M-CSF和staurosporine促进小鼠腹腔巨噬细胞巨涎蛋白摄取ox-LDL

目的：探讨巨噬细胞集落刺激因子（M-CSF）和蛋白激酶C抑制剂(STA)对小鼠腹腔巨噬细胞（MPM）巨涎蛋白摄取氧化低密度脂蛋白(ox-LDL)的影响。 方法： M-CSF与蛋白激酶C抑制剂STA预处理MPM后制备膜蛋白，开展SDS-PAGE电泳及配体印迹试验，测定不同条件下巨涎蛋白结合［125I］ox-LDL的值。 结果： MPM膜蛋白经唾液酸酶预处理后，巨涎蛋白与［125I］ox-LDL的结合值为(2.45±0.46)μg/g细胞蛋白，显著低于对照组的(58.38±1.78)μg/g细胞蛋白。用M-CSF与STA预处理MPM后，处理组与对照组巨涎蛋白结合配体［125I］ox-LDL呈现一趋向饱和的浓度曲线，经线性回归得两类似平行的直线，M-CSF组Bmax(453.59±15.39)μg/g蛋白，显著高于对照组的Bmax(322.77±12.54)μg/g蛋白，而Kd值变化不大。STA处理组Bmax=(362.40±15.31)μg/g蛋白，Kd值为(15.10±2.67)mg/L，对照组Bmax为(264.76±11.29)μg/g蛋白，Kd值为(17.43±2.98)mg/L。 结论： 巨涎蛋白接受M-CSF和STA的上调作用，通过增加其在MPM表面数目而促进对ox-LDL的摄取，促进泡沫细胞的形成。     

鸟类及鼠类的胸腺、腔上囊及脾脏褪黑素受体的鉴定及意义

应用放射配体结合法检测大鼠、小鼠、仓鼠、鸡、鸭、鸽子及鹌鹑的胸腺、腔上囊及脾脏的[~(125)I]褪黑素(M)特异结合位点。结果显示鸟类及鼠类免疫器官均存在[~(125)I]M特异结合位点,并具低结合容量、高亲和力特点;动力学研究表明具可饱和性及可逆性,符合受体的结合及解离过程;特异性研究显示对M及激动剂有高度特异性,符合特异结合位点的全部条件,亚细胞分布的研究表明以细胞核含量最高,线粒体次之,且该结合位点具年龄依赖性降低。结果证实免疫器官存在根黑素受体(MR),免疫组织是M作用的靶器官,M对免疫系统的调节作用是通过免疫组织上MR直接作用的结果。     

氯喹对实验性呼吸窘近综合征大鼠肺中β-受体及cAMP变化的影响

为进一步探讨氯喹对油酸所致的实验性大鼠呼吸窘迫综合征(RDS)的治疗作用机理,我们实验观察了用药前后大鼠肺中β-即受体的变化以及由此引起的cAMP含量的变化. 研究中用放射性配基结合分析法测定β-受体的最大结合容量(Bmax)和解离常数(Kd),以竞争性结合蛋白分析法测定cAMP含量。实验结果显示:正常大鼠组、RDS模型组和氯喹治疗组大鼠肺中的Bmax值分别为490±61、307±55、494±48fmol/mg蛋白;各组Kd值无明显改变;其相应各组大鼠肺中cAMP     

女性器官褪黑素受体鉴定及意义

目的 :已知褪黑素 (Ｍ)对生殖系统有抑制作用 ,提示生殖系统是Ｍ作用的靶器官 ,生殖系统上可能存在褪黑素受体 (ＭＲ)。方法 :ＭＲ检测方法在放射配体结合法。 [1 2 5Ｉ]褪黑素 ( 1 2 5Ｉ·Ｍ ,81 40× 1 0 1 0 Ｂｑ/ｍｍｏｌ)为中科院上海原子能所产品。结果 :1 [1 2 5Ｉ·Ｍ]特异性最大结合量 (Ｂｍａｘ)在卵巢、子宫及输卵管分别为1 2± 0 4,1 0± 0 3,0 6± 0 1ｆｍｏｌ/ｍｇ蛋白。平衡解离常数 (Ｋｄ)分别为 58± 1 1 ,62± 1 4,89± 9ｐｍｏｌ/Ｌ。 2 动力学研究 :随时间增加 ,结合量不断增多 ,至 40ｍｉｎ饱和 ,加入非标记Ｍ…     

