ERK信号通道调控大鼠气道平滑肌细胞的增殖与凋亡

 为了了解ERK信号通道对正常大鼠气道平滑肌细胞(airway smooth muscle cells, ASMCs)增殖与凋亡的调控. 通过对正常大鼠ASMCs原代培养,4～7代用于实验,以ERK激动剂表皮生长因子（EGF）和抑制剂PD98059干预ASMCs生长,采用RT-PCR和免疫荧光染色观察ASMCs上ERK mRNA和蛋白的表达,MTT法、^３Ｈ-TdR掺入法检测ASMC增殖,Hoechst染色和Annexin-Ⅴ FITC PI双染色法检测细胞凋亡,Western免疫印迹检测ERK1/2、磷酸化ERK1/2和procaspase-3蛋白的表达.结果发现ASMCs上存在ERK mRNA和蛋白的表达,与空白对照组比较,PD98059干预后ASMCs的Ａ_４９０值和细胞DNA合成量均减少(Ｐ<0.05),细胞凋亡指数、早期凋亡细胞百分率均增高(Ｐ<0.05),ERK1/2、磷酸化ERK1/2表达和ERK活化率均降低, procaspase-3蛋白的表达增高.EGF干预后ASMCs的Ａ_４９０值和细胞DNA合成量均增高(Ｐ<0.05),细胞凋亡指数、早期凋亡细胞百分率均下降(Ｐ<0.05),ERK1/2、磷酸化ERK1/2表达和ERK活化率均增高, procaspase-3蛋白的表达降低.P+E组无明显差异(P>0.05).ERK信号通道参与大鼠ASMCs增殖和凋亡的调控,ERK对大鼠ASMCs凋亡的调控与procaspase-3蛋白有关,这一发现将有助于对哮喘ASMCs异常增殖调控机制的深入研究.     

IL-19对哮喘模型大鼠气道平滑肌细胞增殖的影响

目的探讨IL-19对哮喘大鼠气道平滑肌细胞（ASMCs）增殖的作用。方法通过对大鼠进行雾化卵清蛋白,制备哮喘模型大鼠。提取哮喘大鼠ASMCs进行培养,分别以1μg/L、10μg/L、100μg/L IL-19干预ASMCs生长。采用流式细胞仪、MTT法检测ASMCs增殖情况,观察不同浓度IL-19对ASMCs增殖的影响。结果与正常鼠相比,慢性哮喘大鼠ASMCs增殖明显,处于S期的细胞比例明显增高。经10μg/L、100μg/L IL-19干预后,慢性哮喘大鼠ASMCs处于S期的细胞比例减少,增殖亦减弱。且两组间比较有明显差异。而经1μg/L IL-19干预后,慢性哮喘大鼠ASMCs处于S期的细胞比例及增殖均无明显变化。结论一定浓度的IL-19可能抑制慢性哮喘大鼠ASMCs的增殖。     

TGF-β_1对不同阶段哮喘模型大鼠气道平滑肌细胞增殖的作用

目的探讨TGF-β1对不同阶段哮喘大鼠气道平滑肌细胞（ASMCs）增殖的作用。方法建立2周、6周哮喘大鼠模型,分别以1μg/L、10μg/L和100μg/LTGF-β1干预ASMCs生长。采用流式细胞仪、MTT法检测ASMCs增殖情况,观察不同浓度TGF-β1对ASMCs增殖的影响。结果 2周和6周哮喘组ASMCs的S期比例、A值分别为（34.31±1.41）%、（35.96±3.46）%;（0.546±0.005）、（0.559±0.009）与对照组（12.24±2.64）%、（0.289±0.009）比较均显著增高（均P〈0.01）。2周、6周哮喘模型组的各TGF-β1干预组ASMCs的S期比例、A值与各自哮喘组比较均显著升高（均P〈0.01）,10μg/L和100μg/LTGF-β1组比1μg/LTGF-β1组对ASMCs增殖作用明显增加（P〈0.01）,10μg/LTGF-β1组和100μg/LTGF-β1组相比,ASMCs增殖细胞占细胞总数的百分比无明显变化,两种浓度增殖作用无有明显差别（P〉0.05）。而2周和6周哮喘组相比,加入不同浓度TGF-β1干预后的差别不大（P〉0.05）。结论与正常鼠相比,2周和6周哮喘大鼠气道平滑肌细胞增殖明显,处于S期的细胞比例明显增高,6周哮喘大鼠气道平滑肌较2周哮喘大鼠增殖更明显。经TGF-β1干预后,2周和6周哮喘哮喘大鼠气道平滑肌细胞处于S期的细胞比例增加,增殖增强,提示TGF-β1可能在哮喘早期阶段即可促进大鼠气道平滑肌细胞增殖,并促进各阶段哮喘大鼠气道平滑肌细胞持续增殖。     

