【作者】 李煌；

【导师】 陈扬熙； 徐芸； 李松；

【作者基本信息】 四川大学 ， 口腔临床医学， 2005， 博士

【摘要】 髁突软骨是口腔颞下颌关节的重要结构,其所具有的生物学特性是口腔正畸功能矫形治疗的基础。作为髁突软骨主要的细胞成分,髁突软骨细胞在维持细胞外基质的完整、适应外周环境刺激以及功能性改建等方面都具有相当重要的作用。 在口腔功能矫形治疗中,压应力是导致髁突软骨改建的关键而敏感的力量之一。压应力作用下,髁突软骨细胞是如何识别应力信号并发生生物应答反应?这一力学信号转导的具体机制仍尚不清楚。因此,压应力作用下髁突软骨细胞早期应答机制的研究已成为口腔科学乃至力学信号转导研究的热点。 力学信号的识别和传递本质上是一系列蛋白质的变化。比较两种不同力学状态下髁突软骨细胞的蛋白质,有助于阐明压应力作用下髁突软骨细胞早期生物学应答反应,发现关键的应力信号转导蛋白分子。蛋白质组学(Proteomics)作为高效率,高通量的蛋白筛选技术,为力学信号转导机制的研究提供了新的思路。蛋白质组学的出现改变了以前只能研究单个蛋白质的状况,建立了以整体观点来解析生命现象的理念,以蛋白质组为基础的生物信息学和实验科学相结合为研究压应力下髁突软骨细胞的早期应答机制提供了一种新的尝试。 本课题体外培养大鼠髁突软骨细胞,利用四点弯曲细胞力学加载装置(Four-Point Bending System)对髁突软骨细胞进行压应力加载,从蛋白质组学的角度对所发生的早期应答反应进行了初步的探讨。运用体外培养技术获得具有稳定生物学表型的髁突软骨细胞;运用流式细更多还原

