中华眼底病杂志

中华眼底病杂志

Delta样配体4对早期糖尿病大鼠视网膜病理结构的影响及其与血管内皮生长因子受体-2的关系研究

查看全文

目的观察Delta样配体4(Dll-4)对早期糖尿病(DM)大鼠视网膜病理结构的影响及其与血管内皮生长因子受体(VEGFR)-2的关系。方法雄性Sprague-Dawley大鼠70只,随机数字表法分为正常组(10只)和DM组(60只)。DM组大鼠经腹腔注射链脲佐菌素诱导DM模型;剔除血糖恢复和死亡大鼠,最终50只大鼠纳入统计。正常组大鼠注射等体积柠檬酸-柠檬酸钠缓冲液。随机数字表法将DM组大鼠分为DM 1m组、DM 2m组、DM 3m组、DM 3m+Anti组、DM 3m+磷酸盐缓液(PBS)组,各组均为10只大鼠。DM 3m+Anti组大鼠玻璃体腔注射抗Dll-4多克隆抗体4 μl,抗体浓度0.25 mg/ml;DM 3m+PBS组大鼠玻璃体腔注射等体积PBS。注射后5 d,处死大鼠。其余各组大鼠不做任何处理,分别于成模后1、2、3个月处死。苏木精-伊红染色观察视网膜各层结构及微血管变化,测量视网膜总厚度;免疫组织化学和荧光定量聚合酶链反应(PCR)检测视网膜中Dll-4、VEGFR-2 mRNA的表达。单因素方差分析比较各组大鼠视网膜中Dll-4、VEGFR-2表达差异;两组间比较采用最小显著差法t检验。结果光学显微镜观察发现,DM 3m组大鼠视网膜神经节细胞层明显水肿,内、外核层变薄,细胞数量减少,排列紊乱,外丛状层(OPL)水肿明显,微血管异常扩张;DM 3m+Anti组大鼠视网膜OPL水肿较DM 3m组减轻,其余各层无明显差异。与正常组比较,DM 3m组、DM 3m+Anti组、DM 3m+PBS组大鼠视网膜总厚度增加,差异均有统计学意义(t=5.596、3.290、4.286,P=0.000、0.008、0.002)。免疫组织化学染色结果显示,正常组大鼠视网膜神经节细胞层可见少量Dll-4阳性表达;神经节细胞层及内、外核层均可见少量VEGFR-2阳性表达。DM 1m组、DM 2m组、DM 3m组大鼠视网膜血管内皮细胞中Dll-4阳性表达逐渐增加。DM 3m+Anti组大鼠视网膜各层及血管内皮细胞中Dll-4阳性表达明显减少,而VEGFR-2表达显著增加。DM 3m+PBS组大鼠视网膜Dll-4、VEGFR-2阳性表达与DM 3m组无明显差异。荧光定量PCR检测结果显示,DM 3m组大鼠视网膜中Dll-4、VEGFR-2 mRNA表达量较正常组明显升高,差异有统计学意义(t=6.705、20.871,P<0.05)。与DM 3m组比较,DM 3m+Anti组大鼠视网膜Dll-4 mRNA表达量降低,VEGFR-2 mRNA表达量升高,差异均有统计学意义(t=2.681、3.639,P<0.05)。DM 3m+PBS组、DM 3m组大鼠视网膜中Dll-4、VEGFR-2 mRNA表达量比较,差异无统计学意义(t=0.513、0.657,P>0.05)。结论DM大鼠早期视网膜病变过程中视网膜血管内皮细胞中Dll-4表达逐渐增加;抑制Dll-4表达,VEGFR-2的表达上调,且OPL水肿减轻。

