| 摘要: |
| 目的探讨在缺氧缺血清环境下，脯氨酸羟化酶抑制剂———二甲基乙二酰甘氨酸（DMOG）促进骨髓间充质干细胞（MSCs）血管新生的作用及其机制。方法MSCs从大鼠骨髓中分离得到，分为：常氧条件下溶剂对照组（N-DMSO）、缺氧条件下溶剂对照组（H-DMSO）、20μMDMOG加药组（D-20滋M）、100滋MDMOG加药组（D-100滋M）及500μMDMOG加药组（D-500滋M），观察以下指标：（1）通过缺氧条件下CCK-8检测DMOG在20μM、100μM及500滋M剂量下对MSCs的保护作用，确定DMOG的最佳剂量；（2）通过Matrigel实验观察DMOG促进MSCs血管新生的作用；（3）通过Westernblot法检测血管新生通路相关蛋白表达。结果（1）不同浓度组DMOG对缺氧损伤MSCs的影响：N-DMSO组OD值3.25±0.05，H-DMSO组2.23±0.14，D-20滋M组2.68±0.43，D-100滋M组3.11±0.25，D-500滋M组3.23±0.04。与N-DMSO组比较，H-DMSO组OD值降低（P＜0.01），与H-DMSO组比较，D-20滋M组、D-100滋M组、D-500滋MOD值均升高（P＜0.05或0.01）。（2）不同浓度组DMOG对正常MSCs的影响：D-20滋M组3.19±0.02，D-100滋M组3.15±0.06，D-500滋M组2.51±0.08。与N-DMSO组比较，D-500滋M组OD值降低（P＜0.01），D-20滋M组、D-100滋M组与N-DMSO组比较差异均无统计学意义（均P＞0.05），提示DMOG在20滋M及100滋M对细胞无毒性。（3）血管新生相关蛋白的表达：与H-DMSO组相比，D-100μM组缺氧3、6及24hHIF-1α均增加（1.44±0.32vs7.79±0.23，2.4±0.28vs3.51±0.79，0.93±0.37vs2.46±0.07，P＜0.05或0.01），pAKT则在缺氧6h增加（0.47±0.15vs0.71±0.03，P＜0.05），VEGF在缺氧6、24h均增加（0.63±0.10vs0.87±0.14，0.42±0.06vs0.70±0.06，P＜0.05或0.01），以缺氧24h最为显著。pmTOR/mTOR及pERK/ERK蛋白两组比较差异均无统计学意义（均P＞0.05）。结论DMOG通过抑制HIF-1α降解及促进AKT磷酸化提高缺氧环境下MSCs的血管新生能力。 |
| 关键词: 骨髓间充质干细胞 二甲基乙二酰甘氨酸 血管新生 缺氧 |
| DOI： |
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| 基金项目:浙江省医药卫生一般研究计划；浙江省卫生高层次创新人才培养工程项目 |
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| DMOG promotes angiogenesis of bone marrow mesenchymal stem cells |
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ZHU Meifei,JIANG Ronglin,LEI Shu,WANG Lingcong,LIU Qian
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the First Affiliated Hospital of Zhejiang Chinese Medical University
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| Abstract: |
| Objective To investigate the effect and mechanism of dimethyloxalyl glycine(DMOG) mediated on angiogen- esis of bone marrow mesenchymal stem cells (MSCs). Methods MSCs were isolated from rat bone marrow and divided into five groups: normoxia solvent control group (N-DMSO), hypoxia solvent control group (H-DMSO), 20滋M DMOG treated group (D-20滋M), 100滋M DMOG treated group (D-100滋M) and 500滋M DMOG treated group (D-500滋M). The following indicators were observed: (1) MSCs were cultured with DMOG (20, 100 and 500滋M) under hypoxic condition. The growth inhibitory effect was observed by CCK-8 and the optimal dosage of DMOG was measured. (2) The angiogenic effect of MSCs was measured by Matrigel method. (3) The expression of angiogenesis related proteins were detected by Western blot. Results (1) Effect of
DMOG on MSCs in different concentration groups：N-DMSO:3.25±0.05, H-DMSO:2.23±0.14, D-20滋M:2.68 ±0.43, D-100滋M: 3.11±0.25, D-500滋M:3.23±0.04. As compared with N-DMSO group, H-DMSO group was significantly decreased(P＜0.01), As compared with H-DMSO group, the OD of D-20滋M, D-100滋M and D-500滋M group were significantly increased (P＜0.05 或 0.01). (2) Effect of DMOG on normal MSCs in different concentration groups: D-20滋M:3.19 ±0.02, D-100μM: 3.15 ±0.06, D-500滋M: 2.51±0.08. As compared with N-DMSO group, D-500滋M group was significantly decreased(P＜0.01), while D-20滋M group, D-100μM group and N-DMSO group had no statistically significant difference (P ＞0.05), suggesting that DMOG with 20μM and 100μM exert none cell toxicity. (3) Expression of angiogenesis related proteins: As compared with H-DMSO group, HIF-1α protein level was significantly increased in D-100滋M group at 3, 6 and 24h after hypoxia (1.44 ±0.32 vs. 7.79 ±0.23;2.4±0.28 vs. 3.51±0.79; 0.93±0.37 vs.2.46±0.07, P＜0.05 or 0.01), pAKT protein level was significantly increased in D-100滋M group at 6h after hypoxia (0.47±0.15 vs. 0.71±0.03, P＜0.05), VEGF protein level was significantly increased in D-100μM group at 6 and 24h after hypoxia (0.63 ±0.10 vs. 0.87 ±0.14; 0.42 ±0.06 vs. 0.70 ±0.06, P＜0.05 or 0.01), especially the 24h after hypoxia. While there was no significant difference between the two groups in protein expression of pmTOR/mTOR and pERK/ERK (P ＞0.05 respectively). Conclusion DMOG inhibits the degradation of HIF-1α and promotes the phosphorylation of AKT to im- prove the angiogenesis of MSCs in hypoxic condition. |
| Key words: Bone marrow mesenchymal stem cells DMOG Angiogenesis Hypoxia |
