【作者】 李大虎；

【导师】 贺福初； 张令强；

【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， 2012， 博士

【摘要】 间充质干细胞（Mesenchymal Stem Cells, MSCs）是一类广泛存在于出生后机体多种组织的多能干细胞，较少涉及到诸如胚胎干细胞的伦理学问题。一般认为在适合条件下，MSCs可以分化为成骨细胞、成脂细胞及成软骨细胞。近些年来又发现了MSCs的一些新功能，例如支持造血、抑制T细胞免疫反应等。不仅如此，一些基于MSCs的细胞疗法已经展开尝试和应用，并取得了良好的进展。目前肥胖、糖尿病以及其他代谢性疾病已经成为世界范围内的首要健康威胁，这些疾病与脂肪细胞的发育和功能异常有着非常密切的联系。成脂分化是MSCs多向分化的重要组成部分，这一过程受到复杂而又精密的调控。简而言之，MSCs受到外界成脂分化信号刺激后，会激活一系列关键转录因子的表达，而这些转录因子继而转录出大量脂肪细胞特异的基因来执行特化的功能，最终完成MSCs向成熟脂肪细胞的转变。目前认为，C/EBPα和PPARγ是成脂分化中的最为关键的转录因子，对它们的转录调控研究有重要的意义。CKIP-1是我们实验室开展研究多年的基因，其蛋白产物N端含有PH结构域，C端含有亮氨酸拉链结构域，认为其主要定位在细胞近质膜区。近年来我们实验室建立了ckip-1^-/-小鼠模型，并在体内水平证明CKIP-1可以增强泛素连接酶Smurf1的活性，抑制成骨前体细胞的分化和矿化能力。因为MSCs是成骨、成脂前体细胞的共同干祖细胞，所以我们在本论文中利用敲除小鼠模型来探讨CKIP-1缺失对MSCs成脂分化的影响。我们采用了从密质骨中分离MSCs的方法，得到了表面抗原均一的高纯度ckip-1^+/+和ckip-1^-/-MSCs，并进行了相关干细胞性能验证。在随后的实验中我们发现：1. ckip-1^-/-小鼠MSCs成脂分化能力增强，关键标志物（marker）基因表达水平显著升高，其中C/EBPα尤为明显，而且这种升高早在转录水平已经发生。我们首先发现CKIP-1蛋白表达在MSCs成脂分化中发生明显变化，随后在比较ckip-1^+/+和ckip-1^-/-MSCs成脂分化能力时发现，ckip-1^-/-MSCs中出现的脂肪细胞显著增多。在将不同培养代数的ckip-1^+/+和ckip-1^-/-MSCs进行成脂分化诱导时，我们发现ckip-1^-/-MSCs显示出更高水平的成脂marker基因表达，其中C/EBPα最为显著。而且通过报告基因实验，我们证明了过表达CKIP-1也可以抑制C/EBPα的下游效应。2.首次证明生理状态下CKIP-1可以定位在细胞核中，并与C/EBPα的转录抑制因子——组蛋白去乙酰化酶HDAC1存在相互作用。目前对C/EBPα的转录调控机制了解得比较清楚的是：成脂分化启动后，其他两个C/EBP转录因子家族成员C/EBPβ与δ会结合在C/EBPα的启动子区激活其转录，而组蛋白去乙酰化酶HDAC1作为转录共抑制子，也会被招募到该区域。HDAC1会降低组蛋白H3及H4乙酰化水平，延缓迟滞RNA聚合酶II对C/EBPα的转录。我们的数据表明CKIP-1可以和上述蛋白复合体中的HDAC1共定位在MSCs的细胞核，并与HDAC1发生内源、外源相互作用，且这种相互作用不需要借助其他分子。3. CKIP-1可以增强HDAC1与C/EBPα启动子区的结合，从而抑制C/EBPα的转录。我们发现与脂肪前体细胞系不同，在MSCs中HDAC1组成型结合于C/EBPα的启动子区，而非被招募；在成脂分化初期，HDAC1与该区结合能力减弱，从其上发生解离。与ckip-1^+/+MSCs相比，在ckip-1^-/-MSCs中，成脂分化启动后结合在该启动子区的HDAC1显著减少，相应地，结合在该区的Ac-H3与RNA聚合酶II显著增多。过表达CKIP-1可以减弱HDAC1与该启动子区的解离，这证明CKIP-1确实可以通过增强HDAC1与该启动子区的结合能力来抑制C/EBPα的转录。4.在高脂饮食下，ckip-1^-/-小鼠体重增长比ckip-1^+/+小鼠更为显著。经过脂肪含量60%的饲料喂养后，ckip-1^-/-小鼠体内主要的脂肪团质量以及瘦素水平也显著高于ckip-1^+/+小鼠，并显示出更为严重的脂肪肝表型。综上，我们发现了CKIP-1基因的另一新功能——抑制MSCs成脂分化，并首次揭示了其在生理条件下的核定位。近期本实验室与中国航天员训练科研中心的合作研究也显示CKIP-1可以抑制心肌肥大，再联系到前期CKIP-1抑制成骨细胞分化的结果，我们认为CKIP-1是一个广谱系抑制干祖细胞分化的基因。此外，对成脂分化而言，前期的认识大多来自脂肪前体细胞系中（如3T3-L1）。我们通过基因敲除小鼠的模型，揭示了CKIP-1可以增强HDAC1对C/EBPα的转录抑制而负调控MSCs的成脂分化，并发现在MSCs中，HDAC1对C/EBPα的转录调控具有自身的特点，这些工作都丰富了对成脂分化中C/EBPα转录调控的认识。另外，我们在实验中还得到了一些意外的发现。虽然小鼠作为应用最为广泛的模式生物，但是依照传统方法从骨髓中分离MSCs存在着许多困难。近些年来有研究者指出小鼠长骨密质骨是MSCs的一个重要来源，并发展出全新的分离方法，而且有数据表明这种方法可以分离到比骨髓中更多更纯的MSCs。根据已有认识，密质骨也存在于顶骨。应用相似的方法，我们从小鼠顶骨密质骨中也分离到了与长骨来源的MSCs细胞形态、表面抗原都高度相似的细胞。尽管如此，我们发现顶骨来源的这些细胞内成骨转录因子osterix表达远高于长骨来源的MSCs。随后我们将这两种不同骨来源的细胞进行了诱导分化实验。与长骨来源的MSCs可以轻易分化为脂肪细胞不同，顶骨来源的细胞丧失了成脂分化的能力。与这一表型相一致，成脂分化的marker基因如PPARγ、C/EBPα及aP-2mRNA水平在顶骨来源的细胞中远低于长骨来源的MSCs。而对于成骨分化而言，经过诱导后，顶骨来源的细胞中成骨分化的marker基因如Runx2，osterix及osteocalcin mRNA水平显著高于长骨来源的MSCs，并展示出显著增强的分化活性及矿化细胞外基质的能力。这些数据说明了这群顶骨来源的细胞更像是分化命运已经决定的祖细胞（committed progenitor cells）而非MSCs。这也提示我们驻留在小鼠机体不同部位密质骨内的间充质干/祖细胞的分化命运已经决定，并不是所有部位的密质骨都可以作为MSCs的来源。更多还原

