| Objective: To investigate the expression of hypoxia inducible factor-lα(HIF-lα) in primary fetal mouse osteoblasts cultured under hypoxia for different time periods,and to assess its relationship with cell apoptosis.Methods: 1. Apply modified tissue mess method to obtain osteoblasts:the calvaria harvested from 19 day fetal mouse was stripped off all soft tissues including the periosteum, rinsed and cut to trivial bone block under sterile condition. Then, the bone block was subcultured in culture flask after digested with 0.25% trypsin for 10min. The acquired cells were purified by"repeated adhensive method".Subsequently the third generation was counted for eight days continuously and the forth generation was identified with alkaline phosphorase stain of calcified nodules.2.The third generation osteoblasts were divided into two groups to be cultured under normoxia and hypoxia containing cobalt chloride (125umol/L) for different time periods (0,1,3,5,7days) to observe cell morphous and count cells.3.Reverse transcription polymerase chain reaction was employed to measure the message RNA expression of HIF-1α,Caspase-3 and Bcl-2 at the above time points for the two groups.4.Western-blotting analysis was used to determine the expression of Caspase-3 and Bcl-2 protein for the two groups.5.Statistical treatment: The above experiment was repeated for three times ,and the experimental data was analyzed by One-Way ANOVA.Result: 1. Through the observation of microscope, the cells obtained from fetal mouse calvaria were fusiform,triangle or polygon with several processes which can connect with another cell process beyond. The nucelus with one to three nucleoli appear round or oval-shape. After subculturing , the cells proliferated slowly at the adaptive stage,then they accelerated to the climax at the sixth day , afterward cell population began to decrease .The fourth generation cells were identified to be osteoblasts by alkaline phosphorase stain of calcified nodule, and positive cells which appeared fusiform and squamelliform were 95% of 200 counted cells.2. The obscure membrane was shrinkage and cytoplasm was dense morpholocally within cells which were disposed by cobalt chloride for three days, and after seven days apoptotic body was identified. The disposed cell counting was to the climax at the fifth day, but obviously less than that in normxia surrounding at the same time point.3. The message RNA expression of HIF-1αand caspase-3 of osteoblasts increase gradually in hypoxia surrounding ,obviously higher than that in normxia surrounding at the same time point(P<0.01), and there was no change in normxia surrounding (P>0.05). Conversely, the message RNA expression of Bcl-2 decrease gradually in hypoxia surrounding , obviously less than that in normxia surrounding at the same time point (P<0.01), and there was no change in normxia surrounding (P>0.05).4. The protein expression of Caspase-3 increase gradually in hypoxia surrounding ,obviously higher than that in normxia surrounding at the same time point(P<0.01),and the protein expression of Bcl-2 was contrary to that of Caspase-3, decreasing gradually in hypoxia surrounding ,obviously less than that in normxia surrounding at the same time point(P<0.01), in addition both protein expressions of Caspase-3 and Bcl-2 were no changes in normxia surrounding (P>0.05).Conlusion: 1. The modified tissue mess method is an ideal one to obtain and culture osteoblasts having typical characteristics.2. Cobalt chloride as a chemical inducer of HIF-1 can induce apoptotic death of osteoblasts.3. HIF-lαmay play a positive role during the apoptotic death of osteoblasts through upregulating the expression of Caspase-3 and downregulating the expression of Bcl-2 at both transcriptional and translational levels,furthermore the mentioned effects are related to the duration of hypoxia. |
