玻璃体内注射色素上皮源性因子对大鼠早期糖尿病视网膜病变的保护作用

目的　观察玻璃体内注射色素上皮源性因子（ｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ-ｄｅｒｉｖｅｄｆａｃｔｏｒ，ＰＥＤＦ）对ＳＤ糖尿病大鼠视网膜ＰＥＤＦ、血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＶＥＧＦ）、炎性相关因子细胞间黏附分子-１（ｉｎｔｅｒｃｅｌｌｕｌａｒｃｅｌｌａｄｈｅｓｉｏｎｍｏｌｅ-ｃｕｌｅ-１，ＩＣＡＭ-１）和单核细胞趋化蛋白-１（ｍｏｎｏｃｙｔｅｃｈｅｍｏｔａｃｔｉｃｐｒｏｔｅｉｎ-１，ＭＣＰ-１）以及细胞通透性相关因子紧密连接蛋白-１（ｚｏｎｕ-ｌａｏｃｃｌｕｄｅｎｓ-１，ＺＯ-１）和蛋白激酶Ｂ（ｐｒｏｔｅｉｎｋｉｎａｓｅＢ，ＰＫＢ，又称Ａｋｔ）表达的影响，探讨ＰＥＤＦ对早期糖尿病视网膜病变的保护作用。方法　选取６～８周龄ＳＤ大鼠６０只，随机分为４组:正常对照组（ＣＯＮ组）、糖尿病组（ＤＭ组）、糖尿病生理盐水注射组（ＤＭ＋ＮＳ组）和糖尿病ＰＥＤＦ注射组（ＤＭ＋ＰＥＤＦ组）。链脲佐菌素诱导建立糖尿病模型后第１周、２周、３周时，ＤＭ＋ＰＥＤＦ组大鼠玻璃体内注射ＰＥＤＦ，而ＤＭ＋ＮＳ组注射同样体积的生理盐水。第４周时处死大鼠，摘出眼球，进行视网膜组织病理学及电镜观察。并采用免疫组织化学方法检测视网膜ＰＥＤＦ、ＶＥＧＦ、ＩＣＡＭ-１、ＭＣＰ-１以及ＺＯ-１、Ａｋｔ的表达情况。结果　糖尿病大鼠均造模成功。４周糖尿病病程尚不能导致明显的视网膜新生血管形成。在透射电镜下，ＤＭ组大鼠视网膜可见神经节细胞水肿，线粒体肿胀变大，基质肿胀明显，可见大部分或全部的嵴消失，部分双侧膜融合，粗面内质网扩张，而玻璃体内ＰＥＤＦ注射可以明显地改善这一病理改变。ＤＭ组大鼠ＰＥＤＦ主要表达于视网膜神经纤维层、神经节细胞层以及内丛状层、光感受器基质以及色素上皮层、脉络膜等部位；ＶＥＧＦ主要表达于神经节细胞层，ＩＣＡＭ-１主要表达在光感受器层，ＭＣＰ-１主要表达在视网膜内层细胞，包括节细胞层和内核层，ＺＯ-１蛋白主要表达于内核层的细胞以及神经节细胞，Ａｋｔ主要表达于内丛状层以及脉络膜的细胞浆中。同ＣＯＮ组相比，ＤＭ组、ＤＭ＋ＮＳ组视网膜中ＰＥＤＦ、ＶＥＧＦ表达差异均无统计学意义（均为Ｐ＞０．０５），而ＩＣＡＭ-１、ＭＣＰ-１表达增加，ＺＯ-１、Ａｋｔ表达减少，差异均有统计学意义（均为Ｐ＜０．０５）。ＤＭ＋ＰＥＤＦ组大鼠视网膜中ＰＥＤＦ、ＶＥＧＦ的表达较ＤＭ组和ＤＭ＋ＮＳ组差异均无统计学意义（均为Ｐ＞０．０５），但ＩＣＡＭ-１、ＭＣＰ-１表达减少，ＺＯ-１、Ａｋｔ表达增多，差异均有统计学意义（均为Ｐ＜０．０５）。结论　ＰＥＤＦ可以明显地改善糖尿病视网膜病变早期视网膜神经节细胞的损害，通过减少炎性因子和增加细胞连接蛋白而减轻血-视网膜屏障的破坏，从而对早期糖尿病视网膜病变起一定的防治作用。

Protective effects of intravitreal injection of PEDF on early stage of diabetic retinopathy

Objective To observe the effects of intravitreal injection of pigment epithelium-derived factor ( PEDF) on the expression of PEDF, vascular endothelial growth factor ( VEGF) . intercellular cell adhesion molecule-l ( ICAM-I ) , monocyte chemotactic protein-l ( MCP-I ) . zonula occludens-l ( ZO-1 ) and protein kinase B ( PKB . also Akt) in the retina of diabetic rats . and discuss the protective effects of PEDF on early stage of diabetic retinopathy. Methods 6 - 8 weeks old male SD rats( n = 60) were randomly divided into four groups: normal control group ( CON group) , diabetic group ( DM group) , diabetic group with NS injection ( DM + NS group) and diabetic group with PEDF injection ( DM + PEDF group) . Diabetes model was induced by intraperitoneal injection of streptozotocin ( STZ) . The intravitreal injection of PEDF was performed at I week,2 weeks ,3 weeks after the DM models was established. Eyeballs were collected after rats were sacrificed at 4 weeks of DM. The expression of PEDF . VEGF. ICAM-I . MCP-I . ZO-1 and Akt in rat retina were assessed by immunohistochemistry , and the ultrastructure of retina was studied by transnussion electron microscopy. Results Diabetic rats were successfully induced. 4 weeks duration of diabetes still could not result in a significant retinal neovascularization. Under transmission electron microscopy , compared with CON rats , retinal ganglion cells edema, swollen and damaged mitochondrial were all found in both DM and DM + NS rats , and these pathological ultrastructural alterations could be partly ameliorated by PEDF injection. In the present study, PEDF was mainly expressed in nerve fiber layer, retinal ganglion cells , inner plexiform layer, photoreceptor matrix and retinal pigment epithelium. While VEGF was mainly expressed in retinal ganglion cell layer. ICAM-I was found expressed in photoreceptor layer. MCPI was expressed in the inner retinal cells .including ganglion cells and inner nuclear layer. ZO-1 was mainly found expressed in inner nuclear layer cells and ganglion cells. Akt was expressed in cells of inner ple)aform layer and choroid. Compared with CON group , the expression of PEDF and VEGF was not statistically significant(P > 0. 05 ) , while the expression of ICAM-I and MCP-I were statistically increased.but the expression of ZOI and Akt were statistically reduced in both DM and DM + NS group ( all P < 0. 05 ) . Compared with those of DM and DM + NS groups, the expression of retina PEDF and VEGF showed no alteration ( all P > 0. 05 ) .while ICAM-I . MCP-I were significantly reduced , but ZO-1 and Akt were significantly increased in DM + PEDF rats ( all P < 0. 05 ) . Conclusion PEDF can prevent retinal ganglion cell damage and vascular hyperpermeability in early diabetic retinopathy by decreasing ICAM-I and MCP-I and increasing ZO-I and Akt expression. PEDF may prevent the progression of early diabetic retinopathy.