姜黄素诱导肿瘤细胞凋亡的研究进展

姜黄素是从姜科植物的根茎姜黄中提取的一种植物多酚,具有抗炎、抗氧化、抗凝、抗动脉粥样硬化、抗肿瘤等药理作用。本文综述了姜黄素诱导肿瘤细胞凋亡的分子生物学机制研究进展。     

凋亡诱导因子在姜黄素诱导的大鼠肺成纤维细胞凋亡中的作用

目的：研究姜黄素对肺纤维化大鼠肺成纤维细胞增殖、凋亡的影响,探讨凋亡诱导因子（AIF）在肺成纤维细胞凋亡中的作用。方法：将体外培养的肺纤维化大鼠成纤维细胞,分别于不同浓度的姜黄素（5、10、20、40μM）和caspase-3抑制剂Z-DEVD-fmk（20μM）孵育,观测细胞生长状态变化。MTT检测成纤维细胞增殖抑制率;流式细胞仪检测细胞凋亡率;Western-Blot测定凋亡诱导因子（AIF）蛋白表达及核转位结果：流式细胞术检测细胞凋亡,5～40μM姜黄素处理12 h,其凋亡率呈浓度依赖,对照组相比,差异显著;而抑制caspase-3并不能完全阻止细胞凋亡。Western-Blot结果显示,姜黄素处理组出现凋亡诱导因子（AIF）蛋白表达与核转位,抑制caspase-3活性后未检测出AIF表达结论：姜黄素可抑制肺成纤维细胞增殖,其诱导大鼠肺成纤维细胞凋亡,可能与线粒体释放AIF有关。     

凋亡诱导因子在姜黄素诱导的大鼠肺成纤维细胞凋亡中的作用

目的:研究姜黄素对肺纤维化大鼠肺成纤维细胞增殖、凋亡的影响,探讨凋亡诱导因子(AIF)在肺成纤维细胞凋亡中的作用.方法:将体外培养的肺纤维化大鼠成纤维细胞,分别于不同浓度的姜黄素(5、10、20、40μM)和caspase-3抑制剂Z-DEVD-fmk(20μM)孵育,观测细胞生长状态变化.MTT检测成纤维细胞增殖抑制率;流式细胞仪检测细胞凋亡率;Western-Blot测定凋亡诱导因子(AIF)蛋白表达及核转位结果:流式细胞术检测细胞凋亡,5～40μM姜黄素处理12 h,其凋亡率呈浓度依赖,对照组相比,差异显著;而抑制caspase-3并不能完全阻止细胞凋亡.Western-Blot结果显示,姜黄素处理组出现凋亡诱导因子(AIF)蛋白表达与核转位,抑制caspase-3活性后未检测出AIF表达结论:姜黄素可抑制肺成纤维细胞增殖,其诱导大鼠肺成纤维细胞凋亡,可能与线粒体释放AIF有关.     

