蛙皮素和生长抑素对人胃癌BGC-823细胞生长的影响及受体后信息传递

目的:观察蛙皮素(BBS)和生长抑素(SS)对人低分化胃癌细胞株BGC-823的生长调控作用,测定细胞内环磷酸腺苷(cAMP)含量、蛋白激酶C(PKC)活性及PKC亚型的表达,探讨受体后信息传导途径．方法:BGC-823细胞在含100 mL／L小牛血清的RPMI 1640培养液中,于37℃,50 mL／L CO2条件下培养,分别加入不同浓度的BBS或SS,应用MTT法观察细胞的增殖程度．应用放射免疫分析方法测定细胞内cAMP含量,应用[γ-32p]ATP掺入外源性底物的方法测定PKC活性,应用Western blot方法分析PKC亚型α,β1,β,及ε的表达．结果:BBS能促进BGC-823细胞的生长,且与剂量呈正相关(r=0．878,P<0．05),这种促生长作用可被其受体拮抗剂所拮抗;BBS能促进细胞内cAMP的产生(r=0．68,P<0．01)及增加PKC活性,PKCα表达明显增加,而β1,β2及ε则无明显表达;SS能显著抑制BGC-823细胞的生长,呈剂量依赖关系(r=0．831,P<0．01);SS能显著抑制BBS的促细胞生长作用,但无明显的剂量依赖关系;SS可使细胞PKC活性明显下降,呈剂量依赖关系(r=-0．74,P<0．01)．结论:经特异性受体介导,BBS能促进胃癌细胞BGC-823的生长;其受体后信息传递途径可能涉及cAMP-PKA系统及DG-PKC系统, PKCα在BBS的受体后信息传递中可能起重要作用:SS可能通过降低PKC活性抑制BGC-823细胞的生长,同时可抑制BBS的促胃癌细胞生长作用．     

Mediation of anaphylaxis-induced jejunal circular smooth muscle contraction in rats

Altered intestinal motility and diarrhea are features of food protein-induced intestinal anaphylaxis in the conscious rat. These experiments were performed to determine the mediator(s) responsible for jejunal circular smooth muscle contraction during this response. Hooded-Lister rats were sensitized by intraperitoneal injection of 10-g egg albumin, and controls were sham-sensitized with saline. Fourteen days later the contractility of the circular muscle in jejunal segments (mucosa intact) was examined in standard tissue baths in response to antigen (Ag) or other agents. While control and sensitized tissues contracted in similar fashion in response to stretch, bethanechol, histamine, or 5-hydroxytryptamine (5HT), Ag contracted only the segments of sensitized animals. The contractile response was: (1) specific to the sensitizing Ag, as bovine serum albumin did not induced contraction and (2) could be passively transferred with serum containing specific immunoglobulin E antibody (IgE-Ab). Concanavalin A, which degranulates both mucosal and connective tissue-type mast cells, and compound 48/80, which degranulates only connective tissue-type mast cells produced contractile responses. Ag-induced contraction was significantly inhibited by the mucosal and connective tissue-type mast cell stabilizer doxantrazole, but not the connective tissue mast cell stabilizer disodium cromoglycate. Diphenhydramine and cimetidine together significantly inhibited histamine-induced contraction, but failed to effect the Ag-induced contraction in sensitized tissues. While the contractile response to 5HT was reduced in the presence of methysergide (5HT₁-receptor antagonist), cinanserin (5HT₂-receptor antagonist), and ICS 205-930 (5HT₃-receptor antagonist), only cinanserin significantly inhibited the contractile response to Ag. Indomethacin significantly inhibited Ag-induced contraction. Ag-induced contraction was resistant to atropine and tetrodotoxin. Thus, food protein-induced alterations in intestinal motility in sensitized rats are due in part to an IgE-mediated mast cell degranulation, 5HT release, prostaglandin synthesis, and contraction of the circular smooth muscle.     

