【作者】 王斌；

【导师】 陈景藻； 郭国祯； 郭鹞； 任东青；

【作者基本信息】 第四军医大学 ， 军事预防医学， 2006， 博士

【摘要】 次声波是频率在0.0001～20Hz范围内的机械振动波。次声波在空气传播中具有传播远、衰减少,穿透力强等物理学特性,次声广泛存在自然界、生活环境、生产环境和军事环境中。一定声压级水平的次声波作用一定时间可致人体功能障碍,甚至器质性损伤。因此,研究次声的发生、传播、生物学作用机制及制订相应的防护措施已越来越受到人们的重视。特别是近年来,一些发达国家投入大量的人力和财力,用于研究次声的生物学效应,并重视作为非致死性新概念武器——次声武器在战争中的作用研究,相关的文献报道也逐年增多。同时,较低声压级水平和较短时间作用可能会具有良好的刺激效应,因此低强度的次声在医疗上的应用也引起了关注,例如研究应用次声治疗慢性胆囊炎、屈光不正等。骨组织作为对应力敏感的组织部分,次声波对骨生成重建的研究目前尚未见报告。本课题观察了较低作用剂量的次声对骨生成的促进作用,并进一步采取体外培养的方式,研究观察较低强度的次声对成骨样细胞增殖分化的影响以及对细胞外基质OPN和ON的mRNA表达和细胞骨架F-actin表达的影响,探讨较低作用剂量的次声促进骨生成的机理。课题共分四部分实验:第一部分次声改善雄性去势大鼠骨质疏松的试验研究目的:观察较低强度的次声波作用于雄性去势大鼠后,其股骨骨质变化的情况。方法:32只3月龄SD雄性大鼠,随机分为正常组、去势组、去势后4Hz次声作用组和去势后20Hz次声作用组,大鼠行双侧睾丸切除术(正常组行假手术)。次声压力舱系统及次声检测系统由第四军医大学与航天工业总公司及中科院声学所协作研制。各组大鼠术后分组置于次声压力舱内进行次声作用,其中前两组无次声输出;后两组次声参数分别为4Hz/100dB和20Hz/100dB;每组30min/d,每周作用5日,连续作用12周后处死。在处死大鼠前的第11d和第3d给与钙黄绿素(5mg/kg)和盐酸四环素(25mg/kg)皮下注射,进行活体荧光标记;麻醉处死后从心脏抽取血液2ml,放射免疫法测定血清骨钙素(BGP)浓度;用DPX-IQ型骨密度仪测定左股骨远端骨密度(BMD);取右侧股骨远端制作脱钙石蜡切片,测定骨小梁面积百分比(Tb·Ar)、骨小梁宽度(Tb·Th)、骨小梁间隙(Tb·Sp)以及骨小梁数目(Tb·N);取右股骨近端制备硬骨切片,荧光显微镜下观察钙黄绿素和四环素标记的双标线,计算骨膜矿化沉积率(MAR)。结果:雄性去势大鼠的血清BGP较正常组降低(p<0.05),MAR减少(p<0.05),骨小梁宽度、条数、小梁百分比减少,小梁间隙增加(p<0.05);4Hz/100dB的次声作用组,显著增加了MAR和小梁宽度、条数等形态学指标(p<0.05),血清BGP也有增加趋势;20Hz作用组仅骨小梁宽度和骨小梁数目较去势组改善(p<0.05),余各项指标未有显著变化;各组股骨远端BMD无显著变化。第二部分次声对成骨样细胞生物学特性的影响目的:研究了100dB次声波对成骨样细胞MC3T3生物学特性的影响。方法:成骨样细胞MC3T3按1×104/ml浓度接种在4个24孔培养板中,分为4组,置于次声压立舱中,分别以不同频率(4Hz、12Hz、20Hz)对细胞进行100dB次声作用,30min/d,对照组无次声输出。观察次声作用5日后细胞增殖、碱性磷酸酶(ALP)活性和骨钙素(OCN)的表达等方面的变化。结果:发现次声作用组MC3T3细胞增殖速度增加,4Hz组和12Hz组细胞密度第3日起增加显著(p<0.05),20Hz组第5日细胞密度增加显著(p<0.05);各频率组和对照组的细胞ALP表达均较低,但无显著差异(p﹥0.05);各频率组细胞OCN表达较对照组明显增高(p<0.01)。第三部分次声作用后MC3T3细胞骨桥素和骨粘连蛋白mRNA表达目的:研究100dB不同频率次声对成骨细胞细胞外基质骨桥素(osteopontin,OPN)和骨粘连蛋白(osteonectin,ON)mRNA的影响。方法:采用4个6孔培养板进行小鼠成骨样细胞系MC3T3细胞的培养,分别设为对照组、4Hz/100dB组、12Hz/100dB组、20Hz/100dB组。各组进行相应参数的次声波作用,对照组无次声输出,30min/d,在第3d次声作用后2h、8h分别进行骨桥素(OPN)及骨粘连蛋白(osteonectin,ON)mRNA原位杂交染色,光学显微镜下进行图像分析,测定细胞阳性物质平均灰度,进行分析。结果:各组次声波作用后2h和4h的时间点,OPN mRNA、ON mRNA含量均较对照组显著升高(p<0.05),各次声作用组间差异不显著(p>0.05);8h后各次声作用组的mRNA含量下降,和对照组差别不显著(p>0.05)。第四部分:次声作用后MC3T3细胞骨架蛋白的动态表达目的:研究4Hz/100dB、12Hz/100dB、20Hz/100dB的次声作用后,小鼠成骨样细胞细胞MC3T3的细胞骨架F-actin表达的改变。方法:将MC3T3细胞接种爬片同第三部分实验,分为对照组和4Hz/100dB、12Hz/100dB、20Hz/100dB的次声暴露组。各组分别接受次声作用30min/d,对照组无次声输出。第3d于暴露后2h、4h、8h不同时间,对细胞进行F-actin的免疫荧光染色,应用激光扫描共聚焦显微镜,测定单个细胞F-actin的平均荧光强度,观察细胞F-actin的表达改变。结果对照组细胞大部分荧光物质呈弥漫状态,胞膜荧光较强,胞浆内少量肌动蛋白纤维丝,方向不规则,长短不一;不同频率次声作用后2h,各次声作用组均可看到胞浆中微丝F-actin明显粗大纤长,荧光物质大多为较长的粗大应力丝,沿细胞纵轴排列较多,数量及荧光强度明显增加(p<0.05);在不同频率次声作用后4h和8h,次声暴露组的细胞F-actin仍处于较高表达状态。不同频率次声作用组细胞的F-actin变化趋势较一致,各组在各时间点未见明显差异(p>0.05)。综上所述,本课题结果提示:1. 4Hz /100dB的次声30min/d共进行12周(每周5日)的作用,可以促进松质骨的生成,增加骨膜矿化沉降率,从而改善了雄性去势所造成的骨质疏松程度。2.体外MC3T3成骨样细胞培养方式发现:100dB次声波30min作用可以促进成骨样细胞的体外增殖和分泌功能,4Hz对细胞体外增殖促进较明显,而20Hz对促进细胞成熟分化作用较明显。3. 100dB的不同频率次声每日30min作用能促进成骨细胞的OPN及ON的mRNA表达一过性增高,次声作用8h后mRNA的表达减弱。4. 100dB的不同频率次声作用30min/d,还可诱导F-actin表达的增强,这种增强改变在次声作用8h后仍未见减弱。5.体外细胞培养实验在4Hz、12Hz、20Hz间未观察到明确的“频率窗”效应。更多还原

【Abstract】 As the audible sound and ultrasound, infrasound is a mechanical vibration wave with frequency in range of 0.0001 to 20Hz. With the physical characteristics of less attenuation in long distance spreading and strong penetration, infrasound is harmful to human and leads to body dysfunction or a even severe damage when the intensity comes to a enough certain degree. So people are becoming increasingly aware of the importance in studying the mechanisms of the occurrence, spreading, biological effects and preventive measures of the infrasound.Infrasound is existed widely in nature. It can be detected in our living condition, productive and military environments. In recent years many developed