Haitao Pan, Qixin Zheng, Xiaodong Guo
Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
J Pept Sci. 2007 Mar;13(3):154-63. doi: 10.1002/psc.826.
A 23-amino acid, bifunctional, integrin-targeted synthetic peptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). The peptide (K)(16)GRGDSPC consists of an amino terminal domain of 16 lysines for electrostatic binding of DNA, and a 7-amino acid integrin-binding domain at the carboxyl terminal. PcDNA3-EGFP plasmids were transfected into BMSCs by (K)(16)GRGDSPC and the positive cells gave out a bright green fluorescence. High levels of gene delivery of pcDNA3-TGF-beta1 plasmids were obtained with 2 to 4 microg/ml DNA concentration, with (K)(16)GRGDSPC at an optimal peptide: DNA w/w ratio of 3:1, with a required exposure time of more than 4 h but shorter than 24 h for BMSC exposure to the peptide/DNA complexes with completely absent serum in the initial stage; with 100 microM chloroquine and at least 8 h exposure for BMSC exposure to chloroquine; with a fusogenic peptide at an optimal (K)(16)GRGDSPC/DNA/fusogenic peptide w/w ratio of 3:1:5; and with Lipofectamine 2000 at an optimal (K)(16)GRGDSPC/DNA/Lipofectamine 2000 w/w ratio of 3:1:2 at a constant DNA concentration of 2 microg/ml. Chloroquine, the fusogenic peptide and Lipofectamine 2000 all significantly promoted gene delivery, but chloroquine was more effective than the fusogenic peptide and had obvious synergistic effects with Lipofectamine 2000. Under optimal conditions, TGF-beta1 gene was transfected into BMSCs without observable toxicity, and the stable expression was examined by RT-PCR and Western blot analysis. The stable transgenic cells showed obvious bands. This novel synthetic peptide, providing a new way for the use of polylysine and RGD motif in DNA vector system, is potentially well suited to ex vivo gene delivery to BMSCs for experimental and clinical applications in the field of bone tissue engineering.
对一种含23个氨基酸的双功能整合素靶向合成肽进行了评估，用于兔骨髓基质细胞（BMSC）的离体基因递送。该肽（K）（16）GRGDSPC由16个赖氨酸的氨基末端结构域用于DNA的静电结合，以及羧基末端的7个氨基酸的整合素结合结构域组成。通过（K）（16）GRGDSPC将PcDNA3 - EGFP质粒转染到BMSC中，阳性细胞发出明亮的绿色荧光。在DNA浓度为2至4μg/ml时，以（K）（16）GRGDSPC在肽与DNA的最佳重量比为3:1时，可实现pcDNA3 - TGF -β1质粒的高水平基因递送，BMSC与肽/DNA复合物接触所需的暴露时间超过4小时但短于24小时，且初始阶段完全无血清；在BMSC接触氯喹时，使用100μM氯喹且暴露至少8小时；在（K）（16）GRGDSPC/DNA/融合肽的最佳重量比为3:1:5时使用融合肽；在DNA浓度恒定为2μg/ml时，以（K）（16）GRGDSPC/DNA/Lipofectamine 2000的最佳重量比为3:1:2时使用Lipofectamine 2000。氯喹、融合肽和Lipofectamine 2000均显著促进基因递送，但氯喹比融合肽更有效，且与Lipofectamine 2000有明显的协同作用。在最佳条件下，TGF -β1基因被转染到BMSC中且未观察到毒性，并通过RT - PCR和蛋白质印迹分析检测到稳定表达。稳定的转基因细胞显示出明显的条带。这种新型合成肽为在DNA载体系统中使用聚赖氨酸和RGD基序提供了一种新方法，可能非常适合用于骨组织工程领域的实验和临床应用中BMSC的离体基因递送。
