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。     

MAPK对胰岛素介导的人血管平滑肌细胞PKCα的影响

目的：在胰岛素的干预下,观察ＭＡＰＫ反义寡核苷酸（ＯＤＮｓ）对人血管平滑肌细胞（ＶＳＭＣ）增殖及ＰＫＣα表达的影响。方法：３ＨＴｄＲ掺入法检测ＶＳＭＣ增殖,逆转录ＰＣＲ、免疫组织化学法检测ＰＫＣα表达。结果：反义ＯＤＮｓ 处理的细胞可显著抑制胰岛素诱导的ＶＳＭＣ的ＤＮＡ合成,ＯＤＮｓ 的上述作用与降低ＶＳＭＣ内ＰＫＣα基因表达有关。结论：胰岛素刺激人ＶＳＭＣ增殖可被ＭＡＰＫ反义寡核苷酸所抑制,可能存在有关胰岛素ＰＫＣＭＡＰＫ激活途径     

缺氧预处理对乳鼠心肌细胞蛋白激酶C活性的影响

在培养的乳鼠心肌细胞缺氧／复氧模型上,观察了缺氧预处理的细胞保护作用及其对细胞蛋白激酶Ｃ活性和蛋白磷酸化的影响。结果表明,ＡＰＣ可减轻心肌细胞的Ｈ／Ｒ损伤;提高细胞存活率,减少细胞脂质过氧化产物生成及细胞内乳酸脱氢酶和蛋白质漏出。模拟ＡＰＣ的短暂缺氧显著激活ＰＫＣ,使心肌细胞内分子量为６６ｋＤ和３１ｋＤ的蛋白条带＾３２Ｐ掺入增加;ＰＫＣ抑制剂Ｈ７完全消除ＡＰＣ对心肌细胞的保护作用,并抑制了短暂缺氧     

pp60c-src在血管平滑肌细胞内丝裂原活化蛋白激酶激活中的作用

观察ｐｐ６０ｃ－ｓｒｃ在血管紧张素Ⅱ（ＡｎｇⅡ）诱导血管平滑肌细胞（ＶＳＭＣｓ）内丝裂原活化蛋白激酶（ＭＡＰＫ）激活中的作用,以了解ＡｎｇⅡ促ＶＳＭＣｓ增殖的信号转导过程。将合成的反义ｃ－ｓｒｃ寡脱氧核苷酸（ｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅ－ｏｔｉｄｅｓ,ＯＤＮｓ）以脂质体包裹转染培养的大鼠ＶＳＭＣｓ,用Ｗｅｓｔｅｒｎ印迹测得细胞裂解液中ｐｐ６０ｃ－ｓｒｃ含量明显下降,免疫沉淀方法测得ｐｐ６０ｃ－ｓ     

用显微切割技术和催化信号扩增法检测何杰金病瘤细胞（H／RS）中的 …

为明确何杰金病（Ｈｏｄｇｋｉｎ’ｓ ｄｉｓｅａｓｅ,ＨＤ）瘤细胞中是否存在人巨细胞病毒（ｈｕｍａｎ ｃｙｔｏｍｅｇａｌｏｖｉｒｕｓ,ＨＣＭＶ）感染,采用显微切割技术结合ＰＣＲ方法检测ＨＤ组织及其瘤细胞Ｈｏｄｇｋｉｎ／Ｒｅｅｄ－Ｓｔｅｒｎｂｅｒｇ（Ｈ／ＲＳ）中的ＨＣＭＶ核酸;采用免疫组织化学催化信号扩增（ｃａｔａｌｙｓｅｄ ｓｉｇｎａｌ ａｍｐｌｉｆｉｃａｔｉｏｎ,ＣＳＡ）法检测ＨＤ中ＨＣＭＶ的立即     

