哮喘小鼠气道平滑肌细胞增殖及细胞周期蛋白D1表达的差异

目的:研究哮喘小鼠气道重建模型中气道平滑肌细胞(ASMC)的细胞增殖、细胞周期及细胞周期蛋白D1(cyclin D1)表达的变化,探讨cyclin D1对支气管哮喘气道重建中平滑肌细胞增殖过程及细胞周期的影响,进而为哮喘的诊断和治疗提供新的依据.方法:用Balb/c小鼠建立哮喘气道重建模型后进行ASMC原代培养,并以原代培养的正常小鼠ASMC为对照组,以四唑盐比色试验(MTF)检测细胞增殖能力,流式细胞术(FCM)测定细胞周期,FCM检测cyclin D1表达.结果:与对照组比较,哮喘组细胞的细胞增殖显著增快(P＜0.05);细胞周期中S期比例明显增高;cyclin D1在胞质中表达明显增加.结论:在哮喘气道重建过程中ASMC经历了一定程度的过增殖过程,cyclin D1的表达增加参与了细胞增殖,气道重建环境可刺激气道平滑肌细胞增殖能力.     

Cigarette smoke extract promotes human pulmonary artery smooth muscle cells proliferation through protein kinase C alpha-dependent induction of cyclin D1

Background Exposure to cigarette smoke stimulates the proliferation of human pulmonary artery smooth muscle cells （HPASMCs） in vivo and in vitro. However, the molecular mechanism remains unclear. This study aimed at investigating the role of signaling pathways involving protein kinase C alpha （PKCα） and cyclin D1 in the cigarette smoke extract （CSE）-induced HPASMCs proliferation.Methods Synchronized HPASMCs were treated with different concentrations of CSE. Cell proliferation was evaluated by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyttetrazolium bromide （MTT） assay and cell counting. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Activation of PKCα was measured by detecting the expression of PKCαprotein in the cytosolic and membrane fractions using Western blotting analysis. Small interfering RNA （siRNA） was used to knockdown PKCα and cyclin D1. The cyclin D1 mRNA was assessed by real-time RT-PCR. The PKCα and cyclin D1protein levels were detected by Western blotting.Results Low concentrations of CSE （1%-10%） stimulated proliferation of HPASMCs, with its maximal effect at 5%.CSE （5%） led to PKCα activation. Inhibition of PKCα activity using G（o） 6976 or siRNA-mediated knockdown of PKCα significantly attenuated CSE-induced cell proliferation and G1/S transition. Cyclin D1, one of key regulators of G1/S transition, was found to be upregulated by 5% CSE at both the mRNA and protein levels. CSE-stimulated cellproliferation and G1/S transition was abolished by cyclin D1 siRNA. Moreover, G（o） 6976 or PKCα siRNA significantlysuppressed CSE-induced upregulation of cyclin D1 at both the mRNA and protein levels.Conclusion PKCα-cyclin D1 pathway at least partially mediates the CSE-induced proliferation in HPASMCs.     

Effect of Cigarette Smoke Extract on the Role of Protein Kinase C in the Proliferation of Passively Sensitized Human Airway Smooth Muscle Cells

Cigarette smoke(CS) is ani mportant risk fac-tor involvedin the development and progression ofasthma[1]. Ovalbumin ( Ova) sensitization inducesairway hyperresponsiveness ( AHR) to inhaled CSin guinea pigs[2]. Airway smooth muscle prolifera-tion plays an i mportant role in airway remodelingin asthma . Protein kinase C (PKC) is one of thecrucial kinases in the intracellular signal transduc-tion pathway .CSEcanincrease PKC's activationinhuman bronchial epithelial cells[3].Because airwaysm…     

