影像科学与光化学 ›› 2018, Vol. 36 ›› Issue (6): 467-477.DOI: 10.7517/issn.1675-0475.180503

• 论文 • 上一篇    下一篇

细胞自发光探针载基因量子点的制备和成像研究

袁晨燕, 安艳丽   

  1. 东南大学 附属中大医院, 江苏 南京 210009
  • 收稿日期:2018-05-29 修回日期:2018-07-28 出版日期:2018-11-15 发布日期:2018-11-15
  • 通讯作者: 袁晨燕
  • 基金资助:
    国家自然基金青年基金项目(81501525)资助

Preperation of Gene-loaded Quantum Dots as Self-illuminating Probes for Cell Imaging

YUAN Chenyan, AN Yanli   

  1. Affiliated Zhong Da Hospital of Southeast University, Nanjing 210009, Jiangsu, P. R. China
  • Received:2018-05-29 Revised:2018-07-28 Online:2018-11-15 Published:2018-11-15

摘要: 本研究的目的是验证量子点携带的荧光素酶在细胞内表达后,能否同时激发作为基因载体的量子点,从而构建基于量子点的活细胞自发光探针。利用壳聚糖修饰量子点构建基因运送载体CS-Qdots并对其进行表征;检测CS-Qdots能否被生物发光细胞在宏观距离上激发;并利用CS-Qdots运送真核细胞荧光素酶报告基因质粒pCMV-luciferase至肝癌细胞内,检测虫荧光素酶基因表达后生物发光波长的改变,在体内外成像中证明CS-Qdots是否被生物发光的能量激发。结果显示:制备的CdTe量子点和壳聚糖修饰后的CS-Qdots纳米颗粒在透射电镜下表现为分散均匀的晶体和球菌,CdTe量子点直径约为5 nm,CS-Qdots纳米颗粒直径约为20~30 nm。动态光散射分析显示,CdTe量子点和CS-Qdots纳米颗粒水合粒子直径分别为102.7±4.4 nm和583.0±13.7 nm。壳聚糖修饰后的量子点表面出现壳聚糖特有的基团,表面电荷也由-16.31 mV转为28.02 mV。光谱分析显示量子点具有很宽的吸收光谱和窄而对称的发射光谱。pCMV-luciferase质粒DNA和CS-Qdots结合后,凝胶阻滞实验显示,当CS-Qdots:pCMV-luciferase>10:1(质量比)时,质粒可与纳米颗粒完全结合。在体外发光细胞激发荧光实验中,CS-Qdots纳米颗粒可以被接种在细胞培养板上的生物发光细胞直接激发。以CS-Qdots作为基因载体运送pCMV-luciferase至肿瘤细胞内表达,在体内外的生物发光成像实验中,均证明成像信号的最大发射波长从生物发光的峰值560 nm跃迁至量子点的发射峰值630 nm,这一结果间接证实了在细胞内表达的生物发光信号可以直接激发作为基因载体的CS-Qdots纳米颗粒。本研究证实CS-Qdots可被细胞内生物发光能量以非结合的形式激发,载基因量子点纳米颗粒pCMV-luciferase/CS-Qdots可作为活细胞自发荧光成像的探针。

关键词: 光学成像, 量子点, 生物发光, 自发光

Abstract: To identify whether the CS-Qdots could act as gene carriers and self-illuminating probes simultaneously as delivering luciferase genes to cells. CdTe quantum dots were prepared and coated with chitosan to construct CS-Qdots nanoparticles. Then the CdTe and CS-Qdots nanoparticles were characterized; the excitation of CS-Qdots tested by bioluminescence in vivo. The reporter genes pCMV-luciferase were deliveried into cells by CS-Qdots and the wavelength of bioluminescence was tested to identify the pCMV-luciferase gene loaded CS-Qdots could act as the gene nanocarriers and self-illuminating probes simultaneously. The morphology of CdTe quantum dots and CS-Qdots nanoparticle were observed by TEM and were found to be spherical and good dispersion. The size of the fluorescent crystal was about 5 nm and the CS-Qdots nanoparticles were about 25-30 nm. When CdTe quantum dots were encapsulated by chitosan, the characteristic peaks of chitosan were observed on the FTIR spectra. The surface charge of CdTe-Qdots was -16.31±0.91 mV and then turned to a positive value of 28.02±1.15 mV. Dynamic light scattering measurements showed that the mean hydrodynamic diameter of CdTe quantum dots was 102.7±4.4 nm, and the mean hydrodynamic diameter of CS-Qdots nanoparticles was 583.0±13.7 nm. Spectral analysis showed that the CdTe quantum dots have wide absorption spectrum and the emission spectrum was narrow and symmetrical. By agarose gel electrophoresis, the plasmid pCMV-luciferase binding affinity of CS-Qdots was tested and the complete retardation was observed over the particle/DNA weight ratio of 10:1. In vivo test CS-Qdots could be directly excited by bioluminescent cells surrounded. When cells were transfected with pCMV-luciferase DNA by using CS-Qdots as gene carriers in vivo or in vitro, the emission peak of bioluminescence could red-shift from 560 to 630 nm, which means the CS-dots could be activated by bioluminescence. It is concluded that the luciferase gene-loaded CS-Qdots could act as wavelength-tunable self-illuminating probes and improve tumor optical molecular imaging.

Key words: optical imaging, quantum dots, bioluminescence, self-illuminating