周期性机械拉伸对大鼠成骨细胞生理活性和力学性质的影响
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摘要
本文采用生物力学、细胞生物学、细胞动力学等方法，研究周期性拉伸刺激对成骨细胞生理活性和力学性质的影响，并对成骨细胞对力学信号的响应机理作了探讨。主要工作和结论如下：
（1）采用消化法从初生大鼠的颅盖骨中分离、培养成骨细胞。
（2）使用四点弯曲加载装置对大鼠颅盖骨分离出的细胞施加周期性拉伸应变，应变水平分别为500με、1000με和1500με，工作频率为0.4Hz，研究周期性拉伸刺激对成骨细胞在不同应变水平和加载时间的条件下的生化响应以及力学性质的影响。结果表明：
a）应变分别为500με、1000με和1500με，频率为0.4Hz的周期性拉伸刺激影响大鼠颅盖骨中分离出的成骨细胞的生理活性。其中500με的应变水平能够促进成骨细胞的增殖、Ⅰ型胶原合成、胞外钙基质分泌、碱性磷酸酶活力和骨钙素合成。而1000με和1500με的应变水平抑制了成骨细胞的增殖、Ⅰ型胶原合成、胞外钙基质分泌、碱性磷酸酶活力和骨钙素合成。1500με的拉伸对成骨细胞的抑制作用比1000με的拉伸更明显。拉伸对成骨细胞生理活性的影响（促进或抑制作用）随加载时间的增加而增加。成骨细胞对周期性拉伸刺激的响应表现出在增殖、基质合成和分化、矿化上的一致变化。
b）利用微管吸吮技术研究应变分别为500με、1000με和1500με，频率为0.4Hz的周期性拉伸刺激对大鼠颅盖骨中分离出的成骨细胞的力学特性的影响。拉伸使成骨细胞的粘附力和铺展面积增加。而且细胞的粘附力和铺展面积随应变的增加而增加。加载时间对细胞的粘附力和铺展面积的影响不明显。
（3）首次设计了应变水平成梯度增加的方式，使成骨细胞在500με下加载2小时后，将应变增加到1000με加载2小时，最后再将应变升高到1500με加载2小时。以这种方式探讨成骨细胞对变化的力学环境的响应情况。结果表明：
a）成骨细胞所处的力学环境从适宜的力学环境（500με）变化到不利的力学环境时（1000με以上），细胞能够将其生理活性迅速地调节至新的力学环境下的状态，表现出细胞增殖、Ⅰ型胶原合成、胞外钙基质分泌、碱性磷酸酶活力和骨钙素合成的急剧变化。说明细胞能够识别不同的力学环境并作出相应的生化活性的调整。
b）应变水平成梯度增加的方式对成骨细胞力学性质的影响表现出与生理活性方面的响应相同的趋势。细胞在力学环境发生变化时粘附力和铺展面积都有明显变化。
（4）胞内钙离子在细胞的力转导过程中其重要作用。本文用荧光探针研究了成骨细胞响应拉伸应变时胞内钙离子浓度的变化。对成骨细胞胞内钙离子浓度的测量表明，在周期性拉伸刺激作用下，成骨细胞表现出胞内钙离子浓度的升高。用钙通道抑制剂（三七总皂苷）处理细胞后再加载的结果证实胞内钙离子浓度的升高部分来源于胞外钙的内流，部分来源于胞内钙库的释放，其中胞外钙的内流起主要作用。
The effects of cyclic stretching on the physiological activity and mechanical property of osteoblasts were studied through biomechanical ,cell biological and cell dynamical methods, as well as the mechanism by which osteoblasts respond to mechanical signal was investigated in the present paper. The main work and conclusions as follows:
(1) Osteoblasts were delievered and cultured from the new-born Wistar rats calvaria by the digesting method.