血管平滑肌细胞细胞周期素依赖性激酶抑制因子的调节

目的 阐明有丝分裂源激活的蛋白激酶(MAPK)和磷脂酶C(PLC)通路在调节细胞周期紊依赖性激酶抑制因子(CKI)p27，p21，p57蛋白表达量中的作用及其对血管平滑肌细胞(VSMC)增殖的影响。方法 分离SD大鼠主动脉中层平滑肌，贴壁法培养平滑肌细胞，无血清培养基培养静止后，分别加入血小板源性生长因子(PDGF)(20ng／m1)、PDGF PD98059(20ng／ml 20mmol／L)、PDGF 硫酸新霉紊(20ng／ml 10mmol／L)等刺激因素，以无血清培养基培养的VSMC作对照。在刺激后90min收集细胞，用免疫沉淀法检测MAPK(p44／p42)活性的改变。在刺激后6和24h收集细胞，用Western蛋白印迹法检测p27、p57和p21蛋白表达量。结果 PDGF刺激后，MAPK活性明显升高(较对照组增加46．6％)，同时VSMC明显增殖。刺激24h后，细胞增殖程度为对照组的1．43倍，p27蛋白的表达量显著下降至对照组的71％；p21和p57蛋白表达量却明显增加；加用PD98058(MAPK抑制剂)和新霉紊(PLC抑制剂)可明显抑制PDGF引起的上述改变，MAPK活性分别较PDGF。刺激组下降了46％和37％，p27蛋白表达量则分别为PDGF刺激组的1．77倍和1．49倍，细胞增殖程度分别降为后者的68％和65％。结论 MAPK(-14／42)变化的幅度是决定CKI表达量和VSMC增殖的关键因素。PLC通路在PDGF刺激VSMC增殖信号转导中同样起关键作用。     

Dendroaspis natriuretic peptide relaxes gastric antral circular smooth muscle of guinea-pig through the cGIvlP/cGMP-dependent protein kinase pathway

AIM: To systematically investigate if cGMP/cGMPdependent protein kinase G (PKG) signaling pathway may participate in dendroaspis natriuretic peptide (DNP)-induced relaxation of gastric circular smooth muscle.METHODS: The content of cGMP in guinea pig gastric antral smooth muscle tissue and perfusion solution were measured using radioimmunoassay,spontaneous contraction of gastric antral circular muscles recorded using a 4-channel physiograph; and Ca2 -activated K currents (Ik(Ca)and spontaneous transient outward currents (STOCs) in isolated gastric antral myocytes were recorded using the whole-cell patch clamp technique.]RESULTS: DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in the perfusion medium.DNP induced relaxation in gastric antral circular smooth muscle,which was inhibited by KT5823,a cGMP-dependent PKG inhibitor.DNP increased IK(Ca)* This effect was almost completely blocked by KT5823,and partially blocked by LY83583,an inhibitor of guanylate cyclase to change the production of cGMP.DNP also increased STOCs.The effect of DNP on STOCs was abolished in the presence of KT5823,but not affected by KT-5720,a PKA-specific inhibitor.CONCLUSION: DNP activates IK(Ca) and relaxes guinea-pig gastric antral circular smooth muscle via the cGMP/PKG-dependent singling axis instead of cAMP/PKA pathway.     

Interaction of Protein Phosphatases and Ethanol on Phospholipase C-Mediated Intracellular Signal Transduction Processes in Rat Hepatocytes: Role of Protein Kinase A

