妊娠小鼠骨组织TRPV5和TRPV6的表达规律及其在胎盘钙转运中的作用探讨

The transient receptor potential vanilloid 5 and 6 (TRPV5, 6) are calcium selective channels that are involved in the absorption and reabsorption of calcium across bone tissue cells and trophoblast cells. The project was designed to reveal the role of TRPV5/6 in the bone metabolism firstly by testing the expression dynamic law of the two proteins on the bone tissue of Kunming Mouse during the pregnancy, and by analyzing the relationship of the levels of protein contents and bone metabolism relevant indicators during the same period. Secondly, the team was intended to investigate the effect of TRPV5/6 on the placenta calcium transport. So the calcium content of the fetus and umbilical vein would be deterninated, as well as the contents of TRPV5/6 in the fetal placenta of mice in late pregnancy. By clearing the relation of TRPV5/6 content in the placenta and maternal bone tissue, we were expected to clear the relation of maternal bone metabolism and placenta calcium transport. In addition, high diet 1,25-(OH)2D level was used to induct high serum 1,25-(OH)2D level on the pregant mice on the clinical animal experiments, so as to investigate the regulating effect of 1,25-(OH)2D on calcium metabolism in pregnancy. On the whole, this program was to clarify the role of TRPV5/6 in calcium transporter and provide basis for the mechanisms of maternal and fetal calcium transport in the pregnancy of the mammal, and to search the effective treatment for pregnancy calcium negative balance.

TRPV5和TRPV6 (transient receptor potential vanilloid type 5、6)是钙离子的高选择性通路蛋白，参与了骨代谢及胎盘钙转运过程中的钙离子跨细胞转运。本项目拟以妊娠期小鼠为研究对象，分析TRPV5、TRPV6蛋白在妊娠母体骨组织动态表达规律，监测同期骨代谢相关指标，揭示TRPV5/6在骨代谢中的作用；同时分析孕中后期胎儿胎盘TRPV5、TRPV6蛋白的动态变化，检测胎儿及胎盘脐静脉钙含量，探讨其与胎儿钙代谢的相关性，揭示胎盘与母体骨组织TRPV5/6的作用及其机制。此外，通过食物添加1,25-(OH)2D建立其高血清水平妊娠小鼠，研究1,25-(OH）2D对TRPV5/6在妊娠期钙代谢中的调控作用。藉此，阐明TRPV5和TRPV6在妊娠期钙代谢中的作用，完善孕期母体和胎儿的钙转运机制，为兽医临床筛选、纠正孕期钙负平衡提供实验依据。

TRPV5 和 TRPV6是钙离子的高选择性通路蛋白。妊娠期胎儿的Ca2+需求依赖于母体-胎儿钙转运，机体的钙主要贮存在骨组织中，胎盘是母体和胎儿沟通的唯一渠道。本项目主要阐释了妊娠期TRPV5和TRPV6在骨组织钙代谢及胎盘组织钙转运中的作用，由此揭示了TRPV5和TRPV6在哺乳动物妊娠期母体-胎儿钙转运中的作用。项目主要开展了以下四方面的研究：1. TRPV5和TRPV6在妊娠昆明小鼠组织中的定位表达；2. TRPV5和TRPV6在妊娠昆明小鼠骨钙代谢及胎盘钙转运中的生理学功能；3. 探讨了1,25-(OH)2D3对TRPV5和TRPV6的影响；4. 通过小鼠胎盘滋养层细胞的分离培养，从细胞水平探讨了TRPV5和TRPV6对昆明小鼠胎盘滋养层细胞钙转运功能的影响。研究结果显示，小鼠骨组织及胎盘组织中均存在TRPV5和TRPV6的表达。且妊娠期骨、胎盘组织中TRPV5、TRPV6mRNA及蛋白水平均呈时序性变化，提示母体妊娠期通过TRPV5和TRPV6介导作用，提高骨钙动员以及胎盘钙转运能力，加强妊娠期间的母体-胎儿钙转运机能。动物临床试验表明1,25-(OH)2D3可以调控妊娠期钙转运通路蛋白TRPV5和TRPV6的表达。细胞水平的基因沉默技术进一步验证了昆明小鼠胎盘滋养层细胞的TRPV5、TRPV6基因介导了细胞内钙转运功能，影响细胞内的钙离子浓度。