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,Plt;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. （Chin J Ocul Fundus Dis，2008，24：255-258）
【摘要】 目的 在实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)模型中,比较常规T2加权成像(T2weighted imaging,T2WI)、钆二乙三胺五醋酸(gadoliniumdiethylenetriamine pentaacetic acid,GdDTPA)和超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)增强图像之间的差异,探讨巨噬细胞在多发性硬化(multiple sclerosis,MS)炎性活动病灶中的细胞学标志。方法 在EAE模型临床症状的亚临床期、初发期、高峰期,13只复发缓解(relapsingremitting,RR)EAE大鼠模型组和13只正常对照组大鼠在注入对比剂之前均行常规T2WI扫描,接着分别在其尾静脉注入GdDTPA后5 min行T1加权成像(T1weighted imaging,T1WI),再注入SPIO,24 h后行T2WI扫描。扫描完毕后立即处死大鼠取脑,行脑组织切片的ED1免疫组织化学染色和Prussian blue染色。结果 EAE模型组大鼠在第11天出现临床症状(初发期),第14天达到高峰期;MRI检查:SPIO增强图像对EAE病灶的显示较常规T2WI和GdDTPA增强图像好。病理学检查:ED1染色,在SPIO显示为低信号的区域内出现了炎症细胞(以巨噬细胞为主)浸润;Prussian blue染色示病灶内巨噬细胞胞质内出现了蓝染颗粒,沉积部位与T2WI上低信号区对应。对照组大鼠均无异常。结论 SPIO较GdDTPA更好地显示EAE模型中炎性活动性病灶内血管周围以巨噬细胞为主的浸润。
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 rAAV2CMVhPEDF into vitreum as treat group,left eyes were injected into rAAV2-CMV-GFP (1times;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 （Plt;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 （Plt;0.05），but all lower obviously than respectively own control group at the same timepoint （Plt;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. （Chin J Ocul Fundus Dis，2008，24：259-264）
Objective To observe the morphological changes of dendrite and soma in retinal ganglion cells (RGCs) which subsisted in early diabetic rats. Methods The RGCs of 3-months-course diabetic rats and coeval normal rats were marked by gene gun techniques. To collect RGCs photographs by Leica microscope with Z axis and CCD camera；to observe the changes of diameter, variance of structural features in dendritic field and somata after classification which according to the size and morphology. Thy-1 antibody marks on the retinal RGCs, taking a photograph under fluorescent microscope, counting the changes of retinal RGCs density in early diabetic rat. Results In three-month diabetic rats,the density of retinal RGCs was decreased obviously. Morphological changes of RGCs in the dendritic fields were observed with gene gun technique. There was no severe variation in all kinds of the bole of cell dendrite, in which some only showed crispation partially and sparseness also twisting in the dendritic ramus. The mean diameter of dendritic field and soma in class A of diabetic rats was (401plusmn;86) mu;m, the mean diameter of dendritic field in control group was (315plusmn;72) mu;m,compared with each other, there is statistically significant differences (t=21.249，Plt;0.001); the mean diameter of soma in class A of diabetic rats was (24plusmn;6) mu;m, the mean diameter of soma in control group was (22plusmn;5) mu;m, compared with each other, there is no statistically significant differences (t=0.927，Pgt;0.05); the mean diameter of dendritic field and soma in class B of diabetic rats were (170plusmn;36)、(14plusmn;2) mu;m respectively, in control group were (165plusmn;36)、(16plusmn;2) mu;m, the mean diameter of dendritic field and soma in class C of diabetic group were(265plusmn;78)、(17plusmn;5) mu;m respectively, in control group were (251plusmn;57)、(17plusmn;4) mu;m , compared with each other, there are on statistically significant differences(t=1.357，0.798，0.835，1.104，Pgt;0.05). Conclusions In short-term diabetes, the survived RGCs show good plasticity in adult diabetic rats, especially in class A. The changes of dendrites were more sensitive than the soma, which could be the leading index of the morphologic changes of RGCs in the early stage. The good plasticity showed by the RGCs and the time window from changing in dendrite to cell death provide us many evidences not only for the research but also for the nerve protection in clinic. (Chin J Ocul Fundus Dis,2008,24:249-254)
Objective To observe the inhibition effect of the hypoxia inducible factor-1alpha; (HIF-1alpha;) specific siRNA on the expression of vascular endothelial growth factor (VEGF) mRNA in retinal tissues in diabetic rat. Methods This is a randomized controlled study. HIF-1alpha; specific siRNA recombinant plasmid was built in pSilencer2.1-U6neo vector. Fifty-four healthy Sprague Dawley (SD) rats were divided into control group (15 rats) and experimental group (39 rats). The experimental rats were induced with streptozotocin injection for diabetic retinopathy model, and then randomly divided into diabetic retinopathy (DR) group (15 rats), vector group (12 rats) and gene therapy group (12 rats). LipofectamineTM2000 mixed with pSilencer2.1-U6neo plasmid or HiF-1alpha; siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively. Nothing was transfected into DR and control group. The expression of VEGF mRNA in retinas was measured by real-time RT-PCR. The inhibition efficiency of VEGFmRNA was calculated at 24, 48, 72 hours and 1 week after injection respectively. Significant differences between groups were evaluated by oneway analysis and LSD-t analysis. Results HIF-1alpha; siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis. Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the control group, increased obviously in the DR and vector group, decreased in the gene therapy group. There was no statistically significant between DR and vector group (t=0.669，0.142,0.151，0.025；P=0.514，0.889，0.882，0.980). The expression of VEGFmRNA in the gene therapy group were obviously decreased compared with DR and vector group (t=8.768, 13.695, 11.285, 8.253;P=0.000). The inhibition efficiency of VEGFmRNA was 32.76%, 43.60%, 47.70%, 50.86% at 24, 48, 72 hours and 1 week after injection. Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-1alpha; siRNA recombinant plasmid.
Objective To observe the opticin expression in the eyes of nonobese diabetes (NOD) mice and nondiabetic NOD mice.Methods Twenty NOD mice were divided into diabetic group (experimental group) and nondiabetic group (control group). All the mice were killed by cervical dislocation method.The eyes were harvested, and the vitreous, retina and sclera were separately collected. Western blot and realtime reverse transcriptionpolymerase chain reaction（RT-PCR）were respectively used to determine opticin protein and OPTC mRNA levels.Results The opticin protein level in the vitreous and retina was lower in the experimental group（t=4.42,4.58;P=0.002,0.002）,but is same in thesclera between the 2 groups（t=0.27,P=0.794）.OPTCmRNA level was vitreousgt;retinagt;sclera. OPTCmRNA levels of vitreous and retina in diabetic group were significantly lower（t=3.30,2.48；P=0.01,0.04）; there was no statistical significant on OPTC mRNA of sclera between two groups（t=0.27,P=0.80）.Conclusion Expression of opticin was suppressed in retina and vitreous of diabetic mice.