大鼠主动脉球囊成形术后尾加压素II受体的变化

目的 :观察大鼠主动脉球囊成形术后尾加压素II (UII)受体特征的变化及血管对UII收缩效应的改变。方法 :测定大鼠主动脉球囊损伤后 [12 5I]-UII配体结合及血管张力。结果 :球囊成形术后 3d、2 1d时 ,血管对UII的反应性增强 (P <0 0 5 ) ,主动脉UII受体最大结合 (Bmax)较对照组分别高 44 %及 36 % [(35 0 3± 2 41)pmol/gproteinand (33 2 6± 2 40 )pmol/gproteinvs(2 4 37± 3 2 0 )pmol/gprotein ,P <0 0 1],受体与配体解离常数 (Kd)无显著差异 (P >0 0 5 )。结论 :血管球囊损伤后UII受体发生上调 ,受体密度增加 ,血管对UII的反应性增强 ,表明UII可能在血管成形术后再狭窄过程中发挥重要作用。     

氧化低密度脂蛋白受体在人血管内皮细胞上的表达及其调控

采用放射配基结合及竞争抑制实验和反转录 聚合酶链反应检测人脐静脉内皮细胞 (HUVECs)上血凝素样的氧化低密度脂蛋白受体 (LOX1)的表达 ,表明HUVECs存在LOX1的基因表达和高亲和力位点Bmax (5 4 5±2 0 3)ng/ 10 6cell,Kd (2 0 1± 0 6 )× 10 -8mol/L ,氧化低密度脂蛋白对其自身受体LOX1的基因表达为正反馈调节 ,应用LOX1抑制剂可抑制此作用。     

分离成年大鼠心肌细胞α_1受体及其磷脂酰肌醇代谢研究

为排除整体心脏组织中其它非心肌成分的影响,本文用胶原酶分离成年大鼠心肌细胞,分析其中α_1受体及其亚型的分布,并研究α_1受体与磷脂酰肌醇代谢的关系。作~(125)I-BE与α_1受体结合的饱和曲线,经Scatchard分析,其Bmax(代表α_1受体的数目)为42.7±0.8fmol/mg蛋白,Kd为58.4±6.3(n=4)。5-MU对~(125)-BE与     

糖尿病患者白细胞糖皮质激素受体及趋化移动改变的研究

我们先前的工作表明糖尿病患者白细胞糖皮质激素受体(GR)的特异结合量(Rs)减少。由于系应用一点分析法,不能区别其减少是由于GR的最大结合容量(Bmax)的改变,还是亲和力(Kd)改变的结果。为此,应用Scatchard分析法测定糖尿病患者外周血白细胞GR的Bmax和Kd,同时测定皮质醇(F)对白细胞趋化移动(ChtM)的抑制率(FI),以判断白细胞GR的改变有无临床及病理学意义。研究对象为糖尿病患者30例,年龄54.6±12.0(33～70)岁;男17、女13例;Ⅰ型10例、Ⅱ型20例。空腹血糖16.5±7.9(6.8～29.0)     

硝苯吡啶对肾性高血压大鼠心肌α_1受体的影响

8月龄肾性(2K,1C)高血压大鼠灌服钙拮抗剂硝苯吡啶10mg/kg/d,共30天,药物降压效果显著,与文献报道一致。同龄正常组(n=9),高血压组(n=6)和用药组(n=7)大鼠断头取出心脏,按常规匀浆离心制得心肌膜标本。用~3H-Prazcsin进行放射配基受体结合分析,数据经计算机软件Scatchard作图计算,结果如下;正常组Bmax=43.31±5.42fmol/mg蛋白,Kd=0.11±0.03nM。高血压组Bmax=54.46±6.16     

狗外周血白细胞糖皮质激素受体的测定

以[~3H]地塞米松(Dex)为配体,建立了狗外周血白细胞糖皮质激素受体(GCR)的放射配体结合测定方法,Scatchard作图分析[~3H]Dex特异结合呈一直线,表观结合容量(R_o)和平衡解离常数(Kd)分别为7.30±1.98fmol/10~6个细胞和4.69±2.65_nM((?)±SD,N=5),特异结合有明显的甾体特异性。用饱和浓度的[~3H]Dex测定了22只无应激条件下成年健康狗外周血白细胞的[~3H]Dex特异结合,结果为3745±436位点/细胞((?)±SD),雄雌之间差异不显著。本方法具有取材方便,重复性好及用血量少等优点,适用于一般实验室。     

Gene structures,biochemical characterization and distribution of rat melatonin receptors

