降钙素基因相关肽通过ERK1/2信号通路对角质形成细胞增殖的影响

目的： 研究降钙素基因相关肽（CGRP）对角质形成细胞增殖活性的影响，并探讨其可能涉及的信号转导通路。方法： ①胸腺嘧啶掺入法（［3H］-TdR）观察CGRP诱导的角质形成细胞株HaCaT细胞增殖，及CGRP受体拮抗剂CGRP8-37、细胞外信号调节激酶ERK1/2特异性抑制剂PD98059对CGRP诱导的增殖活性的影响；②免疫印迹技术观察CGRP诱导后ERK1/2的磷酸化，及CGRP8-37、PD98059对ERK1/2磷酸化的影响。结果： ①CGRP在一定范围内可剂量依赖性地诱导HaCaT细胞增殖，该作用可被CGRP8-37和 PD98059阻断；②CGRP可时间依赖性地诱导HaCaT细胞ERK1/2的磷酸化，CGRP8-37和PD98059可减弱其作用。结论： CGRP可诱导HaCaT细胞增殖，CGRP受体及其相关的ERK1/2信号通路参与其调控机制。     

中华眼镜蛇毒F组份对血小板活化时蛋白酪氨酸磷酸化的影响

目的：观察中华眼镜蛇毒F组份抑制血小板聚集的胞内信号转导机制。方法： 实验分6组：(1)空白组；(2)对照组；(3)-(6)实验组加入浓度为100、30、10、3 mg/L F组份。用比浊法测定血小板聚集率。用蛋白质免疫印迹法（Western blotting）测定血小板内蛋白酪氨酸磷酸化的表达。结果： F组份呈浓度依赖性抑制二磷酸腺苷（ADP）引起的分子量为76、66和37.5 kD蛋白酪氨酸磷酸化的表达，其中30、100 mg/L给药组中，3个分子量蛋白与对照组比较均有显著差异，P<0.05。F组份对ADP引起血小板聚集作用的抑制同降低76、66和37.5 kD 3个分子量蛋白酪氨酸磷酸化的表达呈明显的正相关（r=0.9367,P<0.01）。结论： 中华眼镜蛇毒F组份通过抑制分子量为76、66和37.5 kD蛋白的酪氨酸磷酸化表达，对血小板胞内信号转导过程发生影响，可能是其抗栓机制之一。     

柚皮素通过PI3K/Akt通路拮抗血小板聚集的体外研究

目的:探讨柚皮素拮抗二磷酸腺苷(ADP)诱导的血小板聚集的作用及机制。方法:采用血小板聚集仪观察不同浓度柚皮素对ADP诱导的大鼠血小板聚集的影响。采用荧光显微镜观察柚皮素对血小板在固化纤维蛋白原上扩展功能的影响。采用Western blot检测磷脂酰肌醇3-激酶(PI3K)表达及蛋白激酶B(Akt)磷酸化水平。结果:柚皮素能剂量依赖性地抑制ADP诱导的体外血小板聚集,其半数抑制浓度(IC_(50))值为(132.1±31.7)μmol/L。柚皮素灌胃给药,对ADP诱导的大鼠体内血小板聚集也有明显的抑制作用。柚皮素还能显著抑制血小板在固化纤维蛋白原上的扩展。Western blot结果显示,柚皮素能明显抑制ADP刺激的PI3K表达和Akt磷酸化同时,PI3K广谱抑制剂LY294002能增强柚皮素对Akt磷酸化的抑制作用。结论:柚皮素可能通过抑制血小板PI3K/Akt信号通路发挥抗血小板聚集的作用。     

