The Mechanism Of Osteoblast Apoptosisinduced By AlCl₃ Through Fas And Mitochondria Pathway

Osteoblasts(OBs) were isolated from the calvarium of three-days-old rats and cultured in vitro,and the third generation OB was used in this experiment. Base on the IC50 of Al Cl3 to OB, the OBs were divided into four groups, control-group(CG), low-group(LG), middle-group(MG) and high-group(HG). Each group of OB was exposed to Al Cl3 in final concentration of 0, 0.06, 0.12, 0.24 mg?mL-1, respectively, for 24 h. The apoptosis morphology of OB was detected by acridine orange/ethidium bromide(AO/EB) staining; The mRNA expression of Fas, Fas L, FADD, Bid, Caspase-8, Caspase-9 and Caspase-3 in OB was detected by real-time fluorescence quantitative PCR(q RT-PCR); The protein expression of Fas, Fas L, FADD, Bid, t Bid and Cyt C in OB was detected by Western blost; The activities of Caspase-8, Caspase-9 and Caspase-3 in OB were detected by Caspase activity kit. The results were showed as follows:(1) The early apoptotic OB with bright green punctate nucleus,the late apoptotic OB with bright orange nucleus and the necrotic OB with red nucleus were obviously observed in Al-treated groups. The number of apoptotic OB, necrotic OB and the apoptosis index of OB in Al-treated groups were significantly higher than those in CG. It indicated that Al Cl3 induced OB apoptosis.(2) The mRNA and protein expression of Fas, Fas L, FADD, Caspse8, and the activity of Caspase-8 in Al-treated groups were highter than those in CG(P<0.05, P<0.01).. It indicated that Fas/Fas L signaling pathway was involved in the apoptosis of OB induced by Al Cl3.(3) The Cyt C in cytoplasm increased significantly in Al-treated groups(P<0.01).The mRNA expression and the activity of Caspse9 in Al-treated groups were significantly highter than those in CG(P<0.01). It indicated that the mitochondria signal pathway was involed in apoptosis of OB induced by Al Cl3.(4) The mRNA expression of Bid, the protein expression of Bid and t Bid, the m RNA expression and the activity of Caspse9 were significantly highter than those in in Al-treated groups(P<0.01). It indicated that the mitochondrial apoptotic pathway was regulated by the Fas death receptor pathway in apoptosis of OB induced by Al Cl3.