TLR4-P38MAPK信号通路在LPS诱导BV2细胞中的表达及意义

目的观察LPS诱导小胶质细胞后信号通路Toll样受体4(TLR4)-p38蛋白激酶(p38MAPK)的表达及意义。方法体外培养BV2小胶质细胞,分为对照组、LPS诱导组(LPS刺激12h及24h)及SB203580干预组(LPS+SB203580诱导12h及24h),应用ELISA法检测各组TNF-α、IL-6水平,RT-PCR法检测各组TLR4mRNA和p38MAPK mRNA的表达变化。结果 LPS诱导组细胞分泌TNF-α、IL-6水平显著提高,诱导24h后细胞上清液含量分别为(513.67±14.05)pg/mg和(396.84±15.41)pg/mg。给予SB203580抑制剂后TLR4mRNA和p38MAPK mRNA表达明显减弱,细胞分泌TNF-α、IL-6含量表达与感染组比较也明显降低。结论 LPS刺激小胶质细胞可引起TLR4-p38MAPK信号通路的活化并释放炎性细胞因子,而SB203580则对其有明显的抑制作用,证明TLR4-p38MAPK信号通路与小胶质细胞的炎性活化密切相关。     

肝星状细胞增殖与p38丝裂原活化蛋白激酶信号传导通路的关系

背景：肝星状细胞的激活、增殖导致肝纤维化,p38丝裂原活化蛋白激酶信号通路可参与调控细胞增殖。 目的：探讨SB203580作用于乙醛刺激的大鼠肝星状细胞后p38丝裂原活化蛋白激酶活性变化和细胞增殖变化。 方法：体外培养大鼠肝星状细胞株,在乙醛干预的基础上加入不同浓度的p38特异性抑制剂SB203580进行培养,并设置对照。以Western blot检测磷酸化p38 蛋白表达水平变化,MTT比色法检测细胞增殖。 结果与结论：乙醛刺激后大鼠肝星状细胞内磷酸化p38水平增强,细胞增殖明显。使用5,10,20 μmol/L SB203580能明显抑制乙醛刺激的肝星状细胞增殖(P < 0.05),加大浓度至30 μmol/L时,抑制作用更明显(P < 0.01),抑制率为43.9%,而磷酸化p38水平也降低(P < 0.05)。结果证实,抑制p38丝裂原活化蛋白激酶活性可能影响肝星状细胞的增殖。     

Smad4 siRNA对转化生长因子β诱导Ⅰ型胶原表达的作用

背景：近期研究表明,阻断smad传导通路可能成为一个阻断转化生长因子β引起的肝纤维化的新的靶点。 目的：验证Smad4小分子干扰RNA(siRNA)对转化生长因子β诱导的Ⅰ型胶原的作用。 设计、时间及地点：单一样本观察,于2006-09/2008-10在哈尔滨医科大学药学院完成。 材料：肝星状细胞HSC-T6体外培养,siRNA和荧光素酶报告基因采用Lipofectamine试剂转染,以含巨细胞病毒-β-半乳糖苷酶质粒作为转染阳性对照。 方法：根据不同处理将细胞分为4组,空白对照组;Smad4 siRNA转染组;转化生长因子β处理组;Smad4 siRNA转染后转化生长因子β处理组,将Smad4 siRNA转染入肝星状细胞后行转化生长因子β刺激。各组分别于培养24 h后收集细胞。 主要观察指标：利用荧光素酶报告基因法测定Smad结合转写因子(4XSBE)的活性;反转录-聚合酶链反应法检测Ⅰ型胶原mRNA表达的变化;Western印迹分析观察Smad4蛋白、Ⅰ型胶原蛋白的变化。 结果：Smad4 siRNA有意义的抑制Smad4蛋白表达;Smad4 siRNA抑制转化生长因子β诱导的4XSBE转录报告基因的活性;Smad4 siRNA从mRNA和蛋白水平有效抑制转化生长因子β激活的Ⅰ型胶原的表达。 结论：Smad4 siRNA能够有效地阻断转化生长因子β诱导的Ⅰ型胶原表达。     

