血管内皮生长因子反义寡脱氧核苷酸联合血管生成素-1对糖尿病视网膜病变大鼠视网膜血管渗漏及新生血管生成的影响

Effects of vascular endothelial growth factor antisense oligonucleotide combined with angiopoietin-1 on retinal vascular leakage and neovascularization in diabetic retinopathy rats

目的观察血管内皮生长因子(vascular endothelial growthfactor,VEGF)反义寡脱氧核苷酸(ASODN)联合血管生成素-1(angiogenin-1,Ang-1)对糖尿病视网膜病变(diabetic retinopathy,DR)大鼠视网膜血管渗漏及新生血管生成的影响。方法选取健康雄性SD大鼠60只,随机分为正常对照组(A组)5只和糖尿病组55只,糖尿病组通过链脲佐菌素腹腔注射诱导糖尿病模型成功后再饲养3个月行眼底荧光血管造影检查,确定DR大鼠模型且视网膜病变程度相仿的大鼠,随机抽取45只分为DR对照组(B组,5只)、PBS缓冲液对照组(C组,10只)、VEGFASODN干预组(D组,10只)、Ang-1干预组(E组,10只)、联合干预组(F组,10只)。C组玻璃体内注射PBS缓冲液5μL;D组玻璃体内注射浓度为100μmol·L-1VEGFASODN5μL;E组玻璃体内注射浓度为160mg·L-1Ang-15μL;F组玻璃体内注射100μmol·L-1VEGFASODN及160mg·L-1Ang-1各5μL;3d后再次行上述操作。各组大鼠行眼底荧光血管造影检查,对比观察不同组别大鼠视网膜血管渗漏情况,病理组织切片光学显微镜下突破内界膜的观察视网膜新生血管芽细胞核数。结果 A、B、C、D、E和F组视网膜新生血管渗漏面积分别为0、(20.98±1.14)mm2、(21.47±1.65)mm2、(14.60±1.55)mm2、(13.80±1.19)mm2、(10.81±1.35)mm2;D、E、F组新生血管渗漏面积低于B、C组,差异有统计学意义(F=103.99,P<0.05);F组渗漏面积低于D、E组,差异有统计学意义(F=190.94,P<0.05);B组与C组之间差异无统计学意义(t=0.22,P>0.05)。A、B、C、D、E和F组突破内界膜的新生血管芽细胞核数分别为(1.13±0.31)个、(80.31±5.21)个、(81.08±2.57)个、(37.37±3.41)个、(41.07±2.09)个、(14.41±1.23)个;D、E、F组突破内界膜的血管芽细胞核数均低于B、C组,差异有统计学意义(F=1339.41,P<0.05);F组突破内界膜的血管芽细胞核数低于D、E组,差异有统计学意义(F=714.91,P<0.05);B组与C组之间差异无统计学意义(t=0.35,P>0.05)。结论 VEGF ASODN联合Ang-1玻璃体内注射能明显抑DR大鼠视网膜新生血管的生成,减少视网膜血管渗漏。

Objective To observe the effects of vascular endothelial growth factor （VEGF） antisense oligonucleotide（ASODN）and angiopoietin-1（Ang-1）on retinal vascular leakage and neovascularization in diabetic retinopathy （DR） rats.Methods Sixty healthy male SD rats were randomly divided into normal control group （group A,5 cases） and diabetic group （55 cases）.Streptozotocin was intraperitoneally injected to induce the diabetic model in diabetic group,after fed for 3 months,successful diabetic model were examined by fundus fluorescein angiography （FFA） to confirm the rats with similar retinopathy level,randomly picked up 45 rats and divided into 5 groups：DR control group （group B,5 cases）,PBS buffer control group （group C,10 cases）,VEGF ASODN intervention group （group D,10 cases）,Ang-1 intervention group （group E,10 cases） and combined intervention group （group F,with 10 rats）.PBS buffer of 5 μL was intravitreally injected in group C;100 μmol·L-1 VEGF ASODN of 5 μL was intravitreally injected in group D;160 mg·L-1 Ang-1 of 5 μL was intravitreally injected in group E;100 μmol·L-1 VEGF ASODN and 160 mg·L-1 Ang-1of each 5 μL were intravitreally injected in group F;Then above operation was repeated after 3 days.FFA was performed to observe the retinal vascular leakage,and histopathology specimen was made to observe the number of retinal neovascularization bud nucleus breaking through the internal limiting membrane.Results After intravitreal injection,the area of retinal vascular leakage in group A,B,C,D,E were 0,（20.98±1.14）mm2,（21.47±1.65）mm2,（14.60±1.55）mm2,（13.80±1.19）mm2,（10.81±1.35）mm2,respectively;The areas in group D,E,F were lower than that in group B and C,there was statistical difference（F=103.99,P0.05）;The area in group F was lower than that in group D and E,there was statistical difference（F=190.94,P0.05）;There was no difference between group B and C（t=0.22,P0.05）.The number of retinal neovascularization buds nucleus breaking through the inner limiting membrane in group A,B,C,D,E and F were 1.13±0.31,80.31±5.21,81.08±2.57,37.37±3.41,41.07±2.09 and 14.41±1.23;The number in group D,E,F were lower than that in group B and C,there was statistical difference（F=1339.41,P0.05）;The number in group F was lower than that in group D and E,there was statistical difference（F=714.91,P0.05）;There was no difference between group B and C（t=0.35,P0.05）.Conclusion The intravitreal combined injection of VEGF ASODN and Ang-1 can significantly reduce the retinal vascular leakage,and effectively inhibit the retinal neovascularization.