高转换型肾性骨病中骨保护素及其配体的表达和形态计量学观察

目的 观察高转换型肾性骨病中骨保护素及其配体 (OPG，RANKL)的表达，并与骨形态计量学指标进行相关分析。 方法 选择10例慢性肾衰尿毒症患者和3例正常人进行髂骨活检术，获得骨组织标本。采用免疫组化方法检测OPG和RANKL蛋白质的表达。采用全自动图像分析系统进行骨组织形态计量学测定。结果 10例慢性肾衰尿毒症患者经骨病理学检查证实均为高转换型骨病，以破骨细胞活化形成骨吸收陷窝伴或不伴骨矿化不全为特点。免疫组化显示尿毒症患者骨组织中以RANKL阳性表达为主。与正常对照相比，RANKL阳性表达细胞数目显著增加，OPG阳性表达细胞数目显著减少。尿毒症患者RANKL的阳性表达细胞数目与骨吸收面积和破骨细胞数目呈显著正相关。结论 高转换型肾性骨病中，PTH的溶骨作用可能是通过OPG/RANKL/RANK系统介导的。     

Cannabinoid receptor down-regulation without alteration of the inhibitory effect of CP 55,940 on adenylyl cyclase in the cerebellum of CP 55,940-tolerant mice

The objective of this study was to determine whether the development of tolerance to CP 55,940, a potent cannabinoid agonist, was due to changes in the receptor or second messenger system. ICR mice treated with CP 55,940 (2 mg/kg) twice a day for 6 and one-half days developed a high degree of tolerance to the pharmacological effects of CP 55,940. The ability of CP 55,940 to produce motor hypoactivity, hypothermia and immobility was reduced 163-, 97- and 19-fold, respectively. Evaluation of 3H-CP 55,940 binding to rat brain membranes indicated no difference in receptor affinity between the vehicle- and CP 55,940-treated animals. However, these binding studies revealed a 50% decrease in receptor number in the cerebellum of the CP 55,940-tolerant mice. Although cAMP is generally considered to be the second messenger for cannabinoid receptors, little difference was observed in the inhibitory effects of CP 55,940 on adenylyl cyclase activity in cerebellum between vehicle and drug-treated mice. However, there was an increase in receptor mRNA which suggests a compensation for receptor loss. There are several possible explanations for these results. There may be sufficient spare receptors such that CP 55,940-tolerant mice are capable of producing a maximal effect on the second messenger system. On the other hand, one could conclude that cannabinoid receptor down-regulation does not account for the development of tolerance to all of the effects of CP 55,940 in mice.     

前列腺带性结构及其雄激素、雌激素、表皮生长因子受体分布特征的研究

为了探讨雄激素受体（ＡＲ）、雌激素受体（ＥＲ）、孕激素受体（ＰＲ）和表皮生长因子受体（ＥＧＦＲ）在前列腺带性结构中的分布，应用单饱和剂量测定法，分别测定了前列腺移行带、外周带组织中ＡＲ、ＥＲ、ＰＲ和ＥＧＦＲ的含量。结果表明，在１１例前列腺增生症（ＢＰＨ）患者２２份标本中，移行带和周围带ＡＲ全部阳性；尽管ＡＲ浓度个体差异很大，但是两带之间仍有密切相关性（Ｐ＜０００１）；周围带ＡＲ浓度高于移行带，两带ＡＲ浓度均高于ＰＲ、ＥＲ；ＰＲ和ＥＲ在前列腺带性结构上没有明显差异。ＥＧＦＲ在移行带明显高于周围带。研究证实了ＡＲ、ＥＲ、ＰＲ，ＥＧＦＲ在前列腺移行带和周围带上的分布特征。     

人胎盘微绒毛膜的制备及其转铁蛋白受体研究

作者分别用差速离心法及蔗糖梯度离心法制备胎盘微绒毛膜(PMM),用受体放射分析法研究了PMM的转铁蛋白受体(TfB),结果表明:两种方法制备的PMM均可用于受体分析,但差速离心法更为简便。测定60例产妇PMM的TfR,其数目为3.53±1.98×10~(12)个位点/mg膜蛋白,最大结合容量为6.33±4.21×10~(-12)mol/mg膜蛋白,Kd值为4.95±3.39×10~(-9)mol/L。受体结合反应体系最适实验条件为,膜蛋白浓度每管50μg,标记物浓度每管50 000cpm,孵育30分钟,B/F分离的聚乙二醇浓度为12％(W/V)。对TfR的特性研究表明:TfR与转铁蛋白的结合具有高度亲和力、高度特异性及可饱和性。     

Hematopoietic turnover index in reactive and neoplastic bone marrow lesions: quantification by apoptosis and PCNA labeling

 In order to determine the dynamics of hematopoietic cell turnover, proliferative activity and incidence of apoptosis (programmed cell death) were evaluated in bone marrow trephine biopsies. Selection of patients (20 in each group) included in addition to a control group, idiopathic thrombocytopenia (ITP), reactive thrombocytosis (TH), secondary polycythemia-smokers' polyglobuly (PG), primary (essential-hemorrhagic) thrombocythemia (PTH), polycythemia vera (PV), and finally acute myeloid leukemia (AML). Apoptosis was demonstrated by the in situ end-labeling technique (ISEL) and proliferative activity by applying the monoclonal antibody PC10 raised against proliferating cell nuclear antigen (PCNA). To assess dynamic features of hematopoiesis, an index was calculated consisting of the ratio between PCNA-positive nuclei and the apoptotic cell fraction. This factor was termed the hematopoietic turnover index (HTI). Morphometric analysis revealed that the HTI was significantly increased in AML and PV. According to cell culture studies both disorders are characterized by either a prevalent proliferation of the myeloid or erythroid cell mass. On the other hand, PG, PTH, and TH showed no relevant enhancement of this index in comparison to the control specimen. In vitro experiment results are in keeping with the finding that PG and PTH are not associated with a significant expansion of the erythroid lineage (CFU-E). Similar to ITP and TH, in PTH megakaryocyte proliferation (CFU-MEG) is the predominant feature of cell turnover. Differences between PTH and TH are in line with the reduced in vitro formation of CFU-MEG in the latter disorder. In conclusion, our in situ study on turnover rates of the bone marrow in various neoplastic and reactive lesions extends previous experimental data on hematopoietic cell kinetics. Received: 10 March 1997 / Accepted: 18 May 1997     

