权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Function analysis of Na^+ dependent ion transporters in bone metabolism

Na^依赖性离子转运蛋白在骨代谢中的功能分析

基本信息

批准号：
12671815
负责人：
AMANO Hitoshi
金额：
$ 2.37万
依托单位：
Showa university
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671815/
关键词：
mechanical stress osteoblast RT-PCR osteoclast resorption pit co-culture Na^+Ca^<2+> exchanger NPT2 CSF-1 KB-R7943

项目摘要

Na^+/Ca^<2+> exchanger (NCX) catalyzes the electrogenic exchange of 3 Na^+ for 1 Ca2^+ across the plasma membrane in many mammalian cells. We have previously reported that the Na^+/Ca^<2+> exchange activity had an important role for CSF-1-induced osteoclast differentiation. In the present study, we sought the effect of mechanical stress to confirm the role of NCX1, in osteoblastic bone formation and osteoclastic bone resorption formation.Osteoblast were obtained from calvalia of new bone rat. Mechanical stress induced NCX1 mRNA and F-actin polyme ralization in osteoblast. The application of Gadlinium ion, a blocker of SAchannel, inhibited stretch-activated cell responces.Bone marrow cells were obtained from tibiae and femuor of 5- to 8-week-old male ddy mice. We used Sephadex G-10 column (G10) in order to remove macrophages and stroma cells. Non adherent G10-passed cells were cultured in a-MEM containing the final concentration of 15 % FBS, 100 ng/ml RANKL, 20 ng/ml CSF-1. KB-R7943 inhibited the osteoclast differentiation and bone resorption in vitro dose-dependently. In addition, NCX1 antisense oligo deoxy nucleotide (ODN) reduced the number of tartrate-resistant acid phosphatase-positive multinucleated cells and decreased the amount of NCX1 in protein level. Eight week-old female ddY mice received sham-operation or ovar iectomy (OVX) and were fed a laboratory chow for 4 weeks after the operation. Animals were given subcutaneously 337.5 mg/kg KB-R 7943 or DMSO every other day for 4 weeks. After 4 weeks treatment with KB-R7943, the BMD in the femur of OVX group was significantly decreased compared with sham group. However the BMD in KB-R7943 treated OVX group was quantitatively similar to the sham group. These results suggest that bone resorption was also blocked by treatment with KB-R7943 in vivo.In conclusion, the activation of NCX1 is essential for osteoclast differentiation and activation.

Na^+/Ca^<2+>交换器（NCX）在许多哺乳动物细胞的质膜上催化3na ^+与1ca2 ^+的电交换。我们之前报道过Na^+/Ca^<2+>交换活性在csf -1诱导的破骨细胞分化中起重要作用。在本研究中，我们寻求机械应力的影响，以确认NCX1在成骨细胞骨形成和破骨细胞骨吸收形成中的作用。从新生骨大鼠的破骨处获得成骨细胞。机械应力诱导成骨细胞NCX1 mRNA和f -肌动蛋白聚合。钆离子（schannel阻滞剂）的应用抑制了拉伸激活的细胞反应。从5 ~ 8周龄雄性小鼠的胫骨和股骨中获得骨髓细胞。采用Sephadex G-10色谱柱（G10）去除巨噬细胞和基质细胞。非贴壁g10传代细胞在终浓度为15% FBS、100 ng/ml RANKL、20 ng/ml CSF-1的a-MEM中培养。KB-R7943体外抑制破骨细胞分化和骨吸收呈剂量依赖性。此外，NCX1反义寡聚脱氧核苷酸（ODN）减少了抗酒石酸酸性磷酸酶阳性的多核细胞数量，降低了NCX1蛋白水平。8周龄雌性小鼠分别接受假手术或卵巢切除术（OVX），术后4周喂实验室饲料。小鼠每隔一天皮下注射337.5 mg/kg kb - r7943或DMSO，连续4周。KB-R7943治疗4周后，OVX组股骨骨密度较sham组明显降低。而KB-R7943治疗的OVX组骨密度与假手术组在数量上相似。这些结果表明KB-R7943在体内也能阻断骨吸收。综上所述，NCX1的激活对于破骨细胞的分化和激活至关重要。