Phospholipase C (PLC)-mediated signal transduction processes in rat hepatocytes are subject to modulation by protein phosphatases (PPases) and protein kinases, including protein kinase A (PKA) and protein kinase C. Ethanol (EtOH) stimulates PLC activity in liver cells in the absence of hormones, and EtOH pretreatment inhibits the subsequent stimulation of PLC by hormonal stimuli. There is evidence that protein kinase activities are involved in these actions of EtOH. We investigated the effects of okadaic acid (OKA), a PPase inhibitor, and 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (cpt-cAMP), a cell permeant cAMP analog that activates PKA, on EtOH-induced PLC activation. In addition, we studied the combined effects of cpt-cAMP and EtOH/OKA on vasopressin-induced PLC activation. PLC activation (cytosolic Ca²⁺ mobilization and inositol trisphosphate accumulation) induced by EtOH and vasopressin was inhibited by treatment with OKA, and was potentiated by cpt-cAMP. OKA treatment prevented the effect of cpt-cAMP. Pretreatment with EtOH caused inhibition of vasopressin-induced PLC activation. EtOH also decreased the enhancing effect of cpt-cAMP on the responses to vasopressin. The susceptibility to enhancement by cpt-cAMP plotted as a function of the initial rate of vasopressin-induced Ca²⁺ mobilization in EtOH-treated cells was similar to the pattern observed in OKA-treated cells. These data suggest that interactions of OKA and PKA on EtOH-induced PLC activation occurred at the level of G-protein, and indicate that EtOH may act as an inhibitory agent of PPase.     

Signal transduction of c-Kit receptor tyrosine kinase in CHRF myeloid leukemia cells

Purpose The tyrosine kinase receptor c-Kit (stem cell factor receptor, CD117) is a potential target for signal transduction therapy in different cancers. In this study we investigated c-Kit in CHRF cells, a megakaryoblastic cell line of Acute Myeloid Leukemia (FAB M7).Materials and methods We characterized the interactions between c-Kit and PI 3-kinase (p85) after stimulation with SCF (stem cell factor) as well as the regulation of SHP-1 and SHP-2 associated with Kit in this cell line.Results Stimulation with SCF leads to a significant increase in interaction between Kit and p85 as well as in receptor associated PI 3-kinase activity. Interestingly, using different kinds of substances (AG 1295, CGP 53716) to inhibit the tyrosine kinase activity of c-Kit blocked activation of c-Kit, but the association of p85 still increased after SCF stimulation even when the tyrosine kinase activity of the receptor was completely blocked. In contrast, the other known interaction partners of c-Kit, SHP-1 and SHP-2, exhibited a basal association with c-Kit and no change of the association could be detected after stimulation of CHRF cells with SCF or treatment with the kinase inhibitors.Conclusions Therefore, we suggest that association of p85, SHP-1, and SHP-2 to c-Kit in CHRF cells can, at least in part, occur in a c-Kit kinase-activity independent manner. In contrast, the kinase activity of c-Kit is necessary for the activation of receptor-associated PI 3-kinase.     

Signal Transduction in Ischemic Preconditioning:

Ischemic preconditioning is a phenomenon whereby exposure of the myocardium to a brief episode of ischemia and reperfusion markedly reduces tissue necrosis induced by a subsequent prolonged ischemia. Therefore, it is hoped that elucidation of the mechanism of preconditioning will yield therapeutic strategies capable of reducing myocardial infarction. In the rabbit, the brief period of preconditioning ischemia and reperfusion releases adenosine, bradykinin, opioids, and oxygen radicals that summate to induce the translocation and activation of protein kinase C (PKC). PKC appears to be the first element of a complex kinase cascade that is activated during the prolonged ischemia in preconditioned hearts. Current evidence indicates that PKC activates a tyrosine kinase that leads to the activation of p38 mitogen-activated protein (MAP) kinase or JNK, or possibly both. The stimulation of these stress-activated protein kinases ultimately induces the opening of mitochondrial K(ATP) channels that may be the final mediator of protection by ischemic preconditioning.     

