title: Effect of vascular endothelial growth factor 165 gene transfection on bone defects and its mRNA expression in rabbits.
abstract: BACKGROUND
  Gene therapy has been a hot spot in repair of bone defects in recent years. This study aimed to construct a recombinant plasmid pcDNA3.1-VEGF(165), and to observe the effect of vascular endothelial growth factor 165 (VEGF(165)) gene therapy on bone defects in rabbits.
  
  
  METHODS
  Total RNA was extracted from rabbit bone tissues. VEGF(165) cDNA fragment was prepared by reverse transcription and the gene was cloned by polymerase chain reaction (PCR). Plasmid pMD18-T/VEGF(165) combined with pcDNA3.1 was cloned to reconstruct pcDNA3.1-VEGF(165) plasmid. Thirty New Zealand white rabbits weighing (2.50 +/- 0.13) kg were used to establish models of bone defects (1 cm in length) of the bilateral radii. The bone defects were repaired with absorbable gelatin sponge. After the operation, physiological sodium chloride solution was injected into the injured site in one of the forelegs of the rabbits as the control group, and pcDNA3.1-VEGF(165) plasmid (0.2 ml, 200 ng) was injected into the opposite foreleg as the experiment groups. At weeks 1, 2, 4, 6, 8, and 12 after the treatments, the bones were examined by X-ray, and the specimens of the bone defects were collected, stained with HE, and observed under a light microscope. The expression of VEGF(165) mRNA was examined by real-time quantitative polymerase chain reaction (RQ-PCR).
  
  
  RESULTS
  The pcDNA3.1-VEGF(165) plasmid with a correct sequence was constructed successfully. Postoperative X-ray found no difference between the two groups at week 1. In the experiment group, callus and synostosis were observed after 2 weeks, and osteosis structure was normal at week 12; these phenomena occurred much later in the control group. In the experiment group, HE staining showed a large amount of newly formed blood vessels after 2 weeks, a number of bone trabeculae with osteoblasts proliferation at 4 weeks, and fresh bone cortex and reformed medullary cavity at 12 weeks; whereas in the control group these structures formed in later phases. The VEGF(165) mRNA in the experiment group was expressed at a low level at week 1, reached the peak at weeks 3, and then decreased to a normal level after 6 weeks.
  
  
  CONCLUSIONS
  Local use of pcDNA3.1-VEGF(165) plasmid at bone defects can upregulate the expression of VEGF(165) and accelerate the formation of capillaries and the repair of bone defects. Angiogenesis and osteogenesis can be promoted by a combination of pcDNA3.1-VEGF(165) and gelatin sponge.
authors.affiliation: Department of Orthopaedics, Shandong University, Jinan 250033, China.
authors.forename: Dong-mei
authors.initials: DM
authors.lastname: Zhao
authors.forename: Hai-bin
authors.initials: HB
authors.lastname: Wang
authors.forename: Jia-Feng
authors.initials: JF
authors.lastname: Yang
authors.forename: Shi-qing
authors.initials: SQ
authors.lastname: Wu
authors.forename: Jun-li
authors.initials: JL
authors.lastname: Liu
authors.forename: Fu-yu
authors.initials: FY
authors.lastname: Xu
authors.forename: Li-ping
authors.initials: LP
authors.lastname: Qiu
authors.forename: Jing-long
authors.initials: JL
authors.lastname: Cai
mesh_terms: D000818:Animals; D001847:Bone Diseases / Q000000981:diagnostic imaging / Q000628:therapy*; D015316:Genetic Therapy; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D011859:Radiography; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A