二维回转培养对MLO-Y4骨样细胞PKD2表达定位及胞内钙信号的影响  

The Effects of 2D-Clinorotation on Expression and Location of PKD2 Protein and the Intracellular Ca^(2+) Concentration of MLO-Y4

作　　者：关莹[1] 续惠云[1] 瓮媛媛[1] 商澎[1] 

机构地区：[1]西北工业大学生命学院特殊环境生物物理学研究所空间生物实验模拟技术国防重点实验室,陕西西安710072

出　　处：《现代生物医学进展》2014年第14期2601-2605,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(30700152;31170812);西北工业大学基础研究基金(NPU-FFR-JC201160)

摘　　要：目的:PKD2(polycystin2,多囊肾病蛋白2)能够在细胞膜上形成无选择性的阳离子通道,在肾上皮细胞中PKD2与初级纤毛共定位,通过改变胞内的钙信号过程参与细胞对力学刺激的响应。本实验通过二维回转培养来模拟失重效应,旨在探讨二维回转培养对MLO-Y4骨样细胞PKD2表达定位,及胞内钙信号的影响。初步了解PKD2在小鼠骨样细胞MLO-Y4响应力学刺激过程中起的作用。方法:采用二维回转培养骨样细胞MLO-Y4,用RT-PCR和western blotting检测PKD2的表达,用荧光共聚焦显微镜检测细胞中PKD2与初级纤毛的定位及细胞内钙离子含量。结果:与对照组相比,在二维回转培养后,骨样细胞MLO-Y4的PKD2表达在mRNA和蛋白水平都有明显的下降,PKD2、PKD1(polycystin1,多囊肾病蛋白1)和乙酰化的α-tubulin共定位,同时二维回转培养降低了细胞内钙离子含量。结论:在二维回转培养下,PKD2可能通过调节自身表达来改变细胞膜上PKD通道的数目和开放情况来影响细胞内钙离子含量,参与骨细胞对细胞外应力的感受过程,其详细机制还有待进一步实验研究。这将对探讨骨细胞响应力学刺激的具体机制提供重要的理论依据。Objective： PKD2（polycystin2） could form nonspecific cation channel on the cytomembrane, which is responsible for Ca^2＋penetration when kidney epithelial cell transforms mechanical stimulation into intracellular chemical information. In this study, 2D-clinorotation was used to simulate weightlessness condition. The aim of this study was to investigate the effects of 2D-clinorotation culture on expression and location of PKD2 protein and the intracellular Ca^2＋concentration of mouse osteocyte-like MLO-Y4 cell. Also we want to understand the role of PKD2 in the process of MLO-Y4 responsding to mechanical stimulation. Methods： Mouse osteocyte-like MLO-Y4 cell were cultured under the condition of 2D-Clinorotation.The expression and location of PKD2 was detected by RT-PCR, western-blotting and fluorescent staining, the intracellular Ca^2＋concentration was detected by Ca^2＋staining technique. Results： Under the condition of 2D-Clinorotation culture, the exspression of PKD2 and the intracellular Ca^2＋concentration were distinctly reduced. At the same time, we found that PKD2 co-localized with PKD1（polycystin1） and the specific ciliary axoneme marker-acetylated α-tubulin. Conclusion： the results suggest under 2D-clinorotation culture, mouse osteocyte-like cell could respond to the mechanical stimulation by regulating the expression of PKD2 and the intracellular Ca^2＋concentration. It is helpful to investigate the cellular mechanism of bone cells in responding to mechanical stimulation.

关 键 词：二维回转培养 模拟失重 PKD2 MLO-Y4 

分 类 号：Q291[生物学—细胞生物学]