p38MAPK在小鼠早胚中的表达及其对胚泡发育的作用

目的观察p38丝裂原活化蛋白激酶(p38MAPK)在小鼠早胚中的表达及其作用,探讨p38MAPK与胚泡植入的相关关系。方法取小鼠胚胎的2细胞,4细胞,8细胞,桑葚胚,胚泡等不同发育阶段的早胚,用免疫组化及免疫荧光法测定早胚中p38MAPK的表达及变化情况。用体外胚胎培养技术:将2细胞期小鼠早胚随机分组,接种到添加不同浓度p38MAPK特异性抑制剂SB220025的培养液内进行培养,台盼蓝染色观察p38MAPK特异性抑制剂对小鼠早胚发育的影响。结果 p38MAPK在小鼠胚胎发育各细胞期均呈阳性表达,随着妊娠进展,其表达逐渐加强。体外实验显示:抑制剂组早胚不能发育到胚泡阶段,抑制剂恢复实验发现抑制剂组胚卵仍具存活能力。结论 p38MAPK在小鼠胚胎发育过程中起作用,p38MAPK特异性抑制剂可抑制小鼠早胚的发育。     

内毒素诱导p38MAPK信号转导作用的研究进展

The diseases caused by endotoxin have seriously affected human health. Previous studies have shown that p38 MAPK pathway is involved in the intracellular signal transduction induced by lipopolysaccharide (LPS), which plays an important role in the activation of inflammation-related cells to release inflammation mediator. Recently there have been some progresses in the isoforms distribution, substrate, molecular mechanism of regulating the release of inflammatory mediators, cellular specific activation and levels of p38 MAPK.     

p38MAPK在细胞内的定位及其对LPS刺激的反应

目的研究氯胺酮对内毒素休克小鼠72h存活率的影响.方法实验小鼠随机分为阳性对照组(LPS组,n=23)、氯胺酮1组(K1组,n=22)、氯胺酮2组(K2组,n=23),各组小鼠均经尾静脉注射LPS(25mg/kg),K1、K2组分别在LPS注射前30min肌肉注射氯胺酮(100mg/kg),K2组还在LPS注射后1、4、7h皮下注射氯胺酮(20mg/kg),观察72h存活率.结果3组小鼠的72h存活率分别为21.74%、54.53%、69.57%,K1、K2组值分别显著(P＜0.05)、非常显著(P＜0.01)地高于LPS组.结论氯胺酮可提高内毒素休克小鼠的72h存活率,此作用可能与其抑制LPS刺激后机体的炎症反应有关.     

p38MAPK通路参与炎性痛及电针干预的可能性

丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)是细胞内重要的信号传导系统之一.p38MAPK是丝裂原活化蛋白激酶的重要组成部分之一,在炎症性疼痛的发生发展过程中发挥着重要作用,已有大量资料表明电针具有较好的抗炎镇痛作用,同时有研究发现电针具有调节p38MAPK活化的作用,所以电针在炎性痛疾病中对p38MAPK通路干预存在可能性.     

基于p38-MAPK信号通路探讨和厚朴酚对结直肠癌细胞增殖、凋亡的影响

目的基于p38-丝裂原活化蛋白激酶(p38-mitogen activated protein kinases,p38-MAPK)探讨和厚朴酚(honokiol,HNK)对结直肠癌细胞增殖和凋亡的影响.方法将体外培养HCT116细胞分为对照组(0 μmol/L)、HNK低剂量(20 μmol/L)、中剂量(40 μmol/L)和高剂量组(80μmol/L)。采用CCK-8法和克隆形成试验检测细胞增殖,流式细胞仪检测细胞凋亡,Western印迹检测p38、p-P38、细胞增殖指数67(cell proliferation index 67,Ki67)、B细胞淋巴瘤/白血病-2(B-cell lymphoma-2,Bcl-2)、BCL2相关X蛋内(B-cell lymphoma-2 associated X protein,Bax)和裂解的半胱氨酸天冬氨酸蛋内酶3(cleaved Caspase-3)蛋白表达。将HCT116细胞分为空内对照组(0μmol/L)、HNK组(80μmol/L)、SB203580组(10 μmoL/L)和SB203580+HNK组(SB203580 10μmol/L+HNK 80 μmol/L),检测SB203580对各组细胞增殖、凋亡以及Ki67、cleaved Caspase3蛋白表达的影响。结果与对照组比较,HNK中高剂量组HCT116细胞的相对增殖率和克隆形成率明显降低,细胞凋亡率明显升高,Ki-67、Bcl-2蛋白表达明显下降,p-p38/p38、Bax和cleaved Caspase-3蛋白表达明显升高(P<0.05),且随着HNK剂量增加,其作用明显增加(P<0.05)。与空白对照组比较,SB203580组细胞相对增殖率明显升高,细胞凋亡率明显下降,Ki67蛋内表达明显升高,cleaved Caspase-3蛋白表达明显下降(P<0.05);与SB203580组比较,SB203580+HNK组细胞相对增殖率明显下降,细胞凋亡率明显升高,Ki67蛋白表达明显下降,cleaved Caspase-3蛋白表达明显升高(P<0.05)。结论HNK可以激活p38-MAPK信号通路,抑制HCT116细胞增殖,诱导细胞凋亡。     