The role of protein kinase C in the regulation of cell growth and in signalling to the cell nucleus

The protein kinase C (PKC) family of serine/threonine kinases consists of at least 11 mammalian isoforms, which show slight differences in their molecular structure and enzymatic properties. PKC isoforms are involved in a wide variety of intracellular signalling events and play an important role in tumour promotion and cell growth control in general. Studies of expression levels in cancer cells and studies using overexpression of single isoforms or expression of dominant negative isoforms reveal that, depending on the cellular background, PKC isoforms can either promote or inhibit cell growth. To understand the role of PKC isoforms in growth control, it is essential to understand how PKC functions in the intracellular signalling cascades towards the cell nucleus. Recent work has shown that PKC isoforms can act either in the cytoplasm, and cause nuclear effects indirectly by triggering signalling pathways directed towards the cell nucleus, or, after translocation and activation, can themselves act in the cell nucleus. Received: 8 January 1999 / Accepted: 12 July 1999     

Dendroaspis natriuretic peptide relaxes gastric antral circular smooth muscle of guinea-pig through the cGMP/cGMP-dependent protein kinase pathway

AIM： To systematically investigate if cGMP/cGMPdependent protein kinase G （PKG） signaling pathway may participate in dendroaspis natriuretic peptide （DNP）-induced relaxation of gastric circular smooth muscle.
METHODS： The content of cGMP in guinea pig gastric antral smooth muscle tissue and perfusion solution were measured using radioimmunoassay; spontaneous contraction of gastric antral circular muscles recorded using a 4-channel physiograph; and Ca^2＋-activated K^＋ currents （IK（Ca）） and spontaneous transient outward currents （STOCs） in isolated gastric antral myocytes were recorded using the whole-cell patch clamp technique.
RESULTS： DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in the perfusion medium. DNP induced relaxation in gastric antral circular smooth muscle, which was inhibited by KT5823, a cGMP-dependent PKG inhibitor. DNP increased IK（Ca）. This effect was almost completely blocked by KT5823, and partially blocked by LY83583, an inhibitor of guanylate cyclase to change the production of cGMP. DNP also increased STOCs. The effect of DNP on STOCs was abolished in the presence of KT5823, but not affected by KT-5720, a PKA-specific inhibitor.
CONCLUSION： DNP activates IK（ca） and relaxes guinea-pig gastric antral circular smooth muscle via the cGMP/PKG-dependent singling axis instead of cAMP/ PKA pathway.     

Signal transduction in matrix contraction and the migration of vascular smooth muscle cells in three-dimensional matrix

The interaction of vascular smooth muscle cells (SMCs) and extracellular matrix plays important roles in vascular remodeling. We investigated the signaling pathways involved in SMC-induced matrix contraction and SMC migration in three-dimensional (3D) collagen matrix. Matrix contraction is inhibited by the disruption of actin filaments but not microtubules. Therefore, we investigated the roles of signaling pathways related to actin filaments in matrix contraction. SMC-induced matrix contraction was markedly blocked (-80%) by inhibiting the Rho-p160ROCK pathway and myosin light chain kinase, and was decreased to a lesser extent (30-40%) by a negative mutant of Rac and inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) or p38 mitogen-activated protein kinase (MAPK), but it was not affected by the inhibition of Ras and Cdc42-Wiskott-Aldrich syndrome protein (WASP) pathways. Inhibition of extracellular-signal-regulated kinase (ERK) decreased SMC-induced matrix contraction by only 15%. The migration speed and persistence of SMCs in the 3D matrix were decreased by the inhibition of p160ROCK, PI 3-kinase, p38 MAPK or WASP to different extents, and p160ROCK inhibitor had the strongest inhibitory effect. Our results suggest that the SMC-induced matrix contraction and the migration of SMCs in 3D matrix share some signaling pathways leading to force generation at cell-matrix adhesions and that various signaling pathways have different relative importance in the regulations of these processes in SMCs.     

