褪黑素对小鼠胃癌细胞褪黑素膜受体MT1、MT2表达的影响

目的:探讨褪黑素对小鼠胃癌细胞褪黑素膜受体MT1、MT2表达的影响。方法:应用小鼠MFC前胃癌细胞建立荷胃癌小鼠模型,不同浓度褪黑素干预后,运用免疫组织化学、实时荧光定量PCR、免疫印迹定位定量检测MT1、MT2表达的变化。结果:移植瘤肿瘤细胞表达褪黑素膜受体MT1、MT2,褪黑素促进肿瘤组织膜受体MT1、MT2表达,并呈剂量依赖关系。结论:褪黑素上调褪黑素膜受体MT1、MT2的表达,说明褪黑素可能通过膜受体信号通路抑制胃癌生长。     

MT2受体介导褪黑素对小鼠前胃癌细胞的抑制作用

目的探讨褪黑素(MLT)通过褪黑素膜受体MT2抑制小鼠前胃癌(MFC)细胞增殖及其与丝裂原活化蛋白激酶(MAPKs)、磷脂酰肌醇-3激酶(PI3K)-Akt信号通路的关系。方法应用siRNA技术沉默MT2表达,观察褪黑素对小鼠前胃癌细胞的抑制作用及ERK1/2、Akt的磷酸化的影响。结果 1.siRNA介导的MT2沉默能明显拮抗褪黑素对胃癌细胞增殖的抑制作用;2.沉默MT2可部分阻断褪黑素抑制ERK1/2、Akt磷酸化的作用。结论褪黑素可通过MT2受体抑制ERK1/2、Akt的磷酸化从而抑制胃癌细胞增殖。     

褪黑素体内外对小鼠前胃癌细胞的增殖抑制与凋亡诱导作用

目的 探讨褪黑素(MLT)在体内外对小鼠前胃癌(MFC)细胞的增殖抑制与凋亡诱导作用.方法 体内实验:建立荷胃癌小鼠模型,随机分为5组:A.正常对照组;B.荷瘤空白对照组:每日注射生理盐水100 mg/kg;C.小剂量褪黑素组:荷瘤成功后(接种第6天起)每日腹腔注射MLT 25 mg/kg;D.中剂量MLT组:每日腹腔...     

Pin1抑制剂对小鼠植入前胚体外发育的影响

目的观察Pin1在小鼠植入前胚中的定位分布及其抑制剂胡桃醌(Juglone)对小鼠植入前胚体外发育的影响。方法免疫荧光染色观察不同发育阶段植入前胚中Pin1的分布;收集小鼠1-细胞胚,以KSOM培养液为对照组,以培养液中添加有不同浓度的Juglone为实验组,观察各组1-细胞胚体外发育情况;Real-time PCR检测体外发育2-细胞胚内干细胞转录因子(Sox2、Oct4、c-Myc和Klf4)m RNA表达水平。结果小鼠不同发育阶段植入前胚中都存在Pin1的表达,胞质和胞核内均有分布,且核内荧光着色明显比胞质强;10μmol/L和25μmol/L胡桃醌持续作用93h(注射人绒毛膜促性腺激素后27h~120h)使1-细胞胚阻滞于2-细胞阶段,极少能过渡至4-细胞胚(P0.01)。25μmol/L胡桃醌短时间作用18h(注射人绒毛膜促性腺激素后27~45h)同样使1-细胞胚发育阻滞于2-细胞胚(P0.01);25μmol/L胡桃醌组2-细胞胚内Sox2 m RNA表达水平低于KSOM组(P0.05),而Klf4、cMyc和Oct4 m RNA表达水平无明显变化(P0.05)。结论小鼠1-细胞胚之后,Pin1主要定位在细胞核内,提示其可能参与细胞转录活动。且Pin1抑制剂(胡桃醌)能使2-细胞胚至4-细胞胚过渡率明显降低,并使干细胞转录因子Sox2 m RNA表达下调,提示Pin1在小鼠植入前胚早期发育阶段具有一定作用,其参与调控合子基因组激活与干细胞转录因子Sox2有关。     

