糖尿病大鼠早期视网膜形态观察和Bcl-2、Bax及VEGF表达的意义

Retinal morphology and expression of Bcl-2, Bax and VEGF in early diabetic rats

摘要:

目的：探讨B-细胞淋巴瘤因子(B-cell lymphoma factor，Bcl-2)，Bcl-2相关X蛋白(Bcl2-Associated X protein，Bax)和血管内皮生长因子(vascular endothelial growth factor，VEGF)在早期糖尿病大鼠视网膜上的表达及意义。

方法：用链脲佐菌素(streptozotocin，STZ)腹腔注射(60mg/kg)制作大鼠早期糖尿病模型。于造模后4、8、12wk颈椎脱臼法处死大鼠，取双眼全眼球组织做石蜡切片并做视网膜铺片，通过HE染色观察视网膜各层的形态学和血管分布变化； 取双眼视网膜组织石蜡切片，通过免疫组化法检测Bcl-2、Bax和VEGF在视网膜组织中的表达。行ADP酶视网膜血管染色，观察视网膜血管形态变化。应用激光共聚焦显微镜检测视网膜细胞的形态、细胞中Ca²⁺的荧光强度和分布变化。

结果：糖尿病组造模12wk突破内界膜的内皮细胞核个数呈递增趋势。糖尿病组视网膜中周部和周边部可见无血管区，无血管区面积也明显大于空白对照组，差异有统计学意义(P<0.05)。糖尿病组大鼠VEGF、Bcl-2和Bax光密度值与空白对照组比较，差异均有统计学意义(P<0.05)。糖尿病组大鼠造模4、8、12wk比较，RGCs内钙离子荧光浓度逐渐升高，荧光染色强度比值升高，差异均有统计学意义(P<0.05)。

结论：早期糖尿病大鼠视网膜的Bcl-2和Bax表达非常明显，从而上调 VEGF的表达。Bcl-2、Bax和VEGF是糖尿病视网膜病变中新生血管形成的重要影响因素。

Abstract:

AIM: To investigate the expression and significance of B-cell lymphoma factor(Bcl-2), Bcl2-Associated X protein(BAX)and vascular endothelial growth factor(VEGF)in the retina of early diabetic rats.

METHODS: An early diabetic retinopathy model was made in rat by intraperitoneal injection of streptozotocin(60mg/kg). The model rats were sacrificed at 4,8,12wk after the establishment of the model, and the eyeballs were removed to make paraffin section and retina sheets were prepared. HE staining was used to detected the retinal morphology and vascularity. Immunohistochemistry(IHC)was employed to examine the expression of Bcl-2, Bax and VEGF in the retina. ADP enzyme staining were conducted to evaluate the retinal vascular morphologic change. Nikon-A1 laser confocal microscopy was used to detect the morphology, fluorescence intensity and distribution of Ca²⁺ in retinal cells.

RESULTS: In the diabetic group, the number of endothelial cells in the inner limiting membrane increased 12wk later. In diabetes mellitus group, there was no vascular area in the middle and peripheral retina, and no vascular area was significantly more than that in the blank control group(P<0.05). There were significant differences in the values of VEGF, Bcl-2 and Bax between diabetic rats and control group(P<0.05). Compared with diabetic rats at 4, 8, 12wk, the fluorescence concentration of calcium ions in RGCs increased gradually, and the ratio of fluorescence staining intensity increased significantly(P<0.05).

CONCLUSION: The expressions of Bcl-2 and Bax were significantly increased, which upgraded the expression of VEGF in the retinas of early diabetic rats. Bcl-2, Bax and VEGF might play an important role in the neovascularization of the retinas in early diabetic rats.