柄袋沙蠋体壁和消化系统甲硫氨酸脑啡肽免疫组织化学定位

采用免疫组织化学SABC法,对甲硫氨酸脑啡肽(M-ENK)在柄袋沙蠋体壁及其附属物鳃、疣足和消化系统各器官中的分布进行了研究.结果 表明,柄袋沙蠋体壁、鳃、疣足以及各消化器官的上皮细胞中均有不同程度的M-ENK阳性反应,且体壁、消化道的食管、肠、直肠中阳性反应多集中在上皮细胞的游离端;体壁肌肉中环肌层M-ENK阳性反应比纵肌层强;食道侧囊上皮细胞和结缔组织中也有M-ENK分布.M-ENK在柄袋沙蠋的体壁、鳃、疣足及消化系统各器官中均有分布,且分布密度不同,可能与各部位的功能有关.     

ERK1/2和p-ERK1/2在肺癌组织中的表达及意义

目的研究细胞外信号调节激酶1/2（extracellular regulated kinase 1/2,ERK1/2）及其磷酸化状态（p-ERK1/2）在不同分化程度肺癌中的表达情况,探讨二者与肺癌侵袭、转移的关系。方法采用免疫组化（Envision）法,检测79例肺癌组织及l2例癌旁正常肺组织中ERK1/2和p-ERK1/2的表达。结果ERK1/2在高、中、低分化组表达率分别为13.6%,39.4%,66.7%,p-ERK1/2在高、中、低分化组表达率分别14.3%,27.3%,79.2%（P〈0.05）;无淋巴结转移者阳性率为20%,有淋巴结转移者阳性率为50.1%（P〈0.05）。ERK1/2和p-ERK1/2的表达在不同年龄、性别、肿瘤大小、肿瘤病理类型无显著性差异,而与分化程度有关,其中p-ERK1/2的表达还与有无淋巴结转移有关。结论ERK1/2和p-ERK1/2在肺癌组织中高表达且与分化程度有关。     

P物质与受体nk-1在结肠癌中的表达及定位

目的:了解P物质及其受体神经激肤1(NK-1)在结肠癌中的表达及定位,探讨其在结肠癌发病及临床诊断的意义.方法:应用免疫组化方法检测正常结肠、结肠癌组织及癌旁组织中SP及NK-1的表达.采用jmtjfx10[1].31 统计学软件处理数据,所得数据进行Q检验,结果:①在结肠癌组织中P物质阳性着色于胞浆,呈巢状或弥漫分布.②NK-1受体主要着色于细胞浆内,呈巢状或弥漫分布;少数为于细胞膜.③p物质与其受体NK-1在正常结肠粘膜组织及癌旁组织也有表达,位于细胞浆内,形态多呈圆形、椭圆形或不规则形.④二者在绝大多数结肠组织中呈强阳性表达,显著高于正常结肠粘膜组织和癌旁组织(p<0.01).结论:p物质与其受体NK-1在结肠癌组织中高度表达,提示神经内分泌参与了结肠癌的发生有关,说明P物质及NK-1受体可能参与了结肠癌的发病过程.     

TGF-β1和IGF-1 mRNA在早期胚胎停育绒毛组织中的表达及意义

目的:通过检测胚胎停育患者绒毛组织中TGF-β1和IGF-1 mRNA的表达,探讨其在胚胎停育发病机制中的作用。方法:用半定量逆转录聚合酶链反应(RT-PCR)技术检测正常人流(20例),胚胎停育(25例)绒毛组织中TGF-β1和IGF-1基因在转录水平的表达。结果:(1)与对照组相比,实验组绒毛组织中TGF-β1mRNA表达量降低(P0.05),(2)胚胎停育患者绒毛中IGF-1mRNA表达量升高(P0.05)。(3)绒毛组织中TGF-β1与IGF-1的表达呈负相关(r=-0.793,P0.05)。结论:TGF-β1、IGF-1的表达在基因转录水平发生改变,TGF-β1表达的降低可能上调了IGF-1的表达,提示两者可能共同参与了胚胎停育的发生。     

