粘着斑激酶活性对气道上皮细胞粘附迁移的影响

目的 研究粘着斑激酶(FAK)磷酸化对细胞外基质成份诱导的气道上皮细胞粘附、迁移的影响，探讨FAK活化对气道上皮细胞损伤后修复的影响机制。方法 通过纤维连接蛋白(FN)诱导培养的气道上皮细胞，以计数法测定细胞粘附率，以损伤实验测定细胞迁移速度，以Western blot和免疫共沉淀检测FAK表达水平及其酪氨酸磷酸化程度；以反义FAK寡核苷酸(ODNs)经脂质体转染细胞，观察其对FAK表达和磷酸化、细胞粘附和迁移的影响。结果 FN诱导的气道上皮细胞粘附率和迁移速度明显增高，同时FAK表达水平及其酪氨酸磷酸化程度显著增高；经脂质体转染了反义FAK寡核苷酸的气道上皮细胞粘附率和迁移速度明显降低，同时FAK表达水平及其酪氨酸磷酸化程度降低，且与气道上皮细胞粘附率和迁移速度的降低成显著正相关。结论 FAK是细胞外基质诱导气道上皮细胞粘附、迁移的重要信号分子，其活化促进气道上皮细胞的粘附和迁移，在气道上皮细胞损伤后修复过程中起重要作用。     

姜黄素对肿瘤坏死因子-α诱导的大鼠平滑肌细胞Syndecan-4蛋白及p44/42丝裂原活化蛋白激酶表达的影响

观察姜黄素对肿瘤坏死因子-α（TNF-α）诱导的离体大鼠血管平滑肌细胞（VSMCs）增殖和Syndecan-4蛋白及p44/42丝裂原活化蛋白激酶（MAPK）表达的影响。方法分别用20 ng/ml TNF-α、20 μmol/L姜黄素、20 ng/ml TNF-α联合20 μmol/L姜黄素作用于体外培养的大鼠胸主动脉平滑肌细胞24 h,并设对照组进行比较。MTS/PMS法检测VSMCs的增殖状态,Westernblot蛋白免疫印迹法测定VSMCs中Syndecan-4蛋白及磷酸化p44/42MAPK的表达。结果（1）细胞增殖结果经统计分析,与对照组比较,TNF-α能显著刺激大鼠VSMCs的增殖（P<0.01）,而单独应用姜黄素对大鼠VSMCs增殖无明显作用（P>0.05）。TNF-α联合姜黄素组与 TNF-α组比较,大鼠 VSMCs 的增殖受到抑制（P<0.01）;（2）统计分析蛋白表达结果,与对照组比较,TNF-α组能显著上调大鼠 VSMCs 中 Syndecan-4 蛋白及磷酸化 p44/42MAPK 的表达（P<0.01）,而单独应用姜黄素对大鼠VSMCs中Syndecan-4蛋白及磷酸化p44/42MAPK的表达无明显作用（P>0.05）。与TNF-α组比较,TNF-α联合姜黄素组中大鼠VSMCs中Syndecan-4蛋白及磷酸化p44/42MAPK的表达显著下调（P<0.01）。结论 一定剂量的姜黄素可显著抑制TNF-α诱导的大鼠血管平滑肌细胞增殖和Syndecan-4蛋白及磷酸化p44/42MAPK的表达。     

