摘要
目的探讨小鼠端粒酶逆转录酶（TERT）小分子干扰RNA（siRNA）对小鼠视网膜新生血管形成的抑制作用，及其用于视网膜新生血管疾病治疗的可行性。方法构建TERTsiRNA重组质粒pSIREN—mTERT－1和阴性对照质粒pSIREN—mTERT—N。选择7d龄C57BL／6J小鼠80只随机分为基因治疗组、阴性质粒组、高氧对照组及正常对照组，每组20只。前3组置于75％±2％高氧环境中生活5d，然后回到正常氧环境中。于第12天出氧舱时，分别向基因治疗组、阴性质粒组两组小鼠玻璃体腔内注射上述两种质粒。正常对照组小鼠正常氧环境中饲养。高氧对照组和正常对照组不予玻璃体腔注射。第19天用2％Evens蓝灌注进行视网膜铺片，观察各组小鼠视网膜血管形态变化；反转录－PCR及Real—timePCR检测各组间TERTmRNA和新生血管相关基因的表达变化；组织切片观察并计数突破内界膜的血管内皮细胞数量。对数据采用单因素方差分析进行统计学比较。结果荧光造影视网膜铺片显示，基因治疗组整个视网膜血管分布网基本正常，走形较自然，基本接近正常对照组，未见明显的新生血管丛及大片荧光渗漏，只在视网膜中周部及周边部见少许荧光渗漏，但较阴性质粒组及高氧对照组明显减少。阴性质粒组及高氧对照组视网膜血管紊乱，中周部血管迂曲，伴大片荧光渗漏。RT—PCR及实时PCR显示基因治疗组小鼠视网膜TERTmRNA表达为0．56±0．32，明显少于阴性质粒组及高氧对照组（P〈0．05）。组织切片HE染色观察，基因治疗组仅见1处新生血管芽，偶见突出内界膜的细胞核；阴性质粒组及高氧对照组见散在突出内界膜伸向玻璃体腔的血管芽，内界膜下出现明显的血管内皮细胞增生；光镜下观察突破内界膜新生血管内皮细胞计数，基因治疗组（14．62±1．70）较阴性质粒组（32．38±7．50）及高氧对照组明显减少，差异有统计学意义（P〈0．05）。结论TERT特异的siRNA能有效地抑视网膜新生血管动物模型视网膜中视网膜新生血管的形成，可能会成为一种治疗视网膜新生血管疾病的新方法。
Objective To investigate the inhibitory effect of small interfering RNA （siRNA） targeting TERT on murine retinal neovascularization and explore the feasibility of potential therapeutic approach in retinal vascular disease. Methods Two recombinant plasmids TERT siRNA （pSIREN-mTERT- 1 ） and negative plasmid （pSIREN-mTERT-N） were constructed and 80 seven-day-old C57BL/6J mice were divided randomly into therapeutic group （A）, negative plasmid group （B）, oxygen-induced retinopathy group （C） and normal control group （D）,20 mice in each group. Group A, B and C were exposed to 75%± 2% oxygen for 5 days and then to room air, which induced mice retinal neovascularization. Groups A and B were injected two kinds of the above recombinant plasmid into the murine vitreous on the 12th day. The mice of group D were raised in normal oxygen circumstance. On the 19th day, 2% Evens blue angiography was used to observe the pattern of the retinal vascular. Expression of TERT mRNA were confirmed by reverse-transcription polymerase chain reaction （RT-PCR） and Real-time PCR. Histological observation and vascular endothelial ceils counting were used to examine the effects of siRNA on the retinal neovascularization. Results Retinal flat after Evans blue angiography indicated that the vessels of group A formed a fined radial branching pattern, which was similar to normal mice. In group A, the retinal neovascularization reduced and the structure of retina were more regular than group B and C. At the same time the large vessels were distorted, neovascular clusters proliferated and fluorescence leaked in the middle and periphery area in group B and C. RT-PCR and Real-time PCR showed the expression of TERT mRNA was dowrtregulated in group A compared with groups B and C （ P 〈 0. 05 ）. Paraffin tissue slice with hematoxylin-eosin staining showed that the average counts of vascular endothelial ceils which break through the inner limiting membrane in group A were less than groups B and C, the differences were significant（P 〈 O. 05 ）. Conclusion Pathologic retinal neovascularization can be inhibited by specific TERT siRNA in vivo, which may be a novel efficient strategy azainst oroliferative vasculoDathies.
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2009年第12期1111-1117,共7页
Chinese Journal of Ophthalmology
基金
基金项目：山东省自主创新重大科技专项计划基金（2006GG1102020）
科技部973计划前期研究专项（2007CB516705）
山东省医学科学院课题（2007-29）
