Effect And Mechanism Of Insulin-like Growth Factor-Ⅰ On The Bone Metabolic Disease From Chinese Holstein

Metabolic bone disease, which includes a number of disorders related to the weakening of the bone or impaired regulatory function, is caused by an imbalance in calcium and phosphorus, with a ratio that normally strongly favours an excess of phosphorus. Insulin-like growth factor-I (IGF-1) exert important role in the growth and mineralisation in bone. And it showed correction with the bone metabolic disease from the human clinical date. We hypothesed that IGF-I will exert important effect on metabolic bone disease of dairy cows. Four relative experiments were desiged here toevaluate this effect.1. The relationship of IGF-I and bone metabolism related factors on Chinese HolsteinThe aim of the study was to evaluate the relationship of insulin-like growth factor-I and bone metabolism related factors on Chinese Holstein. Three independent experiments were designed. Experiment I was to detect the level of calcium (Ca), phosphorus (P), parathyriod hormone (PTH), calcitionin (CT) and alkaline phosphatase (ALP) in cows with different age. Experiment II was to detect the level of these indexs in the ones in different lactation stage. And experiment III detected these indexs in health cows and the ones with bone metabolic disease. The results showed that age, the lactation stage, the level of IGF-I, PTH and CT were related to Ca and P metabolism in dairy cows. Serum level of Ca,IGF-1,P,ALP,PTH all showed correction with the bone metabolic disease of cows. And Ca and P showed positive correction with IGF-I, while PTH showed the opposite correction. It could be conclude that IGF-I was corrected to Ca and P metabolim in dairy cattle, and could be used as a marker of bone metabolic disease in cows2. Culture and identification of dairy cattle osteoblasts and its biological characteristicOsteoblastic cells were isolated by trypsin digestion method here. Cells liberated from the bone fragments and adhered on the bottom of the culture flask after 7 days of culture. At the 14^th days'culture, the number of osteoblasts reached the highest level, and the cells had grown to confluence. Cells in culture exhibited ALP activity and were positive to OC in supernate at the 12^th day. The expression of osteocalcin mRNA can be detected with RT-PCR at the 7th day. The results showed that the cells isolated and cultured in this experiment possessed the properties of osteoblast. Pure and active dairy cattle osteoblasts can be obtained by the technique established in this study.3. The stimulatory effect of IGF-I on the proliferation, differentiation, mineralisation, and remodeling of osteoblastic cells from Holstein cattleThis study investigated the effect of exogenous IGF-I on the proliferation and differentiation of osteoblastic cells from Chinese Holstein cattle and the resultant bone-nodule formation and mineralisation in vitro. The MTT assay showed that cell proliferation in the cultures was stimulated by IGF-I at concentrations ranging from 1-200ng/mL, with the maximum effect observed at 100ng/mL. This effect was observed from day 1 and peaked at day 5, but decreased at day 7. At concentrations of 10ng/mL and 100ng/mL, IGF-I significantly induced ALP activity, OC level, matrix calcium content, and nodule formation of the osteoblastic cells by 20% to 180% (P < 0.05 or P < 0.01), compared to the controls. The results by semiquantitive RT-PCR demonstrated that IGF-I with 1-100ng/mL concentrations all showed stimulated RANKL and OPG mRNA expression. Compared with the 0ng/mL group, exposure to 10-100ng/mL groups presented significantly increased ration of OPG/β-actin,RANKL /β-actin, as well as the ration of OPG / RANKL (P < 0. 05). IGF-I ranging from 1-100ng/mL up-regulated OPG protein level in a dose-dependent manner by ELISA, and the 10-100ng/mL groups showed markly enhanced effect compared with 0ng/mL group (P < 0. 05). The results suggested that IGF-I is an anabolic agent for proliferation, differentiation, mineralisation and bone remodeling of dairy cow osteoblasts, and could therefore act as a potential treatment for the metabolic bone diseases in these animals.4. Inhibitory effect of IGF-I on the cow osteoblasts apoptosis induced by serum starvationThe objective of this experiment investigate the effect of serum starvation on the apotosis of osteoblastic cells from Chinese Holstein cattle, and study the effect of IGF-I on this induction. Results showed that the treatment of serum starvation for 2d in the presence of 10^-3 M thymidine induced the apoptosis of cow osteoblasts. In contast to serum starvation group, 100ng/mL IGF-I significantly increased proliferation index (P<0.01), and decreased apoptosis rate (P<0.01) by the Flow cytometry. And the activity of caspase-3 was inhibited by 100ng/mL IGF-I. It suggested that osteoblastic cells from Chinese Holstein cattle could be apoptosed induced by serum starvation for 2d, and IGF-I inhibited this apotosis process.