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[目的]建立一套适合于实验室的高效、快速的体外抗肝癌活性物质筛选模型。[方法]首先,选用乙醇为溶剂对芸香草、半边莲、蜜蒙花、甘草、地榆、柴胡、旱芹和莪术8种中草药植物材料进行浸提。然后通过MTT法对8种中草药体外抗肝癌细胞SMMC-7721的活性进行初测。试验采用二甲基亚砜(DMSO)与甘氨酸-NaOH缓冲液作为溶剂,消除剩余培养基对吸光值的影响。为确保细胞株最大吸收峰位置不变,酶标仪采用570 nm波长测定OD值。为验证筛选出的3种物质对癌细胞的抑制作用,以活性物质浓度为横坐标,细胞生长抑制率为纵坐标,描绘肿瘤细胞生长曲线,倒置显微镜观察细胞不同阶段的生长状况。[结果]倒置显微镜下观察3种细胞阶段的形态:正常状态下,细胞的透明度大、折光性强、轮廓不清;当细胞受污染时,折光性变弱,胞质中常出现空泡、脂滴、颗粒样物质,细胞之间空隙加大,细胞变得不规则,失去原有特点;而受活性物质作用细胞尽管仍然贴壁,但其萎缩,边缘清晰化;三者之间存在一定区别。MTT法培养板定性检测显示,柴胡、莪术、甘草细胞培养孔内的液体颜色都较浅且比较均匀,可见,它们对细胞生长具有明显的抑制作用。而半边莲培养孔内液体颜色深浅不一。还有一些培养孔内液体颜色较深,如卤地菌、蜜蒙花、地榆和旱芹,表明它们对细胞生长没有明显的抑制作用。进一步定量计算表明,浓度为1.0～1.5 mg/ml的甘草乙醇浸提液(ESCG)、莪术乙醇浸提液(ESCC)和柴胡乙醇浸提液(ESCB)对体外培养的肝癌细胞SMMC-7721有较强的抑制增殖作用,抑制率分别为51.6%、48.5%和52.9%。甘草、柴胡和莪术3种物质的抑制作用曲线结果也验证了它们对细胞生长有明显的抑制作用,并且抑制程度与浓度呈正相关。[结论]MTT法筛选可作为重要抗肿瘤的1个基本筛选模型。
[Objective] To establish an efficient and rapid in vitro screening model of active substance against liver cancer suitable for laboratory. [Method] Firstly, ethanol was used as the solvent to extract plant materials from 8 herbs of Rutaceae, Lobelia, Montemon, Licorice, Burnet, Bupleurum, Celery and Curcuma. Then the activity of 8 kinds of Chinese herbal medicine against hepatocellular carcinoma cell SMMC-7721 in vitro was assayed by MTT assay. The experiment used dimethylsulfoxide (DMSO) and glycine-NaOH buffer as the solvent to eliminate the effect of the remaining medium on the absorbance. In order to ensure the maximum absorption peak position of cell lines, microplate reader using 570 nm wavelength determination of OD value. In order to verify the inhibitory effect of the three selected substances on cancer cells, the growth curve of tumor cells was plotted on the abscissa of the active substance concentration and the growth inhibition rate of the cells as the vertical axis, and the growth status of the cells at different stages was observed with an inverted microscope. [Result] Under the inverted microscope, the morphological changes of the three cell stages were observed. Under normal conditions, the cells had a large degree of transparency, strong refraction and an unclear outline. When the cells were contaminated, the refraction became weaker and vacuoles and lipids were often found in the cytoplasm Drops, particulate matter, the gap between the cells increased, the cells become irregular, losing the original features; and by the role of active substances cells still adherent, but its atrophy, marginal clarity; there is a certain difference between the three . The qualitative test of MTT plate showed that the liquid color of Bupleurum, Curcuma and Glycyrrhiza cells were lighter and more uniform, which showed that they had a significant inhibitory effect on cell growth. The lobelia culture hole liquid color depth varies. Some of the darker fluids in the wells, such as Halophytes, Melia, Burnet and Celery, showed no significant inhibitory effect on cell growth. Further quantitative calculations showed that ESCG, ESCC and ESCB at a concentration of 1.0-1.5 mg / ml could inhibit the growth of hepatoma cells SMMC-7721 A strong inhibition of proliferation, inhibition rates were 51.6%, 48.5% and 52.9%. Glycyrrhiza, Bupleurum and Curcuma Curcuma Curcumin results also verified that they have a significant inhibitory effect on cell growth, and the degree of inhibition and concentration was positively correlated. [Conclusion] MTT screening can be used as a basic screening model of important anti-tumor.