【作者】 杨冠；

【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， 2008， 博士

【摘要】 软骨是大部分骨骼的发育引擎,它的发育受到多种信号通路的协同调节。这些信号通路由各种因素驱动,包括细胞因子和激素如Indian hedgehog(Ihh)、甲状旁腺激素相关多肽(parathyroid hormone-related peptide,PTHrP)、转化生长因子β(transforming growth factorβ,TGF-β)超家族分子、成纤维细胞生长因子(fibroblast growth factor,FGF),也包括转录因子如Sox(SRY-related high mobility group-box gene)家族、Runx2(runt-related transcription factor 2)、缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α),以及各种应激因素如内质网应激反应(endoplasmic reticulum stress ,ERSS)等等。这些信号通路涉及调控软骨细胞的增殖、分化、迁移、凋亡以及细胞稳态的维持。当这些过程发生异常时,会使软骨内成骨过程受损,导致各种骨骼疾病的发生。软骨发育障碍是以多发性内生性软骨瘤为特征的软骨发育异常疾病,好发于青壮年,以疼痛、骨骼畸形和病理性骨折为主要临床表现。到目前为止,人们对软骨发育障碍的发生发展过程和分子机制知之甚少。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,它通过负向调控磷脂酰肌醇3-激酶(phosphoinositide 3 kinase,PI3K)信号通路来维持细胞稳态,调节细胞的增殖、分化、迁移、凋亡和黏附。PTEN是最重要的抑癌基因之一,它的缺失和突变与人类遗传性疾病和肿瘤的发生密切相关。然而PTEN与软骨内成骨和软骨发育障碍之间的关系仍未明确。在本研究中,我们通过条件基因敲除技术获得了软骨细胞特异性的PTEN基因敲除小鼠,使我们能够深入探索PTEN调节的PI3K/Akt信号通路在软骨发育和骨骼疾病中的作用。PTEN基因敲除使生长板软骨细胞中Akt得到磷酸化激活,基因敲除小鼠体长增长并发生与人类内生性软骨瘤相类似的软骨发育障碍表型。通过BrdU掺入法跟踪观察发育中的生长板软骨细胞增殖情况,利用原位杂交和BrdU标记-示踪法分析生长板软骨细胞的分化情况,我们发现PTEN基因敲除导致生长板软骨细胞增殖和分化不均一,致使增殖和分化受阻的静息软骨细胞在生长板中央形成瘤核样的结构。瘤核随着生长板的生长逐渐被遗落入骨髓腔内形成位于生长板下端的瘤样软骨结节。组织学观察显示瘤样软骨结节具有典型的内生性软骨瘤病理学特征。除此之外,PTEN基因敲除的生长板排列和分化紊乱,伴有关节变形,关节软骨损耗等病理改变。因此,通过在软骨细胞中特异性地敲除PTEN基因,我们获得了软骨发育障碍的小鼠模型,为我们研究这个疾病提供了理想的遗传学工具。在软骨细胞特异性PTEN基因敲除小鼠体内,瘤核样结构的出现是内生性软骨瘤发生过程中最关键的事件,它们被认为是内生性软骨瘤的雏形。我们发现瘤核内的软骨细胞发生了严重的ERSS。免疫组化和电镜观察显示瘤核内细胞外基质成分的特性和分布发生异常,大量的以2型胶原(type II collagen,Col II)为代表的胞外基质成分堆积在瘤核细胞的内质网腔。伴随着ERSS相关基因免疫球蛋白结合蛋白(binding Ig protein,BiP)表达的上升,软骨细胞的内质网腔发生明显的扩张和破裂,胞外基质的分泌严重受损。我们进一步研究发现只有在低氧环境下,PTEN基因敲除才能导致软骨细胞发生ERSS,同时伴随着软骨细胞分化下降,揭示了PTEN敲除和低氧在ERSS发生过程中的协同作用。在软骨细胞特异性PTEN基因敲除小鼠的生长板软骨中,我们不仅发现了ERSS相关基因表达的上升,也发现了HIF-1α信号通路的活化。免疫组化和原位杂交的结果显示在PTEN基因敲除的生长板中HIF-1α和下游靶基因如血管内皮细胞生长因子( vascular endothelial growth factor , VEGF )、磷酸甘油酸激酶(phosphoglycerate-kinase,PGK)、p21、p57在生长板的中央区域表达异常升高。异常活化的HIF-1α信号通路导致这个区域的软骨细胞增殖停滞,导致软骨周围血管生成增加并侵入生长板。我们还发现在HIF-1α信号通路发生异常活化的部位随后发生了ERSS,形成瘤核。体外实验证实PTEN敲除导致软骨细胞HIF-1α信号通路在低氧条件下发生了过度的活化,且先于ERSS的发生,提示PI3K/Akt信号通路可能通过HIF-1α信号通路调节软骨细胞对低氧的反应,并影响ERSS的发生。在本研究中,我们首次提供了体内的遗传学证据表明PTEN通过抑制HIF-1α信号通路的过度激活,通过抑制ERSS的发生,调节软骨细胞的增殖和分化,在维持生长板软骨的有序结构,抑制软骨发育障碍的过程中发挥着重要作用。我们的研究成果为人们认识软骨发育障碍这一疾病提供了宝贵的参考。更多还原