P38丝裂原活化蛋白激酶在炎症因子诱导糖尿病肾病中的作用

近年来的研究发现,丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)是细胞将信号从细胞膜传递到细胞核的主要通路.其中p38MAPK可通过调节转化生长因子β1、核因子-κB及血管内皮生长因子等炎症因子的表达影响糖尿病肾病的进程.研究p38MAPK及其信号转导通路机制可能为糖...     

p38丝裂原活化蛋白激酶通路在非对称性二甲基精氨酸诱导内皮细胞通透性增高中的作用

目的探讨非对称性二甲基精氨酸(ADMA)对单层内皮细胞通透性的影响,以及氧化应激、p38丝裂原活化蛋白激酶(MAPK)通路在此过程中的作用。方法利用Transwell小室建立单层内皮细胞屏障结构,设立实验组和对照组,实验组经浓度25、50、100、200μmol/L ADMA作用24 h和100μmol/L ADMA分别刺激细胞4、8、16、24 h,或分别经烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶抑制剂、p38 MAPK抑制剂预处理细胞后再加入ADMA刺激。随后用异硫氰酸荧光素(FITC)标记的右旋糖酐漏出法测定单层内皮细胞的通透系数Pa值,并用免疫荧光染色显示细胞骨架及细胞间连接的形态学改变。结果 ADMA呈剂量及时间依赖性的增加单层内皮细胞的通透性,同时促进内皮细胞中应力纤维的形成并破坏细胞间连接。NADPH氧化酶抑制剂和p38 MAPK抑制剂均可对抗ADMA的上述作用。结论 ADMA通过引起氧化应激,激活p38 MAPK通路,改变细胞骨架及细胞间连接的结构,使单层内皮细胞通透性增高。     

高血脂对大鼠脑缺血再灌注损伤后大脑皮质p38丝裂原活化蛋白激酶表达的影响

目的:检测高血脂对脑缺血再灌注损伤后缺血侧大脑皮质p38丝裂原活化蛋白激酶(p38MAPK)表达的影响。方法:喂食高脂饲料建立高血脂动物模型,随后线栓法建立脑缺血再灌注模型。Zea-Longa神经行为学评分法记录大鼠神经行为改变,TTC染色检测脑梗死灶体积,免疫组织化学及免疫印迹检测大脑皮质p38MAPK表达水平。结果:高血脂脑缺血再灌注后神经行为损伤加重,且梗死灶体积较单纯脑缺血再灌注明显扩大。与假手术组比较,单纯脑缺血再灌注组和高血脂脑缺血再灌注组大脑皮质p38MAPK表达明显增加,再灌注2 h时其表达量即开始增高,再灌注24 h时达高峰,而后又降低。相同再灌注时间点,与单纯脑缺血再灌注组比较,高血脂脑缺血再灌注组p38MAPK表达增高。结论:高血脂脑缺血再灌注损伤中,高血脂可上调大脑皮质p38MAPK表达,促进细胞凋亡的发生及加重炎症反应,进而加重缺血再灌注损伤。     

