丹参素促大鼠心肌细胞及心脏干细胞增殖的作用

Effect of Danshensu on growth of myocardial cells and cardiac stem cells

目的:大鼠原代心肌细胞及心脏干细胞(CSCs)培养条件的筛选及丹参素(DS-182)对两种细胞体外增殖的影响。方法:对比组织块培养法及差速培养法,选取适宜培养基、消化液浓度、消化时间、差速贴壁时间及心脏成纤维细胞(CFs)抑制剂等因素。分别用MTT比色法、ELISA法及流式细胞术(FCM)检测丹参素对大鼠心肌细胞及CSCs体外增殖的影响。结果:确定DMEM/F12 1∶1培养基、100 ml/L胎牛血清(FBS)、2.5 g/L胰蛋白酶、5 min/次×5次的消化时间及120 min的差速贴壁时间为最适分离纯化条件。MTT比色法检测显示,与空白对照组相比,经浓度为7.8×10-4～1×10-1mol/L的DS-182处理12、24、48和72 h后,细胞的吸光度(A)值明显增加(P<0.05)。ELISA法检测显示,与空白对照组相比,经浓度为3.9×10-4～1×10-1mol/L的DS-182处理24 h后,细胞的A值明显增加(P<0.05)。FCM检测显示,经浓度为0.1 mol/L的DS-182处理24 h后,c-kit+的细胞数占细胞总数的比例增加了27.0%。结论:改良了一套较为完整的大鼠心肌细胞及CSCs的分离纯化方法。DS-182在一定浓度下(7.8×10-4～1×10-1mol/L)对大鼠心肌细胞及CSCs的增殖具有明显的促进作用。

AIM：To explore the appropriate culture conditions and method for separating myocardial cells and cardiac stem cells （CSCs） from Sprague Dawley neonatal rat hearts and to study the effect of Danshensu （DS182） on the proliferation of the two kinds of cells. METHODS： Different culture mediums, digestive enzyme concentrations, digestion times, differential adhesion times and inhibitors of fibroblasts were selected to optimize the separation and purification methods of rat myocardial cells and CSCs. MTT assay, ELISA, FCM and other methods were used to determine the effect of DS182 on cell proliferation of rat myocardial cells and CSCs. RESULTS： Separation and purification conditions of the two kinds of cells were DMEM/F12 medium 1∶1, 100 ml/L FBS, 25 g/L trypsin, digestion time of 5 min for five times and the differential time of 120 min. MTT assay results showed that compared with those in the blank group, cell A value increased significantly when cells were exposed to the DS182 concentration of 78×10-4-1×10-1 mol/L at 12, 24, 48, 72 h after administration （P〈0.05）. ELISA results showed that cell OD value increased significantly when cells were exposed to the DS182 concentration of 3.9×10-4-1×10-1 mol/L at 24 h after administration （P〈0.05）. FCM results showed that the number of ckit＋ increased by 27.0% when cells were exposed to the DS182 concentration of 0.1 mol/L at 24 h after administration. CONCLUSION： We improved the separation and purification methods of myocardial cells and CSCs. DS182 at certain concentration （78 × 10-4-1×10-1 mol/L） obviously promotes cell proliferation of myocardial cells and CSCs.