络风宁0号方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

Inhibitory Effects of Luofengning-0 Formula on the Growth and Proliferation of Human Coronary Artery Smooth Muscle Cells and Endothelial Cells in Vitro

目的探讨络风宁0号方不同配比对体外培养的人冠状动脉平滑肌细胞(HCASMC)和内皮细胞(HCAEC)生长的影响及剂量依赖关系,明确中药复合物抗再狭窄的有效性和可行性,为中药药物涂层支架的研发提供实验数据支持。方法用体外培养的HCASMC和HCAEC 3~5代,加入96孔细胞培养板,选择抑制HCASMC生长,而对HCAEC无抑制作用的浓度范围(0.2~3.13 mg/ml)作为最佳的水蛭素浓度与1μmol/L紫杉醇组成复合物干预两种细胞,培养48 h后用噻唑蓝比色试验(MTT法)测定各孔吸光值(A值),选择对HCASMC抑制程度最大,而对HCAEC抑制程度最小且与单用紫杉醇比较能最大程度减轻对HCAEC抑制作用的浓度作为络风宁0号方的最佳配比。结果在对HCAEC增殖的影响方面,复合药物组与单药紫杉醇组比较,差异无统计学意义(P>0.05),而对HCASMC的抑制率明显高于单药紫杉醇组(P<0.05),且1μmol/L紫杉醇+0.39 mg/ml水蛭素组可明显降低单药紫杉醇对HCAEC的抑制率。结论选择1μmol/L紫杉醇+0.39 mg/ml水蛭素作为络风宁0号方的最佳配比,可最大限度抑制HCASMC增殖的同时对HCAEC有最小的抑制作用,具备抗再狭窄的有效性和可行性,为新型中药药物涂层支架的研发提供新的思路。

Objective To prove the efficacy of Luofengning- 0 complexes and provide the experimental data for preventing restenosis,we investigated the inhibitory effects of different ratios of Luofengning-0 complexes on the growth of human coronary artery smooth muscle cells( HCASMC) and endothelial cells( HCAEC) cultivated in vitro. Methods The 3 ~ 5 generations of HCASMC and HCAEC were respectively seeded onto 96- well plates,1 μmol / L paclitaxel was added into hirudin of various concentrations to prepare different ratios of Luofengning- 0 complexes. Then HCASMC and HCAEC cells were co- incubated with different ratios of Luofengning- 0 complexes in 96- well culture plate. Every dose was added into 6 wells. The first 5 wells were added with culture medium as blank control group and the remaining 1 well was the non-adjustment well. The second 6 wells were only added with single paclitaxel. After co- incubation for 48 hours,the change of cell growth activity was detected by MTT assay to observe the state of cells. Then the appropriate ratio of paclitaxel to hirudin for maximizing the inhibition of HCASMC and minimizing the inhibition of HCAEC was determined. Results Compared with single paclitaxel group,in the low dose group of LFN- 0 complexes( 1 μmol/L paclitaxel + 0. 39 mg/ml hirudin),the growth of HCAEC( P < 0. 05) were significantly increased and the inhibition ratio of the growth of HCAEC( P < 0. 05) was decreased. Conclusion For the low dose of LFN- 0 complexes can maximize the inhibition of HCASMC and minimize the inhibition of HCAEC,we choose1 μmol / L paclitaxel plus 0. 39 mg / ml hirudin as our final ratio of LFN- 0 complexes. All these proved the rationality of TCM compatibility to reduce the poison theory and provided an objective basis for the new theory of'endogenous collateral wind'.