压应力对成骨细胞的成骨和破骨双向效应的关键信号通路研究

The signaling pathways in osteoblasts induced by mechanical stimulus have been concerned in recent years. Owing to lack of direct evidence from one experimental system, it is inadequate to make the conclusion about the exact biological effect of compressive stress on osteoblasts. Based on the study of a bone compression canine model, we hypothesize that a dual effects of osteoclastogenesis and osteogenesis could be induced by compressive stress and p38 MAPK signaling is the key signaling pathway. In this project, parallel in vivo and in vitro experiments are designed according to the osteoprotegerin/receptor activator of nuclear factor-kappa B ligand/receptor activator of nuclear factor-kappa B (OPG/RANKL/RANK) signaling axis of bone remodeling. After establishment of a jaw compression canine model and a mechanical compressive stress-loaded system of osteoblasts in three-dimensional (3D) culture condition, expression of OPG/OPG mRNA and RANKL/RANKL mRNA is detected by fluorescent immunohistochemistry staining and real-time RT-PCR under a serial of compressive stress, and the conditioned medium of osteoblasts are collected and then incubated with monocyte/macrophage cell line（osteoclast precursor） to determine the inhibition effect on differentiation of the osteoclast precursors by tartrate-resistant acid phosphatase staining. After this, the optimal stress capable of inducing osteoclastogenesis or osteogenesis is screened respectively. Then the possible early signaling pathways of p38 MAPK and ERK1/2 signaling associated with osteogenesis and osteoclastogenesis induced by the optimal stress are measured by immunohistochemistry staining and western blot so as to preliminarily confirm the key role of p38 MAPK signaling from the two aspects of in vivo and in vitro experiments. Finally, the key role of p38 MAPK signaling signaling is confirmed reversely by the application of selective p38 MAPK inhibitor SB203580 and ERK1/2 inhibitor PD98059 and siRNA-p38alpha interfering. The expression of OPG/OPG mRNA, RANKL/RANKL mRNA,total p38 MAPK, phosphorylated p38 MARK, total ERK1/2 and phosphorylated ERK1/2 is determined as above. Based on this project, the biomechanical theory is expected to be enriched by revealing the mechanism of the dual effects of compressive stress on osteoblasts.

骨在机械力作用下的信号转导通路是近年来的研究热点。目前压应力对骨的生物学效应研究还欠充分，缺乏在同一实验体系下发生成骨和破骨作用的直接证据。基于前期骨压缩动物模型研究的基础，我们提出压应力对成骨细胞有成骨和破骨双向效应，且p38MAPK通路是其关键信号通路的工作假设。本项目紧扣骨改建的OPG/RANKL/RANK信号轴心，设计体内外平行实验，建立犬颌骨加压模型和三维培养的成骨细胞加压体系，施加系列压应力，免疫组化、实时定量PCR检测OPG、RANKL表达，筛选产生成骨和破骨的最佳压应力。进而免疫印迹法等检测此最佳压应力下成骨细胞中ERK1/2和p38MAPK等早期信号通路激活情况，从体内外两方面初步确认p38MAPK通路的作用。最后用信号通路特异性抑制剂及siRNA低敲成骨细胞p38MAPK表达，从反向验证p38MAPK通路的作用。从而揭示压应力对骨生物力学效应的机制，丰富骨生物力学理论。

传统观点认为骨在压缩力和牵张力作用下会产生两方面的效应，牵张产生张应力，促进骨生成；压缩产生压应力，促进骨吸收。这是口腔正畸治疗时牙齿移动的生物学基础。但长期在空间站工作的宇航员会出现骨质疏松的症状，提示一定程度的压应力可促进骨生成。因此我们提出，压应力对骨可产生双向效应，即压应力既可产生破骨效应，又可产生成骨效应。.我们的研究包括：①成功建立成骨细胞三维培养及施加压应力的实验体系；在此基础上，②观察压应力诱导成骨细胞的成骨和破骨双向调节效应：从小鼠MC3T3细胞系及小鼠颅骨原代细胞形态、活性、成骨分化水平和对破骨细胞分化的诱导等方面，发现2g/cm2和5g/cm2可能是压应力成骨和破骨双向调节的关键力值。其中2g/cm2时Runx2、Alp及Ocn的mRNA及细胞蛋白表达上调，但RanklmRNA也上调，Rankl/OpgmRNA比值升高，共培养条件下的破骨细胞前体细胞Raw264.7中的TRAP酶活性增加，成骨分化增强，而破骨分化诱导亦增强；5g/cm2时OpgmRNA和蛋白显著上调，Raw264.7中的TRAP酶活性下降，破骨分化诱导受抑制。③压应力条件下成骨和破骨效应诱导的信号通路：在2g/cm2及5g/cm2压应力条件下，蛋白芯片检测发现ERK1/2、P38MAPK及JNK磷酸化水平均随强度增加而表达增强，提示传统MAPK信号通路参与了压应力应答；而mTOR及RSK2在2g/cm2时表达减弱，5g/cm2时恢复原有表达水平，提示其参与了成骨及破骨应答的方向性调节。④Laptm5在成骨和破骨双向调节中的作用：Runx2siRNA和Runx2过表达载体转染MC3T3细胞，发现干扰Runx2可下调Laptm5的表达，Runx2过表达可上调Laptm5的表达。采用ChIP及荧光素酶报告基因载体检测发现Runx2在Laptm5在ATG上游973bp处的P2有结合位点。将启动子载体pGL3-1000bp与Runx2过表达载体共转染细胞，发现Runx2过表达能增强Laptm5转录活性。⑤对Beagle犬牙槽骨CT扫描及植入支抗钉发现，发现犬牙槽骨可供植入支抗钉的位点选择不多，根分叉区可作为植入点选择之一，支抗钉直径越粗稳固性越好。.本项目研究从一个方面揭示骨在压应力条件下的生物学反应机制，对骨折治疗、正畸牙移动、骨质疏松、骨组织工程乃至航天医学等领域的研究都有参考价值。