次声对细胞作用的部分生物力学机制实验研究
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摘要
声波除可听声外还包括超声波和次声波。次声波是频率范围在0.0001～20Hz的机械振动波。次声波有传播远、衰减少,穿透力强等物理学特性,一定声压级水平的次声波可致人体功能障碍,甚至器质性损伤,一般的隔声和吸声材料、建筑物、掩蔽所均难以防护。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。
次声广泛存在自然界、生活环境、生产环境和军事环境中。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,并重视作为新概念武器的次声武器在战争中的作用,相关的文献报道也逐年增多。和其它物理因子一样,次声对机体的原发性作用机制一直为人们所关注。一般认为次声作用于机体组织引起生物共振,从而引起生物学效应。次声是一种机械波,作用于机体组织时可产生一种类似于弹性拉压的机械力。目前越来越多的研究表明:机械应力对细胞生长、分化、凋亡以及基因表达等生理过程和某些病理过程可发挥重要的作用。就细胞而言,在生物共振的基础上,对次声机械力的响应,很可能是引起细胞生物学效应的主要原因,但这种机械力如何被细胞感受并传至细胞内,进而使细胞发生一系列生物学效应,目前国内外还未见相关的报道。因
As the audible sound and ultrasound, infrasound is a mechanical vibration wave with frequency in range of 0.0001to 20Hz. With the physical characteristics of less attenuation in long distance spreading and strong penetration, infrasound is harmful to human and leads to body disfunction or a even severe damage when the intensity comes to a certain degree. We can hardly barricade the interference of infrasound with general sound absorbing material, building and shelter. 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. As to other kinds of physical agents, people have showed solicitude to the primary mechanisms of infrasound. Generally, it is considered that infrasound evokes a biological resonance of bio-organs and gives rise to biological effects. On the other hand, being a mechanical wave, infrasound as an elastic spring forces produces a mechanical stress which something likes a pull and press alternately forces. At present, more and more investigations showed that mechanical stress played a very important role in cells' growth, differentiation, apoptosis and gene expression in some physiological and pathological procedures. Concerning to cells, responding to infrasound forces may be the main reason to create biological effects. However, it is not very clear that how does the infrasound signal be taken over and then transferred to intracellular by the cells to bring about a further series of biological effects. Therefore, it is
important to investigate the changes on force transduction pathway of Extracelllular matrix(ECM) —,Integrin— Cytoskeleton(CSK) as well as cellular membrane structure and intracellular Ca2+ in the experiment to explore the infrasound signal transduction pathway to intracellular to establish the bases for the further studies of infrasound bio-effects. This thesis is composed of four parts as follow:Part one: The effect of infrasound on expression of cytoskeleton filament F-actin of ECV-304 cells and integrin (31 antisense oligonucleotide inhibits the expression of F-actinObjective: To identify the expression of cytoskeleton filament in ECV-304 cells after infrasound exposure and to explore the infrasound signal transduction pathway to intracellular.Methods: After the primary culture on coverglass, cells in experimental groups were exposed to infrasound with different parameter output The cells in control group were managed in the same environment as experimental groups with intensity output of OdB. 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. Phosphorothioate modified antisense and plus-sense oligonucleotides of integrinpi were used to interfere with the expression of F-actin of ECV-304 cells exposed to infrasound with intensity output of 130dB and frequency of 16Hz for 2 hours. The same examination was done after infrasound exposure.Results:1. In control group, we could find under laser scanning confocal microscope that most fluorescein-labelled substance in cells was in diffusion states. Less actin filaments were tenuously short without direction. Less long stress fiber passed through the cells. Most cells with a clear margin appeared in circle, fusiform or polygon shape. Fluorescence of cells membrane showed a little bit stronger than in cytoplasm.'2. Correspondingly F-actin in the cells of experimental groups of 16Hz, 90dB or 11 OdB or 13 OdB were thick long and showed in longitudinal arrangement after once infrasound exposure of 2 hours. Small part of F-actin was arranged in across or network in cells. Less fluorescein-labelled substance in cells was in diffusion states. The intensity of fluorescence was significantly increased(p<0.01). In experimental groups, there was a membrane-like boundary much stronger fluorescence of cells than that in control group. More F-actin expression in experimental groups was being kept obviously for at
