钙结合蛋白S1O0A4基因静默对氧诱导视网膜新生血管的抑制作用及其机制

Silencing of S100A4 gene inhibits oxygen-induced retinal neovascularization in mice

目的观察探讨钙结合蛋白S100A4基因静默对氧诱导视网膜新生血管（RNV）的抑制作用及其机制。方法7日龄C57BL／6J小鼠150只随机分为正常组、正常-病毒对照组、单纯模型组、基因治疗组、空白载体组，每组30只。正常组、正常-病毒对照组小鼠在常氧环境下饲养；其余3组小鼠建立氧诱导视网膜病变模型。小鼠12日龄时，基因治疗组及空白载体组小鼠双眼玻璃体腔注射病毒滴度为1．0×10^9PFU／mI的携带针对S100A4小干扰RNA的重组腺病毒载体（Ad—S100A4-RNAi）和带绿色荧光蛋白的空白腺病毒载体各1．0μl；正常-病毒对照组小鼠玻璃体腔注射同样滴度的等量Ad～S100A4-RNAi。正常组及单纯模型组小鼠不做任何处理。小鼠15日龄时，基因治疗组和正常组作视网膜组织冰冻切片观察病毒转染情况。小鼠17日龄时，各组作视网膜组织切片，计数突破视网膜内界膜的血管内皮细胞核；作全视网膜铺片免疫荧光染色，观察视网膜血管变化；蛋白免疫印迹法（Westernblot）及实时PCR检测小鼠视网膜S100A4、B细胞淋巴瘤／白血病-2基因（bcl-2）、半胱天冬酶（Caspase）-3及环磷腺苷效应元件结合蛋白（CREB）的蛋白和mRNA表达。结果荧光显微镜观察发现，正常组小鼠视网膜未见病毒绿色荧光，仅可见少量自身荧光；基因治疗组小鼠视网膜神经节细胞层、内丛状层、内核层、外丛状层可见病毒绿色强荧光，外核层可见少量弱荧光。单纯模型组突破内界膜的血管内皮细胞核计数较正常组明显增加，差异有统计学意义（t=15．68，P〈0．05）；基因治疗组突破内界膜的血管内皮细胞核计数较单纯模型组、空白载体组明显下降，差异均有统计学意义（t=13．61、14．64，P〈0．05）。与单纯模型组、空白载体组比较，基因治疗组小鼠RNV面积、无灌注区面积明显减少，差异均有统计学意义（P〈0．05）。Westernblot及实时PCR检测结果显示，与单纯模型组、空白载体组比较，基因治疗组小鼠视网膜S100A4、bcl-2、CREB蛋白及mRNA表达明显下调，差异均有统计学意义（P〈0．05）；Caspase-3蛋白及mRNA表达明显上调，差异有统计学意义（P〈0．05）。结论S100A4基因静默可抑制氧诱导RNV。其机制可能与S100A4基因静默下调bcl-2、CREB表达，上调Caspase-3表达作用有关。

Objective To investigate the inhibitory effects and related mechanism of S100A4 gene silencing on oxygen-induced retinal neovascularization. Methods 7-day-old C57BL/6J mice were randomly divided into 5 groups including normal group, normal S100A4 group, oxygen induced retinopathy （OIR） group, OIR-S100A4 group, OIR-green fluorescent protein （GFP） group. To establish the OIR model, mice from all groups except normal one were exposed to （75±2） % oxygen for 5 days and then to room air. In the OIR-S100A4 group and OIR-GFP group, the OIR mice were given an intravitreal injection of 1 μl of 1.0 × 10^9 PFU/ml adenovirus of Ad-S100A4-RNAi or Ad-GFP at P12, and then returned to normoxia for the next 5 days. In the OIR group, OIR was induced in C57bl/6J mice from P7 to P17. In the normal-SlOOA4 group, the normal P12 mice were give an intravitreal injection of 1 μl of Ad-S100A4-RNAi adenovirus, and maintained in room air from P12 to PlT. In normal group, newborn mouse litters were maintained in room air from P0 to P17 without any treatment. Mice in all five groups were euthanized at PlT, and retinas were collected for biochemical assays and morphological study. Retinal neovascularization （RNV） was evaluated by counting the number of pre-retinal neovascular cells and the whole mount immunofluorescent staining of the mouse retina. Protein and mRNA expression levels of S100A4, cAMP responsive element binding protein （CREB）, B cell lymphoma 2 （bcl-2）, Caspase-3 were determined with western blot and real-time PCR. Results The number of pre-retinal neovascular cell nuclei in retinas from OIR-S100A~ group were obviously lower than those in the retinas from OIR group and OIR GFP group （t=13.61, 14.64; P^0.05）. In OIR-S100A4 group, the retinal neovascular tufts area and the vaso-oblitertion area were both significantly smaller than those in OIR group and OIR-GFP group （P〈0.05）. Protein level of CREB and bcl-2 were significantly down-regulated in OIR-S100A4 group than those in OIR and OIR-GFP group （P^0.05）. On the contrary, protein levels of Caspase-3 were upregulated in OIR S100A4 group than those in OIR and OIR-GFP group （P〈0.05）. Conclusion Ad-S100A4- RNAi transfer ameliorates RNV in mouse model of OIR maybe through down-regulating the expression of bcl-2 and CREB, and up-regulating the Caspase-3.