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  本文關(guān)鍵詞：建立MRC-5細胞三維立體培養(yǎng)模型的探索　出處：《中國疾病預(yù)防控制中心》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人胚肺成纖維細胞 三維細胞培養(yǎng) 膠原 PS支架 巨細胞病毒

【摘要】：人胚肺成纖維細胞(human embryonic lung fibroblast,MRC-5)來源于 14 周人胚胎的肺組織,可在體外持續(xù)培養(yǎng)40多代,并且保持正常的二倍體。MRC-5細胞對許多病毒都具有易感性,如:巨細胞病毒、單純皰疹病毒、腸道病毒71型等。當前研究MRC-5細胞主要是基于傳統(tǒng)的二維細胞培養(yǎng)技術(shù),即細胞在塑料培養(yǎng)瓶或培養(yǎng)皿的平面上單層生長。然而,細胞在體內(nèi)生長的狀態(tài)則是三維立體環(huán)境,且具有復(fù)雜和動態(tài)的特性。二維細胞培養(yǎng)由于其自身的局限性,不能充分反映真實組織細胞的生理活性。三維細胞培養(yǎng)彌補了二維細胞培養(yǎng)的缺陷與不足,三維培養(yǎng)模型可在更接近生理環(huán)境下研究細胞生物學(xué)、細胞生理學(xué)及細胞病理學(xué)等。本論文實驗分別以鼠尾I型膠原和聚苯乙烯(polystyrene,PS)為支架構(gòu)建兩種不同的MRC-5細胞三維培養(yǎng)模型,探索在這兩種模型中常規(guī)培養(yǎng)液、血小板衍生生長因子培養(yǎng)液(platelet-derived growth factor,PDGF)和支氣管上皮細胞生長培養(yǎng)液(bronchial epithelial cell growth medium,BEGM)對 MRC-5 細胞生長狀態(tài)的影響,進一步比較這兩種培養(yǎng)模型中MRC-5細胞的生長狀態(tài)的差異,初步觀察在這兩種培養(yǎng)模型中MRC-5細胞感染人巨細胞病毒(human cytomegalovirus,HCMV)的病變狀況。實驗結(jié)果顯示,常規(guī)培養(yǎng)液、PDGF培養(yǎng)液和BEGM培養(yǎng)液均能使細胞在這兩種培養(yǎng)模型中相互交錯生長,形成多層立體的三維網(wǎng)狀結(jié)構(gòu)。其中在三維膠原-MRC-5細胞培養(yǎng)模型中,PDGF培養(yǎng)液有利于早期促進細胞呈樹突狀伸展,常規(guī)培養(yǎng)液和BEGM培養(yǎng)液則更有利于MRC-5細胞在較長時間內(nèi)保持三維立體的網(wǎng)狀結(jié)構(gòu)。常規(guī)培養(yǎng)液可促進細胞在膠原表面聚集,PDGF培養(yǎng)液和BEGM培養(yǎng)液則有利于細胞以單個細胞方式進行遷移。在三維PS-MRC-5細胞培養(yǎng)模型中,與常規(guī)培養(yǎng)液和PDGF培養(yǎng)液相比,BEGM培養(yǎng)液中生長的MRC-5細胞能夠形成更加明顯的三維立體網(wǎng)狀或“鳥巢狀”結(jié)構(gòu),且能長時間保持。與三維膠原基質(zhì)內(nèi)生長的MRC-5細胞相比較,在三維PS支架中生長的MRC-5細胞擴展延伸的速度相對較快,但維持細胞三維立體結(jié)構(gòu)的時間相對較短。在上述兩種培養(yǎng)模型內(nèi)生長的MRC-5細胞均能感染HCMV病毒,并且可呈現(xiàn)明顯的細胞病變效應(yīng)(cytopathic effect,CPE),說明MRC-5細胞沒有因為改變培養(yǎng)體系而改變其對HCMV病毒的易感性。與三維膠原基質(zhì)內(nèi)MRC-5細胞的病變相比較,三維PS支架內(nèi)的MRC-5細胞出現(xiàn)細胞病變的時間相對較早,病變持續(xù)時間相對較短,病變程度相對較高。通過我們的探索研究,利用鼠尾Ⅰ型膠原和聚苯乙烯材料成功地構(gòu)建了兩種不同類型的MRC-5細胞三維培養(yǎng)模型,并且在兩種培養(yǎng)模型中生長的MRC-5細胞對HCMV病毒的易感性沒有發(fā)生改變。這為進一步優(yōu)化MRC-5細胞三維培養(yǎng)模型和利用MRC-5細胞三維培養(yǎng)模型研究病毒感染、增殖及抗病毒藥物等提供實驗依據(jù)。
[Abstract]:Human embryonic lung fibroblast cells (human embryonic lung fibroblast, MRC-5) from the 14 week human fetal lung tissue, in vitro cultured for more than 40 generations continuously, and maintain the normal diploid.MRC-5 cells have susceptibility to many viruses such as cytomegalovirus, Dan Chunpao measles virus, enterovirus 71. Current research MRC-5 cell culture technology is mainly based on the traditional two-dimensional cell, in which cells grown in monolayer culture or plastic bottle dish on the plane. However, the growth of cells in the body state is the three-dimensional environment, and has the characteristics of complex and dynamic. The two-dimensional cell culture because of its own limitations, can not fully reflect the true physiological activity tissue cells. Three dimensional cell culture to make up for deficiencies in the two-dimensional cell culture and the lack of three-dimensional culture model can be closer to the physiological environment of cell biology, cell physiology And cell pathology. The experiment respectively with