同时显示肾微血管和细胞构筑的实验研究

目的：在媒染微血管的同时显示肾内细胞构筑，以了解肾微血管与肾小管之间的相互关系。方法：用单宁酸一氯化铁法(TA—be)媒染肾微血管，再以苏木精、H—E法复染来观察肾小管细胞结构。结果：经复染的2组切片各级血管和细胞均染色良好，血管壁平滑肌纤维、内皮细胞界限清晰，细胞核蓝染，H—E染色的细胞质呈深浅不同的红色。因切片较厚，保证了微血管的立体感，细胞的染色效果却不及薄切片的鲜艳。结论：TA—Fe法配合苏木精或HE复染，可同时显示肾微血管和细胞构筑。     

单宁酸-氯化铁法媒染肺内微血管及细胞的组织学观察

目的　光镜下观察大鼠肺微血管及肺组织细胞。方法　应用单宁酸 氯化铁法 (TA Fe法 )灌流固定大鼠 ,取肺做冰冻切片 ,氯化铁显色 ,光镜下观察。结果　肺内小血管、微血管蜿蜒走行 ,被切成各种断面 ,管壁平滑肌细胞、内皮细胞显示清晰 ,有一定立体感 ;接近被膜的微动脉形成较粗的毛细血管 ,肺泡间毛细血管不易分辨 ,肺间质内有微血管和毛细血管分布 ,微血管周围的肺组织亦被媒染。结论　TA Fe法即可显示肺内微血管 ,又能保存组织细胞结构 ,肺内小动脉和微动脉管壁的平滑肌参与微循环的主动调节机制。     

应用单宁酸-氯化铁法显示小脑不同部位的微血管构筑

目的　光镜下观察 6只大鼠小脑前叶、后叶及蚓部微血管构筑。方法　用单宁酸 -氯化铁法 (TAFM)媒染显示小脑内血管。结果　小脑各部位皮质、髓质动脉均来源于软脑膜动脉和小脑动脉中央支 ,软脑膜动脉分支多以直角进入小脑皮质 ,分别在分子层、蒲肯野细胞层、颗粒层形成毛细血管网 ;小脑后叶髓质中微动脉呈爪状分支 ,小叶两侧皮质深层微动脉或毛细血管可跨过髓质互相沟通。结论　 TAFM显示小脑微血管构筑清晰、立体感强 ,并发现小叶内皮质深层微血管之间有吻合支     

大鼠海马微血管构筑的定量分析及衰老变化

目的:观察并定量分析大鼠海马的微血管构筑及衰老变化。方法:采用单宁酸-氯化铁媒染微血管的方法分别观察青龄、老龄大鼠海马与额叶皮质的微血管构筑,并采用MiVnt图像分析系统对海马与额叶皮质的微血管密度(MVD)及微血管面积密度(MVA)进行定量分析。结果:老龄大鼠海马、皮质的微血管数量显著减少,分布杂乱、扭曲缠结。定量分析显示老龄大鼠海马、皮质的MVD值和MVA值均明显低于青龄大鼠,差异具有统计学意义。结论:老龄大鼠海马及额叶皮质的MVD值、MVA值均明显低于青龄大鼠,这是老年血管性痴呆发生机制的主要形态学依据。     

Protective effect of scutellaria baicalensis stem-leaf total flavonoid on the injury of microvascular architecture and lipid peroxidation induced by cerebral ischemia reperfusion in rats

目的:探讨黄芩茎叶总黄酮(SSTF)预处理对脑局灶性缺血再灌注大脑皮质的保护作用及其机制.方法:线栓法制备局灶性脑缺血再灌注模型,造模成功后24 h测脑梗死体积,单宁酸-氯化铁媒染法显示大脑皮质微血管,并用Mivnt图像分析系统对皮质微血管密度(MVD)及微血管面积密度(MVA)进行定量分析;检测脑皮质组织中超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量.结果:SSTF预处理可改善术后大鼠的MVD和MVA;降低因缺血再灌注引起的脑皮质组织MDA含量增加,提高SOD活力.结论:SSTF预处理可改善脑皮质微血管形态结构,抵制脂质化过氧化反应,减轻自由基损害,对缺血再灌注损伤脑组织具有保护作用.     