P38 MAPK在大鼠脊髓损伤后胶质瘢痕形成早期对GFAP、vimentin表达的调控作用

目的探讨在大鼠脊髓损伤(SCI)后胶质瘢痕形成早期,p38丝裂原活化蛋白激酶(P38 MAPK)对神经胶质纤维酸性蛋白(GFAP)及波形蛋白(vimentin)表达的影响。方法将大鼠随机分为假手术组(sham group)、模型组(model group)、P38 MAPK特异性激动剂anisomycin组(anisomycin group)和P38 MAPK特异性抑制剂SB203580组(SB203580group)。脊髓夹伤模型制作成功后即分别在损伤区硬脊膜下注射0.9%氯化钠溶液、anisomycin和SB203580各10μL,假手术组只打开椎板暴露脊髓,不做其他处理。于术后第1、3、7及14天利用BBB评分评定大鼠后肢运动功能,用Western blot和免疫荧光标记技术检测GFAP和vimentin表达。结果术后第14天,模型组BBB评分显著低于假手术组(P0.01);SB203580组大鼠BBB评分显著高于模型组(P0.05)但仍低于假手术组(P0.01),而anisomycin组则显著低于模型组(P0.05)。术后第7和14天,模型组GFAP、vimentin的表达显著高于假手术组(P0.01);SB203580组GFAP、vimentin的表达均显著低于模型组(P0.05)但仍高于假手术组(P0.05),而anisomycin组GFAP、vimentin的表达均显著高于模型组(P0.05)。结论 SCI后胶质瘢痕形成早期P38 MAPK可调控GFAP、vimentin的表达,抑制P38 MAPK可降低GFAP、vimentin的表达,减轻大鼠SCI后胶质瘢痕形成,促进神经功能的恢复。     

羟考酮通过TLR4/p38MAPK通路抑制小胶质细胞的活化缓解大鼠骨癌痛北大核心CSCD

目的：通过制备骨癌痛大鼠模型,探究羟考酮是否通过调节Toll样受体4(TLR4)、p38丝裂原活化蛋白激酶（p38MAPK）参与调控小胶质细胞活化,缓解大鼠骨癌痛。方法：选取72只SD健康大鼠,随机分为假手术组、骨癌痛组、阳性药物组、羟考酮高浓度组、羟考酮低浓度组、羟考酮+TLR4激活组,每组12只。术前1 h、术后3 d、5 d、7 d、10 d进行动物行为学检测,测定各组大鼠机械缩足反射阈值（MWT）、后足热缩足反射潜伏期（TWL）;ELISA检测各组大鼠脊髓TNF-α、IL-1β水平;免疫荧光染色法检测各组大鼠补体C3受体（OX-42）表达水平;Western blot检测各组大鼠脊髓TLR4、p38MAPK、p-p38MAPK水平。结果：与假手术组相比,骨癌痛组大鼠术后各时间点TWL、MWT值显著降低,大鼠脊髓TNF-α、IL-1β、OX-42水平、TLR4蛋白表达量、p-p38MAPK/p38MAPK显著升高（P<0.05）;与骨癌痛组相比,阳性药物组、羟考酮高浓度组、羟考酮低浓度组大鼠术后各时间点TWL、MWT值显著升高,大鼠脊髓TNF-α、IL-1β、OX-42水平、TLR4蛋白表达量、p-p38MAPK/p38MAPK显著降低（P<0.05）;与羟考酮高浓度组相比,羟考酮低浓度组、羟考酮+TLR4激活组大鼠术后各时间点TWL、MWT值显著降低,大鼠脊髓TNF-α、IL-1β、OX-42水平、TLR4蛋白表达量、p-p38MAPK/p38MAPK显著升高（P<0.05）;阳性药物组与羟考酮高浓度组相比,各指标差异均无统计学意义（P>0.05）。结论：羟考酮可通过抑制TLR4/p38MAPK通路相关蛋白表达和激活减轻炎症反应,抑制小胶质细胞活化,缓解骨癌疼痛。     

白细胞介素1β通过p38信号通路上调系膜细胞表达白细胞介素6

目的：探讨p38信号通路(p38MAPK)在白细胞介素1(IL-1)β上调肾小球系膜细胞表达白细胞介素6(IL-6)中的作用。方法：应用Western Blotting检测p38MAPK在IL-1β诱导的肾小球系膜细胞炎症反应中的活化程度，应用逆转录-聚合酶链反应(RT-PCR)和ELJSA法检测IL-1β诱导的系膜细胞促炎症介质IL-6的表达水平并观察p38MAPK特异性抑制剂SB203580对其mRNA的转录和蛋白质生成的影响。结果：IL-1β以时间和剂量依赖方式刺激系膜细胞引起p38MAPK的活化，并明显上调系膜细胞IL-6表达。SB203580以剂量依赖方式从基因转录和翻译水平显著抑制IL-1β诱导的IL-6表达。结论：p38MAPK在IL-1β诱导的肾小球系膜细胞炎症反应产生炎症介质IL-6中起重要作用。     