G-protein coupled receptors for the pineal hormone melatonin have been partially cloned from rats. However, insufficient information about their cDNA sequences has hindered studies of their distribution and physiological responses to melatonin using rats as an animal model. We have cloned cDNAs of two rat membrane melatonin receptor subtypes, melatonin receptor 1a (MT1) and melatonin receptor 1b (MT2), using a rapid amplification of cDNA end (RACE) method. The rat MT1 and MT2 cDNAs encode proteins of 353 and 364 amino acids, respectively, and show 78–93% identities with the human and mouse counterparts. Stable expression of either rat MT1 or MT2 in NIH3T3 cells resulted in high affinity 2-[¹²⁵I]-iodomelatonin (¹²⁵I-Mel) binding (K _d = 73.2 ± 9.0 and 73.7 ± 2.9 pM, respectively), and exhibited a similar rank order of inhibition of specific ¹²⁵I-Mel binding by five ligands (2-iodomelatonin > melatonin > 6-hydroxymelatonin > luzindole > N-acetyl-5-hydroxytryptamine). RT-PCR analysis showed that MT1 is highly expressed in the hypothalamus, lung, kidney, adrenal gland, stomach, and ovary, while MT2 is highly expressed in the hippocampus, kidney, and ovary. We also performed multi-cell RT-PCR to examine the expression of mRNAs encoding MT1 and MT2 in adult GnRH neurons. MT1 was weakly expressed in male GnRH neurons, and was less expressed in the female neurons. MT2 expression was undetectable in GnRH neurons from either sex. This study delineates the gene structures, fundamental properties, and distribution of both rat melatonin receptor subtypes, and may offer opportunities to assess the physiological significance of melatonin in rats. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Removal of melatonin receptor type 1 increases intraocular pressure and retinal ganglion cells death in the mouse

Previous studies have demonstrated that melatonin is effective in lowering intraocular pressure and that it may also protect ganglion cells. We have recently reported that, in mice lacking the melatonin receptors type 1, 25–30% ganglion cells die out by 18 months of age, suggesting that these receptors might be important for ganglion cells survival. In this study we show that the loss of ganglion cells is specific for melatonin receptors type 1 knock-out since mice lacking the melatonin receptors type 2 did not show any significant change in the number ganglion cells during aging. Furthermore, we report that melatonin receptors type 1 knock-out mice have higher intraocular pressure during the nocturnal hours than control or melatonin receptors type 2 knock-out mice at 3 and 12 months of age. Finally, our data indicate that administration of exogenous melatonin in wild-type, but not in melatonin receptors type 1 knock-out, can significantly reduce intraocular pressure. Our studies indicate that the decreased viability of ganglion cells observed in melatonin receptors type 1 knock-out mice may be a consequence of the increases in the nocturnal intraocular pressure thus suggesting that intraocular pressure levels at night and melatonin signaling should be considered as risk factor in the pathogenesis of glaucoma.     

Circadian rhythms and the effects of exogenous melatonin in the nine-banded armadillo, Dasypus novemcinctus: A mammal lacking a distinct pineal gland

Circadian rhythms of body temperature, activity and oxygen consumption were recorded in the nine-banded armadillo, Dasypus novemcinctus, a mammal lacking a distinct pineal gland. The rhythms were entrained by light-dark cycles and appeared to be phase-coupled. Under constant illumination, the animals displayed free-running circadian rhythms with period lengths of slightly less than 24 hr. Melatonin implants caused a lengthening of the free-running period of activity and body temperature. The occurrence of a “normal” circadian rhythmicity in the armadillo indicates that the pineal organ is not necessary for the circadian organization of this animal whereas melatonin of non-pineal origin may have a role.     

Segmental, coronal and subcellular distribution of 2-[I]iodomelatonin binding sites in the chicken spinal cord

Radioreceptor and autoradiography studies using chicken spinal cords demonstrated that the binding density of 2-[¹²⁵I]iodomelationin ([¹²⁵I]MEL) was significantly higher in the lumbar segment and the specific binding of [¹²⁵I]MEL was localized in the gray matter. Subcellularly, different densities of binding sites were localized in the following order: nuclear > microsomal > mitochondrial > cytosolic. Localization of [¹²⁵I]MEL binding sites in the dorsal gray matter of the chicken spinal cord suggests that melatonin plays a role in regulating the spinal cord functions which may associate with the modulation of temperature and pain transmission and/or visceral and autonomic functions.     

