中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2011年
5期
398-403
,共6页
左玲%栾永昕%裴颖%隋桂芹%苏冠方
左玲%欒永昕%裴穎%隋桂芹%囌冠方
좌령%란영흔%배영%수계근%소관방
血管内皮生长因子受体2%转染%视网膜新生血管化%小鼠
血管內皮生長因子受體2%轉染%視網膜新生血管化%小鼠
혈관내피생장인자수체2%전염%시망막신생혈관화%소서
Vascular endothelial growth factor receptor-2%Transfection%Retinal neovascularization%Mice
目的 评价脂质体介导的血管内皮生长因子受体KDR基因胞外段1-3区(KDRn3)基因转染抑制氧诱导的视网膜新生血管的效果.方法 选1周龄C57Bl/6N小鼠置于氧浓度为75%±2%的氧箱中5 d.回到正常环境中诱导视网膜新生血管模型.在小鼠离开氧箱的当日,向转染组小鼠玻璃体腔注射脂质体pEGFP-N1/KDRn3复合物1 μl;脂质体对照组注射等量脂质体;空白对照组小鼠注射等量PBS.回到正常环境中后5 d,采用视网膜铺片及视网膜冰冻切片在激光共聚焦显微镜下观察绿色荧光蛋白在视网膜的表达;采用荧光标记的右旋糖酐血管灌注下视网膜铺片方法观察视网膜新生血管的分布并测量无灌注区面积;组织学切片观察比较突破视网膜内界膜的血管内皮细胞数量.多组比较采用方差分析,有统计意义后进行两两比较的q检验.结果 转染组视网膜铺片见视网膜局部散在点状绿色荧光信号,空白对照组与脂质体对照组未见绿色荧光信号;视网膜冰冻切片见转染组视网膜神经节细胞层、内核层部分细胞胞质内见绿色荧光表达,空白对照组与脂质体对照组视网膜各层未见绿色荧光表达;视网膜铺片观察可见空白对照组与脂质体对照组在无灌注区边缘均可见新生血管芽及荧光渗漏,转染组见新生血管芽明显减少,生后第17天A、B、C 3组新生血管模型小鼠各组视网膜无灌注区面积分别为(1.33±0.49)、(2.75±0.70)、(2.12±0.35)mm2,组间比较差异有统计学意义(F=17.61,P＜0.01＝.正常对照组及A、B、C组视网膜表面突破内界膜的血管内皮细胞核计数分别为0.20±0.51、13.58±2.48、23.05±3.40及21.70±2.89,组间比较差异有统计学意义(F=1085.25,P＜0.05＝.结论 pEGFP-N1/KDRn3基因转染可不同程度抑制氧诱导C57,Bl/6J小鼠视网膜新生血管的生长.
目的 評價脂質體介導的血管內皮生長因子受體KDR基因胞外段1-3區(KDRn3)基因轉染抑製氧誘導的視網膜新生血管的效果.方法 選1週齡C57Bl/6N小鼠置于氧濃度為75%±2%的氧箱中5 d.迴到正常環境中誘導視網膜新生血管模型.在小鼠離開氧箱的噹日,嚮轉染組小鼠玻璃體腔註射脂質體pEGFP-N1/KDRn3複閤物1 μl;脂質體對照組註射等量脂質體;空白對照組小鼠註射等量PBS.迴到正常環境中後5 d,採用視網膜鋪片及視網膜冰凍切片在激光共聚焦顯微鏡下觀察綠色熒光蛋白在視網膜的錶達;採用熒光標記的右鏇糖酐血管灌註下視網膜鋪片方法觀察視網膜新生血管的分佈併測量無灌註區麵積;組織學切片觀察比較突破視網膜內界膜的血管內皮細胞數量.多組比較採用方差分析,有統計意義後進行兩兩比較的q檢驗.結果 轉染組視網膜鋪片見視網膜跼部散在點狀綠色熒光信號,空白對照組與脂質體對照組未見綠色熒光信號;視網膜冰凍切片見轉染組視網膜神經節細胞層、內覈層部分細胞胞質內見綠色熒光錶達,空白對照組與脂質體對照組視網膜各層未見綠色熒光錶達;視網膜鋪片觀察可見空白對照組與脂質體對照組在無灌註區邊緣均可見新生血管芽及熒光滲漏,轉染組見新生血管芽明顯減少,生後第17天A、B、C 3組新生血管模型小鼠各組視網膜無灌註區麵積分彆為(1.33±0.49)、(2.75±0.70)、(2.12±0.35)mm2,組間比較差異有統計學意義(F=17.61,P＜0.01＝.正常對照組及A、B、C組視網膜錶麵突破內界膜的血管內皮細胞覈計數分彆為0.20±0.51、13.58±2.48、23.05±3.40及21.70±2.89,組間比較差異有統計學意義(F=1085.25,P＜0.05＝.結論 pEGFP-N1/KDRn3基因轉染可不同程度抑製氧誘導C57,Bl/6J小鼠視網膜新生血管的生長.
