原位转染组织因子途径抑制物2基因抑制兔角膜新生血管形成(英文)

Inhibition of corneal neovascularization by tissue factor pathway inhibitor 2 gene transfection in situ

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： 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/TFPI-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 TFP1-2 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. MAIN OUTCOME MEASURES ： ① CNV growth ② Expressions of TFPI-2 and MMPs in corneal tissue RESULTS： Sixty experimental rabbits were involved in the final analysis. ①CNV growth： In the group Ⅲ, when TFPI-2 was transfected in situ, CNV was found and its growth was obviously inhibited. ② Expressions of TFPI-2 and MMPs： The 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. CONCLUSION： The plasmid of pBos-Cite-neo/TFPI-2 contains TFPI-2 gene. The expression of TFPI-2 increases after topical transfection, so CNV growth can be inhibited by inhibiting the activity of MMPs.