维生素D对原代培养哮喘大鼠气道平滑肌细胞增殖的影响

目的:探讨维生素D对哮喘大鼠气道平滑肌细胞(airway smooth muscle cells,ASMCs)的抗增殖机制,并证明细胞周期蛋白D1(cyclin D1)在其抗增殖调控中的作用?方法:原代培养正常对照组和哮喘组大鼠ASMCs,取第3~5代细胞用于实验?总分为以下5组:正常对照ASMCs组?哮喘ASMCs组?哮喘ASMCs + 1 × 10-8 mol/L维生素D组?哮喘ASMCs + 1 × 10-7 mol/L维生素D组?哮喘ASMCs + 1 × 10-6 mol/L维生素D组?用四甲基偶氮唑盐(MTT)法?流式细胞术检测各组ASMCs增殖活性的变化;用逆转录-聚合酶链反应(RT－PCR)和蛋白质印迹(Western blot)检测不同组ASMCs cyclin D1 mRNA和蛋白的表达?结果:① 哮喘ASMCs组G0/G1期细胞比例(80.01 ± 2.55)%明显少于其他各组,S期(8.31 ± 0.42)%和G2/M期(11.21 ± 1.43)%比例增加,MTT法检测其吸光度值增高(0.78 ± 0.04),提示ASMCs增殖明显增强?其中各值分别与正常对照ASMCs组[(90.15 ± 3.02)%?(5.41 ± 0.62)%?(4.38 ± 0.56)%?0.22 ± 0.04]相比,有显著性差异(P < 0.001);同时哮喘ASMCs组与哮喘ASMCs + 1 × 10-6 mol/L维生素D组各值[(89.08 ± 2.81)%?(5.58 ± 0.92)%?(4.61 ± 1.12)%?0.38 ± 0.02]相比,其差异有统计学意义(P < 0.001);与哮喘ASMCs + 1 × 10-7/1 × 10-8 mol/L维生素D组相比,差异也有统计学意义(P < 0.01);②在cyclin D1 mRNA和蛋白表达水平上,哮喘ASMCs组与正常对照ASMCs组?哮喘ASMCs + 1 × 10-6 mol/L维生素D组分别相比较均偏高,差异显著(P < 0.001);③在各不同剂量维生素D作用组,分别在细胞增殖活性检测?cyclin D1 mRNA和蛋白表达水平上,大剂量维生素D组作用均优于最小剂量组,差异有统计学意义(P < 0.001),显示维生素D的抗增殖作用有明显剂量-效应相关性?结论:哮喘组大鼠ASMCs增殖明显,维生素D可以通过调节cyclinD1表达发挥其对哮喘大鼠ASMCs的抗增殖作用?     

Genotoxicity and reduced heat shock protein 70 in human airway smooth muscle cells exposed to cigarette smoke extract

Cigarette smoke is associated with the development of several diseases, such as chronic ob- structive pulmonary disease （COPD）. The purpose of this study was to investigate genotoxicity and heat shock protein 70 （Hsp70） in human airway smooth muscle cells （HASMCs） exposed to cigarette smoke extract （CSE）. HASMCs was exposed to CSE with different doses for 24 h. The level of 8-hydroxydeoxyguanosine （8-OHdG） was determined by using HPLC-ECD, the DNA damage was ana- lyzed by using comet assay, and apoptosis was examined by using Annexin-FITC/PI staining. The pro- duction of Hsp70 after CSE stimulation was tested. Results indicated that CSE significantly increased the level of 8-OHdG, DNA damage and cell apoptosis, and reduced the production of Hsp70. In par- ticular, levels of Hsp70 were inversely correlated with 8-OHdG, DNA damage and cell apoptosis. It was concluded that cigarette smoke induced genotoxicity and decreased the production of cell protective protein Hsp70, which may contribute to the development of some airway diseases.     

PDGF对哮喘大鼠气道平滑肌细胞中ERK通路的影响研究

目的：研究血小板源性生长因子（PDGF）对哮喘大鼠气道平滑肌细胞（ASMC）中ERK信号通路的调控作用。方法建立哮喘大鼠模型,原代分离培养ASMC,设未干预组（A组）、ERK阻断剂组（B组）、PDGF组（C组）和PDGF＋ERK阻断剂组（D组）。A组不加干预;B组又分为B1、B2、B3、B4组,分别加入浓度为0.1、1、5、10μmol/L的ERK阻断剂U0126;C组又分为C1、C2、C3、C4组,分别加入浓度为1、10、25、50μg/L的PDGF同二聚体PDGF- BB;D组加入10μmol/L的U0126和50μg/L的PDGF- BB。以RT- PCR法检测各组ERK1和TGF-β1的mRNA表达,免疫组化法检测A、B4、C4和D组中以上蛋白的表达。结果 RT- PCR提示B1、B2、B3、B4组ERK1 mRNA表达均显著低于A组（均P＜0.01）,U0126以浓度依赖的方式抑制PDGF诱导的ASMC中ERK的活化,C1、C2、C3、C4组ERK1 mRNA表达均显著高于A组（均P＜0.01）,ERK1 mRNA的表达与PDGF- BB存在明显的浓度依赖关系,D组与A组比较无统计学差异（P＞0.05）。免疫组化提示B4组ASMC中ERK1蛋白表达显著低于A组,C4组显著高于A组（均P＜0.01）,D组与A组相比无统计学差异（P＞0.05）;B4组TGF-β1蛋白表达显著低于A组,C4组显著高于A组,同时C4组显著高于B4组（均P＜0.01）,D组与A组相比无统计学差异（P＞0.05）。结论 PDGF可剂量依赖性激活哮喘大鼠ASMC内的ERK通路,同时伴随TGF-β1信号通路的活化。ERK通路参与了ASMC增殖的细胞内信号转导过程,PDGF可能经ERK环节促进TGF-β1通路活化。     