抗血小板膜糖蛋白单克隆抗体SZ-2单链抗体的构建、表达及功能研究

目的：降低鼠源性抗体的免疫原性及分子量，为其进一步研究、应用奠定基础。方法：应用RT—PCR技术，克隆SZ-2重链、轻链可变基因。应用基因重组技术构建SZ-2单链抗体表达载体pET22-2scFv，导入大肠杆菌BL21(DE3)Plys，诱导表达。流式细胞术、ELISA、Western blot检测SZ-2 scFv与血小板结合能力，瑞斯托霉素、凝血酶和ADP诱导血小板聚集试验对表达产物功能进行研究。结果：克隆基因序列符合小鼠轻、重链可变区基因特征；pET22—2scFv表达质粒拼接正确；表达产物以包涵体形式为主，表达量占菌体总蛋白的25％；纯化、复性后证明表达产物具有与血小板结合的活性，并可抑制瑞斯托霉素、凝血酶诱导的血小板聚集。结论：成功表达了SZ—2单链抗体，该小分子抗体具有与血小板结合的活性，并可抑制瑞斯托霉素、凝血酶诱导的血小板聚集。     

ERK1/2信号传导途径参与调控NK细胞的杀伤活性

目的：阐明NK细胞系持续活化的信号通路ERK1／2在调节NK细胞增殖和杀伤活性中可能发挥的作用。方法：制备NK-92和YT细胞的全细胞提取物，进行Westrn blot，检测在细胞系中持续活化的信号传导途径，信号通路特异性阻断剂PD098059阻断ERK1／2活化，MTT方法评价ERK1／2在调节NK细胞杀伤和增殖活性中发挥的作用，RT-PCR检测阻断试剂作用前后杀伤相关分子表达水平的变化。结果：2个代表性的NK细胞系(YT，IL-2非依赖；NK-92，IL-2依赖)中存在ERK1／2(p44／42MAPK)、NF-kB和STAT3信号通路的持续磷酸化，去除IL-2和血清较长时间后仍保持活化状态，而其它信号途径(STAT1、STAT6、PI-3K、p38MAPK)则未见活化。特异性阻断剂PD098059 200μmol／L阻断ERK1／2活化后，NK细胞杀伤。K562靶细胞的能力显著降低。但细胞的增殖活性不受影响。RT-PCR证实，PD098059阻断ERK1／2抑制NK细胞毒活性的同时，杀伤相关分子IFNγ、FasL 、pcrforin的表达水平也不同程度地下调。结论：NK细胞系中持续性活化的ERK1／2主要传递与NK细胞细胞毒性相关的信号，并不参与调节细胞的增殖，该途径通过控制杀伤相关分子IFNγ、FasL、perforin的基因表达从而主导NK对靶细胞的杀伤。     

尿激酶和链激酶对大鼠血小板功能的影响

目的：观察尿激酶（UK）和链激酶（SK）是否能直接激活血小板。方法：在体外制备大鼠血小板，以血小板聚集、血小板释放丙二醛（MDA）及5-羟色胺（5-HT）、血小板内游离钙（[Ca²⁺]_i）为指标，观察UK和SK对血小板的直接作用；在体内观察它们对电刺激诱导大鼠动脉形成血栓的影响。结果：在体外2×10⁶ U/L的UK与SK对凝血酶及ADP诱导的血小板聚集、对凝血酶诱导的血小板释放MDA含量、5-HT含量和血小板[Ca²⁺]_i无明显影响。在体内4×10⁴ U/kg的UK和SK对电刺激诱导的大鼠动脉血栓形成有一定的抑制作用。结论：UK和SK不能直接激活血小板。     

Smad通路参与ERK通路诱导血管平滑肌细胞增殖的过程

目的： 探讨Smad通路是否参与细胞外信号调节激酶(ERK)通路诱导血管平滑肌细胞（VSMCs）增殖的过程及其可能机制。方法：将人脐动脉平滑肌细胞（hUASMCs）分为对照组、血小板源性生长因子（PDGF）组、ERK阻断剂组和PDGF+ERK阻断剂组。用MTT法测hUASMCs的增殖活性(A值)，用免疫组化法测hUASMCs内细胞核增殖抗原（PCNA）、磷酸化ERK和磷酸化Smad蛋白的表达，用RT-PCR法测hUASMCs内Smad2/3 mRNA的表达。结果：PDGF组hUASMCs的增殖活性(A值)及hUASMCs内的PCNA、磷酸化ERK和磷酸化Smad2/3蛋白的表达都明显高于其它各组(P<0.01)；各组hUASMCs内Smad2/3 mRNA的表达没有差异。结论： Smad通路可在蛋白水平参与ERK通路诱导VSMCs的增殖过程。     