预处理保护作用的普遍性及其机理探讨

本工作分别在大鼠的多种器官上观察到缺血预处理（ＰＣ）对缺血／再灌注损伤的保护作用具有器官普遍性,其对血管床的保护是其器官保护的机制之一;在体外培养的乳鼠（兔）心肌细胞及大鼠（兔）胸主动脉血管平滑肌细胞等多种细胞等多种细胞上观察到蛋白激酶Ｃ（ＰＫＣ）激活及ＰＫＣ介导的蛋白磷酸化增强是ＰＣ细胞保护的共有环节。碱性成纤维细胞生长因子可以模拟ＰＣ的细胞保护作用,提示药物预处理对于防治缺血有关的疾病可能上人     

抗原捕获聚合酶链式反应（AC－PCR）直接分型检测单纯疱疹病毒

用抗单纯疱疹病毒（ＨＳＶ）型共同性ｇＣ和ｇＤ羊克隆抗体（ＭｃＡｂ）,包被即Ｅｐｐｅｎｄｏｒｆ管,捕捉ＨＳＶ,同时加入３个引物：一个是ＨＳＶ─１／ＨＳＶ─２型共同性上游引物,另两个分别是ＨＳＶ─１和ＨＳＶ─２型特异性下游引物。借此建立了能直接分型检测ＨＳＶ的抗原捕获聚合酶链式反应（ＡＣ─ＰＣＲ）。ＨＳＶ─１的扩增产物为４７７ｂｐ,ＨＳＶ─２的为３９９ｂｐ两型病毒经ＡＣ─ＰＣＲ扩增后产生分子量不同的ＤＮＡ片段,致使ＡＣ─ＰＣＲ能直接分型检测ＨＳＶ。ＨＳＶ─１和ＨＳＶ─２扩增产物的克隆和序列分析表明,本方法特异性好。用本法检测Ｂａｌｂ／ｃ幼鼠中枢神经系统ＨＳＶ感染的脑标本,进一步证实本方法不仅敏感、特异,而且分型准确。     

巨噬细胞免疫调变信号——PKA与PKC对MAPK信号通路的调节

以前的研究工作表明,细菌脂多糖（ＬＰＳ）可以调变抑制性巨噬细胞为增强Ｔ、Ｂ淋巴细胞及ＮＫ细胞活性,同时又能保持或增强其抗肿瘤效应。忆报道了在这一复杂的免疫调变过程中伴随有蛋白激酶Ｃ（ＰＫＣ）和促分裂原活化蛋白激酶（ＭＡＰＫ）信号转导通路的激活。为了探索免疫调变过程中其他信号对ＭＡＰＫ通路的影响,以ＬＰＳ调变小鼠腹腔抑制性巨噬细胞为模型,研究了ｃＡＭＰ／ＰＫＡ和佛波酯（ＰＭＡ）／ＰＫＣ信号对ＭＡＰＫ     

血管紧张素Ⅱ诱导培养的成年大鼠心肌细胞凋亡及其细胞机制

研究血管紧张素Ⅱ（ＡｎｇｉｏｔｅｎｓｉｎⅡ,ＡｎｇⅡ）诱导培养的成年大鼠心肌细胞（Ａｄｕｌｔｒａｔｖｅｎｔｒｉｃｕｌａｒｍｙ－ｏｃｙｔｅｓ,ＡＲＶＭ）凋亡。酶灌流消化法分离培养ＡＲＶＭ,不同处理后,光镜观察形态改变,琼脂糖凝胶电泳定性分析ＤＮＡ降解程度。结果发现培养的ＡＲＶＭ经１０μｍｏｌ／ＬＡｎｇⅡ处理４８ｈ后,大部分细胞变圆,胞浆浓缩;电泳显示核酸断裂片段“梯形”结构,上述改变在７２ｈ更为明显。上述作用可被氯沙坦、维拉帕米和ｓｔａｕｒｏｓｐｏｒｉｎｅ所取消。结果表明,ＡｎｇⅡ诱导培养的ＡＲＶＭ凋亡由ＡＴ１受体介导、细胞内钙升高和ＰＫＣ激活起重要作用     