姜黄素对肿瘤坏死因子-α诱导的大鼠平滑肌细胞Syndecan-4蛋白及磷酸化p44/42丝裂原活化蛋白激酶表达的影响

目的观察姜黄素对肿瘤坏死因子-α(TNF-α)诱导的离体大鼠血管平滑肌细胞(VSMCs)Syndecan-4蛋白及p44/42丝裂原活化蛋白激酶(MAPK)表达的影响。方法分别用20 ng/ml TNF-α、20μmol/L姜黄素、20 ng/ml TNF-α联合20μmol/L姜黄素作用于体外培养的大鼠胸主动脉平滑肌细胞24 h,MTS/PMS法检测VSMCs的增殖状态,Western blot蛋白免疫印迹法分别测定VSMCs中Syndecan-4蛋白及磷酸化p44/42MAPK的表达。结果(1)统计分析表明,与对照组比较,TNF-α能显著刺激大鼠VSMCs的增殖(P<0.01),单独应用姜黄素对大鼠VSMCs增殖无显著作用(P>0.05)。与TNF-α组比较,姜黄素能显著抑制TNF-α所诱导的大鼠VSMCs的增殖(P<0.01);(2)统计分析表明,与对照组比较,TNF-α组能显著刺激大鼠VSMCs中Syndecan-4蛋白及磷酸化p44/42MAPK的表达(P<0.01),TNF-α的这一作用能被姜黄素所抑制(P<0.01),单独应用姜黄素对大鼠VSMCs中Syndecan-4蛋白及磷酸化p44/42MAPK的表达无显著作用(P>0.05)。结论一定剂量的姜黄素可显著抑制TNF-α诱导的大鼠血管平滑肌细胞增殖和Syndecan-4蛋白及磷酸化p44/42MAPK的表达。     

PP~(60c-Src)在血管紧张素对大鼠血管平滑肌细胞信息转导中的作用

目的 :通过观察 c- Src在 Ang 对大鼠血管平滑肌细胞 (VSMC)丝裂原活化的蛋白激酶 (MAPK)活性和c- fos蛋白表达的影响 ,以进一步了解 Ang 促 VSMC增殖的细胞内信息转导机制。方法 :原代和传代培养 SD大鼠主动脉 VSMC,以脂质体包裹反义 c- Src寡脱氧核苷酸 (Oligodeoxynucleotides ODNs)转染培养的 VSMC以抑制 c- Src蛋白表达和激酶活性。以未转染的 VSMC为对照 ,观察 10 -7mol/ L Ang 刺激对转染的 VSMC的 MAPK活性和 c- fos蛋白表达的影响。蛋白免疫沉淀和酶自身磷酸化率测定 c- Src激酶活性 ;髓鞘碱性蛋白 (MBP)底物磷酸化率测定 MAPK激酶活性 ;Western blot免疫印迹法测定 c- Src和 c- fos蛋白表达情况。结果 :转染不同浓度反义 c- SrcODNs的 VSMC c- Src蛋白含量呈浓度依赖性降低 ,0 .2 μmol/ L、0 .5 μmol/ L、1.0 μmol/ L和 2 .0 μmol/ L分别为对照的 6 8.2 %、34.7%、30 .3%和 15 .8% ,经方差分析具有显著性意义 (P<0 .0 1)。c- Src激酶活性也显著抑制 ;以 Ang 刺激经转染反义 c- Src ODNs的 VSMC,c- Src激酶活性增幅仅为对照组的 8.7% ;MAPK活性仅为对照的 1.6 % ;c- fos蛋白表达的增幅为对照组的 30 .0 %。结论 :Ang 可诱导 VSMC c- Src激活和细胞内信息转导 ,且 Ang 引起的 MAPK和 c- fos     

FIP200对呼吸道上皮细胞细胞粘附迁移的影响

目的研究FIP200（FAK—family interacting protein of 200 kDa）对细胞外基质成份诱导的呼吸道上皮细胞粘附和迁移的影响及其作用机制。方法通过纤连接蛋白（finbronectin，FN）诱导培养呼吸道上皮细胞的粘附迁移，以FIP200反义寡核苷酸（ODN）经脂质体转染细胞；以计数法测定细胞粘附率，以损伤实验测定细胞迁移速度，以Western blot检测FIP200和FAK蛋白表达水平：以免疫共沉淀检测FIP200和FAK结合情况和FAK磷酸化程度。结果与40mg／LFN组相比，经脂质体转染了FIP200反义寡核苷酸的气道上皮细胞粘附率和迁移速度明显增高，FIP200表达水平显著降低，FAK表达水平无明显变化，与FIP200结合的FAK显著降低，FAK酪氨酸磷酸化程度明显增高。结论内源性FIP200和FAK的结合抑制FAK的活化，从而抑制呼吸道上皮细胞的粘附和迁移，内源性FIP200作为FAK的抑制剂而存在；FN能够促使FAK和FIP200结合的解离而活化FAK，从而促进细胞粘附和迁移。     

