摘要
目的:研究缺氧状态下大鼠肺动脉平滑肌细胞(PASMCs)内活性氧(ROS)水平的变化,ROS对细胞外信号调节激酶(ERK)1/2蛋白表达的影响以及ROS和ERK1/2在PASMCs增殖和凋亡关系失衡中的作用。方法:原代培养正常大鼠PASMCs,选用第2-3代用于实验。分别在常氧及缺氧条件下用ROS清除剂tiron、ERK1/2抑制剂PD98059进行分组干预。通过NBT还原法和DCFH-DA荧光探针检测细胞内ROS,免疫荧光法检测磷酸化-ERK1/2(p-ERK1/2)蛋白表达,MTT比色法和增殖细胞核抗原(PCNA)蛋白的免疫细胞化学法检测细胞增殖,原位末端标记法(TUNEL)检测细胞凋亡。结果:(1)缺氧组细胞内ROS水平明显高于对照组(P<0.01);(2)缺氧组的增殖活性与对照组相比显著增高(P<0.01),而凋亡率显著降低(P<0.01),使用tiron后能明显抑制缺氧诱导的细胞增殖(P<0.05),而凋亡率却明显升高(P<0.01);(3)缺氧组p-ERK1/2蛋白表达显著高于对照组(P<0.01),使用tiron后缺氧诱导的p-ERK1/2表达被显著抑制(P<0.01);(4)使用PD98059也能明显抑制缺氧诱导的细胞增殖(P<0.05),而凋亡率也明显升高(P<0.01),缺氧下同时使用PD98059和tiron与单用tiron相比,对细胞增殖和凋亡的影响均无明显差异(P>0.05)。结论:缺氧时PASMCs中生成增多的ROS通过活化下游ERK1/2信号通路,促进PASMCs增殖并抑制凋亡,从而在缺氧性肺动脉重建过程中发挥重要作用。
AIM： To investigate the change of reactive oxygen species （ROS） production in hypoxic pulmonary arterial smooth muscle ceils （PASMCs） of rats, the effect of ROS on the expression of extracellular signal - regulated kinase （ERK） 1/2 protein, and the role of ROS and ERK1/2 in the imbalance between proliferation and apoptosis of PASMCs. METHODS： Primary cultures of PASMCs were established and cells between passages 2 to 3 were used for experiments. PASMCs were treated with tiron, a membrane permeable ROS scavenger, and PD98059, an ERK1/2 inhibitor, under normoxia or hypoxia condition. The ROS production was measured by DCFH - DA and NBT reduction. The expression of phosphorylated - ERK1/2 （ p - ERK1/2） protein was detected by immunofluorescence. Cell proliferation was examined by MTT colorimetric assay and the expression of PCNA. Cell apoptosis was detected by TUNEL. RESULTS： （ 1 ） Compared with control group, the ROS levels in hypoxia group were significantly increased （ P 〈 0. 01 ）. （2） In hypoxia group, the proliferative capacity was higher and the. apoptosis index was lower than those in control group （ P 〈 0. 01 ）. Tiron significantly attenuated hypoxia - induced cell proliferation （P 〈 0. 05） and also significantly raised the apoptosis index in hypoxia cells （P 〈0. 01 ）. （3） The expression of p -ERK1/2 in hypoxia group were higher than that in control group （ P 〈 0. 01 ）, which were significantly suppressed by tiron （ P 〈 0. 01 ）. （4） PD98059 significantly attenuated hypoxia - induced cell proliferation （ P 〈 0. 05 ） and also significantly raised the apoptosis index in hypoxia cells （ P 〈 0. 01 ）. The proliferative capacity and apoptosis index was similar in hypoxia ＋ tiron ＋ PD98059 group to those in hypoxia ＋ tiron group （P 〉 0. 05）. CONCLUSION： The hypoxia -mediated increase in PASMCs proliferation and the decrease in PASMCs apoptosis are related to the overproduction of intracellular ROS through downstream activation of ERK1/2. ROS and ERK1/2 play important roles in the hypoxic remodeling of pulmonary artery.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2009年第1期36-41,共6页
Chinese Journal of Pathophysiology
关键词
活性氧
细胞外信号调节激酶类
缺氧
肺动脉平滑肌细胞
细胞凋亡
细胞增殖
Reactive oxygen species
Extracellular signal -regulated kinases
Hypoxia
Pulmonary arterial smooth muscle cells
Apoptosis
Cell proliferation
