硫化氢改善高糖环境下内皮祖细胞功能活性的机制研究

Mechanisms of hydrogen sulfide on improving endothelial dysdunctions induced by high glucose

目的探讨外源性硫化氢对高糖环境下内皮祖细胞功能活性的影响及机制研究。方法采用密度梯度离心法从25-35岁健康志愿者外周血分离出单个核细胞(HPB-MNC),用EGM-2培养基对EPCs进行培养和诱导分化,培养7d后通过用细胞免疫荧光检测EPCs摄取对乙酰化低密度脂蛋白和结合FITC-UEA-I的特性;将EPCs分别于正常葡萄糖培养液(5.5mmol/L)、高糖培养液(33mmol/L)、高糖(33mmol/L)+不同浓度NaHS(25uM、50uM、100uM、200uM)培养液中培养24h,比较EPCs体外增殖、迁移、黏附能力变化;并建立裸鼠颈动脉内膜拉脱损伤模型,观察高糖对EPCs修复裸鼠颈动脉内皮损伤能力的影响,以及NaHS的保护作用;以及检测NaHS抗高糖环境下内皮祖细胞Akt和eNOS蛋白的表达情况。结果高糖培养液作用EPCs 24h,增殖、迁移、黏附能力明显下降;而当不同浓度NaHS和高糖同时作用于EPCs时,细胞的增殖、迁移、黏附能力得到明显改善(P<0.01);而且,当高糖作用EPCs进行细胞移植,其再内皮化能力明显下降,而当硫化氢和高糖同时作用于EPCs进行移植,其再内皮化能力明显改善,(P<0.01),EPCs表面的Akt/eNOS信号通路明显上调(P<0.01)。结论 NaHS在一定范围内能够明显改善高糖环境下内皮祖细胞的增殖、迁移、黏附能力,并且改善EPCs的血管内皮修复能力,其机制可能与EPCs表面的Akt/eNOS信号通路相关。

Objective To investigate the effect of exogenous Hydrogen sulfide on the proliferation, migration,adhesion and Reendothelialization of EPCs under high glucose. Method Using density gradient centrifugation,the peripheral blood mononuclear cells were isolated from healthy subjects （aged from 25 to 35 years old）,and cultured in EGM-2 medium for 7days. The EPCs were identified by double-positive staining with Dil-ac-LDL and FITC-UEA-1. The expression of endothelial marker proteins was examined by using flow cytometry analysis. The cultured EPCs were treated with concentration of glucose at 5.5,33mmol/L and glusoce at 33mmol/L with NaBS at 25、50、100、200uM for 24h ;the proliferation, migration and adhesion ability of EPCs in vitro were assayed;The carotid arteries endothelium injury were established by guide-wire damage in Male NRMInu/nu athymic nude mice; The effect of high Glucose on the ability of EPCs mediating vascular repair,and the protective effect of NaBS were observed;and we also investigate the expression of Akt and eNOS protein in the EPCs under the high glucose and treatment with NABS. Results Compared to the normal glucose group,the ability of proliferation,migration and adhesion of EPCs in high glucose medium for 24h decreased significantly,while the proliferation, migration and adhesion ability of EPCs were obviously improved （P〈0.01） when different concentrations of NaBS plus high glucose （P〈0.01）. Moreover,the ability of reendothelialization decreased significantly on transplantation EPCs pretreated with high glucose;and the capacity was obviously improved in transplanted EPCs pretreated with hydrogen sulfide and high glucose （P〈0.01）,and the Akt/eNOS signaling pathway on the EPCs surface was significantly upregulated （P〈0.01）. Conclusion NaBS can significantly improve the proliferation,migration and adhesion of endothelial progenitor cells under high glucose condition,and improve the vascular endothelial repair ability of EPCs. The mechanism may be related to the Akt/eNOS signaling pathway.