阿奇霉素对哮喘大鼠气道平滑肌细胞生长的影响

目的 探讨阿奇霉素对哮喘大鼠气道平滑肌细胞（ASMCs）增殖的影响.方法 将20只SD大鼠随机分为对照组和哮喘组（哮喘组制备大鼠哮喘模型后,随机分为不同剂量的阿奇霉素组）,采用CCK-8法测定不同剂量的阿奇霉素组对哮喘大鼠气道平滑肌细胞的药物毒性反应,并分别采用琼脂糖凝胶电泳检测法和激光共聚焦显微镜观察法观察阿奇霉素各组的细胞凋亡情况,同时采用划痕法检测各阿奇霉素组平滑肌细胞的迁移能力.结果 （1）各剂量阿奇霉素组哮喘大鼠气道平滑肌细胞的生长率分别为（72.71±13.16）%、（66.42±4.90）%、（64.92±2128）%、（51.45±2.83）%;（2）与对照组比较,阿奇霉素各组均可以检测到气道平滑肌细胞染色体DNA发生片段化降解;（3）阿奇霉素各组均可显示典型的细胞凋亡形态学特征;（4）划痕法结果显示：阿奇霉素组的迁移数目和距离均较10%FBS血清刺激组减少和缩短（均P〈0.01）.结论 阿奇霉素可抑制哮喘大鼠气道平滑肌细胞增殖、迁移,促进细胞凋亡.     

香烟烟雾提取物对气道平滑肌细胞增殖及内皮素释放的影响

探讨气道平滑肌细胞（ＡＳＭＣ）自分泌的内皮素（ＥＴ）是否介导了吸烟对ＡＳＭＣ的作用。方法在体外培养的家兔ＡＳＭＣ上加入香烟烟雾提取物（ＣＩＳＥ）测定细胞存活率，乳酸脱氢酶（ＬＤＨ）水平，并用Ｈ－ＴｄＲ掺入法及放射免疫方法观察ＣＳＥ对ＡＳＭＣ的增殖作用，及对ＥＴ－１释放的影响。用３Ｈ－ＴｄＲ掺入实验，观察低浓度（５％）ＣＳＥ对ＡＳＭＣ增殖的影响及与ＥＴ的关系。结果１０％和３０％ＣＳＥ对ＡＳＭＣ呈现明显毒性作用，表现为细胞存活率降低，脂质过氧化终产物丙二醛（ＭＤＡ）含量增加和细胞ＬＤＨ漏出随时间进行性增加。５％ＣＳＥ呈时间依赖地刺激ＡＳＭＣ释放ＥＴ－１并促进ＡＳＭＣ（３Ｈ－ＴｄＲ）掺入（较对照组增加５９．９％，Ｐ＜０．０１）。内皮素Ａ受体（ＥＴＡ受体）拮抗剂ＪＫＣ－３０１呈浓度依赖地抑制５％ＣＳＥ促ＡＳＭＣ增殖作用。结论吸烟可以刺激ＡＳＭＣ释放ＥＴ－１且通过ＥＴＡ受体介导ＡＳＭＣ增殖     

