他克莫司缓解大鼠自体移植静脉桥内膜增生的研究

Effect of tacrolimus on intimal hyperplasia in autogenous vein grafts of rats

目的探讨他克莫司对大鼠自体移植静脉移植物(静脉桥)内膜增生的影响。方法 (1)选取28只雄性SD大鼠,建立颈外静脉移植颈总动脉模型,将大鼠随机分为实验组和对照组。实验组大鼠肌注他克莫司0.2 mg/(kg·d),对照组大鼠肌注相同剂量生理盐水。术后2周、4周采集大鼠移植静脉桥,采用HE染色法检测大鼠新生内膜厚度;采用免疫印迹法检测细胞周期蛋白依赖性激酶抑制剂p27表达水平,比较两组大鼠静脉桥组织中p27蛋白表达情况。(2)将人大隐静脉平滑肌细胞分为实验组(根据他克莫司浓度分4个亚组进行干预)和对照组(PBS缓冲液干预)。采用MTT法检测比较各组细胞增殖情况;采用Transwell小室法观察比较各组细胞迁移情况。结果 (1)与对照组比较,术后2周,实验组大鼠移植静脉桥新生内膜厚度明显减少(P<0.05),术后4周时减少更为明显(P<0.01)。经他克莫司治疗后2周和4周实验组大鼠移植静脉桥组织中p27蛋白水平显著高于对照组,差异均有统计学意义(P<0.05或0.01)。(2)不同浓度的他克莫司对HSVSMC增殖均产生抑制作用,随着其浓度增高抑制作用增强,有明显剂量效应关系;半抑制浓度(IC50)为390 nmol·L-1。他克莫司对HSVSMC迁移有明显的抑制作用,他克莫司浓度越高抑制作用越显著,浓度达到2 000 nmol·L-1时,抑制率最高,几乎无细胞迁移。结论他克莫司对自体静脉移植术后静脉桥再狭窄具有明显抑制作用,其机制可能与p27蛋白表达上调,抑制静脉平滑肌细胞增殖与迁移有关。

Objective To study the effects of tacrolimus on intimal hyperplasia in autogenous vein grafts of rats.Methods（ 1） After jugular vein-to-artery bypass grafting surgery,28 male SD rats were randomly divided into the treatment group and control group,treatment group received intramuscular injections of tacrolimus 0. 2 mg /（ kg·d）,while the control group received saline. The vein grafts were harvested after 2 or 4 weeks of operation. HE staining was performed for histological changes of grafted vein intimal membranes; the expression level of cyclin-dependent kinase inhibitor p27 in the vein grafts was detected by western blot,the expression level of p27 was compared between two groups.（ 2） Human saphenous vein smooth muscle cells were treated with tacrolimus（ treatment group,including four subgroups depends on the concentration gradient of tacrolimus） or PBS（ control group）.Cell proliferation was assayed by MTT and migration was observed by Transwell chamber. Results（ 1） After 2 and 4weeks of intervention,intimal thickness in treatment group was decreased significantly in treatment group compared to control group（ P 〈 0. 05）,and the decrease was more significant after 4 weeks of intervention（ P 〈 0. 01）. The expression level of p27 in the treatment group was significantly higher than that in control group in treatment group（ P 〈 0. 05 or 0. 01）.（ 2） Difference concentration of tacrolimus can inhibit cell proliferation,and had the dose-dependent effect on inhibiting the cell proliferation（ P 〈 0. 01）. 50% inhibiting concentration（ IC50） was 390 nmol·L- 1. Tacrolimus can markedly depress the migration of cells,the depression was more significant,when the concentration of tacrolimus was higher,and almost no cell was observed at the level of2 000 nmol · L- 1. Conclusions Tacrolimus can obviously inhibit hyperplasia of intimal thickness after venous transplantation,which may be related to the up-regulated expression of p27 and inhibition of smooth muscle cell proliferation and migration.