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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 screen the culture conditions of rat primary cardiomyocytes and cardiac stem cells (CSCs) and the effect of Danshensu (DS-182) on the proliferation of the two kinds of cells in vitro. Methods: Compared with tissue culture method and differential culture method, the suitable culture medium, digestive juice concentration, digestion time, differential fixation time and cardiac fibroblast (CFs) inhibitor were selected. The effects of Danshensu on the proliferation of rat cardiomyocytes and CSCs were detected by MTT assay, ELISA and flow cytometry (FCM) respectively. Results: The digestion time of DMEM / F12 1: 1 medium, FBS, 2.5 g / L trypsin, 5 min / Optimal separation and purification conditions. MTT colorimetric assay showed that compared with the blank control group, the cell absorbance (A (subscript max)) was significantly decreased after treatment with DS-182 at a concentration of 7.8 × 10-4 ~ 1 × 10-1mol / L for 12,24,48 and 72 h ) Increased significantly (P <0.05). Compared with the blank control group, the A value of the cells was significantly increased (P <0.05) by the ELISA method after being treated with DS-182 at a concentration of 3.9 × 10-4 ~ 1 × 10-1mol / L for 24 hours. FCM results showed that the percentage of c-kit + cells in total cells increased by 27.0% after treated with 0.1 mol / L DS-182 for 24 h. Conclusion: A set of more complete rat cardiomyocytes and CSCs were isolated and purified. The concentration of DS-182 in a certain concentration (7.8 × 10-4 ~ 1 × 10-1mol / L) significantly promoted the proliferation of rat cardiac myocytes and CSCs.