MicroRNA-34a 对小鼠气道平滑肌细胞增殖和ORMDL3 表达的影响

目的 探讨支气管哮喘小鼠模型中microRNA-34a（miR-34a）对气道平滑肌细胞（ASMC）增 殖与血清类黏蛋白1 样蛋白3（ORMDL3）表达的影响。方法 以卵清蛋白（OVA）诱导复制的小鼠哮喘 模型和培养的ASMC 为研究对象,筛选与ORMDL3 密切相关的miRNA ;利用miR-34a-mimic 使miR- 34a 过表达;用苏木精- 伊红染色和MTT 法探究其对ASMC 增殖的影响;实时荧光定量聚合酶链反应和 Western blot 检测ORMDL3 的表达变化。结果 OVA 诱导下小鼠气道组织中miR-34a 被抑制（P <0.05）。 miR-34a-mimic 可减少哮喘小鼠ASMC 异常增殖（P <0.05）;miR-34a-mimic 抑制ASMC 在490nm 下的光 密度值和相对细胞活性（P <0.05）;miR-34a-mimic 下调哮喘小鼠气道组织中ORMDL3 的表达,并抑制体 外培养ASMC 中ORMDL3 的表达（P <0.05）。结论 miR-34a 可抑制ASMC 的增殖和ORMDL 3 基因表达, 为今后哮喘发病机制的研究提供新的思路。     

Valsartan inhibits angiotensin II-induced proliferation of vascular smooth muscle cells via regulating the expression of mitofusin 2

Angiotensin Ⅱ (ANGⅡ) plays an important role in the pathogenesis of atherosclerosis by inducing proliferation of vascular smooth muscle cells (VSMCs).In our study,we observed the effects of valsartan on proliferation of cultured VSMCs treated with or without ANGⅡ by cell counting and methyl thiazolyl tetrazolium (MTT) assay,and detected the expression of mitofusin 2 (Mfn2),a newly discovered cell proliferation inhibitor and a related cell proliferation signaling pathway pro-tein by Western blotting.ANGⅡ at a concentration of 10-6 mol/L significantly stimulated VSMCs proliferation,down-regulated the expression of Mfn2 and upregulated the expression of Raf and ERK1/2.Valsartan inhibited such effects of ANGⅡ at concentrations of 10-5 and 10-6 mol/L,but not at 10-7 mol/L.Valsartan had no significant effect on the proliferation of untreated VSMCs.These results suggest that valsartan inhibits ANGⅡ-induced proliferation of VSMCs in vitro via Mfn2-Ras-Raf-ERK/MAPK signaling pathway.     

Effects of pinacidil on proliferation of cultured rabbit airway smooth muscle cells induced by endothelin-1

It has been found that the potassium channel dysfunction of the membrane of airway smooth muscle cells (ASMCs) is closely associated with proliferation of ASMCs. Preliminary research has demonstrated that pinacidil, an ATP sensitive potassium channel (KATP) opener, could play a remarkable role in the prevention and treatment of antigen induced bronchial asthma in guinea pigs.     

Effect of Cigarette Smoke Extract on the Proliferation of Human Airway Epithelial Cells and Expression and Activation of FAK

The effect of cigarette smoke extract (CSE) on the proliferation of human airway epithelial cells and the possible mechanism was studied. After airway epithelial cells were treated with different concentrations of CSE for 24 h, the cell proliferation was measured by MTT and the distribution of different cell cycles by flow cytometry. The FAK expression level was detected by Western blot and the degree of tyrosine phosphorylation by immunoprecipitation. The results showed that CSE could inhibit the proliferation of human airway epithelial cells, arrest the epithelial cells in G1 phase of cell cycle, dramatically decrease the number of epithelial cells in S and G2 phases； Meanwhile CSE could decrease the expression level of FAK and the degree of its tyrosine phosphorylation. The above effects of CSE were concentration-dependent. The expression of FAK and the degree of its phosphorylation was positively correlated to the increased number of epithelial cells in G1 phase, and negatively to the number of epithelial cells in S and G2 phases. It was concluded that the mechanism by which CSE could inhibit the proliferation of human epithelial cells was contributed to the increased expression and activation of FAK.     

