20(S)-人参皂苷Rg3对乳腺癌MCF-7细胞的诱导凋亡作用

MCF-7 cell apoptosis induced by 20(S)-ginsendoside Rg3 in mammary carcinoma

目的：探讨20（S）-人参皂苷Rg3（SPG-Rg3）对人乳腺癌MCF-7细胞的诱导凋亡作用及其可能机制。方法：人乳腺癌细胞MCF-7细胞株分为SPG-Rg3实验组及空白对照组。采用MTT法观察SPG-Rg3对MCF-7细胞生长的抑制作用，并计算出IC50，依据IC50值确定SPG-Rg3的有效浓度；流式细胞术检测SPG-Rg3作用后MCF-7细胞周期的变化；AO／EB双染从形态学上观察SPG-Rg3对MCF-7细胞凋亡的作用；免疫细胞化学染色和RT-PCR技术分别从蛋白水平和分子水平上检测SPG-Rg3对MCF-7细胞的诱导凋亡作用及其与caspase-8基因的关系。结果：SPG-Rg3 IC50为（155．70±0．71）mg·L^-1,当SPG-Rg3浓度在37．5～600．0mg·L^-1时，MCF-7细胞的生长抑制率随SPG-Rg3浓度的增加而增大，与对照组比较，细胞增殖受到明显抑制（P〈0．05）；MCF-7细胞的生长周期也发生变化，S期细胞数增加，明显高于对照组（P〈0．01），G2／M期细胞数则明显少于对照组（P〈0．01）；并且在G1峰前出现明显的凋亡峰，凋亡细胞数增加。形态学上观察到SPG-Rg3（150mg·L^-1）实验组细胞大部分核着黄色荧光或橘红色荧光，形态呈固缩状、圆珠状或新月形，呈现明显的凋亡改变；caspase-8免疫细胞化学染色可见，对照组MCF-7细胞caspase-8蛋白不表达，SPG-Rg3实验组则呈强表达，两组细胞HSCORE得分为1．894±0．027及2．869±0．043，两组比较差异有显著性（P〈0．01）；提取细胞mRNA并设计caspase-8引物进行RT-PCR，SPG-Rg3实验组细胞caspase-8大量表达，对照组细胞无表达。结论：SPG-Rg3能诱导乳腺癌MCF-7细胞发生凋亡，其机制可能与其活化caspase-8作用有关。

Objective To study the effect of 20 （S） -ginsendoside Rg3 （SPG-Rg3） on apoptosis of human mammary carcinoma line MCF-7 cells and its possible mechanism. Methods Human mammary carcinoma line MCF-7 cells were divided into experiment group of SPG-Rg3 and control group. The inhibition of SPG-Rg3 on the growth of MCF-7 cells was detected by MTT assay, IC50 was calculated to obtain the effective concentration; flow cytometry was used to observe the cell cycle of MCF-7 cells; AO/EB fluorescence double-dye technology was performed to observe the apoptosis of cells in morphology, immunocellularchemistry and RT-PCR were utilized to investigate the apoptosis of MCF-7 cells and its relationship with caspase-8 from protein level and molecule level. Results ＂The IC50 of SPG-Rg3 was （155.7±0.71） mg· L ^-1 , when the concentration of SPG-Rg3 was arranged from 37.5 to 600.0 mg· L ^-1 , the growth inhibitory rate of MCF-7 cells was higher following the increase of its concentration, the cell growth was greatly inhibited compared with control group （P〈0.05）; cell cycle waschanged, the cell number of S period increased compared with control group （P〈0. 01）, the cell number of G2/M period decreased compared with control group （P〈0.01）, in experiment group there was an apoptotic apex before G1 apex, and the number of apoptotic cells increased; most of cell nuleus in SPC-Rg3 （150 mg · L^-1） experiment group appeared yellow or chrysoidine fluorescence, contracted, beaded and crescent; the result of immunocellularchemical staining of caspase-8 indicated that caspase-8 protein had no expression in control group, but stronger expression in SPG-Rg3 group. HSCORE seores of the two groups were 1. 894±0. 027 and 2. 869± 0. 043, the difference was markable （P〈0.01） . MCF-7 cells mRNA was extraited and caspase-8 primers were designed to perform RT-PCR, caspase-8 expressed strongly in experiment group and nothing in control. Conclusion SPG-Rg3 could induce MCF-7 cell apoptosis, and the possible mechanism may be related to aetivating caspase-8.