二氮嗪改善糖尿病小鼠内皮祖细胞功能及其机制

发布时间：2018-04-25 17:08

本文选题：二氮嗪 + 糖尿病　；参考：《第二军医大学》2014年硕士论文

【摘要】：研究目的：糖尿病(Diabetes mellitus，DM)是一种全身系统性代谢紊乱疾病，分为胰岛素依赖型(Insulin-dependent)与非胰岛素依赖型(Insulin-independent)。大血管及微血管病变是糖尿病患者主要的致残致死原因，而内皮功能的损伤(Endothelial dysfunction)则是引起伤口愈合缺陷和血管病变的始发因子。内皮细胞(Endothelial cells，ECs)的活性是维持内皮功能的基础，如果内皮细胞的凋亡率超过正常水平，它就会影响血管的完整性。内皮祖细胞(Endothelial progenitor cells, EPCs)来源于成体骨髓，并可分化为内皮细胞，进而参与血管新生和内皮损伤后的修复过程。已有研究表明，骨髓来源的内皮祖细胞(Bone marrow-derived endothelial progenitor cells, BM-EPCs)可以定向结合到血管再生部位并参与受损内皮的修复。循环中的EPCs的数量与发生血管疾病的概率呈反比。因此EPCs与内皮功能有着至关重要的联系。凝血酶敏感蛋白-1(Thrombospondin-1,TSP-1)是一种有效的内源性的血管生成抑制剂，糖尿病会引起EPCs的TSP-1mRNA的表达量增加, EPCs功能障碍也会导致TSP-1的表达增加。肿瘤抑制基因p53可以激活由THBS1基因编码的TSP-1的启动子，有文献指出：p53可促进TSP-1的产生，使成纤维细胞的血管新生受损。二氮嗪(Diazoxide)是线粒体KATP通道开放剂，可通过使细胞膜超极化，K+外流增多，Ca2+内流减少，具有抑制葡萄糖刺激的胰岛素分泌，从而为胰岛B细胞细胞提供休息，发挥延缓和阻碍糖尿病及其并发症的发展的作用。本课题研究二氮嗪能否加速糖尿病小鼠伤口愈合，改善BM-EPCs功能，并探索其可能机制。研究内容及结果：动物模型：使用链脲佐菌素(Streptozotocin, STZ)诱导的1型糖尿病小鼠模型；细胞模型：以葡萄糖33mM诱导人脐静脉内皮细胞(Human umbilical veinendothelial cells, HUVECs)和BM-EPCs高糖损伤的细胞模型。在动物水平，研究了二氮嗪对糖尿病小鼠内皮功能的影响，从EPCs角度围绕TSP-1和p53两个作用靶点探讨了其机制；在细胞水平，通过细胞功能实验(迁移功能、粘附功能和小管形成功能)来观察二氮嗪对血管生成的影响；并通过测定TSP-1和p53的表达来明确二氮嗪影响血管生成的机制。 1.二氮嗪促进1型糖尿病小鼠伤口愈合选取5周龄的C57BL/6小鼠，18-20g左右，随机分为正常组(Control)、1型糖尿病模型组(STZ)和二氮嗪治疗组(30mg/kg/d灌胃4周)。小鼠麻醉状态下，在其背部切取一个半径6mm的圆形伤口，连续12天每2天观察其伤口愈合的情况。第3、6、9天分别取伤口周围6mm内的皮肤作为标本，CD31免疫组化检测血管密度情况。取2-3mm小鼠的主动脉，通过血管张力系统来检测其血管舒张功能。结果显示：1型糖尿病小鼠的伤口愈合速度明显降低，毛细血管密度显著下降，血管依赖性内皮舒张功能明显下降；二氮嗪治疗后伤口愈合能力增强，血管密度增加，血管依赖性内皮舒张功能增强。 2.二氮嗪改善1型糖尿病小鼠BM-EPCs功能采用Dil-acLDL和FITC-UEA-1双阳性鉴定BM-EPCs。通过CD34和Flk-1双阳性的百分比检测小鼠循环中的EPCs数量。通过Boyden小室和成管实验检测迁移功能和小管形成能力。结果显示：1型糖尿病小鼠循环中EPCs的数目明显降低，二氮嗪治疗后可以显著增加循环中EPCs的数量；1型糖尿病小鼠BM-EPCs的小管形成能力、迁移功能和粘附功能明显下降，二氮嗪治疗后可以显著改善BM-EPCs的小管形成能力、迁移功能和粘附功能。 3.二氮嗪改善1型糖尿病小鼠BM-EPCs功能的可能机制通过免疫印迹法检测1型糖尿病小鼠BM-EPCs中p53和TSP-1表达。结果表明：1型糖尿病小鼠BM-EPCs的p53表达明显升高，二氮嗪治疗后可以显著降低BM-EPCs中p53的水平；1型糖尿病小鼠BM-EPCs的TSP-1的表达明显增加，二氮嗪治疗后可以显著降低TSP-1的表达。 4.二氮嗪改善高糖诱导的BM-EPCs功能障碍及机制正常小鼠骨髓分离的BM-EPCs培养7天后，使用高糖(33mM)诱导细胞损伤模型，二氮嗪(30μM)处理24h后，检测细胞功能和蛋白表达情况。结果显示：高糖诱导的BM-EPCs小管形成能力、迁移功能和粘附功能明显降低，二氮嗪(30μM)处理后可以显著改善高糖诱导对BM-EPCs小管形成能力、迁移功能和粘附功能的损伤。高糖诱导的BM-EPCs中p53和TSP-1的表达明显升高，，而二氮嗪处理后可以下调高糖诱导的BM-EPCs中p53和TSP-1的水平升高。说明高糖诱导引起血管生成抑制因子TSP-1的增加，进而导致BM-EPCs的功能受损，二氮嗪处理后可以显著改善因高糖诱导引起的BM-EPCs功能受损。 5.二氮嗪改善高糖诱导的HUVECs功能障碍使用高糖(33mM)诱导的HUVECs损伤模型，二氮嗪(30μM)处理24h后，检测其细胞功能的变化。结果显示：高糖诱导后会明显降低HUVECs的小管形成能力、迁移功能和粘附功能，二氮嗪(30μM)处理后HUVECs的小管形成能力、迁移和粘附功能显著增强。结论： ATP敏感的钾离子通道开放剂二氮嗪能够显著加速链脲佐菌素诱导的1型糖尿病小鼠伤口愈合速度，促进伤口组织血管新生；这一效应可能与二氮嗪改善糖尿病BM-EPCs功能相关，机制涉及p53/TSP-1通路。
