Ginsenoside Rg1调控异体ASCs旁分泌功能促进糖尿病创面的修复及分子机制

Diabetic Ulcers (DU) is one of the commonly most complications,which it is high incidence, longer treatment and harder healing. It has become the research focus, but the mechanism is unclear. Adipose-derived stem cells(ASCs) can promote repair and regeneration of the injured tissue, while the research about ASCs in DU wounds healing is not deeply opened.Our previous studies have confirmed that the ginsenoside Rg1 (Rg1) can up-regulate the paracrine function and HIF-1 alpha expression of ASCs, then subsequently to enhance the downstream related genes exprseeion and realize its effect on promoting tissue regeneration. These data suggested that Rg1 very likely play an important role in promoting tissue repair and regeneration by HIF-1α regulating the paratine of ASCs. On the basis of our previous research works, we hypothesized that Rg1 could be regulated HIF-1α expression of ASCs to achieve DU wounds healing, which is likely to be its key gene in miRNA31/HIF-1α and PI3K/Akt singaling pathway. We designed a series of experiments to detect the expression of downstream target genes in vivo and in vitro, including constructed wildtype and mutant dual luciferase reporter vectors of FIH-1-3’UTR, miRNA31 expression vector.we verified miRNA31/HIF-1α and then inhibited or activated PI3K/Akt, small hairpin RNA (shRNA) was used to silence the HIF-1α and AKT genes in ASCs. Our aim is illuminated that the molecular mechanisms and efficacy of how to promote wounds healing of DU by using the ASCs with Rg1 pretreament. Finally, we hope that our studies could provide a new approach and theoretical support for clinical application for ASCs widely applying to clinical therapy in regeneration medicine.

糖尿病溃疡（DU）发生率高、难愈合，是晚期糖尿病常见并发症之一，也是当前研究热点，但相关机制尚不明确。研究表明脂肪干细胞（ASCs）能促组织再生修复，但在促DU愈合研究方面欠深入。我们前期证实，人参皂苷Rg1（Rg1）能上调HIF-1α表达、增强ASCs旁分泌及下游相关因子表达促组织再生。提示Rg1可能通过HIF-1α调控ASCs旁分泌在促组织再生中发挥重要作用。本项目拟在此基础上提出Rg1调控ASCs 中miRNA31/HIF-1α、PI3K/Akt通路关键基因HIF-1α表达以促DU愈合的设想，构建FIH-1-3’UTR野生/突变型双荧光素酶及miRNA31表达载体，验证miRNA31/HIF-1α，激活/抑制PI3K/Akt， shRNA沉默ASCs中HIF-1α、AKT，检测下游靶基因，体内外实验阐明Rg1调控ASCs旁分泌促DU愈合的机制，为ASCs治疗DU提供新路径及理论依据。

糖尿病溃疡（DU）发生率高、难愈合，是晚期糖尿病常见并发症之一，也是当前研究热点，但相关机制尚不明确。研究表明脂肪干细胞（ASCs）能促组织再生修复，但在促DU愈合研究方面欠深入。我们前期证实，人参皂苷Rg1（Rg1）能上调HIF-1α表达、增强ASCs旁分泌及下游相关因子表达促组织再生。提示Rg1可能通过HIF-1α调控ASCs旁分泌在促组织再生中发挥重要作用。本项目是应用组织病理学、细胞生物学、分子生物学、干细胞移植与再生医学的基本原理与方法，明确了Rg1 调控ASCs 旁分泌的生物学功能，探索了Rg1 对ASCs 旁分泌功能调节的量效关系及初步调控机制；阐明了miRNA31/HIF-1α、PI3K/Akt 信号通路介导Rg1 干预异体ASCs 提高DU 创面愈合的调控机制；通过体内外实验阐明了shRNA 沉默HIF-1α、AKT 表达后经Rg1 干预异体ASCs 对DU创面愈合的影响及分子机制，为ASCs功能优化调控提供新的治疗策略，对Rg1调控ASCs功能促进软组织创伤修复技术的临床应用和推广提供理论依据。本项目获得的重要结果如下：.（1）体外实验明确不同浓度Rg1可促进ASCs生长增殖及旁分泌功能，并在一定浓度范围内呈正向调节的量效关系。.（2）体外实验证实miRNA31/HIF-1α、PI3K/Akt 是Rg1促进体外ASCs旁分泌功能的重要信号通路。.（3）体内移植实验证实Rg1干预后的异体ASCs 能有效增强其促进糖尿病大.鼠创面的愈合能力；一些关键基因（AKT、HIF-1α、miRNA31、FIH-1）的表达与创面愈合速度及愈合质量成正相关。.（4）体内外正反两方面实验证实miRNA31/FIH-1/HIF-1α及PI3K/Akt/HIF-1α 通路的抑制及下游靶基因的沉默能够降低Rg1促进ASCs的增殖及旁分泌功能效应，说明Rg1促进糖尿病大鼠创面愈合的分子作用机制主要是通过激活miRNA31/FIH-1/HIF-1α及PI3K/Akt/HIF-1α信号通路实现的。