【作者】 翁土军；

【导师】 杨晓；

【作者基本信息】 中国人民解放军军事医学科学院 ， 遗传学， 2009， 博士

【摘要】 坚硬的骨骼能够支撑机体、保护内脏。骨的强度取决于骨量和骨的结构。骨骼是一个动态的、处于新陈代谢的组织——骨重建过程中破骨细胞不断吸收旧骨、成骨细胞不断形成新骨,成年机体通过骨吸收和骨形成的动态平衡维持骨量的稳态。骨重建是由破骨细胞、成骨细胞、骨细胞共同精确调节的动态过程,它们之间相互耦联使骨吸收和骨形成保持动态平衡。成骨细胞和破骨细胞的活性受到生长因子、细胞因子、激素和力学刺激等多种因素的调控。加强成骨细胞的基础研究,对于促进骨形成类药物的研发和骨代谢疾病的防治有重要意义。磷脂酰肌醇3-激酶(phosphoinositide 3 kinase,PI3K)/AKT信号通路通过调节细胞的增殖、分化、迁移和凋亡而具有广泛的生物学效应。但是,PI3K/AKT信号通路在成骨细胞及骨稳态维持中的功能还缺乏足够的体内证据。本研究利用组织特异性条件基因敲除技术,探讨了能影响PI3K/AKT信号通路的两个分子Pten和Gab1基因在成骨细胞中的功能及其对骨代谢的调控。10号染色体缺失的磷酸酶及张力蛋白同源物(Phosphatase and Tensin homolog deleted from chromosome 10,Pten)是一个重要的抑癌分子,它通过负向调控PI3K/AKT信号通路来影响细胞的生物学功能。在本研究中,我们基于Cre-LoxP系统获得了成骨细胞特异性Pten基因敲除小鼠,发现Pten基因敲除小鼠发生骨质硬化症。我们从影像学、组织学、细胞和分子水平研究了Pten基因在骨稳态维持中的功能,并分析了成骨细胞中Pten基因影响骨重建的可能机制。我们利用本室研制的骨钙素启动子指导的Cre重组酶转基因小鼠同Pten条件基因打靶小鼠交配,获得了成骨细胞特异性Pten基因敲除小鼠(Ptenflox/flox; OC-Cre)。检测4月龄小鼠股骨的骨密度,发现无论是雄性还是雌性Pten基因敲除小鼠比同窝同性别对照小鼠的骨密度明显升高。我们用μCT对4月龄小鼠股骨进行了扫描和三维重建,股骨的纵向扫描图显示Pten基因敲除小鼠骨小梁体积增加,靠近膝关节侧松质骨三维重建图显示Pten基因敲除后骨小梁增多变厚,横断面扫描发现Pten基因敲除后皮质骨增厚、骨髓腔变小。接着,我们对小鼠胫骨的骨重建进行了组织和细胞的骨形态计量分析。胫骨塑料切片Von Kossa染色显示,Pten基因敲除后骨小梁增加。骨计量结果显示,与对照小鼠相比Pten基因敲除小鼠骨体积、骨小梁厚度,成骨细胞数量均明显增加。体内钙黄绿素标记显示Pten基因敲除小鼠骨形成率(BFR/BS)和骨矿化率(MAR)较对照小鼠明显提高。这些结果显示Pten基因敲除导致骨形成增加。我们分离原代成骨细胞诱导培养,茜素红染色结果显示矿化结节增多。在前成骨细胞系干涉Pten基因,Westernblot结果显示AKT通路激活,前成骨细胞分化加快。接着我们用抗酒石酸酸性磷酸酶(TRAP)染色鉴定破骨细胞,骨计量结果显示Pten基因敲除小鼠破骨细胞数量(N.Oc/B.Pm)增加。荧光定量PCR结果显示,Pten基因敲除后破骨细胞的功能分子TRAP和组织蛋白酶K(CathK)的表达都明显升高。这些结果表明Pten基因敲除小鼠骨吸收增加。但是,小鼠血清ELISA分析结果显示耦联调控破骨细胞分化的OPG、RANKL没有明显变化,提示成骨细胞Pten基因缺失可能通过别的机制调节破骨细胞分化。荧光定量PCR结果显示,Pten基因敲除小鼠骨组织中单核细胞趋化因子(MCP-2、MCP-5、SDF-1)表达明显升高,提示成骨细胞Pten基因缺失可能通过增加趋化因子的表达招募更多的破骨细胞前体至骨微环境中发育为破骨细胞。Gab1(Grb2-associated binder 1)是一个锚定蛋白,能被多种酪氨酸激酶受体激活,主要通过PI3K/AKT和Ras/MAPK向下传递胞外多种生长因子、细胞因子的刺激。我们首次建立了成骨细胞特异性Gab1基因敲除小鼠(Gab1flox/flox; OC-Cre)模型,发现Gab1基因敲除小鼠发生骨质疏松。本研究通过影像学、组织形态计量、细胞和分子多层面分析证实Gab1基因在维持骨稳态过程中的重要作用,并用三点弯曲力学实验证明Gab1基因敲除小鼠股骨生物力学性能下降。我们检测分析了2月龄和6.5月龄雄性小鼠的骨密度。与同窝对照相比,Gab1基因敲除小鼠的骨密度明显下降。软X线相片显示6.5月龄Gab1基因敲除小鼠椎骨、股骨的骨密度明显加少。μCT扫描和三维重建的结果显示,Gab1基因敲除小鼠骨小梁减少、皮质骨变薄。我们对2月龄小鼠的骨形成和骨吸收进行了细致分析,发现Gab1基因敲除小鼠骨体积明显下降、成骨细胞数量和破骨细胞数量减少,体内钙黄绿素标记显示Gab1基因敲除小鼠骨形成率(BFR/BS)和骨矿化率(MAR)明显降低。这些结果表明Gab1基因敲除小鼠发生低转换率的骨质疏松。通过Gab1基因敲除小鼠原代成骨细胞的研究,发现Gab1基因敲除后成骨细胞矿化减少、凋亡增加、表达RANKL较少从而支持破骨细胞分化的能力减弱。前成骨细胞干涉Gab1基因,也证实Gab1表达下降的细胞矿化减少、凋亡增加。Gab1基因敲除的成骨细胞在IGF-1和insulin刺激时不同时间点p-AKT和p-ERK的水平较野生型明显降低,表明Gab1基因参与IGF-1和insulin对成骨细胞AKT和ERK的激活。综上所述,本研究揭示了能够正、负调控PI3K/AKT信号通路的两个分子Gab1和Pten在骨代谢调控和骨稳态维持中的生理功能。研制成功的Pten基因敲除导致骨质硬化症小鼠,以及Gab1基因敲除导致骨质疏松小鼠为进一步深入研究骨稳态维持的分子机制提供了新的实验动物模型。更多还原

【Abstract】 A rigid skeleton makes it possible to support weight and ensures protection for the organs. Bone strength is determined by bone mass and structure. Bone is a dynamic structure with a continuous tissue renewal called remodeling in which osteoclasts responsible for bone resorption and osteoblasts responsible for bone formation, and the maintenance of bone mass is determined by the coupling of bone resorption to bone formation. Bone remodeling is a danymic process performed by the coordinated activities of osteoclasts, osteoblasts and osteocytes, and tightly controlled by the interaction of these cells. The activation of osteoblasts and osteoclasts is regulated by growth factors, cytokines, hormones and mechanical forces. Further understanding of osteoblastic bone formation is critical for the development of bone anabolic agents and the treatment for osteoporosis.PI3K (phosphoinositide 3 kinase)/AKT