缺氧诱导因子-1在缺氧预处理心肌保护中的作用及其机制研究

目的:研究缺氧诱导因子-1(HIF-1)在缺氧预处理(HPC)心肌细胞保护中的作用及其机制。方法:在培养的SD乳鼠心肌细胞缺氧/复氧(H/R)模型上,观察HPC对于24h后心肌细胞H/R损伤的影响,以MTT法测定心肌细胞存活率,试剂盒测定培养液中乳酸脱氢酶(LDH)活性。制备心肌细胞蛋白提取物,以磷酸化的细胞外信号调节激酶(ERK1/2)抗体测定HPC后不同时间ERK1/2活性,以聚丙烯酰胺电泳迁移实验观察HIF-1α磷酸化,并观察蛋白磷酸酶激动剂BDM和ERKs的上游激酶(MEK1/2)抑制剂PD98059对于HPC诱导的HIF-1α磷酸化以及心肌细胞保护作用的影响。结果:HPC可以提高心肌细胞H/R后存活率、减少LDH漏出,并激活ERK1/2,使HIF-1α发生磷酸化;蛋白磷酸酶激动剂BDM和ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的HIF-1α磷酸化和心肌细胞保护作用。结论:HPC可以提高乳鼠心肌细胞对于H/R的耐受性,其机制涉及ERKs介导的HIF-1α磷酸化。     

儿茶素抑制兔晶状体上皮细胞增殖

目的：探讨在绿茶提取物表没食子儿茶素没食子酸酯（EGCG）抑制兔晶状体上皮细胞增殖过程中， MEK/ERK1/2(mitogen-activated protein kinase kinase, MAPKK, MEK; extracellular signal-regulated kinase，ERK)及c-jun氨基末端激酶（c-Jun NH2-terminal protein kinase， JNK）信号转导通路的作用。 方法： 实验按EGCG浓度分50、100和200 μmol/L 3组。预先分别加入25、50 μmol/L的ERK活性拮抗剂PD980059孵育1 h；用噻唑蓝比色法（MTT比色法）测定晶状体上皮细胞增殖；用蛋白质免疫沉淀法(Western blotting)法检测ERK及JNK的磷酸化水平。 结果： （1）PD980059能明显增强EGCG抑制晶状体上皮细胞增殖的作用，呈浓度依赖性。（2）晶状体上皮细胞内磷酸化的ERK基础水平高。加入EGCG后其活性升高，随后逐渐回降,但始终高于基础水平；ERK的活化水平也随EGCG浓度的增加而增高。（3）细胞内JNK的54 kD亚型磷酸化的基础水平较低而46 kD亚型的较高；呈剂量与时间依赖性。 结论： EGCG可能部分通过调节ERK和JNK的磷酸化水平而抑制晶状体上皮细胞的增殖。     

