氟尿嘧啶联合顺铂对骨肉瘤MG-63细胞的抑制作用及其对TRPV5和TRPV6蛋白表达的影响

doi:10.13481/j.1671-587x.20150620

摘要/Abstract

摘要：

目的:研究氟尿嘧啶联合顺铂对骨肉瘤细胞株MG-63的生长抑制作用,探讨其对瞬时性受体电位通道5(TRPV5)和瞬时性受体电位通道6(TRPV6)蛋白表达的影响。 方法:常规培养MG-63细胞,以5×10⁴mL^-1的细胞密度种植细胞,实验分为对照组、氟尿嘧啶组、顺铂组及氟尿嘧啶联合顺铂组4组。CCK-8法检测不同时间MG-63细胞的生长抑制情况;分别于24、48和72 h采用细胞凋亡-Hoechst染色试剂盒检测MG-63细胞加入不同药物后的凋亡情况;免疫细胞化学染色法检测MG-63细胞在加药前后TRPV5和TRPV6蛋白的表达情况。结果:氟尿嘧啶和顺铂对MG-63细胞的抑制作用均显著,且随着药物浓度的增加和作用时间的延长,细胞中黑色颗粒增多,胞体回缩,细胞老化或死亡。与两药单独或是联用作用于MG-63细胞24 h比较,随着时间的延长,48 h 和72 h两者对细胞的抑制作用明显增强(P<0.05)。与对照组比较,氟尿嘧啶组和顺铂组24、48和72h后凋亡率逐渐增加(P<0.01),随着药物作用时间的延长,细胞凋亡率亦呈升高的趋势。当加入氟尿嘧啶和顺铂72 h后,MG-63细胞表达TRPV5和TRPV6含量较加药前明显降低(P<0.05)。结论:氟尿嘧啶与顺铂联用可以提高MG-63细胞增殖抑制及凋亡作用,同时对MG-63细胞TRPV5和TRPV6表达水平具有明显的抑制作用。

关键词: 氟尿嘧啶, 顺铂, 骨肉瘤, 骨肉瘤细胞株MG-63, 瞬时性受体电位通道

Abstract:

Objective To study the inhibitory effect of fluorouracil combined with DDP on the growth of human osteosarcoma cell line MG-63, and to explore its influence on the expressions of transient receptor potential vanilloid 5 (TRPV5) and transient receptor potential vanilloid 6 (TRPV6) proteins. Methods The MG-63 cells were cultured by the density of 5×10⁴ mL^-1.Fluorouracil group, DDP group, fluorouracil+DDP group and control group containing 10% FBS were set up.The inhibitory rates of growth of MG-63 cells at different time were detected by CCK-8 assay.The apoptosis of MG-63 cells after treated with different drugs was determined by Hoechst staining Kit.The immunocytochemical staining was used to treatent to detect the expressions of TRPV5 and TRPV6 before and after treatment. Results Fluorouracil and DDP both inhibited the growth of MG-63 cells in a time-and dose-dependent manner.There were a lot of black particles in the MG-63 cells and the cells were smaller, aging or death when they were exposed to fluorouracil or DDP.Compared with 24 h group, the inhibitory rates of proliferation of MG-63 cells after treated with the sigle drug of fluorouracil or DDP for 48 and 72 h were increased significantly (P<0.05).Compared with control group,the apoptotic rates of MG-63 cells in fluorouracil group and DDP group 24,48,and 72 h after treatment were increased (P<0.01) in a time-dependent manner.The expression levels of TRPV5 and TRPV6 in MG-63 cells 72 h after treatment of fluorouracil and DDP were decreased significantly compared with before treatment(P<0.05). Conclusion Fluorouracil, DDP and fluorouracil combined with DDP could significantly inhibit the proliferation of MG-63 cells, induce the apoptosis, and decrease the expression levels of TRPV5 and TRPV6.

Key words: fluorouracil, cisplatin, osteosarcoma, osteosarcoma cell line MG-63, transient receptor potential vanilloid

中图分类号:

R738.1

那键, 戴维享, 马超, 张晓东, 邵长青, 刘勇, 王秀力, 刘颖. 氟尿嘧啶联合顺铂对骨肉瘤MG-63细胞的抑制作用及其对TRPV5和TRPV6蛋白表达的影响[J]. 吉林大学学报(医学版), 2015, 41(06): 1201-1206.

