血管新生调控因子及其受体随增龄在小鼠肾组织内的表达

目的 探讨血管内皮生长因子（VEGF）及其受体（VEGFR）、血管生成素（Ang-1、Ang-2）及其受体（Tie2）在增龄小鼠肾脏中的表达，以及它们在肾脏衰老中的作用。 方法 选取4月龄、9月龄、12月龄及20月龄C57小鼠各6只，留取尿液及血液标本，应用常规生化法测定各组小鼠肾功能。用PAS染色对各组小鼠进行肾脏病理染色及分析；荧光染料肾脏灌注对肾小球毛细血管丛密度进行分析。应用免疫组化、免疫荧光、Western印迹及实时定量PCR方法分析肾脏转化生长因子（TGF-β1）及VEGF、VEGFR2（血管内皮生长因子受体2，Flk-1）、Ang-1、Ang-2、Tie2的蛋白及基因表达的变化。 结果 随着增龄，小鼠肾小球硬化指数（GSI）增加，其中20月龄约为4月龄的5倍（P < 0.05）。荧光染料肾脏灌注后见肾小球毛细血管内荧光强度呈减少趋势，20月龄显著低于4月龄（P < 0.05）。免疫组化结果显示，TGF-β1在肾小球及肾小管间质中的表达呈增多趋势，且以肾小球内增多较显著，各组间差异有统计学意义（P < 0.05）。免疫荧光结果显示，Ang-1在肾小球内的表达呈减少趋势，20月龄较4月龄显著减少（P < 0.05）。实时定量PCR结果显示，不同月龄小鼠肾组织内VEGF、Flk-1、Ang-1、Ang-2、Tie2的mRNA表达水平均呈下降趋势，20月龄与4月龄间VEGF、Flk-1及Ang-2差异无统计学意义，而Ang-1和Tie2差异有统计学意义（均P < 0.05）。Western印迹结果显示，4组小鼠肾脏VEGF、Flk-1、Ang-1、Ang-2、Tie2的蛋白表达水平也呈下降趋势，且20月龄与4月龄差异均有统计学意义（均P < 0.05）；TGF-β1的表达量呈增多趋势，其中20月龄较4月龄增多约40%（P < 0.05）。相关分析结果显示，肾小球毛细血管密度、Ang-1、Ang-2、Tie2、VEGF、Flk-1的mRNA及蛋白水平均与Scr呈负相关。 结论 随衰老程度加重，小鼠肾组织内血管内皮生长因子及其受体、血管生成素及其受体的表达减少，且与增龄肾脏的形态学改变及肾功能改变相关。

Expression of angiogenesis regulatory factors and their receptors in mouse kidneys with aging

Objective To investigate the expression of vascular endothelial growth factor (VEGF), angiopoietin and their receptors (VEGFR and Tie2) in aging mice kidney and the possible roles in aging mice. Methods Mice were divided as follows: 4-month old group (n=6), 9-month old group (n=6), 12-month old group (n=6) and 20-month old group (n=6). Paraffin sections of the mice kidneys were stained by PAS. The density of glomerular microvascular was determined by renal perfusion with fluorescent dyes. The level of VEGF, VEGFR2 (Flk-1), Ang-1, Ang-2, Tie2 mRNA expression and protein abundance in kidney was determined by real-time PCR, immunochemistry, immunofluorescence and Western blot. Results Compared with other three groups, in the 20-manth old group, the glomerulosclerosis index (GSI) increased remarkbly (2.48±0.79 vs 0.53±0.19, 0.69±0.18, 1.50±0.70, P<0.05); the fluorescence intensity in glomeruli decreased (P<0.05). lmmunohistochemistry demonstrated that the TGF-131 level in the aging kidneys showed an increase trend in the glomerular tubulointerstitium, and especially in the glomeruli. Real-time PCR results revealed that compared with 4-month old group mice, the mRNA expression of VEGF, Flk-1, Ang-1, Ang-2, Tie2 of the other three groups decreased, the gene levels of VEGF, Flk-1, and Ang-2 fell about 90%, 50% and 80% (all P>0.05), and the gene levels of Ang-1 and Tie2 fell about 75% and 40% in 20-month-old group (all P<0.05). Western blot domonstrated that the protein abundace of VEGF, Flk-1, Ang-1, Ang-2, Tie2 also declined with aging, the protein level of VEGF, Flk-1, Ang-1, Ang-2 and Tie2 dropped by about 35%, 50%, 15%, 13% and 21% respectively in 20-month-old group as compared to 4-month-old group (all P<0.05). Expression of above 5 factors and glomerular fluorescence intensity were negatively correlated with Scr (P<0.05). Conclusions The mRNA expression and protein abundance of VEGF, Flk-1, Ang-1, Ang-2, Tie2 in mice kidneys decreases with aging. Angiogenesis regulatory factors may play important roles in aging progression of the mice kidney.