转前强啡肽基因修饰人骨髓间充质干细胞株的建立

目的 建立前强啡肽(PDP)基因修饰的人骨髓间充质干细胞株并验证其可在体外增强细胞强啡肽的分泌.方法 本实验运用载体Ad5F35-PDP转染人骨髓间充质干细胞hBMSCs,获得分泌强啡肽的干细胞株(hBMSCs-PDP),以转染空载体的细胞(hBMSCs-空载体)和未转染的细胞hBMSCs作为对照,此3组细胞分别采用流式细胞仪检测细胞表面抗原CD29、CD44、CD34、CD45的表达,免疫荧光法检测强啡肽的表达,RT-PCR法检测强啡肽mRNA的表达、Western Blot法测定强啡肽蛋白的分泌.结果 体外实验以流式细胞仪检测此3组细胞的表面标志物CD29、CD44表达均阳性,CD34、CD45表达均阴性且这3组细胞的表达差异比较无统计学意义(P＞0.05).与hBMSCs和hBMSCs-空载体比较,hBMSCs-PDP细胞株强啡肽mRNA的表达和强啡肽蛋白的表达均上调(P＜0.05).结论 本实验成功构建了前强啡肽基因修饰的人骨髓间充质干细胞,并证明转染后的细胞株hBMSCs-PDP可稳定分泌强啡肽并保留干细胞原有的特征表型.     

纳美芬治疗急性脊髓损伤的临床观察

目的 观察纳美芬对急性脊髓损伤患者治疗的临床特点.方法 将62例急性脊髓损伤患者完全随机分为对照组32例和纳美芬组30例.2组患者入院后,对照组仅给予常规治疗,而纳美芬组在常规治疗的基础上予以静脉注射盐酸纳美芬0.5 mg/d,连用14 d治疗.观察患者2周、3个月和6个月随访时的神经功能改善,比较二者的疗效.结果 纳美芬组和对照组的运动功能评分在治疗后2周分别为11.6±4.6 和8.4±3.6,3个月后分别为19.0±5.7和15.3±4.7,6个月后分别为25.7±5.5和21.9±4.9,2组差异均有统计学意义（P〈0.05或P〈0.01）;2组的痛觉评分在治疗后2周分别为8.1±3.0和6.6±3.2,3个月后分别为14.3±3.9和9.6±3.6,6个月后分别为16.3±3.8和11.7±3.9,2组差异均有统计学意义（均P〈0.05）;2组的触觉评分在治疗后2周分别为6.7±2.8和4.8±2.4,3个月后分别为9.1±2.7和7.8±2.3,6个月后分别为11.2±3.2和9.2±2.6,2组差异均有统计学意义（均P〈0.05）.结论 纳美芬能够促进急性脊髓损伤患者的神经功能恢复,改善患者预后.     

Diminution of contractile response by κ-opioid receptor agonists in isolated rat ventricular cardiomyocytes is mediated via a pertussis toxin-sensitive G protein

Opioids directly decrease the contractile response of isolated ventricular cardiomyocytes to electrical stimulation. To investigate whether these effects are mediated via GTP-binding G_i/o proteins we examined the influence of pertussis toxin on the effects of the κ-opioid receptor agonist trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide (U-50,488) methanesulphonate and on the as yet undescribed effects of the opioid peptide dynorphin A (1–8) on contraction. In isolated, electrically driven, rat ventricular cardiomyocytes both agents concentration dependently reduced cell shortening within 15 min, decreasing the contractile response by 79±4% (n=5) and 62±2% (n=6) of control values at maximal effective concentrations of 10 μM (U-50,488) and 1 μM [dynorphin A (1–8)], respectively. Pertussis toxin pre-treatment (200 ng/ml; 4.5–5 h) completely abolished the effects of U-50,488 and dynorphin A (1–8) on the contractile response, indicating that these effects are mediated via G_i/o proteins. In addition, the non-selective opioid receptor antagonist (–)-naloxone and the κ-opioid receptor antagonist nor-binaltorphimine antagonized the effects of U-50,488 and dynorphin A (1–8) on the contractile response. Furthermore, the μ- and δ-opioid receptor agonist (D-Ala², D-Leu⁵)-enkephalin (DADLE) had no effects on contraction. These results indicate that the decrease in cell shortening is due to stimulation of κ-opioid receptors. The direct effect of κ-opioid receptor agonists on the contractile response thus represents an additional mechanism for decreasing cardiac contractility, besides the M-cholinoceptor- or adenosine receptor-mediated pathway. It is conceivable that increased release of endogenous dynorphins from the heart during hypoxia may protect the heart in a similar manner to adenosine. Received: 16 September 1997 / Accepted: 15 June 1998     

强啡肽A(1-17)在脊髓的致瘫作用及其与神经毒作用的相关性

在以行为学为观察指标(甩尾镇痛和斜板实验)的基础上,用组织学方法探讨大剂量强啡肽A在脊髓水平的致瘫作用与其神经毒作用的关系。结果表明,给大鼠蛛网膜下腔(it)注射强啡肽A(1-17)20nmol·L^-1,共10μl,给药后5～10min即引起大鼠后肢不可逆性瘫痪、甩尾反射被抑制长达40h以上。大鼠脊髓组织学检查发现,腰、骶段脊髓前角运动神经元大量坏死或严重变性、以腰段损伤最为显著(运动神经元减少87.2%),其次是骶段(减少69.6%),胸段损伤不明显(减少8.2%)。     