血管紧张素Ⅱ受体1和受体2在小鼠肾脏发育中的表达

目的观察小鼠肾脏发育中血管紧张素Ⅱ受体1(AngiotensinⅡreceptor type1,AT_1)和受体2 (AT_2)的表达特征,探讨小鼠肾脏发育过程中AT_1和AT_2的作用及相互关系。方法应用免疫组织化学技术、免疫印迹法(Western blot)并结合体视学方法检测胚龄12、14、15、16、18d及生后日龄1、3d小鼠肾脏发育中AT_1和AT_2的表达。结果AT_1和AT_2均首先出现在输尿管芽,然后出现在肾小管,生后表达逐渐减弱。早期肾小体内AT_2丰富表达,随着肾小体的成熟表达量逐渐降低。结论在小鼠肾脏发育中,AT_1可能与输尿管芽分支不断延长以及肾小管的增殖密切相关,AT_2可能与输尿管芽和肾小体的相互诱导相关。     

褪黑素体内外对胃癌Th1/Th2/Th17型细胞因子表达的影响

目的 探讨褪黑素（MLT）体内外对胃癌Th1/Th2/Th17型细胞因子干扰素（IFN）-γ、肿瘤坏死因子（TNF）、白细胞介素（IL）-2、IL-4、IL-6、IL-10、IL-17a表达的影响。 方法 1.构建荷胃癌小鼠模型,共32只雄性615小鼠全部荷小鼠前胃癌（MFC）细胞后随机分为4组,分别用0、25、50、100 mg/kg剂量褪黑素进行腹腔注射并测量肿瘤长短径。干预1周后取外周血,剥离肿瘤组织进行称重和测量。2.将MFC接种于6孔细胞培养板中,贴壁24 h后分别用0、2、4、6、8、10 mmol/L浓度褪黑素干预,24 h后形态学观察并收集相应上清液。3.采用ELISA检测外周血清中褪黑素的浓度变化。采用流式液相多重蛋白定量技术流式微珠阵列（CBA）分别检测外周血清、细胞上清液中Th1/Th2/Th17型相关细胞因子的浓度变化。 结果 1.成功建立荷胃癌小鼠模型。与阴性对照组相比, 褪黑素中、高剂量组小鼠外周血清褪黑素浓度明显升高,肿瘤体积明显下降。与阴性对照组相比,中剂量组血清中IL-10浓度明显增加;高剂量组血清IFN-γ、IL-2、IL-10浓度均明显增加。2.褪黑素干预MFC细胞实验中,与空白对照组相比,6、10 mmol/L 褪黑素组中IFN-γ浓度显著降低;4、6、8、10 mmol/L 褪黑素组中IL-6浓度明显降低,而 6 mmol/L 褪黑素组IL-10浓度明显升高。以上差异均具有统计学意义（P<0.05）。 结论 褪黑素体内外对胃癌细胞均有抑制作用且可能通过调节Th1/Th2/Th17细胞相关因子IFN-γ、IL-2、IL-6及IL-10的表达起增强肿瘤免疫作用。     

黄芪多糖联合顺铂对小鼠Lewis肺癌细胞肺转移的影响

目的观察黄芪多糖(APS)联合顺铂(DDP)对小鼠Lewis肺癌细胞肺转移、核因子(NF)-κB、P38、P53及Caspase-9表达的影响。方法将90只Lewis小鼠随机分为模型组、顺铂组(6mg/kg DDP)、APS组(50、100、200)mg/kg,联合用药组[1/2 DDP+APS,即:(3+25、3+50、3+100)mg/kg]。各组均于造模第2天起用药,APS每日1次,DDP每周1次,连续20d。观察肿瘤肺转移情况,采用Real-time PCR法和Western blotting法检测肿瘤组织中NF-κB、P38、P65蛋白和基因,并用免疫组织化学检测Caspase-9的表达。结果与模型组相比,各治疗组均可降低肺转移灶数目(P0.05或P0.01);除P38外,APS中、高剂量组可使小鼠Lewis肺癌组织中NF-κB p65、P53表达降低,Caspase-9表达增高;联合用药高剂量组作用则接近DDP组。结论 APS可抑制小鼠Lewis肺癌细胞的转移,抑制NF-κB和丝裂原活化蛋白激酶(MAPK)信号通路的激活,这可能是其抑制肿瘤转移的机制之一;APS与减半剂量的DDP铂类化疗药物联合使用时,作用增强,APS对DDP有增效减毒作用。     

