CKIP-1基因的敲除小鼠模型建立及其在骨骼、免疫系统中的生理功能研究

【作者】 尹秀山；

【导师】 贺福初； 张令强； 杨晓；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2009， 博士

【摘要】 骨骼系统的发育与功能异常可导致包括骨质疏松在内的多种疾病,骨病已成为我国三大老年病之一,深入研究骨发育的分子机制对于骨病的防诊治具有重要意义。骨发生包括骨组织形成(bone formation)和骨组织吸收(bone resorption)两种基本过程。骨形成后,人的整个一生均通过骨形成及骨吸收构成的骨重塑(bone remodeling)过程保持骨的更新。其中,骨形成由成骨细胞调控,骨吸收则由破骨细胞调控。目前,对破骨细胞的分化及调控机制了解得比较清楚,破骨细胞已成为重要的药靶。相对而言,对控制成骨细胞分化的基因了解较少,基于成骨细胞功能研究的药物研发报道也非常有限,所以,加深对成骨细胞调控的理解是当前的研究重点。由于影响骨骼发育的基因在人类和小鼠中极为保守,因此,小鼠可作为一种理想的研究模型。过去近二十年,转基因和基因敲除小鼠模型的应用深刻地改变了人类对骨生理学的认识,大大加深了人类对骨生物学的理解。但是,目前对于骨骼生理学和骨病遗传学的重要发现绝大多数来自于国外实验室,一个重要的原因是:大量突破性的发现都得益于基因敲除和转基因遗传修饰小鼠模型,而专业方面国内几为空白。基因敲除技术是一种可以定向对生物体进行DNA水平遗传修饰的技术手段,该技术不仅推动了对生命科学重大基本问题的理解,而且对于疾病的治疗提供了模型。近年来,在国家各种基金资助下,国内少数实验室已经初步建立了相应的技术平台,实质性地推动了学术进步,利用遗传学模式动物模型发现新的骨发育调控分子面临新的机遇。本研究正是把握这一机遇,发现了一个新的骨形成负调控分子CKIP-1 (casein kinase 2-interacting protein-1)。CKIP-1为本实验室克隆鉴定并有扎实研究基础和授权发明专利的基因。本研究成功建立了CKIP-1的敲除小鼠模型,并经表型分析发现:(1) CKIP-1基因敲除小鼠可正常出生,表明其非胚胎发育所必需;成体小鼠经软X射线分析表明,敲除小鼠整体骨量增加,颅骨、尾椎骨尤其明显;(2)骨密度仪测量股骨骨矿密度表明敲除小鼠明显高于野生型小鼠;(3)显微CT对股骨远端松质骨三维重建后的定量分析显示CKIP-1敲除小鼠的骨小梁密度、骨连接密度、骨小梁厚度、骨小梁数量均高于野生型,而CKIP-1敲除小鼠的骨小梁间隙低于野生型;(4)钙黄绿素荧光双标技术和骨剂量软件分析表明,敲除小鼠的骨形成速率显著高于野生型;(5)成骨前体细胞经分离体外培养,并行诱导分化及矿化,表明CKIP-1-/-成骨细胞的碱性磷酸酶(ALP)活性显著高于野生型对应细胞;von Kossa和茜素红染色表明,CKIP-1-/-细胞矿化结节的数量极显著地高于野生型对应细胞,显示向细胞外基质沉积I型胶原的能力显著升高。这些数据表明,CKIP-1在生理条件下是成体(而非胚胎期)骨形成的负调控分子,其机制与调控成骨细胞的分化与活性相关。这是CKIP-1生理功能的第一批遗传学证据。进一步的分子机制研究表明CKIP-1的缺失导致骨形成负调控相关的泛素连接酶Smurf1(Smad ubiquitination regulatory factor 1)的E3活性削弱,进而使其底物-磷酸化的MEKK2蛋白水平升高,导致JNK-AP-1通路活性增强,继之上调成骨分化相关基因的转录。至此,我们在自建CKIP-1基因剔除小鼠模型的基础上,发现了CKIP-1在骨稳态调控中的生理功能,同时在整体水平揭示了CKIP-1调控Smurf1活性的生物学意义。目前,绝大多数已被发现的骨发育相关分子都在胚胎期发挥作用,只有少数影响成体的骨量,而后者更容易成为治疗骨质疏松疾病的靶标。我们发现,CKIP-1敲除小鼠年龄越大,骨量的升高与野生型小鼠相比越显著。说明CKIP-1通过在成骨细胞中发挥负调控的作用,使得成体的骨重塑平衡被打破。提示CKIP-1可能作为通过影响成体骨形成而治疗骨质疏松的候选靶标。针对一个候选的药物靶标分子,需要考虑其特异性及副作用,而首应考虑的是免疫系统。因此我们对CKIP-1敲除小鼠免疫系统进行了观察与分析。前期的初步证据表明,CKIP-1在一些静态及激活的免疫细胞亚群中的表达存在显著差别,在此基础上我们对CKIP-1敲除小鼠展开免疫细胞亚群的分析,结果显示,CKIP-1敲除小鼠脾脏中CD4+CD25+细胞的数量增多,骨髓中粒细胞数量增多,这提示CKIP-1可能参与上述免疫细胞亚群的调控;同时发现在LPS刺激下CKIP-1敲除小鼠IL-5和IFN-γ上调。目前,对免疫系统更加深入系统的表型分析和分子机制研究还在进行中。综上,本研究在国际上率先建立了CKIP-1基因敲除的小鼠模型,获得了关于CKIP-1基因生理功能的遗传学证据,发现了CKIP-1在骨形成中的重要负调控功能。并初步探讨了CKIP-1在免疫系统中的可能角色。这些结果加深了对CKIP-1基因的理解,同时为骨质疏松等疾病的防治提供了新的思路。更多还原

