血管内皮生长因子及其受体2与被动致敏的人气道平滑肌细胞增殖相关性研究

目的探讨血管内皮生长因子（VEGF）及其受体2（KDR）在被动致敏的人气道平滑肌细胞（ASNC）中表达变化及其对ASMC增殖的影响。方法培养人ASMC，用支气管哮喘病人血清被动致敏ASMC。用免疫组织化学技术检测ASMC增殖细胞核抗原（POJA）的表达，MTT法检测细胞代谢活性及流式细胞仪分析细胞周期；用RT-PCR及Western blot方法分别检测VEGF和KDR mRNA及蛋白质在不同组人ASMC的表达程度。结果（1）被动致敏组ASMC较对照组和干预组增殖显著增加（P〈0．05）。（2）被动致敏组ASMC的VEGF121、VEGF165和VEGF189的mRNA表达分别较对照组和干预组显著增加（P〈0．05或P〈0．01）。（3）被动致敏组ASMCVEGF及KDR蛋白质的表达较对照组和干预组显著增加（P〈0．05）。直线相关性分析显示，ASMC中PCNA表达与ASMC中VEGF121、165、189及KDRmRNA表达水平呈正相关（r分别为0．73、0．82、0．77、0．70，P〈0．05）；ASMC中PCNA表达与ASMC中VEGF及KDR蛋白质表达水平也呈正相关（r分别为0．69、0．67,P〈0．05）。结论被动致敏的ASMC中VEGF及其受体KDR表达上调。并与ASMC增殖密切相关。该结果提示VEGF及其受体2可能参与了哮喘气道重建中ASMC增殖的过程。     

U46619对培养的人气道平滑肌细胞增殖的作用

目的和方法：研究Ｕ４６６１９（９,１１－ｄｉｄｅｏｘｙ－１１α,９α－ｅｐｏｘｙｍｅｔｈａｎｏ－ｐｒｏｓｔａｇｌａｎｄｉｎＦ２α）对培养的人气管平滑肌细胞增殖的作用。分离人的气管平滑肌细胞并且进行传代培养。在培养基中加入各种浓度的Ｕ４６６１９,计数细胞并且测定［３Ｈ］－胸腺嘧啶核苷（［３Ｈ］－ＴｄＲ）掺入量和三磷酸肌醇［Ｉｎｓ（１,４,５）Ｐ３］累积量。结果：Ｕ４６６１９在一定范围内（１ｎｍｏｌ／Ｌ～１００ｎｍｏｌ／Ｌ）以浓度依赖的方式增加人气管平滑肌的细胞数（Ｐ＜００１）。Ｕ４６６１９也增加［３Ｈ］－ＴｄＲ的掺入量和Ｉｎｓ（１,４,５）Ｐ３的累积量（Ｐ＜００１）。磷脂酶Ｃ抑制剂新霉素阻止Ｉｎｓ（１,４,５）Ｐ３累积量的增加（Ｐ＜００１）,但对Ｕ４６６１９的有丝分裂原的活性没有作用。结论：显示Ｕ４６６１９刺激培养的人气管平滑肌细胞增殖。     

哮喘气道平滑肌细胞表观遗传学调控的研究进展

哮喘气道重塑中一个主要方面即是平滑肌细胞的增生肥大,近些年,人们逐渐发现表观遗传学对气道平滑肌细胞的增殖和分泌炎性因子方面有着重要的调节作用,其中包括DNA甲基转移酶抑制剂可以抑制其表型转换;组蛋白乙酰化与其增生肥大相关;另外,microRNA可以调控哮喘模型中气道平滑肌细胞的各种生理功能,包括抑制其增殖及炎性因子的释放。希望表观遗传学能够成为治疗哮喘的新型靶点。     

