氢吗啡酮对炎性痛大鼠脊髓 ERK1 / 2 信号通路的影响

摘要:目的 探讨氢吗啡酮对炎性痛大鼠脊髓 ERK1 / 2 信号通路的影响。 方法 选择 SPF 级雄性大鼠 30 只,随机分为 3 组,正常组、模型组和氢吗啡酮组;检测各组大鼠热痛阈和机械痛阈;ELISA 检测血清中 TNF-α、IL-6 和 IL-10的含量;Western blot 检测各组大鼠脊髓中 ERK1 / 2 和 p-ERK1 / 2 蛋白表达水平;qRT-PCR 法检测各组大鼠脊髓中ERK1 / 2 mRNA 表达水平。 结果 与正常组相比,模型组大鼠血清炎症细胞因子 TNF-α 和 IL-6 含量明显增高,IL-10 含量明显降低,且术侧后肢热痛 阈 显 著 缩 短 及 机 械 痛 域 明 显 降 低,差 异 均 有 统 计 学 意 义 ( P < 0. 05) ;脊 髓 中ERK1 / 2 mRNA 表达水平、ERK1 / 2 及 p-ERK1 / 2 蛋白表达水平均显著升高,差异有统计学意义( P< 0. 05) 。 注射氢吗啡酮后,与模型组相比,大鼠血清 TNF-α 和 IL-6 含量显著降低而 IL-10 含量显著升高,热痛阈显著延长及机械痛域明显升高,差异均具有统计学意义( P<0. 05) ;氢吗啡酮组 ERK1 / 2 mRNA 表达水平、ERK1 / 2 和 p-ERK1 / 2 蛋白表达水平均明显降低( P<0. 05) 。 结论 氢吗啡酮可有效改善弗氏完全佐剂诱导的大鼠炎性疼痛,其抗炎镇痛机制与降低大鼠机体炎性因子的产生,并抑制 ERK1 / 2 信号通路的活化有关。     

Expression and regulation of metalloproteinases-2, -9 and tissue inhibitors of metalloproteinases in rat corpus luteum

The expression and regulation of metalloproteinases-2, -9 (MMP-2, -9) and their tissue inhibitors TIMP-1, -2, -3 mRNA were studied in this experiment. In the PMSG- hCG primed pseudopregnant rat, MMP-2, -9 mRNA levels were the highest at Day 1, decreased from Day 4, and reached the minimal level at Day 8, then increased at Day 14; no significant changes were observed in TIMP-2 mRNA expression from Day 1 to Day 14; TIMP-3 mRNA expression was the lowest at Day 1, increased from Day 4, reached the maximal level at Day 8, and persisted to Day 14. TNF-αcould significantly increase the expression of MMP-2, -9 and TIMP-1 mRNA in the in vitro perfused pseudopregnant CL, and decrease the expression of TIMP-3 mRNA, but had no effect on TIMP-2 mRNA expression. The results indicate that MMP-2, -9 and TIMP-1, -2, -3 might be involved in the regulation of CL function and maintenance of CL structure via their coordinated gene expression. TNF-α could inhibit luteal regression via increasing MMP-2, -9 and TIMP-1 mRNA in the in vitro perfused pseudopregnant ovary.     

Ras /MAPK / ERK 通过协同 NF- KB 促进肝癌细胞中 cyclinD1 的表达

摘要: 目的 探讨 Ras 癌基因在体内诱导肝肿瘤发生过程中促进 cyclin D1 表达的分子机制。方法 利用 H-ras12V 转基因雄鼠肝肿瘤组织、肿瘤周围组织以及非转基因雄鼠肝组织进行病理分析和总 RNA 及蛋白质的提取。应用 qRT-PCR 和 Western blot 方法检测 cyclin D1 的表达以及调控 cyclin D1 表达的相关信号通路激活状态。采用 miRNA 测序、生物信息学分析和 qRT-PCR 验证方法检测 miR-5102 基因的表达水平。结果 与非转基因雄鼠肝组 织和肿瘤周围组织相比,肿瘤组织中 cyclin D1 基因的 mRNA 和蛋白表达水平显著升高。与非转基因雄鼠肝组织 和肿瘤周围组织相比,在肿瘤组织中 MAPK 信号转导通路中的 ERK 信号分子的蛋白表达水平和磷酸化水平都显 著升高,在肿瘤组织中成高激活状态,NF-KB 信号通路中的抑制因子 IKB 蛋白水平显著降低。miR-5102 的表达水 平没有显著变化。结论 在 Ras 癌基因诱导的肝肿瘤发生过程中,主要通过激活 MAPK/ERK 和 NF-KB 信号通路促进 cyclin D1 的表达。     

