双丹口服液对体外培养心肌干细胞作用的研究

Influence of shuangdan oral liquid on cardiac stem cells cultured in vitro

目的观察双丹口服液(SOL)对体外分离培养的SD仔鼠心肌干细胞(CSCs)的作用。方法分离培养CSCs,将培养的第2代CSCs分为对照组及不同浓度1×10-1mol/L、1×10-2mol/L、1×10-3mol/L、1×10-4mol/L、1×10-5mol/L和1×10-6mol/L的SOL组。培养24 h、48 h后用MTT比色法检测SOL对CSCs的影响。将10-2mol/L SOL组的细胞培养24 h后,用流式细胞仪测定c-kit+/CD45-细胞的比率。结果消化后的心肌组织3 d后,可见成纤维样细胞从组织块儿周围爬出,1周后可见小、圆、亮的细胞出现在组织块周围爬出的成纤维细胞层上。传代培养后,倒置显微镜下观察细胞形态呈较均一的梭型,可见CSCs形成的"太阳状"集落。MTT法检测表明,与对照组相比,SOL作用后的CSCs生长增殖迅速,当SOL的浓度超过1×10-3mol/L时,CSCs增殖更为显著(P<0.05)。流式细胞术检测显示,1×10-2mol/L SOL的细胞培养24 h后,c-kit+/CD45-细胞的比率达到0.976%,与对照组相比(0.301%),差异具有统计学意义(P<0.01)。结论利用酶消化可以成功从SD仔鼠心脏组织中分离培养得到c-kit+的CSCs,SOL对CSCs的体外生长和增殖有一定的促进作用。

AIM To observe the effect of shuangdan oral liquid （SOL） on cardiac stem cells （CSCs） cultured in vitro. METHODS CSCs were isolated and cultured. The second generation CSCs were cultured with different concentrations of SOL （control group; 1×10^-1 mol/L; 1×10^-2 mol/L; 1×10^-3 mol/L; 1×10^-4 mol/L; 1×10^-5 mol/L; 1×10^-6 mol/L） and after 24 h and 48 h, the influence of SOL on CSCs was detected by MTT assay. CSCs were cultured with 10^-2 mol/L SOL and after 24 h the content of c-kit^＋ CD45 cells was measured by flow cytometry. RESULTS Fibroblast-like cells distributed from tissue pieces 3 days after myocardial tissue digestion and small, round, bright cells were observed on the fibroblast layer around the tissue. After subculture, cells showed uniform shuttle type and cardiac stem cells formed “sun-like” colonies under inverted microscope. Compared with the control group, cells cultured in SOL grew and proliferated rapidly. When the concentration of SOL exceeded 1×10^-3 mol/L, cardiac stem cells proliferated more significantly （P〈0.05）. FCM showed that c-kit^＋/CD45 cells reached 0.976% when cultured in SOL （1×10^-2 mol/L） for 24 h, significantly higher than the 0.301% of the cells in normal culture （P〈0.01）. CONCLUSION The c-kit cardiac stem cells can be successfully obtained from the cardiac tissue of New Zealand rats by enzymatic digestion. SOL promotes cardiac stem cell growth and proliferation in vitro.