【作者】 顾建红；

【导师】 刘宗平；

【作者基本信息】 扬州大学 ， 临床兽医学， 2009， 博士

【摘要】 骨代谢是维持骨组织不断更新,保持生命活力的基本过程,这一过程是依靠骨再建（bone remodeling）完成的。大量研究表明,动物骨营养不良的发生主要是破骨细胞（osteoclasts,OC）引起的骨吸收大于成骨细胞（osteoblasts,OB）引起的骨再建。许多研究认为,骨代谢调控因子主要通过调节OB表达核因子κB受体活化因子配体（receptor activator of NF-κB ligand,RANKL）及骨保护素（osteoprotegerin,OPG）,从而间接调控OC的形成及骨吸收功能。维生素D及其活性代谢物是动物钙、磷代谢的重要调节因素之一。然而,钙、磷及维生素D制剂的疗效却参差不齐。因此进一步理解维生素D在骨骼生理和病理学中的确切机制有助于更好地防止代谢性骨病。本文研究了不同浓度1α,25-（OH）₂D₃对体外培养OB增殖、分化及RANKL、OPG蛋白和mRNA表达的影响,并观察了RANKL对体外培养OC形成及骨吸收活性的影响,旨在阐明维生素D调节骨代谢的部分机制。1. 1α,25-（OH）₂D₃对体外培养成骨细胞增殖、分化及周期的影响2.5 g/L胰酶和1 g/LⅡ型胶原酶两步消化法分离3-4日龄SD大鼠乳鼠颅盖骨细胞。采用倒置显微镜、扫描电镜观察细胞形态,碱性磷酸酶（ALP）特异染色鉴定OB。在此基础上,向培养体系中添加不同浓度的1α,25-（OH）₂D₃(0 [无水乙醇溶剂对照]、10^-9、10^-8、10^-7 mol/L)。作用24、48、72 h,MTT法测定OB增殖率、PNPP法测定ALP活性,流式细胞仪测定OB周期。结果显示,10^-9 mol/L 1α,25-（OH）₂D₃作用24、48、72 h均促进OB增殖（P<0.05或P<0.01）,抑制ALP活性（P<0.01）;10^-8、10^-7 mol/L作用24、48 h,OB增殖率与对照组差异不显著（P>0.05）,但24 h时ALP活性均明显升高（P<0.05或P<0.01）,48 h则抑制了ALP活性并使OB滞留在G2/M期（P<0.05或P<0.01）;72 h时10^-7 mol/L组OB增殖率极显著低于其余各组（P<0.01）,并又使ALP活性升高（P<0.01）。说明,低浓度1α,25-（OH）₂D₃(10^-9 mol/L)促进OB增殖,抑制其分化;中高浓度1α,25-（OH）₂D₃(10^-8、10^-7 mol/L)抑制OB增殖,促进其分化,并使细胞滞留在G2/M期。2. 1α,25-（OH）₂D₃对体外培养成骨细胞骨架、GJIC及[Ca²⁺]_i的影响在OB培养的基础上,不同浓度的1α,25-（OH）₂D₃(0 [无水乙醇溶剂对照]、10^-9、10^-8、10^-7 mol/L)作用20 min、24 h,流式细胞仪测定[Ca²⁺]_i;24、48 h,荧光显微镜观察F-actin及细胞间隙连接通讯（GJIC）。结果显示,20 min时,不同浓度1α,25-（OH）₂D₃组[Ca²⁺]_i均显著高于对照组（P<0.05）;24 h时10^-9 mol/L组[Ca²⁺]_i则显著低于对照组（P<0.05）,其余各组间差异不显著（P>0.05）,此时10^-8、10^-7 mol/L组大部分细胞变得扁平,F-actin排列较对照组有序,形成应力纤维;48 h时,对照组及10^-9 mol/L组F-actin表达减少,10^-9 mol/L组GJIC极显著弱于对照组（P<0.01）,而10^-8、10^-7 mol/L组大部分细胞F-actin表达完好,GJIC均极显著强于其余两组（P<0.01）。说明,1α,25-（OH）₂D₃能够影响钙离子通道,同时低浓度1α,25-（OH）₂D₃(10^-9 mol/L)抑制F-actin表达及细胞间隙连接通讯,中高浓度1α,25-（OH）₂D₃(10^-8、10^-7 mol/L)则能维持OB形态,增强细胞间隙连接通讯。3. 1α,25-（OH）₂D₃对体外培养成骨细胞超微形态结构的影响在OB体外培养的基础上,不同浓度1α,25-（OH）₂D₃(0 [无水乙醇溶剂对照]、10^-9、10^-8、10^-7 mol/L)处理48 h,扫描电镜、透射电镜观察OB超微形态结构。结果,与对照组比较,10^-9 mol/L 1α,25-（OH）₂D₃组细胞铺展较好,表面针状突起、胞内线粒体增多;10^-8 mol/L 1α,25-（OH）₂D₃组细胞趋于扁平,表面突起减少,变得细长,内质网增多;10^-7 mol/L 1α,25-（OH）₂D₃组细胞外基质中大量丝状纤维连接成网状,细胞内线粒体较少,出现大量空泡及钙颗粒沉积。说明,低浓度1α,25-（OH）₂D₃(10^-9 mol/L)能促进OB增殖,而中高浓度1α,25-（OH）₂D₃(10^-8、10^-7 mol/L)抑制OB增殖,促进细胞外胶原形成及基质矿化。4. 1α,25-（OH）₂D₃对体外培养成骨细胞RANKL及OPG表达的影响在OB体外培养的基础上,不同浓度1α,25-（OH）₂D₃(0 [无水乙醇溶剂对照]、10^-9、10^-8、10^-7 mol/L)作用24、48、72 h,分别采用ELISA及FQ-PCR法测定RANKL、OPG蛋白及mRNA含量。结果,10^-8、10^-7 mol/L 1α,25-（OH）₂D₃较对照组、10^-9 mol/L组显著或极显著促进RANKL蛋白及mRNA的表达（P<0.05或P<0.01）;10^-9、10^-8 mol/L 1α,25-（OH）₂D₃在不同时间,较对照组显著或极显著促进OPG蛋白及mRNA的表达（P<0.05或P<0.01）,而10^-7 mol/L则极显著抑制OPG mRNA表达（P<0.01）;最终,10^-9 mol/L组48 h时RANKL/OPG比值增高（P<0.05）,10^-8、10^-7 mol/L 1α,25-（OH）₂D₃组RANKL mRNA/OPG mRNA及RANKL/OPG比值则始终高于对照组和10^-9 mol/L组（P<0.01）。说明,1α,25-（OH）₂D₃可剂量依赖性地上调RANKL mRNA/OPG mRNA及RANKL/OPG比值,促进OC的生成及骨吸收功能,增强骨更新。5. RANKL对体外培养破骨细胞形成和活化的影响分离5-6周龄ICR小鼠长骨骨髓细胞,分两阶段培养。第一阶段分三组（A、对照组[不添加任何因子];B、50 ng/mL RANKL;C、25 ng/mL M-CSF）培养3 d,进入第二阶段（Ⅰ、对照组[不添加任何因子];Ⅱ、25 ng/mL M-CSF;Ⅲ、25 ng/mL M-CSF + 50 ng/mL sRANKL）继续培养。倒置显微镜观察细胞形态,酸性磷酸酶（ACP）染色、抗酒石酸酸性磷酸酶（TRAP）染色及扫描电镜观察骨吸收陷窝,鉴定OC的生成及骨吸收活性,同时荧光显微镜观察F-actin。结果,第一阶段培养3 d,对照组和50 ng/mL RANKL组细胞均无贴壁及增殖能力,而25 ng/mL M-CSF明显促进细胞贴壁与增殖。第二阶段培养2 d,25 ng/mL M-CSF + 50 ng/mL RANKL组较25 ng/mL M-CSF组出现更多单核巨细胞,随着时间的延长单核巨细胞增多,出现2个核以上的巨细胞,且M-CSF存在的各组细胞均可表达ACP活性;培养9 d,25 ng/mL M-CSF + 50 ng/mL sRANKL组出现3个核的TRAP阳性OC（10.17±1.55个/孔）,OC数极显著高于对照组（0个/孔）和25 ng/mL M-CSF组（0.67±0.69个/孔）（P<0.01）。同时,sRANKL可诱导F-actin的表达,促进骨吸收陷窝的生成。说明,RANKL在M-CSF存在时可诱导OC的生成及骨吸收活性,但OC数量仍不多。更多还原