趋化因子及趋化因子受体在急性胰腺炎中的研究进展

普遍认为,急性胰腺炎起始于腺泡细胞内的胰蛋白酶原激活,随后引起的炎症反应加剧病情,也是多器官功能衰竭的主要原因。然而,最新的研究表明,急性胰腺炎引起的炎症反应是不依赖于胰蛋白酶原激活的独立病理过程。趋化因子作为能引起细胞趋化的细胞因子,通过与趋化因子受体作用,不但能调控淋巴细胞的生长、成熟和迁移,也参与多种炎症疾病与癌症的病理过程。近年来,多项研究已经阐述趋化因子及趋化因子受体在急性胰腺炎的发病发展过程中起到至关重要的作用。本文总结了CC,CXC和CX3C趋化因子家族成员在参与急性胰腺炎的炎症反应及对胰腺损伤的修复的研究进展,这将为AP临床治疗方案的设计提供新思路。     

TGF-β1和IGF-1mRNA在早期胚胎停育绒毛组织中的表达及意义

目的：通过检测胚胎停育患者绒毛组织中TGF-β1和IGF-1mRNA的表达,探讨其在胚胎停育发病机制中的作用。方法：用半定量逆转录聚合酶链反应（RT-PCR）技术检测正常人流（20例）,胚胎停育（25例）绒毛组织中TGF-β1和IGF-1基因在转录水平的表达。结果：（1）与对照组相比,实验组绒毛组织中TGF-βmRNA表达量降低（P〈0．05）,（2）胚胎停育患者绒毛中IGF—1mRNA表达量升高（P〈0．05）。（3）绒毛组织中TGF-β1与IGF-1的表达呈负相关（F=-0．793,P〈0．05）。结论：TGF-β1、IGF-1的表达在基因转录水平发生改变,TGF-β1表达的降低可能上调了IGF-1的表达,提示两者可能共同参与了胚胎停育的发生。     

TGFβ1及其受体在大鼠整胚及软骨雏形发育过程中的表达

目的 观察TGFβ1及TβRI、TβRII在大鼠整胚及软骨雏形中的表达,初步探讨它们与大鼠整胚及软骨雏形发育间可能的相互关系和作用机制.方法 半定量RT-PCR分析3种因子在13～17 d全胚中的变化情况;免疫组化法观察3种因子在13～17d软骨雏形中的表达及该组织发育结构特点.结果 半定量RT-PCR显示,3种因子在发育13～15 d表达呈递增趋势,16～17 d出现下降.免疫组化显示,TGFβ1主要位于软骨雏形内的软骨骨祖细胞,TβRI、TβRII即可位于软骨骨祖细胞内,也可位于软骨外周的膜上及软骨雏形周围的间质内.结论 TGFβ1及TβRI、TβRII在大鼠整胚及软骨雏形发育过程中可能起着重要的调控作用,尤其是对组织器官形态的发生.     

NFE2L1在食管鳞癌中的表达及临床意义

目的:通过检测食管鳞癌标本中核因子E2相关因子1(NFE2L1)的表达情况,探究NFE2L1在食管鳞癌发生发展中的作用,为食管鳞癌的诊治以及预后评估提供新的思路。方法:收集我校附院2016-2017年经病理组织学检查诊断为食管鳞癌的手术标本40例及其对应的癌旁组织并从NCBI数据库下载GEO测序数据,应用定量PCR、免疫组化和生物信息分析等方法检测分析NFE2L1基因的表达情况。结果:在食管鳞癌组织中,NFE2L1表达阳性31例(77.5%),癌旁组织阳性表达17例(42.5%),两组比较差异显著有统计学意义(P0.001);进一步发现NFE2L1的阳性表达与肿瘤分化程度和淋巴转移相关(P0.05)。但在不同年龄、性别、浸润深度及不同部位之间差异无统计学意义。GEO数据分析结果显示NFE2L1在食管鳞癌组织显著高表达(P0.01),只是未达到显著差异表达的阈值标准(即变化倍数小于2倍)。结论:NFE2L1在食管鳞癌中高表达,表达的高低与食管鳞癌的发生进展密切相关。     

