超声微泡介导Ngb基因过表达对大鼠视网膜急性高眼压损伤的保护研究

Protective effect of UTMD-mediated overexpression of Ngb on rat retina with acute intraocular hypertension

目的探讨超声微泡靶向破坏(ultrasound-targeted microbubble destruction,UTMD)技术介导的脑红蛋白(neuroglobin,Ngb)过表达对大鼠急性高眼压损伤的视网膜保护作用。方法 40只SD大鼠右眼进行基因转染,即利用UTMD将携带外源性Ngb基因的重组质粒转染至大鼠视网膜组织;左眼为对照组(未转染组),即只升高眼压。在转染后48h,采用前房加压灌注升高眼压的方法制作急性高眼压模型,术后5min、10min、30min、60min分别处死各组大鼠,摘取眼球,分离视网膜。Western blotting检测Ngb在急性高眼压时表达的变化情况,测定并比较双眼的caspase-3活性。结果 UTMD介导外源性Ngb在视网膜各层均有表达,对视网膜没有造成损伤。未转染组Ngb表达水平呈时相性变化,在高眼压缺氧5～10min时明显增高,在10min时达到峰值,随后开始下降,在60min时最低。转染组Ngb表达水平明显高于未转染组,在缺氧30min时达到峰值,随后开始下降。随着高眼压时间的延长,caspase-3活性逐渐增加,说明死亡的视网膜神经纤维层细胞越来越多。转染组的caspase-3活性明显低于未转染组,在相同时间点,两组之间差异均有统计学意义(均为P<0.05)。结论 UTMD能够在体内安全、有效地介导Ngb基因转染至视网膜组织中。Ngb过表达可以降低视网膜对缺血缺氧的敏感性,起到视网膜组织保护作用。

Objective To evaluate the protective effect of ultrasound-targeted microbibble destruction（UTMD-MD） mediated overexpression of exogenous neuroglo- bin（Ngb） on rat retina with acute ocular hypertension. Methods A total of 40 SD normal rats were selected, and their eyes were divided into two groups with right eyes as UTMD group（ recombinant plasmid with exogenous Ngb was transfected into rat retinal tissue by UTMD） and left eyes as the control group （ only advanced intraocular pressure）. At 48 hours after UTMD, animal model of acute high intraocular pressure was made in each eye of 40 rats by forcing perfusion of NS into the anterior chamber. At 5 minutes, 10 minutes,30 minutes and 50 minutes,the rats were killed and then their eyeballs were taken and retina were sepsrated. Ngb expression was detected under acute intraocular hypertension by Western blotting and caspase-3 activity was detected and compared between the two groups. Results The expression of UTMD-mediated exogenous Ngb was found in all retina layers and it had no effect on retina. There was timedepended pattern of the expression of Ngb gene in control group. In control eyes, Ngb increased rapidly from 5 minutes to l0 minutes after acute intraocular hypertension and peaked at 10 minutes;then it began to decrease and reached the bottom at 50 minutes. The expression level of Ngb in transfected group was obviously higher than that in untransfected group and peaked at 30 minutes, which then began to decrease. With the time of high intraocular pressure extension, the caspase-3 activity gradually increased which showed that the number of dead nerve fiber layer cells increased. The caspase-3 activity in UTMD eyes was significantly lower than that in control eyes and there was significant difference between them at the same time point （ all P 〈 0.05 ）. Conclusion UTMD can transfect the Ngb gene into SD rat retinal tissue safely and effectively. The overexpression of exogenous Ngb gene can lower the sensibility of retina to shortage of blood and oxygen, and shows a protective effect on retinal tissue.