摘要目的 探讨重组腺病毒介导的人激肽释放酶(human tissue kallikrein 1,hKLK1)基因转移对血小板源性生长因子-BB(platelet derived growth factor-BB,PDGF-BB)诱导下的自发性高血压大鼠(SHR)血管平滑肌细胞(vascular smooth muscle cells,VSMCSHR)增殖的影响及其机制.方法 自行构建重组腺病毒载体,携带目的 基因hKLK1和标志基因强绿色荧光蛋白.细胞计数法和四甲基偶氮唑盐(MTF)比色法检测细胞增殖,流式细胞仪检测细胞生长周期.蛋白免疫印迹法测定细胞周期素依赖性激酶抑制蛋白p27Kip1、p21Cip1的表达.RT-PCR法测定缓激肽B1受体、B2受体的mRNA表达.结果 (1)hKLK1基因转移呈感染复数依赖性(20～100 M0I)抑制PDGF-BB诱导的VSMCSHR生长,100 MOI时抑制率为39.3%;呈时间依赖性抑制VSMCSHR生长,第5天时达高峰,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＜0.001).(3)hKLK1基因转移明显上调PDGF-BB诱导VSMCSHR的p27Kip1、p21Cip1表达,而缓激肽B2受体特异性阻断剂Hoe140明显降低p27Kip1、p21Cip1表达.(4)PDGF-BB诱导VSMCSHR的缓激肽B2受体mRNA表达明显增加(P＜0.001),且Hoe140可明显降低其表达(P＜0.01).结论 hKLK1基因转移可抑制PDGF-BB诱导的VSMCSHR增殖,可上调缓激肽B2受体和细胞周期素依赖性激酶抑制蛋白p27Kip1、p21Cip1表达.

abstractsObjective Tissue kallikrein cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in the proliferation of vascular smooth muscle cells. We investigated the effects of adenovirus-mediated human tissue kallikerin(Ad-hKLK1)gene delivery on the proliferation of vascular smooth muscle cells of SHR(VSMCsSHR)induced by platelet derived growth factor-BB(PDGF-BB). Methods Primary VSMCsSHR were isolated and cultured from thoracic aorta of male SHR. The VSMCsSHR proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazoliuin(MTT). Western blot was used to determine the protein expression of hKLK1, the cycle-independent kinase inhibitors p27Kip1 and p21Cip1 . The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMCsSHR. Results Proliferation of VSMCsSHR induced by PDGF-BB was significantly inhibited post transfection of Ad-hKLK1(20 - 100 MOI)in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 was 100 MOI with peak inhibition rate of 39.3%(cell counting, n = 3,P ＜0.01), 30.2%(MTT, n-3 ,P ＜ 0.01)and 36.4%(peak stunning rate of cell-cycle in phase G0/G1). The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery could be abolished by Hoe140, a bradykinin B2 receptor antagonist. The protein expression of p27Kip1 and p21Cip1 increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects(n = 3,P ＜0.001 ,respectively). PDGF-BB also significantly upregulated the mRNA expression of B2 receptor but not B1 receptor in VSMCsSHR. Conclusion The hKLK1 gene delivery could inhibit PDGF-BB induced proliferation in VSMCsSHR through Bradykinin B2 receptor and up-regulate expression of p27Kip1 and p21Cip1.