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钙敏感受体介导甲状旁腺素和钙在促进骨形成中的作用及其机制研究

The Calcium Sensing Receptor Mediates Bone Turnover Induced by Dietary Calcium and Parathyroid Hormone

作者: 专业:人体解剖学 导师:苗登顺 年度:2009 学位:硕士  院校: 南京医科大学

Keywords

parathyroid hormone, calcium, bone formation, CaSR

        为了研究钙敏感受体能否介导甲状旁腺素(PTH)和钙在促进骨形成中的作用,我们采用PTH+/-CaSR+/-双杂小鼠雌雄交配而获得WT、PTH-/-和PTH-/-CaSR-/-子鼠,并利用正常饮食(1.0%钙、0.6%磷)或高钙低磷饮食(2.0%钙、0.4%磷)喂养PTH+/-CaSR+/-母鼠,观察高钙低磷饮食致母乳成份改变对3周龄子鼠骨形成的影响;同时给予正常饮食喂养的子鼠腹腔注射外源性的PTH(1-34), 80μg/kg体重,1次/日,持续14天,观察外源性PTH对3周龄子鼠骨形成的影响。结果发现:与正常饮食喂养的PTH+/-CaSR+/-母鼠相比,高钙低磷饮食喂养的PTH+/-CaSR+/-母鼠乳汁中钙离子浓度明显升高,而1,25(OH)2D3和PTHrP水平则明显降低。与正常饮食母鼠哺乳的WT子鼠相比,同组的PTH-/-和PTH-/-CaSR-/-子鼠表现为低钙高磷血症,血1,25(OH)2D3降低;高钙低磷饮食母鼠哺乳后,三种基因型子鼠血钙升高,血磷、血1,25(OH)2D3降低,但WT和PTH-/-子鼠升高或降低幅度大于PTH-/-CaSR-/-子鼠;外源性PTH(1-34)处理后,三种基因型子鼠血钙、血1,25(OH)2D3均升高,但WT和PTH-/-子鼠升高幅度大于PTH-/-CaSR-/-子鼠。在正常饮食和高钙低磷饮食母鼠哺乳的PTH-/-和PTH-/-CaSR-/-子鼠中,检测不到血PTH;血PTH水平在高钙低磷饮食母鼠哺乳的WT子鼠低于正常饮食母鼠哺乳的WT子鼠;给予外源性PTH(1-34)后,血PTH水平在PTH-/-和PTH-/-CaSR-/-子鼠中明显低于WT子鼠。无论是正常饮食母鼠哺乳、高钙低磷饮食母鼠哺乳,还是外源性PTH(1-34)给药,与同组的WT子鼠相比,骨长度,骨密度,骨容量,成骨细胞数,Ⅰ型胶原阳性面积,骨矿物质沉积率以及调控和表达成骨细胞活性的基因(Cbfα-1,ALP,Col-1,OCN)和调控成骨细胞活性的蛋白(PTHR,IGF-1)在PTH-/-子鼠中降低,在PTH-/-CaSR-/-子鼠降低更明显;与正常饮食母鼠哺乳的同种基因型子鼠相比,高钙低磷饮食母鼠哺乳后,上述指标在WT和PTH-/-子鼠增加,在PTH-/-CaSR-/-子鼠增加不明显;与正常饮食母鼠哺乳的同种基因型子鼠相比,外源性PTH(1-34)给药后,上述指标在三种基因型子鼠均增高,但在WT和PTH-/-子鼠增高幅度大于PTH-/-CaSR-/-子鼠。体外骨髓间充质干细胞向成骨细胞诱导分化的结果与以上结果具有一致性。无论是正常饮食母鼠哺乳、高钙低磷饮食母鼠哺乳,还是外源性PTH(1-34)给药,与同组的WT子鼠相比,破骨细胞数、破骨细胞面以及RANKL/OPG mRNA比值在PTH-/-子鼠中降低,在PTH-/-CaSR-/-子鼠降低更明显;与正常饮食母鼠哺乳的同种基因型子鼠相比,高钙低磷饮食母鼠哺乳后,上述指标在WT和PTH-/-子鼠降低,在PTH-/-CaSR-/-子鼠降低不明显;与正常饮食母鼠哺乳的同种基因型子鼠相比,外源性PTH(1-34)给药后,上述指标在三种基因型子鼠均增高,但在WT和PTH-/-子鼠增高幅度大于PTH-/-CaSR-/-子鼠。上述结果说明:高钙低磷饮食母鼠哺乳和外源性PTH均具有促骨形成作用,其促进骨形成的作用依赖于钙的补充,经饮食补充的钙和经PTH作用增加的钙都能经CaSR介导刺激骨形成。本研究结果有助于阐明PTH和钙在促进骨形成中的相互作用及机制,为使用适当比例的钙/磷饮食提高儿童的骨峰值和预防骨质疏松,也为PTH和钙剂联合用药治疗骨质疏松及使用CaSR激动剂预防骨质疏松提供理论依据。
    We employed a genetic approach to determine whether the calcium sensing receptor (CaSR) mediates bone turnover associated with dietary calcium (Ca) and parathyroid hormone (PTH). Wild-type (WT) pups and pups with targeted deletion of the PTH (PTH-/-) gene or of both PTH and CaSR (PTH-/-CaSR-/-) genes were nursed by WT dames: 1) on a normal Ca (1.0% Ca and 0.67% Pi) diet or 2) a high Ca (2% Ca and 0.4% Pi) diet. Pups nursed by dames on a normal Ca diet received daily injections of vehicle or of PTH1-34 (80μg/kg) for 2 weeks starting from 1 week of age. Pups fed by dames on a high Ca diet received vehicle only. These regimens induced different bone phenotypic changes, thereby disclosing selective modulation by Ca and PTH mediated through CaSR.Milk Ca content was raised, but milk PTH related protein (PTHrP and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) levels were decreased in PTH+/-CaSR+/- dames fed on a high Ca diet compared to PTH+/-CaSR+/- dames fed on a normal Ca diet. Serum calcium was decreased in 3-week-old PTH-/- pups, and was decreased more dramatically in 3-week-old PTH-/-CaSR-/- pups, whatever they were fed by dams on either the normal diet or the high Ca diet or were administrated with the exogenous PTH, despite it was raised paralleled in the genotype-matched pups fed by dams on the high Ca diet or administrated with exogenous PTH compared to pups fed by dams on the normal diet. Both mutant animals were hyperphosphatemia, whatever they were fed by dams on either the normal diet or the high Ca diet or were administrated with the exogenous PTH, despite it was reduced paralleled in the