【Abstract】 Bone metabolism is the basic process of life to maintain bone tissue updating continuously and to keep vitality of life, and this process depends on the bone remodeling. In various skeletal disease associated with bone loss, such as bone malnutrition, increased osteoclastic bone resorption exceeds formation resulting in low bone mass, skeletal fragility and increased risk of fracture. Recent researchs have showed that many regulating factors for bone metabolism can regulate osteocalsts’formation and activation indirectly by effecting osteoprotegerin （OPG） and receptor activator of NF-κB ligand （RANKL） expressed in osteoblasts. Vitamin D and its active metabolite is one of the important regulators for calcium and phosphorus metabolism. However, the therapeutic effect of calcium, phosphorus and vitamin D praeparatum are variable. Therefore, further understanding of the exact mechanisms of vitamin D in bone physiology and pathology is important to prevent metabolic bone disease. To elucidate the mechanism of bone metabolism accommodated by vitamin D, we investigated the effects on osteoblasts’proliferation, differentiation and the expression of RANKL, OPG in osteoblasts treated by different concentrations of 1α,25-（OH）₂D₃, meanwhile the influences of RANKL on osteoclasts’formation and activation detected by histochemistry staining for Tartrate-resistant acid phosphatase （TRAP） and so on.1. The effects of 1α,25-（OH）₂D₃ on proliferation, differentiation and cell cycle of osteoblasts in vitroTo study the influence of 1α,25-（OH）₂D₃ on proliferation, differentiation and Cell Cycle of Osteoblasts （OB） in vitro. OB were isolated from calvaria bone, then dealt with various concentration of 1α,25-（OH）₂D₃ (0, 10^-9, 10^-8, 10^-7 mol/L). After 24, 48, 72 h cultivation, the proliferation and the activity of alkali phosphatase （ALP） of OB was observed. 48 h incubation later, the changes of cell phase were analyzed using flow cytometer. Compared with the control group, 10^-9 mol/L 1α,25-（OH）₂D₃ promoted proliferation of OB in vitro significantly, and inhibited the ALP activity very significantly. The group with 10^-8, 10^-7 mol/L 1α,25-（OH）₂D₃ had lower proliferation rate of OB than group with 10^-9 mol/L 1α,25-（OH）₂D₃ significantly or very significantly, but stimulated the ALP activity significantly or very significantly within 48 h. At 72 h, 10^-7 mol/L 1α,25-（OH）₂D₃ had the lowest proliferation rate of OB, and the highest ALP activity. 