本文選題：TMJ關(guān)節(jié)盤細(xì)胞　切入點(diǎn)：Nano　出處：《蘭州大學(xué)》2015年碩士論文

【摘要】：目的：利用JPK Nano Wizard 3生物型原子力顯微鏡(Biological Atom Force Microscopy, Bio-AFM)對(duì)山羊顳下頜關(guān)節(jié)盤細(xì)胞表面形貌和生物力學(xué)特性進(jìn)行表征,并通過(guò)激光共聚焦顯微鏡(Laser Scanning Confocal Microscope, LSCM)對(duì)關(guān)節(jié)盤細(xì)胞的肌動(dòng)蛋白、微管蛋白、波形蛋白三種細(xì)胞骨架(Cytoskeleton,CSK)蛋白形態(tài)及蛋白量隨傳代發(fā)生改變的情況進(jìn)行對(duì)比分析,以探討關(guān)節(jié)盤細(xì)胞的生物力學(xué)特性以及原代同傳代關(guān)節(jié)盤細(xì)胞骨架的差異。材料和方法：1.單層培養(yǎng)原代山羊顳下頜關(guān)節(jié)盤細(xì)胞,生理狀態(tài)下用Bio-AFM以接觸模式掃描兩種類型的關(guān)節(jié)盤細(xì)胞表面形貌圖。分別隨機(jī)選取20個(gè)軟骨細(xì)胞樣細(xì)胞以及成纖維細(xì)胞樣細(xì)胞,在每個(gè)細(xì)胞的細(xì)胞核區(qū)和細(xì)胞質(zhì)區(qū)分別獲取力-位移曲線,通過(guò)JPK Data Processing軟件分析各自的楊氏模量和細(xì)胞粘附性能。2.采用免疫熒光雙色復(fù)染法對(duì)各代關(guān)節(jié)盤(P0、P1、P2、P3、P4)細(xì)胞CSK骨架蛋白進(jìn)行免疫熒光染色,通過(guò)LSCM獲取各代關(guān)節(jié)盤細(xì)胞肌動(dòng)蛋白、微管蛋白、波形蛋白三種細(xì)胞骨架形態(tài)圖,同時(shí)分別將各代關(guān)節(jié)盤細(xì)胞三種骨架蛋白的熒光表達(dá)量利用自帶熒光強(qiáng)度測(cè)定軟件進(jìn)行分析對(duì)比。結(jié)果：1.顳下頜關(guān)節(jié)盤細(xì)胞形態(tài)觀察示,原代關(guān)節(jié)盤細(xì)胞多以長(zhǎng)梭形的成纖維細(xì)胞樣細(xì)胞為主,部分呈多角形或圓形的軟骨細(xì)胞樣細(xì)胞,傳代以后關(guān)節(jié)盤細(xì)胞形態(tài)逐漸不規(guī)則,細(xì)胞多角形增多。2. Bio-AFM檢測(cè)示,三角形的軟骨細(xì)胞樣細(xì)胞胞核占據(jù)大部分體積,細(xì)胞骨架結(jié)構(gòu)呈樹(shù)枝狀排列,邊緣伸出許多偽足;呈長(zhǎng)梭形的成纖維細(xì)胞樣細(xì)胞,細(xì)胞核顯著高出胞質(zhì)區(qū),骨架結(jié)構(gòu)排列相對(duì)規(guī)則,細(xì)胞邊緣光滑,較少偽足伸出,兩類細(xì)胞的表面粗糙度沒(méi)有統(tǒng)計(jì)學(xué)差異。力-位移曲線分析得出,兩種類型細(xì)胞胞核區(qū)楊氏模量沒(méi)有差異,而胞質(zhì)區(qū)有差異。但兩種類型的關(guān)節(jié)盤細(xì)胞胞核區(qū)和胞質(zhì)區(qū)粘附性質(zhì)均無(wú)差異。3.免疫熒光染色示,絲狀的肌動(dòng)蛋白主要沿著細(xì)胞膜外圍均勻排列,微管呈放射狀貫穿胞質(zhì),隨著傳代絲狀結(jié)構(gòu)逐漸清晰,形成松散相互交織的網(wǎng)狀；中間纖維相互交織貫穿胞質(zhì),熒光強(qiáng)度不均一,在核周分布較少,胞膜區(qū)密集分布。隨著傳代,三種骨架蛋白逐漸增粗,P4代時(shí)形成較粗的纖維束。三種骨架蛋白的熒光強(qiáng)度整體趨勢(shì)隨著傳代熒光量的表達(dá)遞增,P3代以內(nèi)沒(méi)有統(tǒng)計(jì)學(xué)差異,而P4代同原代相比統(tǒng)計(jì)學(xué)差異明顯。結(jié)論：兩種類型的原代關(guān)節(jié)盤細(xì)胞形貌上雖有很大的差別,但在生物力學(xué)特性上的差異性并不明顯,可能是兩種類型的細(xì)胞在TMJ關(guān)節(jié)盤細(xì)胞發(fā)育階段所起的力學(xué)作用沒(méi)有差異。當(dāng)關(guān)節(jié)盤細(xì)胞傳代至P4代時(shí)肌動(dòng)蛋白、微管蛋白、波形蛋白三種骨架蛋白形態(tài)學(xué)和蛋白熒光量表達(dá)同原代細(xì)胞差異明顯,說(shuō)明TMJ關(guān)節(jié)盤細(xì)胞傳至P4代時(shí)骨架結(jié)構(gòu)發(fā)生了明顯的改變,有可能此時(shí)關(guān)節(jié)盤細(xì)胞已發(fā)生去分化現(xiàn)象,這為以后工程化顳下頜關(guān)節(jié)盤種子細(xì)胞的研究和選擇提供了依據(jù)。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R782.6

【參考文獻(xiàn)】

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1 康宏,包廣潔,董s，

本文編號(hào)：1680478