过氧化物酶体增殖物激活受体δ在原代小鼠成肌细胞分化过程中对肌球蛋白重链亚型的影响#br#

目的 探讨过氧化物酶体增殖物激活受体δ(PPARδ)在成肌细胞分化过程中对肌球蛋白重链(MHC)亚型的影响。方法 分离5只新生小鼠原代成肌细胞,设立对照组、PPARδ激动剂组和PPARδ拮抗剂组;各组细胞在分化培养基中培养2、4、6 d收取细胞样品,MHC免疫荧光鉴定各组细胞分化情况;Real-time PCR及免疫荧光检测MHC各亚型表达水平;Western blotting检测各组细胞MHC蛋白表达水平。结果 成肌细胞分化过程中PPARδ激动剂组形成的肌管和MHC蛋白表达量高于对照组和PPARδ拮抗剂组。通过比较PPARδ激动剂处理后MHC 4个亚型随时间变化规律发现,分化2 d MHCⅠ、MHCⅡa 的上调作用显著;分化4 d MHCⅠ、MHCⅡa、MHCⅡx上调作用显著,其中MHCⅡa上调幅度最大;分化6 d MHCⅠ、MHCⅡa上调作用显著,其中MHCⅡa上调幅度最大。结论 PPARδ可以促进小鼠原代成肌细胞分化,在分化过程中显著上调MHCⅡa表达。     

磷酸化的细胞外信号调节蛋白激酶1/2在正常小鼠脑内的免疫细胞化学定位

细胞外信号调节蛋白激酶(ERK)1/2是重要的信号转导分子。现已知在正常动物的中枢神经系统内有其活化形式即磷酸化的ERK1/2(pERK1/2)分子的存在,但其在小鼠脑内的分布目前还没有全面的观察。本研究用免疫组织化学技术(ABC法)研究了pERK1/2样免疫反应阳性产物在脱臼处死的正常小鼠全脑内的分布。结果发现pERK1/2在正常小鼠脑内有广泛的表达,阳性产物主要存在于神经元,亦见于个别白质内的胶质细胞,脑膜及室管膜细胞也有表达。强阳性表达的核团主要有:岛皮质、视听皮质、边缘前皮质、扣带前皮质、海马垂直部、弓状核、蓝斑和小脑Purkinje细胞;中等阳性表达的核团主要有:感觉运动皮质、外侧隔区、内侧杏仁核、皮质杏仁核和外侧杏仁核、丘脑室旁核前部、视交叉上核、穹隆下器、终板血管器、前腹侧视前核和下丘脑背内侧核;弱阳性表达的核团主要有:视上核、下丘脑室旁核大细胞部、下丘脑后区、顶盖前区、室周灰质腹外侧柱、A5区、孤束核和延髓腹外侧网状结构等。本文结果观察到pERK1/2在正常小鼠脑内广泛存在,提示pERK1/2作为重要的信号转导分子,可能参与许多脑区在正常状态下的功能活动,揭示其分布特点为了解其在脑内的多样性功能提供了形态学依据。     

肾素前体激活人肾系膜细胞ERK1/2诱导TGF-β的表达

目的:观察肾素前体(prorenin)能否激活体外培养的人肾系膜细胞(HRMCs)膜表面的肾素(前体)受体[(P)RR],从而活化细胞外信号调节激酶1/2(ERK1/2)信号通路。方法:体外培养HRMCs。以血管紧张素Ⅱ1型受体(AT1)阻断剂olmsartan和血管紧张素Ⅱ2型受体(AT2)阻断剂PD123319孵育细胞,再以prorenin刺激细胞,并用ERK1/2抑制剂PD98059和HRP(handle region peptide)干预。用免疫蛋白印迹方法(Western blotting)和酶标记免疫吸附测定方法(ELISA)测定目的蛋白水平,以逆转录聚合酶链反应(RT-PCR)方法测定mRNA水平。结果:Prorenin作用于体外培养的HRMCs后,在阻断AT1和AT2受体时,能激活(P)RR使ERK1/2磷酸化增强、转化生长因子(TGF-β)水平增高。这种ERK1/2信号通路的激活是(P)RR激活后效应,并可被ERK1/2信号通道阻断剂PD98059阻断。HRP不能阻断(P)RR激活所致的ERK1/2磷酸化,不能使TGF-β表达降低。结论:在体外培养的HRMCs中,prorenin能够激活(P)RR,使ERK1/2信号通道活化导致TGF-β水平增高。而HRP不能阻断(P)RR激活后ERK1/2的磷酸化及TGF-β表达的增多。     