【Abstract】 Cartilage, which is the principal engine for conducting growth of most mammalian skeletons, is developmentally regulated by multiple signaling pathways whcih are driven by multiple factors including cytokines and hormones such as Ihh (Indian hedgehog), PTHrP (parathyroid hormone-related peptide), members of TGF-β(transforming growth factorβ) superfamily, FGF (fibroblast growth factor), as well as transcription factors such as Sox (SRY-related high mobility group-box gene) family, Runx2 (runt-related transcription factor 2), HIF-1α(hypoxia-inducible factor-1α), and stresses such as ERSS (endoplasmic reticulum stress). They are vitally engaged in the processes of coordinating chondrocytic proliferation, differentiation, migration, apoptosis and cellular homeostasis. Disruption of these processes may impair endochondral ossification, leading to various bone defects. Dyschondroplasia is a defect of cartilage development which is characterized by multiple enchondromatosis within the bone marrow cavity. This defect mainly harms youths by means of pain, skeletal deformities and pathological fractures. However, the molecular mechanisms underlying dyschondroplasia are poorly understood.The tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) is involved in the regulation of cell proliferation, lineage determination, motility, adhesion and apoptosis, as well as in the maintenance of intracellular homeostasis through coordinating the PI3K (phosphatidylinositol-3-kinase) signaling. Loss or mutation of PTEN has been implicated in various hereditary disorders as well as several sporadic human cancers. However, the functions of PTEN in endochondral ossification and dyschondroplasia inhibition remain largely unknown. In this study, we have created chondrocyte-specific PTEN knockout mice (PTENCo/Co;Col2a1-Cre-2) using the Cre–LoxP system. The employment of this genetic model would facilitate our understanding the role of PI3K/Akt signaling in cartilage development and bone defects.Following Akt activation, PTEN mutant mice exhibited excessive body length and dyschondroplasia resembling human enchondroma. By using BrdU incorporation and in situ hybridization/BrdU labeling-chasing assay, we observed asynchronous proliferation and differentiation of the PTEN mutant chondrocytes that led to the formation of neoplastic cores within the central region of growth plates. Abnormal resting chondrocytes within these cores are pinched off from the developing growth plates, gradually forming cartilaginous nodules within the bone marrow cavity. Histological observations showed pathological similarities between these cartilaginous neoplasms and human enchondroma. Additionally, an abnormal arrangement and differentiation of growth plate cartilage coupled with joint deformation as well as depletion of articular cartilage was also observed. Therefore, these knockout mice provided us with powerful genetic models on studying dyschondroplasia.The formation of neoplastic cores which were considered to be the rudiments of enchondromas, was the most remarkable even during the oncogenesis within the PTEN mutant mice. Immunohistochemistry and electron microscopy analyses revealed aberrant properties and distribution of ColII (type II collagen) fibrils within the neoplastic cores. Chondrocytes within the cores experienced severe ERSS characterized by an up-regulation of ERSS related genes as well as engorged and fragmented ER in which extracellular matrix proteins were trapped. We further found ERSS only occurred under hypoxic conditions within the PTEN mutant chondrocytes, leading to impaired maintenance of chondrocytic differentiation. This result suggested the synergistic function of PTEN deficiency and hypoxia in triggering ERSS.An up-regulation of HIF-1αand downstream targets VEGF (vascular endothelial growth factor), PGK (phosphoglycerate-kinase), p21, p57 followed by an emergence of ERSS and neoplastic core was demonstrated via immunohistochemistry and in situ hybridization. Activated HIF-1αsignaling within PTEN mutant growth plates resulted in halted chondrocytic proliferation as well as excessive blood vessel growth and invasion. In vitro study showed that under hypoxia, PTEN mutant chondrocytes exhibited over-reacted HIF-1αsignaling in advance of ERSS, suggesting that PI3K/Akt signaling may regulate the responses of chondrocytes to hypoxia and trigger ERSS via up-regulation of HIF-1αsignaling.In this study, we provided the first genetic evidence to show that PTEN coordinates proliferation and differentiation of growth plate chondrocytes via inhibiting overactive HIF-1αsignaling and ERSS, thereby guides the uniform development of growth plate cartilage and suppresses dyschondroplasia. Our data provided new clues for better understanding molecular mechanisms of dyschondroplasia.更多还原