人表皮生长因子真核细胞缓释型滴眼剂的基因治疗研究

Research on gene therapy of hEGF in rabbit corneal alkali injury

目的:构建人表皮生长因子的真核表达载体,探讨用人表皮生长因子对角膜损伤治疗的可行性。方法:采用基因重组技术将人表皮生长因子基因插入pcDNA.3.1真核表达穿梭质粒,用脂质体包裹制成人表皮生长因子基因缓释型滴眼剂,滴眼治疗家兔角膜碱烧伤。结果:将hEGF-pcDNA3.1重组体进行限制性酶切及PCR扩增可见目的片段;基因治疗组在模型治疗后第7 d,角膜荧光着色变浅,溃疡面变小;第18～21 d溃疡面消失;在基因治疗后第1至28 d,从角膜组织中均可检测出hEGF mRNA,基因治疗组蛋白质表达水平明显升高,第7d达高峰,为224.286 ng/g角膜湿重,持续可表达3周以上;基因治疗眼角膜免疫组织化学染色可在角膜细胞内见hEGF蛋白质表达,第7 d达高峰,细胞转染率高达95%以上,并可持续表达3周以上。结论:用pcDNA.3.1真核表达穿梭质粒作为载体,携带人表皮生长因子基因,用脂质体包裹后制成缓释型滴眼液,以滴眼的方式给药转染角膜,可达到极高的转染率,具有良好的应用前景。

Objective： To construct eukaryotic and probe the feasibility to treat corneal injury expression vector of human epidermal growth factor, by hEGF gene. Methods ： The gene sequence of human epidermal growth factor was constructed by gene engineering technique, the sequence was inserted in pcDNA3. 1 eukaryotic expressing vector. After the hybridized hEGF-pcDNA3. 1 vector was wrapped up by liposome, it was made of a gene Sustained delivery eye drops. The corneas injured by alkali were treated by put drops. Results： The hEGF-pcDNA3.1 recombinant was cut by restriction endonuclease and the sequence of human epidermal growth factor was amplified by PCR. In gene therapy group, the area of cornea ulcer was decreased in 7d and disappeared in 18d to 21d. After the cornea injured by alkali were treated by hEGF gene delivery eye drops from ld to 28d, hEGF mRNA in the corneal tissues were demonstrated. The level of hEGF protein in the cor- neal tissues was significantly increased, on 7d, the level of hEGF protein was the highest, and was 224. 286 ng/g corneal cells were transferred over 95% in 7d and kept over 3 weeks. Conclusion： hEGF gene is carried by pcDNA3.1 eukaryotic expressing plasmid acted as a vector, the gene is wrapped by liposome, and then it is made from a gene sustained delivery eye drops. The models of alkali injury were treated by put drops. The gene may be the therapeutically valuable for the treatment of clinical cornea hurt.