基于转录组测序对2型糖尿病损伤脂肪干细胞修复能力机制的探索研究

Diabetic foot ulcers(DFU), which are the main reasons of amputations in non-traumatic diseases, are common problem in clinic. So far, a large part of global health budget has been expended on it. However, the current treatments for DFU are still unsatisfactory in most of cases. Numerous studies have revealed the positive functions of autologous stem cells for treating DFU. Especially, adipose-derived stem cells (ADSCs), have been considered as a promising approach for this problem. Nevertheless, the repairing capacity of ADSCs derived from diabetics is impaired before graft. Therefore, it is necessary to elucidate the mechanisms of the impairment in order to facilitate the development of stem cell therapy in DFU in the future. Previous studies on this issue are fragmented and lack of comprehensive and systematic methods. Therefore, this project aims to investigate whether the effect of diabetes on the characteristics and functions of ADSCs could be reflected in the transcriptome (mRNA, miRNA, lncRNA) levels. The whole transcriptome sequencing will be used to compare the expression profiles of ADSCs between diabetics and non-diabetics, and bioinformatics analysis is applied to explore the changes of specific transcripts and related signal pathways. Further research activities by use of the nucleic acid transfection, siRNA interference and other techniques to regulate the expression of the key transcripts will be engaged to clarify the function of key transcripts. We are expecting to find the methods to up-regulate the therapeutic capacity of ADSCs in diabetic patients, and thus improve the outcomes of patients with diabetes mellitus in the future.

糖尿病足对全球医疗造成了巨大负担，目前的治疗效果不尽人意。研究证实干细胞尤其是脂肪干细胞（ADSCs）移植能明显促进糖尿病创口愈合，ADSCs移植治疗现已受到广泛推崇并写入了临床指南。然而糖尿病本身损伤了ADSCs的修复能力，这极大地限制了ADSCs的临床应用。以往针对此现象的研究较少，且多为“碎片式”研究，缺乏全面性和系统性。我们前期研究发现糖尿病来源的ADSCs促进创面修复及血管形成能力减弱，我们假设糖尿病对ADSCs修复能力的影响将会在转录组水平得以体现。在此我们利用RNAseq技术比较糖尿病患者和与之匹配的非糖尿病患者的ADSCs的表达谱差异，通过生物信息学分析找出关键转录本和核心信号通路，以期弄清相关机制。进一步通过核酸转染、siRNA干扰等技术调节关键转录本的表达水平，从而阐明关键转录本的功能并提出改善糖尿病患者ADSCs治疗能力的方法，以此来推动ADSCs自体移植治疗的发展。

包括脂肪干细胞（ASC）在内的间充质干细胞在转化医学领域具有巨大的潜力。然而，老年、糖尿病状态，肥胖症，慢性炎症状态和其他代谢性疾病等多种因素可能损害干细胞功能，从而阻碍了干细胞疗法的整体有效性。非编码RNA已被证明在干细胞生物学活动中起着重要作用。然而，ASC中与糖尿病、衰老相关的非编码RNA的整体表达变化和相互作用仍然未知。.我们分别从糖尿病、老年以及年轻供体脂肪组织中分离出ASC（D-ASCs，O-ASCs与Y-ASCs），体外培养后检测对比其细胞功能、衰老程度及潜在修复能力。然后，对三组ASC进行全转录组测序，结合测序数据进行生物信息学分析，以揭示具有显著差异mRNA的功能。由此构建竞争内源性RNA（ceRNA）相关的lncRNA或circRNA。通过PCR的扩大样本验证，最终选择了其中差异表达明显的lncRNA-NONHSAT035482.2进行进一步的功能及机制验证。最后，运用大鼠背部创面模型来评估NONHSAT035482.2敲低后的衰老干细胞的修复能力。研究结果显示，糖尿病供体来源的ASC（D-ASCs）较老年供体的ASC（O-ASCs）及年轻人来源ASC（Y-ASCs）而言，衰老明显，并表现出较差的迁移增殖能力及潜在创面修复能力。通过测序数据分别筛选了D-ASCs与 O-ASCs，O-ASCs与Y-ASCs中差异表达的miRNA，mRNA，lncRNA和circRNA。GO和KEGG分析表明差异表达的mRNA在衰老方面显著富集。进一步根据关键的差异表达mRNA构建了PPI网络以及ceRNA网络。验证 PPI和ceRNA网络中所选的mRNA，lncRNA和circRNA。最终，我们发现并明确lncRNA-NONHSAT035482.2在衰老干细胞中显著上调。在机制上，NONHSAT035482.2可通过海绵吸附miR-143-3p，从而使miR-143-3p对其靶基因ADD3的抑制减弱。动物实验中，敲低NONHSAT035482.2的衰老干细胞在创面模型移植治疗中较对照衰老干细胞表现出更好的促进创面愈合的能力。.总之，此项研究为ASCs衰老、功能障碍的潜在机制提供了新线索，揭示了NONHSAT035482.2/miR-143-3p/ADD3通路可能是调控ASCs衰老所致功能障碍的途径，为逆转ASCs衰老，恢复ASCs治疗能力提供了新的靶点。