IL-1β activates p44/42 and p38 mitogen-activated protein kinases via different pathways in cat esophageal smooth muscle cells

AIM: To examine the pathway related to the IL-1β-induced activation of mitogen-activated protein (MAP)kinases in cat esophageal smooth muscle cells.METHODS: Culture of the esophageal smooth muscle cells from cat was prepared. Specific inhibitors were treated before applying the IL-1β. Western blot analysis was performed to detect the expressions of COX, iNOS and MAP kinases.RESULTS: In the primary cultured cells, although IL-1βfailed to upregulate the COX and iNOS levels, the levels of the phosphorylated forms of p44/42 MAP kinase and p38 MAP klnase increased in both concentration- and time-dependent manner, of which the level of activation reached a maximum within 3 and 18 h, respectively.The pertussis toxin reduced the level of p44/42 MAP kinase phosphorylation. Tyrphostin 51 and genistein also inhibited this activation. Neomycin decreased the density of the p44/42 MAP kinase band to the basal level.Phosphokinase C (PKC) was found to play a mediating role in the IL-1β-induced p44/42 MAP kinase activity.In contrast, the activation of p38 MAP kinase was inhibited only by a pretreatment with forskolin, and was unaffected by the other compounds.CONCLUSION: Based on these results, IL-1β-Induced p44/42 MAP kinase activation is mediated by the Gi protein, tyrosine kinase, phospholipase C (PLC) and PKC. The pathway for p38 MAP kinase phosphorylation is different from that of p44/42 MAP kinase, suggesting that it plays a different role in the cellular response to IL-1β.     

蛋白激酶C在支气管哮喘模型大鼠气道平滑肌细胞增殖中的信号转导机制研究

目的　探讨蛋白激酶C(PKC)信号转导途径在支气管哮喘 (简称哮喘 )大鼠气道平滑肌细胞 (ASMC)增殖中的作用。方法　 (1) 4 8只Wistar大鼠分为哮喘组 (A组 )及对照组 (B组 ) ,根据激发时间 (2、4、8周 )又分别分为A1、A2 、A3 组和B1、B2 、B3 组 ,其中A、B组大鼠各 12只 ,A2 、A3 、B2 及B3 组各 6只。用流式细胞术、四甲基偶氮唑盐 (MTT)法、增殖细胞核抗原 (PCNA)染色等方法观察每组ASMC增殖 ;(2 )用PKC激活剂 12 肉蔻酰 13 乙酸佛波酯 (PMA)及抑制剂Ro 31 82 2 0分别干预A1、B1组ASMC ,观察ASMC增殖的变化 ;(3)用逆转录 聚合酶链测定 (RT PCR)和免疫细胞化学法检测A1、A2 、A3 组和B1组ASMCPKC α的表达。结果　 (1)A组ASMCS期比例、吸光度 (A)值、PCNA表达增高 ,与B组比较差异有显著性 (P <0 0 1)。 (2 )A1组ASMCS期比例、A值、PCNA阳性表达率在干预前分别为 (19± 3) %、0 4 5 9± 0 0 36、(80± 10 ) % ;10nmol/LPMA处理后分别为 (2 7± 4 ) %、0 5 99± 0 0 78、(95± 9) % ;5 0nmol/LPMA处理后为 (14± 3) %、0 346± 0 0 38、(5 3± 8) % ;Ro 31 82 2 0处理后为 (14± 3) %、0 343± 0 0 4 8、(4 9± 8) %。各干预剂处理后与处理前比较差异均有显著性 (P <0 0 1) ;5 0nmol/LPMA处     

Esophageal mucosal mast cell infiltration and changes in segmental smooth muscle contraction in noncardiac chest pain

Mast cells release potent mediators that alter enteric nerve and smooth muscle functions and may contribute to the pathogenesis of functional gastrointestinal disorders. The goal of this study was to determine if mucosal mast cell infiltration was associated with smooth muscle segmental changes in esophageal contraction. All patients with noncardiac chest pain (NCCP) were divided into two groups consisting of patients with non‐erosive reflux disease or functional chest pain (FCP) according to the results of ambulatory 24 hours esophageal pH monitoring and high‐resolution manometry. Pressure–volume (PV) was calculated by multiplying the length of the esophageal segment, duration of the contraction, and mean pressure over the entire space–time box (P mean). Quantification of mast cells was performed in five consecutive nonoverlapping immunostained sections. Spearman correlation analysis showed that the distal segment PV correlated with the mast cell count in all of the patients combined and in patients with FCP with correlation coefficients of 0.509 and 0.436, respectively (P = 0.004 and P = 0.042). Similar findings were observed for the segmental ratio of distal to proximal smooth muscle PV in all patients and in patients with FCP (correlation coefficients 0.566; P = 0.001 and correlation coefficients 0.525; P = 0.012, respectively). Mucosal mast cell infiltration was associated with distal esophageal contraction as a key pathophysiologic factor of NCCP.     

