本文選題：次聲 + 骨質(zhì)疏松　； 參考：《第四軍醫(yī)大學》2006年博士論文

【摘要】： 次聲波是頻率在0.0001～20Hz范圍內(nèi)的機械振動波。次聲波在空氣傳播中具有傳播遠、衰減少,穿透力強等物理學特性,次聲廣泛存在自然界、生活環(huán)境、生產(chǎn)環(huán)境和軍事環(huán)境中。 一定聲壓級水平的次聲波作用一定時間可致人體功能障礙,甚至器質(zhì)性損傷。因此,研究次聲的發(fā)生、傳播、生物學作用機制及制訂相應的防護措施已越來越受到人們的重視。特別是近年來,一些發(fā)達國家投入大量的人力和財力,用于研究次聲的生物學效應,并重視作為非致死性新概念武器——次聲武器在戰(zhàn)爭中的作用研究,相關的文獻報道也逐年增多。 同時,較低聲壓級水平和較短時間作用可能會具有良好的刺激效應,因此低強度的次聲在醫(yī)療上的應用也引起了關注,例如研究應用次聲治療慢性膽囊炎、屈光不正等。骨組織作為對應力敏感的組織部分,次聲波對骨生成重建的研究目前尚未見報告。 本課題觀察了較低作用劑量的次聲對骨生成的促進作用,并進一步采取體外培養(yǎng)的方式,研究觀察較低強度的次聲對成骨樣細胞增殖分化的影響以及對細胞外基質(zhì)OPN和ON的mRNA表達和細胞骨架F-actin表達的影響,探討較低作用劑量的次聲促進骨生成的機理。課題共分四部分實驗: 第一部分次聲改善雄性去勢大鼠骨質(zhì)疏松的試驗研究 目的:觀察較低強度的次聲波作用于雄性去勢大鼠后,其股骨骨質(zhì)變化的情況。 方法:32只3月齡SD雄性大鼠,隨機分為正常組、去勢組、去勢后4Hz次聲作用組和去勢后20Hz次聲作用組,大鼠行雙側(cè)睪丸切除術(正常組行假手術)。次聲壓力艙系統(tǒng)及次聲檢測系統(tǒng)由第四軍醫(yī)大學與航天工業(yè)總公司及中科院聲學所協(xié)作研制。各組大鼠術后分組置于次聲壓力艙內(nèi)進行次聲作用,其中前兩組無次聲輸出;后兩組次聲參數(shù)分別為4Hz/100dB和20Hz/100dB;每組30min/d,每周作用5日,連續(xù)作用12周后處死。在處死大鼠前的第11d和第3d給與鈣黃綠素(5mg/kg)和鹽酸四環(huán)素(25mg/kg)皮下注射,進行活體熒光標記;麻醉處死后從心臟抽取血液2ml,放射免疫法測定血清骨鈣素(BGP)濃度;用DPX-IQ型骨密度儀測定左股骨遠端骨密度(BMD);取右側(cè)股骨遠端制作脫鈣石蠟切片,測定骨小梁面積百分比(Tb·Ar)、骨小梁寬度(Tb·Th)、骨小梁間隙(Tb·Sp)以及骨小梁數(shù)目(Tb·N);取右股骨近端制備硬骨切片,熒光顯微鏡下觀察鈣黃綠素和四環(huán)素標記的雙標線,計算骨膜礦化沉積率(MAR)。 結果:雄性去勢大鼠的血清BGP較正常組降低(p0.05),MAR減少(p0.05),骨小梁寬度、條數(shù)、小梁百分比減少,小梁間隙增加(p0.05);4Hz/100dB的次聲作用組,顯著增加了MAR和小梁寬度、條數(shù)等形態(tài)學指標(p0.05),血清BGP也有增加趨勢;20Hz作用組僅骨小梁寬度和骨小梁數(shù)目較去勢組改善(p0.05),余各項指標未有顯著變化;各組股骨遠端BMD無顯著變化。 第二部分次聲對成骨樣細胞生物學特性的影響 目的:研究了100dB次聲波對成骨樣細胞MC3T3生物學特性的影響。 方法:成骨樣細胞MC3T3按1×104/ml濃度接種在4個24孔培養(yǎng)板中,分為4組,置于次聲壓立艙中,分別以不同頻率(4Hz、12Hz、20Hz)對細胞進行100dB次聲作用,30min/d,對照組無次聲輸出。觀察次聲作用5日后細胞增殖、堿性磷酸酶(ALP)活性和骨鈣素(OCN)的表達等方面的變化。 