靶向性非病毒载体K16GRGDSPC寡肽的合成及转染骨髓基质干细胞的研究  

Synthesis of a novel targeted-nonviral vector K16GRGDSPC oligopeptide and gene delivery to bone marrow stromal cells mediated by the peptide

作　　者：郭晓东[1] 潘海涛[1] 郑启新[1] 杨述华[1] 邵增务[1] 刘勇[1] 李长文[1] 宋玉林[1] 袁泉[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,武汉430022

出　　处：《中华实验外科杂志》2006年第9期1103-1105,共3页Chinese Journal of Experimental Surgery

摘　　要：目的设计合成新型靶向性非病毒载体K16GRGDSPC寡肽,探讨其作为载体介导外源基因转染骨髓基质干细胞(BMSCs)的可行性。方法用固相合成法合成K16GRGDSPC并用质谱仪和高压液相色谱仪进行检测。以其作为载体转染增强型绿色荧光蛋白质粒pcDNA3-EGFP并计数转染效率；转染转化生长因子(TGF)-β1质粒pcDNA3-TGF-β1并通过逆转录-聚合酶链反应(RT-PCR)和TGF-β1敏感细胞株水貂肺上皮细胞(MV1)生长抑制法检测目的基因TGF-β1稳定表达情况及表达产物的生物学活性。结果质谱图显示合成肽的平均分子量为2741．3307 m/z,与理论上计算的平均分子量一致。高压液相色谱仪示合成肽的纯度为94%～95%,满足试验需求。以K16GRGDSPC寡肽为载体转染BMSCs的效率为(18．6±2．1)%,与脂质体(Lipofectamine)介导转染的效率(19．3±2．3)%差异无统计学意义(P＞0．05)；转染TGF-β1基因阳性细胞克隆经RT- PCR扩增有特异性产物,TGF-β1敏感细胞株MV1的增殖受到明显抑制,与对照组比较差异有统计学意义(P＜0．05)。结论所合成的肽是设计的K16GRGDSPC寡肽,以其为载体介导外源基因转染BMSCS是确实可行的,为下一步构建载基因仿生基质材料奠定了基础。Objective To design and synthesize a novel targeted-nonviral vector K16GRGDSPC oligopeptide and investigate the feasibility of gene delivery into bone marrow stromal cells （BMSCs） mediated by this novel peptide, Methods The oligopeptide was synthesized by the method of solid-phase synthesis and examined by mass spectrometer （MS） and high pressure liquid chromatography （HPLC）. Using the peptide as vector the plasmids encoding enhanced green fluorescent protein （pcDNA3-EGFP） and transforming growth factor-beta 1 （pcDNA3-TGF-β1） were separately transfected into rabbit BMSCs. The transfection efficiency of GFP gene was counted and the expression and bioactivity of TGF-β1 gene was examined by reverse transcription-polymerase chain reaction （RT-PCR） and growth inhibition of MV1 TGF-β-sensitive cells. Results MS showed the average molecular weight of synthesized peptide was 2 741. 330 7 m/z,which was identical with that of the designed peptide. HPLC revealed its purity was 94%-95 %, which satisfied the requirement of the experiments. The transfection efficiency of the oligopeptide vector was 18.6%, which had no significant difference with commercial Lipofectamine （ 19.3 % ）. RT-PCR indicated specific amplification products and the growth of MV1 was inhibited greatly as compared with control groups. Conclusion The synthesized peptide was our desired oligopeptide. It＇s sure that this peptide could be used as a novel vector to transfer foreign genes into BMSCs. These provided foundations for next construction of gene-activated bone matrix materials.

关 键 词：非病毒载体 肽类 基因表达 骨髓基质干细胞 

分 类 号：Q78[生物学—分子生物学]