新型纳米载体Superfect^(TM)最适转染条件的研究

Study of the Optimized Transfection Condition of a Novel Nanoscopic Vector,Superfect^(TM)

目的:对新型纳米转染载体SuperfectTM的转染效率和细胞毒性进行初步研究,以探讨其最适的转染条件。方法:以50%融汇率的小鼠黑色素瘤细胞为靶细胞,在不同条件下将,报告基因质粒pEGFP-C1与载体Super-fectTM混合为复合物进行转染。各实验组:不同的质粒剂量(0.25μg,0.50μg)、不同的N/P(N:载体分子表面胺基的数量,P:DNA中磷酸基团的数量)比(2∶1,5∶1,10∶1)及复合物与细胞的孵育时间(3 h,6 h)。各对照组:裸质粒转染组、载体转染组,非转染组为对照。转染72 h后,流式细胞仪检测转染效率,MTT法检测细胞毒性。结果:在不同条件下,SuperfectTM的转染效率有显著性差异(P<0.05),其细胞毒性与载体剂量和孵育时间相关。当质粒剂量为0.5μg,N/P比为5∶1,复合物与细胞孵育3 h时,SuperfectTM不仅具有较高的转染效率,而且表现出较低的细胞毒性。结论:SuperfectTM是一种高效低毒的转染载体,其最适转染条件主要与N/P比及复合物与细胞的孵育时间相关。SuperfectTM在基因治疗领域将具有良好的应用前景。

Objective： To do a primary study of transfection efficiency and cytotoxicity of the novel nanoscopic vector, Superfect^TM , and to discuss its optimized transfection condition and application value in the field of gene therapy. Methods： The enhanced green fluorescent protein expressing plasmid, pEGFP-C1, was transfered into the murine melanoma cells with 50% cell confluency rate under various conditions, i. e. , different DNA doses （0.25 or 0.5.0μg）, N/P（N： number of amidocyanogens on the surface of vector, P： number of phosphate groups of DNA） ratio （2 ： 1, 5 ： 1 or 10 ： 1） and time of complex co-incubating with cells （3 or 6 h）. The control groups were composed of naked gene or vector transfection group and normal group. The efficiency of transfection was examined by flow cytometry and the cytotoxicity was evaluated with MTT assay 72 hours after transfection. Results： There was significant difference among the experiment groups （P〈0.05）. Superfect^TM showed low cytotoxicity in the groups treated with high vector dose and long time of co incubation. Superfect^TM has higher transfection efficiency and lower cytotoxicity in the group of 0.5 μg plasmid, 5 ： 1 N/P ratio and 3 hours＇ incubation with cells. Conclusion： Superfect^TM is the vector with high transfer efficiency and low cytotoxicity. Its optimized transfection condition is mainly related to N/P ratio and time of co-incubation. Superfect^TM possesses well application foreground in the field of gene therapy.