静磁场对成骨细胞功能活性的影响及其机制的初步研究
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摘要
磁场力是正畸中尚待开发的重要力源。随着磁性材料的广泛开发和利用，磁力正畸在国内外得到了广泛开展，但磁力应用的范围、磁力正畸的机理尚不清楚。众所周知，牙—牙槽骨独特的生物学特性是正畸牙得以移动的基础，而成骨细胞是正畸牙移动过程中骨形成的主要效应细胞，并对破骨细胞的分化和成熟具有重要调控作用，成骨细胞在骨改建的过程中处于一个中心调控的地位。目前，关于静磁场作用下成骨细胞功能活性改变及其相应机制的研究，国内外报道甚少。本研究的目的是研究静磁场信号对体外培养成骨细胞形态、功能、信号传导及细胞骨架改建的影响及应答变化，为阐明磁力正畸的应用机理提供实验依据。
本课题利用静磁场加载装置，对体外培养的成骨细胞进行加载，结合分子生物学检测技术和电子扫描电镜、激光共聚焦显微镜等仪器，观察了不同时间、不同磁场强度的静磁场对成骨细胞细胞形态及细胞表面超微结构、细胞增殖活性、碱性磷酸酶活性、细胞内钙离子浓度及细胞骨架表达及改建的影响，初步探讨了磁力正畸过程中不同强度的静磁场对成骨细胞形态及功能活性的作用及其机制。本研究得出以下结论：
1．采用组织块法成功培养出大量生长稳定的成骨细胞。
2．倒置相差显微镜下未能发现静磁场对成骨细胞形态、生长和排列的影响；电子扫描电镜显示本实验所选用的静磁场都引起了成骨细胞细胞表面超微结构的改建：暴露于静磁场后，成骨细胞细胞表面微绒毛增多，并且融合成为囊泡；随着曝磁时间的增加，细胞表面的微绒毛呈层状，囊泡样结构增加，相互融合成较大的囊泡。
3．不同时间、不同强度的静磁场作用对成骨细胞的增殖活性影响不同。50mT的静磁场从24小时到72小时可以持续促进成骨细胞的增殖；而8mT和160mT的静磁场则分别经历了24小时和48小时的潜伏期后，才开始出现促增殖作用。
4．静磁场作用可以增加成骨细胞的碱性磷酸酶活性。与8mT、和160mT的静磁场相比，50mT的静磁场促进成骨细胞碱性磷酸酶活性增加更大；随着静磁场加载时间的延长，碱性磷酸酶活性增加的幅度减小。
5．静磁场可以降低成骨细胞细胞内钙离子的浓度。
6．本实验所选用的静磁场可以导致成骨细胞细胞骨架的改建和重组。静磁场作用后，成骨细胞细胞骨架蛋白表达上调，细胞骨架蛋白向细胞核集中。
综上所述，本研究认为，一定强度的静磁场可以促进成骨细胞的增殖，可以增加成骨细胞的碱性磷酸酶活性。其机制可能是：
1．静磁场作用于成骨细胞的细胞膜，引起细胞膜表面钙泵的活性发生改建，降低了成骨细胞细胞内的钙离子浓度，引起成骨细胞细胞内信号的改变，继而导致成骨细胞生物学活性的改变；
2．静磁场通过直接或间接的作用于成骨细胞细胞骨架，将磁性信号的传递到细胞内，最终导致成骨细胞生物学活性的改建。
本研究提示，在正畸临床应用磁力进行矫治的时候，要选择合适的磁场强度，在骨形成和骨改建过程中，成骨细胞对不同的磁场强度有不同程度的表达反应。本研究丰富了我们对磁力正畸骨改建分子机理的认识，为进一步阐明磁力正畸的应用提供了具有一定参考价值的实验依据。
The orthodontist utilizes the force to make orthodontics, the force to the orthodontics is as the medicine to the physician; and the magnetic force is an important force which is still underdeveloped. With the development and utilization of magnetics, the orthodontics with magnetic force was widely carried out. The movement of the teeth is dependent on the bionomics of the alveolar bone; the osteoblast is the major effector cell and at the same time is the regulator of the osteoclast. Therefore, emphasis paid to the effect and mechanism of the magnetic magnetic field on the osteoblast is considered necessary and important to clarify the mechanism of orthodontics with magnetic force.
