一种新型穿膜肽的固相合成及体外生物学特性研究-第三军医大学学报.doc

新型穿膜肽作为基因运输载体的实验研究[基金项目] [基金项目]国家自然科学基金青年科学基金项目；国家自然科学基金面上项目 [通信作者]任建丽，E-mail: renjianli_1977@163.com 田 菊，王志刚，任建丽，张清凤 （400010重庆，重庆医科大学超声影像学研究所） [摘要] 目的 人工设计合成穿膜肽新序列（CGRLWMRWYSPWARRYGC），并于体外考察其作为基因载体的潜力。方法 采用Nа-芴甲氧羰基 (Fmoc) 法固相合成穿膜肽新序列，并用异硫氰酸荧光素（FITC）标记N端。高效液相色谱及质谱仪检测其纯度及相对分子质量，流式细胞仪及共聚焦显微镜观察其穿膜能力，荧光显微镜和流式细胞仪观察其携带增强型绿色荧光蛋白质粒( pEGFP )进入细胞表达情况，MTT评价穿膜肽与质粒复合物的细胞毒性，琼脂糖凝胶电泳检测穿膜肽与质粒的亲和力及对质粒的保护作用。结果 合成的穿膜肽纯度为96.13%，相对分子质量为2 348.42，流式细胞仪检测显示荧光阳性细胞平均约占98.81%，共聚焦显微镜可见荧光标记的穿膜肽分布于细胞内；穿膜肽携带质粒在人脐静脉内皮细胞中的表达率平均约为11.44%；不同电荷比的复合物对细胞的活性没有明显影响（P＞0.05﹚；琼脂糖凝胶电泳显示当穿膜肽与质粒的电荷比大于2:1时，质粒不能迁移出来，电荷比为4:1、8:1和16:1的复合物能够抵抗DNA酶 I的消化作用，以8:1最为明显。结论 人工设计合成的新型穿膜肽能够作为一种潜在的基因运输载体。 [关键词]固相合成；穿膜肽；基因载体 【中图法分类号】 【文献标志码】A Study on a new cell-penetrating peptides as gene vector Tian Ju, Wang Zhigang, Ren Jianli, Zhang Qingfeng （Institute of Ultrasound Imaging,Chongqing Medical University,Chongqing，400010,China） [Abstract] Objective To design and synthesize a new sequence (CGRLWMRWYSPWARRYGC)of cell-penetrating peptides(CPPs) and to investigate its potentiality as gene vector in vitro. Method The new sequence of CPPs was synthesized by the solid-phase synthetic method with florenl methyoxycarbonyl amino acids. N-terminal fluorescein isothiocyanate-labeled peptide was also synthesized. The purity and molecular weight of CPPs were determined using HPLC and MALDI-TOF-MS. The cell-penetrating activity was examined using flow cytometry and confocal laser scanning microscopy. Transfection effect of CPPs-mediated enhanced green fluorescence protein plasmid (pEGFP) in HUVEC was observed using fluorescent microscopy and flow cytometry. The cytotoxicity of CPPs/plasmid DNA compound was evaluated by MTT assay. The affinity of plasmid DNA and CPPs and the protection of CPPs peptide to plasmid DNA were analyzed by agarose gel electrophoresis. Results The purity of synthesized CPPs was 96.13%, and molecular weight was 2348.42. The results of flow cytometry showed that about 9