老龄大鼠脑缺血／再灌注微血管生成及碱性成纤维细胞生长因子和转化生长因子-β1表达的变化

Angiogenesis of brain after ischemia/reperfusion injury of brain in aged rats and changes in expressions of basic fibroblast growth factor and transformation growth factor-β1

目的 从碱性成纤维细胞生长因子（bFGF）、转化生长因子-β1（TGF-β1）表达方面揭示老年脑缺血/再灌注（I/R）微血管生成的机制.方法 青年SD大鼠按随机数字表法分组,老龄SD大鼠按体重分层随机法分组,分别分为假手术组、缺血（I）3 h及I/R 1、3、6、12 d组,每组6只.采用线栓法制备局灶性脑I/R模型.采用免疫组化和原位杂交技术测定脑微血管密度（MVD）、微血管场面积比、bFGF和TGF-β1的蛋白及TGF-β1 mRNA表达.结果 青年组MVD于I 3 h开始增加,I/R 6 d达峰值,持续增高至I/R 12 d;老龄组MVD自I 3 h持续降至I/R 12 d.青年组微血管场面积比于I/R 1 d明显增加后逐渐下降,12 d明显增高;老龄组微血管场面积比于I/R 1 d达峰值后逐步下降.老龄假手术组MVD（个）较青年假手术组增高（6.88±1.60比5.50±1.53,P＜0.01）,老龄I/R 1、3、6、12 d组MVD、微血管场面积比较青年组同期降低.青年和老龄组bFGF、TGF-β1蛋白表达均逐步增强,I/R 3 d达峰值后逐步减弱.老龄假手术组及I 3 h、I/R 1、3、12 d组bFGF蛋白表达（灰度值）低于青年组同期（176.80±5.10比172.82±1.53,171.81±2.43比167.85±2.41,167.99±5.51比164.90±2.15,152.98±4.11比150.75±1.11,165.67±3.55比161.73±1.29,P＜0.05或P＜0.01）;老龄假手术组及I 3 h、I/R 1、3、6、12 d组TGF-β1蛋白表达（灰度值）均低于青年组同期（182.69±3.12比176.13±4.08,176.89±2.30比170.56±7.47,171.74±2.70比165.43±2.91,157.17±5.20比150.43±4.28,161.72±4.81比155.37±2.92,167.69±2.18比160.28±3.59,均P＜0.01）.青年和老龄组TGF-β1 mRNA表达于I/R 1 d达峰值,随后均逐渐减弱.老龄假手术组和I 3 h、I/R 3、6、12 d组TGF-β1mRNA表达（灰度值）均较青年组同期降低（176.51±9.52比169.09±5.08,176.75±5.74比165.36±4.78,177.33±5.68比165.25±8.14,178.46±4.91比170.51±4.29,203.95±4.51比181.98±5.59,均P＜0.01）.结论 老年脑I/R损伤后脑微血管生成能力明显减弱,其机制可能与促血管生长因子bFGF、TGF-β1蛋白及TGF-β1 mRNA表达减弱有关,增龄因素可能是导致上述因子表达减弱的主要原因之一.

Objective To elucidate the mechanism of angiogenesis after cerebral ischemia/reperfusion （I/R） in the aged rats by observing the changes in expressions of basic fibroblast growth factor （bFGF） and transformation growth factor-β1 （TGF-β1）. Methods Young Sprague-Dawley （SD） rats were classified into groups by random digits table, and aged SD rats were stratified by different body weight. Rats were randomly divided into groups of sham operation, ischemia （I） 3 hours, I/R 1, 3, 6, 12 days, with 6 rats in each group. Focal cerebral I/R model was reproduced by intraluminal filament technique. Microvessel density （MVD） of brain tissue, sum area of lumens were observed, and the expressions of bFGF protein, TGF-β1 protein and TGF-β1 mRNA were assessed with immunohistochemistry and hybridization in situ. Results MVD in young model group began to increase at I 3 hours, peaking at I/R 6 days, maintained up to I/R 12 days. MVD in aged model group began to descend at I 3 hours and continued to I/R 12 days. Sum area of lumens in young model group increased markedly at I/R 1 day, gradually lowered at I/R 1 - 6 days, and increased obviously again at I/R 12 days. Sum area of lumens in aged model group reached peak at I/R 1 day,gradually decreased subsequently. MVD in aged sham operation group were higher than that in young sham operation group （6. 88± 1.60 vs. 5.50± 1.53, P〈0. 01）. MVD and sum area of lumens in aged model group at I/R 1, 3, 6, 12 days were lower than young model group. Expressions of bFGF protein, TGF-β1 protein in young and aged model group were both gradually up-regulated, all of them reaching peak at I/R 3 days,and lowered gradually at I/R 3 - 12 days subsequently. Expressions of bFGF protein （grey level） in both aged sham operation group and those of model group at 1 3 hours, I/R 1, 3, 12 days were lower than those of young sham operation and those of the model group at the same time points （176.80±5. 10 vs. 172. 82±1.53, 171. 81±2. 43 vs. 167. 85±2. 41, 167. 99±5. 51 vs. 164.90±2.15, 152. 98±4.11vs. 150. 75±1.11,165. 67±3. 55 vs. 161.73±1. 29, P〈0. 05 or P〈0. 01）. Expressions of TGF-β1 protein （grey level） in both aged sham operation and those of model group at I 3 hours, I/R 1, 3, 6, 12 days were all lower than those of young sham operation and those of model group at the same time points （182.69±3.12 vs. 176.13±4.08,176.89±2.30 vs. 170.56±7.47, 171.74±2.70 vs. 165.43±2.91, 157.17±5.20 vs. 150.43±4.28,161.72±4.81 vs. 155.37±2.92, 167.69±2.18 vs. 160.28±3.59, all P〈0.01）. TGF-β1 mRNA expressions in both young model group and aged model group reached peak at I/R 1 day, gradually lowered subsequently. Expressions of TGF-β1 mRNA （gray level） in both aged sham operation and those of model group at I 3 hours, I/R 3, 6, 12 days were lower than those of young sham operation and also model group at the same time points （176. 51±9.52 vs. 169.09±5.08, 176.75±5.74 vs. 165.36±4.78, 177.33±5.68vs. 165.25±8.14, 178.46±4.91 vs. 170.51±4.29, 203.95±4.51 vs. 181.98±5.59, all P〈0.01）.Conclusion Angiogenesis is obviously weak after cerebral I/R in the aged, and the mechanism of which might be related to the down-regulation of expressions of bFGF protein, TGF-β1 protein and TGF-β1mRNA. Aging factor may be one of the main reasons which induce the down regulation of expressions mentioned above.