發(fā)布時間：2018-03-15 17:54

  本文選題：軟骨組織工程　切入點：共培養(yǎng)　出處：《華東理工大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：當前,關(guān)節(jié)軟骨損傷的臨床治療手段有限,長期療效也不理想。組織工程為軟骨組織修復(fù)／再生帶來可能,間充質(zhì)干細胞(Mesenchymal stem cells. MSCs)被廣泛應(yīng)用于體外軟骨組織構(gòu)建。發(fā)育過程中,細胞之間相互作用是組織結(jié)構(gòu)形成的關(guān)鍵因素。在軟骨組織工程研究中,對于MSCs與關(guān)節(jié)軟骨組織中的軟骨細胞(Articular chondrocytes, ACs)之間的相互作用,目前的認識還很不清楚。盡管很多研究認為ACs可能指導(dǎo)MSCs的成軟骨分化,但MSCs是否也會影響ACs的行為,還不得而知。體外細胞與細胞共培養(yǎng),可以有效考察細胞間相互作用。因此,本研究將MSCs與ACs進行共培養(yǎng),來詳細探討MSCs對ACs表型的影響作用。 本研究主要包括兩個方面的研究工作,其一,以新西蘭大白兔為實驗動物,分離獲得了兔骨髓間充質(zhì)干細胞(rabbit MSCs, rMSCs)和關(guān)節(jié)軟骨細胞(rabbit ACs, rACs),體外傳代培養(yǎng)考察rACs的生長特性；其二,利用Transwell培養(yǎng)系統(tǒng)考察MSCs對rACs表型的調(diào)控,主要展開了以下幾個層次的探索：(①細胞培養(yǎng)方式,MSCs和rACs分別為二維單層培養(yǎng)或經(jīng)海藻酸鈣水凝膠包埋后三維培養(yǎng)：②rACs預(yù)培養(yǎng)時間,預(yù)先培養(yǎng)1天(T1)或者3天(T3)后與MSCs共培養(yǎng)：③共培養(yǎng)時間：④不同來源的MSCs,包括rMSCs和人羊膜間充質(zhì)干細胞hMSCs;⑤考察了使用MSCs條件培養(yǎng)基(Conditioned medium)與rACs共培養(yǎng)的作用。同時,對rACs (?)勺表征主要包括了三個方面：細胞形態(tài)、基質(zhì)分泌和基因表達。 結(jié)果發(fā)現(xiàn)：rACs在體外傳代培養(yǎng)過程中發(fā)生了顯著的去分化現(xiàn)象,表現(xiàn)在細胞形態(tài)由小且多角形或鋪路石狀,逐步轉(zhuǎn)變大且為成纖維細胞樣的梭形形態(tài),胞外基質(zhì)(氨基聚糖,Glycosaminoglycans, GAG)分泌減少,軟骨相關(guān)特性基因(包括COL2A1, Aggrecan和Sox9等)表達水平下調(diào)；與MSCs共培養(yǎng),促進了二維單層rACs的形態(tài)向梭形轉(zhuǎn)變,而抑制了三維中rACs細胞聚團現(xiàn)象,rACs(?)包外基質(zhì)分泌顯著下降,基因表達也發(fā)生了明顯變化,包括軟骨特性基因的表達下調(diào)：不同來源的MSCs都表現(xiàn)出對rACs表型抑制的作用,盡管hMSCs的作用效果相對較低：最后,MSCs條件培養(yǎng)基也同樣能夠?qū)ACs產(chǎn)生同樣的作用,表明其可能是通過MSCs的旁分泌效應(yīng)來實現(xiàn)。 總而言之,rACs體外培養(yǎng)過程中會逐步去分化,而MSCs的存在能夠加速rACs的去分化,可能是源于MSCs的旁分泌效應(yīng),但是,至于何種分子介導(dǎo)了這樣的作用,還有待進一步的研究。通過本研究工作,我們證實了MSCs對ACs的細胞表型產(chǎn)生顯著地調(diào)節(jié)作用,為基于MSCs進行軟骨組織損傷的修復(fù)/再生的研究提供了重要的科學(xué)啟示。
[Abstract]:At present, the clinical treatment of articular cartilage injury is limited, and the long-term curative effect is not ideal. Mesenchymal stem cells. MSCs (mesenchymal stem cells. MSCs) have been widely used in the construction of cartilage tissue in vitro. The interaction between MSCs and chondrocytes in articular chondrocytes (ACs) is unclear. Although many studies suggest that ACs may direct the chondrogenic differentiation of MSCs, whether MSCs also affects the behavior of ACs. Cell / cell coculture in vitro can effectively investigate the intercellular interaction. In this study, MSCs and ACs were co-cultured to investigate the effect of MSCs on the phenotype of ACs in detail. This study mainly includes two aspects of research work. Firstly, rabbit bone marrow mesenchymal stem cells (RMSCs) and articular chondrocytes rabbit acs, rACs were isolated from New Zealand white rabbits as experimental animals, and the growth characteristics of rACs were investigated in vitro. Secondly, the Transwell culture system was used to investigate the regulation of MSCs on the phenotype of rACs. The following levels of cell culture were explored. The two dimensional monolayer culture and the three-dimensional culture of rACs were respectively two dimensional monolayer culture or three dimensional culture of 2 rACs after embedding with calcium alginate hydrogel. Co cultured with MSCs for 1 day (T1) or 3 days (T3), co-cultured with MSCs for different sources, including rMSCs and human amniotic mesenchymal stem cells hMSCs5, the effect of co-culture with rACs on MSCs conditioned medium was investigated. The characterization includes three aspects: cell morphology, matrix secretion and gene expression. The results showed that there was a significant dedifferentiation in the culture process of the cell, which showed that the cell morphology was small and polygonal or paving stone, and gradually transformed into a large, fibroblast-like fusiform. The secretion of glycosaminoglycans (gag) decreased and the expression of chondrogenic characteristic genes (including COL2A1, Aggrecan and Sox9) down-regulated, co-cultured with MSCs promoted the transformation of two-dimensional monolayer rACs to fusiform. But it inhibited the aggregation of rACs cells in three dimensions. The secretion of extracapsular matrix decreased significantly and gene expression changed significantly, including the down-regulation of cartilage characteristic genes: MSCs from different sources showed an inhibitory effect on the phenotype of rACs. Although the effect of hMSCs was relatively low, the conditioned medium could also produce the same effect on rACs, suggesting that it might be realized by paracrine effect of MSCs. In a word, the MSCs can accelerate the dedifferentiation of rACs, which may be due to the paracrine effect of MSCs. However, which molecules mediate such a function, in the process of culture in vitro, and the existence of MSCs can accelerate the dedifferentiation of rACs, which may be due to the paracrine effect of MSCs. Through this study, we have confirmed that MSCs significantly regulates the cellular phenotype of ACs, which provides important scientific enlightenment for the study of repair / regeneration of cartilage tissue injury based on MSCs.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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相關(guān)期刊論文 前1條

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本文編號：1616318