【作者】 柳东阳；

【导师】 郭定宗；

【作者基本信息】 华中农业大学 ， 临床兽医学， 2012， 博士

【摘要】 斯钙素1(Stanniocalcin-1,STC-1)是一种糖蛋白激素,最早在硬骨鱼体内发现,由鱼类特有腺体----位于肾脏的斯坦氏小体(Corpuscle of Stannius)的I型分泌细胞分泌,STC-1对海水和淡水鱼体内钙和磷酸盐稳态起重要的调节作用。哺乳动物的STC-1蛋白首先在人体内发现。随后在人和小鼠上克隆出了STC-1的cDNA,并在大鼠、犬、羊体内发现该蛋白的存在。哺乳动物的STC-1以肠道和肾脏为主要靶器官,通过抑制肠道对钙吸收和促进肾脏对磷的重吸收来调节体内钙、磷稳态。探索研究STC-1在奶牛钙磷稳态调控中的作用及机制,有助于从细胞水平及分子水平上解析奶牛钙、磷稳态新的作用机制,为奶牛钙磷代谢紊乱疾病防治提供新的靶点。1.斯钙素-1在新生奶牛成骨细胞的定位和表达采用骨膜贴附法培养奶牛成骨细胞,通过碱性磷酸酶(ALP)染色,骨钙素(Osteocalcin, OCN)免疫组化和体外成骨诱导试验,对所培养细胞进行了鉴定。在成骨细胞培养的10d,提取总RNA,设计STC-1特异性引物进行RT-PCR扩增出奶牛STC-1特异性片段。同时采用免疫组化技术检测到STC-1表达于体外培养10d奶牛成骨细胞。结果显示通过骨膜贴附法可以得到奶牛成骨细胞。采用免疫组化和RT-PCR检测到STC-1在奶牛成骨细胞的表达。2.STC-1对奶牛成骨细胞增殖和分化的影响采用不同浓度的rhSTC-1(0ng/m、0.1ng/ml、1.0ng/ml、10.0ng/ml和100.0ng/ml)处理奶牛成骨细胞,MTT法检测细胞增殖,检测成骨细胞标志分子ALP和OCN的表达变化,同时检测体外矿化能力。STC-1可以抑制体外奶牛成骨细胞的增殖,对于ALP和OCN的表达影响具有浓度和时间上差异性,0.1-10.0ng/ml的STC-1可以促进体外成骨细胞的矿化,100.0ng/ml对于体外矿化没有影响。说明STC-1在调节成骨细胞增殖和分化过程中的复杂性。3.CoCl2诱导奶牛成骨细胞缺氧凋亡及对STC-1基因表达的影响本实验探索CoCl2处理奶牛成骨细胞诱导缺氧,在不同时间点(0h,12h,24h,36h,48h)检测细胞凋亡的形态学变化和STC-1等基因的表达变化。结果：CoCl2可以诱导奶牛成成骨细胞发生凋亡,在4个时间点均有明显上升,在24h的表达量最高(P<0.01)。Bax mRNA的表达量在24h最高(P<0.05)。VDR mRNA在4个时间点表达均上升,在36h最高(P<0.01)。结果表明CoCl2诱导奶牛成骨细胞缺氧中,STC-1基因参与了凋亡过程,Bax促进凋亡的发生,VDR也参与这个凋亡过程。4.磁性纳米铁对奶牛成骨细胞的细胞毒性研究采用水相合成法合成MNP,通过TEM、Zeta电势检测仪来检测合成MNP的理化特性。通过MTT法、Prussian Blue Staining、细胞骨架荧光染色和生化试剂盒来检测不同浓度MNP(浓度分组：0μg/ml,50μg/ml,100μg/ml,200μg/ml)对细胞增殖,细胞骨架和细胞内抗氧化酶的影响。通过水相合成法可以合成粒径在8-10nnm的磁性纳米颗粒。奶牛成骨细胞摄取MNP,分布在细胞核周围。随MNP添加浓度增加,细胞活力下降,乳酸脱氢酶释放量增加、细胞骨架F-actin排列出现紊乱、SOD和过氧化物酶的活性降低。说明MNP对奶牛成骨细胞的毒性具有剂量依赖性,作为细胞转染载体,MNP需要进一步的表面修饰以减少毒性。更多还原