Stimulatory effects of a melatonin receptor agonist, ramelteon, on BDNF in mouse cerebellar granule cells

Melatonin receptor activation has been linked to the regulation of neurotrophic factors, including the brain-derived neurotrophic factor (BDNF). To further characterize the effects of melatonin receptor stimulation on neuronal BDNF, we used a clinically available novel agonist for MT1 and MT2 melatonin receptors, ramelteon. Primary cultures of cerebellar granule cells (CGC) have been established as an in vitro model for studying neuronal BDNF. We took advantage of the availability of MT1- and MT2-deficient (knockout; KO) mice to employ primary CGC prepared from wild type (WT), MT1 KO, and MT2 KO mice. We investigated the effects of ramelteon on BDNF protein and mRNA content. Administered in a low nanomolar range, ramelteon increased BDNF protein content in all three types of mouse CGC. This ramelteon-triggered BDNF protein elevation was not preceded by a BDNF mRNA increase. However, it was prevented by treatment of cultures with a protein synthesis inhibitor cycloheximide. These results demonstrated that the MT1/MT2 melatonin receptor agonist ramelteon is capable of increasing BDNF protein in neurons expressing either of the two melatonin receptor types and that this action of ramelteon involves translational mechanisms. Further research is needed to explore the putative influence of ramelteon on BDNF-associated neuroplasticity.     

VEGF Increases Paracellular Permeability in Brain Endothelial Cells via Upregulation of EphA2

Neurological disorders are associated with an increase in the permeability of human brain microvascular endothelial cells (HBMEC). Our previous findings have indicated that EphA2 could increase the permeability of HBMEC. Recent evidence has linked EphA2 and vascular endothelial growth factor (VEGF) to abnormalities in the vascular response. However, it is unclear whether EphA2 is involved in the VEGF‐induced changes in the permeability of HBMEC. Here, changes in permeability were determined by measuring transendothelial electrical resistance (TEER) and the flux of FITC‐dextran. We found that knockdown of EphA2 in HBMEC abolished the VEGF‐induced reduction in TEER and increase in flux of fluorescent dextran. Moreover, VEGF‐induced redistribution of ZO‐1 and the recruitment of detergent‐soluble occludin and claudin‐5 were also prevented. Further results showed that VEGF increased EphA2 expression in a time‐ and dose‐dependent manner, which was inhibited by a neutralizing antibody against VEGFR2 or SU1498. VEGF‐induced EphA2 expression was suppressed in the brain endothelium following treatments with the PI3K inhibitor LY294002, Akt inhibitor or transfection with the dominant‐negative PI3K mutants (Δp110). Similar results were obtained when ERK1/2 activation was inhibited by PD98059 or ERK1/2 siRNA transfection. Our data suggest that VEGF upregulates the expression of EphA2 in HBMEC through binding to VEGFR2 and subsequently activating the intracellular PI3K/Akt and ERK1/2 signaling pathways, which contribute to an increase in paracellular permeability. These data reveal a novel role for VEGF as a regulator of EphA2 expression in the brain endothelial cells and provide insights into the molecular mechanisms of VEGF‐mediated changes in paracellular permeability. Anat Rec, 297:964–972, 2014. © 2014 Wiley Periodicals, Inc.     

Synergistic effects of snail and quercetin on renal cell carcinoma Caki-2 by altering AKT/mTOR/ERK1/2 signaling pathways

Renal cell carcinoma has become the most common subtype of kidney cancer, and has the highest propensity to manifest as metastatic disease. Because of lack of knowledge in events that correlated with tumor cell migration and invasion, few therapeutic options are available. Therefore, in current study, we explore the anti-tumoral effect of a potential chemopreventive natural product, quercetin, combined with anti-sense oligo gene therapy (inhibiting Snail gene). We found that either one of them had the remarkable effects in suppressing cell proliferation and migration, inducing cell cycle arrest and apoptosis in a ccRCC cell line, Caki-2 cells. The combination of both means provides even strong suppressive effects toward these ccRCC cells. Our study, for the first time, provides the possibility of using a novel treatment for renal cancer, by combining natural product and gene therapy.     

