次声对骨细胞成骨效应及相关机制的实验研究

发布时间：2018-05-19 03:31

本文选题：次声 + 骨质疏松　；参考：《第四军医大学》2006年博士论文

【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(p0.05),MAR减少(p0.05),骨小梁宽度、条数、小梁百分比减少,小梁间隙增加(p0.05);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(p0.05),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(p0.05),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(p0.05),20Hz组第5日细胞密度增加显著(p0.05);各频率组和对照组的细胞ALP表达均较低,但无显著差异(p㧐0.05);各频率组细胞OCN表达较对照组明显增高(p0.01)。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(p0.05),各次声作用组间差异不显著(p0.05);8h后各次声作用组的mRNA含量下降,和对照组差别不显著(p0.05)。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(p0.05);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(p0.05)。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。
[Abstract]:The infrasonic wave is a mechanical vibration wave in the range of 0.0001 ~ 20Hz. The infrasonic wave has the characteristics of far transmission, decline and strong penetration in the air transmission, and the infrasound has a wide range of nature, living environment, production environment and military environment.
A certain sound pressure level of infrasonic waves can cause human dysfunction and even organic damage for a certain time. Therefore, the study of the occurrence, transmission, biological mechanism of the infrasound, the mechanism of biological action and the formulation of corresponding protective measures have been paid more and more attention. In recent years, some developed countries have invested a lot of manpower and financial resources for research. The biological effects of infrasound and the role of infrasonic weapons as a non lethal new concept weapon in war are also increasing.
At the same time, lower sound level and shorter time may have a good stimulation effect, so the application of low intensity infrasound in medical treatment also attracts attention, such as the study of the use of infrasound therapy for chronic cholecystitis and ametropia. Bone tissue is a part of the corresponding force sensitive tissue, and the infrasonic wave on bone formation and reconstruction No report has been reported before.
In this study, the effect of infrasound on bone formation was observed, and the effects of lower intensity infrasound on the proliferation and differentiation of osteoblast like cells and the effect on mRNA expression of extracellular matrix OPN and ON and cytoskeleton F-actin expression were investigated. The mechanism of sound promoting bone formation is divided into four parts:
The first part is to study the effect of infrasound on improving osteoporosis in male ovariectomized rats.
Objective: To observe the effect of low intensity infrasonic wave on femur bone changes in male ovariectomized rats.
Methods: 32 male rats of 3 month old SD were randomly divided into normal group, castration group, 4Hz infrasound group after castration and post castrated 20Hz infrasound group. The rats were operated on bilateral testicle resection (normal group). The infrasonic pressure compartment system and infrasound detection system were developed by The Fourth Military Medical University and aerospace Industry General Corporation and the acoustics Institute of Chinese Academy of Sciences. The rats in each group were grouped in the infrasonic pressure compartment for infrasound effect, and the first two groups had no infrasound output, and the second acoustic parameters were 4Hz/100dB and 20Hz/100dB, each group was 30min/d, each week was 5 days, and after 12 weeks of action, 11d and 3D were given to 5mg/kg and tetracycline hydrochloride (25mg/kg). The blood 2ml was extracted from the heart and the concentration of Serum Osteocalcin (BGP) was measured by radioimmunoassay after the anesthesia was executed. The bone mineral density (BMD) of the left femur was measured by DPX-IQ bone densitometer, and the paraffin section of the right femur was made to determine the percentage of bone trabecular area (Tb. Ar), bone small Liang Kuandu (Tb Th), bone, and bone. Small Liang Jianxi (Tb. Sp) and small bone Liang Shumu (Tb. N) were taken from the proximal end of the right femur to prepare the bone slices. The double marking of calcein and tetracycline was observed under the fluorescence microscope, and the mineralization rate of the periosteum (MAR) was calculated.
Results: the serum BGP of male castrated rats decreased (P0.05), MAR decreased (P0.05), bone trabecular width, number of strabeculae, trabeculae percentage decreased, and trabecular space increased (P0.05); 4Hz/100dB's infrasound group significantly increased MAR and trabecular width, P0.05, and serum BGP also increased; 20Hz action group only trabecular bone. The width and trabecular number were improved in the castration group (P0.05), while there were no significant changes in the indexes. There was no significant change in BMD in the distal femur of each group.
The second part is the effect of infrasound on the biological characteristics of osteoblast like cells.
Objective: To study the effect of infrasound 100dB on the biological characteristics of osteoblast like cells (MC3T3).
