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目的:探讨转染内皮抑制素(Endostatin)基因对酸烧伤引起的大鼠角膜新生血管的基因治疗。方法:通过限制性内切酶酶切反应、聚合酶链式反应、DNA测序及用NCBIBLAST软件与基因库序列比较的方法进行鉴定,鉴定后在大肠杆菌中扩增,用质粒纯化试剂盒抽提纯化;用750g/L硝酸银和250g/L硝酸钾混合烧灼液制作大鼠角膜新生血管模型,用结膜下注射脂质体包裹的质粒pBlast- hEndostatin来进行体内基因治疗。结果:实验证实质粒pBlast-hEndostatin含有人endostatin基因。结膜下注射脂质体包裹的质粒Tel押029-82245172Email押IJO.2000@163.compBlast-hEndostatin对酸烧伤引起的炎症性角膜新生血管有明显抑制作用,术后6,10,15d对角膜新生血管面积的抑制率分别为37%,40%,43%;对角膜新生血管密度也有明显的抑制作用,抑制率达40%。对角膜新生血管长度和角膜炎症细胞没有明显抑制作用。角膜新生血管面积与角膜水肿、角膜混浊呈正相关。结论:用结膜下注射脂质体包裹的内皮抑制素基因可以部分抑制酸烧伤引起的大鼠角膜新生血管。其作用机制是转基因产生的内皮抑制素蛋白直接抑制角膜新生血管的形成,而不是通过抑制炎症反应来抑制角膜新生血管的形成。
Objective: To investigate gene therapy of corneal neovascularization induced by acid burns in rats transfected with endostatin gene. Methods: The restriction endonuclease digestion reaction, polymerase chain reaction, DNA sequencing and NCBIBLAST software were compared with the gene bank sequence identification method, identified after amplification in E. coli, extracted with a plasmid purification kit The rat corneal neovascularization model was prepared by mixing 750g / L silver nitrate and 250g / L potassium nitrate mixture, and the in vivo gene therapy was performed by subconjunctival injection of liposome-encapsulated plasmid pBlast-hEndostatin. Results: The experiment confirmed that plasmid pBlast-hEndostatin contains human endostatin gene. Subconjunctival injection of liposome-encapsulated plasmid Tel 0 029-82245172Email charge IJO.2000@163.compBlast- hEndostatin on acid burns caused by inflammatory corneal neovascularization was significantly inhibited, 6,10,15 d after corneal neovascularization Area inhibition rates were 37%, 40%, 43%; corneal neovascularization density also significantly inhibited the inhibition rate of 40%. Corneal neovascularization and corneal inflammation cells did not significantly inhibit the role. Corneal neovascularization area was positively correlated with corneal edema and corneal opacity. CONCLUSIONS: Endothelial inhibin gene encapsulated with liposome in subconjunctival injection can partially inhibit rat corneal neovascularization induced by acid burns. Its mechanism of action is that the genetically engineered endostatin protein directly inhibits the formation of corneal neovascularization rather than inhibiting the formation of corneal neovascularization by inhibiting the inflammatory response.