血小板反应蛋白-1在糖尿病大鼠模型肾损害中的作用及机制初步探讨

目的：观察血小板反应蛋白-1（thrombospondin-1，TSP-1）在链脲佐菌素诱导的糖尿病（diabetes mellitus， DM）大鼠模型肾脏中的表达，初步探讨TSP-1在糖尿病肾病大鼠发病机制中的作用及与糖尿病肾损害的关系。方法：成年雄性SD大鼠80只，随机分为正常对照组（ n＝15）和实验组（ n＝65）。采用STZ 70 mg/kg单次腹腔注射建立DM大鼠模型，按照成模标准去除未成模大鼠。检测各组大鼠空腹血糖（ fasting blood glucose，FBG）、尿蛋白及肾功能；分别于2，4，8周处死各组大鼠，通过HE染色观察各组大鼠肾脏的病理改变，免疫荧光观察TSP-1在各组大鼠肾脏的分布；PCR检测肾组织TSP-1和转录生长因子β1（ transforming growth factor，TGF-β1）基因表达水平；采用酶联免疫吸附法（ ELISA）检测肾组织活性TGF-β1水平；Western杂交检测TSP-1的动态变化。分别对尿蛋白、肾功能、肾脏病理损害、活性TGF-β1与TSP-1蛋白表达的相关性进行分析。结果：HE染色可见DM组大鼠肾小球系膜基质增生，肾小球可见分叶现象、K-W结节等典型的糖尿病肾病损伤特点。与正常对照组相比，DM组大鼠的FBG、尿蛋白、肌酐、尿素氮水平均显著增加（ P﹤0．05），TSP-1在DM组大鼠主要分布于肾小管、肾小球系膜细胞，其蛋白表达在2周时即明显增加，一直持续到第8周。DM 组大鼠肾脏TSP-1的积分光密度（ integrated optical density，IOD）与尿蛋白、肾脏病理损害、肌酐、尿素氮水平、活性TGF-β1浓度均呈正相关。结论：持续高血糖状态可以诱导大鼠肾脏病理损害，引起尿蛋白升高、肾功能异常；高血糖可诱导大鼠肾脏TSP-1蛋白高表达。TSP-1蛋白的高表达可能通过激活TGF-β1信号通路参与DM大鼠蛋白尿的产生及肾损害的发生发展。

Explore the Effect and Mechanism of Thrombos Pondin-1 in the Diabetic Rat Model of Renal Damage

Objective：Study the expression of thrombos pondin-1（TSP-1）in the kidney of diabetes（DM）rat model in-duced by streptozotocin（ STZ）and discussion the role in the pathogenesis of diabetic nephropathy and the relationship with the DM kidney injury. Methods：A total of 80 male Sprague-Dawley rats were randomly divided into normal control group（n=15）and ex-perimental group（n=65）. ExPerimental guoup were treated with an intraperitoneal injection dose（70 mg/kg）of STZ to establish the model of DM. After the injection of STZ,the fasting blood glucose of rats in the experimental group does not meet the standard of model were excluded. The level of the fasting blood glucose（ FBG）,urine protein（ UP）and renal function were monitored in each group;rats in each group were sacrificed at 2,4 and 8 weeks,respectively,use HE staining observe the pathological changes of kid-ney;the expression of TSP-1 was measured by immunohistochemical in the rat kidney;mRNA of TSP-1 and TGF-β1 were reverse transcribed and quantified by PCR;enzyme linked immune sorbent assay（ ELISA）were performed to examine the protein expression levels of active TGF-β1 in kidney. Western Blot to detect the expression of TSP-1 protein in rat kidney. the correlation between u-rine protein,pathological damage,renal function and TSP-1 protein expression were analyzed. Results：HE staining showed mesang-ial and matrix proliferative in DM rats,some typical damage characteristics of diabetic nephropathy can be found in glomerular include leaf Phenomenon,K-W nodules. Compared with normal control group,FBG,UP,creatinine and urea nitrogen was significantly in-creased in DM rats（P﹤0. 05）. TSP-1 is mainly distributed in DM rats in the renal tubules,mesangial cells,its expression was markedly increased at two weeks time,until the eighth week. The integrated optical density（ IOD）of TSP-1 were positively correla-ted with urinary protein,renal pathological damage,creatinine,urea nitrogen and concentration of active TGF-β1 . Conc