PD-L1和CD147在口腔鳞癌中的表达与临床意义

目的:探讨口腔鳞癌组织中免疫共刺激分子PD-L1与细胞外基质蛋白酶诱导因子CD147的表达、两者的相关性及临床意义。方法:应用免疫组化技术检测66例口腔鳞癌组织及36例正常口腔黏膜组织中PD-L1和CD147的表达,分析PD-L1、CD147表达的相关性及二者与口腔鳞癌临床病理参数的关系。结果:PD-L1在口腔鳞癌组织中表达阳性率为68.18%(45/66),正常口腔黏膜组织中表达阳性率仅为16.67%(6/36);CD147在口腔鳞癌组织中表达阳性率为74.24%(49/66),明显高于其在正常口腔黏膜组织中的表达13.88%(5/36)。PD-L1和CD147两者在口腔鳞癌组织中阳性表达率与口腔黏膜组织相比均明显升高(P0.01)。统计学分析显示,PD-L1和CD147在口腔鳞癌组织中的高表达与患者的性别年龄、吸烟史及肿瘤的体积等因素无明显相关,但与TNM分期及鳞癌的组织分化程度紧密相关。口腔鳞癌组织中PD-L1与CD147两者相关性分析r=0.342,P值小于0.01,说明二者的表达呈显著正相关。结论:口腔鳞癌组织中PD-L1与CD147均呈高表达,并且二者的过度表达可能与口腔鳞癌的发生、发展关系密切,合并检测二者可能为OSCC的诊疗及预后指明新的方向,为口腔鳞癌的靶向治疗提供新的靶点。     

癫痫患者额叶及颞叶脑组织A型GABA受体α1亚型基因表达定量研究

为研究A型GABA受体α₁亚型(GABRA1)在癫痫患者病灶额叶及颞叶脑组织中的表达变化与癫痫发病机制的关系, 采用TaqMan探针荧光定量PCR检测GABRA1在癫痫患者病灶额叶和颞叶脑组织及对照组额叶和颞叶脑组织中的表达量的差异. 结果表明, 对照组及癫痫患者额叶GABRA1平均相对表达强度为3.785±1.444和4.399±2.89, 两者间无显著差异(P>0.05). 对照组颞叶脑组织中GABRA1平均表达量是癫痫患者的4.5倍, 对照组及癫痫患者颞叶GABRA1平均相对表达强度为26.802± 8.983和5.986±5.07, 结果具有统计学意义(P<0.05). 提示, 难治性癫痫患者GABRA1表达在不同组织中的改变并非单一性改变, GABRA1表达量的改变导致癫痫的发生存在组织特异性; GABRA1在颞叶脑组织中的表达减少与癫痫的发生, 尤其是难治性癫痫的发生有密切的关系, 可能是难治性癫痫致病的因素之一. 该研究结果为进一步研究癫痫的发病机制及防治提供了科学依据.     

水螅AChE和NPY类似物的定位

乙酰胆碱脂酶(AChE)组织化学定位方法常用于研究动物的神经系统,但迄今未见水螅AChE分布定位的相关报道。本文使用免疫细胞化学、组织化学和亚甲基蓝活染方法,探索AChE、NPY类似物在水螅上的连续分布状态,对阳性标本进行内胚层、外胚层人工分离手术,并与亚甲基蓝染色结果进行比较。本文首次成功地在水螅上定位出AChE,展示了AChE、NPY类似物在水螅上的分布特点。AChE定位在水螅体柱和触手基部外胚层的近外表,呈网状结构,正常水螅上没有发现明显的阳性神经细胞,外胚层表面受损部位内有阳性神经细胞,我们认为是一种临时的生理现象。成熟刺丝囊的刺针内有AChE 产物。NPY免疫组织化学反应(NPYIR)在水螅外胚层近外表处上均有分布,呈网状结构。垂唇和近基盘处NPYIR最强,NPYIR神经细胞仅存在于垂唇和近基盘处的外胚层基部。刺丝囊分化、发育初期NPYIR较强。随刺丝囊发育成熟而逐渐减弱。最后,进行了分析和比较,认为NPY、AChE来源于上皮细胞。水螅刺丝囊的分化和发育与NPY的作用密切相关;刺丝囊发射刺丝的机理与胆碱能的作用可能有联系     