ERK在慢性哮喘大鼠气道平滑肌细胞增殖中的作用

目的：观察细胞外信号调节蛋白激酶(ERK)在慢性哮喘大鼠气道平滑肌细胞的表达，以探讨ERK信号通路在气道平滑肌增殖中的作用。方法：病理图像分析慢性哮喘大鼠气道重塑，免疫组化法检测ERK和PCNA在肺内表达，激光共聚焦显微镜分析ERK1/2、磷酸化ERK1/2和PCNA在气道平滑肌的共表达，免疫印迹和原位杂交检测气道平滑肌中ERK和PCNA蛋白以及mRNA的表达。结果：慢性哮喘大鼠有气道平滑肌层增厚，出现结构重塑。ERK和PCNA在肺内表达增强，同时在气道平滑肌上有ERK和PCNA蛋白与mRNA表达增加。结论：ERK可能是介导慢性哮喘气道重建中平滑肌增殖的重要信号通路之一。     

白三烯D4在促进培养的人气管 平滑肌细胞增殖中的作用

目的和方法:研究白三烯D₄(LTD₄)是否剌激培养的人气管平滑肌细胞(ASMC)增殖。将分离的人ASMC进行传代培养,在培养基中加入各种浓度的LTD₄,计数细胞并测定 [³H]-胸腺嘧啶核苷([³H]-TdR)掺入量和三磷酸肌醇(IP₃)累积量。结果: LTD₄在一定范围内(0.1 nmoL·L^-1~10 nmoL·L^-1)以浓度依赖的方式增加人ASMC(P＜0.01)。LTD₄也增加[³H]-TdR的掺入量和IP₃累积量(P＜0.01)。磷脂酶C抑制剂新霉素(1 μmol·L^-1)阻止IP₃累积量的增加(P＜0.01)。结论:LTD₄剌激培养的人ASMC增殖并且可能在哮喘的气道重塑中起了作用。     

白三烯D_4在促进培养的人气管平滑肌细胞增殖中的作用

目的和方法 :研究白三烯D4 (LTD4 )是否剌激培养的人气管平滑肌细胞 (ASMC)增殖。将分离的人ASMC进行传代培养 ,在培养基中加入各种浓度的LTD4 ,计数细胞并测定 [3 H]-胸腺嘧啶核苷 ([3 H]-TdR)掺入量和三磷酸肌醇 (IP3 )累积量。结果 :LTD4 在一定范围内 (0 1nmoL·L-1～ 10nmoL·L-1)以浓度依赖的方式增加人ASMC(P <0 0 1)。LTD4 也增加 [3 H]-TdR的掺入量和IP3 累积量 (P <0 0 1)。磷脂酶C抑制剂新霉素(1μmol·L-1)阻止IP3 累积量的增加 (P <0 0 1)。结论 :LTD4 剌激培养的人ASMC增殖并且可能在哮喘的气道重塑中起了作用。     

Proliferative effects of eosinophil lysates on cultured human airway smooth muscle cells

BACKGROUND: The hypertrophy/hyperplasia of airway smooth muscle (ASM) cells is one of the characteristic features of bronchial asthma. This structural change leads to the thickening of airway walls resulting in the amplification of airway narrowing. However, the pathogenesis of this structural change has not yet been determined. Eosinophils, which play a pathogenic role in asthma, have been demonstrated to have proliferative effects on fibroblasts and vascular smooth muscle cells. OBJECTIVE: We attempted to investigate the potential of eosinophils to induce the proliferation of ASM cells. METHODS: We examined the effect of lysates of eosinophils purified from peripheral blood of healthy donors on cultured human ASM cell proliferation. RESULTS: Eosinophil lysates significantly induced ASM cell proliferation in time- and dose-dependent manners, reaching a maximum on day 6 at 50% of eosinophil lysates (6.0 +/- 0.7 x 104 [mean +/- SD] /well, n = 5 vs. 4.5 +/- 1.1 x 104/well, n = 5; P < 0.05). This proliferative activity was heat-sensitive and recovered in the soluble fraction of the eosinophil lysates. Furthermore, the molecular weight of the mitogenic activity in the soluble fraction was identified as lower than 10 kDa. The inhibitory activity to ASM cell proliferation was also found in the insoluble fraction of the lysates. CONCLUSION: These results indicate that circulating eosinophils store mitogenic activity for ASM cells, suggesting that eosinophils might contribute to the development of the hyperplasia of ASM cells in asthmatics through the release of the stored mitogenic activity upon stimulation at the site of inflammation.     

