双基因活化的骨软骨组织工程复合体的构建

发布时间：2018-02-21 20:29

本文关键词： 组织工程原位转染基因活性材料骨髓间充质干细胞骨软骨修复软骨下骨　出处：《南京大学》2011年硕士论文　论文类型：学位论文

【摘要】：由于炎症、外伤、肿瘤等各种疾病导致的关节软骨及其下方骨组织破坏甚至大范围的组织缺损,经常会导致关节功能的部分或者全部丧失；而且病损无论原发于关节软骨或骨,到后期均表现为骨软骨同时破坏。前期的组织工程更多的关注了软骨组织的构建,而对具有重要生理作用的软骨下骨关注较少。基因活化材料的原位传输方式,改变了以往基因输送系统中基因成分寻找靶细胞的转染方式,取而代之的是种子细胞或修复细胞在材料内部“捕获”或“遭遇”到DNA成分,因此这种转染方式适用于局部区域修复。本研究利用骨髓干细胞的多向分化性能,原位转染诱导其在同一支架的不同区域分别向软骨细胞和成骨细胞分化,构建骨软骨复合体。在前期工作的基础上,我们选用壳聚糖/明胶制作模拟软骨组织的多孔支架,TGF-β1质粒活化后诱导骨髓干细胞(MSC)向软骨细胞分化；壳聚糖/明胶/羟基磷灰石支架模拟成骨组织,BMP-2质粒活化后诱导MSC向成骨细胞分化；分开培养一周后利用纤维蛋白胶黏合构成骨软骨复合体。通过电镜观察,ELISA检测因子表达,Q-PCR, HE染色,免疫组化染色观察MSC在支架上的生长和分化；体内构建兔膝关节骨软骨全层缺损,植入复合体观察修复效果。扫描电镜显示,我们构建的支架具有很好的成孔性,并且两层之间具有很好的黏合；基因活化材料能够很好的支持MSC的贴附和增殖；ELISA测定TGF-β1和BMP-2因子的表达量显示该系统是一个很好的原位转染系统,基因活化组两种因子都有较高的表达；相比未基因活化的支架,Q-PCR结果显示在软骨层生长的MSC表达的蛋白聚糖和Ⅱ型胶原mRNA量有所上调,成骨层生长的MSC表达的骨桥蛋白,骨连接蛋白和Ⅰ型胶原nRNA量有所上调；免疫组化显示软骨层高表达Ⅱ型胶原而成骨层高表达Ⅰ型胶原,说明在骨软骨复合体上同时诱导了MSC向软骨和成骨细胞分化；利用此复合体进行的兔膝关节全层缺损修复,HE,免疫组化都显示出比单层支架更好的修复效果,促进了软骨和软骨下骨的再生以及与原生组织的整合。本研究说明,双基因活化的双层支架能够在不同层次上同时诱导MSC分别向软骨细胞和成骨细胞分化；此双层复合体能够促进骨软骨缺损的修复。
[Abstract]:Because of inflammation, trauma, tumor and other diseases caused by articular cartilage and underlying bone destruction even large tissue defects, often leads to joint function of all or part of the loss; and whether the primary lesion of articular cartilage or bone, to the late show at the same time. The early destruction of bone and cartilage tissue engineering more attention the cartilage tissue, and plays an important physiological role of the subchondral bone less attention.
In situ gene activation transmission materials, changed the genetic components for gene delivery system targeting cells, instead of the seed cells or repair cells inside the material "capture" or "experience" to the composition of DNA, so this was suitable for local repair.
This research using stem cell differentiation properties of bone marrow, in situ transfection induced in the different areas of the same scaffold to chondrogenic and osteogenic differentiation, construction of bone cartilage complex. On the basis of previous work, we use chitosan / gelatin production simulation of cartilage tissue scaffolds, bone marrow TGF- beta 1 after the activation of stem cells (MSC) differentiation into chondrocytes; chitosan / gelatin / hydroxyapatite scaffold simulating bone tissue, BMP-2 was activated after MSC was induced to differentiate into osteoblasts; separate culture after a week of using fibrin adhesive composition of bone cartilage complex. By electron microscopy, the expression of ELISA, detection of factor Q-PCR, HE staining, growth and differentiation of MSC was observed by immunohistochemistry on the stent; full-thickness osteochondral defects in the rabbit knee joint in vivo implantation of complex construction, to observe the repair effect.
Scanning electron microscopy showed that we constructed with hole bracket as well, and has good adhesion between the two layers; gene activation and proliferation on materials can be well supported by MSC; expression of ELISA was determined by TGF- beta 1 and BMP-2 factor shows that the system is a good primary transfection system expression, gene activation group of two kinds of factors are higher; compared with the stent was not gene activation, Q-PCR results showed that the proteoglycan and collagen type mRNA expression in cartilage growth layer MSC increased expression of osteogenic layer growth MSC osteopontin, osteonectin and collagen type nRNA content increased; immunohistochemistry showed that the expression of type II collagen and cartilage layer of bone high expression of collagen I, in osteochondral and MSC was induced to chondrocytes and osteoblasts of rabbit knee joint; the complex whole layer Defect repair, HE and immunohistochemistry showed better repair effect than monolayer scaffold, promoting the regeneration of cartilage and subchondral bone and integrating with primary tissue.
This study shows that double gene activated bilayer scaffolds can induce MSC to differentiate into chondrocytes and osteoblasts at different levels. The bilayer complex can promote the repair of osteochondral defects.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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