摘要
目的探讨骨桥蛋白(OPN)对缺氧诱导的人肺动脉平滑肌细胞(HPASMCs)增殖的影响,并初步研究其作用机制。方法体外培养HPASMCs细胞,建立低氧模型,随机分为常氧组、低氧组、OPN-siRNA+低氧组和阴性对照(NC-siRNA)+低氧组。实时荧光定量PCR(RT-qPCR)法检测各组HPASMCs细胞OPN和CD44 mRNA表达情况。采用细胞计数试剂盒-8(CCK-8)法检测各组细胞增殖变化情况。流式细胞术检测各组细胞凋亡和周期变化情况。蛋白印迹分析法检测各组细胞OPN、CD44、增殖细胞核抗原(PCNA)、细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白E(Cyclin E)、P27蛋白表达情况。结果与常氧组相比,低氧组HPASMCs细胞存活率、G2/M期比例、PCNA、Cyclin D1、Cyclin E表达及OPN、CD44 mRNA和蛋白表达显著升高(P<0.05),凋亡率、HPASMCs细胞SubG1期、G0/G1期比例及P27蛋白表达显著降低(P<0.05)。与低氧组和NC-siRNA+低氧组相比,OPN-siRNA+低氧组HPASMCs细胞存活率、G2/M期比例、PCNA、Cyclin D1、Cyclin E蛋白表达及OPN、CD44 mRNA和蛋白表达显著降低(P<0.05),凋亡率、HPASMCs细胞SubG1期、G0/G1期比例及P27蛋白显著升高(P<0.05)。结论敲低OPN表达可能通过抑制CD44表达将细胞周期阻滞在G0/G1期,抑制低氧诱导的HPASMCs细胞恶性增殖,促进其凋亡。
Objective To investigate the effect of osteopontin(OPN)on the proliferation of human pulmonary artery smooth muscle cells(HPASMCs)induced by hypoxia and its mechanism.Methods HPASMCs cells were cultured in vitro to establish a hypoxia model.They were randomly divided into normoxia group,hypoxia group,OPN-siRNA+hypoxia group and negative control(NC-siRNA)+hypoxia group.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the mRNA expression of OPN and CD44 in HPASMCs.Cell proliferation was detected by cell counting kit-8(CCK-8)method.Cell apoptosis and cell cycle were detected by flow cytometry.Western blot analysis was used to detect the expression of OPN,CD44,proliferating cell nuclear antigen(PCNA),Cyclin D1,Cyclin E and P27.Results Compared with normoxia group,the cell survival rate of HPASMCs,the proportion of G2/M phase cells,the expression of PCNA,Cyclin D1 and Cyclin E,the mRNA and protein expression of OPN and CD44 were significantly increased in hypoxia group(P<0.05),while the apoptosis rate,the proportions of SubG1 phase and G0/G1 phase,and the expression of P27 protein were significantly decreased(P<0.05).Compared with hypoxia group and NC-siRNA+hypoxia group,the cell survival rate of HPASMCs,the proportion of G2/M phase cells,the protein expression of PCNA,Cyclin D1 and Cyclin E,the mRNA and protein expression of OPN and CD44 were significantly decreased in OPN-siRNA+hypoxia group(P<0.05),while the apoptosis rate,the proportions of SubG1 phase and G0/G1 phase,and P27 protein were significantly increased(P<0.05).Conclusion Knockdown of OPN expression may block the cell cycle in G0/G1 phase by inhibiting the expression of CD44,inhibit the malignant proliferation of HPASMCs induced by hypoxia,and promote its apoptosis.
作者
吴树全
王生兰
WU Shuquan;WANG Shenglan(Department of Emergency,Fifth People’s Hospital of Qinghai Province,Xining 810000,China;Department of Pathophysiology,Medical College of Qinghai University)
出处
《山西医科大学学报》
CAS
2021年第5期593-598,共6页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目(81860076)。
关键词
骨桥蛋白
CD44
人肺动脉平滑肌细胞
增殖
凋亡
细胞周期
osteopontin
CD44
human pulmonary artery smooth muscle cells
proliferation
apoptosis
cell cycle
作者简介
吴树全,男,1973-09生,学士,副主任医师,E-mail:weak666@163.com。
