人主动脉血管平滑肌细胞铁死亡模型的建立

Establishment of Ferroptosis Modelin Human Aortic Vascular Smooth Muscle Cells

目的在人主动脉血管平滑肌细胞(HAVSMCs)中建立铁死亡模型并进行验证。方法主动脉平滑肌细胞来源于在华中科技大学同济医学院附属同济医院心脏大血管外科行心脏移植手术的供体主动脉弓组织,分别使用含Imidazole ketoneerastin(IKE)和胱氨酸缺失(CD)的培养液对HAVSMCs进行铁死亡诱导,并使用Cell Counting Kit-8(CCK-8)试剂盒、人乳酸脱氢酶(LDH)检测试剂盒、细胞流式检测及免疫荧光染色来判断平滑肌细胞的铁死亡模型是否建立成功。结果对HAVSMCs进行铁死亡诱导后,光学显微镜下可观察到细胞出现明显形态学改变,流式细胞学分析显示IKE/CD诱导下碘化丙啶(PI)染色阳性细胞所占比例明显升高;CCK-8和LDH检测结果显示,在IKE/CD诱导下HAVSMCs细胞活力明显降低,细胞损伤率明显增加,而铁死亡抑制剂ferrostatin-1则能逆转铁死亡诱导对细胞的毒性作用;BODIPY-C11细胞流式分析及4-羟基壬烯醛(4-HNE)免疫荧光染色显示,IKE/CD诱导的HAVSMCs中脂质过氧化水平明显上升。结论该研究使用IKE以及胱氨酸缺失培养液成功建立了人主动脉血管平滑肌细胞铁死亡模型,为后续研究提供了实验基础。

Objective To establish and validate a ferroptosis model in human aortic vascular smooth muscle cells(HAVSMCs).Methods HAVSMCs were obtained from aortic arch tissues of donors who underwent heart transplantation at the Cardiothoracic and Vascular Surgery Department,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology.Ferroptosis was induced through imidazole ketone erastin(IKE)and cystine-deficient medium(CD)in HAVSMCs.Cell counting kit-8(CCK-8),human lactate dehydrogenase(LDH)assay kit,flow cytometry and cell immunofluorescence staining were used to evaluate whether ferroptosis was successfully induced in HAVSMCs.Results After the induction of ferroptosis in HAVSMCs for a certain period of time,obvious morphological changes of HAVSMCs were observed under the optical microscope,and the flow cytometry analysis showed that the proportion of propidium iodide(PI)positive stained cells was significantly increased under the induction of IKE/CD.The results of CCK-8 and LDH assays showed that the cell viability of HAVSMCs was significantly reduced,and the level of cell damage was markedly increased under IKE/CD induction.Ferrostatin-1,an inhibitor of ferroptosis,was able to reverse the toxic effects of ferroptosis induction on the cells.The flow cytometry analysis of BODIPY-C11 and immunofluorescence staining of 4-hydroxynonenal(4-HNE)showed a notable increase in lipid peroxidation levels in IKE/CD-induced HAVSMCs.Conclusion In this study,a ferroptosis model was successfully established in human aortic vascular smooth muscle cells with IKE and cystine-deficient medium,which provided an experimental basis for subsequent researches.