【作者】 车明学；

【导师】 谷贵山；

【作者基本信息】 吉林大学 ， 外科学， 2010， 博士

【摘要】 TRPV5是选择性钙离子通道,参与钙离子的吸收和重吸收,其主要表达于肾脏远曲／集合小管、十二指肠及骨破骨细胞的皱褶缘。已有研究显示TRPV5和TRPV6在骨组织均有表达,但两者的生理作用目前还不清楚。阿伦膦酸钠是第三代骨吸收抑制剂,在细胞水平上阿伦膦酸钠主要通过减少破骨细胞数量,抑制破骨细胞皱褶缘的形成,降低成熟破骨细胞溶酶体酶的释放,而降低破骨细胞的功能。本实验引进了TRPV5基因敲除小鼠杂合子,饲养和繁殖按照SPF级要求的动物饲养标准进行,从子代筛选出野生型TRPN5+/+及纯合子TRPV5-/-。为了研究阿伦膦酸钠对TRPV5+/+和TRPV5-/-小鼠骨、肾脏、十二指肠的钙离子通道的表达的影响,本实验将TRPV5+/+和TRPV5-/-小鼠分为四组饲养,其中两组基因型不同的小鼠每周分别给予阿伦膦酸钠2mg/kg一次,连续给予8周,采集血液及尿液标本并做生化分析,小鼠股骨、肾脏、十二指肠制样。从小鼠股骨中提取破骨细胞培养,并对其表面皱褶缘上的钙离子通道进行定量多聚酶链反应分析,应用CT对股骨进行扫描,并对钙离子的吸收峰值进行检测。应用阿伦膦酸钠处理的TRPV5+/+小鼠股骨的厚度增加,而TRPV5-/-鼠股骨厚度由减少转为正常,而肾脏和十二指肠的钙离子通道没有改变。两种基因型小鼠的破骨细胞皱褶缘上的CLC-7蛋白及液泡H+-ATPase的表达未见明显改变,但是阿伦膦酸钠显著增加了TRPV5-/-小鼠的甲状旁腺激素的水平及表达。破骨细胞培养液中TRPV5,液泡CLC-7及液泡H+-ATPase均未受阿伦膦酸钠的影响,TRPV5-/-小鼠的骨髓培养中骨吸收陷窝减少,两者对维生素D的反应未见明显区别。阿伦膦酸钠促使TRPV5+/+小鼠的TRPV5基因上调,而降低TRPV5-/-小鼠破骨细胞的吸收功能说明阿伦膦酸钠在破骨细胞功能中发挥着很重要的作用。经过阿伦膦酸钠处理后,TRPV5+/+处理组小鼠的液泡H+-ATPase和Cl-通道CLC-7mRNA的表达与处理前比较没有改变,相反,阿伦膦酸钠增加了TRPV5-/-小鼠的液泡H+-ATPase和Cl-通道CLC-7的表达。骨再吸收功能被阿伦膦酸钠抑制后,仅有TRPV5的表达进行增量调节,这说明TRPV5在破骨细胞功能中发挥着重要作用。血清高PTH或者PTH任何下游区效应,均是参与维持钙离子的高吸收,然而PTH对钙离子通道表达和钙离子再吸收的刺激反应已被肾脏证实,PTH受体存在于肠道受体,刺激肠道对钙离子的吸收。阿伦膦酸钠介导的对骨再吸收功能的抑制导致钙离子失衡,迫使PTH的诱导作用加长,最终维持血钙水平。阿伦膦酸钠处理的TRPV5-/-小鼠骨质厚度和体积指数明显正常化,且骨吸收标记物DPD在尿液中的排泄量减少。另外由于骨吸收被抑制,所以肾脏钙离子排泄减少,骨中钙离子释放也减少。这些资料提示TRPV5对于破骨细胞的骨再吸收是必需的。TRPV5-/-小鼠破骨细胞皱褶缘表达的液泡H+-ATPase和Cl-通道CLC-7未见改变,两者对破骨细胞的骨再吸收发挥重要作用,这一结果提示TRPV5-/-小鼠仍保留有破骨细胞介导的骨再吸收功能,可能是TRPV6代偿TRPV5的功能。更多还原

【Abstract】 Gene knockout is a self-developed since the late 80s a new type of molecular biology techniques, is the body through a certain way so that a particular gene inactivation or deletion of technology. Gene knockout technology, commonly used in the establishment of a specific gene deletion in animal models, which carry out related research. By gene knockout to determine the nature of specific genes and to study the impact of its body, whether it is understanding the root causes of the disease or to find the target goal of gene therapy are of great significance. At present, the gene knockout mice to study the occurrence of the disease mechanism, diagnosis and treatment of the molecular basis of important experimental material. TRPV5 is a calcium ion-selective channels involved in calcium absorption and re-absorption, its main expression in the kidney distal convoluted/collecting duct, duodenum, and bone osteoclasts fold edge. Alendronate is a third generation of bone resorption inhibitor, at the cellular level, alendronate, primarily by reducing the number of osteoclasts, inhibit osteoclast formation of edge folds, lower mature osteoclast lysosomal enzyme the release, which will reduce osteoclast function. In this study, the introduction of TRPV5 heterozygous knock-out mice, feeding and breeding in accordance with the requirements SPF-class standards of animal husbandry carried out three kinds of offspring phenotype may occur, namely, the wild-type TRPV5+/+, heterozygous TRPV5+/- and homozygous TRPV5-/-, thus a need to conduct genotyping of the offspring, thereby screening out the wild-type TRPV5+/+ and homozygous TRPV5-/-. In order to study of alendronate on TRPV5+/+ and TRPV5-/- mice with bone, kidney, duodenum calcium ion channel expression, this experiment will TRPV5+/+ and TRPV5-/- mice were divided into four groups kept in metabolic cages, in which two groups of mice of different genotypes were given alendronate weekly 2mg/kg once a row for 8 weeks, collecting blood and urine specimens and do biochemical analysis, mouse femur, kidney, duodenum sample preparation. Extracted from mouse femur bone broken cell culture, and its surface folds calcium channel on the edge of a quantitative polymerase chain reaction analysis, application uCT right femur scan, and calcium absorption peak for testing. Application of alendronate treated TRPV5+/+ mice increased the thickness of the femur, while the TRPV5-/- mouse femoral thickness reduced to normal, the expression of both TRPV5 was significantly increased, while the kidney and the duodenum calcium channel has not changed. Two kinds of genotypes of mice osteoclasts folds on the edge of CLC-7 protein and vacuolar H+-ATPase expression in no significant change, however, alendronate significantly increased TRPV5-/- mice, PTH hormone levels and expression. Osteoclast culture medium TRPV5, CLC-7, and the vacuolar H+-ATPase were not affected by the effects of alendronate, TRPV5-/- mice with bone marrow culture to reduce bone resorption, the two pairs of vitamin D, Reaction was no significant difference. Alendronate prompted TRPV5+/+ mice TRPV5 genes increases, which will reduce TRPV5-/- mice osteoclasts absorption shows TRPV5 functions in osteoclasts play a very important role. Studies have shown that TRPV5-/- mice, bone re-absorption process is still ongoing, because alendronate can TRPV5-/- mice, normalization of bone thickness.更多还原