least 8 hours then it gradually decreased. 24 hours later there was not any significantly different in F-actin expression between the experimental groups and the control group. There was not any difference between experimental groups.3. Compared to control group, the expression of F-actin in cells of different frequency groups of 8Hz and 16Hz was significantly increased(pO.OI). The variance pattern of F-actin expression was just same as above in 2. There was not any significantly difference of fluorescence of the cells between experimental groups. Frequency-dependent was not obvious.4. There were some cytomorphological changes in experimental groups after 3times infrasound exposure of 8Hz 16Hz 130dB respectively. Most cells appeared in circle, oblate forms and showed a poor expansion as if it retracted. Less cells appeared in expanded triangle or polygon shapes. Cellular nucleus with a very clear positive IP-stained boundary seemed to be shrink. F-actin with a radiation or longitude arrangement in cytoplasm showed a much stronger fluorescence than that in control or once exposure groups. The thin stress fibers could hardly be observed. Some filaments appeared almost as lump. Cells membrane was interrupted. More F-actin expression in experimental groups was being kept for 8 to 12 hours then it gradually decreased. The cell responsivity in 3 times infrasound exposure group was higher than once exposure group. Cells could be restored at 24 hours after infrasound exposure.5. In sense oligonucleotides group, cells fluorescence was almost in the same level as in control group. Anyway, a weak fluorescence was observed in cells of antisense oligonucleotides group. In simple infrasound exposure group, F-actin was thick long and showed in longitudinal arrangement and the fluorescence was strong. Correspondingly, fluorescence in sense oligonucleotides +infrasound exposure group was almost in the same level as in simple exposure group. However, less F-actin expression in cells of antisense oligonucleotides +infrasound exposure group was observed comparing to simply exposure group and most fluorescein-labelled substance was in diffusion states. Cells in all groups showed not any different in cytomorphology and structure after once exposure.Part two: Expression of integrinp 1 and integrinP 1 mRNA of ECV-3 04 cells after infrasound exposureObjective: To observe the expression of integrinp 1 and integrinP 1 mRNA of EC V-3 04 cell after infrasound exposure and to explore the contribution of integrinP 1 in infrasound signal transduction pathway to intracellular.Methods: Immunohistochemical method and in-situ hybridization were
used to detect the expression of integrinpi and integrinplmRNA of ECV-304 cells after infrasound exposure with intensity output of 90,110,130dB and frequency of 16Hz. Results:1. After once infrasound exposure, integrin(31 expression showed obviously high in experimental groups compared to control group. Positive substance was in cytoplasm and caryon and the karyolemma showed a strong positive stained. Four hours after exposure, the positive substance reached a peak expression and then kept in a high level in 12 hours. At 24 hour more expression could also be observed. Up to 48 hour the expression got close normal.2. After three times exposure most cells appeared in circle, oblate forms and showed a poor expansion. There was an even more positive substance expression in cytoplasm and caryon. Compared to control group expression kept in a high level at 0,4,8,12 and 24 hour(pO.Ol) and when it did not become normal till 48 hours.3. More expression of integrinplmRNA was observed in experimental groups after once infrasound exposure with intensity output of 90,110,130dB and frequency of 16Hz. Positive substance was homogeneously in cytoplasm and karyolemma showed a little bit stronger positive stained. Caryon was in negative stained. In experimental groups cells were keeping in high expression for at least 24 hours(p<0.01). Integrinpi, integrinplmRNA and F-actin in cells of experimental groups showed a synchronized expression.4. There was more expression of integrinplmRNA in cells after three time infrasound exposure than that in only once exposure. More expression could be kept at least for 24 hours.Part three: AFM observation on cells' membrane after infrasound exposureObjective: To Observe the changes of cells' membrane under AFM after infrasound exposure with intensity output of 130dB and frequency of 8Hz and 16Hz to explore the mechanisms of infrasound on cell membrane.Methods: After primary culture, EVC-304 cells, L9129 cells, hippocampus neuron of embryonic rat and two kind of carcinoma cells were exposed to 16Hz 130dB infrasound meanwhile CHO cells and 446cells to 8Hz 130dB for 1 and 3 days and 2 hours each day respectively in different groups to observe the subsequent changes in the membrane of the cells by nano-scale scanning with AFM.
