hEGF在角膜中的基因表达

Gene transfer of human epidermal growth factor in rabbit cornea in vivo

目的 观察人表皮生长因子 (hEGF)基因对角膜细胞的转染情况 ,探讨用hEGF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hEGF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 ,用脂质体包裹注入家兔角膜前房。从RNA、DNA和蛋白质水平分别测定了角膜组织中hEGFmRNA和DNA的表达 ,用免疫组化法测定了hEGF在兔角膜组织中的表达。结果 基因转染 2 4h后在全层角膜细胞内可见hEGFmRNA、DNA和蛋白质表达 ,第 5d达到高峰 ,细胞转染率高达 98%以上 ,随后逐渐降低 ,可持续表达 2 0d以上。结论 用pcDNA3 .1真核表达高效穿梭质粒作为载体 ,携带hEGF基因在脂质体包裹下经前房注射转染角膜 ,可达到极高的转染率 。

Objective To study the transfer by the gene of recombinant human epidermal growth factor(rhEGF) to rabbit cornea cells in vivo and investigate the possibility of treatment of cornea injury by rhEGF gene. Methods The gene sequence of hEGF was constructed by gene engineering technique and inserted in pcDNA3.1 eukaryotic expressing vector.Hybridized rhEGF- pcDNA3.1 vector was wrapped up by liposome and injected into anterior chamber of rabbits.The mRNA and DNA expression of hEGF in the rabbit cornea tissues were determined by RT-PCR and PCR,and its protein expression was observed by immunohistochemical staining. Results After the cornea cells were transferred by rhEGF gene for 24 hours,rhEGF mRNA,DNA and protein were expressed in the corneal tissues of every layer.The cornea cells were transferred over 98% in the 5 th day and then gradually decreased and kept over 20 days. Conclusion rhEGF- pcDNA3.1 vector wrapped by liposome has a high gene transfer rate after its injection into the rabbit anterior chambers.This experiment suggests that hEGF gene may be of a potential therapeutic value for clinical cornea injury.