食管鳞状细胞癌组织中miR-21与ERK1/2表达及临床意义的研究

目的：研究miR-21和ERK1/2在食管鳞状细胞癌(esophageal squamous carcinoma,ESCC)组织中的表达并分析二者的临床意义。方法：原位杂交技术和免疫组化方法分别检测80例食管鳞状细胞癌及癌旁正常组织中miR-21和ERK1/2的表达,Spearman相关统计方法分析miR-21和ERK1/2表达的相关性,并分析二者与ESCC患者临床病理资料的关系。结果：miR-21和ERK1/2在ESCC组织中的阳性表达率和表达量都显著高于癌旁正常组织,尤其在有淋巴结转移的ESCC组织中显著增高,但在ESCC不同性别、年龄、民族、病理类型、肿瘤侵及范围和肿瘤大小间的差异无统计学意义。进一步分析发现miR-21与磷酸化的ERK1/2(p-ERK1/2)在ESCC及癌旁正常组织的表达呈正相关,相关系数为0.570。结论：miR-21和p-ERK1/2与ESCC发生发展密切相关,二者可能协同通过促进肿瘤细胞淋巴结转移增加ESCC的恶性程度。     

肿瘤坏死因子α调控ERK1/2信号通路促进长骨骨样细胞凋亡

文题释义：肿瘤坏死因子α：是一种主要由巨噬细胞和单核细胞产生的促炎细胞因子,并参与正常炎症反应和免疫反应。MAPK信号通路：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、分裂和分化等多种生理及病理过程,ERK是MAPK通路中极其重要的组成部分之一。激活的ERK1/2通过核转位进入细胞核,激活其下游的相关转录因子或激活胞质和胞核激酶等调控细胞的生存、增殖和分化。背景：肿瘤坏死因子α作为促炎因子可诱导成骨细胞凋亡,增强破骨细胞功能,从而造成炎症性骨破坏,但是其作用机制尚不明确。 目的：探讨炎症因子肿瘤坏死因子α对长骨骨样细胞MLO-Y4增殖、凋亡的影响及可能机制。方法：将MLO-Y4细胞分为对照组、肿瘤坏死因子α组、ERK1/2抑制剂组。肿瘤坏死因子α组用含50 μg/L肿瘤坏死因子α的α-MEM完全培养基孵育24 h,ERK1/2抑制剂组用含50 μmol/L PD98059的α-MEM完全培养基孵育24 h,对照组单纯采用α-MEM完全培养基孵育24 h,采用MTT法检测细胞增殖能力,流式细胞术检测细胞凋亡情况,丙二醛、超氧化物歧化酶、谷胱甘肽过氧化物酶试剂盒检测细胞氧化应激水平,用Western blot法测定PCNA、cleaved caspase-3、p-ERK1/2、ERK1/2的蛋白水平。结果与结论：①与对照组相比,50 μg/L肿瘤坏死因子α处理24 h后,细胞增殖能力下降,凋亡率上升;细胞脂质过氧化物丙二醛水平显著增加,而抗氧化物酶超氧化物歧化酶和谷胱甘肽过氧化物酶活性显著降低;②与对照组相比,肿瘤坏死因子α组细胞增殖相关蛋白PCNA的表达显著降低,细胞凋亡相关蛋白cleaved caspase-3的表达显著升高,p-ERK1/2的表达降低,而总蛋白ERK1/2的表达基本保持不变。ERK1/2抑制剂组上述指标与肿瘤坏死因子α组无显著差异;③结果表明,50 μg/L肿瘤坏死因子α可使长骨骨样细胞MLO-Y4增殖能力下降,细胞凋亡增多,其作用机制可能与抑制MAPK-ERK1/2信号通路的活化有关。ORCID: 0000-0003-0863-8618(史方富)中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