Expression of TGF-β2 in LECs of Age-Related Nuclear, Cortex Cataract and the Relationship among TGF-β2, Proliferation,Apoptosis and Transdifferentiation

0 IntroductionAsgue-br-eclaaptseudl acrat acraatcatr ianctc l,ud aems ocnogrte xwh,incuhcl,e acro ratnexdcataract and nuclear cataract are more i mportant .Cortex cataract is different from nuclear cataract inoccurring,developing and clinic display,evenin his-tology. The pathogenesis of age-related cataract hasnot been perfectly described.But it has been verifiedthat thereis alayer oflens epithelial cells (LECs) un-der vertebrates’lens anterior capsule, which is i m-portant in maintaining t…     

1-磷酸鞘氨醇受体1促进心肌梗死后单核/巨噬细胞在梗死心肌组织中浸润和聚集

目的探讨心肌梗死后S1PR1对单核/巨噬细胞功能的影响及作用机制。方法构建急性心肌梗死模型;实验分为实验组(S1PR1激动剂SEW2871处理组)、阴性对照组(DMSO处理组),各组小鼠分别在药物处理后0、5、28 d三个时间点进行对比研究;流式细胞术检测心肌组织、肝脏、肾脏及外周血等组织中单核/巨噬细胞的数量;荧光显微镜下观察心肌组织单核/巨噬细胞荧光表达情况;构建小鼠pCMV.DR8和pMD2.G慢病毒载体并感染小鼠RAW264.7巨噬细胞,筛选最适感染复数(MOI);实验分为S1PR1基因沉默组、过表达组、U0126(ERK信号通路阻断剂)处理组及空白对照组;采用Transwell小室分析细胞迁移情况;通过RAW264.7与HUVECSs共培养检测细胞粘附功能。结果 (1)实验组小鼠心肌组织中单核/巨噬细胞数量明显多于对照组(P 0.05);(2)体外试验显示,SEW2871促进RAW264.7巨噬细胞的粘附和迁移,S1PR1基因敲减后,上述作用明显降低;(3) U0126预处理后,SEW2871的促进细胞粘附和迁移作用显著减弱。结论 S1PR1通过ERK信号通路促进单核/巨噬细胞的粘附和迁移作用。     

Regulation of mouse blastocyst adhesion,outgrowth and secretion of matrix metalloproteinase-2 by cGMP and nitric oxide <Emphasis Type="Italic">in vitro</Emphasis>

Nitric oxide (NO) is a multifunctional messenger molecule produced through oxidation of L-arginine to L-citrulline by enzyme NO synthase (NOS). In the current study, mouse blastocysts were cultured in the different media, and the implantation capacity of blastocyst was evaluated by evaluating the percentage of embryos adhesion and outgrowth after culture for 12, 24 or 48 h. Matrix metalloproteinase-2 (MMP-2) mRNA was detected by RT-PCR, and MMP-2 protein was detected by gelatin zymography. Inhibition of blastocyst adhesion and outgrowth was observed in embryo cultured with 500 μmol/L NOS inhibitor N^G-mono-methyI-L-arginine (L-NMMA) alone; however, 100 μmol/L S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, and 20μmol/L cGMP analogue, 8-Br-cGMP could block this inhibition. The expression and production of MMP-2 in the blastocysts were suppressed by L-NMMA, and SNAP or 8-br-cGMP could reverse this suppression. These results suggest that NO induces embryo implantation by cGMP signaling pathway.     

