脂肪基质细胞脂向分化过程中lncRNA-Adi调控作用和相关机制研究

Adipose tissue engineering could be used to treat cranio-facial soft tissue defects or deficiencies as a best treatment in the future. The regulatory mechanism in ADSCs adipogenic differentiation, especially the epigenetic regulatory mechanism, is the key problem in this research direction. In a previous study, we found a long noncoding RNA which was named with lncRNA-Adi highly expressed differently fore-and aft ADSCs adipogenic differentiation. The following study showed there are some combined motifs between lncRNA-Adi and PPARγ and GATA protein, which are widely accepted as the indispensable key molecules in ADSCs adipogenic differentiation. The above results suggested it very likely that lncRNA-Adi plays an important role by means of controlling PPARγ signal pathway in the regulatory mechanism in ADSCs adipogenic differentiation. So, a systemic study would be performed to explore the expression characteristic, function, target proteins and pathway of lncRNA-Adi in ADSCs adipogenetic differentiation in this project. The results of this study will provide some experimental proofs for understanding the molecular mechanism of ADSCs adipogenetic differentiation and established a new research direction for improve the induction technology of ADSCs adipogenetic differentiation. The results of this study will provide some references to understand the pathogenesis and explore the new therapy method of metabolic diseases, such as diabetes and obesity.

脂肪组织工程有望成为解决颅颌面软组织缺损或不足的最佳方法，极具潜在临床应用前景。在该研究方向，脂肪基质细胞脂向分化调控机制是关键的研究问题之一。本课题组在前期研究中发现lncRNA-Adi在脂肪基质细胞脂向分化前后有显著差异表达，进一步研究发现lncRNA-Adi与脂向分化关键分子——PPARγ及GATA蛋白具有结合位点，该结果提示lncRNA-Adi极可能通过调控PPARγ信号通路在脂肪基质细胞脂向分化过程中发挥调控作用。因此，本课题拟开展系统研究，明确lncRNA-Adi在脂肪基质细胞脂向分化过程中的表达规律、功能、靶蛋白及相关信号通路，从而了解lncRNA-Adi在这一过程中的具体作用及相关机制。该问题的解决可对明确脂肪基质细胞分化调控机制及改善脂向分化诱导技术提供依据，对了解糖尿病、肥胖症等代谢性疾病的发病机制及探索新型治疗方法也有参考价值。

脂肪基质细胞(Adipose-derived mesenchymal stem cells, ADSCs)是再生医学领域最具应用前景的种子细胞来源。本课题组对lncRNA-Adi在ADSCs脂向分化过程中的表达、调控作用及其机制进行了系统性研究，获得了以下原创性研究成果：1.随着ADSCs脂向分化开始，lncRNA-Adi表达水平明显升高，在脂向诱导第3天达到峰值，随后其表达水平逐渐下降，至脂向分化第7天脂肪细胞形成时lncRNA-Adi表达水平仍高于未分化ADSCs。2. lncRNA-Adi高表达于ADSCs脂向分化过程，在ADSCs成骨分化、成软骨分化和成神经分化过程中不表达；在大鼠脂肪和肌肉组织中表达水平较高，在大鼠肺、脑、肾等组织中表达水平低。3.在ADSCs脂向分化过程中干扰lncRNA-Adi显著抑制了细胞脂滴累积、成脂关键基因表达和细胞的增殖，证明lncRNA-Adi作为正向调控分子对ADSCs脂向分化过程中进行调控。4. lncRNA-Adi位于ADSCs细胞质内，其序列不具有Alu元件，且不具有和PPARγ结合的能力。5. lncRNA-Adi与microRNA-449a具有相互作用，并能通过与microRNA-449a的结合作用对ADSCs脂向分化过程中细胞进入细胞周期S期产生调控作用。6. lncRNA-Adi与CDK6 3’UTR竞争性结合microRNA-449a，从而提高CDK6蛋白表达水平，激活pRb-E2F1信号通路，保证细胞顺利进入细胞周期S期完成有丝分裂，同时促进终末分化阶段PPARγ表达，从而对ADSCs脂向分化整个过程发挥调控作用。上述结果证明lncRNA-Adi对ADSCs脂向分化具有正向调控作用，这一作用是通过竞争性结合microRNA-449a，促进CDK6蛋白表达，激活pRb-E2F1信号通路实现的。本课题为深入了解脂向分化过程中的分子调控机制提供了实验依据，为改善脂肪基质细胞脂向分化诱导技术建立了新的研究方向；并对了解糖尿病、肥胖症等代谢性疾病的发病机制及探索其新型治疗方法也有明显的参考价值。