counties throw into a great amount of manpower and money in studying biological effects of infrasound, also they are gradually paying much attention to infrasound doing as a new concept weapon. Articles and reports about the infrasound are being increased year by year.However, the low density infrasound maybe has good effects in medical just like other physiology agents. Some reports founded the low density infrasound can be used in treatment for ametropia, keratitis, anorexia. Being a mechanical wave, infrasound as an elastic spring forces produces a mechanical stress which something likes a pull and press alternately forces. As we kone, bone is very sensitive to strain, it can gain modeling when in the adapted strain windows, but no article about the low density infrasound on bone can be seen in present.In the thesis, we observed the effects of bone modeling by low density infrasound on osteoporosis rats, and then to investigate the cellular proliferation , ALP activity , osteocalin (OCN) synthesis, extracelllular matrix(OPN/ ON ) mRNA level and cytoskeletal F actin expresion in vitro, to explore the infrasound signal transduction pathway into intracellular caused the bio-effects.This thesis is composed of four parts as follow: Part one: Effect of infrasound for the prevention of osteoporosis in orchiectomized SD ratsObjective:To determine the preventive effects of lower density infrasound pressure on bone loss in orchiectomied rats. Method: divided 32 SD male rats into the control group, osteoporosis group, 4Hz infrasound group and 20Hz infrasound group. The rats in last 3 groups had been orchiectomied. Every group was expoused under infrasound of 4, and 20Hz individually 100dB output 30min each day while the control group and the osteoporosis group no infrasound output. After 12 weeks ( every group expoused 5d per week ), we observed the change of serum osteocalcin (OCN), bone mineral density (BMD), mineralization rate (MAR) and hisomorphometric measurement and calculations of distal femur with imaging analysis process.Result: The osteoporosis group was obversly decresed in MAR and hisomorphometric measurement and calculations of distal femur than the control group (p<0.05 ). There was significantly increased in MAR and trabecular area, trabecular thickness, trabecular number in 4 Hz groups vs the osteoporosis group (p<0.05 ). In 4Hz group the serum osteocalcin also increased but no significant difference with the osteoporosis group. But in 20Hz group there were only increased in trabecular thickness, trabecular number ( p<0.05 ), the other measurement were no any differences with the osteoporosis group (p>0.05 ). There were no significant chang in BMD among the osteoprosis group and infrasound groups (p>0.05 ).Part two: Effect on biological characteristics of Osteoblast-like cells exposed by infrasound in vitroObjective:To determine the effects of infrasound pressure on biological characteristics of ostoblast-like cells,MC3T3-E1,in vitro.Method: After primary culture, and devided into 4 groups, the osteoblast-like cells, MC3T3-E1,were exposed by infrasound with intensity output of 100dB and frequency of 4, 12 and 20Hz individually for 30min each day. The cellular proliferation was detected by cell counting method. Biochemical analysis and radio-immunological method were used to measure alkaline phosphatease activity (ALP) and osteocalin (OCN) synthesis after 5 days.Result: The cell proliferation in experimental groups showed obviously faster than that in control group. The cell concentration in 4 and 12Hz groups was significantly increased