Ana-1巨噬细胞分泌exosomes的提取及免疫鉴定

目的:为了证实Ana-1巨噬细胞是否分泌exo-somes。方法:采用分级离心的方法,从Ana-1巨噬细胞培养的上清液提取exosomes。样品用醋酸双氧铀负染,在透射电镜下观察其形态、大小;并分别用Flotillin多克隆抗体、Tsg101单克隆抗体(mAb)、Hsp70mAb和calnexin多克隆抗体,通过免疫印迹法和免疫金标记法进行免疫鉴定。结果:电镜下可以观察到提取的exosomes平均大小为50～100nm。免疫印迹法分析结果显示,提取的exosomes表达Flotillin、Tsg101和Hsp70蛋白,相对分子质量(Mr)分别为45000、43000和70000,但不表达calnexin蛋白;免疫电镜分析显示ex-osomes膜表面和内部有明显的金颗粒。结论:Ana-1巨噬细胞分泌exosomes。     

雌激素改善卵巢切除合并慢性铝中毒对大鼠心脏Cx43表达和金属元素变化的影响

目的 探讨雌激素治疗卵巢切除合并慢性铝中毒大鼠,对心脏间隙连接蛋白43(Cx43)表达和金属离子含量的影响. 方法 40只6月龄雌性SD大鼠,随机分为卵巢假切除组(Sham组),卵巢切除组(OVX组)、卵巢切除合并慢性铝中毒组(OVX+Al组)和卵巢切除合并慢性铝中毒及尼尔雌醇灌胃组(OVX+Al+E2组).于术后3个月,利用离子体发射光谱仪测定心脏金属元素含量,采用免疫组织化学和图像分析法观测心肌细胞Cx43表达.结果 1. Cx43表达:Sham组心房肌Cx43遍布于心肌细胞侧面连接和端闰盘处.心室肌分布于心肌闰盘处.OVX组心房肌的Cx43颗粒部分由细胞侧-侧连接处移至闰盘处或杂乱排列,心室肌Cx43由闰盘处移至细胞侧-侧连接处或杂乱地无序排列,少部分出现在细胞质中,OVX+Al组Cx43排列类似于OVX组,且更加紊乱.OVX+Al+E2组心房肌Cx43回复正常分布,但心室肌未发现回复.2.图像分析结果,各组大鼠心肌细胞Cx43的面积构成比比较无显著差异.3.金属元素含量:在Sham组,金属元素在心脏含量的排列顺序如下:Mg＞Ca＞Fe＞Zn＞Al＞Si＞Cu＞Mn＞Se和Cd.各组间比较具有显著差异的是:与Sham组比较,OVX组Zn降低;OVX+Al+E2组Al、Cd、Si、Se均升高;OVX+Al组与OVX组比较,Si升高、Zn降低.OVX+Al+E2组与OVX+Al组比较, Cd、Mn、Se升高. 结论雌激素改善卵巢切除及加铝后引起的心脏金属元素含量改变和心肌细胞间隙连接蛋白Cx43重构.     

三种含硒化合物对K562细胞的抑制作用

目的 研究比较3种新的含硒化合物在体内、外的抗癌作用和作用机制. 方法 通过四甲基偶氮唑盐(MTT)比色法和移植瘤生长抑制实验及形态学观察、流式细胞术以及激光扫描共焦显微镜,检测分析含硒化合物对K562细胞的抑制作用. 结果 3种含硒化合物能明显抑制K562细胞增殖(P<0.05)和S180、H22移植瘤(n=10)的生长(P<0.01);使K562细胞出现体积缩小,细胞膜完整,染色质高度凝集,边集,核浓染、碎裂,伴有出泡现象和凋亡小体出现等典型的凋亡特征;对K562细胞的周期分布有明显影响,且在大剂量时出现了明显的亚二倍体峰;能够明显增加K562细胞内Ca2+、Mg2+和细胞内活性氧(ROS)的荧光强度(P<0.01),但pH值和线粒体膜电位显著降低(P<0.01). 结论 3种含硒化合物均有体内、体外抗肿瘤作用,其作用机制可能与Ca2+、Mg2+、(活性氧ROS)、pH值和线粒体膜电位(MMP)诱导的细胞凋亡有关.     