ObjectiveTo observe the effect of Delta-like ligand 4 (Dll-4) on the pathological structure of retina in early diabetic rats (DM) and its relationship with vascular endothelial growth receptor-2 (VEGFR-2).MethodsA total of 70 male Sprague-Dawley rats were randomly divided into normal group and DM group, with 10 and 60 rats in each group, respectively. The rats of DM group was induced by intraperitoneal injection of streptozotocin to established DM model. The rats with blood glucose recovery and death were excluded, and the final 60 rats were included in the statistics. Rats in the normal group were injected with an equal volume of citric acid-sodium citrate buffer. Rats in the DM group were divided into DM 1 month (DM 1m) group, DM 2 months (DM 2m) group, DM 3 months (DM 3m) group and DM 3m + Anti group, DM 3m + phosphate buffer solution (PBS) group by random number table method, and 10 rats in each group. In the DM 3m+Anti group, 4 μl of anti-Dll-4 polyclonal antibody was injected into the vitreous cavity, and the antibody concentration was 0.25 mg/ml. The DM 3m+PBS group was intravitreally injected with an equal volume of PBS. Five days after the injection, the rats were sacrificed. Rats in the DM 3m group and the normal group were not treated, and were sacrificed 3 months after the model was established. The structure and microvascular changes of the retina were observed by hematoxylin-eosin staining, and the total thickness of the retina was measured. The expression of Dll-4 and VEGFR-2 in the retina was detected by immunohistochemistry and fluorescence quantitative polymerase chain reaction (PCR). One-way analysis of variance was used to compare the expression of Dll-4 and VEGFR-2 in the retina of each group. The least significant difference t test was used to compare the two groups.ResultsLight microscopy showed that the retinal ganglion cells layer in the DM 3m group were obviously edematous, the inner and outer nuclear layers were thinner, the number of cells was reduced, the arrangement was disordered, the edema of outer plexiform layer was obvious, and the microvessels were abnormally dilated. In the DM 3m+Anti group, the edema of outer plexiform layer was lessened than that of the DM 3m group, and the other layers were not significantly different from the DM 3m group. Compared with the normal group, the total retinal thickness of the DM 3m group, the DM 3m+Anti group and the DM 3m+PBS group increased (t=5.596, 3.290, 4.286; P=0.000, 0.008, 0.002). Immunohistochemical staining showed that a small amount of Dll4 was positively expressed in the retinal ganglion cell layer of the normal group; a small amount of VEGFR-2 was positively expressed in the ganglion cell layer and the inner and outer nuclear layers. The positive expression of Dll-4 and VEGFR-2 in retinal vascular endothelial cells of DM 3m group increased significantly. The expression of Dll-4 was significantly decreased in the retinal layers and vascular endothelial cells of DM 3m+Anti group, while the expression of VEGFR-2 was significantly increased. There was no significant difference between the positive expression of Dll4 and VEGFR-2 in the DM 3m+PBS group and the DM 3m group. The results of real-time PCR showed that the relative expression of Dll-4 and VEGFR-2 mRNA in the DM 3m group was significantly higher than that in the normal group (t=6.705, 20.871; P<0.05). Compared with DM 3m group, the relative expression of Dll-4 mRNA in DM 3m+Anti group decreased, and the relative expression of VEGFR-2 mRNA increased (t=2.681, 3.639;P<0.05). The relative expressions of Dll-4 and VEGFR-2 mRNA in the DM 3m+PBS group and DM 3m group were not statistically significant (t=0.513, 0.657; P<0.05).ConclusionsThe expression of Dll-4 in retinal vascular endothelial cells is gradually increased during the early retinopathy of DM rats. The expression of Dll-4 is inhibited, the expression of VEGFR-2 is up-regulated, and the plexus edema is alleviated.

关键词: 糖尿病视网膜病变; Delta样配体4; 血管内皮生长因子受体2

Key words: Diabetic retinopathy; Detal like 4; Vascular endothelial growth factor receptor 2

引用本文: 余川, 胡迭, 孟旭霞, 周贤慧, 曹博雯, 李晓庆, 陈雅楠. Delta样配体4对早期糖尿病大鼠视网膜病理结构的影响及其与血管内皮生长因子受体-2的关系研究. 中华眼底病杂志, 2018, 34(6): 586-591. doi: 10.3760/cma.j.issn.1005-1015.2018.06.012 复制