改良组织块消化法建立大鼠气道平滑肌细胞模型

目的：建立改进大鼠气道平滑肌细胞（ASMC）的体外培养方法,为相关研究提供实验材料。方法：将组织块连续贴壁进行细胞原代培养,胰酶消化传代培养,差速贴壁进行细胞纯化,形态学及免疫细胞化学染色法进行细胞鉴定。MTT法检测PDGF-BB诱导的ASMC增殖。结果：成功培养大鼠ASMC,以改良组织块消化法最为理想。第四代平滑肌细胞纯度可达95％以上。相差显微镜下培养细胞呈典型“峰谷状”生长。免疫荧光化学染色显示特异性平滑肌肌动蛋白阳性表达。随着PDGF浓度的升高（2-80ng／ml）,MTT比色A490值呈上升趋势。与对照组相比较,80ng／ml、20ng／ml PDGF—BB组有统计学意义（P〈0．01）。结论：改良组织块消化法可缩短培养周期,在充分利用标本的基础上获得大量气道平滑肌细胞。     

三种钠尿肽抑制大鼠肺动脉平滑肌细胞增殖效应的比较

本文比较了心房钠尿肽（ＡＮＰ）、Ｃ－型钠尿肽（ＣＮＰ）、血管钠肽（ＶＮＰ）抑制肺动脉平滑肌细胞（ＰＡＳＭＣｓ）增殖的效应。用蛋白激酶Ｃ激动剂佛波酯（ＰＭＡ）刺激体外培养大鼠ＰＡＳＭＣｓ的增殖,以总蛋白含量和ＭＴＴ比色ＯＤ值为指标,观察三种钠尿肽对ＰＭＡ刺激大鼠ＰＡＳＭＣｓ增殖的影响。结果表明,ＰＭＡ（１０＾－９－１０＾－７ｍｏｌ／Ｌ）显著升高（Ｐ＜０．０５）ＰＡＳＭＣｓ的总蛋白含量和ＭＴＴＯＤ值,     

姜黄素对人食管癌EC9706细胞凋亡的诱导作用

目的:应用姜黄素处理人食管癌EC9706细胞,研究姜黄素对人食管癌EC9706细胞凋亡的诱导作用。方法:应用细胞计数、流式细胞仪、琼脂糖凝胶电泳、Hoechst染色、H.E染色和透射电镜检测经姜黄素诱导处理后人食管癌EC9706细胞的凋亡。结果:经姜黄素诱导处理后,人食管癌EC9706细胞生长抑制率达69.9%;细胞周期检测出现亚二倍体(亚G1期)细胞峰值,细胞凋亡率达23%;琼脂糖凝胶电泳显示出细胞凋亡典型的180-200 bp及其倍体的DNA"梯状"条带;Hoechst染色显示细胞核内出现浓染致密的固缩形态或颗粒状荧光;光镜和电镜下可见典型的细胞凋亡特征:细胞体积缩小,染色体凝集,可见有成群或单独存在的凋亡细胞,电镜下可见凋亡小体存在。结论:姜黄素能够有效诱导人食管癌EC9706细胞的凋亡,从而进一步为食管癌等恶性肿瘤疾病的治疗和凋亡机理的研究提供重要基础和科学依据.     

RyR反义寡核苷酸对气道平滑肌细胞增殖及细胞内钙离子浓度的影响

目的:观察RyR(Ryanodme受体)反义寡核苷酸(ASODN)对大鼠ASMCs(airway smooth muscle cells,气道平滑肌细胞)增殖的抑制作用及对细胞内钙离子浓度的影响.方法:采用胶原酶消化法培养大鼠ASMCs,利用LipofectamineTM2000将正义、反义RyR寡核苷酸导入大鼠ASMCs,MTS/PES法检测不同寡核苷酸对大鼠ASMCs增殖的抑制作用,RT-PCR检测大鼠ASMCs中RyR的mRNA表达,流式细胞仪测定不同寡核苷酸对细胞内钙离子浓度的影响.结果:RyR反义寡核苷酸可抑制大鼠ASMCs的增殖,降低其RyR受体mRNA的表达,并能降低兴奋后的细胞内钙离子浓度的升高.结论:RyR反义寡核苷酸可能通过降低兴奋后的细胞内钙离子浓度来抑制大鼠ASMCs的增殖.     

