软琼脂克隆形成实验评价药物体外抑瘤性与成瘤性

目的：分别采用肿瘤细胞系和转化细胞系摸索软琼脂克隆形成实验的适宜条件，并对冬凌草甲素（oridonin，ORI）的抑瘤性和没食子酸乙酯（ethyl gallate，EG）促瘤性进行评价。方法：1）摸索实验条件：采用6 孔板固态培养法，按不同密度（每孔500~20 000 个）将肿瘤细胞系（Hela，K562 和HepG2）和转化细胞系（Bhas 42）与软琼脂混合铺板，计数克隆形成率。2）HepG2 细胞分别与质量浓度为0.016、0.08、0.4、2、10、50 μg&#183;mL^-1 的ORI 溶液混合后铺板；Bhas 42 细胞预先经质量浓度为0.3、1、3 μg&#183;mL^-1 的EG 溶液处理后，镜下观察细胞达到转化状态后，与软琼脂混合铺板培养。经培养后观察给药处理对克隆形成率的影响。3）利用流式细胞仪分析ORI 和EG 对细胞周期的影响。结果：细胞接种密度为每孔1 000 个左右时，利用肿瘤细胞和转化细胞开展软琼脂克隆形成实验效果较好。ORI 作为抑瘤药物在低浓度条件可显著降低HepG2 细胞的克隆形成率（P<0.01），且对细胞增殖有抑制作用。EG 随给药浓度增加可显著升高Bhas42 的克隆形成率（P<0.01），但对细胞增殖影响不显著。结论：软琼脂克隆形成实验以克隆形成率为检测指标，可较为简便而灵敏地检测药物引起的抑瘤性和促瘤性。该实验结果与细胞周期分析结果相互印证。两者的有机结合，可用于药物对细胞增殖及成瘤性的影响的初步分析。

Objective: To zinvestigate the appropriate conditions for soft agar colony formation assay using tumor and transformed cell lines,and evaluate the tumor suppression effect of oridonin(ORI)and the tumorigenicity of ethyl gallate(EG)respectively. Methods: 1)Experimental conditions:tumor cell lines(Hela,K562 and HepG2) and transformed cell line(Bhas 42)were mixed with soft agar on 6-well culturing plates at different density(500-20 000 cells per well),and the colony formation rates were calculated. 2)HepG2 cells were mixed with ORI at concentrations of 0.016,0.08,0.4,2,10,50 μg&#183;mL^-1;Bhas 42 cells were pretreated with EG at concentrations of 0.3,1,3 μg&#183;mL^-1 and subsequently plated with soft agar while the transformation state was observed. The effects of ORI and EG on colony formation rates were examined. 3)Effects of ORI and EG on cell cycles were analyzed by flow cytometry. Results: Both tumor cells and transformed cells achieved optimized effects when the cells were plated at the density of 1 000 cell per well. ORI as an antitumor drug significantly reduced the clone formation rate(P<0.01)and exhib ited inhibition effect on proliferation of HepG2 cells. EG was able to increase the clone formation rate in a concentration-dependent pattern,but had no effect on the cell proliferation. Conclusion: In the soft agar colony formation assay,clone formation rate is used to estimate the tumor suppression effect and the tumorigenicity induced by drugs in a simple and sensitive manner. Our colony formation assay results are consistent with the cell cycle analysis data. By combining both methods,drug-induced cell proliferation and tumor suppression/tumorgenicity could be effectively predicted.

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