signaling regulate a number of biological processes such as proliferation, differentiation, migration and survival. However, the function of PI3K/AKT signaling on osteoblasts and bone homeostasis in vivo is poorly understood. In the current study, we explored the function of Pten and Gab1 genes which are involved in the regulation of PI3K/AKT signaling in osteoblasts and bone metabolism,.Pten (phosphatase and tensin homolog deleted from chromosome 10) is an important tumor suppressor, participating in the regulation of diverse cellular biological process by suppressing PI3K/AKT signaling. In this study, we created osteoblast-specific Pten knockout mice (Ptenflox/flox;OC-Cre) using the Cre–LoxP system. We found that Pten mutant mice exhibited osteopetrosis phynotype. We studied the function of Pten in osteoblasts and bone homeostasis and explored the underlying molecular mechanism by combining radiological, histological, cellular and molecular methods.Osteoblast-specific Pten mutant mice were obtained by breeding OC-Cre transgenic mice created by ourselves previously with Ptenflox/flox mice. Femurs from Pten mutant mice of both sexes displayed significant increases in bone mineral density (BMD) relative to their wild-type controls at 4 month of age. We performed three-dimensional micro computed tomography (μCT) analysis on femurs from 4-month-old mice. Pten mutant femurs showed pronounced increases in trabecular bone volume in longitudinal section and distal femur region. Trabeculae from mutant femurs were increased and became thicker compared with those of controls. Cortical thickness in the midshaft femurs was increased while bone cavity was decreased significantly. Furthermore, we performed bone histomorphometric analyses of the proximal tibias. Von Kossa staining showed a significantly increased bone volume in the mutant mice. The trabecular bone volume/tissue volume ratio (BV/TV) in tibias of Pten deficient mice was increased relative to that of controls at the age of 4 months. In addition, trabecular thickness (Tb.th) and number of osteoblasts/bone perimeter (N.Ob/B.Pm) were significantly increased in Pten mutants. Double calcein labeling revealed a dramatically increased bone formation rate (BFR) and mineral appositional rate (MAR) in 4-month-old mutant mice compared with controls. These results showed that disruption of Pten in osteoblasts leads to increased bone formation. Additionally, primary calvarial osteoblasts culture exhibited increased bone nodules in Pten mutants compared with controls. We also found that interference of Pten in preosteoblasts increased activation of AKT and accelerated osteoblast differentiation. We next analyzed the osteoclasts using tartrate-resistant acid phosphatase (TRAP) staining. Bone histomorphometric analyses revealed that the number of mature osteoclasts (OcN/BPm) was significantly increased in Pten mutant mice. Real-time PCR analyses displayed that the expression of TRAP and cathepsin K (CathK), which are lysosomal enzymes essential for osteoclastic bone resorption, were markedly increased in femoral bones of Pten mutant mice. These results indicated that disruption of Pten in osteoblasts leads to increased bone resorption. However, the expression levels of osteoprotegerin (OPG) and the ligand receptor activator of NF-κB (RANKL) in serum were comparable between control and Pten