水蛭素对体外培养的人冠状动脉平滑肌细胞和内皮细胞生长的影响

Influence of hirudin on growth of in vitro human coronary artery smooth muscle cells and human coronary artery endothelial cells

目的 探讨水蛭素对体外培养的人冠状动脉平滑肌细胞（HCASMC）和人冠状动脉内皮细胞（HCAEC）生长的影响及剂量依赖关系,明确水蛭素抗再狭窄的有效性和可行性。方法 用体外培养的HCASMC和HCAEC 3-5代,加入96孔细胞培养板,设置调零孔、对照孔、加药孔（6.25、3.13、1.56、0.78、0.39、0.2、0.1、0.05、0.025 mg/ml不同浓度水蛭素共9组,每组6个复孔）,培养48 h后用噻唑蓝比色试验（MTT法）检测不同浓度水蛭素干预下两种细胞的活力和抑制率变化。结果 与对照组相比,低浓度（0.025-0.1 mg/ml）水蛭素在对HCASMC增殖的影响方面无统计学差异（P均〉0.05）,而中高浓度（0.2-6.25 mg/ml）的水蛭素抑制HCASMC增殖,差异有统计学意义（P均〈0.05）,且其抑制率随水蛭素浓度升高而增加。与对照组相比,0.025-3.13 mg/ml的水蛭素在对HCAEC增殖的影响差异无统计学意义（P均〉0.05）,但0.05-0.2 mg/ml的水蛭素对HCAEC有一定的促增殖作用;而高浓度（6.25 mg/ml）的水蛭素对HCAEC增殖的抑制增加,差异有统计学意义（P均〈0.05）,其抑制率达到44.6%。结论 中高浓度水蛭素可呈剂量依赖性抑制HCASMC的生长,低中浓度水蛭素对HCAEC无明显抑制作用,且能在抗血栓的同时保护HCAEC甚至促进HCAEC生长。

Objective To investigate the influence of hirudin on growth of in vitro human coronary artery smooth muscle cells（HCASMC） and human coronary artery endothelial cells（HCAEC） and dose-dependent relation, and indentify the effectiveness and feasibility. Methods HCASMC and HCAEC cultured in vitro（3-5 generations） were respectively seeded onto 96-well plates, and then co-incubated with different doses of hirudin（6.25 mg/ml, 3.13 mg/ml, 1.56 mg/ml, 0.78 mg/ml, 0.39 mg/ml, 0.2 mg/ml, 0.1 mg/ml, 0.05 mg/ml, and 0.025 mg/ml）. After 48 h, the changes of viability and inhibitory rate of HCASMC and HCAEC were detected by using MTT. Results Compared with control group, the influence of low-dose hirudin（0.025 mg/ml-0.1 mg/ml） on HCASMC proliferation had no statistical difference（all P〈0.05）, and that of mid-dose and high-dose of hirudin（0.2 mg/ml-6.25 mg/ml） inhibited HCASMC proliferation（all P〈0.05） and the inhibitory rate increased as the increase of hirudin dose. Compared with control group, the influence of hirudin in dose of 0.025 mg/ml-3.13 mg/ml on HCAEC proliferation had no statistical difference（all P〉0.05）, but hirudin in dose of 0.05 mg/ml-0.2 mg/ml had improvement effect on HCAEC proliferation. The inhibition of hirudin in high dose（6.25 mg/ml） on HCAEC proliferation increased（all P〈0.05） and inhibitory rate reached 44.6%. Conclusion Hirudin in mid-dose and high-dose can inhibit HCASMC growth in and dosedependent manner, while hirudin in low-dose had no significant inhibitory effect on HCAEC growth, and can protect even improve HCAEC growth at the same time of anti-thrombosis