Effect of thrombin on the apoptosis of hippocampal neurons in vitro

Hippocampal neurons were treated by thrombin and thrombin receptor activating peptides (TRAP). Cell survival rate was decreased in a dose-dependent manner by MTT assay. The numbers of apoptotic cell and apoptotic rate of hippocampal neurons treated by different concentrations of thrombin were increased in a dose-dependent manner by terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labeling (TUNEL) method and Flow Cytometry. When the concentration of thrombin is 40 U/mL, TUNEL positive cells and apoptotic rate of hippocampal neurons reached peak value, were 27.3±4.0 and (29.333±4.633)%, respectively. Immunocytochemistry assay show that Bcl-2 protein expression was down-regulated and Bax protein expression was up-regulated with the concentration of thrombin increased. TRAP can mimic the effect of thrombin to induce apoptosis on hippocampal neurons. These data demonstrated that thrombin induced hippocampal neuron apoptosis in a dose-dependent manner through activating protease-activated protein-1 (PAR-1). The change in expression of Bcl-2 and Bax was related with the effect of high concentration thrombin induced apoptosis on hippocampal neurons. Foundation item: Supported by the Natural Science Foundation of Hainan Province (N30215) Biography: YANG Wen-qiong (1968-), female, Ph.D. candidate, research direction: cerebrovascular disease.     

Cloning,expression of<Emphasis Type="Italic">Crocus sativus</Emphasis> phytoene desaturase gene and preparation of antiserum against it

A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET-21a(+) and over-expressed inE. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×10⁵. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma. Foundation item: Supported by the Doctoral Foundation of the Ministry of Education, P. R. China and the Young Science Foundation of Sichuan University (Grant 0020405505012) Biography: Bai Jie (1968-), female, Ph. D candidate, research direction: plant developmental biology and reproductive engineering.     

熏烟加寒燥环境对大鼠基质金属蛋白酶及气道细胞外基质成分的影响

 为了探讨熏烟加新疆寒燥环境对大鼠血清基质金属蛋白酶-9(MMP-9)和肿瘤坏死因子-α(TNF-α)、肺泡灌洗液中透明质酸、层粘连蛋白含量及骨组织中基质金属蛋白酶-1(MMP-1)mRNA含量的影响,利用熏烟并施以寒燥环境刺激的方法建立大鼠模型,使用酶联免疫吸附测定法(Enzyme-Linked Immunoadsordent Assay,ELISA)测其外周血血清中MMP-9和TNF-α以及肺泡灌洗液中透明质酸、层粘连蛋白含量,使用荧光定量PCR法检测骨组织中MMP-1 mRNA的表达。结果表明,大鼠血清中模型组血清MMP-9含量高于对照组(P<0.01),血清中TNF-α、肺泡灌洗液中透明质酸、层粘连蛋白含量模型组高于对照组。骨组织中MMP-1 mRNA表达模型组高于对照组(P<0.05)。熏烟复合模拟新疆的寒燥环境导致的机体血清中MMP-9和TNF-α,肺泡灌洗液中透明质酸、层粘连蛋白增多,骨组织中MMP-1 mRNA表达增高的现象,可能是熏烟复合寒燥环境扰乱机体内环境稳态的微观机制的部分表现。     

发育中大鼠皮层神经元早期无镁细胞外液处理诱导NMDAR亚基mRNA表达的远期变化

以原代培养的大鼠大脑皮层神经元无镁诱导的反复惊厥样放电为模型,根据对培养6d(天)皮层神经元的不同处理分为3组：正常DMEM培养液组（CONT1）、正常细胞外液组（CONT2）和无镁细胞外液组（MGF）。神经元在上述3种液体中孵育3h,然后恢复正常DMEM培养液继续培养,在培养7、12及17d时应用实时定量PCR测定了发育中大鼠皮层神经元无镁细胞外液处理后NMDA受体亚基NR1、NR2A与NR2B mRNA表达的变化。结果显示: MGF组NR1 mRNA表达在培养7d时明显降低,培养12d时明显升高（p<0.05）;NR2A mRNA表达在培养12d时明显降低,培养17d时明显升高（p<0.05）;NR2B mRNA表达在培养7d与17d时明显升高（p<0.05）。而培养17d NR1 mRNA表达、7d NR2A mRNA表达及12d的皮层神经元NR2B mRNA表达3组中两两比较无统计学差异（p>0.05）。MGF组NR1/NR2A、NR1/NR2B和NR1/NR2A/NR2B在培养7、12与17d皮层神经元均有明显的变化。可见早期无镁细胞外液处理可以诱导发育中大鼠皮层神经元远期NMDAR亚基mRNA表达的改变。     

