目的研究4种细胞毒活性测定方法对紫草素体外肿瘤细胞抑制作用的评价效果,对紫草萘醌为代表的天然色素在细胞毒活性评价中常出现的假阴性现象进行对比分析。方法将紫草素与其高敏感株急性早幼粒白血病HL-60细胞和低敏感株人非小细胞肺癌A549细胞体外共培养,通过台盼蓝法、磺酰罗丹明B(SRB)法、Cell Counting Kit-8(CCK-8)法和四甲基偶氮唑盐(MTT)法进行平行实验,测定紫草素在0.4~128μmol/L抑制细胞生长的量效关系曲线。结果台盼蓝法、SRB法、CCK-8法、MTT法测得紫草素对HL-60细胞的半数抑制浓度(IC50)分别为0.57、0.77、1.36、1.01μmol/L,对A549细胞的IC50分别为6.30、10.38、13.48、15.24μmol/L。紫草素浓度分别在3.2、32μmol/L以下时,4种检测方法得到其对2种细胞的抑制率均呈线性增长,超过此浓度即出现差异。其中台盼蓝法与SRB法结果一致性良好,而MTT法与CCK-8法测得的抑制率较低。对于HL-60细胞,紫草素12.8μmol/L时,CCK-8测得抑制率为81%,台盼蓝法为96%;对于A549细胞,紫草素128μmol/L时,MTT法测得抑制率为89%,台盼蓝法为99%。紫草素的吸收光谱与甲臜在波长400~600 nm存在重叠,最大重叠峰在550~570nm,CCK-8试剂与紫草素对HL-60细胞存在协同抑制效应。台盼蓝法结果显示高浓度紫草素几乎完全杀死板孔内细胞,相比对照组差异显著,但MTT法和CCK-8法结果出现假阴性。结论紫草萘醌为代表的天然色素的细胞毒活性评价不建议使用MTT法和CCK-8法,推荐使用SRB法和台盼蓝法。
Objective To study the effects of four cytotoxicity evaluation methods on the inhibition rate of shikonin(SK) in vitro, and to compare the pseudo-negative phenomenon often found in the evaluation of cytotoxic activity of natural pigments represented by naphthoquinones in Lithospermum erythrorhizon. Methods SK was co-cultured with its high-sensitive strain HL-60 cells and low-sensitivity strain A549 cells, trypan blue method, sulforhodamine B(SRB) method, Cell Counting Kit-8(CCK-8) and MTT cytotoxicity test were used for parallel experiments to determine the dose-effect relationship curve of SK(0.4-128 μmol/L) inhibiting the growth of cells. Results The half-inhibitory concentration(IC50) of shikonin on HL-60 cells was determined by trypan blue method, SRB method, CCK-8 method, and MTT method, which was 0.57, 0.77, 1.36, and 1.01 μmol/L, respectively. For A549 cells, the IC50 was 6.30, 10.38, 13.48, and 15.24 μmol/L, respectively. When the concentration of shikonin was below 3.2 μmol/L and 32 μmol/L, the inhibition rate of the two kinds of cells increased linearly by the four methods, followed by differences. Among them, the results of the trypan blue method and the SRB method are in good agreement, while the MTT method and the CCK-8 method have lower inhibition rates. At 12.8 μmol/L, the inhibitory rate of SK on HL-60 cell measured by CCK-8 was 81%, while the inhibitory rate measured by trypan blue method was 96%, and at 128 μmol/L the inhibitory rate of SK on A549 cells measured by MTT method was 89%, however, the inhibitory rate measured by trypan blue method was 99%. The absorption spectrum of SK overlapped with formazan at the wavelength from 400 to 600 nm, with the maximum overlap peak from 550 to 570 nm, and CCK-8 reagent had a synergistic inhibitory effect on HL-60 with SK. The results of trypan blue method showed that SK at the highest dose almost completely killed cells in the plate wells, which was significantly different from the control group, but both MTT method and CCK-8 method resulted in a
Chinese Traditional and Herbal Drugs
柴冰阳(1995-),女,硕士,从事肿瘤药理学研究。Tel:18119257611 E-mail:email@example.com;通信作者:张波(1978-),男,研究生导师,教授,从事系统药理学与中药药理学研究。Tel:(0993)2057670 E-mail:firstname.lastname@example.org.