葛根素通过ERK1/2信号通路促进内皮细胞增殖

目的:体外观察葛根素对人脐静脉内皮细胞(HUVEC)的促增殖作用,并对其机制进行初步探讨。方法:采用四甲基偶氮唑蓝(MTT)法检测不同浓度葛根素对HUVEC细胞增殖的影响;流式细胞术检测细胞凋亡;Western blot检测p-ERK1/2蛋白表达水平。结果:葛根素能剂量依赖性促进HUVEC增殖,在80μM效果最明显,且作用36h时,能显著减少早期凋亡,激活ERK1/2磷酸化。结论:葛根素素对人脐静脉内皮细胞有明显的促进增殖作用,其作用可能与ERK1/2的活化有关。     

汉防己甲素对人鼻咽癌CNE1和CNE2细胞株的放疗增敏作用

目的:探讨最大非毒性剂量汉防己甲素(tetrandrine,Tet)对人鼻咽癌细胞株CNE1和CNE2的放疗增敏机制。方法:分别采用最大非毒性剂量Tet(对CNE1细胞为1.5μmol/L;对CNE2细胞为1.8μmol/L)、4 Gy放疗和最大非毒性剂量Tet联合放疗处理CNE1和CNE2细胞;流式细胞术检测各组细胞周期分布,Western blot检测各组细胞γ-H2AX、cleaved caspase-3、p-CDC25C、CDK1、p-CDK1、cyclin B1、ERK和p-ERK的蛋白水平。结果:最大非毒性剂量Tet联合放疗后可上调CNE1和CNE2细胞中γ-H2AX的表达。放疗组CNE1和CNE2细胞G_2/M期的比例分别为(18.09±0.42)%和(18.48±1.32)%,联合处理组CNE1和CNE2细胞G_2/M期的比例降为(15.88±1.04)%和(13.80±0.82)%,与放疗组比较差异有统计学意义(P0.05)。联合处理可增加放疗所致的cleaved caspase-3的蛋白水平(P0.05)。不同浓度Tet处理CNE1和CNE2细胞后,p-CDC25C和p-CDK1的蛋白水平随Tet浓度增加而升高(P0.05),CDK1的表达无明显改变;最大非毒性剂量Tet不影响p-CDC25C、p-CDK1和CDK1的蛋白水平。在CNE1和CNE2细胞中,联合处理可明显降低放疗引起的p-CDC25C和p-CDK1的蛋白水平(P0.05),上调放疗后cyclin B1的表达,而对总CDK1的表达无明显调节作用;联合处理可显著抑制放疗所致的pERK蛋白水平(P0.05)。结论:最大非毒性剂量Tet可以增加放疗引起的CNE1和CNE2细胞的DNA断裂及细胞凋亡,其放疗增敏的机制可能与Tet调控ERK/CDC25C/CDK1/cyclin B1通路、去除放疗导致的G2/M期阻滞有关。     

Expression of Akt and Mdm2 in human esophageal squamous cell carcinoma

The Akt-Mdm2 pathway plays an important role in carcinogenesis in a variety of malignant tumors. However, the Akt-Mdm2 pathway in esophageal squamous cell carcinoma (ESCC) has not been fully studied. We investigated the proteins and mRNA expression of Akt and Mdm2 to elucidate the roles of these proteins in ESCC. We also examined the effect of Akt knockdown on Mdm2 expression in ESCC cells. ESCC tissue samples were obtained from 23 individuals who underwent surgical resection with no preoperative treatment. Akt1-3 and Mdm2 gene and protein expression were analyzed. The effect of siRNA-mediated Akt knockdown on Mdm2 expression was also studied, using ESCC cell lines. Akt1 and Mdm2 immunoreactivity was detected in 77.8 and 66.7% of tumor specimen from ESCC patients, respectively. Akt1 and Mdm2 mRNA expressions were correlated and significantly elevated in tumor tissue (p < 0.0001 and p < 0.05, respectively). The siRNA-targeted reduction of each Akt isoform reduced Mdm2 protein expression. The overexpression of Akt1 and Mdm2 may be related to esophageal carcinogenesis. Furthermore, Akt expression regulates Mdm2 expression, which may in turn regulate the function of wild-type p53. These results may provide the basis for future preventative or clinical therapies for esophageal cancer.