Regulations of thyroid hormone on cardiac protein kinase C signal pathway in vitro

The experiments were conducted to assess the influences of thyroid hormone on cardiac protein kinase C(PKC) signal pathway with cultured cardiac myocytes and fibroblasts as the models. Cells were pretreated with 1% newborn calf serum (NCS) or angiotensin II (Ang II), and then following by a triiodothyronine (T3) treatment. The PKC activity, PKC_α and PKC_ε expressions were analyzed and compared. In 1% NCS pretreatment, T3 could inhibit PKC activity and PKC_ε expression in cardiac myocytes. The AngII pretreatment led to an increase of PKC activity and PKC_ε expression in cardiac myocytes, and an increase of PKC activity in cardiac fibroblasts. Following by T3 treatment, the increased PKC activity and PKC_ε expression in cardiac myocytes were markedly decreased. In conclusion, whether in 1% NCS or in Ang II pretreatment, T3 could inhibit PKC activity and PKC_ε expression in cardiac myocytes. Foundation item: Supported by the Natural Science Foundation of Hubei Province (98091) Biography: WANG Bao-hua (1974-), female, Ph. D, research direction: cardiovascular pathophysiology.     

The expression of vasa gene during zebrafish ( Danio rerio ) oogenesis

vasa gene expression pattern during oogenesis of zebrafish was examined usingin situ hybridization and fluorescent quantitative RT-PCR. During zebrafish oogensis,vasa mRNA is expressed strongly and uniformly distributed in the cytoplasm in stage II oocytes, followed by a distribution among vacuome in stage III. Later in stage IV and V,vasa mRNA is enriched at the cortex and finally localized at the cortex. The fluorescent quantitative RT-PCR shows that the quantity ofvasa mRNA decreases from stage II to stage III, but remains relatively invariable from stage III to stage V. The observed differences invasa mRNA expression in the different stages of zebrafish oogenesis suggest thatvasa gene plays an important role during oogenesis. Foundation item: Supported by the National Natural Science Foundation of China (30370744, 30150005) Biography: XIANG Fang (1979-), male, Master candidate, research direction: molecular development of animals.     

Colchicine Inhibited the Expression of Tissue Inhibitor of Metalloprotenase-1 and Interleukin-6 in Cultured Activated Hepatic Stellate Cells

Cultured HSCs were treated colchicine with different concentrations for 12 h, respectively. The effects of eolehicine on HSCs growth were measured by MTT assay. Effects of eolchicine on gene expression of HSCs were analysed by using a self-made oligonucleotide mieroarray. Colehieine inhibited HSCs growth in a dose-dependent manner. After 12 h of treatment with 6.25 mg/L of colehicine, the expression of tissue inhibitor of metalloprotenase-1 （TIMP-1） and the expression of interleukin-6 （IL-6） in HSCs were downregulated by 2.3 folds and 2.1 folds, respectively. These results suggest that colchieine＇s beneficial effects may, at least in part, owe to the inhibitory to the proliferation of HSCs and down regulation of the expression of both TIMP1 and IL-6 in HSCs.     

