人外周血晚期内皮祖细胞中的高糖记忆效应

Hyperglycemia metabolic memory effect in late endothelial progenitor cells from human peripheral blood

摘要:

目的：探索人外周血晚期内皮祖细胞（endothelial progenitor cells，EPCs）是否存在高糖代谢记忆。方法：密度梯度离心法分离获取人外周血中的单个核细胞，诱导培养2周时间待细胞呈现立体的鹅卵石状后，即为晚期EPCs，采用细胞免疫化学染色对分离培养的细胞进行鉴定。MTT比色法摸索出高糖作用的浓度和时间梯度后，分3组处理细胞，即正糖组（NG，5.5 mmol/L D-葡萄糖×6 d），高糖组（HG，30 mmol/L D-葡萄糖×6 d），记忆组（MG，30 mmol/L D-葡萄糖×6 d+5.55 mmol/L D-葡萄糖×6 d）处理细胞，采用粘附实验、MTT比色法、EdU实验、Transwell小室实验、成管实验，测定3组细胞的粘附、增殖、迁移、成管功能。结果：粘附结果显示高糖组（38.296±6.615）、记忆组（37.815±6.196）均较正糖组（63.370±10.363）粘附能力下降（P<0.05）；MTT结果显示高糖组（79.041±19.678）、记忆组（81.524±6.710）均较正糖组（100.000±0.000）增殖能力下降（P<0.05）；EdU结果显示高糖组（45.809±7.945）、记忆组（37.740±8.206）均较正糖组（88.858±5.727）增殖能力下降（P＜0.05）；迁移结果显示高糖组（25.111±6.051）、记忆组（25.778±6.037）均较正糖组（64.889±10.729）迁移能力下降（P＜0.05）；成管结果显示高糖组（12.690±3.616）、记忆组（14.198±4.073）均较正糖组（64.407±6.358）成管能力下降（P＜0.05）；记忆组与高糖组相比，粘附、增殖、迁移、成管功能均未能改善（P ＞0.05）。结论：晚期EPCs存在高糖代谢记忆。

Abstract:

Objective：To explore whether there is a hyperglycemia metabolic memory in late endothelial progenitor cells（EPCs） from human peripheral blood. Methods：Peripheral blood mononuclear cells（PBMC） were separated by density gradient centrifugation. Then PBMC was induced and cultivated for two weeks to late EPCs. Immunochemical staining was used to identify cultured cells. MTT was used to explore the gradient of concentration and time of high glucose. Cells were divided into three groups，namely：normal glucose group（NG，5.5 mmol/L treatment for 6 d），high glucose group（HG，30 mmol/L treatment for 6 d），hyperglycemia memory group（MG，30 mmol/L treatment for 6 d then 5.5 mmol/L treatment for 6 d）. Adhesion test，MTT，EdU experiments，Transwell chamber，angiopoiesis experiments were used to measure the ability of adhesion，proliferation，migration，tube formation respectively. Results：The adhesion results showed that the adhesion ability was lower in high glucose（38.296±6.615） group and hyperglycemia memory group（37.815±6.196） than in normal glucose group（63.370±10.363）（P<0.05）. MTT analysis showed that the proliferation ability was lower in high glucose（79.041±19.678） group and hyperglycemia memory group（81.524±6.710） than in normal glucose group（100.000±0.000）（P<0.05）. EdU analysis showed that the proliferation ability was lower in high glucose（45.809±7.945） group and hyperglycemia memory group（37.740±8.206） than in normal glucose group（88.858±5.727）（P<0.05）. The migration results showed that the migration ability was lower in high glucose（25.111±6.051） group and hyperglycemia memory group（25.778±6.037） than in normal glucose group（64.889±10.729）（P<0.05）. The tube formation results showed that the tube formation ability was lower in high glucose（12.690±3.616） group and hyperglycemia memory group（14.198±4.073） than in normal glucose group（64.407±6.358）（P<0.05）. Compared with those of high glucose group，the adhesion，proliferation，migration，tube formation of hyperglycemia memory group was still not improved（P>0.05）. Conclusion：Late EPCs exist hyperglycemia metabolic memory.