内皮素受体反义寡聚核苷酸对内皮素受体基因表达及血管平滑肌细胞增殖的影响

报道了内皮素Ａ型受体反义寡聚核苷酸（ＯＤＮｓ）对大鼠血管平滑肌细胞（ＶＳＭＣ）增殖及内皮素受体基因表达的影响．~３Ｈ－ＴｄＲ参入结果显示,内皮素Ａ型受体反义ＯＤＮｓ处理细胞可显著抑制内皮素诱导的ＶＳＭＣ的ＤＮＡ合成,反转录－ＰＣＲ及受体结合实验结果表明,ＯＤＮｓ的上述作用与降低ＶＳＭＣ内皮素Ａ型受体基因表达活性有关．     

碱性成纤维细胞生长因子对乳鼠心肌细胞缺氧复氧损伤的影响

本实验在培养的乳鼠心肌细胞缺氧复氧损伤模型上观察碱性纤维细胞生长因子对Ａ／Ｒ损伤及蛋白激酶活性的影响,以探讨ｂＥＧＦ作为药物预处理心肌保护的可行性及其机理。结果表明,ｂＥＧＦ预处理呈浓度依赖地提高Ａ／Ｒ后心肌细胞存活率,减少细胞内ＡＴＰ消耗及胞浆乳酸脱氢酶漏出;ＰＫＣ抑制剂Ｈ７完全消除ｇＦＧＦ的上述保护作用;实验结果表明,ｂＦＧＦ可以直接激活心肌细胞ＰＫＣ,其激活时相的变化与缺氧预处理者相近,提示     

Thrombin signal transduction mechanisms in rat vascular smooth muscle cells. Calcium and protein kinase C-dependent and -independent pathways.

Sustained generation of alpha-thrombin and its breakdown forms at sites of thromboses has focused attention on the roles thrombin may play in vascular responses to thrombosis and injury. We have previously shown that alpha-thrombin stimulates many growth signals in cultured rat aortic smooth muscle cells (VSMC). To characterize thrombin growth mechanisms, we studied the effects on cultured VSMC of gamma-thrombin (catalytically active with obstructed anion-binding site required for clotting activity) and D-phenylalanyl-L-prolyl-L-arginine chloromethylketone-alpha-thrombin (catalytically inactive with intact anion-binding exosite) on cultured VSMC. Either derivative alone failed to increase growth, but in combination at 130 nM each, they caused a 75 +/- 5% increase in protein synthesis, similar to that observed with alpha-thrombin. This increase in protein synthesis was related to activation of protein kinase C (PKC) and Na+/H+ exchange, because only in combination could the derivatives increase phosphorylation of a 76,000-dalton PKC substrate and alkalinize the cells. Activation of PKC was correlated with a synergistic effect of the derivatives on diacylglycerol formation at 2 min (maximum, 55 +/- 1% combined increase vs. 24 +/- 9% and 4 +/- 4% individual increases with gamma- and D-phenylalanyl-L-prolyl-L-arginine chloromethylketone-alpha-thrombin alone, respectively, p less than 0.05). The derivatives stimulated PKC without increasing inositol trisphosphate, intracellular Ca2+, or expression of the protooncogene, c-fos. Thus, thrombin stimulation of Na+/H+ exchange, diacylglycerol formation, and growth of VSMC can be distinguished from thrombin mobilization of [Ca2+]i and induction of c-fos mRNA. These data indicate the presence of more than one mechanism for thrombin-mediated signaling events in cultured VSMC. Our results also suggest that various thrombin forms retained in clots may have significant effects on VSMC growth and function.     