外源Smad7转染肝星状细胞及对转化生长因子β1和Ⅰ、Ⅲ型胶原mRNA表达的影响

背景：Smad7是转化生长因子β信号转导途径的主要抑制性蛋白,具有抗纤维化的作用。 目的：构建并鉴定大鼠Smad7真核表达质粒,观察外源Smad7可否有效转染肝星状细胞T6,并进一步研究其对转化生长因子β及Ⅰ、Ⅲ型胶原mRNA 表达水平的影响。 设计、地点：基因重组及细胞观察实验,于新疆石河子大学医学院第一附属医院完成。 材料：pcDNA3.1(+)质粒为课题组保留;大肠杆菌DH5α系石河子大学医学院新疆地方与民族高发病教育部重点实验室所赠;肝星状细胞T6细胞由中国医学科学院肿瘤医院肿瘤研究所提供品。 方法：采用基因重组技术将Smad7cDNA插入真核表达载体pcDNA3.1(+),构建大鼠Smad7真核表达质粒。脂质体介导转染肝星状细胞T6细胞,分为正常对照、空质粒及转染组,G418筛选,挑取阳性细胞。 主要观察指标：反转录-聚合酶链反应法检测各组中Smad7、转化生长因子β及Ⅰ、Ⅲ型胶原mRNA的表达水平。 结果：酶切和测序结果证实Smad7真核表达质粒构建成功。Smad7转染组与正常对照组、空质粒组比较：Smad7 mRNA表达显著增加(P < 0.01);转化生长因子β、Ⅰ型胶原mRNA表达减少(P < 0.01);Ⅲ型胶原mRNA表达差异无显著性意义(P > 0.05)。正常对照组、空质粒组Smad7、转化生长因子β及Ⅰ、Ⅲ型胶原mRNA表达差异无显著性意义(P值均> 0.05)。 结论：大鼠Smad7真核表达质粒构建成功,外源Smad7转染肝星状细胞T6细胞后可有效表达,并能降低转化生长因子β及Ⅰ型胶原mRNA 表达水平。     

结缔组织生长因子及其胶原在硬皮病组织中的表达

背景：近年来，大量文献报道结缔组织生长因子作为转化生长因子β的下游效应因子，是最重要的致纤维化生长因子，在多种纤维化疾病中高表达。 目的：观察结缔组织生长因子在硬皮病组织中的表达情况及与其上、下游效应基因表达的关系，探讨结缔组织生长因子在硬皮病组织中的促纤维化机制。 设计、时间及地点：对照观察实验，于2008-05/11在解放军广州军区广州总医院医学实验科完成。 材料：组织标本均来源于解放军广州军区广州总医院整形外科患者，正常皮肤来自整形手术中切除的多余皮肤。 方法：收集硬皮病皮肤组织、增生性瘢痕组织及正常皮肤组织标本，免疫组化检测结缔组织生长因子的表达，反转录-聚合酶链式反应方法检测结缔组织生长因子、转化生长因子β1、Ⅰ型胶原及Ⅲ型胶原mRNA表达，Western blotting检测结缔组织生长因子蛋白的表达。 主要观察指标：①结缔组织生长因子蛋白在硬皮病皮肤组织中的表达和定位情况。②结缔组织生长因子、转化生长因子β1、Ⅰ型胶原和Ⅲ型胶原mRNA的相对表达量。③比较增生性瘢痕、硬皮病和正常皮肤组织中结缔组织生长因子蛋白表达量的情况。 结果：免疫组织化学染色发现，结缔组织生长因子在基底细胞和角质形成细胞中有强阳性表达，在真皮成纤维细胞中亦有阳性表达。硬皮病皮肤组织及增生性瘢痕组织中结缔组织生长因子、转化生长因子β1、Ⅰ型胶原和Ⅲ型胶原的mRNA过表达，而正常皮肤组织无或仅有极弱表达。Western blotting结果显示硬皮病皮肤组织有明显的结缔组织生长因子蛋白表达带。 结论：在硬皮病纤维化过程中，结缔组织生长因子可能是重要的调控因子之一，结缔组织生长因子可能与转化生长因子β1协同作用促进细胞外基质合成。     

