人组织激肽释放酶基因转移对球囊拉伤后大鼠颈总动脉内膜的影响

Effects of human tissue kallikrein I gene delivery on carotid artery neointima formation after balloon angioplasty in spontaneously hypertensive rats

目的探讨重组腺病毒介导的人组织激肽释放酶1（Ad-hKLK1）基因转移对球囊拉伤后的自发性高血压大鼠（SHR）颈动脉新生内膜增生的影响及其机制。方法取SHR，雄性，采用球囊拉伤法建立大鼠颈动脉再狭窄模型，将存活动物随机分为4组，即假手术组（n=6）、单纯拉伤组（n=8）、拉伤＋注射对照病毒组（n=8）、拉伤＋注射Ad-hKLK1病毒组（n=8）。4周后处死大鼠，测定各实验组再狭窄动脉内膜形态学相关指标。RT-PCR法检测缓激肽受体（B1R和B2R）表达，蛋白免疫印迹法检测细胞周期素激酶抑制蛋白p27Kip1、p21Cip1的表达。结果拉伤＋Ad-hKLK1组大鼠颈总动脉的壁腔面积比W／L明显低于单纯拉伤组（1．056±0．149比1．641±0．194，P〈0．001），抑制率达35．6％，内膜／中膜面积比值显著小于单纯拉伤组（0．77±0．09比1．26±0．051，P〈0．01），抑制率达38．8％（P〈0．001）。拉伤＋Ad-hKLK1组的周期抑制蛋白p27Kip1和p21Cip1表达明显高于单纯拉伤组（0．55±0．05比0．31±0．06，P〈0．001和0．47±0．06比0．26±0．05，P〈0．001）。球囊拉伤后颈动脉的缓激肽B2R的mRNA表达明显高于假手术组（P〈0．01），而各组间的缓激肽B1R的mRNA表达差异无统计学意义。结论hKLK1基因转移可减轻SHR颈动脉球囊损伤后的新生血管内膜形成，注射外源性人组织激肽释放酶基因可上调缓激肽B2RmRNA表达和p27Kip1、p21Cip1的蛋白表达。

Objective To investigate the effects of human tissue kaUikrein 1 （Ad-hKLK1） gene delivery on the neointima formation in carotid arteries of spontaneously hypertensive rats （SHRs）. Methods Carotid artery restenosis was induced in male SHR rats by balloon-injury. Rats were randomly assigned into 4 groups： Sham-operated （ n = 6） ; Angioplasty （ phosphate buffered solution 50 μl, n = 8 ） ; Vector virus （ control virus, 1 ×10^9 IU in 50 μl, n = 8） and Ad-hKLK1 （Ad-hKLK1, 1 × 10^9 IU in 50 μl, n = 8）. Rats were sacrificed 4 weeks later. The wall-to-lumen area ratio and intima/media ratio in carotid artery were assessed by image analysis in HE stained sections. The mRNA bradykinin receptor（ B1R and B2R） expressions were detected by RT-PCR. The protein expression of the cycle-independent kinase inhibitors p27Kip1 and p21Cip1 were determined by Western blot analysis. Results Wall-to-lumen area ratio reduced 35.6% and intima/media ratio reduced 38. 8% in Ad-hKLK1 treated SHRs compared to angioplasty group （ all P 〈0. 001 ）. The expression of p27Kip1 and p21Cip1 increased significantly in Ad-hKLK1 treated SHRs compared with angioplasty rats （ all P 〈 0. 001 ）. The mRNA expression of B2R was significantly upregulated in angioplasty rats compared with sham-operated rats （P 〈 0. 05 ） while mRNA expression of B1R was similar between the 2 groups. Conclusion hKLK1 gene delivery may effectively reduce neointimal formation via downregulating bradykinin B2R and up-regulating the expressions of p27Kip1, p21Cip1 signaling pathways in carotid arteries of SHRs after balloon injury.