阿司匹林对肿瘤坏死因子-α诱导的大鼠平滑肌细胞syndecan-4蛋白及p44/42丝裂原活化蛋白激酶表达的影响

目的观察阿司匹林(ASA)对肿瘤坏死因子-α(TNF-α)诱导的离体大鼠血管平滑肌细胞(VSMCs)syndecan-4蛋白及磷酸化p44/42丝裂原活化蛋白激酶(MAPK)表达的影响。方法体外培养大鼠胸主动脉VSMCs,分别用20 ng/mL TNF-α、1 mmol/L ASA、2 mmol/L ASA、20 ng/mL TNF-α+1 mmol/L ASA、20 ng/mL TNF-α+2 mmol/L ASA作用24 h,并设立相关对照组进行比较。采用MTS/PMS法确定VSMCs的增殖状态,Western blot蛋白免疫印迹法测定VSMCs中syndecan-4蛋白及磷酸化p44/42MAPK表达。结果 (1)与对照组比较,TNF-α组能显著刺激大鼠VSMCs的增殖(P<0.01),单独应用ASA对大鼠VSMCs的增殖无明显作用(P>0.05)。TNF-α联合ASA组与TNF-α组比较,联用能明显抑制TNF-α诱导的大鼠VSMCs增殖(P<0.01)。(2)与对照组比较,TNF-α组能显著刺激大鼠VSMCs中syndecan-4蛋白及磷酸化p44/42MAPK的表达(P<0.01),单独应用ASA对二者的表达无明显作用(P>0.05)。TNF-α联合ASA组与TNF-α组比较,联用可显著降低这两种蛋白的表达水平(P<0.05)。结论一定剂量的ASA可明显抑制TNF-α诱导的大鼠血管平滑肌细胞syndecan-4蛋白及磷酸化p44/42MAPK蛋白的表达。     

韧粘素反义寡核苷酸对血管平滑肌细胞增殖的抑制作用

目的探讨韧粘素 (TN)反义寡核苷酸 (ODN)对培养大鼠血管平滑肌细胞 (VSMC)增殖的抑制作用。方法采用细胞计数及快速竞争性逆转录聚合酶链反应 (RT -PCR)等方法 ,观察不同剂量 ( 5、10、2 0 μg/ml)TN反义ODN和正义ODN对体外培养大鼠VSMC的增殖以及TNmRNA表达水平的影响。　结果TN反义ODN可有效抑制大鼠血管平滑肌细胞的增殖 ,作用呈剂量依赖性 (反义组 5、10、2 0 μg/ml剂量细胞计数分别为 10 5/ml× ( 4 .99± 0 .2 1)、( 4 .0 8± 0 .14 )、( 2 .85± 0 .39) ,对照组为 10 5/ml× ( 5 .95± 0 .41) ,(P <0 .0 5 ) ;TN反义ODN亦可下调TNmRNA表达水平 (反义组 0 .32± 0 .0 5 ,对照组为 0 .5 2± 0 .0 8;P <0 .0 1) ;而TN正义ODN则无上述作用。　结论TN反义ODN可能通过下调TNmRNA表达水平而抑制血管平滑肌细胞的增殖。     

绿茶多酚抑制LDL诱导的血管平滑肌细胞增殖

观察绿茶多酚对低密度脂蛋白(LDL)刺激下离体大鼠胸主动脉血管平滑肌细胞增殖和p44/42MAPK表达的影响。体外培养大鼠胸主动脉血管平滑肌细胞,在LDL(100 μg/ml)刺激时,应用不同剂量的绿茶多酚作用24 h后,采用MTS/PES法确定血管平滑肌细胞的增殖状态。应用流式细胞术测定细胞周期,p44/42磷酸化抗MAPK抗体的蛋白免疫印迹法测定MAPK蛋白表达。与未经LDL处理的平滑肌细胞相比,LDL可以明显刺激大鼠血管平滑肌细胞的增殖,绿茶多酚对LDL刺激下的大鼠血管平滑肌细胞增殖具有明显的抑制作用(P<0.05),并呈现剂量依赖性。流式细胞术检测表明,绿茶多酚可以使LDL刺激下的大鼠血管平滑肌细胞大部分处于G0/G1期,与LDL组相比有明显差异(P<0.05)。LDL对磷酸化p44/42MAPK蛋白表达有显著的增强作用,此作用可被绿茶多酚抑制。绿茶多酚可明显抑制LDL刺激下的大鼠血管平滑肌细胞增殖和磷酸化p44/42丝裂素活化蛋白激酶的表达。     