【Abstract】 Mesoderm-derived mesenchymal stem cells (MSCs) are present in many tissuespostnatally and can differentiate into osteocytes, adipocytes and chondrocytes underappropriate conditions. Aside from their multiple differentiation capacity, MSCs alsoplay important roles in supporting hematopoiesis and in suppressing theimmunoresponse of T lymphocytes. Because of these characteristics, the applicationof MSCs in cytotherapy is promising, such as their use in tissue repair and in reducingimmunological rejection.In present world, improved knowledge of all aspects of adipose biology will berequired to counter the burgeoning epidemic of obesity and relevant metabolicdiseases, which has closed association with abnormal development and function ofadipocytes. The adipocytes constituting white fat tissue are originated from MSCs,which differentiate in response to a series of cues. The extracellular signals aretransduced into nuclear transcription factors via cascades reactions to transcribe manyhundreds of genes, which responsible for establishing the mature fat cell shape andfunction. Transcriptional regulation of adipogenic differentiation is a tightlycontrolled process that is regulated by an elaborate network of transcription factors,cofactors and signalling intermediates from numerous pathways.Among these transcription factors, the most notable are CCAAT/enhancer bindingproteins(C/EBP) and the peroxisome proliferator-activated receptors gamma(PPAR). Actually, it is well accepted that both C/EBPα and PPARγ act as criticalregulators of adipogenesis, since deficiency of either of them shows thedevelopmental defects of white adipose in mouse model. Consequently, in our effortsto gain a complete understanding of the processes regulating the function ofadipocytes, it is important to identify the mechanisms regulating transcription of theseadipogenic transcription factors.CKIP-1is a PH domain containing protein, involving in apoptosis, cell skeletonmaintaining and so on. In recent years, it was found CKIP-1promoted E3ligaseSmurf1’s activities and negatively regulated osteogenesis in mouse model. It is wellknown that osteoblasts and adipoblasts are descended from common progenitors-- MSCs. Yet, the function of CKIP-1in the adipogenic differentiation of MSCs is notknown. In the present study, we isolated and richened MSCs from murine compactbone of CKIP-1wild-type (WT) and knockout (KO) mice, and further identified theirpurity and CFU-F capacity. In the subsequent multiple differentiation assays, wefound that MSCs lacking CKIP-1display enhanced adipogenesis after exposed tostimuli than their wild-type counterparts, and increased transcriptional levels of thecritical adipogenic marker genes, especially for C/EBPα. Due to the HDAC1represscomplex is curial for the transcription of C/EBPα, we investigated the association ofCKIP-1and HDAC1in adipogenesis