Application of Cre-loxP system for elucidation of mechanisms in bone remodeling during tooth movement

当正畸力作用于牙槽骨时，环氧合酶-2被诱导表达，进而合成前列腺素E_2(PGE_2)。据报道，PGE2对骨吸收和骨形成都有很强的刺激作用。这些结果表明，PGE2-PKA信号在正畸牙齿移动中的破骨细胞分化和成骨细胞分化中起重要作用。PGE2-PKA信号区分受压部位的骨吸收和拉伸部位的骨形成的机制在很大程度上还不清楚。本项目的目的是阐明当使用Cre-loxP系统时，体内PGE2-PKA信号调节骨重建的机制。我们建立了在破骨细胞分化早期表达EGFP-Cre融合蛋白的小鼠(RANK-EGFPCre小鼠)。将含有Neo盒的IRES-EGFP-Cre基因插入RANK基因的终止密码子和PolyA序列之间。为检测破骨细胞前体细胞是否表达并发挥作用，将报告基因LacZ导入RANK-EGFPCre小鼠，获得LacZ/RANK-EGFPCre小鼠。对LacZ/RANK-EGFPCre小鼠的股骨进行了组织化学分析。在LacZ/RANK-EGFPCre小鼠的骨髓中检测到大量诱导破骨细胞的LacZ阳性细胞，而在LacZ/野生型小鼠的骨髓中未检测到。Western印迹分析表明，EGFP-Cre蛋白在破骨细胞前体细胞中有表达。总之，RANK-EGFPCre小鼠是分析正畸牙移动过程中破骨细胞前体细胞在牙周组织中分布的有力工具

When orthodontic forces are applied to alveolar bones, cyclooxigenase-2 is inducibly expressed and then prostaglandin E2 (PGE2) is synthesized. PGE2 is reported to have a potent stimulator for both bone resorption and bone formation. These findings suggest that PGE2-PKA signals have an important role in osteoclast differentiation as well as osteoblast differentiation in orthodontic tooth movement. Mechanisms that PGE2-PKA signals discriminate between bone resorption in compressive site and bone formation in tensile site are largely unknown. The purpose of this project is to elucidate mechanisms for regulation of bone remodeling by PGE2-PKA signals in vivo when using Cre-loxP systems. We developed mice expressing EGFP-Cre fusion proteins in early stage of osteoclast differentiation (RANK-EGFPCre mice). IRES-EGFP-Cre cDNA including Neo cassette was inserted between a stop codon and polyA sequences in RANK gene. To test whether cre-recombinase was expressed and worked in osteoclast precursor cells, reporter gene expressing LacZ only after Cre-mediated excision of loxP-flanked DNA sequence was introduce into the RANK-EGFPCre mice, and LacZ/RANK-EGFPCre mice were obtained. Femurs of LacZ/ RANK-EGFPCre mice were histo-chemically analyzed. A lot of LacZ positive cells inducing osteoclasts were detected in bone marrow of LacZ/RANK-EGFPCre mice but not in LacZ/wild type mice. Western blot analyses show that EGFP-Cre protein was expressed in osteoclast precursor cells. Together, RANK-EGFPCre mice are a powerful tool for analysis of distribution of osteoclast precursor cells in periodontal tissue during orthodontic tooth movement