丹参红花提取物改善糖尿病环境下干细胞移植后血管新生功能的机制研究

Therapeutic Angiogenesis is a potential method for treating diabetic foot based on stem cells transplantation, but function and survival of stem cells are damaged in high glucose microenvironment. Results of our studies showed that injection of Salvia miltiorrhiza and Carthamus tinctorius Extract (SCE) could significantly promote angiogenesis in ischemic hind limb of diabetic nude mice. And the data from microRNA microarray showed miR-27b played a role in that. However, the molecule mechanism in detail is unclear. Base on that Endothelial progenitor cells (EPCs) play an important role in vascular self-repair and angiogenesis after vessel endothelium damaged. In this study, we firstly created a cell model that EPCs were challenged in diabetic microenvironment, to explore the protective effects of benefiting SCE in maintaining stem cells’ function and promoting angiogenesis via miR-27b and related signal pathway, and finally such protective effects were confirmed and relative mechanisms were investigated in hind limb ischemia model generating by femoral artery excision in streptozotocin-treated nude mice by combing SCE injection (experimental group) and normal saline (control group) with EPCs, which it is transfected by miR-27b mimic or inhibitor plasmid, transplantation. Blood perfusions, vessel regeneration and microvascular densities were investigated by laser Doppler perfusion imager system, X-ray and immunohistochemistry. Data from our study will not only enrich basic theory system about treatment of TCM combination with stem cell transplantation for angiogenesis, but also provide new evidence for relative clinical application.

以干细胞移植为基础的治疗性血管新生为糖尿病足治疗带来曙光，但机体高糖环境严重影响干细胞的功能和存活。我们前期研究发现，丹参红花提取物（SCE）能显著改善糖尿病后肢缺血血管新生，且miRNA芯片结果提示这可能与血管新生调控因子miR-27b有关，但具体机制不明。基于内皮祖细胞（EPCs）在内皮受损后自身修复和血管新生中的重要作用，本课题拟先构建糖尿病环境下的EPCs细胞模型，探索SCE对该模型的保护作用和miR-27b及其相关调控通路在其中的作用；随后在糖尿病后肢缺血裸鼠模型上，分别移植miR-27b mimic和inhibitor质粒转染的EPCs后，再给予生理盐水（对照组）或SCE（实验组）注射，采用激光多普勒扫描血流灌注情况，X线血管造影观察管样新生及免疫组化分析微血管生成，并探索相关信号通路蛋白变化。为进一步完善中医药联合干细胞移植促进糖尿病足血管新生的理论体系和临床应用提供新依据。

以干细胞移植为基础的治疗性血管新生为糖尿病足治疗带来曙光，但机体高糖环境严重影响干细胞的功能和存活。我们前期研究发现，丹参红花提取物（SCE）能显著改善糖尿病后肢缺血血管新生，且miRNA芯片结果提示这可能与血管新生调控因子miR-27b有关，但具体机制不明。miR27b与血管新生密切相关的一种miRNA，其可能是通过下调TSP-1发挥促血管新生作用。然而，如果在糖尿病下肢缺血的环境下，miR27b/TSP-1信号通路如何发挥作用，尚未可知。因此，我们主要探索糖尿病主要致病因子晚期糖基化终产物亚型之一——CMLs对内皮细胞血管新生能力的影响，并结合miR27b/TSP-1信号通路，探索可能机制。通过移除左侧股动脉的方式构建糖尿病下肢缺血模型，人脐静脉内皮细胞被用于体外实验探索CMLs对血管新生的影响。结果显示，与正常小鼠相比，糖尿病小鼠下肢缺血后血流减少、管腔直径减小、CD31和VEGF以及mi27b表达减少，TSP-1表达上调。CMLs干预脐静脉内皮细胞后，miR27b、VEGF表达下降，TSP-1上调；给予RAGE受体拮抗剂后，趋势逆转。另外，过表达miR27b，能够逆转CMLs下调VEGF的作用，同时促进管样新生。我们数据证实，CMLs能显著损害下肢缺血后血管新生，可能是通过调节内皮细胞内的miR27b/TSP-1信号通路。我们的研揭示了糖尿病下肢缺血后血管新生受损的新机制。