抗血小板糖蛋白Ⅵ单克隆抗体的制备及其功能研究

目的：制备抗血小板糖蛋白Ⅵ（GPⅥ）单克隆抗体，观察其在体外抗血小板黏附和聚集功能。方法：采用基因重组技术体外表达血小板糖蛋白Ⅵ胞外区重组蛋白（rGPⅥ）。以rGPⅥ免疫小鼠，经细胞融合及筛选后制备抗GPⅥ单克隆抗体。采用血小板聚集实验观察该单抗对胶原、Convulxin及ADP诱导的血小板聚集的影响；利用平行板流动小室技术研究在高剪切力条件下该单抗对血小板在胶原表面黏附的抑制效果。结果：正确构建了rGPⅥ表达载体pET-20b（＋）-GPⅥ，rGPⅥ在原核细胞中有效表达。rGPⅥ能够被抗Penta-His单抗和抗GPⅥ多抗识别。制备的抗GPⅥ单克隆抗体SZ118能够识别rGPⅥ，并与血小板有特异的结合能力。SZ118能明显抑制纤维状胶原和Convulxin诱导的血小板聚集，呈抗体剂量依赖性；对ADP诱导的血小板聚集无明显影响。血小板黏附实验表明，SZ118能够明显阻断在高剪切力条件下血小板与纤维状胶原表面的黏附。结论：成功制备抗GPⅥ单克隆抗体SZ118，该抗体与血小板有良好的结合能力，显著抑制胶原诱导的血小板聚集并明显降低血小板与胶原的黏附反应。     

11，12-EET的延迟性心脏保护作用与磷酸化ERK1/2的关系

目的:观察11，12-EET延迟性保护作用对缺血再灌注大鼠心肌ERK活性及磷酸化ERK表达的影响，探讨其在11，12-EET延迟性保护中的作用。 方法:复制大鼠心肌缺血/再灌注模型；观察缺血/再灌注期间心脏收缩期左心室内压上升的最大变化速率（+dp/dtmax）及舒张期左心室内压下降的最大变化速率（-dp/dtmax）；采用免疫共沉淀法测定大鼠心肌组织中细胞外调节激酶（ERK）的活性，采用Western blotting法测定大鼠心肌组织中磷酸化ERK的表达。 结果:I/R组缺血60 min及再灌注30 min两个时段±dp/dtmax均低于sham组（P<0.05），24 hEET+I/R组缺血60 min及再灌注30 min两个时段±dp/dtmax明显高于I/R组（P<0.05），而24hEET+PD+I/R组缺血60 min及再灌注30 min两个时段±dp/dtmax明显低于24hEET+I/R组（P<0.05）。ERK的活性24hEET+I/R高于normal组，24hEET+I/R组低于24hEET+PD+I/R组。磷酸化ERK的表达I/R组高于normal组和sham组，24hEET+I/R高于I/R组，24 hEET+I/R组低于24hEET+PD+I/R组。 结论:外源性11，12-EET具有延迟性心脏保护作用，大量激活磷酸化的ERK参与这种保护作用。     

Involvement of the MAPK-kinase pathway in the PTH-mediated regulation of the proximal tubule type IIa Na+/Pi cotransporter in mouse kidney

Reabsorption of phosphate in the proximal tubule is mainly mediated by the type IIa Na(+)/P(i) cotransporter (NaPi-IIa) and tightly regulated by a variety of factors including dietary phosphate intake and parathyroid hormone (PTH). PTH signals through both apical and basolateral PTH receptors and induces the rapid internalization and subsequent degradation of NaPi-IIa. At least two signalling cascades can be activated by PTH: the PLC/PKC and the cAMP/PKA pathways. Recent evidence from OK cell culture suggested the involvement of MAPK kinases in the PTH action. Here we used freshly isolated coronal mouse kidney slices and incubated them in a physiological buffer in the absence and presence of PTH with inhibitors and activators of the various signalling cascades to further study the events leading to internalization of NaPi-IIa. No alterations in the pattern of immunostaining for alpha-tubulin, actin and several brush border membrane proteins demonstrated intactness of the slices over the experimental period. Application of PTH (100 nM) induced a strong decrease of NaPi-IIa brush border staining and internalization after 45 min of incubation. The localization of the Na(+)/sulphate cotransporter (NaSi), however, was not affected. The internalization of NaPi-IIa could be completely prevented by the PKC inhibitor chelerythrine (1 micro M) or the MAPK-kinase (ERK1/2) inhibitor PD098059 (20 micro M). Without PTH both inhibitors alone had no effect. PTH induced phosphorylation of the ERK1/2 MAPK-kinases which was prevented by PD 098059. Separate activation of the cAMP/PKA pathway by 8-Br-cAMP was completely prevented by PD098059 whereas activation of the PLC/PKC pathway by the PKC activator 1,2-dioctanoyl-sn-glycerol (DOG) and the PKG pathway by 8-Br-cGMP induced internalization of NaPi-IIa which could be only partly blocked by PD 098059. Inhibition by SB203580 or activation by anisomycin of the p38 kinase pathway had no influence on NaPi-IIa localization under control conditions or after PTH stimulation. Furthermore, the PTH-induced decrease in NaPi-IIa protein could be reduced by PD 098059. These results suggest that the ERK1/2 MAPK kinase pathway plays a central role in the signalling of PTH leading to specific internalization and subsequent degradation of the type II NaPi-IIa cotransporter in the proximal tubule.     

