摘要
目的:探讨持续缺氧阵发加剧的缺氧形式(PI hypoxia)导致人肺动脉内皮细胞(HPAECs)受损的具体机制,以及过氧化物酶体增殖物激活受体γ(PPARγ)激动剂罗格列酮可能的保护作用。方法:将HPAECs置于持续15%供氧并间歇低氧至5%的PI hypoxia环境中培养72 h。PPARγ激动剂治疗组的HPAECs培养基中添加了5μM的罗格列酮并同样接受72 h PI hypoxia处理。通过慢病毒感染HPAECs,得到过表达Smad3的HPAECs并接受同样的缺氧处理。在细胞培养结束后使用免疫印迹技术检测细胞裂解中PPARγ的相对含量;高通流式细胞仪测定HPAECs细胞凋亡;CCK-8法测定细胞活力;β-半乳糖苷酶染色衡量细胞老化程度;定量聚合酶链式反应检测端粒相对长度。结果:经历72 h PI hypoxia的HPAECs细胞裂解液中PPARγ表达显著下降。与正常供氧的HPAECs相比,接受72 h PI hypoxia的HPAECs高表达特异性细胞老化因子β-半乳糖苷酶,细胞凋亡显著增加,HPAECs细胞活性明显降低。与PI hypoxia的细胞相比,接受5μM罗格列酮治疗的HPAECs细胞凋亡减轻,细胞活力改善,但是细胞老化程度并没有变化。PI hypoxia环境中培养的HPAECs细胞较正常供氧的HPAECs细胞端粒显著延长,且与接受罗格列酮治疗的细胞端粒长度没有差异。过表达Smad3能显著降低β-半乳糖苷酶,减少细胞凋亡,改善HPAECs细胞活性,但是并没有改变缺氧细胞PPARγ的表达水平。结论:PPARγ激动剂治疗能减轻PI hypoxia诱导的HPAECs细胞凋亡和细胞活力下降,但是并不能改善细胞老化水平。
Objective: To elucidate the mechanisms pertaining to the cellular injury of human pulmonary artery endothelial cells(HPAECs) induced by the persistent hypoxia along with intermittent aggravation(PI hypoxia), and the potential protection by peroxisome proliferator activated receptor γ(PPARγ). Methods:HPAECs were exposed to 72 h of PI hypoxia, which was defined as a persistent hypoxia of 15% FiO_(2) with cyclical oxygen deprivation to 5% FiO_(2). Rosiglitazone(5μM) was applied to the culture medium in the group of cells treated by PPARγ agonist. Over-expressed Smad3 was constructed upon to HPAECs via Lentivirus transfection. After 72 h culture in PI hypoxia, the expression level of PPARγ in the cell lysis was measured via western blot. Apoptosis was evaluated by high-pass flow cytometer, while cellular viability was tested by CCK-8 measures, and senescence of HPAECs were assessed by β-galactosidase staining. Telomere length of HPAECs was evaluated by quantitative polymerase chain reaction.Results: PPARγ expressed low 72 h after PI hypoxic culture. Compared with the HPAECs cultured in normoxia, the HPAECs exposed to PI hypoxia presented with significantly higher density of β-galactosidase(senescent marker), greater portion of cellular apoptosis, and lower viability. The HPAECs treated by 5μM rosiglitazone demonstrated lower apoptosis rates and better viability than those cultured in PI hypoxia without any intervention. The elongation of telomere length was documented in all HPAECs exposed to PI hypoxia, which was not changed by PPARγ treatment.Over-expression of Smad3 significantly reduced the level of β-galactosidase and apopotosis, improved celullar viability, but did not change the expression of PPARγ.Conclusions: PPARγ agonist might mitigate apoptosis and reserve cellular viability of HPAECs in the circumstance of PI hypoxia despite its ineffectiveness on senescence.
作者
黎璟睿
谢江
范正阳
李菲
高杨
LI Jingrui;XIE Jiang;FAN Zhengyang;LI Fei;GAO Yang(The First Clinical Medical College,Lanzhou University,Lanzhou 73000,China)
出处
《心肺血管病杂志》
CAS
2022年第1期92-97,102,共7页
Journal of Cardiovascular and Pulmonary Diseases
基金
国家自然科学基金面上项目(81970079)。
