3-甲基腺嘌呤调控自噬对大鼠内皮祖细胞促进静脉血栓再通的作用及其机制

Methyladenine regulation of autophagy to promote endothelial progenitor cells in rats and its mechanism of venous thrombosis recanalization

目的研究3-甲基腺嘌呤(3-MA)调控自噬对大鼠内皮祖细胞促进静脉血栓再通的作用及其机制。方法将建立的静脉血栓模型的SD大鼠按照体重随机为4组,每组20只。对照组,腹腔注射5 mmol·L^(-1)3-MA 1 m L;空白组,腹腔注射生理盐水1 m L;单用组,腹腔注射内皮祖细胞(EPCs)悬液1 m L;联合组,腹腔注射5 mmol·L^(-1)3-MA 0.5 m L和EPCs悬液0.5 m L;在每组注射移植后的0,7,14,28 d这四个时间点分别取标本。比较各组大鼠血栓机化再通情况;计数各组血栓标本中毛细血管数目,并进行对比;用免疫印迹法测定血管内皮细胞生长因子(VEGF)、碱性成纤维细胞生长因子(b FGF)、血管生成素-1(Ang-1)、膜辅因子蛋白(MCP-1)这四个因子的蛋白表达。结果较对照组和单用组,空白组的切面管腔样结构较多;切面管腔样结构最多为联合组。对照组的毛细血管数目明显多于空白组和单用组(P<0.05);而联合组毛细血管数目明显多于其他3组(P<0.05)。术后第28天,联用组的VEGF、b FGF、Ang-1、MCP-1的蛋白表达量明显高于其他3组(P<0.05),其中对照组、单用组、联合组及空白组的VEGF分别为(0.51±0.24),(0.62±0.14),(2.04±0.15),(0.89±0.28)pg·mL^(-1)。结论 3-MA能适当调控自噬,能提高血栓局部EPCs增殖,促进毛细血管生成及血栓机化和再通。

Objective To study the regulation of autophagy methyladenine on promoting the role of endothelial progenitor cells in rats and mechanism of venous thrombosis recanalization. Methods Eighty SD rats were established in the model of venous thrombosis,and divided into four groups,each group had 20 rats. The control group was injected 1m L concentration of 5 mmol·L^-1 of methyl adenine（3-MA）,the blank group was injected a simple 1 mL saline,the alone group was injected 1m L endothelial progenitor cells（EPCs） suspension,the combination group was injected 0. 5 mL concentration of 5 mmol·L^-1 3-MA and 0. 5 mL EPCs suspension. The samples were taken at four time points of 0,7,14 and 28 d after injection. Compared with each group of rats,the number of capillary vessels was counted. And the protein expression of vascular endothelial growth factor（VEGF）, based fibroblast growth factor（b FGF）, angiopoietin1（Ang-1）, membrane cofactor protein 1（MCP-1） were measured by Western blot method. Changes of proteinexpression in 4 groups was compaered. Results The cross-section of the lumen in blank group was more than the control group and the alone group after administration and the samples of the structure was the most in combined group.The capillary number in control group was significantly higher than that in the blank group and the alone group（P〈0. 05）,while the capillary number in the combined group was significantly higher than that in the other 3 groups（P〈0. 05）. After twenty-eighth days,the protein expression of VEGF,b FGF,MCP-1 and Ang-1 in combined group was significantly higher than that in other three groups（P〈0. 05）. Among them,the control group,single use group,combined group and blank group of VEGF were（0. 51 ＋ 0. 24）,（0. 62 ＋ 0. 14）,（2. 04 ＋ 0. 15）,（0. 89 ＋ 0. 28） pg·mL^-1,respectively. Conclusion 3-MA can be properly regulated autophagy,can improve thrombus local EPCs proliferation and capillary formation and thrombus and recanalization.