【作者】 那键；

【导师】 谷贵山；

【作者基本信息】 吉林大学 ， 外科学， 2009， 博士

【摘要】 新型上皮性钙通道成员TRPV5\TRPV6表现为对钙离子具有高度选择性,对钙离子代谢及骨代谢有着非常重要的作用。但目前TRPV5、TRPV6在成骨细胞上是如何调节成骨细胞的增殖及功能的作用机制、以及其表达的量及其功能的改变受何种因素调节尚不十分清楚。本研究旨在探讨TRPV5及TRPV6在成骨细胞中的表达规律及雌激素、维生素D3的干预作用效果,为认识世界性的医学难题老年性骨质疏松的发病机制提供实验依据。本研究大致分为体内实验及体外实验两部分。体内实验部分:成年雌性去势大鼠抗骨质疏松治疗及相关基因表达。研究女性绝经期后骨质疏松情况常用去势雌性大鼠作为动物模型。我们在此基础上对模拟女性绝经期的去势大鼠进行抗骨质疏松的干预治疗及相关研究。给去势大鼠每周一次注射治疗剂量的苯甲酸雌二醇及维生素D3注射液,共计8周。处死动物后进行骨密度、骨形态计量学、血清骨代谢等指标检测,通过RT-PCR技术检测股骨组织中OPG、RANKL和M-CSF、TRPV5、TRPV6的表达情况。结果表明施加雌激素、维生素D3可在一定程度上拮抗去势大鼠骨质疏松,但是仍然不能完全逆转骨质疏松发生;雌激素的疗效优于维生素D3;在体内去势大鼠实验表明雌激素通过刺激OPG、RANKL的表达通路来介导成骨细胞增生;并且促进钙离子通道TRPV6表达,说明了钙离子和OPG/RANKL作为信号传递递质参与雌激素的成骨作用。体外实验部分:对雌性大鼠颅骨来源成骨细胞的干预试验。雌性大鼠颅骨来源成骨细胞培养并鉴定后,给予传代的成骨细胞以不同浓度的17-β雌二醇、1,25二羟维生素D3干预24小时,应用Western-blot和RT-PCR技术在蛋白质和mRNA水平检测成骨细胞内OPG、RANKL、TRPV5和TRPV6的表达情况,应用激光共聚焦显微镜观察不同浓度雌激素干预后成骨细胞内钙离子含量的变化,间接反映钙离子通道功能及表达情况。结果发现随着雌激素暴露时间的延长,成骨细胞的增殖活性升高,同时雌激素诱导成骨细胞OPG和TRPV5/TRPV6的mRNA及蛋白质表达呈剂量依赖性。体外实验说明OPG和钙离子通道TRPV5/TRPV6在雌激素刺激成骨作用中具有一定作用。利用体外成骨细胞培养观察1,25二羟维生素D3对成骨细胞的作用发现,1,25二羟维生素D3只有在极低的浓度下刺激成骨细胞的增生,并且促进成骨细胞OPGmRNA水平的明显表达,增加了钙离子通道TRPV5/TRPV6的表达,显示出1,25二羟维生素D3的成骨作用需要OPG和钙离子信号通路的参与。更多还原

【Abstract】 Postmenopausal osteoporosis is a heterogeneous disorder characterized by accelerated bone loss after natural or surgical menopause and an increased risk of fractures. The epithelial Ca2+ channel TRPV5/TRPV6 performance to have sensitive selectivity to the calcium ion, thus it has the very important effect on the calcium ion metabolizes and the bone metabolism. The previous study showed TRPV5 was mainly exist in kidney, the TRPV6 was mainly exist in small intestines. TRPV5/TRPV6 has been tested in the osteoblast of bone tissue. The foreign research has already proven to make exchange the calcium ion to keep the extracellular level of calcium. Our experimental term have observed the expression of TRPV5 and TRPV6 in osteoblasts which come from the differentiation of BMSCs. The study on the expression of TRPV5 and TRPV6 in the cultured neonatal rat calvarial osteoblasts and the role of the expression of TRPV5 and TRPV6 in mechanism of post-menopausal osteoporosis has not yet been reported. The present study mainly observed the the expression of TRPV5 and TRPV6 in neonatal rat calvarial osteoblasts and the role of the expression of TRPV5 and TRPV6 in mechanism of post-menopausal osteoporosis. The bone loss in estrogen deficiency results from the increased bone resorption and impaired ability of osteoblastic bone formation. In the other hand, the interventive measures were used to probe into the possible treatment of estrogen and 1,25(OH)2VD3 to post-menopausal osteoporosis.The first section is to study the possible mechanism of estrogen and 1,25 (OH)2VD3 treatment to post-menopausal osteoporosis in vivo experiment. The osteoporosis model was copied by resecting the rat ovarian. Rats of ovariectomy were treated with estrogen or 1,25(OH)2VD3 for anti-osteoporosis for 8 weeks. Rats were sacrificed to detect bone density, bone histomorphometry, and some serum bone metabolic index so on. The RT-PCR technique was used to detected the mRNA expression of OPG, RANKL, M-CSF and TRPV6 in bone of rats of ovariectomy with/without treatment of estrogen / 1,25(OH)2VD3. The results showed, , the treatment of estrogen can protect against osteoporosis to some extent, however, the effect of 1,25(OH)2VD3 against osteoporosis wasn’t significant. Meanwhile, it was showed that estrogen probably promoted the bone formation by stimulating the expression OPG, RANKL pathway and openning of Ca2+ chanel TRPV6.The second section is to study the possible mechanism of estrogen and 1,25(OH)2VD3 treatment to post-menopausal in vitro experiment. As we all known, osteoblast played the important role in the pathogenesis of osteoporosis. In the present study , the neonatal rat calvarial osteoblasts were cultured and subcultured to observed the proliferation and expression of OPG, RANKL, TRPV5 and TRPV6 after exposed to estrogen and 1,25(OH)2VD3. The cells showed the characteristic osteoblast by ALP postive staining and formation of osteoblasts nodule. It was observed that the proliferation of osteoblast activity increased companied with the increase of estrogen concentration. The mRNA and protein expression of OPG,TRPV5 and TRPV6 in osteoblast showed a dose-dependent manner. These results suggested that bone formation induced by estrogen may be mediated by the OPG/OPGL path and calcium ion content regulated by the special Ca2+ chanel TRPV5 and TRPV6. In the other hand, the action of 1,25(OH)2VD3 on osteoblasts was observed and showed that only very low concentrations of 1,25(OH)2VD3 stimulated the proliferation of osteoblasts. Whereas, 1,25(OH)2VD3 promoted the mRNA expression of OPG, TRPV5 and TRPV6 which was similar with the action of estrogen on osteoblast. These results suggested that bone formation induced by 1,25(OH)2VD3 also was mediated by the OPG/OPGL path and the special Ca2+ chanel TRPV5 and TRPV6.更多还原