电刺激对军人血浆褪黑激素昼夜节律的影响

人体的生理与行为节律是由内源性昼夜节律钟产生与调控的。我们通过监测人体体核温度 ,就电针与光照肌对军人血浆褪黑激素节律的影响进行了研究 ,以探索电针调节人体节律的相位反应规律。实验结果显示 ,电针对军人血浆中的褪黑激素节律相位的影响具有明显的依时相性 ,其相位反应曲线呈光性模式。这一发现为治疗睡眠与节律混乱性疾病提供了新的思路。     

Binding characteristics and regional distribution of [I]iodomelatonin binding sites in the brain of the human fetus

The binding and pharmacological characteristics of [¹²⁵I]iodomelatonin binding sites in human fetal brain membrane preparations were determined. Membrane preparations of the whole brain revealed a equilibrium binding constant (K_d) of 17.5 pmol/l with a total number of binding sites (B_max) at 0.8 fmol/mg protein. Among the various brain regions studied, [¹²⁵I]iodomelatonin binding was highest in the hypothalamus, and lowest in the mid-brain, pons-medulla, and cerebral cortex. The K_d of the hypothalamus was calculated to be 26.1 pmol/l and the B_max 5.4 fmol/mg protein. Only 6-chloromelatonin, melatonin and N-acetylserotonin had significant inhibition in the binding. Our results suggest that melatonin receptors are present in the human brain and that melatonin may exert a central action.     

Immunoassay of Melatonin with Enzyme-Labeled Antibodies

This paper reports a non equilibrium competitive enzyme immunoassay method using enzyme-labeled antibodies, for the quantitation of melatonin in chloroform-extracted samples. Its principle is as follows : methoxytryptamine hemisuccinate-human serum albumin conjugate physically absorbed onto a polystyrene sphere and melatonin to be measured, compete for a limited and fixed amount of peroxidase-labeled anti melatonin IgG. After incubation and washings the enzymatic activity bound to the sphere was measured with a chromogenic substrate. This simple method can detect as low as 22 fm of melatonin and is fairly precise. We also present its application to the determination of melatonin in serum and pineal gland.     

Retinal [I]iodomelatonin binding sites in the tree shrew (Tupaiidae)

The characteristics of melatonin-binding sites labelled by [¹²⁵I]iodomelatonin in membrane preparations from the tree shrew retina were determined. Specific binding of [¹²⁵I]iodomelatonin to the membrane preparations of tree shrew retina was rapid, stable, saturable, and reversible. Among the indoles tested only 6-chloromelatonin, melatonin and N-acetylserotonin had significant affinities to the [¹²⁵I]iodomelatonin binding site. Scatchard analysis of the membrane preparations revealed a dissociation constant (K_d) of 51.0 ± 16 pM and total number of binding sites (B_max) of 1.97 ± 0.6 fmol/mg protein.     

人外周血淋巴细胞褪黑素受体的基因与蛋白表达的研究

目的:研究人外周血淋巴细胞褪黑素受体(MR)的基因与蛋白表达的特点。方法:分离健康成年人外周血淋巴细胞,采用异硫氢酸胍-苯酚-氯仿一步法抽提总RNA,分别采用Northern印迹和RT-PCR方法检测MR亚型mt₁和MT₂的mRNA,并将RT-PCR的阳性产物通过自动测序仪测序;同时应用免疫组化染色方法检测mt₁和MT₂受体亚型蛋白在淋巴细胞内的分布特点。结果:Northern印迹方法检测出长约1.1 kb mt₁ mRNA,但未检出长约1.1 kb MT₂ 的mRNA;RT-PCR方法则得到了368 bp mt₁ cDNA片段,无321 bp MT₂ cDNA片段,同时将mt₁阳性条带通过胶回收、测序,结果显示扩增产物与人mt₁受体亚型的基因序列相吻合;免疫组化结果显示:人外周血淋巴细胞存在mt₁受体蛋白,主要分布于细胞膜和细胞浆,呈棕褐色阳性颗粒,而细胞核上未见阳性颗粒,MT₂受体蛋白则未检出。结论:证实正常人外周血淋巴细胞存在mt₁的基因与蛋白的表达,而无MT₂的基因与蛋白的表达,提示外周血淋巴细胞为褪黑素(Mel)作用的外周靶器官,为研究生理及病理情况下Mel对免疫系统的调节奠定了基础。     

Retinal [125I]iodomelatonin binding sites in the tree shrew (Tupaiidae).

The characteristics of melatonin-binding sites labelled by [¹²⁵I]iodomelatonin in membrane preparations from the tree shrew retina were determined. Specific binding of [¹²⁵I]iodomelatonin to the membrane preparations of tree shrew retina was rapid, stable, saturable, and reversible. Among the indoles tested only 6-chloromelatonin, melatonin and N-acetylserotonin had significant affinities to the [¹²⁵I]iodomelatonin binding site. Scatchard analysis of the membrane preparations revealed a dissociation constant (K_d) of 51.0 ± 16 pM and total number of binding sites (B_max) of 1.97 ± 0.6 fmol/mg protein.