【作者】 李素华；

【导师】 郭定宗；

【作者基本信息】 华中农业大学 ， 动物遗传育种， 2008， 博士

【摘要】 奶牛骨代谢性疾病（bone metabolic disease,BMD）是包括软骨症、佝偻病、产后瘫痪、产褥热等一系列由钙磷代谢紊乱引起的营养代谢病。胰岛素样生长因子（insulin-like growth factor,IGF）-I是骨骼生长和矿化中钙磷代谢调节的关键因子,人类临床研究表明其与骨代谢性疾病密切相关。本研究从下述四个方面探讨IGF-I对奶牛骨代谢病中的影响及其机制。1.奶牛血浆IGF-I的测定及其与骨代谢相关因子的关系设计了三个独立的试验以探讨IGF-I与奶牛钙磷代谢相关因子的关系。试验I检测不同泌乳阶段钙、磷、IGF-I、甲状旁腺激素（parathyroid hormone,PTH）、降钙素（calcitionin,CT）等骨代谢相关因子水平,试验Ⅱ检测不同年龄奶牛骨代谢相关因子水平,试验Ⅲ检测奶牛骨代谢病与相关因子的关系。结果显示,奶牛年龄、妊娠状况及血浆IGF-I、PTH、CT水平与机体钙磷水平密切相关,血浆Ca、IGF-I、P、ALP、PTH水平与骨代谢性疾病相关,此外IGF-I与Ca、P正相关（r=0.390,P=0.049;r=0.359,P=0.044）,PTH负相关（r=—0.790,P=0.002）。提示IGF-I与奶牛钙磷代谢密切相关,可以作为诊断奶牛骨代谢病的特异生化标志物。2.奶牛成骨细胞的体外培养、鉴定及生物特性研究采用胰蛋白酶-组织块移性法获取的细胞具有典型的成骨细胞形态和特性:ALP染色呈阳性,活性水平较高;培养7d后可检测到OC mRNA表达,12d后细胞上清液中可检测到OC蛋白;诱导培养2w后细胞可形成钙化结节。提示本实验室建立的方法所培养的细胞具有成骨细胞特性,可获得高纯度、高活性的奶牛成骨细胞。3.IGF-I对奶牛成骨细胞增殖、分化、矿化及重建的影响研究外源性IGF-I对体外培养的奶牛成骨细胞增殖、分化、矿化及重建的影响。结果显示,1-200ng/mL IGF-I均能促进奶牛成骨细胞增殖,100ng/mL组具有最大刺激效应,且IGF-I干预组细胞光密度值（optical density value,OD）从第1d到第5d逐渐增加,第7d降低。与对照组相比,10ng/mL与100ng/mL IGF-I均可显著诱导ALP活性、OC水平以及基质钙含量,钙化结节数量增加20%-180%（P＜0.05或P＜0.01）。半定量RT-PCR结果显示:10、50、100ng/mL IGF-I干预5d OPG/β-actin、RANKL/β-actin比值显著高于0ng/mL组（P＜0.05）;ELISA显示,在1-100ng/mL浓度范围内,IGF-I呈剂量依赖方式上调OPG蛋白表达,其中10、50、100ng/mL干预组与0ng/mL组差异显著（P＜0.05）。提示IGF-I是奶牛成骨细胞增殖、分化以、矿化及重建的代谢促进剂,这可能是IGF-I参与奶牛骨代谢性疾病的重要机制之一。4.IGF-I抑制血清饥饿诱导的奶牛成骨细胞凋亡研究血清饥饿对奶牛成骨细胞凋亡的诱导作用,并探讨IGF-I对血清饥饿诱导的奶牛成骨细胞的影响。结果显示,10^-3 M脱氧胸苷存在时,血清饥饿2d,MTT法检测到细胞存活率为50%,经琼脂糖凝胶电泳可见梯形凋亡条带。流式细胞术分析显示,与血清饥饿组相比,100ng/mL IGF-I显著增高奶牛成骨细胞的增殖指数,降低其凋亡率（P＜0.01）。Caspase-3检测结果显示IGF-I显著抑制csspsse-3的活性（P＜0.05）。结果提示血清饥饿可诱导奶牛成骨细胞凋亡,IGF-I抑制这种凋亡诱导作用。更多还原

【Abstract】 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.更多还原