genotype-matched pups fed by dams on the high Ca diet compared to pups fed by dams on the normal diet. Serum PTH levels were decreased in WT pups fed by dams on the high Ca diet and were raised in WT pups administrated with PTH compared to vehicle treated WT pups fed by dams on the normal diet, and were undetectable in PTH-/- and PTH-/-CaSR-/- pups fed by dams on any diet. With PTH treatment, serum PTH levels were detectable, but was lower significantly in PTH -/- and PTH-/-CaSR-/- pups compared to their WT littermates. Serum 1,25(OH)2D3 was decreased in 3-week-old PTH-/- pups, and was decreased more dramatically in 3-week-old PTH-/-CaSR-/- pups, whatever they were fed by dams on either the normal diet or the high Ca diet or were administrated with the exogenous PTH, despite it was reduced paralleled in the genotype-matched pups fed by dams on the high Ca diet or it was raised paralleled in the genotype-matched pups administrated with exogenous PTH compared to pups fed by dams on the normal diet.In 3-week-old pups receiving vehicle and fed by dames either on a normal or a high Ca diet, bone mineral density (BMD), trabecular volume (BV/TV), the number of osteoblasts, the type I collagen (Col I) positive areas, mineral apposition rate( MAR), the gene expression levels of Cbfα-1, ALP, Col I, OCN genes and the protein expression of PTHR and IGF-1 were all reduced significantly in PTH-/- pups compared to WT pups and were decreased more dramatically in PTH-/-CaSR-/- pups even compared to PTH-/- pups. These bone formation parameters were increased in WT and PTH-/- pups, but not in PTH-/-CaSR-/- pups fed by dames on a high Ca diet compared to the pups fed by dames on a normal Ca diet. These bone formation parameters were increased in 3-week-old WT, PTH-/- and PTH-/-CaSR-/- pups following exogenous PTH treatment for 2 weeks, however, the percentage increase was less in PTH-/-CaSR-/- pups than in WT and PTH-/- pups. The percentage increase in these bone formation parameters was higher in PTH-treated pups than in pups fed by dames on a high Ca diet in both WT and PTH-/- pups.In vehicle treated 3-week-old pups fed by dames on either a normal or a high Ca diet, and in PTH-treated pups fed by dames on a normal Ca diet, the number and surface of TRAP positive osteoclasts were reduced significantly in PTH-/- pups compared to WT pups and were decreased even more dramatically in PTH-/-CaR-/- pups than in PTH-/- pups. However, these parameters were decreased significantly in pups fed by dames on a high Ca diet and increased significantly in PTH-treated pups compared to genotype-matched pups fed by dames on a normal diet. There results indicate that CaSR mediates bone turnover mediated by dietary Ca and can impact on the increased bone turnover elicited by PTH.There results indicate that CaSR mediates bone turnover mediated by dietary Ca and can impact on the increased bone turnover elicited by PTH, suggest that cooperative roles of PTH and calcium on bone formation and imply that dietary calcium and phosphorous in an appropriate proportion play as benefits on neonatal skeletal development and growth. This study provides an experimental and theoretical evidence for the combination of PTH with calcium or CaSR agonist to treat osteoporosis.
        

钙敏感受体介导甲状旁腺素和钙在促进骨形成中的作用及其机制研究

缩写词4-6
中文摘要6-8
英文摘要8-10
前言11-15
材料与方法15-42
结果42-56
讨论56-61
小结61-62
结论和意义62-63
参考文献63-69
附录69-70
综述70-84
    参考文献75-84
致谢84
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