NA Jian, DAI Weixiang, MA Chao, ZHANG Xiaodong, SHAO Changqing, LIU Yong, WANG Xiuli, LIU Ying. Inhibitory effect of fluorouracil combined with DDP on human osteosarcoma cell line MG-63 and its influence in expressions of TRPV5 and TRPV6 proteins[J]. Journal of Jilin University Medicine Edition, 2015, 41(06): 1201-1206.

参考文献

[1] Broadhead ML, Clark JC, Dass CR, et al.Therapeutic targeting of osteoclast function and pathways [J].Expert Opin Ther Targets, 2011, 15(2):169-181.

[2] Jiang L, Tao C, He A, et al.Overexpression of miR-126 sensitizes osteosarcoma cells to apoptosis induced by epigallocatechin-3-gallate [J].World J Surg Oncol, 2014, 12:383.

[3] Tian X, Zhang J, Yan L, et al.MiRNA-15a inhibits proliferation, migration and invasion by targeting TNFAIP1 in human osteosarcoma cells[J].Int J Clin Exp Pathol, 2015, 8(6):6442-6449.

[4] Liu SH, Zhao JH, Deng KK, et al.Effect of radiation on cytotoxicity, apoptosis and cell cycle arrest of human osteosarcoma MG-63 induced by a ruthenium(Ⅱ) complex[J].Spectrochim Acta A Mol Biomol Spectrosc, 2015, 140:202-209.

[5] Duarte IF, Lamego I, Marques J, et al.Nuclear magnetic resonance (NMR) study of the effect of cisplatin on the metabolic profile of MG-63 osteosarcoma cells[J].J Proteome Res, 2010, 9(11):5877-5886.

[6] Ye F, Wang H, Zhang L, et al.Baicalein induces human osteosarcoma cell line MG-63 apoptosis via ROS-induced BNIP3 expression[J]. Tumour Biol, 2015[Epub ahead of print].

[7] 王秀力, 李春梅, 吕茜, 等.胰岛素样生长因子1诱导骨髓间充质干细胞分化为心肌样细胞的实验研究[J].中华心血管病杂志, 2013, 41(2): 150-155.

[8] Fang D, Yang H, Lin J, et al.17β-estradiol regulates cell proliferation, colony formation, migration, invasion and promotes apoptosis by upregulating miR-9 and thus degrades MALAT-1 in osteosarcoma cell MG-63 in an estrogen receptor-independent manner[J].Biochem Biophys Res Commun, 2015, 457(4):500-506.

[9] Huang XC, Maimaiti XY, Huang CW, et al.Synergistic effects of arsenic trioxide combined with ascorbic acid in human osteosarcoma MG-63 cells: a systems biology analysis[J].Eur Rev Med Pharmacol Sci, 2014, 18(24):3877-3888.

[10] Ma J, Huang H, Han Z, et al.RLN2 is a positive regulator of AKT-2-induced gene expression required for osteosarcoma cells invasion and chemoresistance[J].Biomed Res Int, 2015, 2015:147468.

[11] Dai JQ, Huang YG, He AN.Dihydromethysticin kavalactone induces apoptosis in osteosarcoma cells through modulation of PI3K/Akt pathway, disruption of mitochondrial membrane potential and inducing cell cycle arrest[J].Int J Clin Exp Pathol, 2015,8(5):4356-4366.

[12] 刘勇, 郑志超, 赵岩, 等.弥漫浸润型胃癌患者术前多西紫杉醇+顺铂+氟尿嘧啶方案化疗的疗效及安全性观察[J].实用医院临床杂志, 2015,12(2): 65-68.

[13] 陈泽程, 邓燕明, 冯卫能, 等.5-氟尿嘧啶、顺铂联合多西紫杉醇治疗晚期复发鼻咽癌临床观察[J].现代医药卫生, 2015,31(6): 903-905.

[14] Vanoevelen J, Janssens A, Huitema LF, et al.Trpv5/6 is vital for epithelial calcium uptake and bone formation[J].The FASEB J, 2011, 25(9):3197-3207.

[15] Fan H, Shen YX, Yuan YF.Expression and prognostic roles of TRPV5 and TRPV6 in non-small cell lung cancer after curative resection[J].Asian Pac J Cancer Prev, 2014,15(6):2559-2563.