There are few reports about the osteoblast's biological effects of the static magnetic field, and we have not seen the mechanism report of it.
In this study, osteoblast cultures were exposed to static magnetic fields for different times with different density. The effects on the cell shape and the cell surface ultrastructure, cell proliferation, alkaline phosphatase (ALP), intracellular Ca~(2+) concentration, and the cytoskeleton of the static magnetic field were examined with molecular biology technology, electronic scanning microscope(SEM) and Laser Scanning Confocal Microscope(LSCM). The results showed as following:
1. The pieces methods could culture many stable osteoblasts.
2. No changes on the cell shape, cell growth and distribution were observed with inverted phase contrast microscope under the static magnetic field; but the scanning electric microscope observation showed that the static magnetic could result in the rebuilding of the cell surface ultrastructure, compared with the control group, experimental group showed more micirovili in the cell surface and some fused into vesicle.
3. Different static magnetic field makes different proliferation effects on the osteoblast. Compared with the 8mT and 160mT static magnetic fields, which laten 24 hours and 48hours in promoting proliferation, the 50mT could keep on promoting proliferation from 24hours to 72huors.
4. The static magnetic field could increase the ALP activity of osteoblast. Comared with the 8mT and 160mT static magnetic field, the 50mT static magnetic field make larger effect on increasing the osteoblast's ALP activity. With the lasting of the static magnetic field application, the ALP activity increasing extent decreased.
5. The static magnetic field decreases the intracellular Ca~(2+) concentration within our experiment SMF density.
6. The static magnetic field could result in the reorganization and rebuilding of the cytoskeleton of the osteoblast. After the application of the static magnetic field, the cytoskeleton protein upregulated and the
cytoskeleton concentrated to the cellular nucleus.
Based on these findings in the study, it was demonstrated that static magnetic fields with intensities of 8mT, 50mT and 160mT could increase the proliferation and ALP activity of the osteoblast. The static magnetic field may effect on the cell membrance, so as to evoke the Ca~(2+) pump in the membrance and drease the the intracellular Ca~(2+) concentration, thus result in the changes of the osteoblast's biological activity. Another mechanism is through the cytoskeleton, the static magnetic field transmitted the magnetic signal to the inner of the cell, thus lead to the changes of the osteoblast's biological activity.
本课题利用静磁场加载装置，对体外培养的成骨细胞进行加载，结合分子生物学检测技术和电子扫描电镜、激光共聚焦显微镜等仪器，观察了不同时间、不同磁场强度的静磁场对成骨细胞细胞形态及细胞表面超微结构、细胞增殖活性、碱性磷酸酶活性、细胞内钙离子浓度及细胞骨架表达及改建的影响，初步探讨了磁力正畸过程中不同强度的静磁场对成骨细胞形态及功能活性的作用及其机制。本研究得出以下结论：
1．采用组织块法成功培养出大量生长稳定的成骨细胞。
2．倒置相差显微镜下未能发现静磁场对成骨细胞形态、生长和排列的影响；电子扫描电镜显示本实验所选用的静磁场都引起了成骨细胞细胞表面超微结构的改建：暴露于静磁场后，成骨细胞细胞表面微绒毛增多，并且融合成为囊泡；随着曝磁时间的增加，细胞表面的微绒毛呈层状，囊泡样结构增加，相互融合成较大的囊泡。
3．不同时间、不同强度的静磁场作用对成骨细胞的增殖活性影响不同。50mT的静磁场从24小时到72小时可以持续促进成骨细胞的增殖；而8mT和160mT的静磁场则分别经历了24小时和48小时的潜伏期后，才开始出现促增殖作用。
4．静磁场作用可以增加成骨细胞的碱性磷酸酶活性。与8mT、和160mT的静磁场相比，50mT的静磁场促进成骨细胞碱性磷酸酶活性增加更大；随着静磁场加载时间的延长，碱性磷酸酶活性增加的幅度减小。
5．静磁场可以降低成骨细胞细胞内钙离子的浓度。
6．本实验所选用的静磁场可以导致成骨细胞细胞骨架的改建和重组。静磁场作用后，成骨细胞细胞骨架蛋白表达上调，细胞骨架蛋白向细胞核集中。
综上所述，本研究认为，一定强度的静磁场可以促进成骨细胞的增殖，可以增加成骨细胞的碱性磷酸酶活性。其机制可能是：
1．静磁场作用于成骨细胞的细胞膜，引起细胞膜表面钙泵的活性发生改建，降低了成骨细胞细胞内的钙离子浓度，引起成骨细胞细胞内信号的改变，继而导致成骨细胞生物学活性的改变；
2．静磁场通过直接或间接的作用于成骨细胞细胞骨架，将磁性信号的传递到细胞内，最终导致成骨细胞生物学活性的改建。
