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

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

【摘要】：人胚肺成纖維細(xì)胞(human embryonic lung fibroblast,MRC-5)來(lái)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(xì)胞對(duì)許多病毒都具有易感性,如:巨細(xì)胞病毒、單純皰疹病毒、腸道病毒71型等。當(dāng)前研究MRC-5細(xì)胞主要是基于傳統(tǒng)的二維細(xì)胞培養(yǎng)技術(shù),即細(xì)胞在塑料培養(yǎng)瓶或培養(yǎng)皿的平面上單層生長(zhǎng)。然而,細(xì)胞在體內(nèi)生長(zhǎng)的狀態(tài)則是三維立體環(huán)境,且具有復(fù)雜和動(dòng)態(tài)的特性。二維細(xì)胞培養(yǎng)由于其自身的局限性,不能充分反映真實(shí)組織細(xì)胞的生理活性。三維細(xì)胞培養(yǎng)彌補(bǔ)了二維細(xì)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(xì)胞生物學(xué)、細(xì)胞生理學(xué)及細(xì)胞病理學(xué)等。本論文實(shí)驗(yàn)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(xì)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(zhǎng)因子培養(yǎng)液(platelet-derived growth factor,PDGF)和支氣管上皮細(xì)胞生長(zhǎng)培養(yǎng)液(bronchial epithelial cell growth medium,BEGM)對(duì) MRC-5 細(xì)胞生長(zhǎng)狀態(tài)的影響,進(jìn)一步比較這兩種培養(yǎng)模型中MRC-5細(xì)胞的生長(zhǎng)狀態(tài)的差異,初步觀察在這兩種培養(yǎng)模型中MRC-5細(xì)胞感染人巨細(xì)胞病毒(human cytomegalovirus,HCMV)的病變狀況。實(shí)驗(yàn)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(xì)胞在這兩種培養(yǎng)模型中相互交錯(cuò)生長(zhǎng),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(xì)胞培養(yǎng)模型中,PDGF培養(yǎng)液有利于早期促進(jìn)細(xì)胞呈樹(shù)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(xì)胞在較長(zhǎng)時(shí)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(jìn)細(xì)胞在膠原表面聚集,PDGF培養(yǎng)液和BEGM培養(yǎng)液則有利于細(xì)胞以單個(gè)細(xì)胞方式進(jìn)行遷移。在三維PS-MRC-5細(xì)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(zhǎng)的MRC-5細(xì)胞能夠形成更加明顯的三維立體網(wǎng)狀或“鳥(niǎo)巢狀”結(jié)構(gòu),且能長(zhǎng)時(shí)間保持。與三維膠原基質(zhì)內(nèi)生長(zhǎng)的MRC-5細(xì)胞相比較,在三維PS支架中生長(zhǎng)的MRC-5細(xì)胞擴(kuò)展延伸的速度相對(duì)較快,但維持細(xì)胞三維立體結(jié)構(gòu)的時(shí)間相對(duì)較短。在上述兩種培養(yǎng)模型內(nèi)生長(zhǎng)的MRC-5細(xì)胞均能感染HCMV病毒,并且可呈現(xiàn)明顯的細(xì)胞病變效應(yīng)(cytopathic effect,CPE),說(shuō)明MRC-5細(xì)胞沒(méi)有因?yàn)楦淖兣囵B(yǎng)體系而改變其對(duì)HCMV病毒的易感性。與三維膠原基質(zhì)內(nèi)MRC-5細(xì)胞的病變相比較,三維PS支架內(nèi)的MRC-5細(xì)胞出現(xiàn)細(xì)胞病變的時(shí)間相對(duì)較早,病變持續(xù)時(shí)間相對(duì)較短,病變程度相對(duì)較高。通過(guò)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(lèi)型的MRC-5細(xì)胞三維培養(yǎng)模型,并且在兩種培養(yǎng)模型中生長(zhǎng)的MRC-5細(xì)胞對(duì)HCMV病毒的易感性沒(méi)有發(fā)生改變。這為進(jìn)一步優(yōu)化MRC-5細(xì)胞三維培養(yǎng)模型和利用MRC-5細(xì)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(shí)驗(yàn)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(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R329.2

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相關(guān)期刊論文 前2條

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本文編號(hào)：1406719