結果:發(fā)現(xiàn)次聲作用組MC3T3細胞增殖速度增加,4Hz組和12Hz組細胞密度第3日起增加顯著(p0.05),20Hz組第5日細胞密度增加顯著(p0.05);各頻率組和對照組的細胞ALP表達均較低,但無顯著差異(p㧐0.05);各頻率組細胞OCN表達較對照組明顯增高(p0.01)。 第三部分次聲作用后MC3T3細胞骨橋素和骨粘連蛋白mRNA表達 目的:研究100dB不同頻率次聲對成骨細胞細胞外基質(zhì)骨橋素(osteopontin,OPN)和骨粘連蛋白(osteonectin,ON)mRNA的影響。 方法:采用4個6孔培養(yǎng)板進行小鼠成骨樣細胞系MC3T3細胞的培養(yǎng),分別設為對照組、4Hz/100dB組、12Hz/100dB組、20Hz/100dB組。各組進行相應參數(shù)的次聲波作用,對照組無次聲輸出,30min/d,在第3d次聲作用后2h、8h分別進行骨橋素(OPN)及骨粘連蛋白(osteonectin,ON)mRNA原位雜交染色,光學顯微鏡下進行圖像分析,測定細胞陽性物質(zhì)平均灰度,進行分析。 結果:各組次聲波作用后2h和4h的時間點,OPN mRNA、ON mRNA含量均較對照組顯著升高(p0.05),各次聲作用組間差異不顯著(p0.05);8h后各次聲作用組的mRNA含量下降,和對照組差別不顯著(p0.05)。 第四部分:次聲作用后MC3T3細胞骨架蛋白的動態(tài)表達 目的:研究4Hz/100dB、12Hz/100dB、20Hz/100dB的次聲作用后,小鼠成骨樣細胞細胞MC3T3的細胞骨架F-actin表達的改變。 方法:將MC3T3細胞接種爬片同第三部分實驗,分為對照組和4Hz/100dB、12Hz/100dB、20Hz/100dB的次聲暴露組。各組分別接受次聲作用30min/d,對照組無次聲輸出。第3d于暴露后2h、4h、8h不同時間,對細胞進行F-actin的免疫熒光染色,應用激光掃描共聚焦顯微鏡,測定單個細胞F-actin的平均熒光強度,觀察細胞F-actin的表達改變。 結果對照組細胞大部分熒光物質(zhì)呈彌漫狀態(tài),胞膜熒光較強,胞漿內(nèi)少量肌動蛋白纖維絲,方向不規(guī)則,長短不一;不同頻率次聲作用后2h,各次聲作用組均可看到胞漿中微絲F-actin明顯粗大纖長,熒光物質(zhì)大多為較長的粗大應力絲,沿細胞縱軸排列較多,數(shù)量及熒光強度明顯增加(p0.05);在不同頻率次聲作用后4h和8h,次聲暴露組的細胞F-actin仍處于較高表達狀態(tài)。不同頻率次聲作用組細胞的F-actin變化趨勢較一致,各組在各時間點未見明顯差異(p0.05)。 綜上所述,本課題結果提示: 1. 4Hz /100dB的次聲30min/d共進行12周(每周5日)的作用,可以促進松質(zhì)骨的生成,增加骨膜礦化沉降率,從而改善了雄性去勢所造成的骨質(zhì)疏松程度。 2.體外MC3T3成骨樣細胞培養(yǎng)方式發(fā)現(xiàn):100dB次聲波30min作用可以促進成骨樣細胞的體外增殖和分泌功能,4Hz對細胞體外增殖促進較明顯,而20Hz對促進細胞成熟分化作用較明顯。 3. 100dB的不同頻率次聲每日30min作用能促進成骨細胞的OPN及ON的mRNA表達一過性增高,次聲作用8h后mRNA的表達減弱。 4. 100dB的不同頻率次聲作用30min/d,還可誘導F-actin表達的增強,這種增強改變在次聲作用8h后仍未見減弱。 5.體外細胞培養(yǎng)實驗在4Hz、12Hz、20Hz間未觀察到明確的“頻率窗”效應。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R363

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