糖尿病大鼠视网膜血管消化铺片技术的改进及技巧探讨  

Improvements and discussion on technology of diabetic rats' retinal vascular digest preparations

作　　者：何洁[1] 韩静[2] 郝改梅[1] 吴晏[2] 王伟[1] 

机构地区：[1]北京中医药大学基础医学院,北京100029 [2]北京中医药大学科研实验中心

出　　处：《中国中医眼科杂志》2015年第1期1-5,共5页China Journal of Chinese Ophthalmology

基　　金：国家科技部"十二五"支撑计划项目(2012BAI29B07);北京中医药大学在读研究生项目(2014-JYBZZ-XS-024)

摘　　要：目的建立适合视网膜血管的消化方法,探讨一种稳定可重复的视网膜血管消化铺片技术。方法一次性腹腔注射链脲佐菌素建立糖尿病大鼠模型,另设正常组,36周后取材固定,分别采用胶原酶、胰蛋白酶及胃蛋白酶-胰蛋白酶联合消化法三种方法消化视网膜血管,观察并对比三种方法制作的铺片效果;对铺片进行形态学观察,并比较正常组与模型组毛细血管面积密度及内皮细胞/周细胞数比值的变化。结果胶原酶消化程度不足以显示出视网膜血管结构;胰蛋白酶消化法残存较多的神经胶质细胞及其他组织细胞,不能完全显示视网膜血管网;胃蛋白酶-胰蛋白酶联合消化法能完整显示出视网膜血管网,形态结构清晰,可以观察血管的面积分布、管径变化及管壁细胞结构。与正常组相比,模型组视网膜毛细血管排列紊乱,毛细血管面积密度显著升高(P<0.01);内皮细胞增生,周细胞减少,内皮细胞数与周细胞数比值升高(P<0.01)。结论胃蛋白酶-胰蛋白酶联合消化法是最适合视网膜血管的消化方法,结合良好规范的操作技巧可以稳定的重复视网膜血管消化铺片技术。OBJECTIVE To set up a suitable method to digest retina,and to explore a stable and repeatable technology of retinal vascular digest preparations. METHODS A diabetic rats＇ model group was set up by intraperitoneal injection of streptozotocin and fixed after 36 weeks. Another group was set up as control group. Applied colla-genase,trypsin and pepsin-trypsin digestion respectively to digest retinal vessels,then observed and compared the effects of preparations of the three methods; applied morphological observation to preparations and compared capil-lary area density and change of ratio between endothelial cells and pericytes of the model group and normal group.RESULTS Collagenase digestion was not sufficient to exhibit retinal vascular structure; trypsin digestion was not able to exhibit the complete retinal vascular network with much remains of neurogliocyte and other tissue cells;pepsin-trypsin digestion exhibits complete retinal vascular network with clear morphological structure,where the area distribution of vessels,pipe diameter changes,and wall cells structure could be observed. Compared with the normal group,retinal capillary of model group appears disorder and capillary area density significantly increases（P〈0.001）; endothelial cells proliferate and pericytes decrease,making ratio between them increases（P〈0.001）. CONCLUSIONS The pepsin-trypsin digestion was the most suitable method to digest retina,which could repeat retinal vascular digest preparations technology steadily combined with good practices of operating skills.

关 键 词：视网膜血管消化铺片 糖尿病视网膜病变 消化法 

分 类 号：R34[医药卫生—基础医学]