丝裂原活化蛋白激酶在人冠状动脉平滑肌细胞向三维纤维蛋白胶迁移中的作用

目的:观察三维纤维蛋白(Fb)胶对体外培养的人冠状动脉平滑肌细胞(HCASMC)的趋化作用及其信号转导机制.方法:采用胶原酶消化法培养HCASMC,倒置相差显微镜观察其向三维Fb胶中迁移的能力及ERK、p38、JNK信号通路对其迁移能力的影响.Western blot检测Fb对HCASMC p-ERK、p-p38和p-JNK表达的调控.结果:向Fb胶中迁移的HCASMC呈长梭型,细胞数量增加时形成环形管腔样结构.纤维蛋白原(Fg)浓度为0.8 g/L～6.4 g/L时,HCASMC向胶中迁移的数量呈浓度依赖性增加,并随培育时间的延长逐渐增多.用Western Blot分析显示Fb以时间依赖性方式诱导ERK、p38及JNK活化,三者的选择性抑制剂PD98059 50 μmol/L、SB20358010 μmol/L及SP600125 20 μmol/L可分别抑制其活化,但对HCASMC向胶中迁移的抑制能力不尽相同.PD9805950 μmol/L对HCASMC迁移无明显影响,而SB203580 10 μmol/L和SP600125 20 μmol/L均可抑制HCASMC向Fb胶迁移,且后者抑制作用更强.结论:Fb胶通过激活细胞JNK和p38(而不是ERK信号通路)促进HCASMC向Fb胶中迁移,这种机制可能在动脉粥样硬化血栓形成及再狭窄过程中发挥重要作用.     

p38丝裂原活化蛋白激酶在不同细胞内定位的研究

用共聚焦显微镜对不同原代培养细胞的ｐ３８丝裂原活化蛋白激酶（ｍｉｔｏｇｅｎ－ａｃｔｉｖａｔｅｄ ｐｒｏｔｅｉｎ ｋｉｎａｓｅ,ＭＡＰＫ）进行定位,以探讨ｐ３８激活后入核在不同的细胞中是否具有普遍性。发现与单核细胞相似,未受刺激静止的心肌细胞、平滑肌细胞和内皮细胞的ｐ３８荧光强度呈弥散性分布;脂多糖（ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ,ＬＰＳ）刺激后,细胞核区荧光强度均明显增加,胞浆荧光强度均降低     

G蛋白偶联受体激活丝裂原活化蛋白激酶的机理

多种Ｇ蛋白偶联受体的均能激活丝裂原活化蛋白激酶。Ｇｉ蛋白偶联受体主要通过其βγ亚基,依赖Ｒａｓ蛋白途径;在大多数哺乳类细胞中Ｇｓ蛋白偶联受体通过ＰＫＡ途径抑制Ｒａｓ依赖的ＭＡＰＫ活化,但在ＣＯＳ－７细胞,Ｇｓ蛋白偶联受体通过ＰＫＡ途径使表达的ＭＡＰＫ活化;Ｇｑ蛋白偶联受体主要通过ＰＫＣ途径依赖或非依赖于Ｒａｓ使ＭＡＰＫ活化。ＭＡＰＫ信号途径中ＥＧＦ受体,酪氨酸激酶及调节蛋白Ｓｈｃ等联级反应蛋白可能     

丝裂原活化蛋白激酶激活蛋白激酶(MK)研究进展

丝裂原活化蛋白激酶(MAPK)信号通路介导多种重要的细胞生理反应.对下游蛋白激酶的磷酸化是MAPK家族成员发挥生理作用的重要方式.在MAPK的下游存在3个结构上相关的MAPK激活蛋白激酶(MAPKAPKorMK),即MK2,MK3和MK5.在被MAPK激活后,MK可将信号传递至细胞内不同靶标,从而在转录和翻译水平调节基因表达,调控细胞骨架和细胞周期,介导细胞迁移和胚胎发育.最近,在基因敲除研究的基础上,不同MK亚族成员之间的功能区分已经逐渐明晰,使我们对于MK的认识有了长足的进步.     