凝血酶对大鼠脑微血管内皮细胞的影响

目的 :探讨凝血酶 (Thrombin ,TM )对脑微血管内皮的影响。方法 :将大鼠脑微血管内皮细胞进行培养 ,培养液中加入 10U的TM或10U的TM + 0 .4mU的组织蛋白酶G(CaspethsinG ,CATG ) ,相差显微镜动态观察内皮细胞形态的变化 ,免疫组织化学技术检测基质金属蛋白酶 2 (MatrixMetalloproteinase 2 ,MMP 2 )表达的改变。 结果 :TM使内皮细胞发生收缩 ,细胞收缩程度具有时间依赖性 ,使内皮细胞MMP 2表达水平明显增加。TM +CATG加入培养液后 ,细胞形态、MMP 2表达与对照组比较均无明显统计学差异 (P >0 .0 5 )。结论 :TM通过激活蛋白酶激活受体 1(proteaseactivatedreceptor 1,PAR 1) ,使内皮细胞发生收缩 ,促进MMP 2表达 ,是TM增加血脑屏障 (BloodBrainBarrier,BBB)通透性的可能机制。     

用中子活化法对常人与慢性牙周炎患者牙周组织中微量元素分布的研究

中子活化分析是对微量元素进行检测一种比较好的方法,目前国内尚未见其应用与牙周病组织的检测及研究.作者首次应用该种方法在大型及微型核反应堆中对正常人及慢性牙周炎患者牙周组织中的微量元素进行了多元素的测定及分析,初步研究结果提示:人牙周组织内含有AI, Cu,Co、Ca、Fe、Zn,Mg.Na等20种元素,而慢性牙周炎患者牙周组织中Al,Cu,Cr,Zn,Mg,Na等元素均显示高于正常人的趋向,尤以Cu,Cr,Mg三种微量元素明显高于正常人的含量均值,且有显著性差异.病变组织中有些相关元素含量比值出现失平衡,且Fe/Cu,K/Na比值于正常人统计学处理有显著性差异.故提示牙周病的发生与组织中微量元素的改变有一定的关系.     

Protections by non-copper metal ions against copper-mediated inactivation of poliovirion RNA occurring at extraction of virions with phenol

Al3+, Ca2+, Co2+, Cu1+, Fe2+, Fe3+, Mg2+, Mn2+, Ni2+, Pb2+, Sn4+, and Zn2+ were incubated individually with redistilled reagent-grade phenol containing impurities known from previous work to interact with Cu2+ to produce a potent inactivator(s) of the transfectivity of naked poliovirion RNA. Only the mixture with Cu1+ inactivated the RNA. Tests of each of the 11 non-copper test metal ions mixed with Cu2+ before adding the phenol showed that Ca2+ and Mg2+ do not protect, Co2+, Mn2+, Ni2+, Pb2+, and Zn2+ provide moderate protection, and Al3+, Fe2+, Fe3+, and Sn4+ give strong protection against the Cu2+-mediated inactivation. Other points of addition of protective metal ion were tested using Fe3+. Strong protection was afforded even when Fe3+ was added after synthesis of the inactivator(s) from Cu2+ and the active impurities. The relation between Cu2+ and the Fe3+ was shown to be competitive. The hypothesis that ions compete for semi-quinone anion is proposed.     

Metal-catalyzed peroxidation of polyunsaturated fatty acids

In this study malondialdehyde (MDA) was measured as the thiobarbituric acid (TBA) adduct by high performance liquid chromatography with linolenic acid (LA), as well as with LA to which the following metal ions were added: Ag+1, Al+3, As+5, Ba+2, Cd+2, Co+2, Cu+1, Cu+2, Fe+2, Fe+3, Hg+2, Mn+2, Ni+2, Pb+2, and Zn+2. Our results showed that Fe+3 is a particularly potent catalyst of this oxidation reaction. Several other metals also significantly increased the production of MDA over LA alone, as shown by the Mann-Whitney test, including Al+3 and Fe+2 (p less than 0.001), Cu+2, Ba+2, Mn+2 (p less than 0.01), and As+5 (p less than 0.05). Pb+2 almost completely inhibited the production of MDA from LA (p less than 0.001). Addition of the free radical scavenger, butylated hydroxytoluene (BHT), significantly inhibited MDA formation in these latter reactions, indicating they are free-radical mediated. Pre-incubation of the reaction mixtures at 37 degrees C and physiologic pH with both Fe+2 and Fe+3 greatly increased MDA formation, suggesting that in acute metal toxicity the initiating free-radical process might be primarily due to direct metal catalysis of preformed lipid peroxides.     

