吕璐璐;卢华定;陆慧琼;张富程;赵慧清;

• 1:中山大学附属第三医院骨科
• 2:中山大学附属第三医院中心实验室

摘要(Abstract)：

[目的]以透明质酸(HA)修饰的壳聚糖(CS)/质粒DNA(pDNA)纳米粒介导体外基因转染关节软骨细胞与滑膜细胞,以明确其作为非病毒基因载体治疗关节疾病的潜能。[方法]将HA修饰的CS与负载增强型绿色荧光蛋白基因(EGFP)的pDNA以复凝聚法制成纳米粒,以扫描电镜检测纳米粒形态;激光粒度仪测定其粒径、Ze-ta电位及分散度(PDl);凝胶电泳阻滞试验检测HA/CS和pDNA的结合力及pDNA的释放;体外转染兔关节软骨细胞与滑膜细胞,以流式细胞仪及荧光显微镜检测转染效率。[结果]HA/CS/pDNA纳米粒多呈球形,粒径平均为(142.5±20.3)nm,表面Zeta电位平均为(25.99±8.48)mV,分散度平均为(0.283±0.089),可有效保护pDNA免受核酸酶的降解;通过调节pH值至7.5以上或加入壳聚糖酶可促使纳米粒中的pDNA释放;体外转染实验证明HA/CS/pDNA纳米粒能介导pEGFP转染软骨细胞和滑膜细胞并在细胞内表达绿色荧光蛋白,其对软骨细胞的转染能力较强,比裸pEGFP和CS/pEGFP纳米粒有更高的转染效率(P<0.05);但对滑膜细胞的基因转染效率较低,与CS/pEGFP纳米粒无明显差别(P>0.05)。[结论]复凝聚法制备的HA/CS/pDNA纳米粒是一种有效的新型非病毒基因转染载体,在体外可介导基因转染关节软骨细胞和滑膜细胞,其转染效率具有明显的细胞依赖性。

关键词(KeyWords)： 壳聚糖;透明质酸;基因转染;软骨细胞;滑膜细胞

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(编号:30600632);; 广东省科技计划基金资助项目(编号:2010B060900029);; 广东省自然科学基金资助项目(编号:S2011010004808)

作者(Author): 吕璐璐;卢华定;陆慧琼;张富程;赵慧清;

Email:

DOI:

参考文献(References)：

• [1]Steinert AF,Noth U,Tuan RS.Concepts in gene therapy for cartilage re-pair[J].Injury,2008,1:97-113.
• [2]Mao S,Sun W,Kissel T.Chitosan-based formulations for delivery ofDNA and siRNA[J].Adv Drug Deliv Rev,2010,62:12-27.
• [3]汪大彬,文益民,蓝旭,等.聚糖-藻酸盐多通道支架材料细胞相容性研究[J].中国矫形外科杂志,2010,10:836-839.
• [4]Jayakumar R,Chennazhi KP,Muzzarelli RAA,et al.Chitosan conjugatedDNA nanoparticles in gene therapy[J].Carbohydrate Polymers,2010,79:1-8.
• [5]Lu HD,Zhao HQ,Wang K,et al.Novel hyaluronic acid-chitosan nano-particles as non-viral gene delivery vectors targeting osteoarthritis[J].Int J Pharm,2011,420:358-365.
• [6]Campo GM,Avenoso A,Campo S,et al.Small hyaluronan oligosaccha-rides induce inflammation by engaging both toil-like-4 and CD44 re-ceptors in human chondrocytes[J].Biochem Pharmacol,2010,80:480-490.
• [7]Chow G,Knudson CB,Homandberg G,et al.Increased expression ofCD44 in bovine articular chondrocytes by catabolic cellular mediators[J].J Biol Chem,1995,270:27734-27741.
• [8]Li SD,Huang L.Targeted delivery of antisense oligodeoxynucleotide andsmall interference RNA into lung cancer cells[J].Mol Pharm,2006,3:579-588.
• [9]Zhang X,Yu C,Xu S,et al.Direct chitosan-mediated gene delivery tothe rabbit knee joints in vitro and in vivo[J].Biochem Biophys ResCommun,2006,341:202-208.
• [10]de la Fuente M,Seijo B,Alonso MJ.Novel hyaluronic acid-chitosannanoparticles for ocular gene therapy[J].Invest Ophthalmol Vis Sci,2008,49:2016-2024.
• [11]张晓玲,史须,于长隆,等.Chitosan-DAN介导关节基因转移的体外研究[J].中国运动医学杂志,2003,22:240-243.
• [12]Douglas KL,Piccirillo CA,Tabrizian M.Cell line-dependent internali-zation pathways and intracellular trafficking determine transfection effi-ciency of nanoparticle vectors[J].Eur J Pharm Biopharm,2008,68:676-687.
• [13]O’Rorke S,Keeney M,Pandit A.Non-viral polyplexes:scaffold media-ted delivery for gene therapy[J].Prog Poly Sci,2010,35:441-458.
• [14]Liang DC,Liu WG,Zuo AJ,et al.Pre-deliver chitosanase to cells:anovel strategy to improve gene expression by endocellular degradation-induced vector unpacking[J].Int J Pharm,2006,314:63-71.
• [15]曾春,蔡道章,全大萍,等.TGF-β1壳聚糖缓释微球的制备和体外检测[J].中山大学学报:医学科学版,2005,26:347-350.