Musculoskeletal Research Laboratory, St. Michael's Hospital, Division of Orthopaedic Surgery, University of Toronto, Toronto, Ontario, Canada.
J Orthop Trauma. 2010 Sep;24(9):547-51. doi: 10.1097/BOT.0b013e3181ed2ad5.
Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model.
Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS.
The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point after transfection. In addition, human VEGF gene transfection increased osteoblast cell proliferation after 3 days.
These in vitro results suggest that cell-based human VEGF gene therapy is not only effective at causing human VEGF expression, but also enhances endogenous rat VEGF mRNA expression in both fibroblasts and osteoblasts, particularly the rat VEGF164 isoform.
血管内皮生长因子（VEGF）在促进骨折修复过程中的血管生成和骨生成中发挥重要作用。我们之前的研究表明，基于细胞的 VEGF 基因治疗可增强体内兔胫骨节段性骨缺损的骨愈合。本项目旨在研究外源性人 VEGF 对基于细胞的基因转染模型中内源性大鼠 VEGF 信使 RNA（mRNA）表达的影响。
从 Fisher 344 大鼠的真皮组织和骨膜中分别采集大鼠成纤维细胞和成骨细胞。然后使用 Superfect 试剂（Qiagen）将 pcDNA-人 VEGF 转染到细胞中。创建了 4 个实验组：1）成纤维细胞-VEGF；2）成骨细胞-VEGF；3）未转染的成纤维细胞对照；4）未转染的成骨细胞对照。基因转染后 1、3 和 7 天采集培养的细胞。用 Trizol（Invitrogen）提取总 mRNA；通过逆转录-聚合酶链反应测量人 VEGF 和大鼠 VEGF mRNA，并通过 VisionWorksLS 进行定量。
转染的成纤维细胞和成骨细胞在基因转染后 1、3 和 7 天可通过逆转录-聚合酶链反应检测出人 VEGF165 mRNA。人 VEGF165 的水平在第 1 天达到峰值，然后在转染的成纤维细胞和成骨细胞中逐渐降低。在该细胞培养模型中检测到两种内源性大鼠 VEGF 同工型：大鼠 VEGF120 和大鼠 VEGF164。我们比较了转染人 VEGF165 的成纤维细胞或成骨细胞与未转染对照细胞的大鼠 VEGF120 和大鼠 VEGF164 表达水平。在第 1 天（峰值）和第 3 天，转染的成纤维细胞和成骨细胞的大鼠 VEGF164 表达均高于未转染的对照细胞，但在第 7 天则不然。转染的成纤维细胞中大鼠 VEGF120 的表达较低，但转染的成骨细胞中大鼠 VEGF120 的表达较高。此外，人 VEGF 基因转染可在 3 天后增加成骨细胞的增殖。
这些体外结果表明，基于细胞的人 VEGF 基因治疗不仅有效引起人 VEGF 的表达，而且还增强了成纤维细胞和成骨细胞中内源性大鼠 VEGF mRNA 的表达，特别是大鼠 VEGF164 同工型。