【Abstract】 The disorder of bone development and function can lead to many skeletal diseases, such as osteoporosis. Thus, it is critical to understand the molecular details of the anabolic signaling pathways that control bone homeostasis for developing novel bone anabolic agents to reverse osteoporosis.In healthy adults targeted remodeling is a continuous physiological process, and initially bone formation was shown to always follow bone resorption leading to full replenishment of removed bone matrix with newly synthesized bone. Bone remodeling, thus, is understood as a cycle of bone resorption by osteoclasts and formation of new bone by osteoblasts. In contrast to the wealth of knowledge that has been acquired in the osteoclast field, we still know relatively little about osteoblast. There are currently a number of FDA-approved drugs for the prevention and treatment of postmenopausal osteoporosis that work by inhibiting bone resorption .The only FDA-approved agent capable of stimulating new bone formation for reversing bone loss in patients with high risk for osteoporotic fracture is parathyroid hormone (PTH).Since the genes related to bone development are highly conserved between human and mouse, mouse can be used as an ideal animal model. By this means, we have learned a lot about the mechanisms by which the bone is formed and regulated in the past twenty years.Gene targeting is a technique by which targeted gene can be modified accurately at the DNA level. Since the first knockout mice was reported in 1987, gene targeting is now being applied to virtually all areas of biomedicine– from basic research to the development of new therapies. Now deletion of any gene at both temporal and spatial level is feasible. Gene targeting has become one of the most important techniques to uncover the functions of genes in vivo.CKIP-1 (casein kinase 2 interacting protein 1) is implicated in regulation of cell differentiation and apoptosis. All of the previous studies were performed at the molecular and celluler levels. At present, the physiological function of CKIP-1 remains unclear due to lack of genetic evidence. In this study we established the CKIP-1 gene knockout mice model and then performed the phenotype analysis. CKIP-1 deficient mice were born normally, with no apparent gross abnormalities, and were fertile, suggesting that CKIP-1 is not required for embryonic development. Further phenotype analysis showed that compared to wild-type mice, the CKIP-1 deficient mice showed an age-dependent increase in bone mass and bone mineral density. This increase is caused by the enhanced activity of osteoblast differentiation and mineralization. Studies at molecular level showed that the activity of HECT-type E3 ligase Smurf1 decreased significantly in CKIP-1 KO mice. Consistently, the protein level of MEKK2, which is a substrate of Smurf1, was markedly upregulated in the absence of CKIP-1. This leads to the JNK-AP-1 pathway jubilantly regulated. Thus using the CKIP-1 KO mice, we uncovered the physiological role of CKIP-1 in regulation of bone homeostasis, and also confirmed the regulatory role of CKIP-1 on Smurf1 activity in vivo.Our primary data showed that CKIP-1 gene expression was upregulated or downregulated in certain activated immune cells compared with the resting cells. We then further explored the possible role of CKIP-1 in immune regulation using the wild-type and KO mice, we revealed that the number of CD4+CD25+ double positive cells in spleen and granulocytes in bone marrow was increased in CKIP-1 KO mice. These data suggested that CKIP-1 may participate in the regulation of these cells. We also found that the secretion of IL-5 and IFN-γin serum was upregulated when CKIP-1 gene was deficient. Further analysis of CKIP-1 in immune system is under investigation.Taken together, the current study established the CKIP-1 gene knockout mice model and provided the first genetic evidence of physiological function of CKIP-1.Our findings revealed the negative regulatory role of CKIP-1 on bone formation. We also supplied primary analysis of the immune system of CKIP-1 KO mice. These data deepen our understanding of CKIP-1 gene function, and provide new clues for the therapy of bone diseases including osteoporosis.更多还原