Results:1. After infrasound exposure, it was showed on membrane of EVC-304 cells, L9129 cells, CHO cells and hippocampus neuron of embryonic rat that the prominent of the membrane became shorten and the excavation became shallow and the surface of the membrane became smooth in nano-scale scanning with AFM. All these changes might be the characteristics of cell membrane after infrasound exposure.2. The changes were more obvious on cell membrane with three times infrasound exposure than once exposure.3. The structure of the cell membrane is closely related to the function of the cell. Changes on cell membrane in nano-scale might be one of the primary mechanisms of infrasound.Part four: Effects of infrasound on changes of intracellular calcium ion concentration in EVC-304 cells with and without heparin interferenceObjective: To investigate the effects of infrasound on changes of Ca2+ in EVC-304 cells with and without heparin interference to explore the mechanisms of cell injury.Methods: After the primary culture on coverglass , cells with and without heparin interference in experimental groups were exposed to 16Hz 90dB,110dB and 130dB infrasound respectively. Fluo-3/AM fluorescence technique and laser scanning confocal microscope (LSCM) were used for measurement of Ca2+ level. Results:1. There was a significant increase in experimental groups after once or 3 times infrasound exposure compared to control(p<0.01), and there was a significant difference of Ca2+ between 130dB and 90dB group. However, there was no significant difference between 130dB 90dB and 1 lOdB group respectively. It seemed that calcium ion concentration was high in 3 times exposure than once, but there was no significant difference.2. In control groups it showed no significant difference of Ca2+ concentration in cells with or without heparin interference(p>0.05), but in 90dB HOdB and 130dB infrasound exposure groups there was a significant decrease after heparin interference compared to simply infrasound exposure groups with same intensity output(p<0.01).From all above we could get the conclusion that: More F-actin can be found after once infrasound exposure, and the amount of F-actin is closely related to the number of exposure times. The changes of cytoskeleton can be recovered at 24 hours. Cells can be morphologically changed after 3 times
次声广泛存在自然界、生活环境、生产环境和军事环境中。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,并重视作为新概念武器的次声武器在战争中的作用,相关的文献报道也逐年增多。和其它物理因子一样,次声对机体的原发性作用机制一直为人们所关注。一般认为次声作用于机体组织引起生物共振,从而引起生物学效应。次声是一种机械波,作用于机体组织时可产生一种类似于弹性拉压的机械力。目前越来越多的研究表明:机械应力对细胞生长、分化、凋亡以及基因表达等生理过程和某些病理过程可发挥重要的作用。就细胞而言,在生物共振的基础上,对次声机械力的响应,很可能是引起细胞生物学效应的主要原因,但这种机械力如何被细胞感受并传至细胞内,进而使细胞发生一系列生物学效应,目前国内外还未见相关的报道。因
As the audible sound and ultrasound, infrasound is a mechanical vibration wave with frequency in range of 0.0001to 20Hz. With the physical characteristics of less attenuation in long distance spreading and strong penetration, infrasound is harmful to human and leads to body disfunction or a even severe damage when the intensity comes to a certain degree. We can hardly barricade the interference of infrasound with general sound absorbing material, building and shelter. 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. As to other kinds of physical agents, people have showed solicitude to the primary mechanisms of infrasound. Generally, it is considered that infrasound evokes a biological resonance of bio-organs and gives rise to biological effects. On the other hand, being a mechanical wave, infrasound as an elastic spring forces produces a mechanical stress which something likes a pull and press alternately forces. At present, more and more investigations showed that mechanical stress played a very important role in cells' growth, differentiation, apoptosis and gene expression in some physiological and pathological procedures. Concerning to cells, responding to infrasound forces may be the main reason to create biological effects. However, it is not very clear that how does the infrasound signal be taken over and then transferred to intracellular by the cells to bring about a further series of biological effects. Therefore, it is