10^-8, 10^-7 mol/L 1α,25-（OH）₂D₃ caused G2/M arrest significantly or very significantly. These results showed that low dosage of 1α,25-（OH）₂D₃ can promote proliferation and inhibit differentiation, while higher dosage of 1α,25-（OH）₂D₃ can inhibit proliferation, promote differentiation and cause G2/M arrest.2. The effects of 1α,25-（OH）₂D₃ on cytoskeleton, GJIC and [Ca²⁺]_i of osteoblasts in vitroTo study the influence of 1α,25-（OH）₂D₃ on cytoskeleton, gap junction intercellular communication （GJIC） and intracellular Ca2+ ([Ca²⁺]_i) in Osteoblasts in vitro. OB were isolated from calvaria bone. After 20 min and 24 h treated by 1α,25-（OH）₂D₃ (0, 10^-9, 10^-8, 10^-7 mol/L), [Ca²⁺]_i was evaluated. 24 and 48 h incubation later, F-actin and GJIC were observed. Compared with the control group, [Ca²⁺]_i in group with 1α,25-（OH）₂D₃ all increased significantly （P<0.05） at time 20 min. 24 h incubation later, [Ca²⁺]_i in the group with 10^-9 mol/L 1α,25-（OH）₂D₃ was the lowest （P<0.05）. OB in the group with 10^-8 and 10^-7 mol/L 1α,25-（OH）₂D₃ were applanation, stress fibers formed. 48 h later, the expression of F-actin in the control group and group with 10^-9 mol/L 1α,25-（OH）₂D₃ reduced. Compared with the control group, GJIC was weakened after treated with 10^-9 mol/L 1α,25-（OH）₂D₃ very significantly （P<0.01）, while GJIC enhanced in the group with 10^-8 and 10^-7 mol/L 1α,25-（OH）₂D₃ very significantly （P<0.01）. These results demonstrated that [Ca²⁺]_i can be mediated by 1α,25-（OH）₂D₃, higher dosage of 1α,25-（OH）₂D₃ can maintain the morphous of OB and stimulate the communication among OB, while lower dosage of 1α,25-（OH）₂D₃ can inhibit the expression of F-actin and reduce the communication among OB. 3. The effects of 1α,25-（OH）₂D₃ on the morphous and ultrastructure of osteoblasts in vitroTo investigate the effects on the development and ultrastructure of osteoblasts （OB） under different dosages of 1α,25-（OH）₂D₃ in vitro. The morphous and ultrastructure were observed using scanning electron microscope （SEM） and transmission electron microscope （TEM） independently after cultured for 48 h. Compared with the control group, more microvillus and mitochondria were observed in the group with 10^-9 mol/L 1α,25-（OH）₂D₃. In the group with 10^-8 mol/L 1α,25-（OH）₂D₃, osteoblasts became flatter, and contained abundant slender cytoplasmic processes and endoplasmic reticula. Lots of filiform fibers forming network in the Extracellular Matrix of OB, more vacuole and calcium granule, less organelles were observed in the group with 10^-7 mol/L 1α,25-（OH）₂D₃. In conclusion, the present study verified further morphologically that higher concentration of 1α,25-（OH）₂D₃ had obviously facilitative effects on differentiation and functional expression of osteoblasts cultured in vitro, while lower dosage of 1α,25-（OH）₂D₃ stimulate proliferation.4. The expression of RANKL and OPG in osteoblasts treated by 1α,25-（OH）₂D₃ in vitroTo investigate the expression