基于共培养体系考察间充质干细胞调控关节软骨表型的研究

发布时间：2018-03-15 17:54

  本文选题：软骨组织工程　切入点：共培养　出处：《华东理工大学》2012年硕士论文　论文类型：学位论文

【摘要】：当前,关节软骨损伤的临床治疗手段有限,长期疗效也不理想。组织工程为软骨组织修复／再生带来可能,间充质干细胞(Mesenchymal stem cells. MSCs)被广泛应用于体外软骨组织构建。发育过程中,细胞之间相互作用是组织结构形成的关键因素。在软骨组织工程研究中,对于MSCs与关节软骨组织中的软骨细胞(Articular chondrocytes, ACs)之间的相互作用,目前的认识还很不清楚。尽管很多研究认为ACs可能指导MSCs的成软骨分化,但MSCs是否也会影响ACs的行为,还不得而知。体外细胞与细胞共培养,可以有效考察细胞间相互作用。因此,本研究将MSCs与ACs进行共培养,来详细探讨MSCs对ACs表型的影响作用。 本研究主要包括两个方面的研究工作,其一,以新西兰大白兔为实验动物,分离获得了兔骨髓间充质干细胞(rabbit MSCs, rMSCs)和关节软骨细胞(rabbit ACs, rACs),体外传代培养考察rACs的生长特性；其二,利用Transwell培养系统考察MSCs对rACs表型的调控,主要展开了以下几个层次的探索：(①细胞培养方式,MSCs和rACs分别为二维单层培养或经海藻酸钙水凝胶包埋后三维培养：②rACs预培养时间,预先培养1天(T1)或者3天(T3)后与MSCs共培养：③共培养时间：④不同来源的MSCs,包括rMSCs和人羊膜间充质干细胞hMSCs;⑤考察了使用MSCs条件培养基(Conditioned medium)与rACs共培养的作用。同时,对rACs (?)勺表征主要包括了三个方面：细胞形态、基质分泌和基因表达。 结果发现：rACs在体外传代培养过程中发生了显著的去分化现象,表现在细胞形态由小且多角形或铺路石状,逐步转变大且为成纤维细胞样的梭形形态,胞外基质(氨基聚糖,Glycosaminoglycans, GAG)分泌减少,软骨相关特性基因(包括COL2A1, Aggrecan和Sox9等)表达水平下调；与MSCs共培养,促进了二维单层rACs的形态向梭形转变,而抑制了三维中rACs细胞聚团现象,rACs(?)包外基质分泌显著下降,基因表达也发生了明显变化,包括软骨特性基因的表达下调：不同来源的MSCs都表现出对rACs表型抑制的作用,尽管hMSCs的作用效果相对较低：最后,MSCs条件培养基也同样能够对rACs产生同样的作用,表明其可能是通过MSCs的旁分泌效应来实现。 总而言之,rACs体外培养过程中会逐步去分化,而MSCs的存在能够加速rACs的去分化,可能是源于MSCs的旁分泌效应,但是,至于何种分子介导了这样的作用,还有待进一步的研究。通过本研究工作,我们证实了MSCs对ACs的细胞表型产生显著地调节作用,为基于MSCs进行软骨组织损伤的修复/再生的研究提供了重要的科学启示。
[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.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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