应力对体外培养成骨样细胞MG63中膜型基质金属蛋白酶1表达的影响

发布时间：2018-05-09 01:14

本文选题：成骨样细胞MG63 + 压应力　；参考：《吉林大学》2014年硕士论文

【摘要】：目的与背景: 牙槽骨是骨组织中可塑性最强、代谢最活跃者。生理情况下，有牙牙合牙槽骨保持破骨与成骨的动态平衡，使骨组织处于相对稳定的状态。长期缺牙的患者，牙槽骨缺乏牙齿的功能性刺激，而发生进行性、累积性、不可逆转的骨吸收。牙槽骨的不断吸收是引起临床很多修复体失败的主要原因。应力因素是影响剩余牙槽嵴吸收的主要因素，也是我们人为的能够采取干预措施加以改变的。王景云等学者发现，应力与牙槽骨的吸收密切相关；咀嚼压力过大会加速剩余牙槽嵴的吸收；适宜的咀嚼压力能够延缓剩余牙槽嵴的吸收。 Sato等学者从人胚胎cDNA文库中发现了一条新基因，即：膜型基质金属蛋白酶-1（MT1-MMP），其表达于细胞膜表面，能够编码含582个氨基酸的蛋白质。近年来研究发现，作为MMPs家族中被发现的第一个膜型MMPs，MT1-MMP在正常骨骼发育及骨改建过程中具有重要的作用。MT1-MMP表达于成骨细胞和破骨细胞，不仅其自身与正常骨骼组织发育及骨改建有着密切的联系，尚可通过激活其他MMPs及细胞因子而影响骨改建的过程；MT1-MMP能够调控细胞的机械敏感性；在成骨细胞分化为骨细胞的过程中，MT1-MMP作为一种骨细胞生成初期的必须物质具有决定性作用。研究MT1-MMP在骨组织中的作用形式及作用途径，对了解及掌握骨组织相关的疾患及研究防治方法具有重要的指导意义。本实验运用四点弯曲力学加载仪对体外培养的MG-63细胞施加不同强度及不同作用时间的压应力，利用Western Blot及逆转录PCR检测MT1-MMP的表达水平，为研究MT1-MMP在成骨细胞中的作用机制提供了基础实验依据，进一步证实应力作用下的细胞将发生的细胞生物学的改变以及探索咀嚼压力与剩余牙槽嵴吸收的关系奠定理论基础。实验方法：采用四点弯曲力学加载仪对培养在细胞加力板上的MG63细胞施加压应力，频率为0.5Hz，设置梯度变化及单一水平的压应力加载方式，研究压应力在不同力值（0、1000μstrain、2000μstrain、4000μstrain、6000μstrain）及不同时间（0、1h、3h、6h、9h、12h）对MT1-MMP蛋白及基因表达的影响，每组实验重复3次，空白对照组不加力；除应力因素外，其他影响因素基本控制一致。结果： 1、当应变值恒定为2000μstrain时，力学刺激呈时间依赖性的上调成骨样细胞MG63细胞中MT1-MMP蛋白及基因的表达； 2、当应变值为2000μstrain时，力学刺激对成骨样细胞MG63中MT1-MMP蛋白及基因表达作用最强，当应变值为6000μstrain时，MT1-MMP表达最低。结论： 1、在生理范围内，，当对MG63细胞施加恒定的压应力时，MT1-MMP的表达呈时间依赖性的上调； 2、当应力加载时间相同时，MT1-MMP的表达随着力值的增大而增加，但应力超出生理范围时，其表达则降低。 3、适宜的应力在调节骨改建过程中有着重要的作用。
[Abstract]:Purpose and background: Alveolar bone is the most plastic and metabolized bone tissue. Under physiological conditions, the alveolar bone of edentulous teeth keeps the dynamic balance between osteoclast and osteogenesis, which makes the bone tissue relatively stable. In patients with chronic dental deficiency, alveolar bone lacks functional stimulation and develops progressive, cumulative, irreversible bone resorption. The incessant absorption of alveolar bone is the main cause of the failure of many clinical prostheses. Stress factor is the main factor affecting the absorption of residual alveolar ridge. Wang Jingyun and other scholars found that stress is closely related to alveolar bone absorption; masticatory pressure is excessive to accelerate the absorption of residual alveolar ridge; appropriate masticatory pressure can delay the absorption of residual alveolar ridge. Sato and other researchers have found a new gene from the human embryo cDNA library, namely, the membrane matrix metalloproteinase-1 (MMP-1) MT1-MMPN, which is expressed on the surface of the cell membrane and encodes a protein containing 582 amino acids. In recent years, it has been found that MT1-MMP, the first membrane type of MMPsMMP found in MMPs family, plays an important role in normal bone development and bone remodeling. MT1-MMP is expressed in osteoblasts and osteoclasts. Not only is it closely related to normal bone tissue development and bone remodeling, but it can also affect the process of bone remodeling by activating other MMPs and cytokines. MT1-MMP can regulate the mechanical sensitivity of cells. MT1-MMP plays a decisive role in the process of osteoblast differentiation into osteocytes. It is of great significance to understand and master the diseases related to bone tissue and to study the methods of prevention and treatment by studying the forms and ways of action of MT1-MMP in bone tissue. In this study, four point bending mechanical loading apparatus was used to apply compressive stress of different intensity and time to MG-63 cells in vitro. The expression of MT1-MMP was detected by Western Blot and reverse transcription PCR. It provides a basic experimental basis for studying the mechanism of MT1-MMP acting on osteoblasts, further confirms the changes of cell biology under stress and explores the relationship between masticatory pressure and residual alveolar ridge absorption. Experimental methods: A four-point bending mechanical loading instrument was used to apply compressive stress to the MG63 cells cultured on the cell afterloading plate at a frequency of 0.5 Hz, and a gradient change and a single level of compressive stress loading mode were set. The effects of compressive stress on the expression of MT1-MMP protein and gene were studied at different stress values (0 1 000 渭 strain (2000 渭 strain) and 4 000 渭 strain) and at different times. The results showed that the MT1-MMP protein and gene expression were repeated 3 times in each group, but not in the blank control group. Except stress factors, the other influencing factors were basically controlled. Results: 1. When the strain value was 2000 渭 strain, mechanical stimulation up-regulated the expression of MT1-MMP protein and gene in MG63 cells in a time-dependent manner. 2. When the strain value was 2000 渭 strain, the expression of MT1-MMP protein and gene in osteoblast like cell MG63 was the strongest by mechanical stimulation, and the expression of MT1-MMP was the lowest when the strain value was 6000 渭 strain. Conclusion: 1. In physiological range, the expression of MT1-MMP was up-regulated in a time-dependent manner when MG63 cells were subjected to constant compressive stress. 2. When the stress loading time is the same, the expression of MT1-MMP increases with the increase of stress, but when the stress exceeds the physiological range, the expression of MT1-MMP decreases. 3. Suitable stress plays an important role in regulating bone remodeling.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R783

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