脂肪细胞来源的外泌体通过lncRNAs抑制骨髓间充质干细胞成骨分化及其在老年骨质疏松中的作用

Osteoporosis due to aging is a serious public health concern. Approximately over 200 million people worldwide suffer from this disease. It is well established that there are more adipocytes in the bone marrow of aged subjects. And bone marrow derived mesenchymal stem cells (MSCs) display decreased osteogenic capacity and increased adipogenic capacity during aging process, which may plays an important role in etiology of osteoporosis. Previous study reveals that adipocytes are one of the most important components of the bone marrow microenvironment, while its effects and underlying mechanism in MSCs osteogenesis remain largely unknown. Supported by the NSFC（Natural Science Foundation Of China，accomplished in 2014）, our research group have found that the adipocytes and their progenitors in the bone marrow secrete a lot of exosomes, which targets surrounding cells in a paracrine manner. Furthermore, inside the exosomes, there are some adipogenesis/adipocyte highly expressed lncRNAs, including lncRNA HOTAIR. Functional analysis revealed that the adipocyte derived HOTAIR can promote adipogenesis, while inhibit osteogenesis. These preliminary data raise the possibility that adipocytes in the bone marrow might deliver their adipogenic ability through exosomes and encapsuled messages.In the senile osteoporosis, the increased numbers of adipocytes and/or their progenitors in the bone marrow secrete more exosomes, which carries the adipogenic HOTAIR. These exosomes target surrounding MSCs, and in turn lead to decreased osteogenesis and increased adipogenesis. The adipocyte-exosome-adipogenesis forms a vicious cycle and is responsible for the osteoporosis in aging population. In this study, we propose to: 1) confirm the relationship between adipocyte-derived exosomes, their cargo HOTAIR and osteoprosis by using both clinical bone marrow samples and mouse osteoporosis model; 2)determine the function of adipocyte-derived exosomes and their cargo HOTAIR in MSCs osteogenesis and adipogenesis by interfering the exosomes synthesis and secretion and using HOTAIR KO mouse. 3)characterize the co-activators or repressors involved in the above regulation, mainly focusing on the LSD1 (lysine specific demethylase 1) and PRC2 (Polycomb repressive complex 2), which are two well-known histone modifiers involved in the function of HOTAIR. The increased adipocytes in the bone marrow is one of the most dominant traits of senile osteoporosis, which is widely considered as a result of osteoporosis. However its contribution to osteoporosis is ignored. We here explore the effects and functional mechanism how the increased adipocytes affect the osteoporosis, mainly focus on the exosomes and the encapsulated lncRNAs. Our studies will elucidate the function of an important lncRNA HOTAIR in osteogenesis and adipogenesis, and its implication in osteoporosis and other related diseases. The project will definitely illuminate the path of therapy for osteoporosis.

骨髓脂肪细胞比例增高、间充质干细胞(MSCs)成骨能力降低，是老年骨质疏松的重要原因。最新研究表明，脂肪细胞是骨髓微环境重要组成细胞，但其对MSCs的功能调控不清。课题组研究发现，骨髓中脂肪（前体）细胞分泌大量外泌体，通过旁分泌的方式作用于靶细胞；这种脂源性外泌体中含有一种叫做HOTAIR的长链非编码RNA，过表达HOTAIR可促进MSCs成脂分化、抑制其成骨分化。据此，我们推测老年骨髓中，脂肪细胞来源的外泌体携带HOTAIR，以旁分泌的方式抑制MSCs成骨、促进其成脂，形成恶性循环，是老年骨质疏松的重要原因。本项目拟通过临床骨髓样本和小鼠模型明确脂肪细胞分泌的外泌体及其中的HOTAIR对MSCs成骨成脂分化的调控机制，综合HOTAIR敲除小鼠体内功能实验、细胞生物学、分子生物学和生物信息学等方法探明HOTAIR调控成骨成脂分化的分子机制，有望发现老年骨质疏松的新机制，为其防治提供新思路。

老年个体骨髓微环境中脂肪细胞比例增高可能是间充质干细胞(MSCs)成骨能力降低，易发骨质疏松的重要原因。本项目基于前期研究基础，探讨脂肪细胞源性外泌体对MSCs成骨分化的调控及具体机制。主要发现包括：1）脂源性外泌体可促进MSCs成脂分化，抑制其成骨分化。2）长链非编码RNA是脂肪组织来源的外泌体的重要组分，脂肪组织来源的外泌体中富含lncRNA HOTAIR；3）HOTAIR可通过协同调控一系列成骨分化相关基因表达（如C/EBPα、PPARγ、Runx2和Osterix等核心转录因子）抑制MSCs成骨分化；4）NF-κB活化可能是脂肪细胞中HOTAIR表达升高的原因。上述研究表明，脂肪细胞来源的外泌体通过向MSC传递HOTAIR抑制其成骨分化，参与老年骨质疏松，该途径是老年骨质疏松防治的重要靶点。. 在此基础上，我们还探讨了雌激素缺乏条件下骨质疏松的外泌体机制及基于外泌体的靶向递药策略。主要发现包括：1）发现miR-92a在正常和雌激素缺乏的骨髓组织中表达异常及其对MSCs成骨分化能力的调控，是潜在的防治雌激素缺乏相关骨质疏松的靶点；2）构建了一组靶向骨髓不同细胞类型的外泌体，为精准干预骨髓微环境提供了支撑；3）创建了外泌体-脂质体融合的CRISPR/Cas9基因编辑系统，并高效递送至MSCs，为任意过表达或干涉候选基因奠定了基础；4）探讨了脂肪及巨噬细胞外泌体在牙周炎条件下对MSC的调控。上述研究一方面从疾病机制发现出发寻找靶点，另一方面，通过递药策略改良，为体内干预提供可能；有望为骨质疏松等骨代谢相关疾病提供新思路。. 在该项目支持下，共发表SCI论文6篇；其中以通讯作者发表于Adv Sci，Curr Stem Cell Res Ther，Biochem Biophys Res Commun 和Bone Res；以第一作者兼共同通讯作者发表于ACS Nano；以共同通讯作者发表于Int Immunopharmacol。项目进行期间，申请国家发明专利2项（已授权1项），实用新型专利1项（已授权1项）；培养毕业硕士3名，在站博士后1名，在读博士2名，在读硕士1名。

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