摘要
目的建立糖尿病微血管病变大鼠模型,模拟糖尿病视网膜及肾微血管病变的生化及病理改变。方法健康雄性Sprague-Dawley大鼠40只,随机分为空白组、模型组,分别为10、30只。空白组、模型组大鼠分别给予普通饲料、高脂高糖饲料喂养5周后,模型组大鼠按体重35mg/kg剂量经腹腔单次注射1%链脲佐菌素(STZ)建立2型糖尿病模型。建模后持续喂养至第10周(建模后4周),观察大鼠一般情况,采集样本进行后续实验。采集大鼠动脉血、尿样标本检测血肌酸酐(CREA)、三酰甘油(TG)、总胆固醇(TC)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)及尿微量白蛋白、尿肌酐(UCr)、尿糖;计算肾脏指数、微量尿白蛋白/尿肌酐比值(UACR)。光相干断层扫描血管成像(OCTA)观察大鼠视网膜浅层毛细血管丛血管变化及无灌注区情况;苏木精-伊红、高碘酸-雪夫染色观察大鼠肾脏、视网膜形态结构变化;免疫荧光染色观察大鼠视网膜Miller细胞神经纤维及细胞核表达情况;透射电子显微镜观察视网膜组织超微结果。组间比较采用独立样本检验。结果建模后4周,与空白组比较,模型组大鼠体重显著降低,随机血糖显著升高,差异有统计学意义(t=5.755、-51.291,P<0.05);肾脏指数、尿糖、UACR显著升高,UCr显著降低,差异有统计学意义(t=10.878、137.273、3.482、-6.110,P<0.05);CREA降低,TG、TC、HDL-C、LDL-C升高,差异均有统计学意义(t=28.012、33.018、118.018、13.585、16.480,P<0.05)。OCTA检查可见视网膜浅层毛细血管无灌注区。光学显微镜观察发现,模型组大鼠视网膜内界膜肿胀、增厚,表面不平,内、外核层细胞排列紊乱,密度降低;肾小球充血伴皮质管状上皮肿胀,肾小球系膜区增宽,基底膜增厚。免疫染色结果显示,模型组大鼠视网膜内、外丛状层呈片状强绿色荧光表达,内、外核层呈散在点状绿色荧光表达。透射电子显微镜发现,模型组大鼠视网膜微血管基底膜轻度增厚,血管内皮细胞水肿,内皮细胞核及周细胞核固缩、核膜皱缩,线粒体轻度肿胀,空泡化。结论高糖高脂喂养联合单次腹腔注射STZ 35mg/kg可成功建立2型糖尿病微血管病变模型。
Objective To establish a rat model of diabetic microangiopathopathy and simulate the biochemical and pathological changes of diabetic retinal and renal microangiopathopathy.Methods Forty healthy male Sprague-Dawley rats were randomly divided into blank group and model group(10 and 30 rats,respectively).After the rats in blank group and model group were fed ordinary diet and high-fat and high-sugar diet for 5 weeks,respectively,the rats in model group were injected with 1%streptozotocin(STZ)through the abdominal cavity at the dose of 35 mg/kg to establish a type 2 diabetes model.After modeling,the rats were continuously fed until the 10th week(4 weeks after modeling),the general conditions of the rats were observed,and samples were collected for follow-up experiments.Serum creatinine(CREA),triglyceride(TG),total cholesterol(TC),high density lipoprotein(HDL-C),low density lipoprotein(LDL-C),microalbuminuria,urinary creatinine(UCr)and urine sugar were detected.Calculate the kidney index and microalbumin/urinary creatinine ratio(UACR).Optical coherence tomography angiography(OCTA)was used to observe the vascular changes and non-perfusion area of retinal superficial capillary plexus.The morphological and structural changes of kidney and retina were observed by hematoxylin-eosin and periodate Scheff staining.The expression of nerve fibers and nucleus of Muller cells in rat retina was observed by immunofluorescence staining.Ultrastructural results of retina were observed by transmission electron microscope.Independent sample t test was used for comparison between groups.Results Four weeks after modeling,compared with blank group,the body weight of rats in model group was significantly decreased,and random glucose was significantly increased,with statistical significance(t=5.755,-51.291;P<0.05).Renal index,urinary glucose and UACR were significantly increased,while UCr was significantly decreased,with statistical significance(t=10.878,137.273,3.482,-6.110;P<0.05).CREA decreased,TG,TC,HDL-C,LDL-C increased,and the differences were statistically significant(t=-28.012,33.018,118.018,13.585,16.480;P<0.05).0CTA examination showed that there was no perfusion area of shallow retinal capillaries.The optical microscope showed that the inner boundary membrane of retina in model group was swollen and thickened,the surface was uneven,the inner and outer nuclear layer cells were disordered and the density decreased.Glomerular congestion was accompanied by cortical tubular epithelial swelling,widening of the mesangial area,and thickening of the basement membrane.The results of immunostaining showed that the inner and outer plexiform layers of the retina showed lamellar strong green fluorescence expression,and the inner and outer nuclear layers showed scattered dot green fluorescence expression.Transmission electron microscopy showed that the basal membrane of retinal microvessels in model group was slightly thickened,vascular endothelial cells edema,endothelial nucleus and perinucleus contraction,nuclear membrane contraction,mild mitochondrial swelling,vacuolation.Conclusion High-glucose and high-fat feeding plus a single intraperitoneal injection of STZ 35 mg/kg can successfully establish a microangiopathic model of type 2 diabetes.
作者
郑菀睿
王瑞
罗向霞
周茹玉
杨芮
曾敏
洪卓民
顾丽萍
Zheng Wanrui;Wang Rui;Luo Xiangxia;Zhou Ruyu;Yang Rui;Zeng Min;Hong Zhuomin;Gu Liping(Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;Department of Ofice of Academic Research,Lanzhou 730000,China;Department of Pathology,Gansu Provincial Hospital of Traditional Chinese Medicine,Lanzhou 730050,China)
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2023年第9期760-766,共7页
Chinese Journal of Ocular Fundus Diseases
基金
国家自然科学基金(81960888)。
关键词
糖尿病微血管病变
动物模型
评价
Diabetic microvascular complications
Animal model
Evaluation