TLR4-/- 抑制小鼠氧诱导视网膜新生血管生成的作用机制

目的通过建立氧诱导视网膜血管病变（OIR）模型，探讨Toll样受体4(TLR4)在视网膜新生血管中的作用及其机制。方法使用7日龄新生TLR4基因敲除（TLR4-/-）C57BL／6J小鼠和7日龄新生C57BL／6J小鼠建立OIR模型。分别于小鼠第12天（P12）、17天（P17）和21天（P21）时通过小鼠视网膜荧光灌注铺片观察新生血管面积，评估新生血管情况。通过免疫荧光染色,观察TLR4在小鼠视网膜中的表达情况。通过Real-Time PCR检测两组OIR小鼠P12和P17视网膜组织中核因子-κB(NF-κB)、血管内皮细胞生长因子(VEGF)和促炎因子白细胞介素-6(IL-6)的mRNA表达水平。结果在OIR模型中，通过视网膜荧光灌注铺片发现P12两组小鼠视网膜出现无血管区，P17均出现无血管区和新生血管簇，而且新生血管面积达到最高峰。P12、P17和P21时，TLR4-/- OIR小鼠中的视网膜新生血管面积均明显比正常OIR小鼠减少，差异有统计学意义（P<0.05)。通过免疫荧光检测发现TLR4在小鼠视网膜上广泛表达，与未建立OIR模型的正常小鼠相比，在P17时正常OIR小鼠视网膜的TLR4表达明显升高。对比正常OIR小鼠，TLR4-/- OIR小鼠视网膜组织的NF-κB、VEGF和促炎因子IL-6的表达水平明显降低，差异均有统计学意义（P<0.05)。结论TLR4在视网膜新生血管形成过程中起到重要作用,TLR4基因缺失通过抑制NF-κB信号通路，减少血管生成因子和炎性因子的释放，抑制OIR小鼠视网膜新生血管的生成。

Mechanism of inhibitory effect of TLR4-/- on oxygen-induced retinal neovascularization in mice

ObjectiveTo investigate the role and mechanism of TLR4 in retinal neovascularization by establishing a model of oxygen-induced retinal angiopathy (OIR). Methods The OIR model was established by using 7-day-old TLR4 gene knockout C57BL/6J mice as the experimental group and 7-day-old C57BL/6J mice as the control group. The area of neovascularization was observed by retinal fluorescence perfusion on the 12th day (P12), 17th day (P17) and 21st day (P21). The expression of TLR4 in mouse retina was observed by immunofluorescence staining. The mRNA expression levels of NF-κB, VEGF and pro-inflammatory factor IL-6 in P12 and P17 retinas of the two groups were detected by real-time PCR. ResultsIn the OIR model, through the method of fluorescence perfusion retinal preparation, we found that the retina of P12 mice showed non-vascular area, on P17 showed non-vascular area and neovascularization cluster, and the neovascularization area reached the peak. On P12 and P21, the area of retinal neovascularization in TLR4-/- OIR mice was significantly lower than that in normal OIR mice. Through immunofluorescence detection, it was found that TLR4 was widely expressed in the retina of mice. Compared with the normal mice without OIR, the expression of TLR4 in the retina of normal OIR mice was significantly increased on P17. Compared with normal OIR mice, the expression levels of NF-κB, VEGF and pro-inflammatory factor IL-6 in retina of TLR4 knockout OIR mice were significantly reduced. ConclusionTLR4 plays an important role in retinal neovascularization. TLR4-/- inhibits the formation of retinal neovascularization in OIR mice by inhibiting NF-κB signal pathway and reducing the release of angiogenic factors and inflammatory factors. This will provide a new strategy for the prevention and treatment of retinal neovascular diseases.