【Abstract】 The condylar cartilage plays an important role in Temporomandibular joint (TMJ). The successful orthopedic treatment is on the base of the physiological feature of the condylar cartilage. Condylar chondrocytes, the mainly cell type normally resident within condylar cartilage, which are responsible for the synthesis and maintenance of a viable extracellular matrix, adapted to cope with the physical pressures of its environment, functional remodeling under the outside stimulation.In oral orthopedic treatment, the compressive stress is one of the key and sensitive strength which results in the functional remodeling of the condylar cartilage.Under the compressive stress, how the condylar chondrocytes recognize the signal of the compressive stress? How the condylar chondrocytes take place the biological response? The mechanism of the stress -signal transduction is not clear until now. Therefore, the study of the early-responsing mechanism of the condylar chondrocyte under the compressive stress has become the focus in the area of the orthodontic science and stress-signal transduction science."Proteomics", which proposed in 1994 by Wilkins, provide a new direction of the study of the stress-signal transduction. However, the processes which recognize the stress-signal are the change of a series of proteins indeed. Compare the proteins of the condylar chondrocytes in different conditions: stressed and unstressed group, which is helpful to find the key signal transduction protein and describe the early-responsing mechanism of the condylar chondrocyte under the compressive stress. The development of the "Proteomics" allows people to change the condition which focus on the single protein, give people the idea which analysis thelife in the view of "the whole body". Therefore, the method of the combination of bioinformatics and experimental science which on the base of the "Proteomics", will give us a new idea to study the early-responsing mechanism of the condylar chondrocyte under the compressive stress.Methods: The fetal rat condylar chondrocytes were cultured in vitro, then subjected to mechanical compressive stress by self-made four-point bending system at a 0.5Hz frequency for Omin, 15min, 30min, 60min, 120min, 240min, respectively. In each time-phase, the cells were loaded with compressive stress at 2000|j.strain and 4000nstrain respectively. The cell proliferation of the chondrocyte was analyzed by flow cytometry and MTT method.As the certain time point at 2000ustrain and 4000ustrain for 60min, two dimentional electrophoresis (2D-PAGE) and matrix-assisted laser-desorption ionization time of flight massspectrometry (MALDI-TOF-MS) were used to separated and identified proteins in the whole cell lysate.According to the results of MALDI-TOF-MS, Laser Scanning Confocal Microscope (LSCM) was used to oberserve the change of cytokeletonal protein in different times. Actin, vimentin, stress-70 protein/GRP75 protein were observed by Western Blotting. Raf kinase inhibitor protein (RKIP) was measured by Real- time PCR and Western Blotting.Results:1. The shape of the rat condylar chondrocytes is polygon or astero-shape. The cell body spread out which is full of energy. The stainning of thetype of II collagen by IHC shows positive.2. With 2000|4.strain compressive stress loading, the cell proliferation did not change obviously within 60 mins, but it increased with loading times. With 4000ustrain compressive stress loading, the percentage of the S-stage in the cell cycle was inhibited in 60min.3. With 2000ustrain compressive stress loading for 60min, the expressional level of the protein spot of the whole cell lysate did not change obviously. With 4000(istrain compressive stress loading for 60min, the protein of the whole cell lysate changed significantly, which the expressional level of some proteins down regulate.4. Under 4000ustrain compressive stress, the difference protein spots were indetified by MALDI-TOF-MS. It included the protein related to signal transduction which named RKIP/PEBP, the protein related to cytoskeleton which named actin, vimentin, vimentin-Rho-GTPase; the protein related to glycometabolism which named Alpha enolase, Stress-70 protein/75 kDa glucose regulated protein, and others as 10- formyltetra-hydrofolate dehydrogenase, and Zinc finger protein 265.5. With 4000ustrain compressive stress loading for Omin, 15min, 30min, 60min, 120min, and 240min, the cytoskeletonal protein such as actin, vimentin expression downregulate in the early times, then upregulated. The changes of Stress-70 protein/GRP75 expressional level are the same.6. With 4000ustrain compressive stress loading for Omin, 15min, 30min, 60min, 120min, and 240min, RKIP/PEBP mRNA and protein expressional level were not coincidence with each other. Its protein expression downregulate in the beginning of loading times, which regulate RKIP mRNA expression by "feed-back", upregulate the RKIP mRNA expression. On the contrary, The RKIP/PEBP protein expressional level recovered in 240min which affected by theincreased of RKIP/PEBP mRNA expression.Conclusions:1. There are stable biological features in the fetal rat condylar chondrocytes which is original from the reliable tissue source. It can satisfy our subsequent research.2. The level of 2000|istrain compressive—stress is probably eaqual to the hydrostatic pressure in the physiological level.It indicates that hydrostatic pressure in the physiological level is the foundation of the condylar chondrocyte’s normal function. The level of 4000ustrain compressive — stress is a suitable outside-mechanical-stimulation, It indicates that the stress-signal was recognized and responsed by the condylar chondrocyte.3. With 4000ustrain compressive — stress loading, the early-responsing mechanism of the condylar chondrocyte mainly showed the inhibitory reaction. With the loading time increased, the "self-protection /regulation"plays an important role in the arly-responsing mechanism of the condylar chondrocyte, which regulate the proteins expression and make the chondrocyte defense the outside stimulation.4. With 4000|j.strain compressive — stress loading, the early-responsing mechanism of the condylar chondrocyte is very complicated which involved in different proteins. The cytoskeletonal protein, the energy metabolism protein, and the protein related to signal-path plays an important role.5. The cytoskeletonal protein, which names Actin and vimentin, are chiefly responsible for the compressive stress. The cytoskeleton recognize the stress stimulate at first, and then transmit the stress-signal and provocate the corresponed signal transduction protein molecule.6. The changes of Stress-70 protein/GRP75 give us hints that the mitochondrium of the condylar chondrocyte is subject to temple injure. Therfore, the energy supply is insufficient, the level of metabolism and the protein expression down-regulate.7. As the endogenous inhibitor in the signal transduction path, RKIP was the key target of the compression-signal transduction path. There is bi-directional signalling between MAPK cascades and cytoskeleton, which plays an important role in the early-responsing mechanism of the condylar chondrocyte更多还原