熊果酸对人骨肉瘤细胞U2-OS增殖及凋亡的影响研究  

Effect of ursolic acid on proliferation and apoptosis of human osteosarcoma cell line U2-OS

作　　者：黄世福[1] 蔡风[2] 程昭贤 周荣平[2] 郝亮[2] 

机构地区：[1]吉水县人民医院骨科,江西吉水331600 [2]南昌大学第二附属医院骨科,南昌330006

出　　处：《中国修复重建外科杂志》2017年第11期1371-1376,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81760487);江西省中医药科技计划项目(2015A024)~~

摘　　要：目的探讨熊果酸对人骨肉瘤细胞U2-OS增殖以及凋亡的影响,分析其作用机制。方法取人骨肉瘤细胞株U2-OS分为4组,分别采用浓度为0、10、20、40μmol/L的熊果酸进行培养。培养0、24、48、72 h,应用细胞计数试剂盒8(cell counting kit 8,CCK-8)检测细胞增殖能力变化;培养48 h,使用流式细胞术测定熊果酸对骨肉瘤细胞周期和凋亡的影响,应用实时荧光定量PCR和Western blot检测周期蛋白cyclin D1的表达和凋亡相关蛋白Caspase-3的表达和活化情况。结果 CCK-8检测示,培养0、24 h时各组吸光度(A)值比较,差异无统计学意义(P>0.05);48、72 h时随浓度增加A值逐渐减小,各组间比较差异均有统计学意义(P<0.05)。流式细胞术检测示,随熊果酸浓度增加,U2-OS细胞的G_1期增加、S期及G_2/M期减少,细胞凋亡率逐渐增加,各组间比较差异均有统计学意义(P<0.05)。与0μmol/L组相比,10、20、40μmol/L组cyclin D1 m RNA及蛋白相对表达量下调(P<0.05);各组间Caspase-3 m RNA相对表达量比较差异无统计学意义(P>0.05);但随熊果酸浓度增加,Caspase-3前体蛋白下降,活化的Caspase-3增加,各组间差异均有统计学意义(P<0.05)。结论熊果酸能有效抑制U2-OS细胞增殖,诱导cyclin D1表达下调导致细胞在G_0/G_1期阻滞,同时使Caspase-3活化增加进而促进细胞凋亡。Objective To investigate the effect of ursolic acid on the proliferation and apoptosis of human osteosarcoma cell line U2-OS and analyze its mechanism. Methods Human osteosarcoma cell line U2-OS was divided into 4 groups, which was cultured with ursolic acid of 0, 10, 20, and 40 μmol/L, respectively. At 0, 24, 48, and 72 hours after being cultured, the cell proliferation ability was detected by cell counting kit 8（CCK-8）. At 48 hours, the effects of ursolic acid on cell cycle and apoptosis of U2-OS cells were measured by flow cytometry. Besides, the expressions of cyclin D1 and Caspase-3 were detected by real-time fluorescent quantitative PCR and Western blot. Results CCK-8 tests showed that the absorbance（A） value of each group was not significant at 0 and 24 hours（P0.05）; but the differences between groups were significant at 48 and 72 hours（P0.05）. Flow cytometry results showed that, with the ursolic acid concentration increasing, the G_1 phase of U2-OS cells increased, the S phase and G_2/M phase decreased, and cell apoptosis rate increased gradually. There were significant differences between groups（P0.05）. Compared with the0 μmol/L group, the relative expressions of cyclin D1 m RNA and protein in 10, 20, and 40 μmol/L groups significantly decreased（P0.05）; whereas, there was no significant difference in relative expression of Caspase-3 m RNA between groups（P0.05）. However, with the ursolic acid concentration increasing, the relative expressions of pro-Caspase-3 protein decreased and the relative expressions of activated Caspase-3 increased; there were significant differences betweengroups（P0.05）. Conclusion Ursolic acid can effectively inhibit the proliferation of osteosarcoma cell line U2-OS,induce the down-regulation of cyclin D1 expression leading to G_0/G_1 phase arrest, increase the activation of Caspase-3 and promote cell apoptosis.

关 键 词：熊果酸 骨肉瘤 U2-OS细胞 细胞增殖 细胞凋亡 

分 类 号：R738.1[医药卫生—肿瘤]