仲富瑞, 程宦立, 张 浩, 杜毅超,b, 胡启辉, 付文广,b, 夏先明,b

西南医科大学附属医院 a. 肝胆外科； b. 四川省院士(专家)工作站， 四川 泸州 64600

Effectofkaempferolontheproliferation,migration,invasion,andapoptosisofhumanhepatomaBel-7402cells

ZHONGFuruia,CHENGHuanlia,ZHANGHaoa,DUYichaoa,b,HUQihuia,FUWenguanga,b,XIAXianminga,b.

(a.DepartmentofHepatobiliarySurgery,b.Academician(Expert)WorkstationofSichuanProvince,TheAffiliatedHospitalofSouthwestMedicalUniversity,Luzhou,Sichuan64600,China)

Abstract:ObjectiveTo investigate the effect of kaempferol on the proliferation, migration, invasion, and apoptosis of human hepatoma Bel-7402 cells and related molecular mechanism.MethodsHepatoma Bel-7402 cells cultured in vitro were randomly divided into control group and low-, middle-, and high-concentration experimental groups. The experimental groups were treated with low-, middle-, and high-concentration kaempferol (25, 50, and 100 μmol/L), and the control group was treated with an equal volume of dimethyl sulfoxide. CCK-8 assay was used to observe the effect of kaempferol on the viability of Bel-7402 cells; plate colony formation assay was used to evaluate the effect of kaempferol on cell colony formation ability; wound healing assay and Transwell chamber were used to observe the effect of kaempferol on cell migration and invasion; Western blot was used to measure the expression of apoptosis- and cycle-related proteins. A one-way analysis of variance was used for comparison between multiple groups, and the least significant differencet-test was used for further comparison between two groups.ResultsAfter 24 hours of treatment, the cell viability was 100.00%±2.72% in the control group and 75.70%±2.42%, 62.79%±2.45%, and 43.41%±2.11%, respectively, in the low-, middle-, and high-concentration experimental groups, and compared with the control group, the experimental groups had a significant reduction in cell viability (allP<0.05). The number of cell colonies was 923.3±35.2 in the control group and 682.7±24.4, 464.0±22.0, and 327.3±14.0, respectively, in the low-, middle-, and high-concentration experimental groups, and compared with the control group, the experimental groups had a significant reduction in cell colony formation ability (allP<0.05). After 24 hours of treatment, the relative migration rate was 100.00%±1.11% in the control group and 63.33%±1.16%, 51.72%±3.23%, and 37.18%±2.71%, respectively, in the low-, middle-, and high-concentration experimental groups, and the number of transmembrane cells was 212.0±3.0 in the control group and 134.0±2.0, 71.0±2.0, and 34.0±1.0, respectively, in the low-, middle-, and high-concentration experimental groups; compared with the control group, the experimental groups had significant reductions in relative migration rate and number of transmembrane cells (allP<0.05). After 48 hours of treatment, compared with the control group, the low-, middle-, and high-concentration experimental groups had a significant reduction in the expression of the anti-apoptotic protein Bcl-2 (allP<0.05), a significant increase in the expression of the pro-apoptotic protein Bax (allP<0.05), and a significant reduction in the expression of CyclinD1 (allP<0.05).ConclusionKaempferol can inhibit the proliferation, migration, and invasion of human hepatoma Bel-7402 cells and promote the apoptosis of such cells, possibly by regulating the apoptosis proteins Bax and Bcl-2 and downregulating the expression of CyclinD1.

Keywords：kaempferol; liver neoplasms; cell proliferation; cell movement; apoptosis

原发性肝癌是人类消化系统最常见的癌症之一[1]，在我国尤为高发,具有恶性程度高、复发率高、转移率高的特点[2]。随着手术切除、射频消融、靶向治疗等治疗手段不断发展，肝癌患者的预后得到了一定改善，但治疗后的复发率仍较高，整体的5年生存率仍不理想[3]，因此寻找有效地抑制肝癌细胞增殖和转移的天然药物，无疑具有重大意义。山萘酚(Kaempferol, KA)是一种从传统中药赶黄草中提取的天然黄酮类化合物，现代药理研究[4]发现KA具有抗氧化、抗炎、抗癌等多种药理活性，且有研究证明KA能够通过不同的机制抑制卵巢癌[5]、结直肠癌[6]等癌细胞增殖转移，促进癌细胞凋亡。但KA在肝癌中的基础研究较少，故本研究通过体外实验，探讨KA对肝癌Bel-7402细胞体外克隆形成、增殖、侵袭迁移、凋亡的影响，也将阐述可能的分子机制，以期为KA用于新药开发及肝癌的临床治疗提供科学依据。

1 材料与方法

1.1 材料 肝癌Bel-7402细胞为本实验室保存，高糖DMEM培养基和胎牛血清购自美上海中乔新舟生物科技有限公司，山萘酚、结晶紫购于上海麦克林生化科技有限公司，CCK-8试剂盒、BCA蛋白浓度测定试剂盒、ECL化学发光试剂盒、一抗Bcl-2、Bax、cyclin D1、GAPDH及二抗均购自上海碧云天生物科技有限公司。

1.2 研究方法

1.2.1 细胞培养 将肝癌Bel-7402细胞置于含10%胎牛血清、1%青链霉素的高糖DMEM培养液中放于恒温培养箱(5%CO2、37 ℃)中常规传代培养，维持细胞处于对数生长期。……