摘要目的 观察糖尿病大鼠视网膜中以及双靶点干预后血管内皮生长因子(VEGF)和结缔组织生长因子(CTGF) mRNA表达变化.方法 Sprague-Dawley大鼠48只,随机分为正常对照组(CON1组)和糖尿病(DM)组.经鼠尾静脉注射链脲佐菌素制作糖尿病大鼠模型.建模后8、10、12周取大鼠视网膜标本行逆转录实时定量聚合酶链反应(RT-PCR)检测VEGF、CTGF mRNA表达变化.根据上述结果,选取相同条件的大鼠60只,随机选择50只大鼠依照上述方法建立糖尿病大鼠模型；10只大鼠为正常对照组(CON2组).建模后第10周,将糖尿病大鼠随机分为双靶点干预组、ranibizumab单靶点干预组、CTGF小发夹RNA(shRNA)单靶点干预组、DM未干预组.干预1周后取各组大鼠视网膜标本,行实时定量RT-PCR检测VEGF、CTGF mRNA表达变化.结果 建模后第8周,DM组大鼠视网膜CTGF mRNA水平显著增高且一直持续到第12周,与CON1组比较,差异均有统计学意义(t=-2.49、-2.67、-2.42,P＜0.05)；第8周时DM组大鼠视网膜VEGF mRNA水平与CON1组比较,差异无统计学意义(f=-0.443,P=0.669)；第10周时显著上调且一直维持到第12周,与CON1组比较,差异有统计学意义(t=-2.35、-2.57,P＜0.05).Ranibizumab单靶点干预组大鼠视网膜VEGF mRNA表达水平较DM未干预组显著降低,差异有统计学意义(t=-3.44,P＜0.05),与CON2组比较,差异无统计学意义(f=-1.37,P＞0.05)；CTGFmRNA表达显著高于DM未干预组,差异有统计学意义(t=2.48,P＜0.05).CTGF shRNA单靶点干预组大鼠视网膜CTGF、VEGF mRNA表达均较DM未干预组显著降低,差异有统计学意义(t=0.23、-2.92,P＜0.05).双靶点干预组大鼠视网膜VEGF、CTGF mRNA表达均下调,与DM未干预组比较,差异均有统计学意义(t=-6.09、-5.11,P＜0.001)；与CON2组比较,差异无统计学差异(t=-1.16、1.139,P＞0.05).结论 早期DM大鼠视网膜内VEGF、CTGF基因表达即升高,且CTGF升高较VEGF早.Ranibizumab结合CTGF shRNA双靶点干预能同时降低VEGF、CTGF基因在DM大鼠视网膜中的表达.

abstractsObjective To observe the effects of dual targets intervention on the expression of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in diabetic rat retina.Methods Forty-eight Sprague-Dawley rats were randomly divided into control group (CON1 group) and diabetes mellitus group (DM group).The rats of DM group were induced with streptozotocin injection creating a diabetic model.Retinas were obtained at eight,10,12 weeks after DM induction from both groups.CTGF and VEGF mRNA levels were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR).Based on the results of above experiments,60 rats with same conditions were selected.Fifty rats were induced with streptozotocin injection creating a diabetic model,and 10 rats comprised the control group (CON2 group).Then the 50 diabetic rats were randomly divided into ranibizumab and CTGF shRNA dual targets intervention group,ranibizumab single-target intervention group,CTGF shRNA singletarget intervention group and non-intervention group.Retinas were obtained at one week after intervention from all the groups.CTGF and VEGF mRNA levels were examined by RT-PCR.Results The levels of CTGF mRNA were significantly higher in DM group than that in CON1 group at the 8th weeks after DM induction,and this up-regulation was maintained through the 12th week (t=-2.49,-2.67,-2.42;P＜0.05).There was no difference on VEGF mRNA levels between DM group and CON1 group at the 8th weeks after DM induction (t=-0.443,P =0.669).VEGF mRNA levels of DM group started to be significantly elevated over those in the CON1 group at the 10th week,and remained to be higher at the 12th week (t=-2.35,-2.57; P＜0.05).The VEGF mRNA of ranibizumab single-target intervention group was significantly lower than that in non-intervention group (t=-3.44,P=0.014),which was similar to CON2 group (t =-1.37,P＞ 0.05) ; however,the CTGF mRNA level was significantly increased as compared to the non-intervention group (t =2.48,P ＜ 0.05).In the CTGF shRNA single-target intervention group,the levels of CTGF and VEGF mRNA were decreased as compared to the nonintervention group (t =0.23,-2.92; P＜ 0.05).In the ranibizumab and CTGF shRNA dual targets intervention group,the levels of CTGF and VEGF mRNA were decreased as compared to the nonintervention group (t=-6.09,-5.11; P＜0.001),which was similar to CON2 group (t=-1.16,1.139; P＞0.05).Conclusions Both CTGF and VEGF gene expression are up-regulated in early diabetic rat retina,and the level of CTGF increased earlier than VEGF.Ranibizumab combined with CTGF shRNA could simultaneously reduce the level of CTGF and VEGF mRNA in diabetic rat retina.