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周期蛋白D1 参与香烟烟雾提取物促进人肺动脉平滑肌细胞增殖和迁移

向敏, 徐永健^*, 刘先胜, 曾大雄

华中科技大学同济医学院附属同济医院呼吸内科，武汉 430030

摘要

本文旨在探讨周期蛋白D1 (cyclin D1)在香烟烟雾提取物(cigarette smoke extract, CSE)所致人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells, HPASMCs)增殖和迁移中的作用。构建反义cyclin D1 基因真核表达载体(pIRES2-EGFP-ascyclin D1)，采用脂质体介导基因转染法将空载体(pIRES2-EGFP)和pIRES2-EGFP-ascyclin D1 导入正常HPASMCs后，分别进行CSE 干预。细胞随机分为6 组：对照组、空载体组、反义cyclin D1 组、5% CSE 组、空载体+5% CSE组、反义cyclin D1+5% CSE 组。用实时荧光RT-PCR 和Western blot 法分别检测cyclin D1 mRNA 和蛋白的表达，采用流式细胞术、四甲基偶氮唑盐比色法(MTT)、增殖细胞核抗原(PCNA)染色法测定细胞增殖能力，Transwell 小室法检测细胞迁移能力。结果显示，反义cyclin D1 基因真核表达载体pIRES2-EGFP-ascyclin D1 成功构建，并成功转染入HPASMCs，转染后HPASMCs 中cyclin D1 的mRNA 和蛋白表达水平较对照组均显著下降(P<0.05)。与对照组比较，5% CSE 组cyclinD1 的mRNA和蛋白表达水平均明显升高(P<0.05)，细胞增殖和迁移能力显著增强(P<0.05)。与5% CSE 组比较，反义cyclinD1+5% CSE 组cyclin D1 mRNA 和蛋白表达水平均明显下降(P<0.05)，细胞增殖和迁移能力显著降低(P<0.05)。上述结果提示，CSE 可通过上调cyclin D1 表达促进HPASMCs 增殖和迁移，反义cyclin D1 基因真核表达载体可抑制CSE 介导的HPASMCs 增殖和迁移，提示cyclin D1 在CSE 所致HPASMCs 增殖和迁移中发挥重要调控作用。

关键词： 香烟; 周期蛋白D 1 ; 肺动脉; 平滑肌细胞; 细胞增殖; 细胞迁移

分类号：R563 ；R332

[Cyclin D1 is involved in human pulmonary artery smooth muscle cells proliferation and migration induced by cigarette smoke extract.] [Ariticle in Chinese]

XIANG Min, XU Yong-Jian^*, LIU Xian-Sheng, ZENG Da-Xiong

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

Abstract

The present study was aimed to investigate the role of cyclin D1 in human pulmonary artery smooth muscle cells (HPASMCs)proliferation and migration induced by cigarette smoke extract (CSE). The eukaryotic expression vector of antisense cyclin D1 gene(pIRES2-EGFP-ascyclin D1) was recombinated. The recombinant and empty vector were separately transfected into normal HPASMCsusing liposome. Then the cells were treated with or without 5% CSE. The cells were randomly divided into six groups: control group,vector group, antisense cyclin D1 group, 5% CSE group, vector+5% CSE group and antisense cyclin D1+5% CSE group. Theexpressions of cyclin D1 mRNA and protein were detected by real-time fluorescence RT-PCR and Western blot, respectively. Theproliferation of HPASMCs was examined by cell cycle analysis, MTT assay and proliferation cell nuclear antigen (PCNA) immunocytochemicalstaining. The migration of HPASMCs was measured by Transwell cell test. The results showed that the eukaryoticexpression vector of antisense cyclin D1 gene was constructed and transfected into HPASMCs successfully. The cyclin D1 mRNA andprotein levels in antisense cyclin D1 group were significantly lower than those in control group (P<0.05). In 5% CSE group, the cyclinD1 mRNA and protein levels were elevated significantly compared with those in control group (P<0.05), and the indicators of cell proliferation and migration were increased as well (P<0.05). The cyclin D1 mRNA and protein levels, the indicators of cell proliferationand migration in antisense cyclin D1+5% CSE group were remarkably lower than those in 5% CSE group (P<0.05). These resultssuggest that CSE could promote HPASMCs proliferation and migration through up-regulation of cyclin D1 expression. PIRES2-EGFPascyclinD1 could attenuate CSE-induced proliferation and migration of HPASMCs by suppressing the expression of cyclin D1, whichimplicates that cyclin D1 might be involved in the process of HPASMCs proliferation and migration stimulated by CSE.

Key words: tobacco; cyclin D1; pulmonary artery; smooth muscle cells; cell proliferation; cell migration

收稿日期：2010-01-14　　录用日期：2010-02-23

通讯作者：徐永健　　E-mail: yjxu@tjh.tjmu.edu.cn

引用本文：

向敏, 徐永健, 刘先胜, 曾大雄. 周期蛋白D1 参与香烟烟雾提取物促进人肺动脉平滑肌细胞增殖和迁移[J]. 生理学报 2010; 62 (2): 156-162.

XIANG Min, XU Yong-Jian, LIU Xian-Sheng, ZENG Da-Xiong. [Cyclin D1 is involved in human pulmonary artery smooth muscle cells proliferation and migration induced by cigarette smoke extract.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (2): 156-162 (in Chinese with English abstract).