(2) In order to investigate the effects of cyclic stretching on the biochemical responses and mechanical property of osteoblasts at different strain magitude and loading time, a four-point bending apparatus was used to apply cyclic stretching on osteoblasts .The applied strain magnitudes were 500με、1000μεand 1500μεrespectively and working frequency was 0.4Hz. The results showed that:
a) Cyclic strtching at 500με,1000μεand 1500μεwith the frequency of 0.4Hz affected the physiological activity of osteoblasts delivered from rat calvaria. Stretching at 500μεincreased cell proliferation, collagen type I secretion, extracellular calcium deposition, alkaline phosphatase (ALP) activity and osteocalcin production. On the contrary, stretching at 1000μεand 1500μεinhabited cells proliferation, collagen type I secretion , extracellular calcium deposition ,ALP activity and osteocalcin production. The inhabiting effects of steteching at 1500μεwere more significant than that of 1000με. The promoting or inhabiting effects of stretching on osteoblasts increased with the loading time. Furthermore, the reponses of osteoblasts to cyclic stimulation expressed accordant changes in prolliferation, matrix production, differentiation and mineralization.
b)The micropipette aspiration technique was used to study the effects of cyclic stretching on the mechanical property of rat calvirial osteoblasts which were loaded at 500με,1000μεand 1500μεrespectively with the frequency of 0.4Hz. The stretching increased cells adhesive forces and spreading areas. Furthermore, cells adhesive forces and spreading areas
increased with the strain magnitude. Loading time had no significant effect on cells adhesive forces and spreading areas.
(3) A new loading method, step by step increased stretching (step stretching in shortened form) was also designed and used in this article. Cells were stretched at 500μεfor 2h firstly, followed by stretching at 1000μεfor 2h. Then the strain level was raised to 1500με and cells were stretched for 2h. The step by step increased stretching methods was used to investigate osteoblasts response to the changing mechanical environment. The results showed that:
a) When the stretching on osteoblasts were changed from an appropriate stimulation (500με) to an inhabiting stimulation (above 1000με), osteoblasts changed their physiological behaviors to adapt to the new mechanical environment. Cells proliferation, collagen typeⅠsecretion , extracellular calcium deposition ,ALP activity and osteocalcin production changed rapidly, suggesting that cells could distinguish different mechanical environment and adjust their biochemical responses accordingly.
b) The effects of step loading on osteoblasts mechanical property had the same trends as physilogical activity. Cells modified their adhesive forces and spreading areas when the mechanical environment was changed.
(4) The intracelluler calcium plays an important role in cell mechaotransduction. The fluorescence probe (fluo-3/AM) was used to investigate the changes of intracelluler calcium concentration when osteoblasts responded to stretching. The results showed that osteoblasts that were stretched increased their intracelluler calcium concentration. Cells were stretched after being treated with the panax notoginseng saponins, a Ca2+ channel inhibitor. The results showed that the rise of intracelluler calcium concentration was due to the influx of extracellular calcium and the release of intracellular calcium store, in which the influx of extracellular calcium play the main role.
（1）采用消化法从初生大鼠的颅盖骨中分离、培养成骨细胞。
（2）使用四点弯曲加载装置对大鼠颅盖骨分离出的细胞施加周期性拉伸应变，应变水平分别为500με、1000με和1500με，工作频率为0.4Hz，研究周期性拉伸刺激对成骨细胞在不同应变水平和加载时间的条件下的生化响应以及力学性质的影响。结果表明：
a）应变分别为500με、1000με和1500με，频率为0.4Hz的周期性拉伸刺激影响大鼠颅盖骨中分离出的成骨细胞的生理活性。其中500με的应变水平能够促进成骨细胞的增殖、Ⅰ型胶原合成、胞外钙基质分泌、碱性磷酸酶活力和骨钙素合成。而1000με和1500με的应变水平抑制了成骨细胞的增殖、Ⅰ型胶原合成、胞外钙基质分泌、碱性磷酸酶活力和骨钙素合成。1500με的拉伸对成骨细胞的抑制作用比1000με的拉伸更明显。拉伸对成骨细胞生理活性的影响（促进或抑制作用）随加载时间的增加而增加。成骨细胞对周期性拉伸刺激的响应表现出在增殖、基质合成和分化、矿化上的一致变化。
b）利用微管吸吮技术研究应变分别为500με、1000με和1500με，频率为0.4Hz的周期性拉伸刺激对大鼠颅盖骨中分离出的成骨细胞的力学特性的影响。拉伸使成骨细胞的粘附力和铺展面积增加。而且细胞的粘附力和铺展面积随应变的增加而增加。加载时间对细胞的粘附力和铺展面积的影响不明显。
（3）首次设计了应变水平成梯度增加的方式，使成骨细胞在500με下加载2小时后，将应变增加到1000με加载2小时，最后再将应变升高到1500με加载2小时。以这种方式探讨成骨细胞对变化的力学环境的响应情况。结果表明：
a）成骨细胞所处的力学环境从适宜的力学环境（500με）变化到不利的力学环境时（1000με以上），细胞能够将其生理活性迅速地调节至新的力学环境下的状态，表现出细胞增殖、Ⅰ型胶原合成、胞外钙基质分泌、碱性磷酸酶活力和骨钙素合成的急剧变化。说明细胞能够识别不同的力学环境并作出相应的生化活性的调整。