[Abstract]:The purpose of the study is:
Diabetes mellitus (DM) is a systemic metabolic disorder that is divided into insulin dependent (Insulin-dependent) and non insulin dependent (Insulin-independent). Major vascular and microvascular lesions are the main cause of death in diabetic patients, and endothelial dysfunction (Endothelial dysfunction) is caused by the injury of endothelial function (Endothelial dysfunction). The initiation factor of wound healing defects and vascular lesions. The activity of Endothelial cells (ECs) is the basis for maintaining endothelial function. If the apoptosis rate of endothelial cells exceeds the normal level, it will affect the integrity of blood vessels. Endothelial progenitor cells (Endothelial progenitor cells, EPCs) are derived from adult bone marrow and can be differentiated into internal cells. The number of Bone marrow-derived endothelial progenitor cells (BM-EPCs) derived from bone marrow can be directed to vascular regeneration sites and involved in the repair of damaged endothelium. The number of EPCs in the cycle and the incidence of vascular disease in the circulation have been studied. The rate is inversely proportional. Therefore, EPCs has a vital link with endothelial function. -1 (Thrombospondin-1, TSP-1) is an effective endogenous angiogenesis inhibitor. Diabetes causes an increase in the expression of TSP-1mRNA in EPCs, and EPCs dysfunction also leads to an increase in the expression of TSP-1. The tumor suppressor gene p53 can be stimulated. The promoter of TSP-1, encoded by the THBS1 gene, indicates that p53 can promote the production of TSP-1 and damage the angiogenesis of fibroblasts. Two azazazine (Diazoxide) is a mitochondrial KATP channel opening agent, which can induce the hyperpolarization of the cell membrane, the increase of K+ Exodus, the decrease of the flow in Ca2+, and the secretion of insulin to inhibit glucose stimulation. The islet B cell cells provide a rest and play a role in retarding and hindering the development of diabetes and its complications. This topic studies whether two azozine can accelerate wound healing in diabetic mice, improve the function of BM-EPCs, and explore its possible mechanism.