丝裂原活化蛋白激酶信号通路相关研究

丝裂原活化蛋白激酶信号通路是生物体内重要的信号转导系统之一,参与介导细胞生长、发育、分裂、分化等多种生理反应过程。在哺乳动物细胞中存在5个MAPK亚族,分别是ERK1／2、JNK、p38、ERK3／4和ERK5。MAPK通常定位于细胞质中,受激活后移行进入细胞核,并产生相应的生理作用。     

丝裂原活化蛋白激酶信号通路与受精

受精是雌雄生殖细胞相互融合形成合子的过程 ,是生命的开端。有性生殖的生命体通过受精 ,使基因组由生殖细胞的单倍体恢复为体细胞的二倍体 ;同时 ,精子与卵子的结合激活了以母源信息形式贮存在卵子中的发育程序 ,使卵子由受精前的生命活动抑制状态转变为活跃状态 ,即活化卵子。受精涉及到两个不同个体间异源细胞的识别、融合、信号转导和分裂周期调节 ,因此久已成为发育生物学、细胞生物学和生物化学的重要研究课题。多种蛋白激酶参与对受精过程的调节。作者和其他人的研究均发现丝裂原活化蛋白激酶 (ｍｉｔｏｇｅｎａｃｔｉｖａｔｅｄｐｒ…     

β肾上腺素受体的丝裂原活化蛋白激酶信号途径

β肾上腺素受体（β－AR）除了通过经典的信号途径介导细胞生物功能外,还可以激活丝裂原活化蛋白激酶（MAPK）信号途径,活化后的MAPK参与调节多种细胞生物学活动。然而,将β-AR与MAPK信号联系起来的分子机制还需要进一步的研究。     

丝裂素活化蛋白激酶参与内皮素刺激的兔主动脉平滑肌细胞增生

内皮素（ｅｎｄｏｔｈｅｌｉｎ,ＥＴ）是已知的体内活性最强的缩血管物质,其缩血管作用由Ｇ蛋白偶联受体所介导。但ＥＴ强大的促血管平滑肌细胞（ＶＳＭＣ）增生效应的机理尚未完全阐明。本研究选用培养的兔胸主动脉ＶＳＭＣ,探讨丝裂素活化蛋白激酶（ＭＡＰＫ）在ＥＴ促细胞增生中的作用。结果表明：ＥＴ－１呈时间和浓度依赖性地促进细胞摄取￣３Ｈ－ＴｄＲ和激活ＭＡＰＫ,此作用可被蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴＰ）,Ｈ－７和ＥＴ＿Ａ受体拮抗剂ＢＱ１２３所抑制,但不被酪氨酸激酶抑制剂ＨｅｒｂｉｍｙｃｉｎＡ（Ｈｅｒｂ）所抑制,用ＰＫＣ激动剂ＰＭＡ（Ｐｈｏｒｂｏｌｍｙｒｉｓｔａｔｅａｃｅｔａｔｅ）预处理ＶＳＭＣ,使其ＰＫＣ活性下调,可显著减弱ＥＴ－１对ＭＡＰＫ的激活能力。本结果提示：（１）ＭＡＰＫ参与ＥＴ－１所致的ＶＳＭＣ增生;（２）ＥＴ－１促细胞增生与激活ＭＡＰＫ的作用是由ＥＴ＿Ａ受体和ＰＫＣ介导的。     