b）应变水平成梯度增加的方式对成骨细胞力学性质的影响表现出与生理活性方面的响应相同的趋势。细胞在力学环境发生变化时粘附力和铺展面积都有明显变化。
（4）胞内钙离子在细胞的力转导过程中其重要作用。本文用荧光探针研究了成骨细胞响应拉伸应变时胞内钙离子浓度的变化。对成骨细胞胞内钙离子浓度的测量表明，在周期性拉伸刺激作用下，成骨细胞表现出胞内钙离子浓度的升高。用钙通道抑制剂（三七总皂苷）处理细胞后再加载的结果证实胞内钙离子浓度的升高部分来源于胞外钙的内流，部分来源于胞内钙库的释放，其中胞外钙的内流起主要作用。
The effects of cyclic stretching on the physiological activity and mechanical property of osteoblasts were studied through biomechanical ,cell biological and cell dynamical methods, as well as the mechanism by which osteoblasts respond to mechanical signal was investigated in the present paper. The main work and conclusions as follows:
(1) Osteoblasts were delievered and cultured from the new-born Wistar rats calvaria by the digesting method.
(2) In order to investigate the effects of cyclic stretching on the biochemical responses and mechanical property of osteoblasts at different strain magitude and loading time, a four-point bending apparatus was used to apply cyclic stretching on osteoblasts .The applied strain magnitudes were 500με、1000μεand 1500μεrespectively and working frequency was 0.4Hz. The results showed that:
a) Cyclic strtching at 500με,1000μεand 1500μεwith the frequency of 0.4Hz affected the physiological activity of osteoblasts delivered from rat calvaria. Stretching at 500μεincreased cell proliferation, collagen type I secretion, extracellular calcium deposition, alkaline phosphatase (ALP) activity and osteocalcin production. On the contrary, stretching at 1000μεand 1500μεinhabited cells proliferation, collagen type I secretion , extracellular calcium deposition ,ALP activity and osteocalcin production. The inhabiting effects of steteching at 1500μεwere more significant than that of 1000με. The promoting or inhabiting effects of stretching on osteoblasts increased with the loading time. Furthermore, the reponses of osteoblasts to cyclic stimulation expressed accordant changes in prolliferation, matrix production, differentiation and mineralization.
b)The micropipette aspiration technique was used to study the effects of cyclic stretching on the mechanical property of rat calvirial osteoblasts which were loaded at 500με,1000μεand 1500μεrespectively with the frequency of 0.4Hz. The stretching increased cells adhesive forces and spreading areas. Furthermore, cells adhesive forces and spreading areas
increased with the strain magnitude. Loading time had no significant effect on cells adhesive forces and spreading areas.
(3) A new loading method, step by step increased stretching (step stretching in shortened form) was also designed and used in this article. Cells were stretched at 500μεfor 2h firstly, followed by stretching at 1000μεfor 2h. Then the strain level was raised to 1500με and cells were stretched for 2h. The step by step increased stretching methods was used to investigate osteoblasts response to the changing mechanical environment. The results showed that:
a) When the stretching on osteoblasts were changed from an appropriate stimulation (500με) to an inhabiting stimulation (above 1000με), osteoblasts changed their physiological behaviors to adapt to the new mechanical environment. Cells proliferation, collagen typeⅠsecretion , extracellular calcium deposition ,ALP activity and osteocalcin production changed rapidly, suggesting that cells could distinguish different mechanical environment and adjust their biochemical responses accordingly.
b) The effects of step loading on osteoblasts mechanical property had the same trends as physilogical activity. Cells modified their adhesive forces and spreading areas when the mechanical environment was changed.
(4) The intracelluler calcium plays an important role in cell mechaotransduction. The fluorescence probe (fluo-3/AM) was used to investigate the changes of intracelluler calcium concentration when osteoblasts responded to stretching. The results showed that osteoblasts that were stretched increased their intracelluler calcium concentration. Cells were stretched after being treated with the panax notoginseng saponins, a Ca2+ channel inhibitor. The results showed that the rise of intracelluler calcium concentration was due to the influx of extracellular calcium and the release of intracellular calcium store, in which the influx of extracellular calcium play the main role.
引文
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