体外培养小脑浦肯野神经元转染及条件的优化

Experimental study on optimization of transfection on Purkinje cells of cerebellum in vitro

目的探讨用化学转染试剂体外转染小脑浦肯野神经元的最佳转染条件。方法体外分离培养E18 d的昆明小白鼠的小脑浦肯野神经元,用lipofectamine^(TM)2000、磷酸钙沉淀试剂、Effectene转染试剂包被质粒分别转染体外培养7 d的浦肯野神经元。另外,用lipofectamine^(TM)2000包被质粒,分别转染7、14、21 d以及28 d浦肯野神经元。每组分为5个样本。48 h后观察绿色荧光蛋白的表达情况,并用台盼兰检测细胞的存活率。结果(1)3种转染试剂的最高转染率分别为lipofectamine^(TM)2000(19.63±5.76)%、磷酸钙沉淀法(1.84±1.85)%、Effectene(1.48±1.38)%。lipofectamine^(TM)2000与其他两种方法的转染率均有显著差异。(2)2μl的lipofectamine^(TM)2000的转染效率为(15.11±4.01)%,细胞存活率与对照组没有明显差异,为(92.98±2.10)%。(3)培养早期(7 d、14 d)的浦肯野神经元损伤小,与对照组没有明显差异。结论该实验首次证实了lipofectamine^(TM)2000能有效地转染体外培养的小脑浦肯野神经元。不同的发育阶段对转染效率也有较大的影响,lipofectamine^(TM)2000比较适合早期的浦肯野神经元转染。

Objective To discuss the best condition of chemical transfection agents for transfection on Purkinje cells of cerebellum in vitro. Methods Cerebellum Purkinje cell of Kun Ming mice at embryo 18 d were isolated and cultured in vitro. Plasmid covered with lipofectamineTM 2000, calcium phosphate precipitation reagents, effectene transfection reagents were transfected to cerebellum Purkinje cell after cultured 7 d in vitro respectively. At the same time, plasmid covered by lipofectamineTM 2000 was transfected to Purkinje cells after having been cultured for 7 d, 14 d, 21 d and 28 d respectively. Each group had 5 samples. Expression of green fluorescent protein was observed after 48 h of transfection, and the survival rate of Purkinje cell was checked by trypan blue. Results （ 1 ） Maximal transfection rates were （ 19. 63 ± 5.76） % for lipofectamine^TM2000, （ 1.84 ± 1.85 ） % for calcium phosphate transfection, （ 1.48 ±1. 38 ） %for effectene transfection. Method of lipofectamine^TM2000 appeared obvious different as compared with other methods. （2） Transfection rate of 2 μl lipofectamine^RM2000 amounted to （15.11 ± 4.01 ）%, survival rate were （92.98 ±2.10）% which had no obvious difference compared with contrast group. （3） Purkinje cell got less damage in earlier phase （7 d, 14 d）, there was no differences as compared with contrast groups. Conclusion This experiment certifies first time lipofectamineTM 2000 can transfect Purkinje cells of cerebellum in vitro. Different culture phase has certain influence on transfection rate. Lipofectamine^TM2000 is more suitable for transfection on Purkinje cells at earlier phases.