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背景:有研究表明,基质金属蛋白酶所参与的细胞外基质降解在角膜新生血管形成过程中起关键作用,组织因子途径抑制物2是新近发现的一种新型的丝氨酸蛋白酶抑制物,能有效抑制基质金属蛋白酶的活性。选用组织通道途径抑制物组织因子途径抑制物2作为治疗基因,是否能对角膜新生血管形成产生影响?目的观察局部转染组织因子途径抑制物2基因对实验兔角膜新生血管形成的影响。设计:随机对照观察。单位:武汉协和医院外科实验室和中山大学附属第三医院中心实验室。材料:本实验于2004-06/2006-03在武汉协和医院外科实验室及中山大学附属第三医院中心实验室完成。选用健康纯种成年新西兰大白兔60只,体质量2.5~3.0kg,雌雄不限。术前经裂隙灯检查均无明显眼前段病变。pBos-Cite-neo/组织因子途径抑制物2质粒(由协和医院血液科仲任博士惠赠),过氧化物酶阻断剂、非免疫性羊血清、鼠抗人MMP-1,2,3单克隆抗体、生物素标记的羊抗鼠IgG二抗(Santacruz公司)。方法:实验干预:硝酸银烧灼法建立各组实验兔兔角膜新生血管模型,随机摸球法将模型兔分为Ⅰ,Ⅱ,Ⅲ组,每组20只,分别结膜下注射生理盐水、空白质粒和真核表达质粒pBos-Cite-neo/组织因子途径抑制物2。实验评估:采用裂隙灯显微镜下观察角膜新生血管生长情况;采用RT-PCR方法检测造模2周后各组实验兔角膜组织组织因子途径抑制物2的表达;采用免疫组织化学测定造模后3,5,7,9,14d角膜组织基质金属蛋白酶蛋白的表达。主要观察指标:①各组实验兔角膜新生血管生长情况。②实验兔角膜组织组织因子途径抑制物2及基质金属蛋白酶的表达情况。结果:纳入实验兔60只均进入结果分析。①角膜新生血管生长情况:Ⅲ组原位转染组织因子途径抑制物2后兔角膜新生血管形成及生长明显受抑制。②角膜组织因子途径抑制物2及基质金属蛋白酶表达:Ⅲ组实验兔角膜组织组织因子途径抑制物2基因表达高于Ⅰ,Ⅱ组(P<0.01);Ⅲ组角膜组织MMP-1,2,3表达较Ⅰ,Ⅱ组降低,以MMP-1,3下降显著。结论:质粒pBos-Cite-neo/组织因子途径抑制物2含有组织因子途径抑制物2基因,局部转染后角膜组织表达组织因子途径抑制物2升高,通过抑制基质金属蛋白酶的活性而抑制角膜新生血管生长。
BACKGROUND: Studies have shown that extracellular matrix degradation mediated by matrix metalloproteinases plays a key role in the formation of corneal neovascularization. Tissue factor pathway inhibitor 2 is a newly discovered serine protease inhibitor that can effectively inhibit matrix Metalloproteinase activity. The use of tissue channel inhibitor inhibitor of tissue factor pathway 2 as a therapeutic gene can affect corneal neovascularization.Objective To investigate the effect of local transfection of tissue factor pathway inhibitor 2 on corneal neovascularization in experimental rabbits. Design: Randomized controlled observation. Unit: Union Hospital of Wuhan and Sun Yat-sen University Third Affiliated Hospital Laboratory. MATERIALS: The experiment was performed at the Central Laboratory of Third Affiliated Hospital of Sun Yat-sen University, Union Hospital, Wuhan Union Medical College from June 2004 to March 2006. Selection of healthy purebred adult New Zealand white rabbits 60, body weight 2.5 ~ 3.0kg, male or female. Preoperative examination by slit lamp no obvious anterior segment lesions. pBos-Cite-neo / Tissue Factor Pathway Inhibitor 2 Plasmid (Presented by Dr. Zhong Ren, Department of Hematology, Union Hospital), Peroxidase Blocking Agent, Non-immune Sheep Serum, Mouse Anti-Human MMP- Clone antibody, biotinylated goat anti-mouse IgG secondary antibody (Santacruz). Methods: Experimental intervention: the corneal neovascularization model was established by silver nitrate burning method in each group. The rabbits were randomly divided into Ⅰ, Ⅱ and Ⅲ groups with 20 rats in each group. The rats were injected subconjunctivally with normal saline and blank plasmid And eukaryotic expression plasmid pBos-Cite-neo / tissue factor pathway inhibitor 2. Experimental evaluation: The growth of corneal neovascularization was observed under a slit lamp microscope. The expression of tissue factor pathway inhibitor 2 in corneal tissue of rabbits in each group was detected by RT-PCR 2 weeks later. Immunohistochemistry was used to detect the changes of corneal neovascularization , 5, 7, 9 and 14 days after the operation. The expression of matrix metalloproteinase protein in corneal tissue was detected. MAIN OUTCOME MEASURES: ① Corneal neovascularization in experimental rabbits in each group. ② The expression of tissue factor pathway inhibitor 2 and matrix metalloproteinase in corneal tissue of rabbits. Results: 60 rabbits were included in the analysis of results. ① corneal neovascularization: group Ⅲ in situ transfection of tissue factor pathway inhibitor 2 rabbit corneal neovascularization and growth was significantly inhibited. ② The expression of inhibitor of corneal tissue factor pathway 2 and matrix metalloproteinase: The expression of tissue factor pathway inhibitor 2 in corneal tissue of group Ⅲ was higher than that of group Ⅰ and Ⅱ (P <0.01) 3 expression than Ⅰ, Ⅱ decreased, with MMP-1,3 decreased significantly. CONCLUSION: Plasmid pBos-Cite-neo / Tissue factor pathway inhibitor 2 contains the Tissue factor pathway inhibitor 2 gene. After local transfection, the tissue factor pathway inhibitor 2 is expressed in the cornea and inhibits the activity of the matrix metalloproteinase Neovascularization.