Bim蛋白介导肾小管近端上皮细胞凋亡的位点及分子治疗机制研究

As the leading course of end-stage renal disease, diabetic nephropathy (DN) is recognized as a disease most resulting from the lesion of the glomerulus. While in our preliminary work, it was confirmed that the apoptosis of proximal tubule epithelial cells (PTEC) occurred much earlier than glomeruli injury in the development of diabetic nephropathy. Further studies showed that the increased activity of proapoptosis protein Bim (Bcl-2 interacting mediator) was closely related to the lesion of PTEC. Though the BH3 domain has been identified as the main fragment linked with the proapoptosis activity of Bim protein, the exactly mechanism is remain unclear. Based on the sequence homology analysis, there are two highly conserved sequences -133 to -128 motif and -160 to -148 motif, have been screening out. So, we are going to clarify which segment of these two sequences induces the apoptosis of PTEC. Furthermore, we will set up a polypeptide pool and select the specific antagonist to inhibit the apoptosis of PTEC. Finally, we will test the therapeutic efficacy of the selected peptide in rat model of diabetic nephropathy. . The significance of the study is that it found a new mechanism and potential target of diabetic nephropathy on the basis of long term investigation; try to interfere it with specific polypeptide. This study will let usto get a better understanding of DN.

糖尿病肾病（DN）是糖尿病主要并发症，发病机制尚未完全清楚。既往研究多集中于肾小球病变。但近来证实，肾小管也参与DN的发病。我们前期研究发现，在DN发病中肾小管病变早于肾小球病变，在尿蛋白正常时已出现肾小管近端上皮细胞（PTEC）凋亡。同时证实，促凋亡分子Bim蛋白是介导PTEC凋亡的关键因子，BH3结构域是Bim介导凋亡的主要位点，并找到BH3上两个高度保守的片段，由此提出假说：Bim是通过BH3功能区片段介导PTEC凋亡，干预该位点能抑制PTEC凋亡，治疗DN。为证实该假说，该研究拟采用定向突变技术明确BH3调控PTEC凋亡的具体片段，并构建短肽库，从中筛选出能干预该功能区片段的短肽，用该短肽抑制PTEC凋亡和DN进展。该课题有充分的前期研究基础，以肾小管病变为切入点发现了DN新的发病机制，并首次探索用短肽拮抗剂更加精确、靶向的进行干预，为DN的治疗提供全新的思路和治疗靶点。

Bim通过BH3功能区片段介导PTEC凋亡，本课题应用Clustalx和DNAman软件进行同源比对及蛋白质结构分析，选出两个高度保守的功能区片段 LSAMAS（-133 至-128 片段），IAQELRRIGDEFN(-160 至-148 片段)，因在晶体结构显示的Bim BH3中，只存在IAQELRRIGDEFN，IAQELRRIGDEFN位于活性空腔内，经文献依据，选定IAQELRRIGDEFN并进行延长，最终选定Bim BH3功能区序列为DMRPEIWIAQELRRIGDEFNAYYARR。通过高通量筛选技术寻找能与上述功能区序列特异性结合的短肽，并运用计算机模拟技术、Biacore系统（检测亲和力）、蛋白质生物化学、细胞生物学实验等, 验证所筛选的短肽能否抑制PTEC凋亡和DKD进展及其作用机制进行了研究。首先采用高通量筛选技术，以前面确定的Bim BH3功能区序列为抗原，用随机混合肽库做筛选（7 肽库混 12 肽库），采用ELISA法筛选出能特异性结合功能区多肽的8个短肽。应用计算机模拟技术，使用Discovery Studio2016模块ZDOCK实现短肽与DMRPEIWIAQELRRIGDEFNAYYARR蛋白质对接计算，验证结合能力，并结合ELIAS结果筛选出亲和力高的两个短肽，分别与DMRPEIWIAQELRRIGDEFNAYYARR进行Biacore检测。后选定亲和力最高的短肽进行细胞功能学实验，验证抑制PTEC凋亡；并在早期糖尿病肾病大鼠体内进行了初步实验。综上所述，前期研究证实，高糖通过Bim介导PTEC凋亡。本研究证实，Bim的BH3区域的DMRPEIWIAQELRRIGDEFNAYYARR是其功能区片段，并通过高通量筛选、ZDOCK和Biacore检测等方法，筛选出能结合该功能片段的特异性结合短肽，在体外验证了其抗PTEC凋亡的效果，为将来DKD的治疗开发了新的、潜在的治疗靶点。项目资助发表SCI论文9篇，中文核心期刊3篇，培养学生2人。项目投入经费52.0000万元，支出51.5834万元。剩余经费0.4166万元，剩余经费计划用于本项目后续支出。