The application of the transcription factor related to urinary stone formation to elucidation and gene therapy

尿路结石形成相关转录因子在阐明及基因治疗中的应用

• 批准号: 14571513
• 负责人: TOZAWA Keiichi
• 金额: $ 2.05万
• 依托单位: Nagoya City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571513/
• 关键词: osteopontin; focal adhesion kinase; autocrine; MDCK cells; ノックアウトマウス; 尿路結石症; 遺伝子治療

【Purpose】The formation of urinary tract stones is a self-defense reaction to eliminate oxalic acid. Does there exist an OPN-secreting system where the self-stimulation of OPN (adhesion) triggers the secretion of OPN? We speculated that the expression of the OPN protein may be self-regulated by autocrine mechanism. Therefore, we examined the expression of OPN with the phosphorylation of FAK using MDCK and NRK-52E. Introduction Focal adhesion kinase (FAK), a phosphoenzyme, is also a substrate for tyrosine phosphorylation by pp^<60V-Src>. In the renal tubule cells, signal transmission is considered to occur and the expression of various genes in the cell regulated by the tyrosine-phosphorylation of proteins such as FAK and cell structural proteins with the adherence of integrin and osteopontin (OPN). Recently, many results about OPN on stone formation have been reported as follows;1)Osteopontin (OPN) plays an important role in the formation of calcium stones.2)Integrin□V□3,the ligand of O … More PN is expressed in the canine distal renal tubule (MDCK) cells. We speculated that this phosphorylation pathway would be activated and OPN secretion is induced more and more in stone formation.【Methods】MTT assay;MDCK cells (cell density 1 x 10^7/ml) were cultured for 4 hours on a 96-well plate coated with OPN protein at a concentration of 1 -1000 ng/ml. Then, the number of adherent cells was examined by an MTT assay.Western Blotting, Northern Blotting;MDCK and NRK-52 cells (1 x 10^7/ml) stimulated for 4 hours with 1 -1000 ng/ml OPN protein. Then proteins and mRNA were extracted and Western blotting and Northern blotting were performed. (Before Western blotting, the cells were washed with PBS to remove the OPN protein).Immunoprecipitation;MDCK cells (cell density 1 x 10^7/ml) stimulated for 4 hours with OPN protein at a concentration of 1 -1000 ng/ml. Then, immunoprecipitation was performed using an FAK antibody.Phosphorylation Assay;The protein obtained by immunoprecipitation was subjected to Western blotting with anti-phosphotyrosine antibody (x1000).【Results】1)The number of MDCK cells adhering to the dish increased with the concentration of the OPN protein coating the plate.2)The phosphorylation of FAK was promoted by the cell (integrin) -extracellular matrix (OPN) adherence.3)The expression of OPN-mRNA and OPN protein increased with the concentration of OPN stimulating the MDCK cells and NRK-52 cells.【Discussion】The expression of OPN protein on the MDCK cells was increased by the stimulation by the adherence of OPN t6 the cells. An autocrine mechanism was found to exist, and the phosphorylation of FAK was suggested to be involved in the signal transmission in the cell. This autocrine mechanism is suspected to be involved in stone formation. Less

【目的】尿路结石的形成是一种清除草酸的自卫反应。是否存在一个OPN分泌系统，其中OPN的自我刺激（粘附）触发OPN的分泌？我们推测OPN蛋白的表达可能通过自分泌机制进行自我调节。因此，我们使用MDCK和NRK-52E检测了OPN与FAK磷酸化的表达。Focal adhesion kinase （FAK）是一种磷酸化酶，也是pp^<60V-Src>磷酸化酪氨酸的底物。在肾小管细胞中，信号传递和细胞内各种基因的表达被认为是由FAK和细胞结构蛋白的酪氨酸磷酸化以及整合素和骨桥蛋白（OPN）的粘附所调节的。近年来，关于OPN对结石形成的影响的研究结果有：1)骨桥蛋白（Osteopontin， OPN）在钙结石的形成中起重要作用。2)整合素□V□3，O的配体。更多PN在犬肾远端小管（MDCK）细胞中表达。我们推测这一磷酸化途径在结石形成过程中被激活，OPN分泌越来越多。【方法】MTT法；MDCK细胞（细胞密度为1 × 10^7/ml）在包被OPN蛋白的96孔板上培养4小时，浓度为1 -1000 ng/ml。然后用MTT法检测贴壁细胞的数量。Western Blotting, Northern Blotting；用1 -1000 ng/ml的OPN蛋白刺激MDCK和NRK-52细胞（1 × 10^7/ml） 4小时。提取蛋白和mRNA，进行Western blotting和Northern blotting。（Western blotting前，用PBS洗涤细胞，去除OPN蛋白）免疫沉淀；MDCK细胞（细胞密度为1 × 10^7/ml）用浓度为1 -1000 ng/ml的OPN蛋白刺激4小时。然后，使用FAK抗体进行免疫沉淀。磷酸化检测;免疫沉淀获得的蛋白用抗磷酸酪氨酸抗体（x1000）进行Western blotting。【结果】1)随涂层OPN蛋白浓度的增加，MDCK细胞贴壁数量增加。2)细胞（整合素）-细胞外基质（OPN）粘附促进了FAK的磷酸化。3) OPN- mrna和OPN蛋白的表达随OPN浓度刺激MDCK细胞和NRK-52细胞而增加。【讨论】OPN黏附于MDCK细胞后刺激细胞内OPN蛋白表达增加。发现存在自分泌机制，提示FAK的磷酸化参与了细胞内的信号传递。这种自分泌机制被怀疑参与了结石的形成。少

项目成果

Kenjiro Kohri et al.: "Identification of promoter regions involved in cell-and development stage-specific osteopontin expression in bone kidney, placenta, and mammary gland : an analysis of transgenic mice"Journal of Bone and Mineral Research. 19. 78-88 (

Kenjiro Kohri 等人：“骨肾、胎盘和乳腺中参与细胞和发育阶段特异性骨桥蛋白表达的启动子区域的鉴定：转基因小鼠的分析”《骨与矿物研究杂志》。

Kenjiro Kohri et al.: "Medical prevention of urolithiasis"Japanese Journal of Urological Surgery. 16. 1133-1139 (2003)

Kenjiro Kohri等人：“尿石症的医学预防”日本泌尿外科杂志。

郡 健二郎 他: "尿路結石症の予防医学"泌尿器科外科. 16. 1133-1139 (2003)

Kenjiro Gun 等人：“尿石症的预防医学”《泌尿外科》16. 1133-1139 (2003)。

Takahiro Yasui, et al.: "Ostepontin regulates adhesion of calcium oxalate crystals to renal epithelial cells"International Journl of Urology. 9(2). 100-109 (2002)

Takahiro Yasui 等人：“骨桥蛋白调节草酸钙晶体与肾上皮细胞的粘附”国际泌尿学杂志。

Kenjiro Kohri et al.: "Identification of promoter regions involved in cell-and development stage-specific osteopontin expression in bone, kidney, placenta, and mammary gland : an analysis of transgenic mice"Journal of Bone and Mineral Research. 19. 78-88

Kenjiro Kohri 等人：“骨、肾、胎盘和乳腺中参与细胞和发育阶段特异性骨桥蛋白表达的启动子区域的鉴定：转基因小鼠的分析”《骨与矿物研究杂志》。