连晓鹏　马　捷.山西医科大学，山西太原　03000；.山西医科大学第二医院，山西太原　03000

重组人促红细胞生成素抑制细胞自噬减轻心肌微血管内皮细胞氧化损伤

连晓鹏1马捷2
1.山西医科大学，山西太原030001；2.山西医科大学第二医院，山西太原030001

目的 探讨重组人促红细胞生成素（recombinant human erythropoietin，rhEPO）对大鼠心肌微血管内皮细胞（cardica microvascular endothelial cells，CMECs）氧化损伤的保护作用和可能机制。方法 组织块贴壁法培养CMECs，倒置显微镜形态学观察，免疫荧光法鉴定。H2O2建立氧化损伤模型，取2～3代CMECs，随机分为对照组、200 μmol/L H2O2组、200 μmol/L H2O2+5 U/mL rhEPO组、200 μmol/L H2O2+10 U/mL rhEPO组、200 μmol/L H2O2+ 20 U/mL rhEPO组。应用MTT法测定细胞存活率，化学比色法测定上清液中乳酸脱氢酶（LDH）含量，Western-blot检测LC3、Beclin1和p-mTOR含量。结果 大鼠CMECs呈梭形、多角形等不规则形态，形成单层紧密鹅卵石样排列，抗体阳性90%以上；与对照组比较，H2O2处理组存活率降低（P<0.05），LDH释放量增加（P<0.05），rhEPO预处理后，与单纯H2O2组比较，上述指标可逆转（P<0.05）；与对照组比较，H2O2处理组LC3Ⅱ和Beclin1表达水平增高（P<0.05），rhEPO预处理后,LC3Ⅱ和Beclin1含量减低，与单纯H2O2组比较差异有统计学意义（P<0.05）；与对照组比较，单纯H2O2组p-mTOR表达减少（P<0.05），余H2O2组p-mTOR表达增多（P<0.05），rhEPO预处理后，与单纯H2O2组比较，诱导p-mTOR表达增高（P<0.05）。结论 rhEPO抑制氧化损伤CEMCs的过度自噬，提高细胞存活率，这种保护作用可能与rhEPO激活PI3K/AKT/mTOR通路有关。

重组人促红细胞生成素；心肌微血管内皮细胞；氧化损伤；自噬

[Abstract]Objective To explore the protective effect and mechanism of recombinant human erythropoietin on cardiac microvascular endothelial cells against oxidative damage.Methods The tissue block method was applied for CMECs. The cells were observed under inverted microscope and identified by immunofluorescence.To establish oxidative injury modle was by H2O2and CMECs at passage 2 to 3 were divided randomly into control group,200 μmol/L H2O2group, 200 μmol/L H2O2+5 U/mL rhEPOgroup,200 μmol/L H2O2+10 U/mL rhEPOgroup,200 μmol/L H2O2+20 U/mL rhEPO group.MTT assay was used to determine Cell viabilily.Chemical colorimetry were used to measure LDH of supernatant. Western-blot was used to evaluate protein expression of LC3,Beclin1 and p-mTOR.Results The cultured CMECs of rat showed fusiform shape or polygon,and the monolayer cultures displayed a typical cobblestone or paving-stone morphology and the positive rate was 90%or more.Compared with control group,the cell viabilily rate was reduced and LDH release quantity was increased(P<0.05)and after pretreated by rhEPO,compared with pure H2O2group,the above indexes were reversed(P<0.05);Compared with control group,protein expression of LC3Ⅱand Beclin1 were increased (P<0.05);after pretreated by rhEPO,compared with pure H2O2group,the protein expression were lowered(P<0.05);Compared with control group,p-mTOR of the pure H2O2group was reduced(P<0.05),but the other of H2O2group were increased(P<0.05),after pretreated by rhEPO compared with pure H2O2group,the protein expression were increased(P< 0.05).Conclusion RhEPO inhibits excessive autophagy of CMECs in oxidative damage and improves the cell viabilily rate.The protective effect may be related to activation of PI3K/AKT/mTOR signal away.

[Key words]Recombinant human erythropoietin;Cardiac microvascular endothelial cells;Oxidative damage;Autophagy

先天性心脏病是小儿时期最常见的心脏病，目前外科治疗是最有效的矫正手段。体外循环是手术中重要辅助手段，但这个过程伴有心脏的缺血再灌注损伤。其主要机制是氧自由基产生的氧化损伤[1]。氧化损伤不仅引起细胞凋亡、坏死，还能引起过度自噬[2]。自噬（autophagy）是利用细胞溶酶体降解回收利用受损、变性、衰老的蛋白质，实现蛋白和能量更新代谢，对细胞维持稳态具有非常重要意义[3]。有研究表明，氧自由基刺激不同信号转导的炎性因子诱导细胞自噬，损伤内皮细胞等[4]，CMECs是氧自由基等最早作用的靶细胞[5]，其损伤也是心肌细胞损伤的始动环节。……