精氨酸加压素对星形胶质细胞水孔蛋白-4表达的调节及脑水肿形成影响的研究

目的研究精氨酸加压素(AVP)对星形胶质细胞水孔蛋白-4(AQP4)表达的调节,以及p38 MAPK信号通路在AQP4表达过程的作用,明确AVP及AQP4在脑水肿发生过程中的作用。方法大鼠大脑皮质分离星形胶质细胞,星形胶质细胞经分别用AVP、V1a受体(V1aR)拮抗剂和SB 203580进行处理,采用免疫组织化学技术及RT-PCR对AQP4 mRNA进行检测,Western blot检测p38 MAPK信号通路在AVP诱导AQP4表达中的活化程度。结果500nmol/L的AVP处理6h后,AQP4 mRNA表达开始升高(P<0.01),到12h达高峰(P<0.01),24h后仍维持在较高的水平(P<0.05)。加入p38 MAPK抑制剂SB 203580干预后,AQP4 mRNA表达水平与对照组比较差异不显著(P>0.05);AVP处理15min后p38 MAPK磷酸化水平开始增加,30min达高峰,持续到60min开始下降。V1aR拮抗剂处理后p38 MAPK磷酸化水平整个时间段均未出现明显变化。结论AVP通过激活V1aR引起p38MAPK信号通路活化从而诱导AQP4 mRNA高表达,从基因水平对AQP4进行调节,可能在脑水肿发生中,尤其是在星形胶质细胞水肿形成中起重要作用。V1aR拮抗剂及p38 MAPK抑制剂能抑制AQP4 mRNA的表达,避免星形胶质细胞肿胀。     

p38MAPK在体外培养星形胶质细胞缺氧/复氧损伤中的表达

目的 建立SD大鼠星形胶质细胞缺氧复氧损伤模型,探讨p38MAPK活性变化与星形胶质细胞损伤的关系.方法 体外培养新生SD大鼠星形胶质细胞,实验设正常对照组（N）、SB203580组（SB组,10 μmol/L）、缺氧/复氧组（H/R组）和缺氧/复氧组＋SB203580阻断p38MAPK组（H/R＋SB组）.应用MTT法、WB法、ELISA法检测缺氧4 h、8 h、复氧6 h、12 h、24 h、48 h时细胞存活率,p38MAPK、p-p38（磷酸化p38MAPK）及TNF-α的变化.结果 培养星形胶质细胞GFAP阳性表达率大于97%.缺氧/复氧使星形胶质细胞活力降低,SB203580阻断p38MAPK细胞活力高于H/R组,各组星形胶质细胞总p38MAPK水平无显著变化,缺氧复氧干预后p-p38表达上调,TNF-α水平显著增高.用SB203580阻断p38MAPK通路后,SB＋H/R组较H/R组p-p38、TNF-α水平降低.SB组总p38MAPK、p-p38、TNF-α水平与N组比较无显著变化.结论 p38MAPK信号通路参与了星形胶质细胞缺氧复氧损伤过程.     

实验性脑出血后ICAM-1和IL-1β的表达与p38 MAPK通路关系研究

目的 探讨脑出血后血肿周围组织p38丝裂原活化蛋白激酶(p38MAPK)在脑出血血肿周围组织炎性因子表达的关系.方法 健康雄性Wistar大鼠30只,将动物随机分成脑出血加SB203580组和脑出血组,采用免疫组织化学方法观察干预前后不同时间点细胞间黏附分子-1(ICAM-1)和白介素-1β(IL-1β)的变化.结果 免疫组化染色显示SB203580干预组ICAM-1、IL-1β在大鼠脑出血血肿周围组织中的表达均较对照组明显降低,差异有显著意义.结论 SB203580抑制IL-1β和ICAM-1的表达,p38MAPK信号通路在脑出血后血肿周围组织损伤中发挥重要的作用.     

p38MAPK信号通路与应力介导成肌细胞的凋亡*

背景：在体内条件下,细胞力学的功能研究因其所处生理环境的复杂性、实验条件的不易控制而很难得到满意结果。 目的：在成功构建成肌细胞体外培养-力学刺激模型的基础上,研究p38MAPK信号通路在成肌细胞凋亡中的作用及其机制。 方法：将体外培养的C2C12细胞分为对照组和SB203580组,SB203580组中加入 20 mmol/L的p38MAPK抑制剂SB203580。应用细胞应力加载装置Flecell Strain Unit-5000T给细胞提供15%的力值,分别施加0,6,12,24 h的周期性张应力。每分钟10个循环,每循环包括3 s牵张,3 s松弛。Hoechst 33258染色观察细胞的形态学变化;流式细胞仪检测细胞凋亡情况;RT-PCR法检测促凋亡基因bax mRNA的表达;Western blot检测信号通路中p38MAPK和p-p38MAPK蛋白的表达。 结果与结论：随着加力时间的延长,细胞逐渐出现核固缩及凋亡小体,凋亡率增加(P < 0.05),bax mRNA表达增多(P < 0.05);细胞p38MAPK和p-p38MAPK蛋白均在加力6 h达到最低,此后逐渐升高。p38MAPK抑制剂SB203580可抑制加力引起的细胞凋亡,减少bax mRNA及p38MAPK和p-p38MAPK蛋白的表达(P < 0.05)。说明p38MAPK信号通路在应力介导的成肌细胞凋亡中起到重要的作用。     