of MSCs. It is found that CKIP-1can localize inthe nucleus and interact with HDAC1directly. This is the frist time that CKIP-1hasbeen observed in the nucleus under physiological conditions. Our date also show thatHDAC1constitutively binds the promoter of C/EBPα in MSCs, but dislodges duringthe early phase of adipogenesis. In the presence of CKIP-1, the dislodgment ofHDAC1from the promoter of C/EBPα is weakened, which suppresses thetranscription of C/EBPα and adipogenesis. Furthermore, CKIP-1deficiency leadsmore Ac-histone3and RNA pol II in the promoter of C/EBPα during adipogenesis ofMSCs, but less HDAC1. Moreover, on high fat diet, CKIP-1KO mice showaccelerated body weight gain, increased total fat mass and severe fatty liverphenotype than their WT counterparts. Taken together, we conclude that CKIP-1is anovel negative regulator of adipogenesis of MSCs.In addition, we observed some interesting phenomenons by chance. MSCs culturewas first developed by Friedenstein et al. from bone marrow, and since then, bonemarrow has become the common source for isolating MSCs. Thus far, MSCs havebeen isolated from the bone marrow of many species, including humans, rats and pigs.In contrast to other species, murine MSCs cannot be easily harvested from the bonemarrow due to contamination by hematopoietic cells, and this contamination isdifficult to eliminate using the characteristic plastic adherence of MSCs. Recentstudies showed that the compact bone tissue of long bones is a novel source of MSCsfor both humans and mice. These studies developed a novel method to obtainhigh-purity murine MSCs by culturing collagenase-digested compact bone fragments.Some procedures in this method help avoid hematopoietic cell contamination, such asby removing bone marrow before collagenase digestion and discarding the releasedcells after collagenase digestion. Furthermore, subsequent experiments showed thatcompact bone is a richer source of MSCs than the marrow plug within it. Based on these findings, we postulated that murine MSCs could be isolated from the compactbone of the calvaria, which has a relatively low bone marrow content. In this study,we found that cells migrating from the calvaria possess morphological characteristicsand surface antigen profiles similar to those of MSCs derived from long bones.However, these calvaria-derived cells highly expressed the osteogenic transcriptionfactor osterix. The calvaria-derived cells lost their adipogenic capacity but gained ahigher osteogenic capacity. These results suggest that not all types of murine compactbones in the body are sources of MSCs and that the differentiation fate ofmesenchymal stem/progenitor cells in different types of compact bones is alreadycommitted. The cells that migrate from the calvaria should be considered progenitorcells rather than MSCs.更多还原