rat tail collagen type I and polystyrene (polystyrene, PS) to construct two MRC-5 three-dimensional cell culture model for different support, explore the common medium in the two model, platelet-derived growth factor (platelet-derived growth medium factor, PDGF) and bronchial epithelial cells the growth medium (bronchial epithelial cell growth medium, BEGM) growth effect on MRC-5 cells, further comparison of the two kinds of culture model of MRC-5 cells in the growing state of difference, preliminary observation on these two kinds of cultured human cytomegalovirus MRC-5 cell infection model (human cytomegalovirus, HCMV). The experimental results of lesions show that the conventional culture medium, PDGF medium and BEGM medium can make the cell culture model in the two mutually staggered growth, formation of multi layer three-dimensional mesh structure Which in three-dimensional collagen -MRC-5 cell culture model, PDGF culture medium is conducive to the early promotion of dendritic cells were stretched, common culture medium and BEGM culture medium is more conducive to MRC-5 cells maintain a three-dimensional reticular structure in a long time. The conventional culture medium could promote the accumulation of cells in collagen surface, PDGF medium and BEGM medium is conducive to the cells in a single cell by way of transfer. In the 3D PS-MRC-5 cell culture model, compared with the conventional culture medium and PDGF culture medium, BEGM culture of MRC-5 cells grown in liquid to form a three-dimensional network more obvious or "bird nest" structure, and can be kept for a long time. With the growth of three-dimensional collagen matrix the growth in MRC-5 cells compared to MRC-5 cells in three-dimensional PS scaffold extended relatively fast speed extension, but maintaining cells in three-dimensional structure in a relatively short time. The two culture model the growth of MRC-5 cells could be infected with the HCMV virus, and showed obvious cytopathic effects (cytopathic effect, CPE), indicating that MRC-5 cells did not change because the culture system and change its susceptibility to HCMV virus. MRC-5 cells and three-dimensional collagen matrix in the lesions compared to 3D PS stent. MRC-5 cytopathic time relatively early, in the relatively short duration of disease, a relatively high degree. Through our research, the successful construction of the two kinds of different types of MRC-5 three-dimensional cell culture model using rat tail collagen and polystyrene material, and there is no change in the growth of the susceptibility of two kinds of culture MRC-5 cell model of HCMV virus. This in order to further optimize the three-dimensional culture model of MRC-5 cells and MRC-5 cells using three-dimensional culture model to study the virus infection and proliferation It provides experimental basis for antiviral drugs and so on.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R329.2

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本文編號：1406719