목적 평개지질체개도적혈관내피생장인자수체KDR기인포외단1-3구(KDRn3)기인전염억제양유도적시망막신생혈관적효과.방법 선1주령C57Bl/6N소서치우양농도위75%±2%적양상중5 d.회도정상배경중유도시망막신생혈관모형.재소서리개양상적당일,향전염조소서파리체강주사지질체pEGFP-N1/KDRn3복합물1 μl;지질체대조조주사등량지질체;공백대조조소서주사등량PBS.회도정상배경중후5 d,채용시망막포편급시망막빙동절편재격광공취초현미경하관찰록색형광단백재시망막적표체;채용형광표기적우선당항혈관관주하시망막포편방법관찰시망막신생혈관적분포병측량무관주구면적;조직학절편관찰비교돌파시망막내계막적혈관내피세포수량.다조비교채용방차분석,유통계의의후진행량량비교적q검험.결과 전염조시망막포편견시망막국부산재점상록색형광신호,공백대조조여지질체대조조미견록색형광신호;시망막빙동절편견전염조시망막신경절세포층、내핵층부분세포포질내견록색형광표체,공백대조조여지질체대조조시망막각층미견록색형광표체;시망막포편관찰가견공백대조조여지질체대조조재무관주구변연균가견신생혈관아급형광삼루,전염조견신생혈관아명현감소,생후제17천A、B、C 3조신생혈관모형소서각조시망막무관주구면적분별위(1.33±0.49)、(2.75±0.70)、(2.12±0.35)mm2,조간비교차이유통계학의의(F=17.61,P＜0.01＝.정상대조조급A、B、C조시망막표면돌파내계막적혈관내피세포핵계수분별위0.20±0.51、13.58±2.48、23.05±3.40급21.70±2.89,조간비교차이유통계학의의(F=1085.25,P＜0.05＝.결론 pEGFP-N1/KDRn3기인전염가불동정도억제양유도C57,Bl/6J소서시망막신생혈관적생장.
Objective To evaluate the effect of liposome mediated plasmids KDRn3 injected into the vitreous to inhibit experimental retinal neovascularization. Methods One-week-old C57BL/6N mice were exposed to 75% ± 2% oxygen for 5 days, then returned to the room air to induce retinal neovascularization. Cationic liposome mediated KDRn3 comp-lex (1 μl) was injected into the vitreous in the treatment group. PBS 1 μl or liposome were injected in the control group. The pEGFP-N1/ KDRn3 expression was observed by using fluorescence microscope. Retinal neovascularization was evaluated by counting the number of vascular endothelial cell nuclei on the vitreal side of the inner limiting membrane of the retina and measuring the areas of non-perfusionsin in central retina. Results KDRn3 protein was expressed both in the ganglion layer and in the inner layer. Retinal wholemount preparation of retinal neovascular animal model showed that prominent neovascular tuft and fluorescein leakage and large areas of non-perfusionsin in central retina. Fewer neovascular tufts and fewer areas of non-perfusionsin could be seen after pEGFP-N1/KDRn3 injection. There were statistic differences between control group and pEGFP-N1/ KDRn3 injecting group with the number of vascular endothelial cell nuclei on the vitreal side of the inner limiting membrane of the retina(0. 20 ±0. 51, 13. 58 ±2. 48,23. 05 ±3. 40,21. 70 ± 2. 89;F = 1085. 25, P ＜ 0. 05 ) and the areas of non-perfusionsin in central retina [(1. 33 ± 0. 49 ) , ( 2. 75 ± 0. 70 ) , ( 2. 12 ± 0. 35) mm2; F = 17. 61 , P ＜ 0. 01] . Conclusion pEGFP-N1/KDRn3 gene transfer can inhibit retinal neovascularisation in C57Bl/6J mice of ischaemia-induced retinal neovascularisation on some extent.
