中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (12): 2120-2124.doi: 10.3969/j.issn.1673-8225.2012.12.007

• 纳米生物材料 nanobiomaterials • 上一篇    下一篇

转化生长因子β1基因缓释的壳聚糖纳米粒制备及体外检测***☆

卢华定，吕璐璐，赵慧清，王  昆   

  1. 中山大学附属第三医院骨科，广东省广州市  510630
  • 收稿日期:2011-08-09 修回日期:2011-10-29 出版日期:2012-03-18 发布日期:2012-03-18
  • 作者简介:卢华定☆，男，1971年生，浙江省温岭市人，汉族，博士，副教授，主要从事关节疾病基因治疗及软骨组织工程的研究。 johnnielu@ 126.com
  • 基金资助:

    国家自然科学基金资助项目(30600632)；广东省科技计划项目(2010B060900029)；广东省自然科学基金资助项目(S2011010004808)。

Preparation and examination in vitro of transforming growth factor beta 1 gene releasing chitosan nanoparticles

Lu Hua-ding, Lü Lu-lu, Zhao Hui-qing, Wang Kun   

  1. Department of Orthopaedics, the Third Affiliated Hospital of Sun Yat-san University, Guangzhou  510630, Guangdong Province, China
  • Received:2011-08-09 Revised:2011-10-29 Online:2012-03-18 Published:2012-03-18
  • About author:Lu Hua-ding☆, Doctor, Associate professor, Department of Orthopaedics, the Third Affiliated Hospital of Sun Yat-san University, Guangzhou 510630, Guangdong Province, China johnnielu@126.com
  • Supported by:

    National Natural Science Foundation of China, No. 30600632*; Science and Technology Project of Guangdong Province, No. 2010B060900029*; Natural Science Foundation of Guangdong Province, No. S2011010004808*

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摘要：

背景：壳聚糖作为一种非病毒载体，具有低毒性、低免疫原性、良好的生物相容性以及带可高正电荷密度的特性，易与带负电荷的DNA通过静电作用形成相互作用体避免核酸酶的降解。
目的：构建负载重组人转化生长因子β1基因的壳聚糖纳米粒，检测其体外缓释转化生长因子β1基因及对软骨细胞基因转染等性能。
方法：将壳聚糖与负载增强型绿色荧光蛋白基因和转化生长因子β1基因的质粒DNA(pDNA)以复凝聚法制成壳聚糖/ pEGFP-TGF-β1纳米粒。
结果与结论：制备的壳聚糖/pEGFP-TGF-β1纳米粒呈球形，粒径、表面电位与pH值相关，随着pH值升高，粒径增大，表面电位减少。纳米粒可有效保护pDNA免受核酸酶的降解。纳米粒的pDNA包封率为(87.5±2.3)%；pDNA可从纳米粒中缓慢释放。体外转染实验证实壳聚糖/pEGFP-TGF-β1纳米粒能转染软骨细胞并在细胞内表达绿色荧光蛋白。提示壳聚糖/ pEGFP-TGF-β1纳米粒能有效保护pDNA免受核酸酶降解，具有良好的缓释转化生长因子β1基因的能力，并能介导基因转染软骨细胞。
关键词：转化生长因子β1；壳聚糖；软骨细胞；组织工程；基因载体
doi:10.3969/j.issn.1673-8225.2012.12.007

关键词: 转化生长因子&beta, 1, 壳聚糖, 软骨细胞, 组织工程, 基因载体

Abstract:

BACKGROUND: As non-viral gene vectors, chitosan have the low cytotoxicity, low immunogenicity, good biocompatibility, and high positive charge density. It can easily interact with negatively charged DNA through electrostatic interactions to form chitosan-DNA nanoparticles which can prevent nuclease degradation.
OBJECTIVE: To prepare chitosan nanoparticles carrying recombinant human transforming growth factor β1 (TGF-β1) gene and to detect the sustained release efficiency and gene transfect against chondrocytes in vitro.
METHODS: The chitosan/plasmid enhanced green fluorescent protein-TGF-β1 (chitosan/pEGFP-TGF-β1) nanoparticles were prepared by a complex coacervation method with chitosan and plasmid DNA (pDNA) which loaded EGFP gene and recombinant human TGF-β1 gene.
RESULTS AND CONCLUSION: The prepared chitosan/pEGFP-TGF-β1 nanoparticles were spherical. Their particle size and Zeta potential were highly correlated with pH value. With the increase of pH value, the particle size increased and the Zeta potential decreased. The nanoparticles could effectively protect pDNA from degradation against nucleases. The encapsulation rate of pEGFP-TGF-β1 was (87.5±2.3)%; and pEGFP-TGF-β1 could be slowly released from the nanoparticles. Gene transfection in vitro proved that chitosan/pEGFP-TGF-β1 nanoparticles were efficient in transfecting chondrocytes and the expression of green fluorescent proteins was observed. Chitosan/pEGFP-TGF-β1 nanoparticles can effectively protect pDNA from nuclease degradation, have controlled release ability of TGF-β1 gene, and can mediate gene transfection against chondrocytes.

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