丹参酮ⅡA对高糖培养的大鼠血管平滑肌细胞p38MAPK信号通路的影响

目的:观察丹参酮ⅡA对高糖诱导的血管平滑肌细胞增殖的影响,分析该作用与氧化应激以及丝裂原活化蛋白激酶p38(p38 mitogen-activated protein kinases,p38 MAPK)信号通路的关系。方法:取第3代原代培养的大鼠血管平滑肌细胞建立高糖诱导细胞增殖模型,实验分为正常对照组、高糖组和丹参酮ⅡA组,BrdU掺入法测定血管平滑肌细胞的DNA合成水平;黄嘌呤氧化酶法、硫代巴比妥酸法分别测定细胞培养上清中超氧化物歧化酶(SOD)、丙二醛(MDA)含量;免疫印迹法(Westernblot)检测磷酸化p38MAPK蛋白表达。结果:高糖诱导的血管平滑肌细胞呈快速增殖状态,在10~25 mmol.L-1间增殖效应呈剂量依赖性;丹参酮ⅡA能抑制高糖诱导的血管平滑肌细胞增殖,提高高糖培养的细胞中SOD水平,降低MDA含量,减少p-p38MAPK蛋白的表达。结论:丹参酮ⅡA抑制高糖诱导的血管平滑肌细胞增殖,这可能与其能减少血管平滑肌细胞中的氧自由基,抑制p38MAPK信号通路有关。     

蠲痹颗粒醇提物调控MAPK信号转导抑制T细胞活化效应机制的研究*

目的 探讨蠲痹颗粒醇提物（JBKL）在丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号转导通路中抑制T细胞活化的效应机制，揭示扶阳学派应用扶阳理论治疗类风湿关节炎（rheumatoid arthritis，RA）的现代免疫学机制。方法 噻唑蓝（MTT）比色法检测JBKL对伴刀豆球蛋白（Con A）、脂多糖（LPS）诱导T、B脾淋巴细胞增殖活性及细胞毒性的影响；建立体外抗CD3单克隆抗体（Anti-CD3 mAb）诱导的T细胞活化模型，MTT检测JBKL对T细胞体外活化增殖的影响，酶联免疫吸附法（ELISA）测定干扰素-γ（IFN-γ）、白细胞介素-17A（IL-17A）、IL-10和IL-6在细胞上清中的含量；蛋白质印迹法（Western blot）检测体外纯化CD4+ T细胞中ERK和p38蛋白的磷酸化水平。结果 JBKL在浓度30 μg/mL时显著抑制Con A、Anti-CD3 mAb和LPS诱导的T细胞和B细胞增殖功能；JBKL在浓度（3~30） μg/mL时对正常脾淋巴细胞无明显的细胞毒性作用，明显下调IFN-γ、IL-17A和促炎因子IL-6的含量，而促进IL-10的分泌；JBKL在浓度100 μg/mL时抑制MAPK信号通路，可降低纯化CD4+ T细胞磷酸化ERK和p38蛋白的表达。结论 JBKL阻碍T细胞介导的免疫反应，减少MAPK信号通路磷酸化ERK和p38蛋白表达，可能是其治疗RA的重要分子机制之一。     

Activated focal adhesion kinase involved in adhesion and migration of vascular smooth muscle cells stimulated by fibronectin

