新型靶向性非病毒载体K16GRGDSPC转染骨髓基质细胞的条件优化  

Condition Optimization of Transforming Growth Factor-beta 1 Transfection to Marrow Stromal Cells Mediated by a New Targeted-nonviral Vector

作　　者：潘海涛[1] 郑启新[1] 郭晓东[1] 刘勇[1] 李长文[1] 宋玉林[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：《华中科技大学学报（医学版）》2006年第3期369-372,376,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.30200063;No.3047083)

摘　　要：目的 优化新型靶向性非病毒基因载体——含RGD肽K16GRGDSPC介导转化生长因子β1（TGF-β1）基因转染骨髓基质细胞的转染条件。方法 固相合成法合成K16GRGDSPC寡肽并用质谱仪和高压液相色谱仪进行检测。在保持其它因素一致的条件下，通过变化单一因子来筛选某个最佳参数。结果 合成肽与设计肽的分子量一致，纯度为94％～95％，满足实验要求。在寡肽载体介导转染的过程中，溶酶体抑制剂氯喹起着不可或缺的作用，而且在氯喹溶液中的暴露时间至少应维持在8h以上；在载体／DNA复合物中暴露时间过短（〈4h）或过长（〉24h）对转染都有不利影响；血清对载体／DNA复合物与细胞的结合有明显抑制作用；质粒含量在2～4μg时转染效果最佳；质粒／载体质量比为1：2～1：4时有较高的转染效率；加入转铁蛋白也能显著提高基因的转染和表达，且在25μg时转染效率达到最大。结论 通过优化转染条件可提高寡肽载体的转染效率，可为下一步进行骨基质材料表面基因修饰研究提供基础。Objective To optimize the transfection conditions of transforming growth factor-beta 1 （TGF-β1） gene mediated by a new targeted-nonviral vector- oligopeptide K16GRGDSPC to marrow stromal cells. Methods Oligopeptide K16GRGDSPC was synthesized by solid-phase synthesis and identified by mass spectrometer and high pressure liquid chromatography, Optimal conditions were screened out by changing single factor, Results The molecular weight of the synthesized peptide was identical with that of the designed peptide and its purity was 94 % to 95 %, which satisfied the requirement of the experiments, During the course of gene transfection mediated by the oligopeptide vector, it was found that chloroquine was essential and the time exposure to chloroquine should he maintained for at least 8 h, When the time of the cells exposure to the vector/DNA complexes was shorter than 4 h or longer than 24 h, it was adverse to the gene transfection, Serum could significantly inhibit the binding of the vector/DNA complexes to the cells, When the amount of DNA was 2-4 μg and the weight/ weight ratio of vector to DNA was 2-4 to 1, the optimal transfection results were obtained, Transferrin also increased the gene transfection and gene expression and the optimal transfection efficiency was obtained when transferrin was added with an amount of 25 μg, Conclusion The transfection efficiency mediated by the new oligopeptide vector could be enhanced by optimizing the transfection conditions. It would provide foundations for next research on gene modification on bone matrix materials,

关 键 词：靶向性非病毒载体 含RGD肽 转染 优化 

分 类 号：Q789[生物学—分子生物学]