| Many studies have shown that lead have toxic in body systems and organs,such as neurological, cardiovascular system and bone metabolism. Lead has impacted on calcium metabolism, BGP, the endocrine system. It not only has interfered with bone formation and resulted in functional decline, but aslo caused by a unique type of disease or pathological changes. Osteoblasts are bone-forming cells and responsible for the synthesis, secretion, and mineralization of bone matrix. It plays a very important role in bone tissue growth and development, the balance of bone metabolism, bone repair and maintain. Isolating and culturing osteoblasts in vitro provides conditions for toxicology. High purity and vitality osteoblasts are the premise for study. To study the effects of lead toxicity on osteoblasts, osteoblasts were isolated from novo-rat cranial bones and cultured in vitro. These results provided rationale for the normal physiological function of bone.1.Isolation and identification of rat osteoblastsTo establish the skill of isolation osteoblasts in order to study the effect of lead injection on the level of cell of bone metabolism diseases. Mouse primary osteoblasts were obtained from calvaria of SD novo-rat that digested by trypsin and collagenase and identified by staining of alkaline phosphatase (ALP) and mineralized matrix (alizarin red). It was concluded that the modified twice-enzyme isolation method was an ideal technique to obtain and culture osteoblasts with typical characteristics and high purity for the experiment.2.Effects of lead on the osteoblasts cultured in vitroThe mOBs were divided into four groups: [Pb 0], [Pb 20], [Pb 40], and [Pb 80] to receive 0μM, 20μM, 40μM and 80μM of Pb2+ as Pb acetate solution respectively. Then shape and growth of OB were observed under inverted microscope. Cell proliferation was observed by microtitration (MTT), ALP activity was detected through PNPP method. The results indicated that, among three treated groups, Pb exposure induced gap of the cells was observed to scale-up in the group [Pb 20]. Moreover, most of cells collapsed and died in group [Pb 80] when compared to control. In addition, Pb exposure caused marked the inhibited proliferation of osteobals in the group [Pb 80] (P<0.01). Among three treated groups there was descrease in ALP activity (P<0.05). It was concluded that lead could resulte in the damage patterns and reduce the ability of bone formation.3.Effects of lead on the osteoblasts CaMIn order to understand the effects of lead on the osteoblasts CaM, the mOBs were divided into four groups: [Pb 0], [Pb 20], [Pb 40] and [Pb 80] to receive 0μM, 20μM, 40μM and 80μM of Pb2+ as Pb acetate solution respectively. After 24h, the results show that Pb exposure caused marked increase in CaM protein level and CaM mRNA level in group [Pb 80] and in group [Pb 40]. It is concluded that there were increase in CaM protein levels and CaM mRNA level.4.Effects of lead on the osteoblasts Phosphatidylcholine Phospholipase C(PC-PLC)To study effects of lead on the osteoblasts PC-PLC through different concentrations of lead (0, 20, 40, 80μmol/L) were added into culture solution. After 24h, results showed the decline in PC-PLC protein level. PC-PLC activities also significantly decreased (P<0.01). It is concluded that Pb lead to a decrease in PC-PLC activity and protein level in a dose-dependent manner.In conclusion, we isolated and identificated of rat osteoblasts, and found the effects of lead on cell proliferation and ALP activities of the osteoblasts cultured in vitro. Our studies first indicated that lead may play an important role in the mOBs through the osteoblasts CaM and PC-PLC.
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