Transforming growth factor-beta: Signal transduction via protein kinase C in cultured embryonic vascular smooth muscle cells

Summary Transforming growth factor-beta (TGF-β), an ubiquitous regulatory peptide, has diverse effects on the differentiation and behavior of vascular smooth muscle cells (VSMC). However, the molecular mechanism through which TGF-α exerts its effects remains obscure. We investigated the phosphoinositide/protein kinase C [PKC] signaling pathway in the action of TGF-β on cultured embryonic avian VSMC of differing lineage: a) thoracic aorta, derived from the neural crest; and b) abdominal aorta, derived from mesenchyme. The second messenger responsible for activation of PKC is sn-1,2-diacylglycerol [DAG]; TGF-β increased the mass amounts of DAG in the membranes of neural crest-derived VSMC concurrent with translocation of PKC from the soluble to the membrane fraction, but TGF-β had no effect on the DAG or PKC of mesenchyme-derived VSMC. TGF-β potentiated the growth of platelet-derived growth factor (PDGF)-treated, neural crest-derived VSMC; but abolished PDGF-induced growth of mesenchymal cells. It is concluded that molecular and functional responses of VSMC to TGF-β are heterogeneous and are functions of the embryonic lineage of the VSMC.     

The protective effects of puerarin in cardiomyocytes from anoxia/reoxygenation injury are mediated by PKCε

Puerarin is an isoflavone isolated from traditional Chinese medicine Ge‐gen (Radix Puerariae). Clinical studies have confirmed the cardioprotective effects of puerarin; however, the mechanisms underlying these effects are still unclear. On the basis of previous findings, we hypothesized that puerarin protects cardiomyocytes from ischemia–reperfusion injury via the protein kinase C epsilon (PKCε) (a critical cardioprotective protein) signalling pathway. Neonatal rat primary cardiomyocytes were preconditioned with puerarin or puerarin plus εV1‐2, a selective PKCε inhibitor, prior to anoxia/reoxygenation (A/R) treatment. Western blot analysis showed that expression and activity of PKCε protein in puerarin preconditioned group were both increased compared with the control or A/R group. Subsequent assays showed that preconditioning with puerarin could increase the viability of neonatal rat primary cardiomyocytes treated with A/R, decreased the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, cell necrosis and apoptosis induced by A/R injury. However, the protective effects of puerarin completely disappeared in the group pretreated with puerarin plus εV1‐2. Thus, for the first time, we revealed the protective effects of puerarin in cardiomocytes from anoxia/reoxygenation injury are mediated by PKCε. Copyright © 2014 John Wiley & Sons, Ltd.     

The role of actin depolymerizing factor in advanced glycation endproducts-induced impairment in mouse brain microvascular endothelial cells

Orexin-A, which is an endogenous neuropeptide, is reported to have a protective role in ischemic stroke. High-concentration glutamic acid (Glu) induced by hypoxia injury in ischemic stroke can be inhibited by glial glutamate transporter GLT-1 which is only expressed in astroglia cells. A previous study reported that Orexin-A may regulate GLT-1 expression. However, the role of orexin-A in the regulation of GLT-1 in ischemic stroke still remains unclear. In this study, we aimed to investigate the effect and the underlying mechanism of orexin-A on Glu uptake in astrocytes in vitro and this effect on protecting the neurons from anoxia/hypoglycemic injury. The expression of GLT-1 significantly increased in the astrocytes with orexin-A treatment under anoxia/hypoglycemic conditions, promoting the uptake of Glu and inhibiting the apoptosis of co-cultured cells of astrocytes and neurons. However, these effects were significantly weakened by treatment with orexin-A receptor 1 (OX1R) antagonist. Orexin-A significantly up-regulated the expressions of PKCα and ERK1/2 under anoxia/hypoglycemic conditions in astrocytes, whereas the OX1R antagonist markedly reversed the effect. Furthermore, PKCα or ERK1/2 inhibitor significantly constrained the GLT-1 expression in astrocytes and facilitated the apoptosis of co-cultured cells, and GLT-1 overexpression could reverse those effects of PKCα or ERK1/2 inhibitor. Taken together, orexin-A promoted the GLT-1 expression via OX1R/PKCα/ERK1/2 pathway in astrocytes and protected co-cultured cells against anoxia/hypoglycemic injury.     

