粘附因子Pinch1/2对骨稳态的调控作用及机制研究

STUDY ON THE ROLE AND MECHANISM OF FOCAL ADHESION PROTEINS PINCH1/2 IN THE REGULATION OF BONE HOMEOSTASIS

王依姝

11649007

博士

生物医学工程

肖国芝

2020-06-02

2020-07-27

哈尔滨工业大学

深圳

胶体半导体量子点具有发光波长可调，发光色纯度高，发光波段覆盖可见光区域的优点，在显示和照明领域有广泛的应用。所谓光学微腔，是指在一维或多维方向上尺寸与光波波长相当的微型光学谐振器。微腔结构的量子点发光二极管（quantum dot light-emitting diodes, QLEDs）器件，由于微腔效应，其发光效率相比传统的以ITO 作底电极的底发射器件提高了，电致发光（electroluminescence, EL）光谱的半高宽也相较于非微腔结构底发射器件更窄。基于磷化铟（InP）的QLEDs 器件，其量子点不含镉，不会对人体和环境产生危害，是未来环境友好型显示器件的发展方向。但是基于InP 的QLEDs器件的发光效率相对较低（1.2%），开启电压大（5.4V），发光光谱的半高宽也较宽（full width at half maximum, FWHM）（48 nm），这些问题正好可以利用微腔来改善。本论文首先制备了基于硒化镉（CdSe）的用银作正极和负极的微腔结构的QLEDs 器件，对比分析了微腔结构QLEDs 器件的性能与传统的以ITO 作底电极的底发射器件的性能差异。通过优化腔长，微腔结构的器件的发光效率是底发射器件效率的两倍多，而且发光光谱半高宽从36 nm 减小到了28 nm。骨质疏松症是一种全身性骨骼疾病，其特征是骨量降低和骨组织微结构的退化，继而引发骨骼脆性增加和骨折风险增大。骨质疏松性骨折将消耗巨大的医疗和财政资源。机体通过不断进行骨重塑来维持骨稳态，该过程中破骨细胞分解旧骨质，成骨细胞负责骨质重建。骨细胞是骨组织中数量最多的细胞，可以分泌表达多个重要的骨源性因子，如RANKL、OPG、Sclerostin等调节破骨细胞和成骨细胞的形成和功能。但是目前为止，调控骨细胞功能和存活的关键因子尚不清晰。PINCH蛋白是重要的粘附因子。哺乳动物细胞具有两种功能性PINCH蛋白：PINCH1和PINCH2，它们在细胞骨架组织和细胞外基质粘附、迁移、增殖和存活中发挥重要作用。但PINCH1和PINCH2在调节骨稳态中的作用尚未明确。因此，本文将深入研究PINCH对骨稳态的调控，以期获得骨质疏松的预防和治疗策略的新线索。本文以骨细胞Pinch敲除的小鼠模型为研究对象分析Pinch在调控骨稳态中的作用。使用Micro-CT分析该动物模型的骨量，发现骨细胞Pinch缺失会导致小鼠骨量特别是骨密度降低。骨量受成骨细胞介导的骨形成和破骨细胞介导的骨吸收的共同影响。在研究中发现，钙黄绿素实验结果证实骨细胞Pinch缺失会导致明显的骨形成速率下降即骨细胞Pinch缺失会导致小鼠成骨细胞功能发生改变。TRAP染色的结果显示，破骨细胞的活性在实验组与对照组直接无差异，即骨细胞Pinch缺失并不影响骨吸收，尽管骨细胞Pinch缺失会导致Rankl表达上调，但Opg也出现了等比例的上调。骨细胞中单独敲除Pinch1或者全身敲除Pinch2对骨量均无影响，提示Pinch1和Pinch2在调控骨稳态的过程中存在功能代偿。骨细胞Pinch缺失小鼠的骨丢失是由于骨形成的改变导致的。本文进一步了研究Pinch调控骨稳态的分子机制。结果显示，Pinch1可以与Igf1r直接结合，这一定程度上影响Sclerostin的表达。我们同时在体外验证了这个结果，在MLO-Y4细胞中使用CRISPR-Cas9的技术敲出了Pinch1得到了同样的结果。骨细胞中Sclerostin的高表达只在骨骼局部，这一方面影响骨髓间充质干细胞的分化，使其成脂分化能力增强，成骨分化能力减弱。骨髓间充质干细胞的这种变化可能是由于骨髓间充质干细胞中Yap1/Taz表达量降低导致。另一方面骨细胞Pinch缺失抑制了原代成骨细胞中的Wnt/β-Catenin信号通路，Wnt/β-Catenin信号通路是调控骨形成最重要的信号通路之一。就骨细胞本身而言，Pinch缺失导致细胞凋亡增加但并不影响骨细胞的自噬，这是整合蛋白的表达降低影响的。骨细胞是长骨的机械感应装置，机械应力是维持骨稳态的重要因素。为研究Pinch是否参与骨细胞的机械信号传导，我们采用了小鼠后肢减载和尺骨加力两个模型。在小鼠后肢减载模型中，骨细胞Pinch缺失小鼠出现了更严重的骨丢失。在小鼠尺骨加力模型中，对照组小鼠的皮质骨受机械力的刺激会出现骨基质厚度增加，但是这种改变在骨细胞Pinch缺失小鼠中被削弱。小鼠尺骨加力后，对照组小鼠骨形成能力增强的表型在实验组中丢失。这些结果均提示骨细胞Pinch缺失后，骨细胞对机械信号的响应变弱。综上所述，本文阐述了Pinch蛋白在调节骨稳态中起关键作用，本研究的成果提示骨细胞Pinch蛋白可作为防治骨质疏松症的潜在靶分子。