本研究提示，在正畸临床应用磁力进行矫治的时候，要选择合适的磁场强度，在骨形成和骨改建过程中，成骨细胞对不同的磁场强度有不同程度的表达反应。本研究丰富了我们对磁力正畸骨改建分子机理的认识，为进一步阐明磁力正畸的应用提供了具有一定参考价值的实验依据。
The orthodontist utilizes the force to make orthodontics, the force to the orthodontics is as the medicine to the physician; and the magnetic force is an important force which is still underdeveloped. With the development and utilization of magnetics, the orthodontics with magnetic force was widely carried out. The movement of the teeth is dependent on the bionomics of the alveolar bone; the osteoblast is the major effector cell and at the same time is the regulator of the osteoclast. Therefore, emphasis paid to the effect and mechanism of the magnetic magnetic field on the osteoblast is considered necessary and important to clarify the mechanism of orthodontics with magnetic force.
There are few reports about the osteoblast's biological effects of the static magnetic field, and we have not seen the mechanism report of it.
In this study, osteoblast cultures were exposed to static magnetic fields for different times with different density. The effects on the cell shape and the cell surface ultrastructure, cell proliferation, alkaline phosphatase (ALP), intracellular Ca~(2+) concentration, and the cytoskeleton of the static magnetic field were examined with molecular biology technology, electronic scanning microscope(SEM) and Laser Scanning Confocal Microscope(LSCM). The results showed as following:
1. The pieces methods could culture many stable osteoblasts.
2. No changes on the cell shape, cell growth and distribution were observed with inverted phase contrast microscope under the static magnetic field; but the scanning electric microscope observation showed that the static magnetic could result in the rebuilding of the cell surface ultrastructure, compared with the control group, experimental group showed more micirovili in the cell surface and some fused into vesicle.
3. Different static magnetic field makes different proliferation effects on the osteoblast. Compared with the 8mT and 160mT static magnetic fields, which laten 24 hours and 48hours in promoting proliferation, the 50mT could keep on promoting proliferation from 24hours to 72huors.
4. The static magnetic field could increase the ALP activity of osteoblast. Comared with the 8mT and 160mT static magnetic field, the 50mT static magnetic field make larger effect on increasing the osteoblast's ALP activity. With the lasting of the static magnetic field application, the ALP activity increasing extent decreased.
5. The static magnetic field decreases the intracellular Ca~(2+) concentration within our experiment SMF density.
6. The static magnetic field could result in the reorganization and rebuilding of the cytoskeleton of the osteoblast. After the application of the static magnetic field, the cytoskeleton protein upregulated and the
cytoskeleton concentrated to the cellular nucleus.
Based on these findings in the study, it was demonstrated that static magnetic fields with intensities of 8mT, 50mT and 160mT could increase the proliferation and ALP activity of the osteoblast. The static magnetic field may effect on the cell membrance, so as to evoke the Ca~(2+) pump in the membrance and drease the the intracellular Ca~(2+) concentration, thus result in the changes of the osteoblast's biological activity. Another mechanism is through the cytoskeleton, the static magnetic field transmitted the magnetic signal to the inner of the cell, thus lead to the changes of the osteoblast's biological activity.
引文
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