Homologous Desensitization of the D1 Dopamine Receptor

Preincubation of D384 cells, derived from the human astrocytoma cell line G-CCM, with dopamine resulted in a time-dependent attenuation of cyclic AMP responsiveness to subsequent dopamine stimulation. This effect was agonist specific because the prostaglandin E1 (PGE1) stimulation of cyclic AMP of similarly treated cells remained unchanged. The attenuation by dopamine was concentration dependent with a maximum observed at 100 microM. A comparison of dopamine concentration-response curves of control and dopamine-preincubated cells revealed no change in the Ka apparent value, but a marked attenuation of the maximal response. Preincubation of cells with dopamine in the presence of D1 but not D2 selective antagonists partially prevented the observed attenuation. Attenuations in dopamine responsiveness were also obtained when D384 cells were preincubated with D1 but not D2 receptor agonists. The level of attenuation attained related to agonist efficiency in stimulating cyclic AMP: SKF38393 less than 3,4-dihydroxynomifensine less than fenoldopam less than 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene = dopamine. However, increasing the efficiency of 3,4-dihydroxynomifensine stimulation of cyclic AMP, using the synergistic effect of adding a low concentration of forskolin, produced no further change in the attenuation of the subsequent response to dopamine. Thus, the D1 dopamine receptors expressed by D384 cells undergo homologous desensitization. Uncoupling of the D1 dopamine receptor appears to be independent of cyclic AMP formation, analogous to a mechanism proposed for the beta-adrenergic receptor.     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.     

Development of High Affinity Selective VIP1 Receptor Agonists

Gourlet, P., A. Vandermeers, P. Vertongen, J. Rathe, P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. Development of high affinity selective VIP₁ receptor agonists. Peptides 18(10) 1539–1545, 1997.—The biological effects of VIP are mediated by at least two VIP receptors: the VIP₁ and the VIP₂ receptors that were cloned in rat, human and mice. As the mRNA coding for each receptor are located in different tissues, it is likely that each receptor modulates different functions. It is therefore of interest to obtain selective agonists for each receptor subtype. In the present work, we achieved the synthesis of two VIP₁ receptor selective agonists derived from secretin and GRF. [R¹⁶]chicken secretin had IC₅₀ values of binding of 1, 10,000, 20, and 3000 nM for the rat VIP₁-, VIP₂-, secretin- and PACAP receptors, respectively. This peptide, however, had a weaker affinity for the human VIP₁ receptor (IC₅₀ of 60 nM). The chimeric, substituted peptide [K¹⁵,R¹⁶,L²⁷]VIP(1-7)/GRF(8-27) had IC₅₀ values of binding of 1, 10,000, 10,000 and 30,000 nM for the rat VIP₁-, VIP₂-, secretin- and PACAP receptors, respectively. Furthermore, its also showed an IC₅₀ of 0.8 nM for the human VIP₁ receptor and a low affinity for the human VIP₂ receptor. It is unlikely that this GRF analogue interacted with a high affinity to the pituitary GRF receptors as it did not stimulate rat pituitary adenylate cyclase activity. The two described analogues stimulated maximally the adenylate cyclase activity on membranes expressing each receptor subtype.     