Inhibitory effects of ethanol on rat mesangial cell proliferation via protein kinase C pathway

A large body of evidence has shown that ethanol inhibits the cell growth and cell proliferation in a variety of cell types. However, it has not been studied whether ethanol inhibits the proliferation of mesangial cells (MC) in the kidney. We examined the effects of ethanol on cell proliferation in cultured rat MC. Treatment with ethanol (10-200 mM) for 48 hr inhibited [(3)H]thymidine incorporation into MC in a concentration-dependent manner. The same concentrations of ethanol also inhibited the increase in cell number of MC. GF109203X and chelerythrine chloride, inhibitors for protein kinase C, eliminated the inhibitory effects of ethanol; and protein kinase C activator, PMA, mimicked the effects of ethanol. In contrast, neither the protein kinase A inhibitor H-89 nor the protein kinase G inhibitor KT5823 had any effect. These findings suggest that ethanol has inhibitory effects on the proliferation of MC, probably via activation of the protein kinase C pathway.     

Phosphatidylethanol in high density lipoproteins increases the vascular endothelial growth factor in smooth muscle cells

To study whether qualitative changes in high density lipoprotein (HDL) phospholipids mediate part of the advantageous effects of ethanol on atherosclerosis, we investigated whether HDL associated phosphatidylethanol (PEth) affects the secretion of vascular endothelial growth factor (VEGF) from cultured human smooth muscle cells. Serum-starved human umbilical vein HUVS-112D smooth muscle cells were incubated in the presence of PEth–HDL, HDL, or buffer. The phosphorylation of protein kinase C (PKC) and mitogen activated protein kinase (p44/42 MAPK) was determined by specific antibodies against phosphorylated and total proteins. VEGF concentrations were measured from cell culture medium of the cells. PEth increased the secretion of VEGF into the culture medium of HUVS cells. PEth–HDL increased the PKC phosphorylation by 2.1-fold and p44/42 MAPK phosphorylation by 3.3-fold compared with HDL, indicating that PEth-containing HDL particles influence vascular smooth muscle cells by PKC and p44/42 MAPK signalling. This may mediate the effects of ethanol on vascular wall by increasing the VEGF secretion from smooth muscle cells. The secreted VEGF may inhibit the formation of neointima and in doing so helps prevent atherosclerosis.     

细胞外信号调节激酶1/2在血管内皮细胞凋亡中的表达变化及作用

研究背景树突状细胞(DCs)是体内功能最强大的抗原递呈细胞,具有独特的激活初始T淋巴细胞的功能,是启动和调控特异性免疫应答的中心环节。近年来的大量基础和临床研究证实,动脉粥样硬化(As)是一种慢性炎症和免疫性疾病,DCs直接或间接参与As的发生发展。以DCs为作用靶点可能是干预As的有效方法。丹参酚酸B(Sal B)是自中药丹参中提取出的水溶性单体,是丹参的重要药理活性成份,化学结构明确,性质稳定。以往的研究证实,Sal B对心、脑、肝、肾等多个器官具有重要的保护作用。近期的研究还发现,Sal B具有抗炎症、抗免疫反应的作用,能够影响As的发生发展,但具体作用机制和靶点还不明确。目的从免疫炎症的角度探讨Sal B对氧化低密度脂蛋白(ox-LDL)诱导的人单核细胞源DCs免疫功能成熟的影响,进一步研究其作用机制,为临床As的防治提供新靶点和新思路。方法培养人单核细胞源DCs,Sal B预处理后,再与ox-LDL共孵育。流式细胞术检测DCs表面分子(CD40、CD1a、CD86和HLA-DR)的表达,ELISA法检测细胞培养上清液细胞因子(IL-12和TNF-α)的浓度,Western blot法检测PPARγ和...     