麻黄碱抑制支气管平滑肌细胞增殖的机制

BK通道(large-conductance Ca2 -activated K channel)是一种大电导、电压和钙离子依赖的钾通道,存在于哺乳动物几乎所有的组织中.β1亚基是首次从平滑肌细胞中分离到的一种BK通道辅助亚基.麻黄作为一种β肾上腺素受体激动剂,是一味治疗哮喘的常用中药.利用RT-PCR和膜片钳等技术,研究麻黄治疗哮喘的作用机制.结果表明:麻黄碱(ephedrine,Eph)作为麻黄的一种主要成分,显著抑制支气管平滑肌细胞(bronchial smooth muscle cells,BSMCs)的增殖和BK通道β1亚基的表达,但是对BK通道的电流没有显著影响.     

Effects of Trimethoxystilbene on Proliferation and Apoptosis of Pulmonary Artery Smooth Muscle Cells

Proliferation of pulmonary artery smooth muscle cells (PASMC) is an important contributor to the progress of pulmonary arterial hypertension (PAH). Anti-inflammatory therapies may have therapeutic applicability for PAH. Resveratrol (RES) has prominent anti-inflammatory effects in vitro, but in vivo its beneficial effects are limited by short systemic half life and poor lipotrophy. A derivative of RES, trimethoxystilbene (TMS), has higher lipotropy and extended half life compared with RES, and can potentially overcome the limitations of RES. In the present study, we studied the effects of TMS and RES on proliferation and apoptosis of PASMC stimulated with Tumor Necrosis Factor-??. The effects on PASMC proliferation were quantified by MTT, while apoptosis was assessed by flow cytometry (Annexin V/propidium iodide assay). We observed strong inhibitory effects of TMS on the growth of PASMC, and these effects were 20 times more potent than those of RES. We further documented induction of apoptosis in PASMC treated with TMS, again, to a higher degree than with RES. In conclusion, TMS is more potent than RES in the inhibition of proliferation and induction of apoptosis of PASMC, demonstrating its potentially beneficial role for treating PAH.     

番茄红素对血管平滑肌细胞增殖与凋亡的影响

目的:VSMCs增殖是动脉粥样硬化的主要病理过程之一.本研究通过观察番茄红素对人血管平滑肌细胞(VSMC)增殖和凋亡的影响,并探讨与Toll样受体4(TLR-4)有关的信号通路相关分子机制,旨在为番茄红素治疗动脉粥样硬化提供理论基础.方法:番茄红素处理体外培养的VSMC,分别应用MTT及流式细胞术分析处理后的VSMC增殖及凋亡情况;采用荧光定量PCR和Western blot检测处理后VSMC的TLR-4及骨髓分化分子88 (MyD88)表达水平的变化;ELISA检测番茄红素对VSMC肿瘤坏死因子α(TNF-α)及白细胞介素6 (IL-6)分泌量的影响.结果:番茄红素处理后VSMC细胞增殖速度降低,凋亡增加;番茄红素处理后VSMC内TLR-4及MyD88的表达降低,TNF-α及IL-6分泌减少.结论:番茄红素可抑制VSMC生长增殖;促进凋亡,从而发挥治疗动脉粥样硬化的作用,其机制可能与TLR-4及MyD88表达降低、TNF-α及IL-6分泌减少有关.     