Research content and results:
Animal model: the model of type 1 diabetic mice induced by Streptozotocin (STZ); cell model: cell model induced by glucose 33mM (Human umbilical VeinEndothelial cells, HUVECs) and BM-EPCs high glucose injury with glucose 33mM. At animal level, the endothelium of diabetic mice was studied. The effect of function was discussed from the EPCs point of view around the two targets of TSP-1 and p53. At the cell level, the effects of two azazazine on angiogenesis were observed by cell function experiments (migration function, adhesion function and tubule formation function), and the mechanism of two azazazine to influence angiogenesis was determined by measuring the expression of TSP-1 and p53.
1. two azazazine promotes wound healing in type 1 diabetic mice
5 weeks old C57BL/6 mice, 18-20g, were randomly divided into normal group (Control), type 1 diabetes model group (STZ) and two azinazine treatment group (30mg/kg/d gavage 4 weeks). Under the anesthetic state of the mice, a circular wound with a radius of 6mm was cut on the back of the mice, and the wound healing was observed every 2 days for 12 days. 6mm on day 3,6,9 took the surrounding 6mm respectively. The blood vessel density was detected by CD31 immunohistochemistry. The vasodilatation function of the aorta of 2-3mm mice was measured by vascular tension system. The results showed that the wound healing speed of type 1 diabetic mice decreased significantly, the capillary density decreased significantly, and the vascular dependent endothelial diastolic function decreased significantly; two After nitrozine treatment, wound healing ability was enhanced, vascular density increased and vascular dependent endothelial function increased.
2. two azazazine improves BM-EPCs function in type 1 diabetic mice
The Dil-acLDL and FITC-UEA-1 double positive identification of BM-EPCs. was used to detect the number of EPCs in mice circulated by the percentage of CD34 and Flk-1 positive. The migration function and tubule formation ability were detected by Boyden compartment and tube test. The results showed that the number of EPCs in the cycle of type 1 diabetic mice decreased significantly, and two azazazine could be significant after the treatment of azazazine. The number of EPCs in the circulation was increased; the tubuloforming ability of BM-EPCs in type 1 diabetic mice, the migration function and adhesion function decreased significantly. After two azazine treatment, the tubuloforming ability, migration function and adhesion function of BM-EPCs could be significantly improved.
3. possible mechanism of two azazine improving BM-EPCs function in type 1 diabetic mice
The expression of p53 and TSP-1 in BM-EPCs of type 1 diabetic mice was detected by Western blot. The results showed that the p53 expression of BM-EPCs in type 1 diabetic mice increased significantly, and the level of p53 in BM-EPCs could be significantly reduced after two azazazine treatment. The TSP-1 expression of BM-EPCs in type 1 diabetic mice was significantly increased, and the TSP-1 of two azazazine could significantly reduce TSP-1. Expression.
4. two azazine improves BM-EPCs dysfunction induced by high glucose and its mechanism
7 days after BM-EPCs culture of normal mice bone marrow separation, the cell damage model was induced by high glucose (33mM), and two azazazine (30 mu M) was treated with 24h to detect the cell function and protein expression. The results showed that high sugar induced BM-EPCs tubule formation ability, migration function and adhesion function decreased significantly, and two azazazine (30 micron M) treatment could be significantly improved. High sugar induced BM-EPCs tubule formation, migration and adhesion damage. The expression of p53 and TSP-1 in BM-EPCs induced by high glucose was significantly increased, and two azazazine treatment could reduce the level of p53 and TSP-1 in high glucose induced BM-EPCs. It indicated that high glucose induced the increase of angiogenesis inhibitor TSP-1, and thus lead to BM-. The function of EPCs is impaired. Two azazine treatment can significantly improve BM-EPCs damage induced by high glucose.
5. two azazazine improves HUVECs dysfunction induced by high glucose
Using high glucose (33mM) induced HUVECs damage model, two azazazine (30 mu M) treated 24h, the changes in cell function were detected. The results showed that high glucose induced obviously lower tube formation ability, migration function and adhesion function, two azazazine (30 micron M) treatment, HUVECs tubule formation ability, migration and adhesion function significantly enhanced.
Conclusion:
ATP sensitive potassium channel opener, two azazazine, can significantly accelerate the healing speed of streptozotocin induced wound healing in type 1 diabetic mice and promote angiogenesis in wound tissue. This effect may be related to the improvement of diabetic BM-EPCs function by two azinazine, and the mechanism involves the p53/TSP-1 pathway.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

【参考文献】