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目的观察黄酮金雀异黄素(genistein,Gen)抑制人肝癌HepG2细胞的增殖和诱导其分化的作用。方法体外培养HepG2细胞,以全反式维甲酸(RA)为阳性对照,MTT比色法测定Gen对HepG2细胞增殖的影响;细胞计数法测定HepG2细胞存活率;瑞氏-姬姆萨染色法和考马斯亮蓝染色法分别观察Gen对HepG2细胞形态、细胞微管蛋白排列的影响;放免法测定HepG2细胞AFP的分泌量;酶促反应试剂盒检测细胞中γ-GT、ALP;Diamondstone分光光度法测定细胞中TAT的含量。结果MTT法和细胞计数法检测结果都表明,不同浓度(10μmol·L-1、100μmol·L-1)的Gen诱导人肝癌HepG2细胞48h,细胞增殖活力明显降低,并呈剂量依赖性下降。10μmol·L-1Gen孵育HepG2细胞48h后细胞形状由圆形转变为长梭形、细胞相互分散、细胞核变小、核仁的数目减少;微管微丝增多,排列更整齐,细胞趋向成熟分化。以10μmol·L-1Gen分别处理HepG2细胞24,48,72和96h后,显著地降低了γ-GT的活性和AFP的表达,增加了TAT和ALP的活性。结论Gen能抑制人肝癌HepG2细胞的生长和增殖,并能诱导其向成熟细胞分化。
Objective To observe the effect of genistein (Gen) on the proliferation and differentiation of HepG2 human hepatocellular carcinoma cells. Methods HepG2 cells were cultured in vitro. All-trans retinoic acid (RA) was used as a positive control. The effect of Gen on the proliferation of HepG2 cells was determined by MTT assay. The survival rate of HepG2 cells was determined by cell counting method. Wright-Giemsa staining and Coomassie brilliant blue staining were used to observe the effect of Gen on HepG2 cell morphology and tubulin distribution; AFP secretion was measured by radioimmunoassay; γ-GT and ALP were detected by enzymatic reaction kit; Diamondstone spectrophotometry TAT content in cells. Results The results of MTT assay and cell counting assay showed that Gen-induced human hepatocellular carcinoma HepG2 cells treated with 10 μmol·L-1 and 1100 μmol·L-1 of GenBank at 48 h significantly decreased the proliferation of cells in a dose-dependent manner. HepG2 cells were incubated with 10μmol·L-1Gen for 48h, the shape of the cells changed from round to long fusiform, the cells were dispersed, the nucleus became smaller and the number of nucleolus decreased. The number of microtubules was increased, arranged neatly, and the cells tended to mature and differentiated. HepG2 cells were treated with 10μmol·L-1Gen for 24,48,72 and 96 h, respectively, which significantly reduced the activity of γ-GT and the expression of AFP, and increased the activity of TAT and ALP. Conclusion Gen can inhibit the growth and proliferation of human hepatoma HepG2 cells and induce its differentiation into mature cells.