低氧预处理尿源性干细胞用于促进糖尿病肾病的肾组织修复及其作用机制的研究

Diabetic nephropathy (DN) is one of the most common complications of chronic renal failure. Current therapies have not successfully achieved full recovery of renal function yet. Stem cells-based therapy provides alternative in treatment of DN. Many studies have showed that transplantation of stem cells enhanced renal function and decreased the blood sugar levels, which might be via their trans-differentiation or paracrine effects to stimulate endogenous cells participate into renal tissue repair. However, it needs the invasive procedures to obtain the stem cells. Our group first demonstrated the presence of stem cells in human voided urine (i.e. urine derived stem cells, USCs) , and then fully characterized these cells. USCs possess the multipotent differentiation capacity, proangiogenic paracrine effects and immunomodulatory properties. Our recent data demonstrated that USCs significantly enhanced renal function by protecting the implanted cells from inflammatory injury, and reducing the pool of macrophages and amount of interstitial collagen deposition within the kidney tissue after implantation of human USCs in an athymic rodent model of chronic renal failure. More recently, our pilot study has demonstrated that USCs collected from the patients with DN (D-USCs) can be cultured but they are slow in growth and enter apoptosis in vitro. Preconditioning hypoxia (5%O2 for 7 days) significantly improved D-USCs survival, promoted cell proliferation and increased antioxidative potential by expression of Redox responsive transcript factor (FOXO3a) and Hypoxia-inducible factor 1-alpha (HIF1α). Thus, we hypothesize that hypoxia preconditioning will be particularly beneficial to USCs from diabetic patients (HP-D-USCs), reversing the oxidative damage from their in vivo environment; these HP-D-USCs can improve renal function via inhibiting inflammatory responses and glomerular remodeling in vivo To test these hypotheses, we propose the following Specific Aims in this proposal: 1. Determine whether HP-D-USCs improve cell survival, proliferation via activation of Akt singling pathway; 2. Determine whether hypoxia preconditioning promotes trans-differentiation, paracrine action, anti-inflammatory and anti-oxidative effect of D-USCs via activating CRCX4-CRCX singling pathway; 3. Determine the therapeutic impacts of HP-D-USCs in improvement of renal function and histological structures, and in decrease blood sugar levels via paracrine effect and anti-oxidative potential, which might provide a very promising cell source in treatment of diabetic nephropathy.

糖尿病肾病是导致慢性肾功能衰竭的常见原因之一。目前应用的治疗措施不能理想地控制糖尿病肾病的发生与发展。干细胞治疗通过细胞分化和旁分泌作用改善肾功能并缓解高血糖症状, 但是这些干细胞需要通过创伤方式获得。我们在世界上首次从人尿液中分离出一种来源于肾脏并具有自我更新和多分化潜能的尿源性干细胞（USCs），它还具有抑制炎症和调节免疫等作用。我们研究显示，将人USCs移植到慢性肾功能衰竭大鼠后，能阻止肾脏细胞损伤，减少炎症反应及胶原沉积，改善肾功能。我们还发现：从糖尿病肾病患者尿液也能提取USCs，经低氧处理后细胞增殖和抗氧化能力基本恢复。本研究将通过体内和体外实验探讨经低氧处理的患者USCs是否通过激活Akt通路，提高细胞活性，增强细胞旁分泌和抗炎抗氧化损伤作用而改善肾功能。本研究从患者USCs这个新视点揭示低氧处理的USCs在糖尿病肾病修复中的作用和机制，为糖尿病肾病的治疗提供新思路。

通过收集健康志愿者和糖尿病肾病患者的尿液，通过无创的方式分离提取获得尿源性干细胞（USC and D-USC），并将两组细胞分别置于常氧状态（20% O2）和低氧状态（3% O2）进行培养，从干细胞干性、增殖、凋亡、旁分泌作用等方面检测，结果显示D-USC与USC同样具有干细胞的相关特性，可以自我增殖及分泌营养因子，但与USC相比，D-USC的增殖减弱，凋亡增加，分泌功能减低，通过低氧环境培养，D-USC的相关生物学功能有提高。以NOD/SCID为模型建立Ⅰ型糖尿病肾病模型，以SD大鼠为模型建立Ⅱ型糖尿病肾病模型，将USC移植进入动物模型体内并进行相应的生化和组织学检测，结果提示USC对肾脏的纤维化有明显的修复作用。