SRPK1介导的JAM-A异常剪接在糖尿病创面延迟愈合中的作用及机制研究

糖尿病（DM）创面难愈是目前临床和基础研究的重点和难点，表皮细胞功能异常是糖尿病创面难愈的重要原因之一。申请者前期研究发现链接粘附分子JAM-A在创面愈合中起重要作用。预实验发现正常表皮细胞中JAM-A RNA可通过其3’UTR发挥ceRNA功能，调控创面愈合关键因子LGR5。但是在DM创面中，缺失ceRNA功能结构的3’UTR短缩型JAM-A剪接体（JAM-A 3S）的生成明显增多，且与剪接因子激酶SRPK1的表达密切相关。我们据此提出假说：高糖环境下SRPK1特异性活化造成JAM-A异常剪接生成JAM-A 3S，后者不能保护LGR5，造成表皮细胞迁移功能障碍导致DM创面愈合延迟。本课题拟进一步利用体外培养细胞、转基因小鼠DM模型和临床样本，通过RIP、RNA pull down、CRISPR靶向敲除等手段证实该假说，为DM创面愈合研究提供新的实验参考，并为其治疗提供新的作用靶点。

Nonhealing wounds of diabetes mellitus (DM) have always been the basic and clinical research emphases and hotspot, the dysfunction of epidermal cell is one of the causes of that. Our previous studies has found that the expression of junction adhesion molecule-A（JAM-A）was increased and played an important role in skin wound healing. In the preliminary experiment, JAM-A was found to function at RNA level in epidermal cells, the 3’UTR of JAM-A regulated the expression of wound healing key factor LGR5 by competing endogenous RNAs (ceRNA).However, the main change of JAM-A in DM wounds newly generated epidermis is an increase in the percentage of 3'UTR shortening JAM-A spliceosome (JAM-A 3S).Moreover, the splicing factor kinase SRPK1 is up-regulated in DM wound and closely related to JAM-A3S production. We speculate that the activation of SRPK1 in DM wound with a high glucose environment may cause abnormal splicing of JAM-A, leading to the increasing production of JAM-A 3S. Furthermore, JAM-A mRNA could not be used as ceRNA to protect the normal expression of target gene, e.g., LGR5, affecting the migration function of epidermal cells and induce the delayed wound healing. Therefore, we simulate DM by application of high glucose (HG) and employ RIP, RNA pull down, EMSA and CRISPR/Cas9 targeted knockout techniques to explorer the process and possible mechanism of SRPK1’s activation and JAM-A’s splicing. We would also build human epidermal cell line with different segments JAM-A modification to clarify the effect of JAM-A 3S on epidermal cell function. Furthermore, the DM models of transgenic mice and clinical samples would be employed to clarify the role of JAM-A in epidermal cells dysfunction of DM wounds, and providing a noval potential target for DM wounds healing.