CAJ | 학술논문

目的观察重组腺相关病毒(rAAV)介导的血管内皮生长因子(VEGF)反义核苷酸(rAAV-aVEGF165)对糖尿病大鼠视网膜VEGF表达的影响.方法 40只Sprague-Dawley大鼠用链尿佐菌素(STZ)腹腔注射诱导糖尿病大鼠模型.去除实验中途死亡和血糖恢复的大鼠.共计32只大鼠纳入研究,随机平均分为实验组和对照组,每组各16只.实验组和对照组大鼠玻璃体腔分别注射rAAV-aVEGF165(10<'10pfu/ml)、磷酸盐缓冲液各10 μl,建模后1、5个月免疫组织化学及Western blot评价视网膜VEGF的表达,并对视网膜血管进行透射电子显微镜观察.结果 1个月时,实验组和对照组大鼠视网膜VEGF表达极低;5个月时.实验组大鼠视网膜VEGF表达较同期对照组降低.差异有统计学意义(t=23.87,P<0.01).透射电子显微镜观察显示,实验组视网膜未见明显异常改变.而对照组视网膜微血管腔明显狭窄,基底膜明显增厚,密度不均.并出现断裂,内皮细胞明显肿胀,突起增多,管腔明显狭窄.周细胞内细胞器结构模糊,细胞核固缩,呈凋亡状态,异染色质明显分布不均,浓集.结论 rAAV-aVEGF165能下调视网膜VEGF的表达,从而阻止糖尿病视网膜病变的发生、发展;rAAV是眼反义基因治疗的有效载体.
목적 관찰중조선상관병독(rAAV)개도적혈관내피생장인자(VEGF)반의핵감산(rAAV-aVEGF165)대당뇨병대서시망막VEGF표체적영향.방법 40지Sprague-Dawley대서용련뇨좌균소(STZ)복강주사유도당뇨병대서모형.거제실험중도사망화혈당회복적대서.공계32지대서납입연구,수궤평균분위실험조화대조조,매조각16지.실험조화대조조대서파리체강분별주사rAAV-aVEGF165(10<'10pfu/ml)、린산염완충액각10 μl,건모후1、5개월면역조직화학급Western blot평개시망막VEGF적표체,병대시망막혈관진행투사전자현미경관찰.결과 1개월시,실험조화대조조대서시망막VEGF표체겁저;5개월시.실험조대서시망막VEGF표체교동기대조조강저.차이유통계학의의(t=23.87,P<0.01).투사전자현미경관찰현시,실험조시망막미견명현이상개변.이대조조시망막미혈관강명현협착,기저막명현증후,밀도불균.병출현단렬,내피세포명현종창,돌기증다,관강명현협착.주세포내세포기결구모호,세포핵고축,정조망상태,이염색질명현분포불균,농집.결론 rAAV-aVEGF165능하조시망막VEGF적표체,종이조지당뇨병시망막병변적발생、발전;rAAV시안반의기인치료적유효재체.
Objective To observe the influence of rAAV-mediated antisense vascular endothelial growth factor (rAAV-aVEGF165) on the expression of retinal VEGF in diabetic rats. Methods 40 Sprague-Dawley rats induced diabetic rat model by intraperitoneal injection with streptozotocin (STZ). 32 rats were involved in study besides death and blood sugar recovery in experimental process, 16 spragud- Dawleg (SD) rats were received intravitreal injection with rAAV-aVEGF165 (1010 pfu) as experimental group, another group of Sprague-Dawleg (SD) rats were injected with phosphate buffered saline (PBS) as control group. One and five month after model establishment, the expression of retinal VEGF was evaluate by immunhistochemistry and Western blot; the retinal vasular was examined by transmission electron microscopy. Results On 1 month,the expression of retinal VEGF was lowest in each group. On 5 month, the expression of retinal VEGF was decreased in experimental group which compared to control, the difference are statistically significant (t=23.87, P < 0.01). The transmission electron microscopy results showed that retina has no obvious chages in experimental group, however,contral group showed fragmental thickening and splitting of basement membrane, swelling and deformation of endothelia cells, fingerlike prcess into the capillary cavity,and uneven distibution of heterochromatin in pericytes. Conclusion rAAV-aVEGF165 can reduce the expression of retinal VEGF thereby preventing occurrence and development of diabetic retinopathy, rAAV is an effective vectors of eye antisense gene.