p38MAPK在非对称性二甲基精氨酸所致内皮细胞骨架改变过程中的作用

目的：探讨非对称性二甲基精氨酸（asymmetricdimethylargine,ADMA）对人脐静脉内皮细胞（humanumbilicalveinendothelialcells,HUVECs）的细胞骨架改变的影响及p38丝裂原激活蛋白激酶（p38mitogen—activatedproteinkinase,p38MAPK）在该过程中的作用。方法：体外进行HUVEC培养,实验分为正常对照组、sB203580组、ADMA组（量效关系组、时效关系组）及sB203580＋ADMA组（SB203580＋ADMA量效关系组及SB203580＋ADMA时效关系组）。对各组细胞进行免疫荧光染色,利用激光扫描共聚焦显微镜观察肌动蛋白（F-actin）形态变化,图像分析软件行F-actin荧光灰度值分析,流式细胞仪行F．actin荧光定量分析。结果：ADMA可诱导HUVECs应力纤维形成,导致F．actin荧光灰度值、荧光定量增加;而SB203580可抑制ADMA的作用。结论：ADMA可呈时间及浓度依赖性地导致细胞骨架改变。p38MAPK特异性抑制剂SB203580可抑制ADMA对内皮细胞骨架的改变,提示p38MAPK参与了ADMA所导致的HUVECs内皮细胞的骨架改变。     

p38有丝分裂原激酶活化参与继发性脊髓损伤后神经细胞凋亡的实验研究

探索脊髓损伤(SCI)后p38有丝分裂原激酶(p38MAPK)的表达及其与神经细胞凋亡的关系。采用Allen’s法建立大鼠急性SCI动物模型,用蛋白印迹法和免疫组织化学染色方法检测SCI后p38MAPK和caspase-3表达的变化;采用实时定量PCR法检测SCI后caspase-3 mRNA的表达;用原位末端标记法(TUNEL)检测SCI后神经细胞凋亡。结果表明SCI后总p38MAPK表达无变化,但p38MAPK磷酸化明显增多,于伤后6h达高峰;伤后24hcaspase-3表达明显增加。TUNEL检测显示,伤后6h受损伤的脊髓组织有少许凋亡细胞出现,24h细胞凋亡最明显。鞘内注射p38MAPK抑制剂SB203580,明显减少caspase-3的表达,并减少细胞凋亡。以上结果显示SCI后损伤局部p38MAPK激活诱导了caspase-3基因表达,导致神经细胞凋亡;抑制p38MAPK可以阻止SCI后继发的神经细胞凋亡。     

p38 MAPK signaling mediates retinoic acid-induced CD103 expression in human dendritic cells

Retinoic acid (RA) is an active derivative of vitamin A and a key regulator of immune cell function. In dendritic cells (DCs), RA drives the expression of CD103 (integrin α_E), a functionally relevant DC subset marker. In this study, we analyzed the cell type specificity and the molecular mechanisms involved in RA-induced CD103 expression. We show that RA treatment caused a significant up-regulation of CD103 in differentiated monocyte-derived DCs and blood DCs, but not in differentiated monocyte-derived macrophages or T cells. DC treatment with an RA receptor α (RARα) agonist led to an increase in CD103 expression similar to that in RA treatment, whereas RARA gene silencing with small interfering RNA blocked RA-induced up-regulation of CD103, pointing to a major role of RARα in the regulation of CD103 expression. To elucidate RA-induced signaling downstream of RARα, we used Western blot analysis of RA-treated DCs and showed a significant increase of p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, DCs cultured with RA and a p38 MAPK inhibitor had a significantly reduced expression of CD103 compared with DCs cultured with RA only, indicating that p38 MAPK is involved in CD103 regulation. In summary, these findings suggest that the RA-induced expression of CD103 is specific to DCs, is mediated primarily through RARα and involves p38 MAPK signaling.     

Activation of the p38 MAP kinase pathway is required for foam cell formation from macrophages exposed to oxidized LDL

Endocytosis of oxidized low density lipoproteins (oxLDL) by macrophages, mediated by scavenger receptors, is thought to play a central role in foam cell formation and, thus, in the pathogenesis of atherosclerosis. OxLDL activates several MAP kinases, including the ERK, JNK and p38 MAP kinases, but the role of these activations in oxLDL uptake has not been studied. In the present investigation, we find that SB203580, a specific inhibitor of p38, blocks oxLDL-exposed J774 cells from becoming foam cells. Inhibition of foam cell formation by blockade of the p38 pathway is, at least in part, due to inhibition of oxLDL-induced up-regulation of the scavenger receptor CD36. Using pharmaceutical inhibitors and dominant active MAP kinase kinases, we demonstrated that activation of the p38 pathway, but not the ERK or JNK pathways, is necessary and sufficient to transactivate PPARgamma, a nuclear receptor that has recently been shown to play a pivotal role in oxLDL-induced CD36 expression. Our results for the first time demonstrate a regulation of CD36 by p38, and the importance of the p38 pathway in regulation of foam cell formation.     

