生物活性玻璃纤维的基因转染研究

doi:10.15541/jim20140074

无机材料学报 ›› 2014, Vol. 29 ›› Issue (10): 1023-1028.DOI: 10.15541/jim20140074 CSTR: 32189.14.10.15541/jim20140074

生物活性玻璃纤维的基因转染研究

刘惠^1,2,3, 陈晓峰^1,2,3, 李贤^1,2,3, 李玉莉^1,2,3, 林泽枫^1,2,3

(1. 华南理工大学材料科学与工程学院, 广州 510640; 2. 国家人体组织功能重建工程技术研究中心, 广州 510006; 3. 广东省生物医学工程重点实验室, 广州 510006)

收稿日期:2014-02-19 修回日期:2014-04-03 出版日期:2014-10-20 网络出版日期:2014-09-22
作者简介:刘惠(1989–), 女, 硕士. E-mail: half0614@yeah.net
基金资助:
国家重点基础研究发展计划(973计划)(2011CB606204);国家自然科学基金(51172073, 51202069);高等学校博士学科点专项科研基金(20110172110002);中央高校基本科研业务费专项资金(2014ZM0009, 2013ZM004, 2012ZP0001)

Gene Transfection of Bioactive Glass Fibers

LIU Hui^1,2,3, CHEN Xiao-Feng^1,2,3, LI Xian^1,2,3, LI Yu-Li^1,2,3, LIN Ze-Feng^1,2,3

(1. School of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, China; 2. National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China; 3. Key Laboratory of Biomedical Engineering in Guangdong, Guangzhou 510006, China)

Received:2014-02-19 Revised:2014-04-03 Published:2014-10-20 Online:2014-09-22
About author:LIU Hui. E-mail: half0614@yeah.net
Supported by:
National Key Basic Research Program (973 Program) of China (2011CB606204);National Natural Science Foundation of China (51172073,51202069);Specialized Research Fund for Doctoral Program of Higher Education of China (20110172110002);Fundamental Research Funds for the Central University (2014ZM0009, 2013ZM004, 2012ZP0001)

摘要/Abstract

摘要：

生物活性玻璃由于含有钙、磷成分, 因此在基因转染方面具有应用潜能。本研究通过溶胶-凝胶结合静电纺丝技术制备了具有连续介孔或分级纳米孔的微纳米生物活性玻璃纤维(BGF), 并将其用于基因转染。实验结果显示, BGF在无血清培养基中可大量释放Ca²⁺和PO₄^3-, 这些释放的离子在沉积的同时可装载质粒DNA, 起到基因转染载体的作用, 转染效率与BGF之间具有剂量依赖性。当质粒浓度为1 μg/mL、BGF浓度为1000 μg/mL时, 细胞的转染效率可达对照组(脂质体转染试剂)的50%以上。其转染机理与传统的磷酸钙基因转染类似, 而其较好的离子溶出保障了其使用的稳定性, 有望替代纳米磷酸钙转染体系用于基因传输。

关键词: 生物活性玻璃, 纤维, 基因转染, 磷酸钙

Abstract:

Bioactive glasses (BGs) exhibit potential applications for gene transfection because of their composition including Ca and P. Here, bioactive glass fibers (BGFs) with mesopores or hierarchical nanopores, were prepared by electrospinning process using BGs Sol-Gel precursor and its effect on mediating gene transfection was investigated. The results indicate that BGF acts as a gene vector by releasing Ca²⁺ and PO₄^3-, and then reunioning them along with plasmid DNA in Dulbecco's Modified Eagle’s Medium (DMEM). BGFs have a dose-dependent manner in transfection efficiency. When using 1 μg/mL plasmid, the transfection efficiency of BGF with concentration at 1000 μg/mL is higher than 50% of lipofectamine LTX_PLUS. The transfection mechanism of BGF is similar to that of calcium phosphate (CaP) system. Furthermore, BGF’s sufficient ions releasing ensures stability and effectiveness to be applied in gene transfection, which makes BGF a promising candidate for gene delivery in replace of traditional gene carrier, the nano-calcium phosphate system.

Key words: bioactive glass, fiber, gene transfection, calcium phosphate

中图分类号:

TQ174

刘惠, 陈晓峰, 李贤, 李玉莉, 林泽枫. 生物活性玻璃纤维的基因转染研究[J]. 无机材料学报, 2014, 29(10): 1023-1028.

LIU Hui, CHEN Xiao-Feng, LI Xian, LI Yu-Li, LIN Ze-Feng. Gene Transfection of Bioactive Glass Fibers[J]. Journal of Inorganic Materials, 2014, 29(10): 1023-1028.

图/表 8

图1 生物活性玻璃纤维的制备流程图

Fig. 1 Schematic of the BGF preparation process

图2 生物活性玻璃纤维的SEM形貌(a)及其不同内部结构的TEM(b, c)以及矿化6 h后的TEM(d)照片

Fig. 2 SEM image (a) and TEM images of different inner structure (b,c) and structure after mineralization for 6 h (d) of BGFs

图3 生物活性玻璃络合质粒DNA后的琼脂糖凝胶电泳结果

Fig. 3 Agar gel electrophoresis of pAcGFP-OA after complexing with BGF A-D: BGF concentrations at 1000, 700, 400 and 100 μg/mL, respectively

图4 含绿色荧光蛋白质粒的BGF稀释不同浓度后转染293T细胞的荧光显微镜照片

Fig. 4 Fluorescent images of 293T cell after transfection with GFP plasmid-contained BGF at different concentrations a-i: BGF concentration at 100, 200, 400, 600, 800, 1000, 1300, 1650 and 2000 μg/mL, respectively;. j: Merged image from fluorescence and bright field images of 293T cell after transfection by GFP plasmid-contained BGF at 400 μg/mL; k: Image of positive control transfected by Lipofectamine. Bars in all images are 100 μm

图5 不同组成的纤维SEM照片和等量纤维转染293T细胞的荧光显微镜照片

Fig. 5 SEM images of fibers with different compositions and fluorescence microscope (FM) images of 293T cells transfected by equivalent fibers

图6 不同生物活性玻璃的SEM照片和等量材料转染293T细胞的荧光显微镜照片

Fig. 6 SEM images of different type of bioactive glasses and fluorescence microscope (FM) images of 293T cells transfected by equivalent fibers

表1 293T细胞转染效率的流式细胞仪结果

Table 1 Transfection efficiency of 293T cells measured by flow cytometry

Concentration (BGF) /(μg· mL^-1)	0	200	400	600	800	1000	600	600	0(Lipofectamine)
Amount of GFP Plasmid /(ng·mL^-1)	1	1	1	1	1	1	2	4	1
Transfection efficiency /%	0	0.12	0.68	5.74	14.40	21.30	10.40	15.70	38.30

图7 不同形貌(F纤维, S球形, I不规则)生物活性玻璃在DMEM中浸泡6 h后, 溶液中的离子浓度曲线。

Fig. 7 Ion release profiles of different types of BGs after soaking in DMEM for 6 h F: Fiber; S: microsphere; I: Irregular

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生物活性玻璃纤维的基因转染研究

Gene Transfection of Bioactive Glass Fibers

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