Objective To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth mu scle cells (SMCs) adhesion and migration stimulated by fibronectin.Methods Adhesion and migration of cultured SMCs were stimulated by different concentrati ons of fibronectin (FN), FAK and its phosphorylation were detected by immunoprec ipitation and Western blot. FAK antisense oligodeoxynucleotides (ODNs) were tra nsfected into SMCs by cationic lipid to investigate its modulatory effects on ty rosine phosphorylation. SMCs adhesion and migration were also measured by morph ological enumeration and modified Boyden Chambers, respectively.Results FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN. FAK phosphorylation were detected only at 20 μg/ml FN or more. FAK antisense ODNs were transfected efficiently by cationi c lipid and FAK phosphorylation was inhibited substantially. The SMCs migration rate in the 5-60μg/ml FN groups was reduced by 17.89%-27.67%. Cell migrat ion stimulated by FN at 10, 20, 40 and 60μg/ml were reduced by 23.26%, 21.6 3%, 19.31% and 17.88%, respectively (P&lt;0.05).Conclusions FAK phosphorylation and FAK-mediated signal transduction play important roles i n SMCs adhesion and migration stimulated by ECM. The process can be inhibited e ffectively by FAK antisense ODNs.     

绿茶多酚抑制LDL诱导的血管平滑肌细胞增殖

The proliferation and migration of vascular smooth muscle cells (VSMCs) is one of the major mechanisms of intimal thickening in atherosclerosis and post-angioplasty restenosis. Elevated plasma levels of low-density lipoprotein (LDL) have been implicated in the pathogenesis of atherosclerotic vascular diseases. The purpose of this study was to determine the effects of green tea polyphenols on the proliferation and p44/42 mitogen-activated protein kinase (MAPK) activity in rat VSMCs simulated by native LDL. Rat aortic VSMCs were cultured and treated with LDL (100 microg/ml) in the absence or presence of green tea polyphenols, and the cell proliferation was subsequently quantified by non-radioactive MTS/PES assay and the cell cycle analyzed by flow cytometry. The p44/42 MAPK activity was evaluated by immunoblotting using anti-p44/42 phospho-MAPK antibody. Compared with the cells without polyphenol treatment, the proliferation of the VSMCs induced by LDL was dose-dependently inhibited by green tea polyphenols (P<0.05), with more numerous cells in G(0)G(1) phase (P<0.05) as shown by flow cytometry analysis. LDL significantly enhanced the p44/42 MAPK activity, an effect obviously inhibited by green tea polyphenols (at 100 microg/ml). These results suggest that green tea polyphenols can inhibit high levels of LDL-induced proliferation of phosphorylated p44/42 MAPK expression in rat VSMCs. Green tea polyphenols may, therefore, offer vascular protection by inhibiting VSMC growth in response to hypercholesterolemia.     

绿茶多酚抑制晚期糖基化终产物诱导的大鼠血管平滑肌细胞增殖

目的 本研究观察绿茶多酚对晚期糖基化终产物(AGEs)刺激下离体大鼠胸主动脉血管平滑肌细胞增殖的影响。方法 体外培养大鼠主动脉血管平滑肌细胞(VSMCs)应用不同浓度的AGEs分别刺激VSMCs并设立对照组进行比较．将不同剂量的绿茶多酚与AGEs共同作用并设立对照组进行比较，采用非放射性的MTS／PES法确定血管平滑肌细胞的增殖状态。结果 与对照组相比，晚期糖基化终产物对血管平滑肌细胞增殖具有明显的刺激作用。在晚期糖基化终产物刺激下．绿茶多酚可明显抑制血管平滑肌细胞的生长。AGEs对p44／42MAPK磷酸化蛋白表达有显著的增强作用，此作用可被绿茶多酚抑制。结论 本研究提示绿茶多酚可抑制晚期糖基化终产物诱导的血管平滑肌细胞增殖及p44／p42 MAPK磷酸化蛋白的表达。     

