【作者】 李莉；

【导师】 高大中；

【作者基本信息】 重庆医科大学 ， 内科学， 2005， 硕士

【摘要】 目的:研究不同剂量的吲哚-3-甲醇(indole-3-carbinol, I3C)对球囊损伤术后大鼠颈动脉壁 Akt-1、NF-κB、Bcl-X/L、Bax 和 Bcl-2 蛋白表达的影响,探讨 I3C 诱导血管损伤后平滑肌细胞(vascular smooth musclecells,VSMCs)凋亡的分子机制,为利用 I3C 防治经皮冠状动脉介入术(perutaneous corcnary intervention, PCI)后再狭窄(restenosis,RS)提供理论基础。 方法:建立 35 只雄性 SD 大白鼠(体重 300-400g)颈动脉损伤的动物模型,采用完全随机分组设计,分为对照组和实验组。对照组:5只动物,颈动脉球囊损伤术后不给药;实验组:30 只动物,球囊损伤术后给药量为 12.5mg/d,25mg/d,50mg/d,给药时间分别为 4 天,7天,共 6 个亚组,每个亚组 5 只动物。所有动物均在术后 14 天取材,免疫组化 SABC 法检测血管壁细胞 Akt-1、NF-κB、Bcl-X/L、Bax 和Bcl-2 蛋白的表达,计算机图像分析 Akt-1、NF-κB、Bcl-X/L 蛋白表达的平均光密度值和 Bax、Bcl-2 蛋白表达的平均评分值。 结果: 1 Akt-1 蛋白表达的部位主要集中在大鼠颈动脉中层平滑肌细胞中。对照组 Akt-1 蛋白的表达值较高,各实验组 Akt-1 蛋白均有降低,其中以 50mg 4 天组、25mg 7 天组、50mg 7 天组蛋白表达值降低显著(0.0725 ±0.00581 vs. 0.0590 ±0.00235,P<0.01;0.0725 ±0.0058 vs.0.0646 ±0.00257,P<0.05;0.0725 ±0.00581 vs. 0.0517 ±0.00207,重庆医科大学硕士研究生学位论文 3P<0.01)。同一剂量的 I3C (50mg)作用下,4 天组和 7 天组的 Akt-1 蛋白的表达值有差异(0.0590 ±0.00235 vs. 0.0517 ±0.00207,P<0.01)。2 NF-κB 蛋白在对照组和各实验组大鼠颈动脉壁细胞均有表达,阳性颗粒可以出现在胞浆中或是胞核中。各实验组 NF-κB 蛋白表达值均较对照组减低,除 12.5mg 4 天组 NF-κB 蛋白表达值与对照组比较无差异外(0.0947 ±0.00252 vs. 0.1003 ±0.00700,P=0.516),其他各实验组与对照组比较均有显著性差异(P<0.05 或 P<0.01)。同一给药剂量不同给药时间组的比较中, 仅 25mg 作用 4 天和作用 7 天(0.0872 ±0.00146 vs. 0.0816 ±0.00211,P<0.01)显示有统计学意义。3 Bcl-X/L 蛋白阳性颗粒主要出现在胞浆中,而且表达的部位主要集中在血管中层平滑肌细胞中。 对照组 Bcl-X/L 蛋白表达的平均光密度值较高(0.0809 ±0.00170),各实验组均有降低,以 50mg 4 天组(0.0593 ±0.00111)、25mg 7 天组(0.0766 ±0.00059)、50mg 7 天组(0.0516 ±0.00235)的平均光密度值与对照组比较有显著性差异(P<0.01)。同一剂量(50mg)实验 4 天组和实验 7 天组之间 P <0.01,其他同一剂量不同给药时间之间的平均光密度值比较无意义。4 Bax 蛋白在球囊损伤后的大鼠颈动脉壁细胞对照组和各实验组均有表达,表达的部位主要集中在血管中层平滑肌细胞中。各实验组Bax 蛋白的表达值和对照组平均评分值比较均有增加,其中 50mg 4 天组、25mg 7 天、50mg 7 天作用后,Bax 蛋白表达的平均评分值增加明显(5.8000±0.46368 vs. 2.4400 ±0.35777, P<0.01;3.0600±0.18166 vs.2.4400 ± 0.35777, P<0.05 ; 5.9800 ± 0.41473 vs. 2.4400 ± 0.35777,P<0.01 )。相同剂量的 I3C 作用下 Bax 蛋白表达 4 天组和 7 天组无显著差异。5 Bcl-2 蛋白表达各实验组与对照组比较其蛋白表达的平均评分值均有减低,但除外 12.5mg 4 天组(4.1200 ±0.47645)、12.5mg 7 天组更多还原