mutant mice, indicating that increased osteoclast numbers in Pten mutant mice is driven by other regulator but not by RANKL or OPG. Real time PCR showed that Pten muant bone enhanced expression of the monocyte chemoattractants (MCP-2, MCP-5, SDF-1), which may recruit osteoclast precursors to bone and influence osteoclastogenesis.The Grb2-associated binder 1 (Gab1), which serves as a scaffolding adaptor protein, is phosphorylated by diverse receptor tyrosine kinases, and subsequently activate PI3K/AKT and Ras/MAPK (mitogen activated protein kinase) pathways transmitting key signals from cytokine and growth factor. We generated osteoblast-specific Gab1 mutant mice (Gab1flox/flox;OC-Cre) and provided the first evidence that Gab1mutant mice developed osteoporosis. In this study, we showed that the molecular scaffold Gab1 was critical for normal postnatal bone homeostasis verified by combined radiological, histological, cellular and molecular methods. Three-point bending test demonstrated that femurs from Gab1 deficiency mice exhibited decreased biomechanical properties. Bone mineral density (BMD) from femurs of mutant mice is significant decreases relative to that of controls at 2 months and 6.5 months old. Soft x-ray analyses of vertebrae and femora revealed that Gab1 mutant mice exhibited lower bone mineral density than their wild-type littermates at 6.5 months of age.μCT analysis revealed that decreased trabecular bone volume and cortical thickness from Gab1 mutant femurs compared with controls. We also performed analyses about bone formation and bone resorption at 2-month-old proximal tibias. Bone histomorphometric analyses revealed that both bone formation and bone resorption parameters, the trabecular bone volume/tissue volume ratio, the number of osteoblasts/bone perimeter (N.Ob/B.Pm and the percentage of bone surface covered by mature osteoclasts (OcS/BS), were significantly decreased in in Gab1 mutant mice compared with controls. Double calcein labeling revealed a dramatically declined bone formation rate/bone surface (BFR/BS) and mineral appositional rate (MAR) in 2-month-old mutant mice compared with controls. These data showed that Gab1 mutant mice developed low turnover osteoporosis. In vitro primary osteoblasts culture systems showed that Gab1 deficiency in osteoblasts caused three cell autonomous abnormalities: suppressed osteoblast mineralization, increased susceptibility to apoptosis and decreased RANKL expression to support osteoclastogenesis. We also found that interference of Gab1 in preosteoblasts increased susceptibility to apoptosis and decreased osteoblast mineralization. In addition, we found that phosphorylation of AKT and ERK was significantly decreased in mutant osteoblasts under stimulation by IGF-1 and insulin, suggesting that Gab1 has a critical role in mediating insulin and IGF-1 stimulated activation of AKT and ERK in osteoblasts.In conclusion, we revealed the physiological functions of Pten and Gab1 genes, which are able to regulate PI3K/AKT pathway by a negative or positive way, in the maintenance of bone homeostasis. The osteoblast-specific Pten knockout mice which developed osteopetrosis and osteoblast-specific Gab1 knockout mice which exhibited osteoporosis will serve as new animal models for further investigation on molecular mechanisms underlying the maintenance of bone homeostasis.更多还原