Osteopontin antisense oligonucleotide inhibits adhesion of calcium oxalate crystals in Madin-Darby canine kidney cell
Osteopontin antisense oligonucleotide inhibits adhesion of calcium oxalate crystals in Madin-Darby canine kidney cell
复制标题
DOI：
10.1016/s0022-5347(01)62604-1
复制
发表时间：
1998-10-01
影响因子：
6.6
通讯作者：
Kurita, T
中科院分区：
文献类型：
--
作者：
Yamate, T;Kohri, K;Kurita, T
目的：我们先前认为骨桥蛋白(OPN)在尿石症早期沉积钙晶体与细胞黏附的过程中起重要作用。为了进一步证实这一理论，我们试图在翻译水平上抑制OPN的表达，并检测其对晶体形成和黏附过程的细胞生物学影响。材料和方法：我们在Madin Darby犬肾(MDCK)细胞中合成了与OPN编码序列的适当部分相对应的反义和正义寡核苷酸。在脂质体DOTAP的辅助下，将反义和正义寡核苷酸导入融合单层培养的MDCK细胞中。进一步孵育后，用免疫荧光显微镜观察细胞内OPN表达的抑制情况，并通过钙-45掺入结石和扫描电子显微镜观察草酸钙晶体的形成。结果：当反义寡核苷酸浓度高于20mM时，OPN的合成受到抑制。在20mM以上的浓度范围内，加入浓度依赖的寡核苷酸可抑制~(45)Ca在结石中的掺入，在50mM时，~(45)Ca的掺入被抑制了90%以上。结论：OPN是细胞外基质中的主要成分，参与远端肾小管细胞内钙晶体的形成和黏附，提示OPN在尿石症早期刺激结石晶体沉积和黏附中起重要作用。
Purpose: We previously suggested that osteopontin (OPN) plays an important role in the process of deposited calcium crystals adhesion to cells in the early stages of urolithiasis. To further confirm this theory, we tried to inhibit OPN expression at the translational level and examined its cellular biological consequence on the formation and adhesion process of crystals.Materials and Methods: We synthesized antisense and sense oligonucleotide corresponding to an appropriate part of the coding sequence for OPN in Madin Darby canine kidney (MDCK) cells. With the aid of lipofection reagent DOTAP, antisense and sense oligonucleotide were introduced into MDCK cells grown in a confluent monolayer. After further incubation, inhibition of OPN expression in the cells was assessed by immunofluorescence photomicrography, and formation of calcium oxalate crystals was quantitated by incorporation of Ca-45 into the stone and visualized by scanning electron microscopy (SEM).Results: Antisense oligonucleotide at concentrations higher than 20 mu M inhibited synthesis of OPN. Incorporation of 45Ca into the calculus stone was inhibited by the addition of oligonucleotide in a concentration dependent manner in a range above 20 mu M. More than 90% of incorporation was inhibited at 50 mu M as compared to control. Inhibition of calcium crystal formation was confirmed by SEM.Conclusions: OPN was shown as a major component in the extracellular matrix involving the formation and adhesion of calcium crystals in the distal renal tubular cells, suggesting that OPN plays an important role in stimulating deposition and adhesion of calculus crystals to cells in the early stages of urolithiasis.