胰岛素影响自发性高血压大鼠血管平滑肌细胞增殖及肌动蛋白分布的丝裂原激酶的机制探讨

血管平滑肌细胞增殖的同时伴有细胞内肌动蛋白的改变,这种改变受PKC-MAPK信号转导途径调控,但目前机制尚不清楚。为探讨胰岛素对PKC-MAPK信号转导途径参与调控血管平滑肌细胞增殖及细胞内肌动蛋白分布的影响,本研究用PKC抑制剂预处理SHR在鼠体外培养的血管平滑肌细胞,观察预处理的血管平滑肌细胞经胰岛素刺激后细胞内DNA的合成、MAPK的活性、表达及细胞内肌动蛋白的分布。发现,胰岛素刺激后可使血管平滑肌细胞增殖,同时伴有[^3H]TdR掺入增加、MAPK活性及表达与对照组比较明显升高。这些作用可被PKC抑制剂阻断。胰岛素在刺激血管平滑肌细胞增殖的同时也使细胞内肌动蛋白重新分布,这一效应也可被PKC抑制剂阻断。 上述结果提示,胰岛素使血管平滑肌细胞增殖的效应可能与MAPK信号转导途径有关。     

Minimally modified low density lipoproteins induce aortic smooth muscle cell proliferation via the activation of mitogen activated protein kinase

We have investigated the effects of modifying LDL by Cu++ and various hemoglobin preparations on aortic smooth muscle cell proliferation and on the activation of mitogen activated protein kinase. We found that at very low concentrations (10 g/ml), LDL modified by all of the above agonists markedly stimulated cell proliferation (5–10 fold). This was accompanied by a 2–3 fold stimulation in mitogen activated protein kinase (MAPK) activity. We conclude that modification of LDL under situations that are closer to those found in vivo (i.e. hypoxic conditions), may involve the activation of MAPK as a common biochemical mechanism of action. This in turn, contributes to aortic smooth muscle cell proliferation.     

白介素-10抑制TNF-α诱导的血管平滑肌细胞增殖

研究观察了重组人白介素 10 (rhIL 10 )对肿瘤坏死因子 (TNF α)刺激的离体大鼠胸主动脉血管平滑肌细胞增殖、细胞周期及对p4 4 /p4 2丝裂素活化蛋白激酶的影响。实验培养大鼠主动脉血管平滑肌细胞 ,采用MTS/PES法确定血管平滑肌细胞 (vascularsmoothmusclecells,VSMCs)的增殖状态 ;应用流式细胞术测定细胞周期 ;利用p4 4 / 4 2磷酸化抗MAPK抗体的蛋白免疫印迹法测定MAPK蛋白表达。结果显示 :( 1)TNF α处理组与对照组相比 ,TNF α对VSMC增殖具有明显的刺激作用 (P <0 0 5 )。rhIL 10单独应用对VSMCs生长没有影响 (P >0 0 5 )。在TNF α刺激下 ,低至 10ng/ml的rhIL 10可抑制VSMCs的生长 (P <0 0 5 )。流式细胞术测定的结果显示 ,rhIL 10分别可使TNF α作用下的VSMC大部分处于G0 /G1期 ,与对照组相比有明显差异 (P <0 0 1)。 ( 2 )TNF α对p4 4 /p4 2MAPK蛋白表达有显著的增强作用 ,此作用可被rhIL 10抑制。结果提示 ,rhIL 10可抑制TNF α诱导的VSMC增殖及p4 4 /p4 2丝裂素活化蛋白激酶的表达     

Immunodetection of activated mitogen-activated protein kinase in vascular tissues

Mitogen-activated protein kinase (MAPK) is a key modulator of cytoplasmic-nuclear signal transmission, and for this reason measurement of MAPK activity has become very popular. Monitoring of MAPK activity may be particularly relevant to the cardiovascular system where it has already been shown that the stimulation of cardiomyocytes and smooth muscle cells by stretch and by growth factors activates MAPK. Since both growth factors and mechanical stress are causal agents for certain pathologies, enhanced MAPK activity may be a good predictor of disease progression. A variety of methods have been designed to measure the activation of this enzyme including an in vitro assay coupled to either gel electrophoresis or binding to P81 paper, an activity gel assay to detect p42/44 isoforms and, more recently, monitoring MAPK phosphorylation using immunoblot detection. The validity of the latter method is based on the correlation between MAPK activity and the degree of phosphorylation. The antibodies have also been of use in the detection of MAPK translocation in cell monolayers. In this report, we discuss the advantages and disadvantages of all MAPK detection methods and demonstrate an additional application for the MAPK antibodies using an in vitro restenosis model. In addition, the utility of MAPK measurements to smooth muscle pathophysiology and vascular injury (as a predictor of injury) has been assessed.     