SH-SY5Y细胞α7尼古丁受体蛋白抑制对tau蛋白及p38 MAPK通路的影响及其与AD发病机制的关系

目的研究α7尼古丁受体(nAChR)蛋白抑制对SH-SY5Y细胞tau蛋白磷酸化水平的影响及其与p38 MAPK通路的关系,探讨α7 nAChR调节tau蛋白磷酸化的相关机制。方法用α7 nAChR阻断剂MLA阻断SH-SY5Y细胞α7 nAChR蛋白的活化及其表达,用p38 MAPK阻断剂SB203580阻断SH-SY5Y细胞p38 MAPK信号通路蛋白的活化及其表达,Western blotting方法测定tau蛋白、p-tau(S404)、p-tau(S214)、α7 nAChR、p38 MAPK及p-p38 MAPK(Thr180/Tyr182)蛋白表达水平。结果细胞经MLA处理后,p-tau(S404)和p-tau(S214)蛋白水平明显升高(P0.01),p-p38 MAPK和α7 nAChR蛋白水平明显降低(P0.01),tau蛋白和p38 MAPK蛋白水平保持不变;经SB203580处理后,SB203580及MLA共同处理后均引起p-tau(S404)、p-tau(S214)、p-p38 MAPK和α7nAChR蛋白水平显著降低(P0.01),tau蛋白和p38 MAPK蛋白水平无变化。结论α7 nAChR可通过阻断p38MAPK信号传导通路抑制tau蛋白过度磷酸化。     

Characteristics of the nerve growth factor receptors and a nerve growth factor receptor--nerve growth factor covalent complex

Nerve growth factor is a polypeptide hormone that is required for the normal growth and development of the embryonic sensory and sympathetic nervous systems. On these cells, there are two different receptors for the nerve growth factor. Recently, these receptors have been isolated from three cell types and shown to have essentially the same binding characteristics. Molecular weights for receptors from two of these cell types, embryonic sensory and rat pheochromocytoma cells, have been determined. In addition, the formation of a covalent nerve growth factor, nerve growth factor receptor complex, has been investigated on embryonic sensory and sympathetic neurons. The formation of this covalent complex containing 125I-beta nerve growth factor is prevented by the addition of excess unlabeled nerve growth factor or by the addition of sodium fluoride and dinitrophenol. This complex forms at 4 degrees, 22 degrees, and 37 degrees, indicating that it is occurring on the cell surface. A disulfide bond is involved in the formation of this covalent complex.     

Epidermal growth factor influences the neurotrophic/differentiating action of nerve growth factor

Exposure of naive PC12 cells, sympathetic neurons from rat superior cervical ganglia, and brain-derived septal neurons to epidermal and nerve growth factors simultaneously resulted in some alteration of cellular events induced by nerve growth factor alone. A more pronounced decline of catecholamine content, no additional change in acetylcholinesterase activity, and additive stimulation of RNA and protein syntheses were found in PC12 cells. Earlier elevation of the enzyme activity was observed in sympathetic but not in septal neurons. Epidermal growth factor appeared to support independently the same level of acetylcholinesterase activity in septal neurons as revealed for nerve growth factor during the first week and cell survival throughout 2 weeks of observation. The data obtained indicate that epidermal growth factor augments temporarily some effects of nerve growth factor, thus supporting the idea of an important role of mitogenic growth factors in neural development as complementary and/or substitutive regulators of nerve cell differentiation and survival.     

