重组腺相关病毒载体介导的色素上皮衍生因子对糖尿病大鼠视网膜的影响

目的以重组腺相关病毒载体（rAAV）介导色素上皮衍生因子（PEDF）转染糖尿病大鼠视网膜，观察其表达及其对血管内皮生长因子（VEGF）和视网膜微血管的影响。方法 雄性 Wister大鼠链脲佐菌素腹腔注射诱导糖尿病模型。随机分为1（DM1）、3（DM3）、6（DM6）个月组。右眼玻璃体注射rAAV2-CMV-hPEDF作为治疗组，左眼注射rAAV2-CMV-GFP作为自身对照眼。正常大鼠右眼假注射，左眼不注射。应用半定量逆转录聚合酶链反应和Western blot测定VEGF和PEDF的mRNA和蛋白的表达情况，应用视网膜血管铺片观察视网膜微血管的变化。结果注射后大鼠治疗眼视网膜hPEDF mRNA表达不断增强，持续到6个月，达到顶峰。PEDF蛋白表达亦不断增加，与同时间点对照眼相比差异有统计学意义（t=4.292，10.721，16.692；P＜0.01）。治疗组各时间点视网膜VEGF mRNA及蛋白表达量相互间无统计学意义（t=1.621，0.698，0.758；P＞0.05），均显著高于正常对照组（t=5.171，6.857，7.542；P＜0.05），但与同时间点自身对照眼相比表达显著降低（t=2.343，3.263，4.455；P＜0.01）。糖尿病大鼠治疗眼与自身对照眼视网膜微血管形态在1个月时与正常对照组视网膜相比无显著差异，但3个月与6个月时治疗眼与自身对照眼相比视网膜毛细血管损伤改变明显减轻。结论 rAAV2-CMV-hPEDF玻璃注射可增加糖尿病大鼠视网膜PEDFmRNA和蛋白水平表达，改善其早期视网膜微血管的损害，并抑制VEGF的表达，其调节在mRNA水平。 （中华眼底病杂志，2008，24：259-264）

Effect of recombinant adeno-associated virus mediated pigment epithelium derived factor on retina of diabetic rats

Objective To observe the expression of the pigment epithelium derived growth factor (PEDF) in retina and the effects of PEDF for vascular endothelial growth factor (VEGF) and retinal microvascular after recombinant adeno-associated virus mediated pigment epithelium derived factor (rAAV-mediated-PEDF)transferred retina of diabetic rats. Methods Male Wister rats were induced diabetes with intraperitoneal injection of streptozocin, then divided into 1 month group (DM1), 3month group (DM3) and 6 month group randomly. In diabetic rats, right eyes were injected rAAV2-CMV- hPEDF into vitreum as treat group,left eyes were injected into rAAV2-CMV-GFP (1×1011 v.g/ml) as self-control group. In normal rats, right eyes were punctured but not injected (CONf), left eyes let it be without any disposal. The levels of VEGFmRNA and hPEDFmRNA in retina were evaluated using RT- PCR in different period. The protein levels of VEGF, PEDF within the retina were determined using western blot. The change of retinal capillary were observed through retinal vascular flattening. Result The expression of hPEDFmRNA in retina was enhanced persistence after rAAV2-CMV-hPEDF injected, achieved climax until 6 months. The levels of PEDF were also incerased consecutive, the differences were statistically significant in treatment group compared with own control group (P<0.01). Levels of mRNA and protein of VEGF at different time-points among therapy group were not statistically significant, all obviously higher than normal (P<0.05),but all lower obviously than respectively own control group at the same time-point (P<0.01). The morphology of retinal capillary was not different significant with normal rats in 1 month diabetic rats. Morphology changes of therapy groups were less than those of respective own control group in DM3 and DM6. Conclusion Intravitreous injection rAAV2-CMV- hPEDF can increase expression of mRNA and protein of PEDF,alleviate lesion of retinal microvascular in early period of diabetic rats and supress expression of VEGF in retina of diabetic rats. The regulation occur on mRNA level.