蛋白激酶C和蛋白酪氨酸激酶介导脂多糖及细胞因子诱导大鼠血管平滑肌细胞一氧化氮的生成

采用Spprague-Dawley大鼠胸主动脉中膜、外膜和培养的血管平滑肌细胞(VSMCs)作材料,鉴定不同类型的血管组织经炎性介质刺激后其一氧化氮(NO)的产生来源,闻明蛋白激酶C(PKC)和蛋白酪氨酸激酶(PTK)介导大鼠VSMCs生成NO的调控机制。大鼠VSMCs经脂多糖(LPG)和细胞因子(TNF-α,IL-1β)处理后,以剂量依赖方式促进NO释放。采用Western Blot证实经刺激的VSMCs伴有iNOS表达上调。进一步实验表明PKC和PTK参与LPS和细胞因子诱导NO生成的胞内信号转导。用PKC抑制剂H7与VSMCs共培育,H7能明显减少LPS、TNF-α和IL-1β诱导细胞NO的形成。白屈菜赤碱亦可抑制NO的生成,但HAl004对VSMCs的NO生成无抑制作用,提示PKC参与NO的生成与调控。PTK抑制剂genistein和tyrphostin AG18均能抑制由LPS、TNF-α和IL-1β引发VSMCs释放NO,同时伴iNOS蛋白表达下调,而PKC抑制剂不能阻断iNOS的表达。上述观察结果提示,PKC介导LPS和细胞因子诱导细胞合成NO可能是通过iNOS翻译后加工;而PTK则以上调iNOS表达而促增NO生成。     

Effect of cell passage and density on protein kinase G expression and activation in vascular smooth muscle cells

It has been shown that rat aortic smooth muscle cells (AoSMCs) lost PKG-I expression when propagated repetitively or grown at low densities. Conversely, AoSMCs isolated from PKG-I deficient mice are indistinguishable from those isolated from normal mice in morphology and growth characteristics. In this study, human AoSMCs were grown from passage 9 (p9) to passage 15 (p15) and rat AoSMCs were isolated and cultured from p1 through p15. Western blotting and immunofluorescence microscopy showed little difference in PKG-I expression among different passages. Next, rat AoSMCs of p4 were grown and harvested at different cell densities. Western blotting again showed little difference among cells seeded or harvested at different densities. To test the effect of cell passage on PKG-I activation, rat AoSMCs of p4 and p11 were treated with cGMP and analyzed by Western blotting for phosphorylated vasodilator-stimulated phosphoprotein (P-VASP). The results showed that p4 had higher level of PKG-I activation than p11.     

Role of integrin-linked kinase in vascular smooth muscle cells: regulation by statins and angiotensin II

Our goal was to characterize the role of integrin-linked kinase (ILK) in vascular smooth muscle cells (VSMC), which play a crucial role in atherogenesis. Transfection of VSMC with wild-type and dominant-negative ILK cDNA constructs revealed that ILK mediates migration and proliferation of VSMC but has no effect on VSMC survival. The pro-atherogenic mediator angiotensin II increases ILK protein expression and kinase activity while statin treatment down-regulates ILK in VSMC. Functionally, ILK is necessary for angiotensin II-mediated VSMC migration and proliferation. In VSMC transduced with dominant-negative ILK, statins mediate an additive inhibition of VSMC migration and proliferation, while transfection with wild-type ILK is sufficient to overcome the inhibitory effects of statin treatment on VSMC migration and proliferation. In vivo, ILK is expressed in VSMC of aortic sections from wild-type mice where it is down-regulated following statin treatment and up-regulated following induction of atherosclerosis in apoE-/- mice. These data identify ILK as a novel target in VSMC for anti-atherosclerotic therapy.     

Aspirin-induced AMP-activated protein kinase activation regulates the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effects via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.