Li Juanjuan, Li Yunpeng, Zhang Meixia, Hu Zhulin
Department of Ophthalmology, Second People’s Hospital of Yunnan Province, Kunming, China.
Mol Vis. 2012;18:1354-60. Epub 2012 May 30.
To investigate the inhibitory effect of Ras-related C3 botulinum toxin substrate 1-small interfering RNA (Rac1-siRNA) on retinal neovascularization in a rat model.
Rac1-short hairpin RNA (shRNA) was synthesized, constructed, and transfected into HeLa cells. Reverse transcription polymerase chain reaction was then conducted to test for Rac1 gene expression. Retinal vein obstruction was performed in 25 Sprague-Dawley rats using the retinal photodynamic method. The vitrea bulbus of the eye in the shRNA rats group was transfected with the Rac1-shRNA vector, the other eye in the blank control group was transfected with the blank vector, and the interference control group was prepared by transfecting the Rac1-shRNA vector. Two weeks after transfection, the neonatal vessels were tested using fluorescein isothiocyanate-dextran retinal angiography. The number of endothelial cells beyond the internal limiting membrane was counted with hematoxylin and eosin staining.
A large area of neovascularization and fluorescein isothiocyanate leakage was found in the positive control group. However, a small area of neovascularization and a little fluorescence leakage were found in the shRNA group, whereas the retinal vessels were normal in the negative control interference group. In the shRNA interference group, the mean number of endothelial cells beyond the internal limiting membrane was significantly higher than that in the positive control group or the interference negative control group (p<0.05).
Silencing Rac1 expression with RNA interference inhibits retinal neovascularization in rats.
研究Ras相关的C3肉毒杆菌毒素底物1小干扰RNA(Rac1-siRNA)对大鼠视网膜新生血管形成的抑制作用。
合成、构建Rac1短发夹RNA(shRNA)并转染至HeLa细胞。随后进行逆转录聚合酶链反应检测Rac1基因表达。采用视网膜光动力法对25只Sprague-Dawley大鼠造成视网膜静脉阻塞。shRNA大鼠组的眼球玻璃体腔转染Rac1-shRNA载体,空白对照组的另一只眼转染空白载体,干扰对照组通过转染Rac1-shRNA载体制备。转染两周后,采用异硫氰酸荧光素-葡聚糖视网膜血管造影检测新生血管。用苏木精和伊红染色计数内界膜外的内皮细胞数量。
阳性对照组出现大面积新生血管形成和异硫氰酸荧光素渗漏。然而,shRNA组仅出现小面积新生血管形成和少量荧光渗漏,而阴性对照干扰组的视网膜血管正常。在shRNA干扰组,内界膜外内皮细胞的平均数量显著高于阳性对照组或干扰阴性对照组(p<0.05)。
RNA干扰沉默Rac1表达可抑制大鼠视网膜新生血管形成。