中文摘要：

目的：探讨重组腺病毒介导的人组织激肽释放酶（hKLK1）基因转移对血小板源性生长因子-BB（PDGF-BB）诱导下的自发性高血压大鼠（SHR）血管平滑肌细胞（VSMCSHR）增殖和迁移的影响。方法：自行构建双顺反子重组腺病毒载体,携带强绿色荧光蛋白（EGFP）标志基因和目的基因hKLK1;用细胞计数法和四甲基偶氮唑盐（MTT）比色法检测细胞增殖,流式细胞仪检测细胞生长周期;蛋白免疫印迹法（Western blotting）测定细胞周期素依赖性激酶抑制蛋白p27^Kip1、p21^Cip1的表达。采用改良Boyden微孔膜双槽法测定VSMCSHR迁移。结果：（1）hKLK1基因转移呈感染复数依赖性（20-100MOI）抑制PDGF-BB诱导的VSMCSHR生长,100MOI时抑制率为39.3%;呈时间依赖性抑制VSMCSHR生长,第5d时达高峰,抑制率为35.2%。（2）hKLK1基因转移可显著抑制PDGF-BB诱导的VSMCSHR增殖,峰值抑制率为30.2%（P〈0.01）;细胞周期阻滞于G0/G1期的VSMCSHR明显增多,最大阻滞率为36.4%（P〈0.01）,而缓激肽B2受体特异性阻断剂Hoe140逆转了hKLK的抑制作用。（3）hKLK1基因转移明显上调PDGF-BB诱导VSMCSHR的p27^Kip1、p21^Cip1表达,Hoe140明显降低p27^Kip1、p21^Cip1表达。（4）hKLK1基因转移可明显抑制PDGF-BB诱导的VSMCSHR细胞迁移,抑制率为34.6%,且Hoe140不影响该抑制作用。结论：hKLK1基因转移可抑制PDGF-BB诱导的VSMCSHR增殖,主要由缓激肽B2受体介导的,通过上调细胞周期素依赖性激酶抑制蛋白p27^Kip1、p21^Cip1表达的途径。而hKLK1基因转移抑制VSMCSHR迁移效应可能不通过B2受体。

英文摘要：

AIM：To investigate the effects of adenovirus-mediated human tissue kallikerin （Ad-hKLK1） gene delivery on the proliferation,migration of VSMCSHR induced by platelet derived growth factor-BB （PDGF-BB）. METHODS： The VSMCSHR proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazoliuin （MTT）. The migration was assessed by modified Boyden chamber assay. Western blotting was used to determine the expressions of the cycle-independent kinase inhibitors p27^Kip1 and p21^Cip1.RESULTS： Proliferation of VSMCSHR induced by PDGF-BB was inhibited after transfection of Ad-hKLK1 （20-100 MOI） in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 fell on 100 MOI,with the peak inhibition rate of 39.3% （cell counting,n=3,P〈0.01）,30.2% （MTT,n=3,P〈0.01）,peak stunning rate of cell-cycle in phase G0/G1 at 36.4%. The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery were significantly abolished by Hoe140,a bradykinin B2 receptor antagonist. Migration of VSMCSHR induced by PDGF-BB was inhibited after hKLK1 gene delivery,with the peak inhibitory rate of 34.6% （n=6,P〈0.01）. However the inhibitory effects of migration were not blocked by Hoe140. The protein expression of p27^Kip1 and p21^Cip1 increased significantly after the hKLK1 gene delivery,whereas Hoe140 nearly completely blocked these effects （n=3,P〈0.01,respectively）.CONCLUSION： The hKLK1 gene delivery may inhibit the proliferation and migration of VSMCSHR induced by PDGF-BB. Bradykinin B2 receptor probably mediates the up-regulating expression of p27^Kip1 and p21^Cip1 that contributes to the inhibitory effects of proliferation of hKLK1. However,the inhibitory effects of migration by hKLK1 gene delivery may not be mediated by bradykinin B2 receptor.