山羊颞下颌关节盘细胞特性的原子力显微镜表征及骨架蛋白的研究

发布时间：2018-03-29 09:13

本文选题：TMJ关节盘细胞　切入点：Nano　出处：《兰州大学》2015年硕士论文

【摘要】：目的：利用JPK Nano Wizard 3生物型原子力显微镜(Biological Atom Force Microscopy, Bio-AFM)对山羊颞下颌关节盘细胞表面形貌和生物力学特性进行表征,并通过激光共聚焦显微镜(Laser Scanning Confocal Microscope, LSCM)对关节盘细胞的肌动蛋白、微管蛋白、波形蛋白三种细胞骨架(Cytoskeleton,CSK)蛋白形态及蛋白量随传代发生改变的情况进行对比分析,以探讨关节盘细胞的生物力学特性以及原代同传代关节盘细胞骨架的差异。材料和方法：1.单层培养原代山羊颞下颌关节盘细胞,生理状态下用Bio-AFM以接触模式扫描两种类型的关节盘细胞表面形貌图。分别随机选取20个软骨细胞样细胞以及成纤维细胞样细胞,在每个细胞的细胞核区和细胞质区分别获取力-位移曲线,通过JPK Data Processing软件分析各自的杨氏模量和细胞粘附性能。2.采用免疫荧光双色复染法对各代关节盘(P0、P1、P2、P3、P4)细胞CSK骨架蛋白进行免疫荧光染色,通过LSCM获取各代关节盘细胞肌动蛋白、微管蛋白、波形蛋白三种细胞骨架形态图,同时分别将各代关节盘细胞三种骨架蛋白的荧光表达量利用自带荧光强度测定软件进行分析对比。结果：1.颞下颌关节盘细胞形态观察示,原代关节盘细胞多以长梭形的成纤维细胞样细胞为主,部分呈多角形或圆形的软骨细胞样细胞,传代以后关节盘细胞形态逐渐不规则,细胞多角形增多。2. Bio-AFM检测示,三角形的软骨细胞样细胞胞核占据大部分体积,细胞骨架结构呈树枝状排列,边缘伸出许多伪足;呈长梭形的成纤维细胞样细胞,细胞核显著高出胞质区,骨架结构排列相对规则,细胞边缘光滑,较少伪足伸出,两类细胞的表面粗糙度没有统计学差异。力-位移曲线分析得出,两种类型细胞胞核区杨氏模量没有差异,而胞质区有差异。但两种类型的关节盘细胞胞核区和胞质区粘附性质均无差异。3.免疫荧光染色示,丝状的肌动蛋白主要沿着细胞膜外围均匀排列,微管呈放射状贯穿胞质,随着传代丝状结构逐渐清晰,形成松散相互交织的网状；中间纤维相互交织贯穿胞质,荧光强度不均一,在核周分布较少,胞膜区密集分布。随着传代,三种骨架蛋白逐渐增粗,P4代时形成较粗的纤维束。三种骨架蛋白的荧光强度整体趋势随着传代荧光量的表达递增,P3代以内没有统计学差异,而P4代同原代相比统计学差异明显。结论：两种类型的原代关节盘细胞形貌上虽有很大的差别,但在生物力学特性上的差异性并不明显,可能是两种类型的细胞在TMJ关节盘细胞发育阶段所起的力学作用没有差异。当关节盘细胞传代至P4代时肌动蛋白、微管蛋白、波形蛋白三种骨架蛋白形态学和蛋白荧光量表达同原代细胞差异明显,说明TMJ关节盘细胞传至P4代时骨架结构发生了明显的改变,有可能此时关节盘细胞已发生去分化现象,这为以后工程化颞下颌关节盘种子细胞的研究和选择提供了依据。
[Abstract]:Aim: to characterize the surface morphology and biomechanical properties of goat temporomandibular joint (TMJ) disc cells by JPK Nano Wizard 3 biological Atom Force microscopy (Bio-AFM). The morphology and protein quantity of actin, tubulin and vimentin in three kinds of cytoskeleton CSKs were analyzed by laser confocal microscopy (LSCM). In order to study the biomechanical characteristics of the disc cells and the difference of the cytoskeleton between the primary and the same generation, the materials and methods: 1. Monolayer culture of goat temporomandibular joint disc cells. In physiological condition, the surface morphologies of two types of articular disc cells were scanned by Bio-AFM in contact mode. Twenty chondrocyte-like cells and fibroblast-like cells were randomly selected. The force-displacement curves were obtained in the nuclear and cytoplasmic regions of each cell, respectively. The JPK Data Processing software was used to analyze the Young's modulus and cell adhesion. 2. Immunofluorescence staining was used to detect the CSK cytoskeleton protein of P0P1P2P2P3P4) cells from each generation of articular disc cells by immunofluorescence staining, and the actin of each generation was obtained by LSCM. Tubulin, vimentin, cytoskeleton morphology, At the same time, the fluorescence expression of three kinds of cytoskeleton proteins in each generation of articular disc cells were analyzed and compared by using the software of fluorescence intensity measurement. Results: 1. The morphology of temporomandibular joint disc cells was observed. The primary disc cells were mainly long fusiform fibroblast-like cells, some of which were polygonal or round chondrocyte-like cells. After passage, the morphology of articular disc cells was gradually irregular, and the number of polygonal cells increased by .2. Bio-AFM assay showed that, The nucleus of triangular chondrocyte-like cells occupies most of the volume, the cytoskeleton structure is dendriform, the edges protrude many pseudopods, and the long fusiform fibroblast-like cells, the nucleus is significantly higher than the cytoplasmic region, The cytoskeleton structure is relatively regular, the cell edge is smooth, the pseudopodia is less outstretched, the surface roughness of the two kinds of cells has no statistical difference, the analysis of the stress-displacement curve shows that there is no difference in the Young's modulus in the nuclear region of the two types of cells. However, there was no difference in adhesion properties between the nuclear and cytoplasmic regions of the two types of articular disc cells. Immunofluorescence staining showed that the filamentous actin was mainly arranged evenly along the periphery of the cell membrane, and the microtubules were radially penetrated through the cytoplasm. With the passage of the filamentous structure becoming clear, forming a loose interwoven mesh, the intermediate fibers interweave through the cytoplasm, the fluorescence intensity is not uniform, the distribution around the nucleus is less, and the cell membrane area is densely distributed. The three kinds of skeleton protein gradually thickened and formed coarse fiber bundles at P4 generation. The overall fluorescence intensity trend of the three kinds of skeleton proteins showed no statistical difference within P3 generation with the increase of the fluorescence expression of the three kinds of skeleton proteins. Conclusion: although there is a great difference in the morphology of the primary articular disc cells between the P4 generation and the primary generation, there is no obvious difference in biomechanical properties between the two types of primary articular disc cells. It is possible that there is no difference between the two types of cells at the developmental stage of the TMJ disc cells. When the disc cells are subcultured to the P4 passage, actin, tubulin, and tubulin, The morphological and fluorescent expression of vimentin was significantly different from that of primary cells, which indicated that the cytoskeleton structure of TMJ articular disc cells changed obviously when it was transferred to P4 passage, and it was possible that the dedifferentiation of disc cells had taken place at this time. This provides a basis for the later research and selection of seed cells of temporomandibular joint disc.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R782.6

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