周期蛋白D1在蛋白激酶C调控哮喘大鼠气道平滑肌细胞增殖中的作用

目的：观察蛋白激酶C(PKC)激活后哮喘大鼠气道平滑肌细胞(ASMCs)周期蛋白D1(cyclin D1)的表达变化及两者表达相关性，探讨cyclin D1在PKC调控哮喘大鼠ASMCs增殖中所起的作用。方法:卵清蛋白（OVA）吸入制备SD大鼠2周哮喘模型，原代培养ASMCs。采用正常大鼠（N组）和模型大鼠（A组）第3-6代细胞，分别应用PKC特异性激活剂12-肉豆蔻酰-13-乙酸佛波酯（PMA）及抑制剂Ro-31-8220干预ASMCs, 根据不同处理分为4组：（1）空白组；（2）10 nmol/L PMA组；(3)10 nmol/L PMA+5 μmol/L Ro-31-8220组； (4)5 μmol/L Ro-31-8220组。采用流式细胞术，四甲基偶氮唑盐（MTT）法，增殖细胞核抗原（PCNA）染色等方法观察药物对ASMCs增殖的影响。RT-PCR方法检测PKC-α和cyclin D1 mRNA表达水平，Western blotting方法检测PKC-α和cyclin D1蛋白表达水平。线性相关分析评价PKC-α和cyclin D1表达水平的关系。结果:（1）在哮喘组，与空白对照比较，PMA组、Ro-31-8220组S+G2/M期比例、吸光度A值、PCNA阳性表达率，差异显著（P<0.01）。在正常组，与空白对照相比，PMA组、Ro-31-8220组S+G2/M期比例、吸光度A值、PCNA阳性表达率差异均显著（P<0.01）。（2）哮喘组内PMA组、Ro-31-8220组PKC-α以及cyclin D1 mRNA 和蛋白表达水平与空白对照比较，差异显著（P<0.01）。正常组和哮喘组变化趋势一致。(3)在mRNA水平，大鼠ASMCs PKC-α和cyclin D1的表达呈明显正相关(r=0.476，P<0.05)，在蛋白水平两者的表达亦呈明显正相关(r=0.899, P<0.01)。结论:PKC-α能促进正常大鼠和哮喘大鼠ASMCs增殖，其在基因和蛋白的表达水平与cyclin D1的表达呈正相关，提示PKC-α可能通过调节cyclin D1而影响ASMCs的增殖。     

TRPC6在PDGF诱导的大鼠气道平滑肌细胞增殖中的作用

 目的：探讨经典瞬时受体电位通道6 (TRPC6) 对血小板源性生长因子 (PDGF) 诱导的气道平滑肌细胞 (ASMCs) 增殖的影响。方法：组织贴块联合酶消化法培养原代大鼠ASMCs。间接免疫荧光法鉴定平滑肌细胞及检测TRPC6在ASMCs上的表达。CCK-8法检测PDGF诱导ASMCs的增殖。Real-time PCR 检测PDGF作用后TRPC6 mRNA的表达。Western blotting检测PDGF作用后TRPC6蛋白的表达。CCK-8法检测TRPC6阻断剂对PDGF 诱导ASMCs增殖的作用。结果：细胞免疫荧光显示：TRPC6广泛存在于气道平滑肌细胞。CCK-8法检测细胞的增殖发现,20 μg/L PDGF作用后 ASMCs发生增殖 (P<0.05);PDGF与TRPC6阻断剂SKF96365共同作用于ASMCs,ASMCs的增殖较单独使用PDGF组减弱 (P<0.05),且减弱的程度具有剂量及时间依赖性。Real-time PCR结果显示：PDGF分别作用于ASMCs 12 h、24 h和48 h后,TRPC6 mRNA表达与相应的对照组比较明显升高 (P<0.05)。Western blotting检测结果显示：PDGF分别作用ASMCs 24 h和48 h后,TRPC6蛋白表达与相应的对照组比较明显升高 (P<0.05)。结论：TRPC6参与了PDGF诱导ASMCs增殖的过程,PDGF促进ASMCs增殖可能与其上调TRPC6 mRNA和蛋白表达相关。     