Ethanol Feeding Causes Inactivation of Both State 1 and State 2 Rat Hepatic Asialoglycoprotein Receptors

Previous studies have shown that ethanol feeding in rats causes inactivation and redistribution of ˜50% of the total asialoglycoprotein receptors (ASGPRs) in hepatocytes (Tworek et al., J. Biol. Chem. 271:2531, 1996), and that two equal populations of hepatic ASGPRs mediate ligand uptake and processing via two functionally different pathways (Weigel in Glycoconjugates: Composition, Structure and Function , Marcel Dekker, 1992, p. 421). The purpose of this study was to determine if ethanol feeding causes preferential inactivation of only one of these two ASGPR populations, which have been designated state 1 and state 2 ASGPRs. The state 2, but not state 1, ASGPRs are inactivated in isolated hepatocytes by a variety of drugs and inhibitors. State 2 ASGPRs can also be inactivated in permeable cells by ATP treatment and then reactivated by treatment with fatty acyl coenzyme As. In the present study, permeable cell assays for state 2 ASGPR inactivation and reactivation were used to assess whether hepatocytes from ethanol-fed rats contain inactive state 2 ASGPRs. The results show that preferential inactivation of one ASGPR population does not occur after ethanol feeding. That inactive ASGPRs could not be reactivated by treatment with palmitoyl-coenzyme A to a greater extent in ethanol-fed versus control cells indicates there is not a larger pool of inactivated state 2 ASGPRs in treated cells. We conclude that ethanol feeding causes equal inactivation of both state 1 and state 2 ASGPRs. Ethanol feeding may represent the first treatment found to inactivate state 1 ASGPRs.     

Alterations of G-protein Coupling Function in Phosphoinositide Signalling Pathways of Rat Hippocampus by Ischaemic Brain Injury

The activation of membrane-associated phospholipase C is rapidly and transiently induced in the central nervous system by a variety of stimuli. Ischaemic brain injury is one of the situations that leads to a dramatic increase in polyphosphoinositide (PPI) turnover. In this study, stimulation of PPI hydrolysis by glutamate (500 μM) was measured in hippocampal slices from rats up to 21 days after an ischaemic insult of 30 min. Ischaemia was induced using the four-vessel occlusion method. PPI hydrolysis elicited by glutamate was significantly increased in the slices prepared from ischaemic rats 24 h after reperfusion, the accumulation of inositol phosphates (InsP_s) and inositol 1,4,5-trisphosphate (InsP₃) was 614±74% ( n = 8) and 182±11% ( n = 9) of the basal level respectively. This potentiation was also observed 21 days after ischaemia. Hyper-responsiveness to glutamate was also accompanied by an increase in AIF⁻₄-stimulated formation of [³H]inositol phosphates. In addition, global ischaemia did not change either high-affinity [³H]glutamate binding in hippocampal membranes or the stimulation of PPI hydrolysis by carbachol or noradrenaline in hippocampal slices. The present results suggest that the increased responsiveness to glutamate is the result, at least in part, of functional changes at the G-protein level, and may contribute to the pathophysiology of ischaemic brain injury or to the regenerative phenomena that accompany ischaemic damage.     

Pharmacological Characterization of Bradykinin Receptors Coupled to Phosphoinositide Turnover in SV40-Immortalized Human Trabecular Meshwork Cells

This study sought to pharmacologically characterize bradykinin receptors on SV40-immortalized human trabecular meshwork (HTM3) cells. Phosphoinositide (PI) turnover studies were conducted using [³H]myo-inositol-labeled HTM3 cells and anion exchange chromatography to quantify [³H]inositol phosphates generated in response to bradykinin (BK) and various BK analogs. The blockade of these responses was studied using two potent and receptor-subtype selective antagonists. BK and T-kinin (Ile-Ser-BK; TK) induced a 4.2–4.4 fold stimulation of PI turnover above base levels at 1–10 μM. Several other peptides unrelated to BK, including angiotensin II, endothelin, cholecystokinin, bombesin and peptide YY tested at 1–10 μMwere essentially inactive. The molar potencies (EC₅₀) of BK, TK and close analogs were: BK=4.5±0.5 nM(n=6), Lys-BK=6.5±0.7 nM(n=3), TK=38.8±6.6 nM(n=8), Met-Lys-BK=41.5±13.4 nM(n=4), Des-Arg⁹-BK=2093±626 nM(n=4). All the latter BK-related peptides>were full agonists. The actions of BK and TK were potently and competitively antagonized by Hoe-140 (molar potency=0.6–1 nM;pA₂n=8.97–9.21,n=3–4) and byD-Arg⁰[Hyp³,-Thi^5,8,-DPhe⁷]-BK (molar potency=251 nM;-log potency, pK_b=6.6), two selective B₂-type BK antagonists. In conclusion, rank order of potency of BK agonists and the blockade of BK- and TK-induced PI turnover by the selective antagonists are consistent with the classification of the BK receptors on HTM3 cells as the B₂-receptor subtype.