2-甲氧基雌二醇对恶性黑素瘤B16细胞株增殖与凋亡的影响

Effects of 2-methoxyestradiol on the proliferation and apoptosis of B16 malignant melanoma cells

目的 探讨2-甲氧基雌二醇（2-ME）对小鼠恶性黑素瘤B16细胞增殖与凋亡的影响,并初步探讨其机制.方法 选用小鼠B16细胞进行体外培养,实验分为阴性对照组和药物处理组,药物处理组加入2-ME,使其终浓度分别为5、10、20、40 μmol/L,阴性对照组不加2-ME.作用不同时间后,倒置相差显微镜下观察细胞形态变化;根据磺酰罗丹明B方法测得的各组A490值绘制生长曲线;采用磺酰罗丹明B法检测黑素瘤细胞活性;流式细胞仪检测细胞周期及凋亡;逆转录PCR和实时PCR法分析凋亡诱导基因（gadd45b）及原癌基因（c-myc）的表达情况.结果 重复测量的方差分析显示,5、10、20、40 μmol/L的2-ME作用于黑素瘤细胞不同时间后对其增殖的抑制作用差异有统计学意义（F=1170.94,P＜ 0.01）;上述浓度的2-ME作用24、48、72 h对黑素瘤细胞增殖的抑制作用差异亦有统计学意义（F=1843.04,P＜ 0.01）;药物浓度和培养时间存在交互作用（F=272.79,P＜ 0.01）.10、20、40μmol/L 2-ME作用48 h后,B16细胞的凋亡率分别上升至（4.13±1.12）％、（11.25±2.38）％、（19.46±2.9）％,与阴性对照组[（0.23±0.5）％]相比,差异均有统计学意义（P＜0.01）;细胞出现G0/G1期阻滞,对照组细胞、10、20、40 μmol/L 2-ME处理组细胞G0/G1期比例分别为（44.1±3.4）％、（59.5±5.6）％、（63.4±8.2）％、（70.8±4.4）％,且随着浓度的增加,G0/G1期细胞明显增加,各处理组及对照组间比较,G0/G1期细胞比例差异有统计学意义（F=13.56,P＜ 0.05）.与阴性对照组相比,20 μmol/L和40 μmol/L 2-ME作用24 h可升高gadd45b的表达（均P＜ 0.01）,10、20、40μmol/L 2-ME均可降低c-myc基因的表达,差异均有统计学意义（均P＜0.05）.结论 2-ME在体外能够抑制小鼠恶性黑素瘤B16细胞的增殖,能够使c-myc基因的表达降低,使gadd45b基因的表达升高.

Objective To investigate the effects of 2-methoxyestradiol （2-ME） on the proliferation and apoptosis of a mouse malignant melanoma cell line B16,and to explore their mechanism.Methods B16 cells were cultured in vitro,and divided into a negative control group receiving no treatment and several intervention groups treated with 2-ME at final concentrations of 5,10,20,40 mmol/L,respectively.After different durations of treatment,inverted phase-contrast microscopy was conducted to observe the morphologic change of B16 cells,sulforhodamine B （SRB） assay to evaluate proliferative activity and to draw growth curve of B16 cells according to the absorbance value at 490 nm,flow cytometry to detect cell cycle and apoptosis,and reverse transcription PCR and real-time PCR were performed to measure the expressions of the apoptosis-inducing gene gadd45b and proto-oncogene c-myc.Results As repeated measures analysis of variance showed,there were significant differences in the inhibitory effect on B16 cell proliferation among different concentrations （5,10,20,40 mmol/L） and different treatment durations （24,48,72 hours） of 2-ME （F =1170.94,1843.04,respectively,both P 〈 0.01）,and there was a significant interaction effect between these concentrations and treatment durations （F =272.79,P 〈 0.01）.After 48-hour treatment with 2-ME at 10,20 and 40 mmol/L,the apoptosis rate of B16 cells was increased to （4.13 ± 1.12）%,（11.25 ± 2.380）% and （19.46 ± 2.9）% respectively,compared to （0.23 ± 0.5）% in the negative control group （all P〈 0.01）; the proportion of B16 cells in G0/G1 phase was increased to （59.5 ± 5.6）%,（63.4 ± 8.2）% and （70.8 ± 4.4）% respectively,compared to （44.1 ± 3.4）% in the negative control group.There was a significant difference in the proportion of B16 cells in G0/G1 phase among the negative control group and intervention groups （F =13.56,P 〈 0.05）.Moreover,the mRNA expression of gadd45b was significantly enhanced after 24-hour treatment with 2-ME at concentrations of 20 and 40 mmol/L （both P〈 0.01）,while that of c-myc was significantly weakened after treatment with 2-ME at 10,20 and 40 mmol/L （all 〈 0.05） compared with the negative control group.Conclusion 2-ME can inhibit the proliferation of B16 cells in vitro,upregulate the expression of gadd45b gene and downregulate the expression of C-myc gene.