瘦素对大鼠气道平滑肌细胞增殖的影响

目的:探讨瘦素对大鼠气道平滑肌细胞(ASMCs)增殖的影响及其可能的作用机制.方法:体外培养大鼠的ASMCs,分别用RT-PCR和Western blot测定ASMCs中瘦素受体mRNA和该细胞上瘦素受体蛋白的表达.不同浓度的瘦素(0～100 μgL)干预培养的ASMCs不同时间(1～72 h)后,以CCK-8法测定ASMCs的增殖情况.不同浓度的瘦素作用于ASMCs 48 h后,Western blot测定磷酸化细胞外调节蛋白激酶(p-ERK)和磷脂酰肌醇3激酶(PI-3K)的表达.结果:不同浓度的瘦素作用不同时间后,均可促进大鼠ASMCs增殖,并呈浓度依赖性(r=0.837,P＜0.01)和时间依赖性(r=0.874,P＜0.01).Western blot的结果显示,不同浓度的瘦素干预后,大鼠ASMCs中p-ERK、PI-3K蛋白的表达较对照组显著增加(P＜0.05),并与瘦素的浓度呈正相关(前者r=0.793,P＜0.01,后者r=0.746,P＜0.01).结论:大鼠ASMCs表面有瘦素受体表达.瘦素可促进体外培养的大鼠ASMCs增殖,其机制可能与激活p-ERK和PI-3K有关.     

Fibroblast growth factor 9 signaling inhibits airway smooth muscle differentiation in mouse lung

In mammalian lungs, airway smooth muscle cells (airway SMCs) are present in the proximal lung adjacent to bronchi and bronchioles, but are absent in the distal lung adjacent to terminal sacs that expand during gas exchange. Evidence suggests that this distribution is essential for the formation of a functional respiratory tree, but the underlying genetic mechanism has not been elucidated. In this study, we test the hypothesis that fibroblast growth factor 9 (Fgf9) signaling is essential to restrict SMC differentiation to the proximal lung. We show that loss of Fgf9 or conditional inactivation of Fgf receptors (Fgfr) 1 and 2 in mouse lung mesenchyme results in ectopic SMCs. Our data support a model where FGF9 maintains a SMC progenitor population by suppressing differentiation and promoting growth. This model also represents our findings on the genetic relationship between FGF9 and sonic hedgehog (SHH) in the establishment of airway SMC pattern. Developmental Dynamics 238:123–137, 2009. © 2008 Wiley‐Liss, Inc.     

香烟提取物对支气管哮喘大鼠气道平滑肌细胞增殖的影响

目的： 探讨香烟提取物（CSE）对支气管哮喘(简称哮喘)大鼠气道平滑肌细胞（ASMCs）增殖作用及可能机制。方法: 16只SD大鼠随机分为对照组和哮喘组,各8只。原代培养大鼠ASMCs,取第3-6代细胞,分为对照组、对照+CSE组、哮喘组、哮喘+CSE组、哮喘+CSE+嘧啶基-苯磺酰胺（GW8510,细胞周期蛋白依赖激酶-4抑制剂）组、哮喘+GW8510组。用流式细胞术、四甲基偶氮唑盐（MTT）法及增殖细胞核抗原（PCNA）免疫细胞化学技术检测ASMCs增殖;用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹（Western blotting）检测细胞周期蛋白D1(cyclin D1)的表达。结果: （1）哮喘组ASMCs与对照组ASMCs相比,在S+G2/M期比例、吸光度（A）值和PCNA表达率上明显增高,差异显著（P＜0.01）。（2）哮喘组ASMCs S+G2/M期比例、吸光度（A）值和PCNA表达率分别为(18.30±1.12)%、0.512±0.110、(55.1±3.7)%;哮喘+CSE组分别为（32.12±1.17）%、0.801±0.210、（90.2±7.3）%;哮喘+CSE+GW8510组分别为(17.21±0.95)%、0.508±0.009、(54.3±4.8)%;哮喘+GW8510组分别为(11.16±1.48)%、0.345±0.078、(40.6±5.4)%。除哮喘组、哮喘+CSE+GW8510组两组比较差异无显著外,其余两两比较差异均显著（P＜0.01）。（3）哮喘组、哮喘+CSE组、哮喘+CSE+GW8510组、哮喘+GW8510组ASMCs cyclin D1 mRNA A值比值和蛋白表达A值比值分别为0.236±0.045、0.271±0.002;0.369±0.124、0.379±0.002;0.231±0.075、0.261±0.002;0.165±0.064、0.193±0.002。除哮喘组、哮喘+CSE+GW8510组两组比较差异无显著外,其余两两比较差异均显著（P＜0.01）。结论: 正常与哮喘大鼠ASMCs在CSE干预后增殖明显加快,cyclin D1表达明显增加。CSE可能是通过cyclin D1参与调控哮喘大鼠ASMCs的增殖。     

