大鼠平滑肌细胞增殖与西洛他唑抑制作用的体内实验

Proliferation of rat vascular smooth muscule cells in vivo and the inhibitory effect of cliostazol

目的:以球囊损伤SD大鼠模型颈动脉内膜增生为基础,比较西洛他唑给药组和生理盐水对照组大鼠血管内膜增生强度和平滑肌细胞增殖能力方面的差异。方法:①实验于2004-01/12在解放军沈阳军区总医院全军心血管病研究所完成。实验选用健康雄性SD大鼠,体质量300～500g。②建立动物模型:腹腔麻醉36只大鼠后,暴露左颈总动脉及颈内、外动脉,自颈外动脉向近心端插入2FFogarty球囊导管至颈总动脉起始部,球囊充水0.10～0.15mL,慢速回拉至颈内外动脉分叉处,反复3次,完成后结扎颈外动脉。③36只SD大鼠随机分为2组,每组18只,分别为生理盐水组,西洛他唑给药组(7.5×10-7mol/L)。给药方式:灌肠给药,5mL/次,术后即刻开始,持续给药28d。④观察并比较两组大鼠损伤血管壁各成分结构变化图像定量分析结果。应用免疫组化分析大鼠动脉血管壁增殖情况,并应用反转录聚合酶链式反应分析两组损伤的动脉半胱天冬酶3在mRNA水平表达变化。结果:大鼠36只均进入结果分析。①大鼠动脉血管壁增殖情况免疫组化分析结果:西洛他唑给药组Ki67免疫组化染色阴性,细胞增殖能力下降;生理盐水组Ki67免疫组化染色阳性,细胞增殖能力旺盛。②损伤血管壁各成分结构变化图像定量分析结果:西洛他唑给药组大鼠颈动脉血管壁损伤后28d内膜增生面积明显小于生理盐水组犤(0.015±0.002),(0.197±0.004)mm2,t=299.06,P<0.01犦;管腔面积明显大于生理盐水组犤(0.193±0.008),(0.011±0.008)mm2,t=118.21,P<0.01犦。两组大鼠损伤动脉中膜增生面积差异不明显(P>0.05)。③两组损伤的动脉半胱天冬酶3在mRNA水平表达反转录聚合酶链式反应分析结果:西洛他唑给药组平滑肌细胞中半胱天冬酶3表达明显高于生理盐水对照组。结论:①通过在体大鼠颈动脉内膜增生模型建立,有效地模拟了西洛他唑抑制体内血管平滑肌细胞增殖的发生过程。②西洛他唑能够有效抑制体内血管平滑肌细胞增殖并诱导其凋亡,抑制血管损伤后新生内膜的形成,为临床防治增生性血管疾病的提供理论依据。

AIM： To compare the intensity of vascular intima hyperplasia and the ability of vascular smooth muscle cell proliferation between the cilostazol treated group and saline control group based on the carotid artery intima hyperplasia of SD rats with balloon injury. METHODS： ①The experiment was carried out in the Military Institute of Cardiovascular Disease, Chinese PLA General Hospital of Shenyang Military Area Command between January and December 2004. ②Healthy ＇male SD rats of 300-500 g were made into animal models： After intraperitoneal anesthesia, 36 rats were treated with exposure of left common carotid artery, internal and external carotid artery, 2F Fogarty tube was inserted from external carotid artery towards the proximal end till the initial part of common carotid artery, the balloon was filled with 0.10-0.15 mL water, and then pulled back slowly to the crotch of internal and external carotid artery, which was repeated for 3 times, and then external carotid artery was ligated. ③ Thirty-six SD rats were randomly divided into saline group （n =18） and cilostazol treated group （n=18, 7.5×10^-7 mol/L）. The rats were adnfinistrated with clusis, 5 mL for each time, immediately after the surgery for 28 days. ④The results of the imaging quantitative analysis of the structural changes of each component in the injured vascular wall were observed and compared in the two groups. The proliferation of arterial vascular wall was analyzed with immunohistochemistry, the changes of the caspase-3 mRNA expression in the injured arteries in both groups were analyzed with reverse transcription-polymerase chain reaction. RESULTS： All the 36 rats were involved in the analysis of results.① The immunohistochemical results of the proliferation of arterial vascular wall： In the cilostazol treated group, the immunohistochemical staining of Ki67 was negative, the ability of cell proliferation was decreased; In the saline group, the immunohistochemical staining of Ki67 was positive, the ability of cell proliferation was high. ② The results of the imaging quantitative analysis of the structural changes of each component in the injured vascular wall： The area of intima hyperplasia at 28 days after the injury of arterial vascular wall was obviously smaller in the cilostazol treated group than in the saline group [（0.015±0.002）, （0.197±0.004） mm^2, t=299,06, P 〈 0.01], and the lumen area was obviously larger in the cilostazol treated group than in the saline group [（0.193±0.008）, （0.011±0.008） mm^2, t=118,21, P 〈 0.01], There was no obvious difference in the area of media hyperplasia of the injured artery between the two groups （P 〉 0.05）. ③The caspase-3 mRNA expression in the smooth muscle cells was obviously higher in the cilostazol treated group than in the saline group, CONCLUSION： ① The occurrence process of in vivo inhibition of cilostazol to the proliferation of vascular smooth muscle cells is effectively imitated by the establishment of models of in vivo rat carotid arterial intima hyperplasia. ② Cilostazol can effectively inhibit the in vivo proliferation of vascular smooth muscle cells, induce its apoptosis, and inhibit the formation of new intima after vascular injury, so it provides a theoretical evidence for the clinical prevention and treatment of proliferative vascular diseases.