摘要
目的观察不同浓度辛伐他汀对体外培养的糖尿病大鼠内皮祖细胞（EPC）数量和功能的影响。方法实验研究。雄性成年Wistar大鼠20只，通过腹腔内注射链脲佐菌素（STZ）建立糖尿病大鼠模型。建模后第12周脱颈处死大鼠，提取大鼠股骨和胫骨骨髓，利用密度梯度离心方法分离单核细胞进行培养。将培养的细胞随机分为正常对照组和干预组，正常对照组用EGM-2培养液培养，干预组分别在EGM-2培养液中加入0．01、0．1、1、10μmol／L的辛伐他汀进行培养。培养后7d，利用Dil标记的乙酰化低密度脂蛋白和异硫氰酸荧光素标记的荆豆凝集素-1共同染色方法鉴定EPC，同时利用CD34和CDl33共同标记法，以流式细胞仪鉴定EPC。倒置相差显微镜下计数EPC克隆形成单位，采用MTr比色法、Transwell小室和黏附能力测定实验观察EPC的增殖、迁移和黏附能力。摘除大鼠眼球行HE染色和视网膜病理组织学检查。组间骨髓EPC数量、增殖、迁移及黏附能力的比较采用单因素方差分析，组间各指标的多重比较采用LSD—t检验。结果EPC集落计数结果显示，干预组EPC集落数量[（16．5±1．6）、（19．0±2．3）、（21．9±2．0）、（13．9±2．4）＋／200倍视野]较正常对照组[（14．0±2．4）个／200倍视野]增加（F=20．493，P〈0．01）。EPC增殖实验、迁移实验及黏附实验结果显示，干预组EPC增殖能力（0．11±0．01、0．13±0．02、0．20±0．04、0．07±0．01）较正常对照组（0．07±0．01）提高（F=46．849，P〈0．01）；干预组EPC迁移能力[（10．5±1．6）、（12．9±2．2）、（15．9±2．4）、（9．4±1．4）＋／200倍视野]较正常对照组[（8．9±1．2）个细胞／200倍视野]提高（F=20．442，P〈0．01）；干预组EPC的黏附能力[（10．6±1．9）、（15．1±2．7）、（19．0±3．9）、（7．9±1．2）＋／200倍视野]较正常对照组[（7．5±1．2）＋／200倍视野]提高（F=33．013，P〈0．01）。光镜下观察可见，糖尿病大鼠视网膜厚度变薄，细胞排列紊乱，部分血管扩张，有向外突出、破溃的趋势。透射电镜下观察可见，毛细血管内皮细胞水肿，线粒体肿胀，毛细血管基底膜增厚，管腔狭窄和闭塞。结论较低浓度辛伐他汀可呈剂量依赖性提高体外培养的糖尿病大鼠骨髓EPC数量，增强EPC黏附、迁移和增殖功能。
Objective To observe the effect of simvastatin on the amount and function of endothehal progenitor cells from bone marrow in diabetic rats. Methods Experimental study. Twenty male Wistar rats were induced with streptozotocin （STZ） injection for the establishment of diabetic retinopathy model. Mononuclear cells were collected by density gradient centrifugation from the bone marrow of rats. The isolated cells were cultivated in dishes coated with fibronectin. Cultured cells were divided into normal control （CON） group and the intervention group. Cells in the CON group were cultured with regular culture medium and the intervention group was treated with O. 01, O. 1, 1 and 10 μmol/L of simvastatin. Immuno- fluorescence staining and flow cytometry were used to indentify EPC. The colony number of EPC was assayed by CFU counting. Proliferation, migration and adhesion function of EPC were assayed by MTT chromatometry, modified Boyden chamber assay and adhesion activity assay, respectively. All eyeballs were examined by histopathological examination stained by hematoxylin and eosin （HE） and electron microscopic examination. The amount, proliferation, migration and adhesion function of EPC were analyzed by one-way ANOVA and LSD-t methods. Results The cell clusters number of bone marrow-derived EPC was significantly increased in intervention groups [ （ 16. 5 ± 1.6 ）, （ 19.0 ± 2. 3 ）, （ 21.9± 2. 0 ）, （ 13.9 ± 2.4） n/200 fields in cultures treated with O. 01,0. 1, 1 and 10 μmol/L of simvastatin] compared with that in the CON group [ （14.0 ± 2. 4 ）n/200 fields ]. The proliferation ability of EPC was increased in intervention groups （0. 105 ± 0. 014, 0. 133 ± 0. 024, 0. 202 ± 0. 039 and 0. 068± 0. 011 ） compared with that in the CON group（0. 072± 0. 011 ）. The migration ability of EPC was increased in intervention groups [ （ 10. 5 ± 1.6）, （ 12. 9 ± 2. 2 ）, （ 15.9 ± 2.4 ）, （ 9.4± 1.4 ） cells/200 fields ] compared with that in the CON group [ （ 8.9±1. 2） cells/200 fields ]. The adhesion ability of EPC was increased in intervention groups [ （ 10. 6± 1.9）, （ 15.1 ± 2.7 ）, （ 19. 0± 3.9 ）, （7.9 ± 1.2 ） cells/200 fields ] compared with that in the CON group [ （7. 5 ± 1.2） cells/200 fields ]. The thickness of retina was reduced and retinal cells became disorganized in diabetic rats. Transmission electron microscopy showed capillary lumen stenosis and retinal microaneurysms with severe local tissue ischemia such as vacuolar degeneration perivascular tissue. Conclusion Simvastatin can enhance the amount and function of EPC from bone marrow in diabetic rats in a dose-dependent manner.
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2012年第11期1015-1020,共6页
Chinese Journal of Ophthalmology
基金
天津市应用基础及前沿技术研究计划（10JCZDJC20300）
