关键词: 骨肉瘤, Ⅱ型谷氨酰胺转氨酶, bcl-2相关x蛋白质, 细胞色素C, 细胞凋亡

Abstract:

Abstract:Objective To investigate the anti-apoptosis effect of transgiutaminase 2(TG2) in osteosarcoma cell line MG-63 and to explore the mechanism of inhibiting apoptosis of tumor cells. Methods The TG2-tgrgeted siRNA was designed,and the hypoxia culture model of MG-63 cells  was established by a hypoxia incubator and the cells were divided into four groups: normal oxygen group,the  cells were cultured under normal oxygen;hypoxia group,the  cells were cultured in hypoxic incubators;control siRNA hypoxia group, the cells were cultured in hypoxic incubators after transfection of control siRNA; TG2 siRNA hypoxia group ,the cells were cultured in hypoxic incubators after transfection of TG2 siRNA.The expressions of Bax and cytochrome C(Cyt C) and the apoptotic rates were observed at different  time  (6,12,24,48,and 72 h) after hypoxia culture.Microtiter plate assay was performed to monitor the intracellular TG2 activity.RT-PCR was used to detect the mRNA expressions of TG2 and Bax.The expressions levels of TG2,Bax and Cyt C were observed by immunohistochemical staining and Western blotting method.The  apoptotic rates were analyzed using flow cytometry.Results Compared with normal oxygen group,the activity of TG2,the mRNA and protein expression levels of TG2 in hypoxia group and control siRNA hypoxia group were increased remarkably with the prolongation of  the hypoxia time (P<0.01);the expression level of Bax  protein  was decreased significantly(P<0.01),but the  expression level of Bax mRNA  had no significant change(P>0.05);the expression  levels of Cyt C protein   in cytoplasm and mitochondria and the apoptotic rates had no  markedly changes(P>0.05).Compared with hypoxia group and control siRNA hypoxia group,the expression levels of mRNA and protein of TG2 in TG2 siRNA hypoxia group were decreased significantly at different time points(P<0.01);the protein expression levels of Bax and Cyt C in cytoplasm and the apoptotic rates were increased markedly(P<0.01);the expression level of Cyt C in mitochondria was decreased(P<0.01).Conclusion TG2 can inhibit the apoptosis of tumor cells through down-regulating the  Bax expression and preventing Cyt C releasing into the cytoplasm.

Key words: osteosarcoma, transgiutaminase 2, bcl-2-associated x protein, cytochrome C, apoptosis