绿茶多酚抑制晚期糖基化终产物诱导的大鼠血管平滑肌细胞增殖

目的本研究观察绿茶多酚对晚期糖基化终产物(AGEs)刺激下离体大鼠胸主动脉血管平滑肌细胞增殖的影响。方法体外培养大鼠主动脉血管平滑肌细胞(VSMCs)应用不同浓度的AGEs分别刺激VSMCs并设立对照组进行比较,将不同剂量的绿茶多酚与AGEs共同作用并设立对照组进行比较,采用非放射性的MTS/PES法确定血管平滑肌细胞的增殖状态。结果与对照组相比,晚期糖基化终产物对血管平滑肌细胞增殖具有明显的刺激作用。在晚期糖基化终产物刺激下,绿茶多酚可明显抑制血管平滑肌细胞的生长。AGEs对p44/42 MAPK磷酸化蛋白表达有显著的增强作用,此作用可被绿茶多酚抑制。结论本研究提示绿茶多酚可抑制晚期糖基化终产物诱导的血管平滑肌细胞增殖及p44/p42 MAPK磷酸化蛋白的表达。     

血管紧张素Ⅱ诱导血管平滑肌细胞肥厚过程中MAPK对细胞周期素依赖性激酶抑制因子的调节作用

OBJECTIVE: To investigate the role of mitogen-activated protein kinase (MAPK) in the regulation of cyclin-dependent kinase inhibitors (CDKI) in the process of vascular smooth muscle cell (VSMC) hypertrophy induced by angiotensin II stimulation. METHODS: The medial layer of male SD rat aorta was isolated for VSMC culture. After cultured in serum-free medium to arrest the cell growth, VSMCs were stimulated with angiotensin II (1x10(-6) mol/L) or/and phorbol myristate acetate (PMA, 20 nmol/L), with the cells cultured in serum-free medium serving as control. MAPK activity of the cells was assayed 90 min after stimulation with immunoprecipitation test, and the expression levels of CDKI p27, p57 and p21 were determined by Western blotting 6 and 24 h after stimulation respectively. RESULTS: Compared with the control level, MAPK activity of the VSMCs was up-regulated by 238% by treatment with angiotensin II alone which, however, did not inhibit p27 expression or induce VSMC proliferation. Costimulation with angiotensin II and PMA slightly inhibited p27 expression but VSMC proliferation was still not observed. CONCLUSION: MAPK pathway is an important channel for extracellular proliferative and hypertrophic signal transduction into the nucleus of VSMCs.     

Proliferation of renal mesangial cells induced by very low density lipoprotein is mediated by p42/44 mitogen activated protein kinase

Background The plasma concentration of very low density lipoprotein （VLDL） is negatively correlated to renal function in glomerular diseases. Effects of VLDL on renal function have been partially attributed to the proliferation of mesangial cells. This study examined the potential role of the p42/44 mitogen activated protein kinase （MAPK） in mesangial cell proliferation induced by VLDL. Methods Mesangial cells were treated with VLDL at different concentrations or for different time. The cell cycle of the mesangial cells was analyzed by Xl-r assay and flow-cytometry; MAPK activity was also assayed. In some experiments, cells were treated with VLDL together with or without 0.1 pmol/L PD 98059. Results Ten to 500 μg/ml VLDL stimulated the proliferation of mesangial cells cultured in vitro in a concentration-dependent manner. The effect was associated with an increase in p42/44 MAPK activity. Increased proliferation of mesangial cells by VLDL was significantly attenuated by PD98059, a specific p42/44 MAPK inhibitor. Conclusion These results indicate that the p42/44 MAPK pathway is an important regulator of mesangial cell proliferation and of renal functions.     

肝硬化大鼠血管平滑肌细胞Gq蛋白信号转导功能的研究

目的 为观察肝硬化大鼠动脉血管平滑肌细胞对血管紧张素Ⅱ（ＡＴ－Ⅱ）是否存在受体后Ｇｑ蛋白信号转导的异常。方法 采用总胆管结扎制血大鼠肝硬化模型，分离主动脉血管平滑肌细胞后进行细胞培养，并予免疫组化鉴定。分别采用细胞感应微生理探测系统、三磷酸肌醇（ＩＰ３）分析法、Ｗｅｓｔｅｒｎ印迹法检测血管平滑肌细胞ＡＴ－Ⅱ受体介导的细胞外液酸化速率（ＥＣＡＲ）、ＩＰ３生成、Ｐ４２／４４丝裂原激活的蛋白激酶（ＭＡＰ