摘要
目的观察人表皮生长因子基因对角膜细胞的转染情况,探讨用人表皮生长因子基因对角膜损伤治疗的可行性。方法利用基因重组技术构建了人表皮生长因子全基因序列,将该基因序列插入 pcDNA3.1真核表达高效穿梭质粒,用脂质体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。在治疗后的不同天数,分别测定了角膜组织中人表皮生长因子 mRNA 和 DNA 的表达,用 ELISA 法测定了人表皮生长因子在兔角膜组织中的含量。结果在基因转染后24h 至28天,角膜组织分别用 RT-PCR 和 PCR 均可检测出人表皮生长因子 mRNA 和 DNA 表达。用 ELISA 法测定人表皮生长因子蛋白质表达水平。结果表明,在转染后24h,实验组分别与对照组和空白组平行对比,实验组蛋白质表达水平明显升高(P<0.01),第7天达高峰,为224.286mg/g 角膜湿重,随后逐渐降低,持续可表达20天以上;在转染后24h,免疫组织化学染色在全层角膜细胞内亦可见 hEGF 蛋白质表达,第7天达到高峰,细胞转染率高达98%以上,随后逐渐降低,持续可表达20天以上。结论用 pcDNA3.1真核表达高效穿梭质粒作为载体,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 observe the transfection of corneal cells by the gene of HEGF,and probe the feasibility to treat corneal injury by HEGF gene.Methods The gene sequence of human epidermal growth factor was constructed by gene engineering technique,the sequence was in- serted in pcDNA3.1 eukaryotic expressing vector.After the hybridized rhEGF-pcDNA3.1 vector was wrapped up by liposome,it was made of a gene sustained delivery eye drops.The cornea injured by alkali were treated by put drops.The mRNA and DNA expressing of human epidermal growth factor in the rabbit cornea tissues were determined by RT-PCR and PCR,and the protein expressing of it was observed by ELISA assay and its protein expression was observed by immunohistochemical method.Results After the cornea injured by alkali were treated by rhEGF gene delivery eye drops from 24h to 28 days,rhEGF mRNA and DNA in the corneal tissues were demonstrated by RT-PCR and PCR.The level of rhEGF protein in the corneal tissues was measured by ELISA assay.The results showed,after rhEGF gene was transferred 24h,the level of rhEGF protein in experimental group compared with in control group and blank group was significantly increased(P<0.01).In the 7th day,the level of rhEGF protein was the highest,was 224.286 ng/g corneal wet weight.Soon afterwards,the level was gradually decreased,but it was kept over 20 days,and protein were expressed in the cornea tissues of every layers.The cornea cells were transferred over 98% in the 7th day and then gradually decreased and kept over 20 days.Conclusion rhEGF gene is carried by pcDNA3.1 eukaryofic expressing plasmid acted as a vector, the gene is wrapped by liposome,then it is made from a gene sustained delivery eye drops.The models of alkali injury were treated by put drps. The gene may be the therapeutically valuable for the treatment of clinical cornea hurt.
出处
《医学研究通讯》
2005年第2期7-10,共4页
Bulletin of Medical Research
基金
河南省科研事业发展计划资助项目(0141163216)