Inducing dopaminergic differentiation of expanded rat mesencephalic neural stem cells by ascorbic acid in vitro

Ascorbic acid (AA) induced differentiation of neural stem cells (NSCs) into dopaminergic (DAergic) neurons is reported.NSCs derived from rat mesencephalon were maintained and expanded in a defined medium containing mitogens of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).Compared with the control, ascorbic acid treatment led to more DAergic neuronal differentiation as indicated by the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT), which are specific markers of dopamine neurons.AA induction also enhanced expression of Nurr1 and Shh.PD98059, an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, could block AA-induced Nurr1, TH and DAT mRNA expression.The results might suggest a new strategy to provide enough dopaminergic cells for the therapy of Parkinson's disease (PD), and Nurr1 and ERK signaling pathway might participate in the AA-induced DAergic differentiation.     

rhEPO对预处理低氧损伤胶质细胞MMP-9表达的影响

 探讨人促红细胞生成素（rhEPO）对低氧损伤胶质细胞的影响及对金属蛋白酶-9（MMP-9）表达的影响。通过体外纯化培养第3代星形胶质细胞,将其分为正常组、低氧组、rhEPO预处理组;以四唑蓝（MTT）比色法测定低氧培养12、24、36h细胞的存活率,倒置显微镜和透射电镜观察低氧对胶质细胞形态的影响;免疫荧光和逆转录-聚合酶链反应（RT-PCR）方法研究低氧对星形胶质细胞MMP-9表达的影响。结果显示:低氧组星形胶质细胞在低氧培养下出现细胞肿胀,且随时间的延长而加重,rhEPO预处理组在各时间点细胞肿胀明显轻于低氧组。rhEPO能减轻细胞超微结构的改变及降低MTT比值。RT-PCR及免疫荧光检测表明：低氧组MMP-9 mRNA及蛋白的表达在低氧各时间点均高于正常组,在24h达到最高,在36h开始降低（P<0.05）;rhEPO预处理组MMP-9mRNA及蛋白的表达变化在12、24、36h较低氧组低（P<0.05）。由此得出结论：rhEPO通过抑制MMP-9的表达促进低氧条件下星形胶质细胞的存活。     

Correlation of matrix metalloproteinase－2, －9, tissue inhibitor－1 of matrix metalloproteinase and CD44 variant 6 in head and neck cancer metastasis

This study aimed to explore the molecular mechanism in tumor invasion and metastasis. The expression of matrix metalloproteinase-2, -9 (MMP-2, MMP-9), tissue inhibitor-1 of matrix metalloproteinase (TIMP-1), cell adhesion molecule 44 variant 6 (CD44v6), HER2/neu and p53 was investigated in 154 patients with head and neck squamous cell carcinoma (SCC) by ABC and ImmunoMax immunohistochemical method. Their clinical relevance and correlation were analysed. The expression of MMP-2, MMP-9, TIMP-1, CD44v6, HER2/neu and p53 was found in cancer cells in 87.01%, 85.71%, 68.18%, 98.05%, 55.19% and 50.65% cases respectively. Linear regression and correlation analysis revealed that there was close positive relationship (P<0.05) between the expression of MMP-2 and MMP-9, TIMP-1 and CD44v6, HER2/neu and MMP-9, MMP-2 and p53. Up-regulation of MMP-2 was accompanied by advanced T stage (P<0.01). There was also a trend of MMP-2 expression being related with tumor metastasis. Increased expression of HER2/neu was found in patients with tumor recurrence(P<0.05). The expression of TIMP-1 was higher in laryngeal cancer than that in pharyngeal cancer, and higher in keratinizing and non-keratinizing SCC than that in basaloid SCC(P<0.05). These findings suggested that MMP-2 and MMP-9, HER2/neu and MMP-9, MMP-2 and p53 had a coordinate function in aggression of tumor; that MMP-2 had a more important function than MMP-9 in tumor invasion and metastasis; and that HER2/neu might serve as a biomarker for poor prognosis in HNSCC.     

Solid-phase preparation and characterization of Chitosan

0　IntroductionChitosanhasbeenwidelyusedinpharmaceu tical,agriculturalandindustrialfieldbe causeofitsintriguing properties[1 ] .Chemicalmethodstopreparechitosanfromchitinhavebeenbroadlystudiedincludingsolutionmethod ,meltedalkalinemethod,microwavesemi drymethod ,improvedLinelbergmethod ,dissolution precipita tionmethodandsoon ,inwhichalkalinewasallusedasthecatalyst[2 ] .Biologicalmethodwasapromisingwayinthatitproducedlittleenviron mentalpollutionanditsreactionconditionsweremild .However,thism…