from the 3rd day than in control group(p<0.05). Anyway, the same tendency could be observed in 20Hz group till the 5th day (p<0.05). There were not any differences between control group and experimental groups in ALP activity(p>0.05). The secretion of OCN was greatly increased in experimental groups(p<0.01).Part three: Effects of infrasound on osteonectin and osteopontin mRNA level of osteoblast-like cells MC3T3 in vitroObjective: To determine the effects of lower density infrasound pressure on osteopontin(OPN) and osteonectin(ON)mRNA level of osteoblast-like cells MC3T3.Method: After primary culture, the cells were exposed by infrasound with intensity output of 100dB and frequency of 4, 12 and 20Hz individually for 30min each day. After the 3rd day, .Result: Those infrasound exposured groups were significantly increased in the mRNA level of ON and OPN vs. the control group in 2h and 4h (p<0.05). There was no any differences between every groups and the control group in 8h in the mRNA level of OPN and ON (p>0.05).Part four: The effect of low density infrasound on filament F- actin of osteoblast MC3T3Objective:To study the alterations in expression of F-actin of osteoblast MC3T3 exposed by low density infrasound.Methods: After the primary cultured on coverglass , and divided into four groups just like the experiment part three, the cells in experimental groups were exposed by infrasound with intensity output of 100dB and frequency of 4Hz, 12Hz and 20Hz individually for 30min/d. The cells in control group were managed in the same environment as experimental groups with intensity output of 0dB. Laser scanning confocal microscope was used to examine the changes of F-actin after immunofluorescent staining and the photos were taken for further analysis of the cells` average fluorescence by spectrofluorimetric quantification.Results: In control group, most fluorescein-labelled substance was in diffusion states. Less actin filaments were tenuously short without direction. Correspondingly F-actin in the cells of experimental groups was thick long and showed in longitudinal arrangement after the exposure. The intensity of fluorescence was significantly increased. In every experiment group, there was a membrane-like boundary strong fluorescence. . More F-actin expression in experimental groups were being kept obviously increasing in 2h , 4h and 8h than the control group (p<0.05). However, there was not any significantly different in F-actin expression between the experimental groups ( p>0.05). Anyway cells in experimental groups showed a synchronous change.Conclusion:1. It could prevent bone loss in orchiectomied rats by expoused with 4 Hz/100dB infrasound 30min/d. There was significantly increased in MAR and trabecular area, trabecular thickness, trabecular number in 4Hz/100dB groups vs the osteoporosis group.2. It could promote proliferation of the osterblast-like cells by exposured with 4Hz/100dB infrasound for 30min each day in vitro. and promote secretion of the cells by 20Hz/100dB infrasound for 30min/d.3. The infrasound of 4Hz/100dB, 12Hz/100dB and 20Hz/100dB, 30min/d could promote the mRNA level of 0PN and 0N. This effects at last for 4 hours. There were no obviously different among 4Hz, 12Hz and 20Hz.4. F-actin expression can be increased in osteoblast MC3T3 exposed 30min/d with infrasound whioch intensity output of 100dB and frequency of 4Hz, 12Hz, 20Hz . The expression increasing of F-actin still can be founded in 8h after exposed under the low density infrasound.5. No special frequency effects can be founded in those cells-cultured expriments.更多还原