Dexniguldipine hydrochloride,a protein-kinase-C-specific inhibitor,affects the cell cycle,differentiation, P-glycoprotein levels,and nuclear protein phosphorylation in friend erythroleukemia cells

Dexniguldipine hydrochloride (DNIG) is a potent antineoplastic agent with well-documented anti-(protein kinase C) activity and an ability to reverse multidrug resistance. Given the importance of protein kinase C (PKC) activity in proliferation and differentiation, we examined the effect of DNIG on several parameters of Friend erythroleukemia cell (FELC) activity. Particular attention was paid to proliferation, hexamethylene-bisacetamide-(HMBA)-induced differentiation, nuclear localization of protein kinase C, and nuclear protein phosphorylation. P-glycoprotein expression was also followed as an indicator of changes in multidrug resistance. At 2.5 M, DNIG caused a significant decrease in the rate of FELC proliferation, while maintaining a cellular viability of greater than 80%, whether exposure to the drug was continuous over 96 h or took the form of a 6-h pulse/chase. DNA synthesis was decreased in cells exposed to DNIG for 20 h. Flow cytometry showed a marked increase in the percentage of cells in S phase of the cell cycle. Phosphorylation studies revealed decreased phosphorylation of two nuclear proteins (80 kDa and 47 kDa) following a 4-h exposure to the drug. HMBA-induced differentiation was significantly inhibited with continuous exposure to DNIG, and this effect appears to be a pre-commitment one, as 6-h pulse/chase exposures also resulted in inhibition of differentiation. Cells induced to differentiate with HMBA also demonstrated a decrease in the quantity of the 80-kDa phosphoprotein. western blotting revealed that, even in the face of decreased phosphorylation, exposure to this PKC inhibitor resulted in an increase in the amount of nuclear PKC. Finally, levels of P-glycoprotein were decreased in the presence of this drug. Our work identifies several effects of the PKC inhibitor DNIG on FELC and suggests several roles for PKC in regulating FELC proliferation and differentiation. Additionally, these results suggest that this PKC inhibitor may increase the effect of other chemotherapeutic drugs, particularly S-phase-specific ones, by increasing the length of S phase and decreasing multidrug resistance. The possibility of combination therapy with DNIG and other antineoplastic agents should be investigated further in light of these findings.Abbreviations DNIG dexniguldipine hydrochloride - FELC Friend erythroleukemia cells - HMBA hexamethylene bisacetamide - MDR multidrug resistance - PKC protein kinase C Financial support: this work was supported in part by a grant from the Ladies Leukemia League to J. R. J. and a grant from the NIH, DK40501, to B. S. B.     

