脂质体介导质粒pcDNA3.1/ KDRn3基因在转染小鼠视网膜组织中的表达及定位

目的：将真核表达质粒pcDNA3.1/ KDRn3以脂质体介导转染C57BL/6J小鼠视网膜组织，观察其表达及定位。方法：首先对真核表达质粒pcDNA3.1/KDRn3进行扩增和酶切鉴定，然后将20只健康C57BL/6J小鼠随机分成对照组及玻璃体腔注射后2、5、7和14 d组，每组4只鼠8眼。注射组每眼玻璃体腔注射脂质体pcDNA3.1/ KDRn3复合物1 μL，分别于注射后2、5、7及14 d摘除眼球，制成石蜡切片，用KDRn3蛋白免疫荧光染色在激光共聚焦显微镜下观察KDRn3蛋白表达及定位。结果：Xho Ⅰ和BamHⅠ进行双酶切,切下900 bp大小片段,与目的片段大小一致,表明该质粒确实为KDRn3。注射后2 d在视网膜神经节细胞层细胞胞浆内可见红色荧光信号，注射后5和7 d组视网膜神经节细胞层、内核层部分细胞胞浆内可见红色荧光信号；注射后14 d视网膜神经节细胞层、部分内核层细胞胞浆内可见红色荧光信号，但发出红色荧光信号的细胞数明显减少。　结论：阳离子脂质体可有效将pcDNA3.1/ KDRn3基因转染小鼠视网膜组织，并得到持续表达。

Expression and location of pcDNA3.1/ KDRn3 gene in retina of mice mediated by liposome transfection

Objective To observe the expression and location of the plasmid pcDNA3.1/ KDRn3 after transfected into the retina of C57BL/6J mice by using liposome. Methods The KDRn3 gene was amplified by PCR and then identified by Enzymatic digestion.20 C57BL/6J mice were randomly divided into 5 groups(n=4),four groups were intravitreally injected with the optimal liposome plasmid mixture,the other was control group. 2,5,7,and 14 d after injection,the localization of pcDNA3.1/ KDRn3 was observed by using immunfluorescence staining method. Results A 900 bp gene part was digested with Xho Ⅰ and BamHⅠwhich agreed with the length of KDRn3.The red fluorescence of KDRn3 protein expressed in the retinal ganglion layer 2 d after injection;after 5 d or 7 d,it expressed both in the ganglion layer and in the inner layer.After 14 d,it showed the weakened expression.Conclusion Cationic liposome can mediate pcDNA3.1/KDRn3 gene into the retina of the C57BL/6J mice effectively and it can express for a long time.