脂质体介导含RGD序列内皮抑素基因抑制角膜新生血管的研究

目的观察脂质体介导的含RGD序列的内皮抑素（RGD—ES）抑制兔碱烧伤角膜新生血管（CNV）的效果。方法碱烧伤后诱导兔产生CNV，36只兔（72只眼）随机分为4组。在碱烧伤后，分别球结膜下注射0．2mL的50g／L脂质体pCI—RGD～ES转染液（A组）、50g／L脂质体pCI—ES转染液（B组）、空白载体转染液（C组）、PBS缓冲液（D组）。每周注射2次，共4次。动态观察CNV的生长状况。第3、7、14天免疫组织化学法检测血管内皮生长因子（VEGF）的表达，并在显微镜下进行微血管计数。结果在模型制作后的各个时间点，A组和B组CNV的面积明显小于D组，差异有统计学意义（P〈0．01），但各时间点c组和D组间CNV面积的比较差异无统计学意义（P〉0，05）。在各时间点，A组和B组角膜的微血管数量均明显少于D组（P〈0．01），其中A组角膜VEGF表达及微血管数量最少。但各时间点c组与D组间角膜VEGF的表达及微血管数量的比较差异无统计学意义（P〉0．05）。VEGF的表达定位于角膜上皮细胞、炎性细胞及新生血管内皮细胞细胞质内。结论RGD—ES抑制CNV的活性增强，脂质体是眼病基因治疗的理想载体，且脂质体本身对CNV无抑制作用。

Inhibition of corneal neovascularization by liposome mediated plasmid encoding endostatin with RGD sequence

Background It has been demonstrated that αvβ3/αvβ5 and α5β1 integrins are overexpressed in neovascular tissue.Consequently,peptide containing the RGD (Arg-Gly-Asp) sequence,which exists in ligands of integrins,is effective in targeting therapeutic reagents to neovascular endothelium.ObjectivePresent study aims to investigate the antiangiogenetive effects of liposome mediated plasmid encoding endostatin with RGD sequence on alkali burn-induced corneal neovascularization (CNV) in rabbits.MethodsCNV models induced by alkali burn were established in 72 eyes of 36 New Zealand white rabbits by putting the filter paper with 1 mol/L NaOH at the central cornea for 20 seconds.The animal models were divided into four groups randomly.0.2mL of liposome and plasmid encoding RGD-ES complex (liposome mediated pCI-RGD-ES injection group),liposome and plasmid encoding ES complex (pCI-ES injection group),liposome and carrier plasmid (pCI) complex (pCI-ES injection group),and PBS were subconjunctivally injected respectively in the models from four groups twice a week for two weeks.The growth status of CNV was observed on day 1,3,7,14 after alkali burn under the slim lamp microscope.Experimental animals were sacrificed on the 3rd,7th and 14th day and the expression of VEGF in CNV was detected by immunohistochemistry.The number of corneal microvessels was counted based on the number of CNV cross-section under the light microscope.ResultsCNV area was significantly smaller in liposome mediated pCI-RGD-ES injection group and pCI-ES injection group compared with PBS group at different time points (P＜0.01),but no significant difference was seen in CNV area between pCI-ES injection group and PBS group at different time points (P＞0.01).The changes of number of corneal microvessels was followed a similar fashion as the change of CNV area.Expression of VEGF in cornea was obviously stronger in pCI-ES injection group and PBS group than liposome mediated pCI-RGD-ES injection group and pCI-ES injection group.ConclusionEndostatin with RGD sequence could effectively inhibit corneal neovascularization,and liposome is proved to be a potent carrier in gene transfer.