摘要
目的观察重组腺病毒-p21（rAd—p21）对氧诱导小鼠视网膜新生血管（RNV）的抑制作用。方法将56只健康7日龄C57BL／6J小鼠随机分为对照组、磷酸盐缓冲液（PBS）组、rAd-p21组及rAd-无目的基因对照（rAd—NC）组，每组14只。PBS组、rAd—p21组及rAd～NC组建立氧诱导RNV模型，并于小鼠11日龄时玻璃体腔分别注入1p1PBS、rAd—p21及rAd—NC。对照组不做任何处理。小鼠17日龄时，每组处死4只小鼠，摘取眼球分别作荧光视网膜铺片和切片，观察小鼠RNV发生情况；Image-Proplus6．0软件测量分析无灌注区面积；同时提取视网膜总RNA和总蛋白，应用逆转录聚合酶链反应（RT-PCR）及蛋白免疫印迹法（Westernblot）检测p21、细胞周期素依赖蛋白激酶2（CDK2）mRNA及蛋白在视网膜组织中的表达。结果荧光及光学显微镜观察发现，rAd—p21组小鼠视网膜无灌注区、新生血管及突破视网膜内界膜的血管内皮细胞核较PBS组和rAd—NC组减少；tAd—p21组无灌注区面积较PBS组和rAd—NC组明显减少，组间差异有统计学意义（F=101．634，P〈O．05）。RT—PCR及Westernblot检测结果显示，rAd—p21组p21mRNA和蛋白表达明显高于对照组、PBS组及rAd—NC组，组间差异有统计学意义（F=839．664、509．817，P〈0．05）；rAd—p21组CDK2mRNA和蛋白表达明显低于对照组、PBS组及rAd—NC组，组间差异也有统计学意义（F=301．858、592．882，P〈0．05）。结论rAd-p21可通过上调p21表达、降低CDK2表达，抑制RNV生成。
Objective To observe the the inhibitory effect of recombined adenovirus mediated delivery of p21 （tAd-p21） on oxygen-induced retinal neovascularization in mice. Methods A total of 56 C57BL/6 mice at the age of seven days were divided into control group, phosphate buffer solution （PBS） group, rAd- p21 group and rAd-no purpose gene control （rAd-NC） group, 14 mice in each group. The retinal neovascularization of PBS, rAd-p21and rAd-NC group were induced by oxygen, and received an intravitreal injection 1 /zl PBS, rAd-p21 and rAd-NC at postnatal day 11, respectively. The rats of control group were not intervened. At postnatal day 17, RNV was determined by retinal flat mounts and retinal section; non- perfusion areas of retina were analyzed by Image-Pro plus 6.0 software; reverse transcription-polymerase chain reaction （RT-PCR） and Western blot was used to measure the mRNA and protein expression of p21 and CDK2. Results Compared with PBS and rAd-NC groups, the retinal non-perfusion areas, neovascularization and the numbers of endothelial cell nuclei breaking through the internal limiting membrane in rAd-p21 group were reduced significantly. Non-perfusion areas of retina in rAd-p21 group was less than that in PBS and rAd-NC groups, the difference among these three groups was significantly （F= 101. 634, P〈0.05）. Compared with the other three groups, the level of p21 mRNA and protein in rAd-p21 group increased significantly （F=839. 664, 509. 817; P〈0.05） ; the level of CDK2 mRNA and protein in rAd-p21 group decreased significantly （F=301. 858, 592. 882; P〈0.05）. Conclusion rAd-p21can inhibit oxygen- induced retinal neovascularization, up-regulated p21 expression and down-regulated CDK2 expression may be the mechanism.
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2012年第5期493-497,共5页
Chinese Journal of Ocular Fundus Diseases
关键词
视网膜新生血管化/预防和控制
基因转移技术
转染
动物实验
Retinal neovascularization/prevention ~ control
Gene transfer techniques
Transfeetion
Animal experimentation