罗红霉熬caveolin-1-p—ERK1／2对离体哮喘大鼠气道平滑肌细胞cyclinD1表达的影响

目的探讨罗红霉素（RXM）经caveolin-1-P-ERKl／2对离体哮喘大鼠气道平滑肌细胞（ASMCs）cyclinDl表达的影响。方法将30只SPF级雄性SD大鼠随机均分为对照组和哮喘组,以卵自蛋白致敏和激发复制哮喘气道重塑模型;采用Im—age—ProPlusVersion60图像分析软件测定支气管壁和支气管平滑肌层厚度;透射电镜观察两组大鼠ASMCs微囊表达情况;CCK-8检测不同浓度RXM[A组（只含0．1％DMSO）、R10组（含RXMl0．g／ml＋0．1％DMSO）、R25组（含RXM25ug／ml＋01％DM—S0）、R50组（含RXM50ug／ml＋0．1％DMSO）、R100组（含RXM100tJg／ml＋0.1％DMSO）1干预哮喘ASMCs后细胞的增殖情况,Westernblot测定caveolin-1、P—ERK、cyclinDl的蛋白表达。结果哮喘组支气管壁和支气管平滑肌层明显增厚,而微囊表达匮乏。哮喘组大鼠支气管壁与平滑肌层厚度均高于对照组（均P〈0．01）。各RXM干预组ASMCsA值均低于A组,其中R100组明显低于A组（P〈0．05）。R100组caveolin-1表达明显高于A组及R10组,cyclinDl表达明显低于A组及Rt0组,差异均有统计学意义（P〈0,05或O．01）;R25、R50、R100组P—ERK表达均明显低于A组,差异均有统计学意义（P〈0．05或001）。哮喘ASMCs增殖活性与caveolin-1蛋白表达呈负相关（P〈0．05）,与P—ERK及cyclinDl蛋白表达呈正相关（P〈005或0．01）;caveolin-1与P—ERK蛋白表达呈负相关（P〈0．01）,与cyclinDl蛋白表达呈正相关（P〈001）;P—ERK与cyclinD1蛋白表达呈正相关（P〈0．01）。结论RXM可能通过上调caveolin-1表达、抑制ERK1／2活化,进而影响cyclinD1的表达,从而抑制哮喘大鼠ASMCs增殖。     

姜黄素抑制大鼠气道平滑肌细胞增殖的作用

目的 研究姜黄素对气道平滑肌细胞(airway smooth muscle cells,ASMCs)增殖的作用.方法 体外培养大鼠原代ASMCs.CCK-8法检测血小板源生长因子(platelet-derived growth factor,PDGF)和PDGF加姜黄素处理的ASMCs A150的光密度值,以观察PDGF诱导的增殖和姜黄素的抗增殖作用.Western blotting 检测ERK1/2蛋白的表达水平.结果 与对照组(1.04±0.12)比较,CCK-8法检测给予PDGF 2 d后,10ng/mL PDGF组(1.16±0.14)和50 ng/mL PDGF组(1.30±0.15)的A150的光密度值均显著增加.P＜0.01.给予姜黄素处理12、24和36 h后,与对照组比较,11.25μmol/L组(21.57%、43.55%和65.99%)、16.88μmol/L组(39.64%、57.44%和77.80%)和25.3μmol/L组(44.50%、53.68%和92.68%)的细胞平均抑制率均增加显著,P＜0.05.姜黄素(25μmol/L)加PDGF(25 ng/mL)处理20、40和60 min后ERK1/2蛋白表达水平均显著降低.结论 姜黄素对增殖的ASMCs有抑制作用,可能与抑制了ERK1/2的信号通路活化有关.     

西罗莫司对大鼠血管平滑肌细胞增殖、起始识别复合物1表达的影响及机制探讨

目的探讨西罗莫司对体外培养的大鼠血管平滑肌细胞(VSMCs)增殖、起始识别复合物1(ORC1)表达的影响及机制。方法细胞随机分为对照组和实验组,每组3瓶/孔,血清饥饿法培养细胞24h使其同步化。对照组采用含10%胎牛血清的DMEM培养液培养,实验组采用含10μmol/L西罗莫司+10%胎牛血清的DMEM培养液培养。继续培养0～48h,采用免疫细胞化学法检测增殖细胞核抗原(PCNA)表达,电子显微镜观察细胞超微结构,RT-PCR法检测ORC1mRNA的表达,蛋白质印迹法检测P53、cyclin D1、cyclin A和ORC1蛋白的表达。结果与对照组相比,实验组48hPCNA阳性表达率降低[(20±2.1)%vs(80±3.0)%,P<0.05],P53蛋白表达升高(P<0.05);细胞核有固缩的迹象;实验组cyclin D1蛋白表达轻微升高(P>0.05),48hcyclin A蛋白的表达下降(P<0.05)。对照组ORC1mRNA的表达在0～12h升高,24～48h降低,高峰期在12h。实验组细胞ORC1mRNA的表达始终处于高水平。与同时间对照组比较,实验组48hORC1mRNA的表达升高(P<0.05)。蛋白质印迹分析结果与之相似。结论西罗莫司抑制VSMCs增殖,同时促进其凋亡;其作用环节可能是通过阻止细胞由G0/G1期向S期转化,诱导细胞处于静止状态;ORC1的作用环节位于西罗莫司作用点的上游,进一步支持ORC1参与细胞复制起始过程。