Airway smooth muscle modulation and airway hyper-responsiveness in asthma: new cellular and molecular paradigms

There is growing evidence indicating the existence of a causal relationship between abnormal airway smooth muscle (ASM) function and airway hyper-responsiveness, a poorly understood feature of asthma that can be defined as an excessive bronchospastic response. In recent years, there has been a veritable explosion of articles suggesting that ASM exposed to proasthmatic cytokines can elicit a hyper-responsive state to contractile G-protein-coupled receptor (GPCR) agonists. Aberrant airway responsiveness could result from abnormal calcium signaling, with changes occurring at various levels of GPCR-associated signal transduction. This review presents the latest observations describing novel mechanistic models that could explain the involvement of ASM in airway hyper-responsiveness. This review will discuss the role of ASM in β₂-agonist-mediated bronchial hyper-responsiveness and the clinical significance of cell–cell contact between ASM and mast cells recently described to be intimately infiltrated within the ASM tissues in asthmatic patients. The possibility that allergens could trigger airway hyper-responsiveness by directly acting on ASM via activation of immunoglobulin E receptors, FcεRI and FCεRII will also be discussed. These important findings further support the notion that targeting ASM could offer new treatment for many features of asthma, including airway hyper-responsiveness. Future therapeutic intervention includes: the prevention of ASM–inflammatory cell physical and/or functional interaction, the inhibition of Immunoglobulin E receptor-dependent signal transduction, and the abrogation of cytokine-dependent pathways that modulate receptor-associated calcium metabolism.     

Platelet-derived growth factor and transforming growth factor-β modulate the expression of matrix metalloproteinases and migratory function of human airway smooth muscle cells

Background Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been suggested to be involved in the pathogenesis of asthma. Their expression in airway smooth muscle (ASM) cells could be involved in collagen turnover and migration of these cells and thus may contribute to airway remodelling.
Objective To examine the effect of pro-fibrotic growth factors TGF-β and platelet-derived growth factor (PDGF) on the expression of MMPs/TIMPs in cultured human ASM cells and to examine the role of MMP in the migration of ASM cells.
Methods ASM cells were stimulated with TGF-β and/or PDGF. Expression and activity of MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-2 were evaluated by quantitative RT-PCR, Western blot and zymography. Modified Boyden-chamber migration assay was performed to investigate the effect of secreted MMP-3 and TIMP-1 on ASM-cell migration.
Results PDGF strongly up-regulated the expression of MMP-1 at mRNA and protein levels. PDGF, when combined with TGF-β, caused synergistic up-regulation of MMP-3. TIMP-1 was additively up-regulated by TGF-β and PDGF. These growth factors had no effect on the expression of MMP-2 and TIMP-2. U0126, an extracellular signal-regulated kinase (ERK) pathway inhibitor, inhibited the up-regulation of MMP-1 by PDGF. The synergistic/additive up-regulation of MMP-3 and TIMP-1 was inhibited by U0126 and SB431542, a Smad pathway inhibitor. Supernatant from ASM cells in which MMP-3 production was knocked down by RNA interference showed a decreased migratory effect on ASM cells, whereas supernatant from cells with suppressed TIMP-1 expression resulted in increased migration.
Conclusion Our results suggest that PDGF with/without TGF-β could facilitate migration of ASM cells by modification of MMP–TIMP balance through the ERK pathway.