Inhibition of corneal neovascularization by tissue factor pathway inhibitor 2 gene transfection in situ / 中国组织工程研究

ABSTRACT

BACKGROUND: Some studies have demonstrated that the degradation of extracellular matrix (ECM), which matrix metalloproteinases (MMPs) participates in, plays a key step in the corneal neovascularization (CNV). Tissue factor pathway inhibitor 2 (TFPI-2), a new type serine proteinase inhibitor found recently, can effectively inhibit the activity of MMPs. Whether TFPI-2 gene transfection can influence CNV is unclear.OBJECTIVE: To investigate the effect of TFPI-2 gene transfection on CNV.DESIGN: Randomized controlled experiment.SETTING: Laboratory for Department of Surgery, Wuhan Union Hospital; Central Laboratory, the Affiliated Third Hospital of Sun Yat-sen University.MATERIALS This study was carried out in the laboratory for Department of Surgery of Wuhan Union Hospital and State Central Laboratory of the Third Hospital Affiliated to Sun Yat-sen University between June 2004 and March 2006. Sixty healthy purebred adult New Zealand rabbits of either gender, weighing 2.5 to 3.0 kg, were involved. Preoperatively, no obvious anterior segment ocular lesion was found by slit-lamp examination. pBos-Cite-neo/TFPl-2 was kindly gifted by Dr. Zhong Ren (Department of Hematology, Union Hospital). Peroxydase blocking agent, nonimmune goat serum,mouse anti-human MMP-1, 2 and 3 monoclonal antibodies, biotin labeled goat-anti-mouse IgG second antibody (Santa cruz Company) were used in this study.METHODS: Experimental intervention Experimental rabbit models of CNV were created in each group by silver nitrate cautery. Then, the rabbit models were randomized into 3 groups and 20 rabbits for each group. Different reagents were subconjunctivally injected via many points in each group saline in the group Ⅰ, empty vector in the group Ⅱ, plasmid encoding TFPI-2 in the group Ⅲ. Experimental evaluation CNV growth was observed under the slit-lamp biomicroscope.The expression of TFPI-2 in each rabbit model was detected by reverse transcription-polymerase chain reaction (RT-PCR) method 2 weeks after modeling; the expression of MMPs in corneal tissue was detected by immunohistochemical method at 3,5,7,9 and 14 days after modeling.TFPI-2 gene expression was significantly higher in the group Ⅲ than in the group Ⅱ and group Ⅰ (P ＜ 0.01); The MMP-1, 2, 3 expressions in the corneal tissue were significantly lower in the group Ⅲ than in the group Ⅱ and group Ⅰ,respectively, especially MMP-1, 3.