山奈酚对大鼠肝癌细胞CBRH7919的增殖抑制及诱导凋亡作用

Influences of Kaempferol on Inhibiting Proliferation and Inducing Apoptosis of Rat Hepatoma Cell Line CBRH7919

【目的】观察山奈酚对大鼠肝癌细胞CBRH7919增殖抑制、诱导凋亡和细胞周期的影响。【方法】选用CBRH7919肝癌细胞株,传代培养24 h后,加入不同浓度山奈酚处理,采用四甲基偶氮唑盐(MTT)法检测细胞存活率,4,′6-二脒基-2-苯基吲哚(DAPI)染色观察细胞凋亡形态,彗星电泳技术和流式细胞术检测药物对细胞DNA的作用。【结果】山奈酚体外能抑制大鼠肝癌细胞CBRH7919的增殖,以6.25、12.5、25、50、100μmol/L浓度的山奈酚处理细胞72 h,对CBRH7919细胞的增殖抑制率显著升高,呈明显的剂量效应关系。用50、100μmol/L浓度的山奈酚作用于细胞24、48、72、96 h,对细胞的增殖抑制作用显著增强,呈明显的时间效应关系,即药物作用时间越长,抑制作用越强。DAPI染色显示不同浓度山奈酚组的细胞均有明显的细胞凋亡特征。彗星电泳显示30、60、120μmol/L浓度的山奈酚作用于细胞72 h后,细胞后面均呈现长度不等的拖尾,平均光密度值较空白对照组显著降低(P<0.01),彗星尾距较空白对照组显著增加(P<0.01),且两者改变与药物浓度相关。流式细胞仪检测结果显示30、60、120μmol/L浓度的山奈酚作用于细胞72 h出现亚二倍体凋亡峰,细胞周期被阻滞于S期,不同浓度山奈酚组凋亡率较空白对照组均明显增高。【结论】山奈酚可能通过抑制大鼠肝癌细胞CBRH7919的增殖及诱导其凋亡而产生抗肿瘤作用。

Objective To observe the effect of kaempferol on inhibiting proliferation and inducing apoptosis of rat hepatoma cell line CBRH7919.Methods After subculture for 24 hours,rat hepatoma cell line CBRH7919 was treated with different concentrations of kaempferol.In-vitro cell survival rate was detected by methyl thiazolyl tetrazolium（MTT） assay,the apoptosis of CBRH7919 was observed with 4＇,6-diamidino-2-phenylindole（DAPI） staining method,and the effect of kaempferol on the proliferation and apoptosis of CBRH7919 was investigated with comet electrophoretic technique and flow cytometry.Results Kaempferol in the dose of 6.25,12.5,25,50,and 100 μmol·L-1 inhibited the proliferation of CBRH7919 after treatment for 72 hours,and the effect was in a dose-dependent manner.After treatment with 50 and 100 μmol· L-1 kaempferol for 24,48,72 and 96 hours,the inhibitory effect on CBRH7919 proliferation showed a time-dependent manner.The results of DAPI staining method showed obvious apoptosis in different concentrations of kaempferol groups.The results of comet assay showed that the length of the trailing of CBRH7919 was different in 30,60 or 120 μmol·L-1 kaempferol groups after treatment for 72 hours,the average optical density was lower and the trailing length was longer in kaempferol groups than those in the blank control group（P0.01）,and the effect was in a dose-dependent manner.The flow cytometry assay illustrated that subdiploid apoptotic peak was presented in 30,60 or 120 μmol·L-1 kaempferol groups after treatment for 72 hours,the cell cycle of kaempferol was arrested in the S phase,and the apoptotic rate was higher in kaempferol groups than that in the blank control group（P0.01）.Conclusion The anti-tumor mechanism of kaempferol is probably related with proliferation inhibition and apoptosis induction of rat hepatoma cell line CBRH7919.