【摘要】 目的:检测转铁蛋白能否增强新型靶向性非病毒载体K16GRGDSPC寡肽介导外源基因对骨髓基质干细胞的转染和表达,优化寡肽载体的转染条件,为下一步利用寡肽载体构建载基因基质材料进行骨缺损修复提供实验依据。方法:实验于2004-10/2005-01在协和医院骨科实验室完成。①用固相合成法合成K16GRGDSPC寡肽并用质谱仪和高压液相色谱仪进行检测。②新西兰大白兔麻醉后无菌条件下自股骨大转子处抽取骨髓2mL,1500g离心收集细胞,加入含体积分数0.2新生牛血清的DMEM混悬细胞于培养瓶中常规培养。48h后更换培养基,除去未贴壁细胞,细胞生长至85%融合后传代。③应用转化生长因子真核表达载体pcDNA3-TGFβ1和增强型绿色荧光蛋白真核表达载体pcDNA3-EGFP,观察转铁蛋白对K16GRGDSPC寡肽介导上述两种质粒转染骨髓基质干细胞的转染及表达效率的影响。结果:①寡肽的分子量和纯度检测:质谱图显示肽平均分子质量为2741.3307m/z,与理论上计算设计合成肽的平均分子质量一致,高压液相色谱仪示合成肽的纯度为94%～95%。②转铁蛋白、寡肽载体、质粒DNA三者形成复合物的电泳鉴定结果:将1μg寡肽载体分别与10μg或20μg转铁蛋白及2μgpcDNA3-TGFβ1混合后电泳,结果显示三者混合物的电泳迁移率均低于不加转铁蛋白的空白对照,且加20μg转铁蛋白较加10μg转铁蛋白的电泳迁移率更低。说明带正电荷的转铁蛋白与寡肽载体和质粒DNA形成了复合物。③转铁蛋白增加K16GRGDSPC寡肽转染效率:2μgpEGFP质粒与6μgK16-GRGDSPC寡肽加5～45μg转铁蛋白转染骨髓基质干细胞,转染效率随转铁蛋白浓度的增加而逐步提高,在25μg时转染效率达到最大,进一步增高转铁蛋白浓度,转染效率并不增加。④转化生长因子β1活性检测结果:体外以6μgK16GRGDSPC寡肽加25μg转铁蛋白介导2μgpcDNA3-TGFβ1转染骨髓基质干细胞,转染72h后测得明显高于未加转铁蛋白的转化生长因子β1的表达量[(64.4±4.3),(48.6±3.5)μg/L,P<0.01]。结论:转铁蛋白能与载体K16GRGDSPC和质粒DNA形成复合物,且显著增强K16GRGDSPC寡肽介导外源基因在骨髓基质干细胞中的转染和表达。更多还原

【Abstract】 AIM: To detect the enhancement of oligopeptide K16GRGDSPC-mediated gene transfection and expression in bone marrow stromal cells by transferrin and optimize the conditions for gene transfection, so as to provide experimental foundations for next studies of constructing gene activated bone matrix scaffolds to restore bone defect. METHODS: The experiment was conducted at the laboratory for Department of Orthopedics of Union Hospital from October 2004 to January 2005. ①Oligopeptide K16GRGDSPC was synthesized with solid-phase batch peptide synthesizer and was detected with mass spectrograph and high performance liquid chromatography. ②Under aseptic condition, bone marrow of 2 mL was extracted from the greater trochanter of femur of the anesthetized New Zealand rabbits , and then the cells were collected after centrifugation of 1 500 g.DMEM containing 0.2 volume fraction of new-born fetal bovine serum was added in the culture flask for routine culture. Culture medium was changed 48 hours later and the unattached cells were removed. Passage was performed when 85% cells confluenced. ③Eukaryotic expressing vector containing enhanced green fluorescent protein (pcDNA3-EGFP)and transforming growth factor β1 (pcDNA3-TGFβ1) were used to detect the effect of transferrin on the efficiency of gene transfection and expression in bone marrow stromal cells mediated by the two above-mentioned plasmids.RESULTS: ① Molecular weight of oligopeptide and purity detection : Mass spectrometry showed the average molecular weight of the peptide was 2 741.3307 m/z, which was just the average molecular weight of predesigned peptide. High performance liquid chromatography showed the purity was about 94% to 95%. ②The electrophoresis identification results of composite of transferrin, oligopeptide vector and plasmid DNA : Electrophoretic mobility of the composite of 1 μg oligopeptide vector, 2 μg pcDNA3-TGFβ1 and 10 μg or 20 μg transferrin was lower than that of the control group . And 20 μg transferrin added resulted in smaller mobility as compared and 10 μg transferrin added, suggesting that transferrin with positive charge and oligopeptide vector and plasmid DNA formed composite. ③Transferrin enhanced the transfection efficiency of oligopeptide K16GRGDSPC:The transfection efficiency of 2 μg pEGFP plasmid, 6 μg oligopeptide K16GRGDSPC and 5-45 μg transferrin was increased gradually with the increase of the level of transferrin, reached the top when the level of transferrin was 25 μg . The transfection efficiency was not increased when the level of transferrin was further enhanced. ④ Detected results of transforming growth factor : 6 μg K16GRGDSPC oligopeptide and 25 μg transferrin mediated 2 μg pcDNA3-TGF β1 was used to transfect bone marrow stromal cells , after transfection for 72 hours, the expression of transforming growth factor β1 was significantly higher as compared with non- transferrin added (64.4±4.3),(48.6±3.5) μg/L,P < 0.01.CONCLUSION: Transferrin , vector K16GRGDSPC and plasmid DNA can form a complex and transferrin can significantly increase oligopeptide K16GRGDSPC-mediated gene transfection and expression in bone marrow stromal cells .更多还原