抗氧化应激对糖尿病小鼠骨髓干细胞凋亡的影响及机制探讨

Research has shown that the implantation of bone marrow stem cells (BMSCs) produces poor angiogenic effect when with diabetes. However, the precise mechanisms remain unclear. Our preliminary research data showed that, compared with healthy mice, the expression of reactive oxygen species (ROS) in BMSCs, gamma-H2AX (γ-H2AX), senescence-associated-beta-galactosidase (SA-beta-Gal) and the apoptosis of BMSCs was up-regulated to different degrees, with the expression of phosphorylated-Protein kinase B(p-Akt) and B-cell lymphoma-2(Bcl-2) protein down-regulated significantly. Therefore, we conjecture that the oxidative stress induced by high glucose would promote the apoptosis of BMSCs, thus antioxidants could inhibit the same process. To verify this hypothesis, Streptozotocin (STZ) induced diabetic mice models are used. The treated group are given an antioxidant therapy, which is a daily subcutaneous injection of N-acetyl-L-cysteine (NAC) for 4 weeks. Then we collect CD117+ BMSCs, analyze the survival, early apoptosis, DNA damage and senility of those BMSCs in comparison with the untreated group. We also culture BMSCs by high glucose in order to reveal the molecular mechanisms based on the phosphatidylinositol 3-kinase/Protein kinase B/ B-cell lymphoma-2（PI3K/Akt/ Bcl-2）signal pathway. This study will lay the theoretical foundation for the application of implantation of BMSCs for ischemic diseases with diabetes.

骨髓干细胞移植治疗在合并糖尿病情况下的血管新生效果欠佳，迄今具体机制未明。我们前期研究发现与正常小鼠相比较，糖尿病小鼠骨髓干细胞血清活性氧族（ROS）水平升高，DNA损伤标志蛋白γ-H2AX表达、衰老标志蛋白SA-beta-Gal表达及细胞凋亡显著增加，p-Akt及Bcl-2蛋白表达明显下降，因此我们推测高血糖引起的氧化应激会导致骨髓干细胞凋亡，给予抗氧化应激治疗可以抑制其凋亡。为验证此推测，本研究拟给予链脲佐菌素诱导的糖尿病小鼠腹腔注射抗氧化剂N-乙酰半胱氨酸（NAC），4周后采集分离CD117+骨髓干细胞，通过与非治疗组比较，分析NAC治疗后骨髓干细胞的存活率、凋亡、DNA损伤以及衰老状况，并通过体外高糖培养骨髓干细胞、基于PI3K/Akt/ Bcl-2信号通路探讨其相关分子机制。本研究将为骨髓干细胞移植在糖尿病合并缺血性疾病方面的治疗奠定理论基础。

骨髓干细胞治疗在合并糖尿病情况下的血管新生效果欠佳, 考虑与高血糖引起的氧化应激导致细胞损伤相关，本研究探讨了抗氧化剂N-乙酰半胱氨酸（N-acetyl-L-cysteine；NAC）对糖尿病小鼠CD117+骨髓干细胞凋亡的影响并探讨其机制。我们建立了STZ糖尿病小鼠模型，分别给予小鼠腹腔注射NAC (观察组) 和NaCl (对照组)，4周后采用ELISA法测定以上2组小鼠的尿8-Isoprostane（氧化应激标志物），评估NAC治疗效果；免疫磁珠分离法采集CD117+骨髓干细胞，采用免疫荧光法检测两组CD117+骨髓干细胞增殖情况；流式细胞术检测两组CD117+骨髓干细胞中活性氧 (ROS) 水平以及细胞早期凋亡状况。使用Western blot方法检测核因子红细胞介素2相关因子2 (nuclear factor erythroid 2-related factor 2, Nrf2) 的表达。采用实时荧光定量方法观察SOD1、SOD2、GPX1、Txnrd1、GCL、HO-1在两组细胞中的相对表达情况。我们发现与对照组比较，观察组尿8-Isoprostane水平显著性降低 (P<0.05)，细胞增殖率显著性升高(P<0.05)， CD117+细胞凋亡率显著性降低 (P<0.01)；与对照组相比，NAC治疗组CD117+骨髓干细胞的ROS表达显著性降低、Nrf2表达明显增强。此外、 NAC治疗组SOD1、SOD2、GPX1、Txnrd1、GCL以及HO-1的相对表达量与对照组相比显著性降低(P<0.01)。因此，我们认为NAC可通过减轻糖尿病小鼠的氧化应激来抑制CD117+骨髓干细胞的凋亡。本项目为骨髓干细胞移植在糖尿病合并缺血性疾病患者方面的应用奠定理论基础，同时为NAC抗氧化应激治疗的临床应用提供参考。