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利拉鲁肽调节Sirtuin表达对高糖诱导的人内皮集落形成细胞生物学行为的影响
作者:张路 唐康 周波 
单位:400016 重庆 重庆医科大学附属第一医院内分泌科(张路、唐康、周波) 
关键词:内皮集落形成细胞 利拉鲁肽 沉默信息调节因子 血管内皮生长因子 血管生成素 
分类号:R587.1
出版年,卷(期):页码:2019,44(5):361-368
摘要:

 [摘要]  目的  探索利拉鲁肽是否通过调节Sirtuin影响高糖诱导的人内皮集落形成细胞(ECFC)的生物学行为。方法  采用密度梯度离心法从外周血中分离获取单个核细胞,用EBM-2诱导培养出ECFC,随机分为正糖组(5.5 mmol/L D-葡萄糖)、渗透压组(5.5 mmol/L D-葡萄糖+24.5 mmol/L甘露醇)、高糖组(30 mmol/L D-葡萄糖),均处理6d。随后采用EdU实验、Transwell实验、成管实验、β-半乳糖苷酶实验分别测定ECFC的增殖、迁移、成管及衰老情况,采用免疫印迹法测定Sirtuins(Sirtuin1–7)、血管内皮生长因子(VEGF)、血管生成素的表达水平。研究利拉鲁肽对ECFC的影响时,将细胞分为4组,除上述正糖组与高糖组外,还设有正糖利拉鲁肽干预组(5.5 mmol/L D-葡萄糖+100 nmol/L利拉鲁肽)、高糖利拉鲁肽干预组(30 mmol/L D-葡萄糖+100 nmol/L利拉鲁肽),均处理6d,观察4组细胞的生物学行为变化并测定VEGF和血管生成素的表达水平。结果  EdU实验显示高糖组的细胞增殖率明显低于正糖组[(30.16±12.36)% vs. (88.00±13.77)%]Transwell实验显示高糖组的细胞迁移能力明显低于正糖组(25.11±6.05 vs. 64.89±10.73),成管实验显示高糖组细胞成管能力明显低于正糖组(27.50±3.90 vs. 69.61±5.48)β-半乳糖苷酶染色实验显示高糖组细胞衰老率明显高于正糖组[(87.63±9.63)% vs. (71.35±7.58)%],差异均有统计学意义(P<0.05)。高糖情况下,Sirtuins家族表达普遍降低,VEGF和血管生成素表达均显著下降(P<0.05)Sirtuin1的表达随利拉鲁肽浓度的升高而升高。予以适宜浓度的利拉鲁肽干预后,VEGF和血管生成素的表达均明显增加(P<0.05)EdU实验显示高糖利拉鲁肽干预组的细胞增殖率明显高于正糖组[(54.09±27.29)% vs. (29.01±7.56)%]Transwell实验显示高糖利拉鲁肽干预组的细胞迁移能力明显高于高糖组(32.25±4.99 vs. 21.75±3.10),成管实验显示高糖利拉鲁肽干预组的细胞成管能力明显高于高糖组(69.61±8.11 vs. 39.85±5.78)β-半乳糖苷酶染色实验显示高糖利拉鲁肽干预组的细胞衰老率明显低于高糖组[(52.06±7.94)% vs. (69.03±1.57)%],差异均有统计学意义(P<0.05)结论  高糖可降低ECFC的增殖、迁移和成管能力,增加细胞衰老比例,降低SirtuinsVEGF和血管生成素的表达;利拉鲁肽可逆转高糖导致的ECFC生物学行为改变,同时上调Sirtuin1VEGF及血管生成素的表达。

 [Abstract]  Objective  To explore whether liraglutide could affect high glucose induced-biological behavior of human endothelial colony forming cell (ECFC) by regulating sirtuin. Methods  Mononuclear cells in human peripheral blood were isolated by density gradient centrifugation. ECFCs were induced and cultured with EBM-2 complete medium. The cells were randomly divided into three groups: normal glucose group (NG, 5.5 mmol/L D-glucose for 6 days), high glucose group (HG, 30 mmol/L D-glucose for 6 days), and osmotic pressure group (OSM, 5.5 mmol/L D-glucose+24.5 mmol/L mannitol for 6 days). The proliferation, migration, tube formation and senescence of ECFCs in the three groups were tested by EdU, Transwell, tube formation and SA-β-gal experiments respectively. The protein expression levels of Sirtuins (Sirtuin1-7), vascular endothelial growth factor (VEGF) and angiogenin were measured by Western blotting. Except NG and HG groups mentioned above, NG + liraglutide intervention group (5.5 mmol/L D-glucose+100 nmol/L liraglutide) and HG+liraglutide intervention group (30 mmol/L D-glucose+100 nmol/L liraglutide) were added in order to observe the effects of liraglutide on ECFC biological behavior and the expression of VEGF and angiogenin. The intervention time in all groups is 6 days. Results  EdU, Transwell and tube formation experiments showed that the proliferation rate, migration ability and tube formation ability of ECFCs were lower in HG group [(30.16±12.36)%, 25.11±6.05, 27.50±3.90, respectively] than in NG group [(88.00±13.77)%, 64.89±10.73, 69.61±5.48, respectively], while the SA-β-gal experiment showed the senescence rate of ECFCs was higher in HG group than in NG group [(87.63±9.63)% vs. (71.35±7.58)%], all with statistical significance (P<0.05). Under high glucose conditions, the expression of Sirtuins family was generally reduced, and the expressions of VEGF and angiogenin significantly decreased (P<0.05). The expression of Sirtuin1 showed an upward trend with the increase of liraglutide concentration. After intervention with appropriate concentration of liraglutide, the expressions of VEGF and angiogenin significantly increased (P<0.05). EdU experiment showed the proliferation rate of ECFCs was higher in HG+liraglutide intervention group than in NG group [(54.09±27.29)% vs. (29.01±7.56)%], the Transwell and tube formation experiments showed that the migration ability and tube formation ability of ECFCs were higher in HG+liraglutide intervention group (32.25±4.99, 69.61±8.11) than in HG group (21.75±3.10, 39.85±5.78), and the SA-β-gal experiment showed the senescence rate of ECFCs was lower in HG+liraglutide intervention group than in HG group [(52.06±7.94)% vs. (69.03±1.57)%], all with statistical significance (P<0.05). Conclusions  High glucose may accelerate ECFCs senescence while decrease the ability of proliferation, migration and tube formation of ECFCs, accompanied by down-regulation of Sirtuins, VEGF and angiogenin. Liraglutide may reverse the changes of ECFCs biological behavior induced by high glucose and up-regulate the expression of Sirtuin1, VEGF and angiogenin.

基金项目:
国家自然科学基金(81370940)
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