Methods: the osteoblast like cell MC3T3 was inoculated in 4 24 hole culture plates at 1 x 104/ml concentration, divided into 4 groups and placed in the subvocal chamber for 100dB infrasound at different frequencies (4Hz, 12Hz, 20Hz), 30min/d, and no infrasonic output in the control group, and observed the cell proliferation, alkaline phosphatase (ALP) activity and Osteocalcin (OCN) after the infrasound action for 5 days. A change in expression, etc.
Results: the proliferation rate of MC3T3 cells in the infrasound group was increased, the cell density in group 4Hz and 12Hz increased significantly (P0.05) for third days, and the cell density increased significantly on fifth days in 20Hz group (P0.05), and the expression of ALP in each frequency group and control group was lower, but there was no significant difference (P? 0.05), and the expression of OCN in each frequency group was significantly higher than that in the control group (P0.01).
The third part is the expression of osteopontin and osteonectin mRNA in MC3T3 cells after infrasound.
Objective: To study the effects of infrasound at different frequencies on the extracellular matrix osteopontin (OPN) and osteonectin (ON) mRNA of osteoblasts in 100dB.
Methods: the mouse osteoblast like cell line MC3T3 cells were cultured with 4 6 Hole culture plates. The control group, 4Hz/100dB group, 12Hz/100dB group and 20Hz/100dB group were set respectively. Each group performed the infrasonic action of the corresponding parameters, the control group had no infrasound output, 30min/d, 2h, 8h, 2h, 8h, and 8h, respectively after the 3D infrasound. Ectin, ON) mRNA was stained by in situ hybridization, and image analysis was carried out under optical microscope.
Results: the time points of 2H and 4h after each group of sound waves, OPN mRNA and ON mRNA content were significantly higher than those of the control group (P0.05), and there was no significant difference between the various infrasound groups (P0.05), and the mRNA content of each infrasound group decreased after 8h, and there was no significant difference between the control group and the control group (P0.05).
The fourth part: dynamic expression of MC3T3 cytoskeletal protein after infrasound.
Objective: To investigate the changes of cytoskeleton F-actin expression in MC3T3 cells of mouse osteoblast like cells after infrasound of 4Hz/100dB, 12Hz/100dB and 20Hz/100dB.
Methods: the MC3T3 cells were inoculated with third parts of the experiment, which were divided into the control group and the infrasonic exposure group of 4Hz/100dB, 12Hz/100dB and 20Hz/100dB. Each group received infrasound 30min/d, and the control group had no infrasound output. 3D was exposed to 2h, 4h, and 8h at different time after exposure. The immunofluorescence staining of F-actin was performed on the cells, and the laser scanning confocal microscopy was used. The mean fluorescence intensity of F-actin in a single cell was measured by micromirror, and the expression of F-actin was observed.
Results most of the fluorescent substances in the control group were diffused, the fluorescence of the cell membrane was stronger, and a small amount of actin fibers in the cytoplasm were irregular and different in the direction. After the infrasound of different frequencies, 2h could be seen in the cytoplasm of the cytoplasmic microfilament F-actin. Most of the fluorescent substances were long large stress wires and along the cells. The number and fluorescence intensity increased significantly in the longitudinal axis (P0.05), and the cell F-actin in the infrasound exposure group was still higher after the different frequencies of infrasound 4H and 8h. The change trend of F-actin in the cells of different frequency infrasound groups was the same, and there was no significant difference between each group at each time point (P0.05).
To sum up, the results of this topic are as follows:
The effect of the infrasound 30min/d of 1. 4Hz /100dB for 12 weeks (5 days a week) can promote the formation of cancellous bone and increase the mineralization rate of the periosteum, thus improving the degree of osteoporosis caused by the male castration.
2. the culture of MC3T3 osteoblast like cells in vitro found that the action of 100dB infrasound 30min can promote the proliferation and secretion of osteoblast like cells in vitro, and 4Hz promotes the proliferation of cells in vitro, while 20Hz has a significant effect on promoting the maturation and differentiation of cells.
3. 100dB different frequency infrasound daily 30min action can promote the mRNA expression of OPN and ON of osteoblasts in a transient increase, and the expression of mRNA decreases after infrasound effect 8h.
4. 100dB of different frequencies infrasound 30min/d can also induce F-actin expression enhancement, which is not weakened after infrasound 8h.
5. in vitro cell culture experiments showed no clear "frequency window" effect between 4Hz, 12Hz and 20Hz.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

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