important to investigate the changes on force transduction pathway of Extracelllular matrix(ECM) —,Integrin— Cytoskeleton(CSK) as well as cellular membrane structure and intracellular Ca2+ in the experiment to explore the infrasound signal transduction pathway to intracellular to establish the bases for the further studies of infrasound bio-effects. This thesis is composed of four parts as follow:Part one: The effect of infrasound on expression of cytoskeleton filament F-actin of ECV-304 cells and integrin (31 antisense oligonucleotide inhibits the expression of F-actinObjective: To identify the expression of cytoskeleton filament in ECV-304 cells after infrasound exposure and to explore the infrasound signal transduction pathway to intracellular.Methods: After the primary culture on coverglass, cells in experimental groups were exposed to infrasound with different parameter output The cells in control group were managed in the same environment as experimental groups with intensity output of OdB. 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. Phosphorothioate modified antisense and plus-sense oligonucleotides of integrinpi were used to interfere with the expression of F-actin of ECV-304 cells exposed to infrasound with intensity output of 130dB and frequency of 16Hz for 2 hours. The same examination was done after infrasound exposure.Results:1. In control group, we could find under laser scanning confocal microscope that most fluorescein-labelled substance in cells was in diffusion states. Less actin filaments were tenuously short without direction. Less long stress fiber passed through the cells. Most cells with a clear margin appeared in circle, fusiform or polygon shape. Fluorescence of cells membrane showed a little bit stronger than in cytoplasm.'2. Correspondingly F-actin in the cells of experimental groups of 16Hz, 90dB or 11 OdB or 13 OdB were thick long and showed in longitudinal arrangement after once infrasound exposure of 2 hours. Small part of F-actin was arranged in across or network in cells. Less fluorescein-labelled substance in cells was in diffusion states. The intensity of fluorescence was significantly increased(p<0.01). In experimental groups, there was a membrane-like boundary much stronger fluorescence of cells than that in control group. More F-actin expression in experimental groups was being kept obviously for at
least 8 hours then it gradually decreased. 24 hours later there was not any significantly different in F-actin expression between the experimental groups and the control group. There was not any difference between experimental groups.3. Compared to control group, the expression of F-actin in cells of different frequency groups of 8Hz and 16Hz was significantly increased(pO.OI). The variance pattern of F-actin expression was just same as above in 2. There was not any significantly difference of fluorescence of the cells between experimental groups. Frequency-dependent was not obvious.4. There were some cytomorphological changes in experimental groups after 3times infrasound exposure of 8Hz 16Hz 130dB respectively. Most cells appeared in circle, oblate forms and showed a poor expansion as if it retracted. Less cells appeared in expanded triangle or polygon shapes. Cellular nucleus with a very clear positive IP-stained boundary seemed to be shrink. F-actin with a radiation or longitude arrangement in cytoplasm showed a much stronger fluorescence than that in control or once exposure groups. The thin stress fibers could hardly be observed. Some filaments appeared almost as lump. Cells membrane was interrupted. More F-actin expression in experimental groups was being kept for 8 to 12 hours then it gradually decreased. The cell responsivity in 3 times infrasound exposure group was higher than once exposure group. Cells could be restored at 24 hours after infrasound exposure.5. In sense oligonucleotides group, cells fluorescence was almost in the same level as in control group. Anyway, a weak fluorescence was observed in cells of antisense oligonucleotides group. In simple infrasound exposure group, F-actin was thick long and showed in longitudinal arrangement and the fluorescence was strong. Correspondingly, fluorescence in sense oligonucleotides +infrasound exposure group was almost in the same level as in simple exposure group. However, less F-actin expression in cells of antisense oligonucleotides +infrasound exposure group was observed comparing to simply exposure group and most fluorescein-labelled substance was in diffusion states. Cells in all groups showed not any different in cytomorphology and structure after once exposure.Part two: Expression of integrinp 1 and integrinP 1 mRNA of ECV-3 04 cells after infrasound exposureObjective: To observe the expression of integrinp 1 and integrinP 1 mRNA of EC V-3 04 cell after infrasound exposure and to explore the contribution of integrinP 1 in infrasound signal transduction pathway to intracellular.Methods: Immunohistochemical method and in-situ hybridization were
