特异性谷氨酸受体拮抗药非诺班对骨肉瘤LM7细胞增殖与凋亡的影响及其机制

Effects of fenobam,the specific glutamate receptor antagonist,on the proliferation and apoptosis of osteosarcoma LM7 cell and the mechanism

目的探讨特异性谷氨酸受体拮抗药非诺班对骨肉瘤细胞LM7增殖及凋亡的影响。方法用不同剂量的谷氨酸抑制剂受体非诺班作用于骨肉瘤细胞系LM7,采用细胞免疫荧光染色实验及细胞集落形成实验观察不同浓度的药物对骨肉瘤细胞增殖情况的影响,采用细胞TUNNL染色实验观察骨肉瘤细胞系在不同药物浓度下的凋亡情况。Western blot法检测PI3K/AKT信号通路相关蛋白的表达情况。结果对照组细胞活性为(99.8±0.2)%,100μmol/L非诺班治疗组细胞活性为对照组的(98.3±3.4)%,200μmol/L组细胞活性为对照组的(68.6±5.6)%,300μmol/L组细胞活性为对照组的(46.4±3.5)%。随着非诺班浓度的增加,Ki-67荧光染色阳性的细胞数减少,对照组及100、200、300μmol/L组依次为(2750±32)、(2720±45)、(1280±47)、(695±50)个;LM7细胞形成的集落数相应减少;同时TUNEL荧光染色阳性的细胞数逐渐增加,对照组及100、200、300μmol/L组分别为(14±5)、(23±6)、(2653±274)、(3152±179)个。Western blot结果显示非诺班抑制PI3K及AKT蛋白的磷酸化。结论特异性谷氨酸受体拮抗药非诺班能有效抑制骨肉瘤细胞的增殖,促进其凋亡,该过程与抑制PI3K/AKT信号通路有关,为临床治疗骨肉瘤提供可能的线索。

Objective To explore the effect of fenobam,the specific glutamate receptor antagonist,on the proliferation and apoptosis of osteosarcoma LM7 cell,and to investigate the underlying mechanism.Method osteosarcoma LM7 cells were treated with fenobam at different dose,immunofluorescence and colony formation assay was used to determine the effects of fenobam on cell proliferation,besides,cell apoptosis were observed by TUNEL staining.The phosphorylation of AKT and PI3K protein were evaluated by Western blot.Result The cell viability in control group was（99.8 ±0.2）%,based on this,the cell viability ratio of 100 μmol/L fenobam group to control group was（98.3 ± 3.4）%,and was（68.6±5.6）% for 200 μmol/L group to control group,and was（46.4±3.5）% for 300 μmol/L group vs control.As the concentration of fenobam increased,the number of cells with positive staining of Ki-67 was markedly decreased,and were（2750 ± 32）,（2720 ± 45）,（1280 ± 47） and（695 ± 50） in control group,and 100,200,300 μmol/L fenobam treated group;meanwhile,the colony formation of LM7 cells was also reduced accordingly; in the meantime,number of cells with positive TUNEL staining was mounting,with the number of apoptosis cells being（14 ± 5）,（23 ± 6）,（2653 ± 274） and（3152 ±179） in control group and in 100,200 and 300 μmol/L fenobam group,respectively.Western blot indicated that fenobam could significantly inhibit the phosphorylation of AKT and PI3K protein.Conclusion Fenobam,the specific glutamate receptor antagonist can effectively inhibit the proliferation of osteosarcoma cells,promoting the apoptosis,and the process is related to the inhibition of PI3K/AKT signaling pathway,which provides potential clues for the clinical treatment of osteosarcoma.