胃肠运动自主节律性的产生机制

ICC作为胃肠运动自主节律性的起搏细胞已得到广大研究者的承认 ,但这种自主节律性的产生机制仍不清楚。随着对ICC认识的逐步深入 ,已经发现一些离子流 ,如Ca2 + 、Na+ 、K+ 、Cl-与ICC的起搏密切相关。ICC的起搏电流可能是由Ca2 + 调节的非电压依赖性非选择性阳离子通道所介导的多种阳离子组成 ,Ca2 + 起重要的调节作用。本文对此领域的研究进展进行综述 ,期望为深入理解胃肠运动调控机制及其功能性疾病的发病机理提供参考资料。     

Determination of nitrate in blood by gas chromatography and gas chromatography-mass spectrometry

We devised a sensitive and simple method for determining nitrate in whole blood, using an extractive alkylation technique. Nitrate in whole blood was reduced to nitrite by hydrazine sulfate in the presence of Cu2+ and Zn2+ as catalysts, and alkylated with pentafluorobenzyl bromide using tetradecyldimethylbenzylammonium chloride as the phase-transfer catalyst. The obtained derivative was analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with electron-capture detection. The detection limit of nitrate in whole blood was 0.01 mM. The calibration curve was linear over the concentration range from 0.02 to 1.0 mM for nitrate in whole blood. The accuracy and precision of the method were evaluated and the relative standard deviations were found to be within 10%. Using this method, the blood nitrate levels of two victims who committed suicide by inhaling automobile exhaust gas were determined.     

Lead and zinc block a voltage-activated calcium channel of Aplysia neurons.

1. The effects of Pb2+ and Zn2+ on the peak of the voltage-activated calcium current of Aplysia neurons were examined. Calcium currents were reversibly blocked by Pb2+ at concentrations that did not significantly affect potassium and sodium currents and by Zn2+ at concentrations associated with a delay and reduction of peak sodium and potassium currents. 2. The block by both was concentration dependent, and percentage blockade was reduced in elevated Ca2+. The threshold Pb2+ concentration for blockade in 20 mM Ca artificial sea water (ASW) was approximately 1 microM, whereas for Zn2+ it was 2 mM. The Hill coefficient for Pb2+ action was near 1.0 under all conditions, whereas for Zn2+ it was 1.4-1.6. 3. With addition of Pb2+, the voltage at which peak calcium current was generated shifted to hyperpolarized voltages, an effect similar to that caused by reduction of Ca2+ concentration in the absence of Pb2+. Zn2+ shifted the voltage at which peak current was generated in a depolarizing direction. 4. Pb2+ did not significantly change inactivation but shifted the voltage dependence of activation to hyperpolarized voltages in a dose-dependent manner. Zn2+ shifted both activation and inactivation in a depolarizing direction in a dose-dependent fashion. 5. The blockade of calcium currents by Pb2+ but not Zn2+ was highly voltage dependent and increased with depolarization. 6. Our results suggest that Pb2+ is a specific, potent, competitive, and reversible blocker of calcium currents. These observations are consistent with a competition by Pb2+ with Ca2+ at a binding site within the calcium channel. In contrast, the blockade of calcium currents by Zn2+ is probably through actions at fixed charge sites external to the channel.     