碳酸锂通过蛋白激酶C/丝裂原活化蛋白激酶信号调节海马神经元延迟整流钾通道

碳酸锂可以用于治疗创伤和神经退行性疾病导致的脑部损伤.研究表明其保护效应与蛋白激酶C(PKC)和胞外信号调节激酶(ERK)有关.研究表明PKC激动剂PDBu可以抑制延迟整流钾通道(IK)电流并使其激活电压曲线向超极化方向移动.碳酸锂(50 μmol/L)可以抑制PDBu的反应.进一步的研究表明,预先加入MEK/ERK抑制剂U0126(20 μmol/L),碳酸锂不能逆转PDBu对,IK的作用.因此,PKC和丝裂原活化蛋白激酶(MAPK)/ERK级联反应通路可能在钾离子通道的磷酸化调节中起作用.另外,AC-cAMP和GC-cGMP的交互作用也可能参与碳酸锂对PKC激活作用的调节,成为其神经保护作用的机制之一.     

The various effects of fractionated oxidized low density lipoproteins on the growth of smooth muscle cells in culture

The effects of fractionated oxidized low density lipoproteins (oxidized LDL) on the growth of vascular smooth muscle cells (VSMC) and their relationship to the formation of lysophosphatidylcholine (lyso-PC) as well as the activation of protein kinase C (PKC) were studied. VSMC were isolated from porcine aorta by explant culture. LDL was isolated from porcine blood by sequential ultracentrifugation and oxidized LDL was obtained by incubating LDL with 5 µM CuSO₄ at 37° C for various lengths of time. Our results showed that LDL oxidized for 12 h and eluted from fast protein liquid chromatography at 43 min inhibited the growth of VSMC, and that LDL oxidized for longer than 48 h and eluted at 48 min stimulated the growth of VSMC. The formation of lyso-PC in the oxidized LDL correlated well with its stimulatory effect, suggesting that lyso-PC is responsible for the mitogenic effect of oxidized LDL. This stimulatory effect of oxidized LDL was inhibited by staurosporine, a PKC inhibitor. Treatment with oxidized LDL increased the activity of membrane PKC, but it decreased that of cytosolic PKC, suggesting the translocation of PKC from cytosol to the membrane in the presence of oxidized LDL. These results suggested that the oxidized LDL-stimulated VSMC growth was mediated by the formation of lyso-PC and the activation of PKC.     

Enhancement by alpha-tocopheryl hemisuccinate of nitric oxide production induced by lypopolysaccharide and interferon-gamma through the upregulation of protein kinase C in rat vascular smooth muscle cells.

The effect of alpha-tocopheryl hemisuccinate (TS) on lipopolysaccharide (LPS)/interferon-gamma (IFN)-induced nitric oxide production in rat vascular smooth muscle cells (VSMC) was examined. The LPS/IFN-induced NO production was enhanced by TS but not by the other alpha-tocopherol (alpha-T) derivatives alpha-tocopheryl acetate (TA) and alpha-tocopheryl nicotinate (TN), or alpha-T itself. alpha-T, TA and TN inhibited the enhancement by TS of LPS/IFN-induced NO production. The enhancing effect of TS was observed in the presence of LPS, but not IFN, suggesting that TS participates in the LPS-stimulated signal pathway leading to NO production. Protein kinase C (PKC) inhibitors, but not protein kinase A inhibitors, inhibited the enhancing effect of TS on LPS/IFN-induced NO production. Furthermore, TS enhanced the amount of PKCalpha in VSMC. From these results, we concluded that the enhancing effect of LPS/IFN-induced NO production was caused by upregulation of PKC in VSMC.