神经激肽B受体在小鼠消化道内的定位

采用免疫组织化学ABC染色方法研究了神经激肽B受体（NK3r)在小鼠消化道的分布。MK3r样阳性的神经无胞体及神经纤维主要分布在十二指肠,空肠,回肠及结肠的粘膜下层神经丛和肌间神经丛,NK3r样阳性产物在食管,胃和直肠的神经丛中未见分布;NK3r样阳性产物大部分避限于神经细胞表面,也存在于胞和一些轴突内部,并在胞质中较细胞表面染色浅。。统计结果表明NK3r样免疫阳性神经元占肠神经系统神经元总数的0．5－1％,提示小鼠消化道内NK3r样阳性神经元可能参与消化功能的调节。     

FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors

Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M₁-, M₃-, and M₅-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70 ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M₁- and M₅-acetylcholine receptors and the amplitude of these signals was larger at the M₁-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M₁-, M₃- and M₅-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation.     

Immunofluorescence and Histochemical Methods for Neural M1 Pyruvate Kinase Localization

Abstract: The distribution of pyruvate kinase (ATP pyruvate phosphotransferase, EC 2.7.1.40) in the nervous system has been studied by both immunofluorescence and a histochemical procedure using nitro blue tetrazolium. The localization in various parts of rat central nervous system in situ , cerebellar and cerebral cortex, was compared to that found in vitro in cultures of cerebellum, spinal ganglia, cerebral astrocytes, and skin fibroblasts. (1) Pyruvate kinase was found predominantly in the cytoplasm of neuronal cell bodies. (2) Large neurons were better visualized than small ones. (3) No glial localization was clearly demonstrated in situ , although this does not rule out the presence of some M₁ pyruvate kinase. (4) Regions expected to be rich in nerve terminals, such as the cerebellar glomeruli or the cerebellar molecular layer, showed intense staining even when the cell bodies themselves were negative. This was expected, owing to the previous demonstration of the presence of M₁ pyruvate kinase in nerve ending by subcellular fractionation methods. (5) The localization was similar in situ and in tissue culture, except that nerve processes were better seen in the latter and astrocytes were sometimes stained in vitro. (6) Variation in intensity of staining was observed in similar cell types in the same section or in the same culture. This could represent different metabolic or functional or maturational states.     

Role of CREB in the regulatory action of sarsasapogenin on muscarinic M1 receptor density during cell aging

This work studied the role of cyclic AMP responsive element binding protein (CREB) in the up-regulation of M₁ muscarinic acetylcholine receptor (M₁ receptor) density by sarsasapogenin (ZMS) in CHO cells transfected with M₁ receptor gene (CHOm1 cells). During cell aging, sarsasapogenin elevated M₁ receptor density as well as CREB and phosphor-CREB (pCREB) levels. CREB peaked earliest, followed by pCREB and M₁ receptor density peaked last. When CREB synthesis was blocked by antisense oligonucleotides, the elevation effect of sarsasapogenin on M₁ receptor density was abolished. These results suggest that sarsasapogenin up-regulates M₁ receptor density in aged cells by promoting CREB production and phosphorylation. Furthermore, the results support the hypothesis that pCREB regulates M₁ receptor gene expression through heterodimer formation.     

巴西橡胶树胶乳磷脂酶A_2的分离纯化及部分酶学性质鉴定

通过丙酮沉淀、DEAE-纤维素离子交换柱层析和Sephadex G-100凝胶过滤柱层析等分离纯化技术,对巴西橡胶树胶乳C-乳清磷脂酶A2进行分离纯化。用SDS-PAGE测定其亚基的相对分子量。测定该酶最适温度和pH,动力学常数Km和Vmax。并测定Ca2+和La3+对酶活性的影响。结果显示:该酶被纯化了49.47倍,产率为5.12%。SDS-PAGE检测为单一条带,其亚基相对分子量约43kDa。最适反应温度为37℃,最适反应pH为8.0,Km为0.44mmol·L-1,Vmax为7.22μmol.(mL.min)-1。最适Ca2+浓度为50μmol·L-1,稀土元素La3+离子对磷脂酶A2活性有抑制作用,但加入Ca2+后可缓解La3+对磷脂酶A2活性的抑制作用。胶乳C-乳清磷脂酶A2与其他植物磷脂酶A2在Ca2+的依赖性上存在差异。研究结果为今后探索橡胶树胶乳磷脂酶A2的催化机理、调节机理及生理功能等奠定了基础。