Role of the human CD38 molecule in B cell activation and proliferation

Human CD38 is a surface molecule which has been attributed the function of a signaling channel leading to cellular activation and proliferation, an ectoenzyme with multiple function as well as an inducer of Ca²⁺ mobilization from cytoplasmic stores. The effect mediated by CD38 have been studied in different cell populations: the results obtained in human B cells are apparently contradictory, with CD38 simultaneously leading to apoptosis in early B cells while increasing survival in cells derived from lymph node germinal center. Other effects recently reported concern a different potential in terms of signaling in early B cells and derived cell lines or in more detailed disease models of human leukemia, namely B chronic lymphocytic leukemia cells.
To complete the picture of the effects mediated by CD38 in the B cell compartment, we have studied the signals elicited by ligation of the human molecule in mature B cells from circulating pool and also from spleen of normal individuals. The information obtained completes the picture of CD38 and mature B cells, where we also studied the contribution of relevant cytokines involved in maintenance and differentiation of these normal cells, namely IL-1 α, IL-2, IL-4 and IL-6. Our results indicate that human CD38 plays a key role as a co-receptor in mature B cells from normal individuals.     

桑色素对小鼠T淋巴细胞体外活化、增殖和细胞周期的影响

目的:研究桑色素(morin)对小鼠T淋巴细胞活化、增殖和细胞周期的影响。方法:以刀豆蛋白A(ConA)刺激培养的淋巴结来源的小鼠淋巴细胞,再以不同终浓度的morin与T细胞共培养,利用流式细胞术(FCM),检测早期T细胞活化的标志CD69分子的表达,以羧基荧光素双醋酸盐琥珀酰脂(CFDA-SE)染色检测T细胞的增殖;以碘化丙锭(PI)染色分析T细胞的细胞周期。结果:小鼠T细胞培养6 h后,未经ConA刺激的对照组中CD69 T的细胞比率为(2.97±0.12)%,经ConA刺激的CD69 T细胞的比率明显增高,达到(72.52±0.66)%,与对照组相比差别明显(P<0.01)。终浓度为25、50、100μmol/L的morin均下调CD69 T细胞的比率,其中,100μmol/L的morin抑制作用最强,为(48.95±0.81)%,与对照组比较具有统计学意义(P<0.01)。CFDA-SE染色分析显示,ConA组培养48 h和72 h的T细胞的增殖指数(PI)分别为(1.58±0.04)和(1.95±0.02),各浓度的morin对ConA刺激的T细胞增殖,具有明显地抑制作用,以100μmol/L的morin抑制作用最明显。培养48 h的ConA组T细胞的PI为(1.02±0.02)、培养72 h的ConA组T细胞的PI为(1.03±0.01),与相应时间的对照组比较,均有统计学意义(P<0.01)。PI染色后流式细胞术分析的结果表明,ConA组处于S期的T细胞的比率为(27.05±0.39)%,显著高于对照组的比率(5.10±0.07)%。morin组中S期的细胞比率较高。结论:Morin可显著抑制ConA刺激的T细胞活化及增殖;其对增殖的抑制作用主要表现为S期的细胞的阻滞。     

小鼠p38MAPK重组腺病毒载体的构建及其在成骨细胞系中的表达

目的:利用AdEasyTM system构建携带小鼠p38MAPK基因的重组腺病毒,感染成骨细胞系MC3T3-E1,检测外源p38MAPK在细胞中的表达。方法:用PCR的方法扩增p38MAPK基因,将其克隆到pMD18-T载体中,进行测序。经BglⅡ和HindⅢ双酶切后接入pShuttle-CMV穿梭载体,构建重组腺病毒的穿梭质粒pShuttle-CMV-p38MAPK。将经PmeI线性化的pShuttle-CMV穿梭载体与pAdEasyTMDNA电穿孔共转化BJ5183重组细菌,获取重组腺病毒质粒Ad-p38MAPK,再将经PacI线性化的Ad-p38MAPK重组病毒骨架质粒转染AD293包装细胞,包装并扩增病毒。用Ad-p38MAPK感染小鼠MC3T3-E1成骨样细胞,以Western blot法检测p38MAPK在小鼠成骨细胞中的表达。结果:构建并包装表达p38MAPK蛋白的重组腺病毒,该重组腺病毒在体外能有效感染小鼠成骨细胞系MC3T3-E1并高表达p38MAPK蛋白。结论:成功地构建了携带小鼠p38MAPK基因的重组腺病毒,为研究p38MAPK在成骨细胞中的作用奠定了实验基础。