Identification of basic fibroblast growth factor as a cholinergic growth factor from human muscle

Dissociated embryonic chick ciliary ganglion cells in culture were used as a bioassay to isolate a cholinergic growth-promoting protein from extracts of autopsied adult human muscle. An active protein was purified after acid and salt precipitation of extract, cation exchange, molecular sieving, heparin affinity chromatography, and in some cases, SDS-PAGE. This protein increased levels of choline acetyltransferase activity and ACh synthesis with time in culture. The protein was identified as basic FGF by several criteria. It shared the high affinity for heparin and was the same approximate molecular weight, 18 kD, as basic FGF. Activity was removed from solution by antibodies specific for basic FGF. Recombinant human basic FGF was equally effective in stimulating CAT activity, but was not additive with our purified protein at saturating concentrations. Basic FGF was also found in extracellular matrix and conditioned medium from cultured embryonic chick muscle. The activity could be released from extracellular matrix by treatment with heparinase or high salt extraction. Basic FGF stimulates neurite outgrowth as well as the capacity for transmitter synthesis. Thus, basic FGF is present in embryonic and adult muscle and capable of acting as a growth regulator for cholinergic neurons.     

Macrophage colony-stimulating factor in nerve growth factor preparations

Following a report that nerve growth factor preparations have granulocyte-colony-stimulating activity, we investigated the presence of colony-stimulating factors in 7s mouse submaxillary nerve growth factor and its subunits. Macrophage colonies were formed in mouse bone marrow cultures after exposure to preparations of 7s nerve growth factor, the gamma subunit, and, to a small extent, the alpha subunit; the beta subunit, which is responsible for the nerve growth function, did not stimulate colony growth. Furthermore, the esteropeptidase activity of the gamma subunit was not detected in preparations of macrophage colony-stimulating factor purified from the giant cell tumor (GCT) cell line. Immunoprecipitation of radiolabeled gamma subunit with a polyclonal antibody to L-cell macrophage colony-stimulating factor showed a protein band that could represent the gamma subunit of nerve growth factor. Separation of the macrophage activity from the esteropeptidase activity of the gamma subunit was accomplished on the basis of molecular size. Thus, macrophage colony-stimulating factor was a contaminant of nerve growth factor produced by the mouse submaxillary gland and copurified with the gamma subunit.     

Nerve growth factor and the neurotrophic factor hypothesis

The discovery of nerve growth factor (NGF) over 40 years ago led to the formulation of the “Neurotrophic Factor Hypothesis”. This hypothesis states that developing neurons compete with each other for a limited supply of a neurotrophic factor (NTF) provided by the target tissue. Successful competitors survive; unsuccessful ones die. Subsequent research on NTFs has shown that NTF expression and actions are considerably more complex and diverse than initially predicted. Even for NGF, different regulatory patterns are seen for different neuronal populations. As would be predicted by the “Neurotrophic Factor Hypothesis”, NGF levels critically regulate basal forebrain cholinergic neuron size and neurochemical differentiation. In contrast, the level of trkA, the NGF receptor, regulates these properties in caudate-putamen cholinergic neurons. Understanding NTF regulation and actions on neurons has led to their use in clinical trials of human neurological diseases. NTFs may emerge as important therapies to prevent neuronal dysfunction and death.     

Astroglial expression of hepatocyte growth factor and hepatocyte growth factor activator in human brain tissues

Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes, and also has multifunctional effects on some other cells in various organs. A HGF activating protease, HGF activator (HGFA) has recently been identified as a key enzyme that regulates the activity of HGF in vivo. HGFA appears to be associated with the cell surface. We examined HGFA immunolabelling in the brains of neurologically normal and Alzheimer disease (AD) cases. Furthermore, we identified the expression of the mRNA for HGF and HGFA by in situ hybridization histochemistry. The HGFA antibody stained only astrocytes in the white matter in all the brain tissues. Expression of the mRNAs of HGF and HGFA was also seen in white matter astrocytes. These results suggest that, in human brain, secreted pro-HGF from astrocytes might be activated by HGFA on/or near the astrocytic cell surface.     

Overexpression of nerve growth factor and basic fibroblast growth factor in AIDS dementia complex.

Although neurotrophic factors are currently considered as treatment for neurodegenerative diseases, little is still known about their presence in the central nervous system under pathological conditions. We investigated the expression of the neurotrophic molecules NGF, bFGF, BDNF and IGF-1 in brain tissue of patients suffering from AIDS dementia complex. In contrast to IGF-1 and BDNF, NGF and bFGF mRNA levels were significantly elevated. Strong NGF immunoreactivity was found in perivascular areas and was colocalized with infiltrating macrophages, whereas intense bFGF staining was found in cells with characteristic astrocytic morphology. These data suggest that the induction of NGF and bFGF alone appears to be insufficient as a compensatory mechanism to prevent ADC.