【Abstract】 1. Expression and Localization of Stanniocalcin1in Bovine OsteoblastsAs a novel glycoprotein hormone, Stanniocalcin-1(STC-1) was first identified in teleost species, and it is involved in the regulation of mineral homeostasis in bony fish and mammals. STC-1can not only affect the mammals bone development, but also protect neurons from the damage of ischemia, and stimulate the angiogenic response. Although it is widely expressed in rodent skeletons, whether this hormone is expressed in the skeleton of ruminant animals, like bovines, is still unknown. Here, we investigated the expression of STC-1in bovine osteoblasts by using Immunocytochemical staining and RT-PCR. Our results show that the mRNA and protein of STC-1are expressed in the bovine osteoblasts during later differentiation periods (10th day) in vitro. The present data support the notion that STC-1may play a role in the process of bovine bone development.2. The effect of STC-1on the proliferation differentiation, and mineralization of bovine osteoblastsSTC-1have been affected the proliferation and differentiation of the rodents osteoblasts, but the effect of STC-1for the ruminant osetoblasts is unknown. In this study, the bovine osteoblasts were treated by STC-1in different concentration. Cell viability was determined by MTT assay. Alkaline phosphatase(ALP)and Osteocalcin(OCN), specific marker of osteoblasts, were investigated by ALP kit and RIA, respectively. The ability of mineralization for bovine osteoblasts in vitro was determined by Alizarin red S staining. The results showed that Stanniocalcin1can inhibit the proliferation of bovine osteoblasts, but the effect on the ALP and OCN concentration were vary dependent on the STC-lconcentratin and the length of treating time.3. STC-1involved in the bovine osteoblasts hypoxic apoptosis inducing by Cobalt chlorideIn this study, we aimed to identify the morphological change and expression of STC-1mRNA for bovine osteoblasts at0h,12h,24h,36h,48h after apoptotic inducing by Cobalt chloride. Our results show that the number of apoptotic cells gradually increased when that the extending of inducing time. Expression level of the STC-1was elevated in different time point compare with the control group, and highest level (P<0.01) was occur in24h after inducing by CoCl2.Th expression level of Bax and VDR also elevated in testing group, and the highest level of Bax and VDR were in24h、36h time point, respectively. This results indicate that STC-land VDR were involved in osteoblasts apoptosis which inducing by Cobalt chloride, and Bax can promote the apoptosis.4. Cytotoxicity of Magnetic Nanoparticles (Fe3O4) for bovine osteoblastsMagnetic Nanoparticles (MNP,(Fe3O4)could be used as gene transfection shuttle under external magnetic field more effective than other transfection systems, but the MNP showing the cytotoxity. As the key step of exogenous STC-1transfected into bovine osteoblasts, we should be asses the cytotoxicity of MNP for osteoblasts firstly. MNP were synthesized by aqueous chemical co-precipitation method, and the physicochemical characterization of MNP were characterized by SEM, Zeta potential meter and external magnetic field, respectively. Cell viability was determined by MTT method. Nanoparticle uptake by osteoblasts was studied using Prussian blue staining. Cytoskeleton stained by Phalloidin-FITC. LDH, SOD and Peroxidase activity were measured by using assay Kit. This results show that proliferation of osteoblasts were inhibited by MNP in concentraton-dependently manner, and the cytoskeleton was disrupted, LDH leaking level were elevated. SOD and Peroxidase activity were decreased. Our results suggest that MNP have a cytotoxicity for osteoblasts.更多还原