蛋白激酶C-核因子κB信号转导通道对人肺动脉平滑肌细胞增殖和血管内皮生长因子表达的影响

目的　探讨蛋白激酶C(PKC) 核因子κB(NF κB)信号转导通道对人肺动脉平滑肌细胞 (HPASMCs)增殖和血管内皮生长因子 (VEGF)表达的影响。方法　体外培养HPASMCs ,用工具药PKC激活剂 12 肉豆蔻酰 13 乙酸佛波酯 (PMA)和NF κB抑制剂二硫代氨基甲酸吡咯烷 (PDTC) ,将HPASMCs分为对照组、PMA组和PMA PDTC组在常氧和缺氧条件下培养。逆转录 聚合酶链反应 (RT PCR)检测VEGFmRNA表达 ,Westernblot法检测VEGF和NF κB的抑制蛋白IκBα蛋白表达 ,免疫细胞化学法检测NF κBp6 5的表达和定位 ,流式细胞术检测细胞周期时相分布。结果　( 1)NF κBp6 5胞核染色阳性率、IκBα蛋白相对表达量及细胞周期G2 /M % :常氧或缺氧PMA组与相应对照组、PMA PDTC组比较差异均有显著性 (P均 <0 0 5 ) ;缺氧PMA组与常氧PMA组比较差异有显著性 (P <0 0 5 )。 ( 2 )VEGFmRNA和蛋白表达 :常氧对照组、PMA组、PMA PDTC组组间差异均无显著性 (P均 >0 0 5 ) ;缺氧PMA组均高于缺氧对照组、缺氧PMA PDTC组、常氧PMA组 ,差异均有显著性 (P均 <0 0 5 )。 ( 3)缺氧PMA组NF κB胞核染色阳性率、VEGF蛋白相对表达量、G2 /M %之间均呈正相关 (r =0 5 87～ 0 710 ,P均 <0 0 5 )。结论　常氧培养HPASMCs存在PKC NF κB信号转导通道 ;     

丝裂素活化蛋白激酶的激活和转核与大鼠 血管平滑肌细胞增殖关系的研究

目的 探讨丝裂素活化蛋白激酶（MAPK）激活、转核与血管紧张素Ⅱ（AngⅡ)刺激血管平滑肌细胞（VSMC）增殖间的关系。方法 本实验采用培养大鼠胸主动脉VSMC。用^3H-胸腺嘧啶核苷（^3H-TdR)掺入法测定DNA合成,用p43/p44磷酸化抗MAPK抗体的蛋白免疫印迹法测定MAPK蛋白量,用免疫细胞化学技术观察MAPK活化并转位入细胞核的过程。结果 （1）AngⅡt和PD98059的上述作用都呈剂量依赖性。（2）AngⅡ对MAPK蛋白表达有显著增强作用。此作用同样被PD98059以剂量依赖方式抑制。（3）AngⅡ刺激5min后,MAPK出现在VSMC的细胞浆中,30min时MAPK进入细胞核,3h后MAPK染色从核内消失,上述MAPK转核过程被PD98059抑制。结论 本实验证实人细胞核,3h后MAPK染色从核人消失,上述MAPK转核过程被PD98059抑制。结论 本实验证实AngⅡ能激活培养大鼠主动脉VSMC的MAPK,活化的MAPK从细胞浆转位进入细胞核导致VSMC增殖。     

细胞外信号调节激酶1/2在血管内皮细胞凋亡中的表达变化及作用

目的观察细胞外信号调节激酶1/2(ERK1/2)在血管紧张素Ⅱ诱导的内皮细胞中不同时点的表达变化,为阐明血管内皮细胞凋亡对动脉粥样硬化的诊治具有重要意义。方法制备血管紧张素ⅡRPMI1640培养液(10-6mol/L)培养人脐静脉内皮细胞,采用四甲基偶氮唑蓝比色法测定内皮细胞存活率,通过AnnexinV-FITC/PI双染流式细胞仪检测细胞凋亡率、Hochest33258荧光染色观察凋亡细胞形态学的变化,利用RT-PCR法分析凋亡调控基因Bcl-2、Bax mRNA表达变化,Western-Blot测定磷酸化ERK1/2水平。结果血管紧张素Ⅱ诱导内皮细胞的凋亡率明显高于对照组(P<0.01),与对照组相比,Bcl-2 mRNA表达呈持续性降低;Bcl-2/Bax比值下降,ERK1/2磷酸化水平于12 h明显增加,18 h达到高峰(P<0.01),24 h下降至稳定,总ERK1/2蛋白水平无明显变化。结论 ERK1/2信号转导途径参与血管紧张素Ⅱ诱导内皮细胞的凋亡发生、发展过程,并可能通过调控内皮细胞Bcl-2/Bax比值来实现。