反义癌基因c－jun对大鼠血管平滑肌细胞增殖的影响

构建可表达原癌基因ｃ－ｊｕｎ正义和反义ＲＮＡ的真核细胞表达载体,将其导入体外培养的大鼠血管平滑肌细胞（ＶＳＭＣ）后,经Ｎｏｒｔｈｅｒｎ分析证实,两种插入方向的ｃｊｕｎ基因均在ＶＳＭＣ中大量表达;￣３Ｈ－ＴｄＲ参入实验表明,ｃ－ｊｕｎ反义ＲＮＡ对ＶＳＭＣ的增殖具有显著抑制作用．提示用反义核酸抑制ｃ－ｊｕｎ表达对治疗某些由于ＶＳＭＣ增殖所引起的心血管病可能具有一定意义．     

The Pro-Proliferative Effects of Nicotine and Its Underlying Mechanism on Rat Airway Smooth Muscle Cells

Recent studies have shown that nicotine, a major component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. Cigarette smoking can promote a variety of pulmonary and cardiovascular diseases, such as chronic obstructive pulmonary disease (COPD), atherosclerosis, and cancer. A predominant feature of COPD is airway remodeling, which includes increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodeling in COPD have not yet been fully elucidated. Here, we show that nicotine induces a profound and time-dependent increase in DNA synthesis in rat airway smooth muscle cells (RASMCs) in vitro. Nicotine also significantly increased the number of RASMCs, which was associated with the increased expression of Cyclin D1, phosphorylation of the retinoblastoma protein (RB) and was dependent on the activation of Akt. The activation of Akt by nicotine occurred within minutes and depended upon the nicotinic acetylcholine receptors (nAchRs). Activated Akt increased the phosphorylation of downstream substrates such as GSK3β. Our data suggest that the binding of nicotine to the nAchRs on RASMCs can regulate cellular proliferation by activating the Akt pathway.     

A Synthetic Chloride Channel Relaxes Airway Smooth Muscle of the Rat

Synthetic ion channels may have potential therapeutic applications, provided they possess appropriate biological activities. The present study was designed to examine the ability of small molecule-based synthetic Cl^– channels to modulate airway smooth muscle responsiveness. Changes in isometric tension were measured in rat tracheal rings. Relaxations to the synthetic chloride channel SCC-1 were obtained during sustained contractions to KCl. The anion dependency of the effect of SCC-1 was evaluated by ion substitution experiments. The sensitivity to conventional Cl^– transport inhibitors was also tested. SCC-1 caused concentration-dependent relaxations during sustained contractions to potassium chloride. This relaxing effect was dependent on the presence of extracellular Cl^– and HCO₃ ⁻. It was insensitive to conventional Cl^– channels/transport inhibitors that blocked the cystic fibrosis transmembrane conductance regulator and calcium-activated Cl^– channels. SCC-1 did not inhibit contractions induced by carbachol, endothelin-1, 5-hydroxytryptamine or the calcium ionophore A23187. SCC-1 relaxes airway smooth muscle during contractions evoked by depolarizing solutions. The Cl^– conductance conferred by this synthetic compound is distinct from the endogenous transport systems for chloride anions.     

同型半胱氨酸对大鼠血管平滑肌细胞增殖的作用

血中同型半胱氨酸（ｈｏｍｏｃｙｓｔｅｉｎｅ,ＨＣＹ）浓度的升高已成为动脉粥样硬化发生的一个独立危险因子．为进一步阐明ＨＣＹ促进血管平滑肌细胞（ｖａｓｃｕｌａｒｓｍｏｏｔｈｍｕｓｃｌｅｃｅｌｓ,ＶＳＭＣｓ）增殖,从而引起动脉粥样硬化发生的机理．本实验采用细胞计数、３Ｈ－ＴｄＲ参入、细胞周期分析、Ｎｏｒｔｈｅｒｎ杂交方法证明,一定剂量的ＨＣＹ可促进离体培养的ＷＫＹ大鼠血管平滑肌细胞增殖,使其ＤＮＡ合成增加,细胞周期中Ｓ期细胞所占比例增加４３％,并促进ｃ－ｍｙｃ与ｃ－ｆｏｓ原癌基因ｍＲＮＡ表达增加．提示ＨＣＹ可能通过促进ＶＳＭＣｓ增殖而诱发动脉粥样硬化