一氧化碳在缺氧条件下抑制肺平滑肌细胞的增殖。

Carbon monoxide inhibits proliferation of pulmonary smooth muscle cells under hypoxia.

作者信息

Zhen Guohua, Xue Zheng, Zhang Zhenxiang, Xu Yongjian

机构信息

Department of Respiratory Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

出版信息

Chin Med J (Engl). 2003 Dec;116(12):1804-9.

PMID:14687464

Abstract

OBJECTIVE

To investigate the expression of inducible heme oxygenase (HO-1) gene in pulmonary artery smooth muscle cells (PASMCs) exposed to hypoxia, and the influence of carbon monoxide (CO) on the proliferation of PASMCs under hypoxic conditions.

METHODS

Primary culture of rat PASMCs were passed every 3 days, and the 3 - 5 passages were used. After exposure to hypoxic conditions (95% N2, 5% CO(2)) 0, 12, 24 and 48 hours, the level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR). The volume of COHb in the medium was measured spectrophotometrically. The cyclic guanosine mono-phosphate (cGMP) concentration of cell extracts was determined by radioimmunoassay. PASMCs were divided into 5 groups, cultured under normoxia and hypoxia and treated with hemin, hemoglobin (Hb) and exogenous CO respectively. Then 3-(4, 5-cimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay and immunocytochemical staining were used to study the energy metabolism and the expression of proliferating cell nuclear antigen (PCNA) in PASMCs. Flow cytometry was used to analyze the cell cycle of PASMCs.

RESULTS

After exposure to hypoxic conditions for 12, 24, and 48 hours, the HO-1 mRNA increased by 2.7%, 5.7% and 27.1% respectively (P < 0.01). The carboxy-hemoglobin (COHb) in the medium increased by 13.8%, 31.0% and 93.1% (P < 0.01); the cGMP concentrations were 2.7, 4.0 and 6.8-fold compared with the control group (P < 0.01 and P < 0.05). In comparison with the control group, the value of MTT colorimetric assay, the immunocytochemical staining of PCNA and the percentages of PASMCs in S and G2M phases in the hypoxic group were significantly higher (P < 0.01). After treatment with Hemin and CO, the results of the above analysis decreased significantly (P < 0.01 and P < 0.05), but increased significantly after treatment with Hb (P < 0.01 and P < 0.05).

CONCLUSIONS

The expression of HO-1 gene in PASMCs is upregulated by hypoxia and the production of endogenous CO is elevated as well. The endogenous CO suppresses the proliferation of PASMC in an autocrine way. Both the induction of endogenous CO by Hemin and the treatment with exogenous CO can suppress the proliferation of rat PASMCs of under hypoxic conditions.

摘要

目的

研究缺氧环境下肺动脉平滑肌细胞（PASMCs）中诱导型血红素加氧酶（HO-1）基因的表达情况，以及一氧化碳（CO）对缺氧条件下PASMCs增殖的影响。

方法

大鼠PASMCs原代培养，每3天传代一次，取3 - 5代细胞使用。分别在缺氧环境（95% N2，5% CO₂）中暴露0、12、24和48小时后，采用逆转录聚合酶链反应（RT-PCR）检测HO-1 mRNA水平。用分光光度法测定培养基中碳氧血红蛋白（COHb）的含量。采用放射免疫法测定细胞提取物中环磷酸鸟苷（cGMP）的浓度。将PASMCs分为5组，分别在常氧和缺氧条件下培养，并分别用血红素、血红蛋白（Hb）和外源性CO处理。然后采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐（MTT）比色法和免疫细胞化学染色法研究PASMCs的能量代谢及增殖细胞核抗原（PCNA）的表达情况。采用流式细胞术分析PASMCs的细胞周期。

结果

在缺氧环境中暴露12、24和48小时后，HO-1 mRNA分别增加了2.7%、5.7%和27.1%（P < 0.01）。培养基中的碳氧血红蛋白（COHb）分别增加了13.8%﹑31.0%和93.1%（P < 0.01）；cGMP浓度分别是对照组浓度的2.7倍、4.0倍和6.8倍（P < 0.01和P < 0.05）。与对照组相比，缺氧组MTT比色法测定值、PCNA免疫细胞化学染色结果以及PASMCs在S期和G2M期的比例均显著升高（P < 0.01）。用血红素和CO处理后，上述分析结果显著降低（P < 0.01和P < 0.05），但用Hb处理后显著升高（P < 0.01和P < 0.05）。