脂多糖对体外培养的人冠状动脉平滑肌细胞生长的影响——对建立支架内再狭窄炎症细胞模型的探索

Influence of lipopolysaccharide on growth of in vitro human coronary artery smooth muscle cells: an investigation on establishing in-stent restenosis inflammatory cell model

目的探讨不同浓度脂多糖（LPS）作用不同时间对体外培养的人冠状动脉平滑肌细胞（HCASMC）生长的影响,分析其应用于HCASMC的无毒性反应浓度范围,为开展介入术后再狭窄相关研究建立HCASMC炎症活化模型提供实验数据支持。方法体外培养的HCASMC 3-5代,加入96孔细胞培养板,用噻唑蓝比色实验（MTT法）检测不同浓度（0μg/ml,0.01μg/ml,0.1μg/ml,0.5μg/ml,1μg/ml,5μg/ml,10μg/ml,100μg/ml）LPS在不同作用时间（24 h,46 h,72 h）下对HCASMC活力影响,酶标仪492 nm处测定各组A值,细胞活力=（实验组A值-调零孔A值）/对照组A值。结果 LPS在低于100μg/ml浓度范围内,干预HCASMC时间短于48 h未出现细胞毒性,多表现为促细胞增殖效应,而干预时间大于48 h则显现出较为明显的细胞毒性,且细胞活力随LPS浓度的增大而减低;干预72 h后LPS浓度在0-0.01μg/ml范围内有促增殖作用,0.1-0.5μg/ml浓度则产生轻微的细胞抑制作用,但细胞毒性不明显,在1-100μg/ml浓度下HCASMC毒性反应逐渐出现,且HCASMC活力随LPS浓度的升高而减低。同一浓度水平的LPS随着作用时间延长,HCASMC的细胞活力逐步下降,且作用时间越长下降越明显。结论LPS在浓度0.1μg/ml以下未出现明显的细胞毒性,其中LPS浓度0.01μg/ml干预24 h,人冠状动脉平滑肌细胞增殖最显著,可作为建立炎症诱导人冠状动脉平滑肌细胞活化模型的最佳条件。

Objective To investigate the influence of lipopolysaccharide（LPS） in different doses on growth of in vitro human coronary artery smooth muscle cells（HCASMC） at different times, analyze non-toxic concentration range of LPS to HCASMC, and provide test data for establishing HCASMC inflammation activation model in studies related to restenosis after interventional therapy. Methods HCASMC was cultured in vitro for 3-5 generations, and seeded onto 96-well plates. The influences of LPS in different doses（0 μg/m L, 0.01 μg/m L, 0.1 μg/m L, 0.5 μg/m L, 1 μg/m L, 5 μg/m L, 10 μg/m L and 100 μg/m L） on viability of HCASMC at different time points（24 h,46 h,72 h） were detected by using MTT assay. The value A was detected by using ELISA at point of 492 nm, and cell viability=（value A of test group-value A of zero hole）/value A of control group. Results When LPS dose was lower than 100 μg/m L and interventional time was shorter than 48 h, there was no cytotoxicity observed to HCASMC and there was promotion effect on proliferation. When interventional time was longer than 48 h, there was significant cytotoxicity observed and viability of HCASMC would decrease as LPS dose increased. After intervention for 72 h, LPS had promotion effect on proliferation in doses from 0 μg/m L to 0.01 μg/m L, and mild inhibitory effect in doses from 0.1 μg/m L to 0.5 μg/m L but cytotoxicity was not significant. The toxic reaction gradually appeared when LPS dose was from 1 μg/m L to 100 μg/m L, and viability of HCASMC decreased as LPS dose increased. When LPS doses were the same, viability of HCASMC gradually decreased as interventional time was prolonged, and the longer the interventional time was, the more significant the decrease of HCASMC was. Conclusion LPS had no significant cytotoxicity when its dose is 0.1 μg/m L, and when its dose is 0.01 μg/m L, the proliferation of HCASMC will be more significant after intervention for 24 h, which can be taken as the best condition for establishing HCASMC inflammation activation model.