Nerve endings in human sympathetic ganglia

Forty-eight human sympathetic ganglia from 22 sympathectomies were examined ultrastructurally after one of three different fixations: (1) glutaraldehyde + osmium tetroxide, (2) glutaraldehyde + potassium dichromate + osmium tetroxide, or (3) potassium permanganate. Three different kinds of synapsing nerve ending could be identified after all fixation schedules. Type 1: “Cholinergic,” containing small, agranular vesicles 40–60 nm in diameter (75% of all vesicles) and some large granular vesicles (100 nm in diameter). The number of type 1 profiles decreased with increasing age. Type 2: “Adrenergic,” containing small granular vesicles 40–70 nm in diameter (over 90% of the vesicles). Type 3: “Nonadrenergic, noncholinergic,” characterized by large opaque vesicles 80–160 nm in diameter (over 50% of all vesicles). The frequencies of the types were counted after potassium permanganate fixation: type 1–71%, type 2–23%, and type 3–6%. In addition, at least two types of nerve profiles were observed which did not form synapses: (1) profiles entirely filled with mitochondria (mitochondrial accumulation), and (2) large nerve profiles full of different kinds of vesicles, myelin figures, and mitochondria (axonal dilatation). It is concluded that the sympathetic ganglion cells receive cholinergic and adrenergic innervation and also a third type of innervation, the possible peptidergic nature of which is discussed.     

Selective dissolution of AuCu and AuAg alloys in vitro and in vivo

AuCu and AuAg alloys (30 at.% Au) have been immersed in aqueous solutions of chloride and sulphide with additions of protein (mucin). The surfaces of the alloys were subsequently analysed by means of ESCA and compared with previous and similar tests without addition of mucin. No noticeable effect of the mucin addition was observed on the surface composition of the alloys after exposure to the various solutions. Cu in contrast to Ag was found to be selectively dissolved from the 2 or 3 uppermost atomic layers in chloride as well as sulphide solutions (pS > 4), both with and without additions of mucin, leaving behind a surface enriched from 10 to 25 at.% in Au. Testing of the same alloys in vivo for 8 d revealed the same tendencies to selective dissolution of Cu. The AuAg alloy was tarnished in the sulphide solutions and in vivo.     

In situ imaging of intracellular calcium with ischemia in lung subpleural microvascular endothelial cells

We propose that generation of reactive oxygen species (ROS) during ischemia is associated with an increase in intracellular calcium ([Ca2+]i) in pulmonary capillary endothelial cells. We used an isolated rat lung model and epifluorescence microscopy to evaluate [Ca2+]i in subpleural microvascular endothelial cells in situ by ratio imaging of the fluorophores, Calcium Green and Fura Red (CG/FR). Lungs were ventilated continuously under control (continuously perfused) or global ischemia (no perfusion) and thus remained adequately oxygenated even with ischemia. Ischemia for 5 min led to increase in CG/FR, indicating increase in [Ca2+]i in endothelial cells in situ; CG/FR remained elevated during a subsequent 10 min of ischemia. Ca(2+)-free perfusion and gadolinium (100 microM) inhibited the increase in [Ca2+]i, while thapsigargin (250 nM) had no effect. These results indicate that increase in endothelial cell [Ca2+]i with ischemia was due to influx from the extracellular medium. Perfusion with N-acetyl-L-cysteine (20 mM) or diphenyleneiodonium chloride (10 microM) prevented the ischemia-mediated [Ca2+]i increase, suggesting a role for ROS in the Ca2+ changes with ischemia. Membrane depolarization by perfusion with high potassium (K+) or glyburide also resulted in increased [Ca2+]i whereas the K(+)-channel agonist cromakalim, inhibited ischemia-mediated Ca2+ influx. We conclude that increased ROS generation with 'oxygenated' lung ischemia is associated with influx of Ca2+ and an increase in endothelial cell cytosolic calcium concentration.     

Relationship between magnesium, carcinogenic metals and ionic permeability of the human amnion

The effects of the well established carcinogenic metals or metalloids (As, Be, Cr, Ni), hypothetically carcinogenic but well-established cocarcinogenic metals (Cd, Pb, Co) and weak co-carcinogenic metals (Al, Cu, Fe, Zn) and the antagonism between Mg and these metals were studied on the ionic transfer through the isolated human amnion. The ionic transfer is estimated by the measure of the membrane potential (delta psi), the cationic number transference t+ and the ionic fluxes (F1, F2). All carcinogenic metals increase delta psi (+) and reduce t+, F1 and F2. Mg has an opposite action with regard to Cd, Pb and Ni, but may act as a co-carcinogenic agent by increasing the effects of Al, Cu and Zn.