Vitamin D is a lipid soluble vitamin that is mostly used to treat bone metabolism-related diseases. In this study, the effect of Cd toxicity in vitro on osteogenic differentiation derived from BMSCs and the alleviating effect of lα, 25-(OH)2D3 were investigated. Cell index in real time was monitored using a Real-time cell analyzer (RTCA) system. The activity of alkaline phosphatase (ALP), and the calcified nodules and the distribution of Runx2 protein were detected using ALP staining, alizarin red staining, and immunofluorescence, respectively. Furthermore, the mitochondrial membrane potential and the apoptotic rate of BMSCs, the mRNA levels of RUNX2 and type Ⅰ collagen alpha2 (COL1A2) genes, and the protein expression of Col1 and Runx2 were detected using flow cytometry, qRT-PCR and western blot, respectively. The proliferation of BMSCs and osteogenic differentiation were enhanced after treatment with different concentrations of lα, 25-(OH)2D3 compared with the control group. However, 5 μmol/L Cd inhibited the proliferation of BMSCs. In addition, 10 nmol/L lα,25-(OH)2D3 attenuated the toxicity and the apoptosis of BMSCs treated by Cd, and also promoted the osteogenic differentiation including the activity of ALP, and the protein expression of Col1 and Runx2. lα, 25-(OH)2D3 can alleviate cadmium-induced osteogenic toxicity in White Leghorn chickens in vitro.

骨是镉毒性作用的主要靶器官之一,但其对鸡骨髓基质细胞(bone marrow stromal cells,BMSCs)增殖和成骨分化的毒性作用仍不清楚.本研究利用差速贴壁纯化法获得鸡BMSCs,加入不同浓度镉处理不同时间,采用CCK-8法检测细胞增殖,碱性磷酸酶(alkaline phosphatase,ALP)和茜素红染色鉴定成骨分化,RT-PCR检测成骨相关基因(COL1、OSX、RUNX2、ALP、OCN、OPG、OPN、RANKL)表达变化.结果显示,1～10 μmol/L的镉显著促进BMSCs增殖,20 μmol/L的镉显著抑制其增殖(P＜0.05);5 μmol/L以上的镉可显著抑制ALP活性,并以浓度依赖方式极显著抑制成骨相关基因(COL1、OSX、RUNX2、ALP、OCN、OPG、OPN)mRNA表达,上调RANKL mRNA表达(P＜0.01).表明,一定浓度镉可抑制鸡BMSCs体外增殖及其向成骨细胞(OB)的分化.

Bones play an important role in maintaining the level of calcium in blood. They provide support for soft tissues and hematopoiesis and undergo continuous renewal throughout life. In addition, vitamin D is involved in regulating bone and calcium homeostasis. Galectin-3 (Gal-3) is a β-galactoside-binding protein that can regulate bone cell differentiation and function. Here, we aimed to study the regulatory effects of Gal-3 on vitamin-D-regulated osteoclastogenesis and bone resorption in chicken. Gal-3 expression in bone marrow stromal cells (BMSCs) from 18-day-old chicken embryos was inhibited or overexpressed. BMSCs were then co-cultured with bone marrow monocytes/macrophages (BMMs) with or without addition of 1α,25(OH)2D3. The results showed that 1α,25(OH)2D3 upregulated the expression of Gal-3 mRNA and receptor activator of nuclear-factor κB ligand (RANKL) expression in BMSCs and promoted osteoclastogenesis, as shown by the upregulated expression of osteoclast (OC) markers (CtsK, CAII, MMP-9, and TRAP) and increased bone resorption, a method for measuring the bone resorption area in vitro. Knockdown of Gal-3 by small-interfering RNA (siRNA) in BMSCs downregulated the expression of RANKL mRNA and attenuated the effects of 1α,25(OH)2D3 on osteoclastogenesis and bone resorption. Conversely, overexpression of Gal-3 in BMSCs enhanced the effects of osteoclastogenesis and bone resorption by increasing the expression of RANKL mRNA. These results demonstrated that Gal-3 mediates the differentiation and bone resorption of osteoclasts regulated by 1α,25(OH)2D3.

猫泛白细胞减少症又称猫瘟热,多发于2～6月龄未防疫的猫,对宠物猫危害极大. 导致猫泛白细胞减少症的病原为细小病毒科细小病毒属的猫细小病毒(FPV).感染FPV的猫多表现为精神萎顿,食欲减退或废绝,呕吐,下痢,体温升高(＞39.7℃),白细胞数减少. 本病一年四季均可发生,以冬春季节发病率较高,不同性别的猫发病率有明显差异.猫瘟热传染性强、发病急,一岁以内、未进行过免疫或免疫不完全的幼猫比成年猫更易患病,且病情更加严重,死亡率更高.处于病毒血症期的感染动物可通过分泌物、呕吐物、尿液及粪便排出大量病毒.临床康复的水貂和家猫可在一年内持续向外排毒,是猫瘟热的主要传染源.

为探究1α,25-(OH)2D3对鸡骨髓间充质干细胞(BMSCs)和骨髓单核巨噬细胞(BMMs)体外共培养体系中破骨细胞(OC)形成的影响,笔者对1α,25-(OH)2D3处理后的细胞进行抗酒石酸酸性磷酸酶染色(TRAP)、骨吸收功能鉴定、OC标志基因mRNA和蛋白表达的检测.结果 表明:1α,25-(OH)2D3能够上调BMSCs中核因子κB受体活化因子配体(RANKL)的表达,下调骨保护素(OPG)的表达.1α,25-(OH)2Ds处理共培养细胞5d,OC数目较对照组明显增多,骨吸收活性较对照组极显著增强,基质金属蛋白酶9(MMP-9)、Ⅱ型碳酸酐酶(CAⅡ)、组织蛋白酶K(CtsK)及TRAP等标志性蛋白的mRNA和蛋白表达极显著高于对照组(P＜0.01),其中10-8 mol·L-11α,25-(OH)2D3效果最明显.结果 表明,10-8mol·L-1 1α,25-(OH)2D3能够介导鸡BMSCs和BMMs共培养体系中OC的形成,该OC具有骨吸收活性,为进一步研究OC在家禽骨骼及钙磷代谢紊乱性疾病中的作用机制奠定基础.

目的 研究1α,25-(OH)2D3对体外培养小鼠破骨细胞(osteoclast,OC)分化及骨吸收的直接影响.方法 采用核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)和巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)诱导体外培养C57BL/6J小鼠OC前体分化成OC.RTCA检测细胞增殖、抗酒石酸酸性磷酸酶染色检测OC数量、Western blot检测OC骨吸收功能相关蛋白和转录因子蛋白表达水平、免疫荧光和扫描电镜观察OC骨架和形态、骨细胞培养板分析骨吸收陷窝面积,观察10 nM 1α,25-(OH)2D3对OC形成及骨吸收功能的影响.结果 10 nmol/L 1α,25-(OH)2D3对细胞增殖无显著影响,但能显著抑制OC形成,抑制骨吸收功能相关蛋白和转录因子蛋白的表达,阻碍OC骨架肌动蛋白环和伪足小体的形成,减弱OC骨吸收功能.结论 1α,25-(OH)2D3能抑制体外培养小鼠OC的形成及骨吸收功能,在钙磷代谢紊乱及骨骼疾病中对OC具有直接调控作用.