ERK5对体外血小板活化及在体血栓的影响

目的:探讨细胞外信号调节激酶5(ERK5)对体外血小板聚集及在体血栓的影响及机制。方法:采用Western blot对人血小板中ERK5的表达及其在血小板活化后的磷酸化水平进行检测;采用血小板聚集仪检测ERK5特异性抑制剂XMD8-92对血小板聚集及致密颗粒释放的影响;采用Fe Cl3颈动脉血栓模型检测ERK5对在体血栓的影响;采用Western blot检测XMD8-92对蛋白激酶B(Akt)和人第10号染色体缺失的磷酸酶及张力蛋白同源蛋白(PTEN)磷酸化的影响。结果:人血小板中存在ERK5的稳定表达,其磷酸化水平在血小板活化后显著升高(P0.05)。XMD8-92可抑制多种血小板激活剂引起的血小板聚集和致密颗粒释放(P0.05)。Western blot结果表明,XMD8-92可通过下调PTEN Ser370位点磷酸化而增强PTEN的活性,从而抑制Akt的磷酸化,这种抑制效果也通过血小板特异PTEN基因敲除小鼠得到了验证。在体血栓研究表明,XMD8-92经尾静脉给药,可显著延长小鼠第一次颈动脉血栓的形成时间。结论:ERK5可通过影响PTEN的磷酸化调节Akt的活化,进而影响到体外血小板的聚集和在体血栓的形成。     

Association of decreased phosphorylation of ERK-2 with costimulation of rat T cell activation by MEK-1 inhibitors and TGF-beta1

Transforming growth factor-beta (TGF-beta) is usually known as an immunosuppressive cytokine, but we and others have shown stimulatory effects of TGF-beta on activation of Th2 T-lymphocytes. In the present investigation we have studied the effect of TGF-beta1 on phosphorylation of ERK, a MAP-kinase downstream of the Ras pathway. ERK is phosphorylated by MEK-1 and PD098059 and U0126 are specific inhibitors for this kinase. We demonstrate in the present study that these inhibitors abrogate the inhibitory effect of adh-splc (adherent-spleen cells) on activation of primary rat T-cells and induce a strong costimulatory effect almost as strong as we have previously shown with TGF-beta1. When TGF-beta1 is acting stimulatory on T-cell activation, it decreases phosphorylation of ERK-2 and thereby its activation. To investigate whether TGF-beta1 and MEK-1 inhibitors influence the same pathways, we compared their effects on cytokine profiles associated with SEA-induced rat T cell activation. TGF-beta1 induced IL-10 production, slightly decreased TNF-alpha production and decreased IFN-gamma production. The PD098059 inhibitor decreased both IFN-gamma and TNF-alpha production and together with TGF-beta1, it totally blocked IFN-gamma, TNF-alpha and IL-10 production. Thus TGF-beta1 and PD098059 showed overlapping but not identical effects on the cytokine pattern.     

