A23187 诱导HUVECs 释放微囊泡的提取与鉴定及对H9c2 细胞的作用研究

尚曼，张琦，张梦晓，王瑶，陈艳，吴艳娜，宋君秋，刘艳霞
作者简介：尚曼（1988-），女，助理实验师，心血管药理学
通信联系人：刘艳霞（1963.6-），女，教授，心血管药理学.

（天津医科大学基础医学院药理学教研室，天津 300070）
摘要：目的：采用钙离子载体A23187 诱导人脐静脉内皮细胞释放微囊泡，对其进行分离提取和鉴定，并研究内皮微囊泡对H9c2 心肌细胞的影响。方法：10 μM A23187 作用于人脐静脉内皮细胞30 min 后，超速离心细胞培养上清液，分离提取内皮微囊泡。采用1 μm、2μm 标准微球及PE-CD144 抗体，通过流式细胞仪对内皮微囊泡进行粒径及表型鉴定。将不同浓度的内皮微囊泡与H9c2 心肌细胞共同培养6 h，比色法检测H9c2 细胞存活率及乳酸脱氢酶活性；采用Hoechst 33258 染色从形态学观察H9c2 细胞凋亡。结果：A23187 可诱导人脐静脉内皮细胞产生微囊泡，超速离心的方法分离提取内皮微囊泡，流式检测证实其粒径﹤1 μm 且呈CD144+。不同浓度的内皮微囊泡均显著降低H9c2 细胞存活率(P<0.05)，提高H9c2细胞LDH 活性(P<0.05)，具有浓度依赖性。Hoechst 33258 染色发现随内皮微囊泡浓度增加，细胞核凝聚，细胞碎片增多。结论：人脐静脉内皮细胞钙超载时会释放产生微囊泡，并发现内皮微囊泡对心肌细胞具有损伤作用。
关键词：A23187；人脐静脉内皮细胞；内皮微囊泡；流式细胞仪；钙超载 ；损伤

 中图分类号：R966
Isolation and identification of endothelial microvesicles induced by A23287 and its effects on H9c2 cells
SHANG Man, ZHANG Qi, ZHANG Mengxiao, WANG Yao, CHEN Yan, WU
Yanna, SONG Junqiu, LIU Yanxia
(College of Basic Medicine, Tianjin Medical Universtiy, Tianjin 300070)
Abstract: IObjective: To isolate and identify endothelial microvesicles (EMV) induced by ionophore A23187, and investigate the effects of EMV. Methods: EMV were isolated from HUVECs culture supernatant by ultracentrifugation after a 30 min treatment with 10 μM 30 ionophore A23187. EMV were characterized using 1 μm, 2 μm latex beads and PE-CD144 antibody by flow cytometry. EMV of different concentrations were co-cultured with H9c2 cardiomyocyte for 6 h. Cell viability and activity of LDH was tested by colorimetry. Moreover, apoptosis of H9c2 cells was observed through Hoechst 33258 staining. Result: Microvesicles could be induced by A23187 on HUVECs, and isolated by ultracentrifugation. Flow Cytometry identified that vesicles induced by A23187 were CD144 positive with a size of < 1 μm. EMV at different concentrations could significantly reduce the viability of H9c2 cells, and increase LDH activity of H9c2 cells in a dose dependent manner (P<0.05). Fragmented or condensed nuclei could be observed with the increasing concentration of EMV through Hoechst 33258 staining. Conclusion: Microvesicles can be released from HUVECs in the process of calcium overload, and it was discovered that EMV exerted damage effects on H9c2 cells.
Key words: A23187; human umbilical vein endothelial cells; endothelial microvesicles; flow cytometry; calcium overload; injury