of RANKL, OPG and RANKL mRNA, OPG mRNA, osteobalsts obtained from Sprague Dawley rats were treated with different concentrations of 1α,25-（OH）₂D₃. The expression of RANKL and OPG was detected by the method of Immunohistochemistry and ELISA. RANKL mRNA and OPG mRNA were determined through FQ-PCR. Compared with the control group and the group with 10^-9 mol/L 1α,25-（OH）₂D₃, 10^-8 and 10^-7 mol/L 1α,25-（OH）₂D₃ can significantly or very significantly induce the expression of RANKL and RANKL mRNA. 10^-9, 10^-8 mol/L 1α,25-（OH）₂D₃ can stimulate the expression of OPG and OPG mRNA significantly or very significantly, while 10^-7 mol/L 1α,25-（OH）₂D₃ can inhibit the expression of OPG mRNA significantly. The ratio of RANKL/OPG in group with 10^-9 mol/L 1α,25-（OH）₂D₃ was higher than control group at the 48th hour. However the expression of RANKL/OPG and RANKL mRNA/OPG mRNA in the group with 10^-8, 10^-7 mol/L were higher than the control group and the group with 10^-9 mol/L 1α,25-（OH）₂D₃ all the time. These results showed that 1α,25-（OH）₂D₃ can enhance bone turnover through facilitating the formation and activity of osteoclasts via enhance RANKL mRNA/OPG mRNA and RANKL/OPG dose dependently.5. The effects of RANKL on osteoclasts’formation and activation in vitroTo investigate the effects of RANKL on osteoclasts’formation and activation in vitro, bone marrow cells were isolated from 5 to 6 weeks old ICR mice. The first step of culture with different cytokines （A: the control group without any cytokines; B: 50 ng/mL RANKL; C: 25 ng/mL M-CSF） was followed by the second step （Ⅰ: the control group without any cytokines;Ⅱ: 25 ng/mL M-CSF;Ⅲ: 25 ng/mL M-CSF + 50 ng/mL RANKL）. The morphology was observed by phase-contrast microscope. Osteoclasts’shap and activation were identified by acid phosphatase （ACP） staing, tartrate resistant acid phosphatase （TRAP） staining and observating of F-actin, detection of resorption lacunae through scanning electron microscopy. The cells at the control group and the group with 50 ng/mL RANKL had no ability of adherence and proliferation, while 25 ng/mL M-CSF could promote cells’adherence and proliferation at the 3rd day of the first step. After 2 days incubation at the second step, there were more mononuclear giant cells treated by 25 ng/mL M-CSF + 50 ng/mL RANKL than that treated by M-CSF alone. However, all the cells formed under 25 ng/mL M-CSF had ACP activity. 9 days incubation later, the number of osteoclasts with three nucleus in the group with 25 ng/mL M-CSF + 50 ng/mL RANKL （10.17±1.55/well） was more than those in the control group （0/well） and the group with 25 ng/mL M-CSF （0.67±0.69/well） very significantly. RANKL could induce the expression of F-actin, and facilitate the formation of bone resorption lacunar. These results demonstrated that RANKL could induce osteoclats’formation and activation at presence of M-CSF, but the number of osteoclasts was still parum.更多还原