Bischoff R, Cordier Y, Perraud F, Thioudellet C, Braun S, Pavirani A
Transgene S.A., 11 rue de Molsheim, Cedex, 67082 Strasbourg, France.
Anal Biochem. 1997 Dec 1;254(1):69-81. doi: 10.1006/abio.1997.2401.
Transfection of satellite cells from dog muscle (myoblasts) in primary culture has been optimized with respect to the position of the cholesteryl moiety along the polyamine chain of spermidine or spermine. Spermidine or spermine were derivatized with cholesterylchloroformate giving rise to three isomers in the case of spermidine and two isomers for spermine that were separated by reversed-phase high-performance liquid chromatography (rp-HPLC). The position of the cholesteryl moiety was assigned by 13C-NMR and coelution with synthetic isomers of defined structure. The isomeric cationic lipids were evaluated for their transfection activity in myoblasts from dog muscle and a human lung epithelial cell line (A549) using plasmid DNA expressing the luciferase reporter gene. The results showed that the position of the cholesteryl moiety is of critical importance for efficient transfection of myoblasts in primary culture with isomers having a derivatized secondary amine being significantly more effective than those with a derivatized primary amine. On the contrary, differences in the A549 cell line were less pronounced and did not follow the same pattern. The results show that slight structural differences between cationic lipids lead to significantly different transfection efficiencies for myoblasts in primary culture. This may also represent an advantage in view of cell or organ targeting.
针对胆固醇部分沿亚精胺或精胺的多胺链的位置，对原代培养的犬肌肉卫星细胞（成肌细胞）的转染进行了优化。用胆固醇氯甲酸酯对亚精胺或精胺进行衍生化，亚精胺产生三种异构体，精胺产生两种异构体，通过反相高效液相色谱（rp-HPLC）进行分离。胆固醇部分的位置通过13C-NMR以及与确定结构的合成异构体共洗脱来确定。使用表达荧光素酶报告基因的质粒DNA，评估了这些异构阳离子脂质对犬肌肉成肌细胞和人肺上皮细胞系（A549）的转染活性。结果表明，胆固醇部分的位置对于原代培养的成肌细胞的有效转染至关重要，具有衍生化仲胺的异构体比具有衍生化伯胺的异构体明显更有效。相反，在A549细胞系中的差异不太明显，且不遵循相同模式。结果表明，阳离子脂质之间的微小结构差异导致原代培养的成肌细胞的转染效率显著不同。从细胞或器官靶向的角度来看，这也可能是一个优势。
