中文摘要：

目的评价聚酰胺-胺型树枝状高聚物（PAMAM-dendrimer，PAMAM-D）作为靶向表皮生长因子受体（EGFR）的荧光标记反义寡核苷酸（ASODN）转染人脑恶性胶质瘤体外细胞系U251的可行性及优化方案。方法应用电泳结合实验筛选PAMAM-D与反义EGFR寡核苷酸最佳结合N／P比值，通过荧光显微镜动态观察、逆转录-聚合酶链反应（RT-PCR）评价PAMAM-D转染相同剂量ASODN至人脑恶性胶质瘤体外细胞系U251的效果，噻唑蓝（MTT）染色法间接反映各转染试剂的细胞毒性。结果（1）电泳结合实验显示电荷比（N／P）大于16：1时ODN才能与PAMAM完全结合。（2）动态荧光显微镜观察发现PAMAM-D-ASODN呈颗粒状分布在细胞质内，而oligofectamin-ASODN形态均匀。（3）RT-PCR结果显示PAMAM-D和oligofectamin介导的靶向EGFR的ASODN转染的U251细胞的EGFR表达明显下调；MTT法分析证明PAMAM的细胞毒性与oligofectamin的基本相当（P〉0．05），PAMAM-D-ODN的毒性随电荷比的增大而增大。结论PAMAM-D可以作为寡聚核苷酸的投递载体成功转染人脑胶质瘤细胞。

英文摘要：

Objective To investigate the feasibility and optimize proposal of PAMAM-D as a gene carrier to delivery antisense oligodeoxynucleotide （ASODN） targeting EGFR on human U251 giloma cell line. Methods The PAMAM-D and ASODN were combined in different N/P ratios over electricity effect to screen the optimized N/P ratio by using the electrophoresis. Equal dose of ASODN was transfected into human U251 giloma cell line to determine the transfection effect of PAMAM-D. Dynamic fluorescent microscopic observation, RT-PCR and MTT method were conducted to compare the transfection difference. Results （ 1 ） The electrophoresis combination showed that the PAMAM-D could completely combine with ODN when charge ratio was beyond 16： 1 ; （2） Dynamic fluorescent microscopic observation revealed that the PAMAM-D-ASODN was distributed in particle in cytoplasm while the oligofectamin- ASODN was well-distributed; （3） RT-PCR demonstrated that the EGFR expression of U251 cells transfected with PAMAM-D and oligofectamin mediated targeting EGFR, was down -regulated remarkably. MTT proved that cell cytotoxicity of PAMAM was equel to that of oligofectamine. Conclusion PAMAM-D was a superior carrier to transfect ODN into human U251 giloma cell.