG/RANKL; promoting osteogenic differentiation in MC3T3-E1 cells.2. COX-2 signal involved in the effects of LMHFV on the biological characteristics of MC3T3-E1 cells. PKA signal, ERK1/2 signal and p38 signal involved in the induction of COX-2 protein by LMHFV in MC3T3-E1 cells.The autocrine effect of PGE2 production in MC3T3-E1 cells played an important role on the effect of LMHFV induce COX-2 protein expression in MC3T3-E1 cells through PGE2/cAMP/PKA signal pathways.更多还原