摘要
目的:观察刚断乳雄性SD大鼠单侧输尿管完全梗阻模型中血管内皮生长因子对肾小管周围毛细血管形态学变化的调节作用,探讨血管内皮生长因子在肾病进展中的生物活性作用。方法:实验于2005-03/2006-05在苏州大学儿科研究所完成。①实验材料:30只清洁级刚断乳雄性SD大鼠,体质量50～70g。②实验过程:结扎SD大鼠左侧输尿管建立单侧输尿管完全梗阻模型。于术后0,1,7,14,28d,分别随机选择6只模型大鼠,收获肾脏标本。③实验评估:采用苏木精-伊红染色观察肾积水的严重程度;Masson染色观察肾小管间质纤维化程度;PAS染色观察肾小管萎缩程度;免疫组织化学方法检测肾小管周围毛细血管的密度和血管内皮生长因子的表达水平;原位末端标记法对肾小管周围毛细血管和肾小管上皮细胞进行原位凋亡测定;透射电镜显示超微结构变化;血管内皮生长因子蛋白的表达强度和间质纤维化程度采用Leica图像分析系统检测。结果:①梗阻第1周,肾小管上皮细胞胞浆里的血管内皮生长因子染色在局部有增强,胎肝激酶1阳性肾小管周围毛细血管数量变化不显著,肾小管上皮细胞凋亡很少见,间质纤维化轻。第2,4周,血管内皮生长因子表达逐渐下降,直至在一些肾小管内完全消失。与此同时,胎肝激酶1阳性的肾小管周围毛细血管数量减少,肾小管扩张或萎缩明显,间质纤维化严重。②电镜显示肾小管上皮细胞、肾小管周围毛细血管内皮细胞的死亡形式主要为凋亡。③原位末端标记法显示肾小管上皮细胞凋亡在第14天达到高峰,然后迅速下降。④在梗阻第2周时,原位末端标记法阳性的肾小管周围毛细血管内皮细胞数与血管内皮生长因子表达面积百分比负相关(r=-0.668,P<0.05);肾小管周围毛细血管密度与血管内皮生长因子表达面积百分比正相关(r=0.707,P<0.05),而与肾小管上皮细胞凋亡负相关(r=-0.863,P<0.01)。结论:肾小管周围毛细血管减少与肾小管上皮细胞内血管内皮生长因子的表达不足相关,并与肾小管上皮细胞凋亡相关。
AIM： To investigate the morphologic changes of peritubular capillary （PTC） by vascular endothelial growth factor （VEGF） during the course of complete unilateral ureteral obstruction （UUO） in weanling male Sprague-Dawiey （SD） rats, and explore the bio-active role of VEGF in the nephropathy. METHODS： The experiment was conducted in the Laboratory of Pediatrics, Children＇s Hospital Affiliated to Soochow University from March 2005 to May 2006. （1）A total of 30 SD rats were adopted, aged 50-70 g. （2）Complete UUO was induced in weanling male SD rats by ligating the left ureter. Each six rats ware randomly selected to yield kidneys at days 0, 1, 7, 14, 28 postoperatively. （3）The severity of hydronephrotic kidneys was evaluated on routine hematoxylin-eosin stained sections, and the tubulointerstitial fibrosis was analyzed on Masson trichrome stained sections. Renal tubular atrophy was assessed on periodic acid Schiff stained sections. Morphologic changes in PTC density and the expression of VEGF ware immunohistochemically detected, and the apoptosis of PTC endothelial cell and renal tubular epithelial cell ware examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL） technique, and electron-microscopic studies was used for the ultrastructure. For quantification of VEGF expression and tubulointerstitial fibrosis, areas with positive staining were measured using Leica image analysis. RESULTS： （1）In the first week of UUO, the immunohistochemical labeling of tubular VEGF was intensified, associated by deformation and dilation of adjacent fetal liver kinase （FLK）-1-positive PTC lumina, mild renal tubular cell apoptosis and minimal tubulointerstitial fibrosis. In the second week, renal tubular VEGF labeling and the FLK-1 labeled PTC number began to decline. By the 4^th week, the number of FLK-1-positive PTC lumina was significantly decreased in areas of marked renal tubular atrophy and tubulointerstitial scarring.（2）Electron microscopic studies demonstrated PTC endothelial cell and tubular epithelial cell apoptosis.（3）Renal tubular epithelial cell apoptosis （as detected in TUNEL） peaked on the 14^th day after ureteral ligation, and thereafter decreased rapidly. （4）By the 2^nd week of UUO, there was a negative correlation between the number of TUNEL-positive PTC cells and the VEGF labeling area percentage （r =-0.668, P 〈 0.05）. The density of PTC demonstrated a positive correlation with the VEGF labeling area percentage （r =0.707, P 〈 0.05）, but a negative correlation with the renal tubular epithelial cell apoptosis （r =-0.863, P 〈 0.01）. CONCLUSION： The depletion of VEGF in renal tubular cells is crucial to the loss of PTC. PTC. regression might contribute to renal tubular cell apoptosis.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第49期9821-9825,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
