体外培养包埋透明质酸/壳聚糖/质粒DNA纳米粒的壳聚糖支架负载软骨细胞  

Porous chitosan scaffolds with embedded hyaluronic acid/chitosan/plasmid DNA nanoparticles co-cultured with chondrocytes in vitro

作　　者：卢华定[1] 吕璐璐[1] 赵慧清[1] 戴驭虎[1] 

机构地区：[1]中山大学附属第三医院骨科,广州510630

出　　处：《中华实验外科杂志》2014年第2期362-364,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81272040、30600632）;广东省自然科学基金资助项目（S2011010004808）

摘　　要：目的 观察包埋透明质酸（HA）/壳聚糖（CS）/质粒DNA （pDNA）纳米粒的壳聚糖支架对软骨细胞生物学性状的影响及基因转染能力,探讨其作为基因活化软骨组织工程支架的可行性.方法 构建包埋HA/CS/pDNA纳米粒的壳聚糖支架,负载兔关节软骨细胞体外培养,1～2周后通过扫描电镜、组织学、免疫组织化学、倒置相差显微镜及激光共聚焦显微镜检测支架对软骨细胞表型及功能的影响,以及基因转染的能力.结果 支架孔径为100～300 μm,孔孔隙率为（86.0±1.2）％;软骨细胞在包埋HA/CS/pDNA纳米粒的支架中贴附良好,维持表型稳定,1～2周时实验组分泌蛋白多糖及Ⅱ型胶原等软骨细胞外基质较对照组明显增多,1周时测得支架中的HA/CS/pDNA纳米粒介导基因转染软骨细胞并表达绿色荧光蛋白.结论 包埋HA/CS/pDNA纳米粒的壳聚糖支架细胞相容性良好,能转染培养的软骨细胞表达绿色荧光蛋白.Objective To evaluate the feasibility and the value of porous chitosan scaffolds with embedded hyaluronic acid/chitosan/plasmid DNA （HA/CS/pDNA） nanoparticles as a gene-activated scaffold for the cartilage tissue engineering after an observation of how they affect the cell behaviors and gene transfection capability towards chondrocytes in vitro.Methods Porous chitosan scaffolds with embedded HA/CS/pDNA nanoparticles were constructed,and then chondrocytes were seeded and cultured in a threedimensional environment for 1-2 weeks.The effects of the gene activated scaffold on the cell phenotype,proliferation,synthesis of the extracellular matrix,and the ability of gene transfection towards cultured chondrocytes were observed by the scanning electron microscopy,histology,immunohistochemistry,phasecontrast microscopy and laser scanning confocal microscopy.Results The porosity of the scaffold was （86.0 ± 1.2）% with pores ranging in size from 100-300 μm.Chondrocytes which attached to the wall of pores of the scaffold had largely maintained round morphology and could secrete the extracellular matrix such as proteoglycan and collagen Ⅱ.The HA/CS/pDNA nanoparticles could mediate gene transfection towards chondrocytes and express enhanced green fluorescent protein （EGFP）.Conclusion Porous chitosan scaffolds with embedded HA/CS/pDNA nanoparticles have a good cellular compatibility,can mediate gene transfect towards cultured chondrocytes to express EGFP,and are expected to be an ideal gene activated scaffold for cartilage tissue engineering.

关 键 词：壳聚糖 软骨细胞 基因活化基质 转染 组织工程 

分 类 号：R318.08[医药卫生—生物医学工程]