流体剪切力致破骨细胞钙响应在骨代谢中的意义探讨

Bone and Joint bracket constitute the human body. Normal bone metabolism play important role in human life. The balance of osteoblastic bone matrix formation and osteoclast bone matrix absorption is an important part of bone metabolism. Many studies have demonstrated that calcium signaling and mechanical factors have a significant impact on bone metabolism, but the mechanism is not fully understood..This object pays more attention to osteoclasts,due to their unique absorption function in the bone matrix. Our previous study showed that fluid shear stress(FSS) can induce calcium response in osteoclast, and is closely related to cell differentiation. For the understanding of its mechanism, seven signaling pathways have been blocked to be compared with normal group. The conclusion was that FSS may activate mechanosensitive calcium channel(MSCC) firstly. Then extracellular calcium influx and phospholipase C (PLC) promote calcium release from endoplasmic reticulum (ER), subequently, forming a calcium peak. Although we carried out the research, we still don't know if FSS have influence to osteoclast function and bone metabolism..In this project, our team plans to use flow chamber system, intracellular calcium staining, long-term cell culture system and animal experiments to validate the importance of calcium signaling on bone metabolism. Furthuremore, it may provide some evidence for regulating bone metabolism.

骨与关节构成人体支架，维持正常骨代谢对人体生命活动具有重要意义。成骨细胞骨基质形成和破骨细胞骨基质吸收是骨代谢的重要环节，许多研究表明力学因素和钙信号对骨代谢有重要影响，但具体机制尚不完全清楚。.破骨细胞作为唯一具有骨基质吸收功能细胞，是本项研究的重点。申请者之前的研究表明流体剪切力可促进破骨细胞产生钙响应，且与剪切力大小和细胞分化状态密切相关。为认识其发生机制，阻断了可能产生钙信号7种途径，得出流体剪切力作用于破骨细胞后可能通过活化细胞表面机械敏感钙离子通道促进细胞外钙离子内流，进而活化磷酸酯酶C,促进内质网内钙离子释放进而形成钙响应峰。但对流体剪切力能否通过钙响应影响破骨细胞功能及骨代谢尚不清楚。.本项目拟利用流动腔技术、钙离子染色、破骨细胞长时程培养技术和动物实验进一步验证此过程对骨代谢的重要性，阐明相关规律，为临床上通过剪切力和钙信号调控骨代谢提供实验依据。

.力学因素影响骨代谢相关疾病进程，认识相关机制对此类疾病防治有重要意义。本课题在执行过程中明确了早期或晚期分化阶段破骨细胞在流体剪切力（Fluid shear stress, FSS）刺激下主要通过STIM1或TRPV4促进细胞外Ca2+内流。长时程FSS刺激（20 dyne/cm2×2小时/天×12天）可抑制破骨细胞形成和骨基质吸收功能，且主要是由于FSS激活TRPV4通道后破骨前体细胞内钙储备减少，进而融合形成破骨细胞和骨基质吸收能力减弱。.为进一步认识力学刺激、TRPV4和骨代谢疾病之间关系，我们使用正常大鼠和TRPV4低表达大鼠来设计对照、尾吊和尾吊后再运动（每天尾吊23小时后运动1小时）等动物模型，结果表明正常大鼠尾吊后再运动中不出现明显骨质疏松，而TRPV4低表达大鼠组中出现明显骨质疏松。TRPV4激动剂GSK1016790A可部分缓解大鼠骨质疏松，而对大鼠股骨髓腔内注射质粒病毒降低TRPV4表达可对治疗骨质硬化症有一定帮助。.实验中构建了将抗体、多相DNA和confocal相结合的MD HCR用于破骨细胞TRPV4高效检测，这一方法也可用于其他细胞膜蛋白的高灵敏度检测，对临床诊治相关疾病有一定帮助。STIM1在伴有成骨细胞化生的口腔癌组织中多呈高表达并且会促进肿瘤进展。本课题基本完成从FSS致破骨细胞钙响应机制探讨到动物实验相关工作，并发现通过激活或抑制TRPV4通道功能可缓解大鼠骨质疏松症和骨质硬化症状，为骨代谢相关疾病的诊治提供参考。.