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基因治疗：, X1 Q, q: M G5 S$ \7 R
目前干细胞转染效率很低（脂质体法）。电穿孔和病毒转染可能提高转染效率，但是电穿孔对细胞活力影响大，病毒转染安全性值得考虑。于是，一些研究人员正在开发智能化的纳米材料提高基因转染效率，同时可用于示踪细胞在体内的运输。
8 c1 ~" O1 g" `0 O' F' b h 超顺磁纳米颗粒就是一种理想的载体。在转染方面，经过层层组装，形成从内到外的颗粒/质粒/特定化学分子结构。经过化学修饰过的纳米颗粒（表面包裹双亲性分子）可通过两种途径入胞。其一，直接跨膜入胞,进入胞浆，表面修饰物在胞浆内解体而暴露质粒；其二，通过细胞内吞方式入胞，但是存在被溶酶体降解的风险。在示踪方面，细胞内的磁性纳米颗粒可用于MRI成像，便于跟踪干细胞在机体内的定位。3 F- M o1 f0 E
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磁性纳米颗粒用于细胞转染文章详情可见：. m2 y( D. D7 [/ M i% X9 M9 P
http://www.nature.com/nprot/journal/v2/n10/full/nprot.2007.352.html9 c- w$ V! o7 w/ J
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Generation of magnetic nonviral gene transfer agents and magnetofection in vitro) z: |& _! A( |4 B& b7 P; F ^
Nature Protocols 2, 2391 - 2411 (2007) " R( a% K( z5 I8 {7 t/ \. g
Olga Mykhaylyk, Yolanda Sánchez Antequera, Dialekti Vlaskou & Christian Plank
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This protocol details how to design and conduct experiments to deliver nucleic acids to adherent and suspension cell cultures in vitro by magnetic force–assisted transfection using self-assembled complexes of nucleic acids and cationic lipids or polymers (nonviral gene vectors), which are associated with magnetic (nano) particles. These magnetic complexes are sedimented onto the surface of the cells to be transfected within minutes by the application of a magnetic gradient field. As the diffusion barrier to nucleic acid delivery is overcome, the full vector dose is targeted to the cell surface and transfection is synchronized. In this manner, the transfection process is accelerated and transfection efficiencies can be improved up to several 1,000-fold compared with transfections carried out with nonmagnetic gene vectors. This protocol describes how to accomplish the following stages: synthesis of magnetic nanoparticles for magnetofection; testing the association of DNA with the magnetic components of the transfection complex; preparation of magnetic lipoplexes and polyplexes; magnetofection; and data processing. The synthesis and characterization of magnetic nanoparticles can be accomplished within 3–5 d. Cell culture and transfection is then estimated to take 3 d. Transfected gene expression analysis, cell viability assays and calibration will probably take a few hours. This protocol can be used for cells that are difficult to transfect, such as primary cells, and may also be applied to viral nucleic acid delivery. With only minor alterations, this protocol can also be useful for magnetic cell labeling for cell tracking studies and, as it is, will be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized transfection efficiency in any cell type. |
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发表于 2010-4-24 17:28
发表于 2010-4-24 23:21