Osteoporosis is a systemic skeletal disease characterized by the decrease of bone mass and the microarchitectural deterioration of bone tissue, leading to an increment of bone fragility and fractures risk. Nowadays, it causes huge financial burden and consumes a lot of medical resources with ageing of the population. The body maintains bone mass homeostasis through continuous bone remodeling, in which osteoclasts break down the old bone matrix while osteoblasts complete bone reconstruction. Osteocytes are the most abundant cells in bone tissues which derived from bone marrow mesenchymal stem cells, and they make a great contribution to bone homeostasis maintainence in physiological and disease states. Osteocytes can secrete and express many key factors, such as RANKL, OPG, and Sclerostin, etc. controlling the formation and function of osteoclasts and osteoblasts. Osteocytes are long-lived cells and some will die by apoptosis, however, the key factors controlling their function and survival are still unknown until now. Mammalian cells have two functional PINCH proteins, PINCH1 and PINCH2, which play significant roles in cell and extracellular matrix adhesion, cell migration, proliferation, and survival. The role of PINCH1 and PINCH2 in regulating bone homeostasis is unclarified. This thesis aims to study role regulation of PINCH on regulation of bone homeostasis to obtain new clues to the prevention and treatment strategies for osteoporosis.To exam the role of PINCH in regulating bone homeostasis, mouse model with osteocyte Pinch deletion is analyzed. Micro-CT examination of bone mass showed that osteocyte Pinch deletion lead to significant bone loss especially bone mineral density. Bone mass is controlled by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. In our study, we found that bone formation was significantly reduced by the loss of Pinch in osteocyte measured by calcein double labelling, but bone resorption was not affected detected by TRAP staining. Although the Pinch deletion in osteocytes lead to an up-regulation of Rankl expression, Opg also has a proportional up-regulation. Furthermore, single knockout of Pinch1 or global knockout of Pinch2 didn’t affect bone mass, indicating that Pinch1 and Pinch2 had functional compensation in regulating bone remodeling. Bone loss in osteocytic-Pinch deletion mice was due to changes in bone formation.The molecular mechanism of Pinch regulating bone homeostasis was further explored in the thesis. We found that Pinch1 could directly bind to Igf1r to partially affect Sclerostin expression. This result was also verified by CRISPR-Cas9 technology to knockout Pinch1 in MLO-Y4 cells in vitro. The high expression of Sclerostin in osteocytes affected the differentiation of bone marrow mesenchymal stem cells, as well as inhibited the Wnt/β-Catenin signaling pathway in primary osteoblasts, which played an important role in bone formation. Meanwhile, the deletion of osteocyte Pinch changed the microenvironment of the bone marrow cavity, resulting in enhanced adipogenic differentiation and weakened osteogenic differentiation of mesenchymal stem cells, and this could be partially explained by the decreased expression of Yap1 / Taz in bone marrow mesenchymal stem cells. As for the osteocytes, Pinch deletion resulted in increased apoptosis without affecting autophagy caused by the low expression of integrin.Osteocytes are mechanical sensing devices for long bones. To investigate whether Pinch is involved in the mechanical signal transduction of osteocytes, two mouse models, of hindlimb unloading and ulnar loading model, were utilized in the experiments this study. In the hindlimb unloading model, mice with osteocyte Pinch deletion exhibited more severe bone loss. However, in the ulnar loading model, the thickness of the bone matrix could be increased to cortical bone in control mice, and this increase was lost in mice with osteocyte Pinch deletion. Thus the response of osteocytes to mechanical signal was decreased by osteocytic Pinch deletion. In summary, this thesis describes the key role of Pinch protein in regulating bone homeostasis and bone mechanical transduction. Our results indicates that modulate osteocyte Pinch expression could be a potentially strategy for prevention and treatment of human metabolic bone disease.

骨质疏松 骨细胞 粘附因子Pinch1/2 Sclerostin 骨生物力学

Osteocyte Pinch1/2 Bone homeostasis Sclerostin

中文

联合培养

南方科技大学

王依姝. 粘附因子Pinch1/2对骨稳态的调控作用及机制研究[D]. 深圳. 哈尔滨工业大学,2020.