【作者】 翟必华；

【导师】 刘宗平；

【作者基本信息】 扬州大学 ， 临床兽医学， 2005， 硕士

【摘要】 骨细胞生物学功能(成骨细胞骨形成、破骨细胞骨吸收)对机体骨代谢平衡和骨骼的健康发育至关重要。多数疾病通过增加破骨细胞的数量和(或)增强破骨细胞的活性而引起骨量减少,因此正确理解破骨细胞形成和活化的调控机制具有重要的理论和实践意义。钙、磷是体内必需的矿物元素,也是体内含量最多的矿物元素。摄入充足的Ca、P对保持动物的骨骼完好至关重要。本研究利用成骨细胞和脾细胞共培养以诱导破骨细胞生成的方法,探讨了Ca、P因子对破骨细胞生成和活化的影响,为Ca、P的合理利用提供了理论依据。此外,本研究利用激素诱导脾细胞转化为破骨细胞的方法,证明了破骨细胞形成和活化确实涉及两种机制,为以后治疗骨质疏松症和骨营养不良性疾病提供了理论依据。 1.钙磷因子对体外培养破骨细胞影响的研究 通过成骨细胞与脾细胞共培养的方法在体外诱导脾细胞转化为破骨细胞,在转化过程中加入高、中、低浓度的钙磷因子及不同比例浓度的钙磷双因子,采用形态学观察、抗酒石酸酸性磷酸酶(TRAP)染色计数、扫描电镜观察象牙片吸收陷窝方法检测钙磷因子作用下的破骨细胞的生成和活化。结果表明,中、高浓度的钙磷因子对破骨细胞的生成和活化均有显著抑制作用(P<0.05),高浓度钙因子与对照组比较,差异极显著(P<0.01),Ca:P为2:1时对破骨细胞生成和活化的抑制作用最强。 2.骨保护素对体外培养破骨细胞影响的研究 通过成骨细胞与脾细胞共培养的方法在体外诱导脾细胞转化为破骨细胞,在转化过程中加入不同浓度的OPG,采用形态学观察、抗酒石酸酸性磷酸酶(TRAP)染色计数、扫描电镜观察象牙片吸收陷窝方法检测OPG作用下破骨细胞的生成和活化。结果表明,随着添加OPG浓度的增加,破骨细胞TRAP(+)数逐渐减少,各试验组与对照组相比,差异均极显著(P<0.01)。吸收陷窝的数目和面积也逐渐减少,OPG浓度为50ng/ml、75ng/ml已观测不到吸收陷窝。 3.破骨细胞形成和活化的两种调控机制研究 提取四周龄C57雌性小鼠的脾细胞,将鼠重组粒细胞—巨噬细胞集落刺激因子(GM—CSF)和TNFα(±IL-1α)加入到体外培养的脾细胞中,同时加入OPG和sRANKL以区别RANKL/RANK/OPG机制。破骨细胞的形成和活化采用形态学、TRAP染色、骨吸收陷窝方法进行鉴定。结果表明,TNFα(±IL-1α)确实能诱导脾细胞融合产生TRAP阳性的多更多还原

【Abstract】 Osteocyte’s biological function is very important to metabolic balance and health of bone. Many diseases caused low bone mass through increasing the number of osteoclasts and (or) reinforcing the activation of osteoclasts, therefore, understanding the regulative mechanisms involved in osteoclasts formation and activation has the value both in theory and in practice. Calcium and phosphorus are necessary to body. The amount of them is also the most in body. Intaking enough calcium and phosphorus is vital to maintain bone’s health, In our work, we study effects of calcium and phosphorus on the formation and activation of osteoclasts, which is induced by a co-culture system with rat spleen cells and osteoblasts, as thus, we offer theory basis to rational utilization of calcium and phosphorus. Moreover, we also prove that they are two mechanisms involved in the osteoclasts formation and activation through the way of cell culture in vitro, so we should take two mechanisms into account in treating osteoporosis and bone malnutrition.1. The effects of calcium and phosphorus on osteoclasts formatio and activationOsteoclasts are obtained by a co-culture system with rat spleen and osteoblast. In the conversion process, different strengths of calcium, phosphorus and different concentration ratios of calcium to phosphorus were added to the co-culture system. The formation and activation of osteoclasts is detected by ways of morphologic observation, cytochemical staining for tartrate-resistant acid phosphatase (TRAP) that is a marker of osteoclasts and detection of lacunar resorption through scanning electron micrograph. The results show that high concentration of calcium and phosphorus inhibit the formation and activation of osteoclasts (P<0.05) . Moreover, we also find that experimental group, in which the ratio of calcium to phosphorus is 2:1, has the most powerful inhibitory action to osteoclasts formation and activation.2. The effects of osteoprotegerin(OPG) on osteoclasts formatio and activationOsteoclasts are obtained by a co-culture system with rat spleen and osteoblast. In the conversion process, different strengths of OPG were added to the co-culture system. The formation and activation of osteoclasts is detected by ways of morphologic observation, cytochemical staining for tartrate-resistant acid phosphatase (TRAP) that is a marker of osteoclasts and detection of lacunar resorption through scanning electron micrograph. The results show that the number of TRAP(+) osteoclasts is decrease when concentration of OPG is increase, the number and area of lacunar resorption is as also. Discrepancy between experimental group and control group has statistical significance(P<0.01). In experimental group (50ng/ml、 75ng/ml), we can not detect lacunar resorption.3. Studies on two regulative mechanisms involved in osteoclast formation and activationGM-CSF and TNF α ( ± IL-1 a ) were added to spleen cells obtained from C57 female rat, meanwhile, OPG and sRANKL were also added to these cultures to distinguish the pathway of osteoclastogenesis. The formation and activation of osteoclasts is detected by ways of morphologic observation, cytochemical staining for tartrate-resistant acid phosphatase (TRAP) that is a marker of osteoclasts and detection of lacunar resorption through scanning electron micrograph. The results indicate that in the presence of GM-CSF, TNF α is sufficient for inducing osteoclast differentiation from spleen cells and that TNF α acts synergistically with IL-1 α to stimulate lacunar resorption. This process is distinct from the RANKL/RANK/OPG signalling pathway.更多还原