中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2009年
12期
1111-1117
,共7页
闵晓洁%董晓光%周庆军%刘廷%银红梅%谢立信
閔曉潔%董曉光%週慶軍%劉廷%銀紅梅%謝立信
민효길%동효광%주경군%류정%은홍매%사립신
端粒%未端转移酶%RNA%小分子干扰%视网膜新生血管化%基因疗法%小鼠
耑粒%未耑轉移酶%RNA%小分子榦擾%視網膜新生血管化%基因療法%小鼠
단립%미단전이매%RNA%소분자간우%시망막신생혈관화%기인요법%소서
Telomerase%RNA%small interfering%Retinal neovaseularization%Gene therapy%Mice
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TERT siRNA重组质粒pSIREN-mTERT-1和阴性对照质粒pStREN-mTERT-N.选择7 d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5 d,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-time PCR检测各组间TERT mRNA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TERT mRNA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牛血管内皮细胞计数,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鼎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.
目的 探討小鼠耑粒酶逆轉錄酶(TERT)小分子榦擾RNA(siRNA)對小鼠視網膜新生血管形成的抑製作用,及其用于視網膜新生血管疾病治療的可行性.方法構建TERT siRNA重組質粒pSIREN-mTERT-1和陰性對照質粒pStREN-mTERT-N.選擇7 d齡C57BL/6J小鼠80隻隨機分為基因治療組、陰性質粒組、高氧對照組及正常對照組,每組20隻.前3組置于75%±2%高氧環境中生活5 d,然後迴到正常氧環境中.于第12天齣氧艙時,分彆嚮基因治療組、陰性質粒組兩組小鼠玻璃體腔內註射上述兩種質粒.正常對照組小鼠正常氧環境中飼養.高氧對照組和正常對照組不予玻璃體腔註射.第19天用2%Evens藍灌註進行視網膜鋪片,觀察各組小鼠視網膜血管形態變化;反轉錄-PCR及Real-time PCR檢測各組間TERT mRNA和新生血管相關基因的錶達變化;組織切片觀察併計數突破內界膜的血管內皮細胞數量.對數據採用單因素方差分析進行統計學比較.結果 熒光造影視網膜鋪片顯示,基因治療組整箇視網膜血管分佈網基本正常,走形較自然,基本接近正常對照組,未見明顯的新生血管叢及大片熒光滲漏,隻在視網膜中週部及週邊部見少許熒光滲漏,但較陰性質粒組及高氧對照組明顯減少.陰性質粒組及高氧對照組視網膜血管紊亂,中週部血管迂麯,伴大片熒光滲漏.RT-PCR及實時PCR顯示基因治療組小鼠視網膜TERT mRNA錶達為0.56±0.32,明顯少于陰性質粒組及高氧對照組(P＜0.05).組織切片HE染色觀察,基因治療組僅見1處新生血管芽,偶見突齣內界膜的細胞覈;陰性質粒組及高氧對照組見散在突齣內界膜伸嚮玻璃體腔的血管芽,內界膜下齣現明顯的血管內皮細胞增生;光鏡下觀察突破內界膜新牛血管內皮細胞計數,基因治療組(14.62±1.70)較陰性質粒組(32.38±7.50)及高氧對照組明顯減少,差異有統計學意義(P＜0.05).結論 TERT特異的siRNA能有效地抑視網膜新生血管動物模鼎視網膜中視網膜新生血管的形成,可能會成為一種治療視網膜新生血管疾病的新方法.
목적 탐토소서단립매역전록매(TERT)소분자간우RNA(siRNA)대소서시망막신생혈관형성적억제작용,급기용우시망막신생혈관질병치료적가행성.방법구건TERT siRNA중조질립pSIREN-mTERT-1화음성대조질립pStREN-mTERT-N.선택7 d령C57BL/6J소서80지수궤분위기인치료조、음성질립조、고양대조조급정상대조조,매조20지.전3조치우75%±2%고양배경중생활5 d,연후회도정상양배경중.우제12천출양창시,분별향기인치료조、음성질립조량조소서파리체강내주사상술량충질립.정상대조조소서정상양배경중사양.고양대조조화정상대조조불여파리체강주사.제19천용2%Evens람관주진행시망막포편,관찰각조소서시망막혈관형태변화;반전록-PCR급Real-time PCR검측각조간TERT mRNA화신생혈관상관기인적표체변화;조직절편관찰병계수돌파내계막적혈관내피세포수량.대수거채용단인소방차분석진행통계학비교.결과 형광조영시망막포편현시,기인치료조정개시망막혈관분포망기본정상,주형교자연,기본접근정상대조조,미견명현적신생혈관총급대편형광삼루,지재시망막중주부급주변부견소허형광삼루,단교음성질립조급고양대조조명현감소.음성질립조급고양대조조시망막혈관문란,중주부혈관우곡,반대편형광삼루.RT-PCR급실시PCR현시기인치료조소서시망막TERT mRNA표체위0.56±0.32,명현소우음성질립조급고양대조조(P＜0.05).조직절편HE염색관찰,기인치료조부견1처신생혈관아,우견돌출내계막적세포핵;음성질립조급고양대조조견산재돌출내계막신향파리체강적혈관아,내계막하출현명현적혈관내피세포증생;광경하관찰돌파내계막신우혈관내피세포계수,기인치료조(14.62±1.70)교음성질립조(32.38±7.50)급고양대조조명현감소,차이유통계학의의(P＜0.05).결론 TERT특이적siRNA능유효지억시망막신생혈관동물모정시망막중시망막신생혈관적형성,가능회성위일충치료시망막신생혈관질병적신방법.
Objective To investigate the inhibitory effect of small interfering RNA (siRNA) targeting TERT on routine 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 cells 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 urea in group B and C.RT-PCR and Real-time PCR showed the expression of TERT mRNA was downregulated 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 cells which break through the inner limiting membrane in group A were less than groups B and C,the differences were significant(P＜0.05).Conclusion Pathologic retinal neovascularization can be inhibited by specific TERT siRNA in vivo,which may be a novel efficient strategy against proliferative vasculopathies.