used to detect the expression of integrinpi and integrinplmRNA of ECV-304 cells after infrasound exposure with intensity output of 90,110,130dB and frequency of 16Hz. Results:1. After once infrasound exposure, integrin(31 expression showed obviously high in experimental groups compared to control group. Positive substance was in cytoplasm and caryon and the karyolemma showed a strong positive stained. Four hours after exposure, the positive substance reached a peak expression and then kept in a high level in 12 hours. At 24 hour more expression could also be observed. Up to 48 hour the expression got close normal.2. After three times exposure most cells appeared in circle, oblate forms and showed a poor expansion. There was an even more positive substance expression in cytoplasm and caryon. Compared to control group expression kept in a high level at 0,4,8,12 and 24 hour(pO.Ol) and when it did not become normal till 48 hours.3. More expression of integrinplmRNA was observed in experimental groups after once infrasound exposure with intensity output of 90,110,130dB and frequency of 16Hz. Positive substance was homogeneously in cytoplasm and karyolemma showed a little bit stronger positive stained. Caryon was in negative stained. In experimental groups cells were keeping in high expression for at least 24 hours(p<0.01). Integrinpi, integrinplmRNA and F-actin in cells of experimental groups showed a synchronized expression.4. There was more expression of integrinplmRNA in cells after three time infrasound exposure than that in only once exposure. More expression could be kept at least for 24 hours.Part three: AFM observation on cells' membrane after infrasound exposureObjective: To Observe the changes of cells' membrane under AFM after infrasound exposure with intensity output of 130dB and frequency of 8Hz and 16Hz to explore the mechanisms of infrasound on cell membrane.Methods: After primary culture, EVC-304 cells, L9129 cells, hippocampus neuron of embryonic rat and two kind of carcinoma cells were exposed to 16Hz 130dB infrasound meanwhile CHO cells and 446cells to 8Hz 130dB for 1 and 3 days and 2 hours each day respectively in different groups to observe the subsequent changes in the membrane of the cells by nano-scale scanning with AFM.
Results:1. After infrasound exposure, it was showed on membrane of EVC-304 cells, L9129 cells, CHO cells and hippocampus neuron of embryonic rat that the prominent of the membrane became shorten and the excavation became shallow and the surface of the membrane became smooth in nano-scale scanning with AFM. All these changes might be the characteristics of cell membrane after infrasound exposure.2. The changes were more obvious on cell membrane with three times infrasound exposure than once exposure.3. The structure of the cell membrane is closely related to the function of the cell. Changes on cell membrane in nano-scale might be one of the primary mechanisms of infrasound.Part four: Effects of infrasound on changes of intracellular calcium ion concentration in EVC-304 cells with and without heparin interferenceObjective: To investigate the effects of infrasound on changes of Ca2+ in EVC-304 cells with and without heparin interference to explore the mechanisms of cell injury.Methods: After the primary culture on coverglass , cells with and without heparin interference in experimental groups were exposed to 16Hz 90dB,110dB and 130dB infrasound respectively. Fluo-3/AM fluorescence technique and laser scanning confocal microscope (LSCM) were used for measurement of Ca2+ level. Results:1. There was a significant increase in experimental groups after once or 3 times infrasound exposure compared to control(p<0.01), and there was a significant difference of Ca2+ between 130dB and 90dB group. However, there was no significant difference between 130dB 90dB and 1 lOdB group respectively. It seemed that calcium ion concentration was high in 3 times exposure than once, but there was no significant difference.2. In control groups it showed no significant difference of Ca2+ concentration in cells with or without heparin interference(p>0.05), but in 90dB HOdB and 130dB infrasound exposure groups there was a significant decrease after heparin interference compared to simply infrasound exposure groups with same intensity output(p<0.01).From all above we could get the conclusion that: More F-actin can be found after once infrasound exposure, and the amount of F-actin is closely related to the number of exposure times. The changes of cytoskeleton can be recovered at 24 hours. Cells can be morphologically changed after 3 times
引文
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