糖尿病视网膜病变大鼠骨髓内皮祖细胞数量和功能变化

摘要

目的 观察在大鼠糖尿病视网膜病变(DR)模型中骨髓内皮祖细胞(EPCs)功能变化,探讨EPCs在DR发病机制中的作用.方法 实验研究.雄性成年Wistar大鼠50只,以随机数字表法,随机分为正常对照(CON)组(14只鼠)和糖尿病(DM)组(36只鼠).根据病程,将DM组大鼠再分为糖尿病1个月(DM1)组、糖尿病3个月(DM3)组、糖尿病6个月(DM6)组,每组各12只.采用密度梯度离心法从各组大鼠骨髓获取单核细胞,将其接种于纤维连接蛋白包被的培养板;培养7d后,利用Dil-acLDL和FITC-UEA-I细胞荧光染色法及流式细胞仪CD34和CD133抗体标记法鉴定EPCs.在倒置相差显微镜下对EPCs计数克隆形成单位(CFU),以评估骨髓EPCs的数量水平；采用MTT比色法、Transwell小室和黏附能力测定实验观察EPCs的增殖、迁移和黏附能力.同时于各相应时间点摘除大鼠眼球行苏木精-伊红(HE)染色,采用免疫组织化学法分析血管内皮生长因子(VEGF)在大鼠视网膜中的表达.对比分析EPCs的数量和功能变化及其与DR形态学变化的相互关系.组间骨髓EPCs数量、功能及视网膜VEGF表达阳性率比较,采用单因素方差分析和LSD-t检验法.结果 DM1、DM3、DM6组大鼠骨髓来源EPCs-CFU为(9.6±1.8,11.5±2.5,14.0±2.4个/×200倍视野)较CON组(16.6±2.7个/×200倍视野)减少,差异有统计学意义(F=13.311,P＜0.01)；DM1、DM3、DM6组EPCs增殖能力(0.133±0.027,0.106±0.016,0.072±0.011 0D)较CON组(0.203±0.068 0D)降低,差异有统计学意义(F=16.091,P＜0.01)；DM1、DM3、DM6组EPCs迁移能力(11.9±2.2,10.1±1.8,8.9±1.2个/×200倍视野)较CON组(14.8±2.6个/×200倍视野)降低,差异有统计学意义(F=12.506,P＜0.01)；DM1、DM3、DM6组EPCs黏附能力(14.8±3.2,10.5±2.0,7.5±1.2个/×200倍视野)较CON组(17.9±4.8个/×200倍视野)降低,差异有统计学意义(F=17.087,P＜0.01).光学显微镜下,与CON组比较,DM1、DM3组视网膜视网膜厚度变薄,细胞排列紊乱,细胞核肿胀、体积增大,DM6组视网膜厚度更薄,细胞排列紊乱更加明显,部分血管扩张.VEGF在CON组大鼠视网膜中表达为阴性,DM1组阳性率为(18.6±2.74)％,DM3组阳性率约为(34.3±2.21)％,DM6组阳性率为(58.73±2.48)％.结论 糖尿病大鼠骨髓EPCs数量较正常大鼠降低,生物学功能减退.随着DR的进展,EPCs数量逐渐回升.%Objective To observe the change in the number and function of endothelial progenitor cells (EPCs) from bone marrow in rats with diabetic retinopathy (DR); to discuss the role of EPCs in the pathogenesis of DR.Methods In an experimental study,50 male Wistar rats were divided into a control (CON) group (14 rats) and diabetes (DM) group (36 rats).The rats in the DM were further divided into 3 groups of 12 rats each based on the time periods when analysis was performed:1 month (DM1),3 months (DM3) and 6 months (DM6).Mononuclear cells were collected by density gradient centrifugation from the bone marrow of the rats.The isolated cells were cultivated in dishes coated with fibronectin.Immunofluorescence staining and flow cytometry were used to identify EPCs.The number of EPCs in the colony was assayed by CFU counting; proliferation,migration and adhesion function of EPCs were assayed by MTT chromatometry,modified Boyden chamber assay and adhesion activity assay.All eyeballs were examined by hematoxylin and eosin (HE) staining and vascular endothelial growth factor (VEGF) by immunity set expression.The correlation between changes in EPCs and DR morphological changes was analyzed.The changes in the number and function of EPCs from bone marrow and expression of VEGF in the retina were analyzed by single factor analysis of variance and LSD-t test.Results The number of cell clusters in bone marrow-derived EPCs was significantly reduced in the DM1,DM3,DM6 groups (9.6±1.8,11.5±2.5,14.0±2.4 n/×200 fields) compared to the CON group (16.6±2.7 n/×200 fields).The ability of EPCs to proliferate was reduced in the DM1,DM3,DM6 groups (0.133±0.027,0.106±0.016,0.072±0.011 OD) compared to the CON group (0.203±0.068 OD).The ability of EPCs to migrate was reduced in the DM1,DM3,DM6 groups (11.9±2.2,10.1±1.8,8.9±1.2 n/×200 fields) compared to the CON group (17.9±4.8 n/×200 fields).The adhesion ability of EPCs was reduced in the DM1,DM3,DM6 groups (14.8±3.2,10.5±2.0,7.5±1.2 n/×200 fields) compared to the CON group (17.9±4.8 n/×200 fields).Accompanied by responsive pathological changes of retinal structure and vessels,the thickness of the retina was reduced and retinal cells became disorganized in the DM1 and DM3 groups.Endothelial cells expressed edema in the DM6 group.The expression of VEGF in the retina of rats was 0,18.6％±2.74％,34.3％±2.21％,58.73±2.48％,in CON,DM1,DM3,DM6 groups respectively.Conclusion The number and biological dysfunction of EPCs from bone marrow were reduced in diabetic rats.With the development of DR,the number of EPCs increased gradually.