A clinical and experimental study of platelet function in chronic renal failure

Coagulation and platelet function studies were performed on 24 normal subjects and 29 patients with chronic renal failure due to various causes. Thrombocytopenia was uncommon in the uraemic patients but there was reduced platelet retention in glass bead columns and platelet aggregation with adenosine diphosphate (ADP) and thrombin was slower and less complete than normal. The rate of platelet disaggregation in uraemic patients was significantly reduced. The abnormalities tended to be more severe in more uraemic subjects. In normal subjects no inter-relationships were observed between the various measurements of platelet activity. In patients there were significant interrelationships between the measurements of platelet aggregation with ADP and thrombin and between the measurements of aggregation and retention in glass bead columns. It is suggested that if a common pathway is involved in these reactions it is adversely affected in uraemia.Plasma coagulation defects were uncommon and present in only five of the uraemic subjects. Impaired prothrombin consumption apparently due to defective platelet function was present in half the patients but was not detected by a kaolin activation method. Although platelet coagulation function was activated during ADP aggregation and disaggregation in normal and uraemic subjects, it did not correlate in the latter with impairment of aggregation. It is suggested that aggregation and activation of platelet coagulant activity are not necessarily related aspects of platelet function. An effect of uraemic plasma on normal platelets was demonstrated by mixing experiments consistent with a humoral cause for the uraemic platelet defects.     

Change in activity of factor XIII in the plasma during platelet aggregation

During platelet aggregation induced by ADP or thrombin, a reduction in the activity of factor XIII is observed in platelet-enriched rat plasma. On the addition of active factor XIII (XIIIa) to this plasma, besides the increased ADP-induced aggregation, activity of factor III in the plasma is reduced. In the case of thrombin-induced platelet aggregation, addition of factor XIIIa is accompanied by a marked decrease in its activity in the plasma. The degree of aggregation under these circumstances is lower than in control samples. The observed differences in the character of aggregation taking place in the presence of factor XIIIa, when different aggregants (ADP and thrombin) are used, are evidently due to interaction between active factor XIII and thrombin added to the plasma as the aggregant.     

西咪替丁对血小板功能及血栓形成的影响

目的:观察西咪替丁(Cim)对血小板功能及血栓形成的影响。方法:在体外,Cim与血小板温育后,测量血小板聚集、血小板生成丙二醛(MDA)、血小板内游离钙[Ca²⁺]_i及血栓素B₂(TXB₂)含量。用电刺激大鼠颈动脉诱导血栓形成并观察Cim的促进作用。结果:Cim使ADP诱导的血小板聚集增加,凝血酶诱导的血小板[Ca²⁺]_i、MDA升高,TXB₂下降,使电刺激大鼠颈动脉闭塞时间(OT)缩短。结论:Cim增强血小板功能,促进血栓形成。     

Specific inhibitors of mitogen-activated protein kinase and PI3-K pathways impair immune responses by hemocytes of trematode intermediate host snails

To characterize molecular mechanisms regulating snail cellular immune responses, the contributions of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3-K) were examined in hemocytes of the trematode intermediate host snails Biomphalaria glabrata and Lymnaea stagnalis. Simultaneous measurement of phagocytosis/encapsulation and H2O2 production by hemocytes in the presence or absence of specific signal transduction inhibitors was used to assess the role of extracellular-signal regulated kinases 1 and 2 (ERK1/2), p38, JNK and PI3-K. Hemocyte spreading was significantly reduced in a dose-dependent manner by the ERK inhibitor, PD098059, and by wortmannin, a potent PI3-K inhibitor. The JNK inhibitor, SP600125, and the p38 kinase inhibitor, SB203580, had no effect on hemocyte spreading. Sheep red blood cell phagocytosis was significantly impaired by PD098059, SP600125, and SB203580. Hydrogen peroxide production during phagocytosis was severely inhibited by PD098059. Additionally, PD098059, but not the other inhibitors, significantly impaired the cellular encapsulation of trematode larvae and H2O2 production during encapsulation. These results suggest that MAPK and PI3-K signal transduction pathways play a pivotal role in the immune responses of snail hemocytes. PI3-K and ERK appear to strongly regulate cell motility. ERK, JNK and p38 contribute to phagocytosis-mediated signal transduction. ERK also play a major role in oxidative burst activation and the encapsulation of trematode larvae by snail hemocytes.