【Abstract】 Objective: To investigate the effects of indole-3-carbinol (I3C) on theexpressions of Akt-1, NF-κB, Bcl-X/L, Bax and Bcl-2 proteins in carotidartery of rats after balloon lesions, and to study the molecular mechanismsof I3C inducing the apoptosis of vascular smooth muscle cells (VSMCs)after blood vessel injury, the study will provide a theoretical basis forpreventing coronary artery restenosis after percutaneous coronaryintervention (PCI) in using I3C. Methods: 35 SD rat models (weight: 300- 400g) with carotid lesionswere divided randomly into 2 groups: control group (balloon lesions buttreated with nothing, n=5) and experimental group (treated with I3C, n=30).The experimental group was subdivided into 6 groups by different doses(12.5mg/d, 25mg/d and 50mg/d respectively) and different treatment time(4 days and 7days respectively). 14 days later after operation, the targetsegments were acquired. Then the protein expression changes of Akt-1,NF-κB, Bcl-X/L, Bax and Bcl-2 were detected in the carotid wall cells withSABC method, and the mean optical density values of Akt-1, NF-κB andBcl-X/L and the mean scores of Bax, Bcl-2 were measured by computerimage analysis. Results: 1. Protein Akt-1 was mainly expressed in the middle layer of ratscarotid artery. The expression values of Alt-1 were increased in controlgroup, however, in experimental group, decreased in different degree,especially in 50mg 4-day-group, 25mg 7-day-group and 50mg 7-day-group(0.0725 ±0.00581 vs. 0.0590± 0.00235, P<0.01; 0.0725 ±0.00581 vs.0.0646 ±0.00257, P<0.05; 0.0725 ±0.00581 vs. 0.0517 ±0.00207, P<0.01).In the same doses of I3C (50mg/d), there were differences in 4-day-groupand 7-day-group (0.0590 ±0.00235 vs. 0.0517 ±0.00207, P<0.01). 2. Protein NF-κB were both expressed in the control group and theexperimental group, its positive particles can be found in cytoplasm ornucleus of rat carotid artery wall cells. In each sub-group of theexperimental group, the expressions of NF-κB were lower than thecorresponding control group. There were significant difference between thecontrol group and the experimental group (P<0.05 或 P<0.01) excepting the12.5mg 4-day-group (0.0947 ±0.00252 vs. 0.1003 ±0.00700,P=0.516).Comparison with the sub-groups of the experimental group in the samedoses and the different treatment time, there was only statisticalsignificance between the 25mg 4-day-group and 25mg 7-day-group (0.0872±0.00146 vs. 0.0816 ±0.00211, P<0.01). 3. Protein Bcl-X/L was mainly detected in the smooth muscle cellcytoplasm of rat carotid artery, and it located chiefly in the middle layersmooth muscle cell of the carotid artery. In the control group, the meanoptical density value of Bcl-X/L was 0.0809 ±0.00170, and the mean valuein the experimental group was decreased. There was significant difference(P<0.01) between the control group and the 50mg 4-day-group (0.0593±0.00111), 25mg 7-day-group (0.0766 ±0.00059) and 50mg 7-day-group(0.0516 ±0.00235). In the experimental sub-groups treated with the samedoses but not the same treatment time, there were not statistic differenceapart from the comparison of 50mg 4-day-group and 7-day-group (P﹤0.01). 4. Protein Bax were both expression in the control group and theexperimental group after balloon lesions in carotid artery of rats, and itlocated chiefly in the smooth muscle cells in the middle layer of the carotidartery. In each experimental sub-groups, the expression of Bax wasincreased more than the control group, especially in the 50mg 4-day-group,25mg 7-day-group and 50mg 7-day-group (5.8000 ±0.46368 vs. 2.4400±0.35777, P<0.01; 3.0600 ±0.18166 vs. 2.4400 ±0.35777, P<0.05; 5.9800±0.41473 vs. 2.4400 ±0.35777, P<0.01). In the same I3C doses, there wasnot significant difference in the expression of Bax between 4-day-groupand 7-day-group. 5. Protein Bcl-2 was decreased in the experimental group comparedwith the control group. There was significant difference between them withthe exception of 12.5mg 4-day-group (4.1200 ±0.47645) and 12.5mg7-day-group (3.9200 ±0.42071). In the更多还原