Opiate agonists microinjected into the nucleus accumbens enhance sucrose drinking in rats

Previous studies have indicated that central opioid peptides and opiate receptors play an important role in the modulation of ingestive behaviors. The nucleus accumbens (Acb), a forebrain region involved in reinforcement, contains high levels of opiate receptors. The present investigation was undertaken to determine the relative involvement of opiate receptor subtypes in sucrose drinking. Morphine (0, 0.5, 5 μg/0.5 μl), the mu agonist D-Ala2,NMe-Phe4,Glyo15-enkephalin (DAMGO; 0, 0.025, 0.25 and 2.5 μg/0.5 μl), the delta agonist D-Pen2,5-enkephalin (DPEN; 0, 0.031, 0.31,3.1 μg/0.5 μl), and the kappa agonists U50488H (0, 0.0186, 0.186, 1.86 μg/0.5 μl), and dynorphin (0, 0.05, 0.5, 5 μg/0.5 μl) were microinfused into Acb. Intake of 5% sucrose, drinking duration, locomotion, rearing, and grooming were measured in a 30-min session in rats previously adapted to sucrose. After microinjection into Acb, morphine induced a robust increase in both sucrose intake and drinking duration at the low dose. DAMGO enhanced sucrose drinking at lower doses, and suppressed drinking at the highest dose. DPEN also increased sucrose intake in a dose-dependent manner. U50488H and dynorphin had no effect on sucrose drinking. In addition, it was demonstrated that intra-Acb administration of DAMGO specifically enhanced palatable sweet solution drinking, leaving water intake unchanged. Although mu and delta agonists tended to increase spontaneous motor activity, the pattern of effects indicated that increases in ingestion could not be simply attributed to general arousal. These findings demonstrate that both mu and delta receptors within the accumbens may have an important modulatory role in ingestion of palatable substances. Received: 14 October 1996 / Final version: 14 February 1997     

强啡肽对实验性神经细胞老化过程中M胆碱能受体位点数影响的研究

目的　探讨强啡肽 (DYN)对实验性神经细胞 M受体位点数的影响。方法　采用小鼠神经母细胞瘤细胞 (NBA2 )无血清培养建立神经细胞老化实验研究模型。以 M受体放射性配基分析术研究 DYN对实验性神经细胞的 M受体位点数的影响。结果　两种不同浓度的 DYN皆可使M受体位点数下降 (P<0 .0 1 ) ,以高浓度组更显著。结论　 DYN使 M受体位点数下降的作用可能是延缓实验性神经细胞老化的机制之一。(最终浓度为 0 .1nmol/L) ,以测定非特异性结合。各管加入2 0 0 μl膜蛋白液 ,37℃振荡温育 4 0 min。冰冷缓冲液终止反应。6 9型纤维滤膜三层抽滤并淋洗 ,滤膜烘干 ,投入 5 ml闪烁液(含 0 .4 % PPO的三甲苯 )中 ,以 YSL- 76型液闪计数器做固相测定。1.8　 数据处理　实验得到特异性结合的技术率 ,被测定效率除 ,等于特异性结合衰变率 ,再除以放射性配基活度 ,即等于特异性结合位点数〔7〕。采用 POMS方差分析 ,经 IBM微机处理数据。2　结果与讨论DYN对 NBA2 细胞 M胆碱能受体位点数的影响 ,见表 1。表 1　 DYN对 M受体位点数的影响 (nmol/10 6 细胞 )组别 n M受体位点数 (x± s)BSD组 5 449.796± 356.730AID组 5 486.941± 2 4 7.1 65DYN- 7组 5 1 4 5.71 4± 77.82 61 )DYN- 8组 5 1 51 .873± 95.80 61 )　　注 :DY     

Opiate modulation of dynorphin conversion in primary cultures of rat cerebral cortex

Rat brain cortical cells in primary culture were used to investigate long-term effects of opiates on endopeptidases acting on dynorphin peptides. Enzyme activity in the soluble fraction of the cells converted dynorphin B to Leu-enkephalin-Arg⁶ and to a lesser extent to Leu-enkephalin. Five day treatment with 10 μM morphine increased the conversion to Leu-enkephalin-Arg⁶ by 370%. This effect was prevented by the presence of naloxone in the culture medium. The opiate-inducible activity was directed to the Arg-Arg bond in dynorphins with preference for dynorphin B>-neoendorphin>>dynorphin A. The K_m for the generation of Leu-enkephalin-Arg⁶ from dynorphin B was 40 μM. Enzyme activity was inhibited by dynorphin fragments, in the following order of potency: dynorphin A(1–13)>A(2–13)>A(1–17)>A(2–17) and by SH-reagents, suggesting the presence of a cysteine-protease. The opiate-stimulated dynorphin-converting enzyme (DCE)-activity affects the balance between dynorphin peptides (selective for κ-opioid receptors) and enkephalin peptides (selective for δ-opioid receptors). Since both types of opioid peptides can influence the development of opiate tolerance, the change in the extent of this transformation may be functionally important.     

The effects of morphine treatment and morphine withdrawal on the dynorphin and enkephalin systems in sprague-dawley rats

The effect of morphine tolerance and withdrawal on prodynorphin peptides was studied in relevant brain areas and in the pituitary gland of male Sprague-Dawley rats, and compared with effects on the proenkephalin-derived peptide Met-enkephalin. After 8 days of morphine injections (twice daily), dynorphin A and B levels increased in the nucleus accumbens and dynorphin A levels increased also in the striatum. Morphine treatment increased striatal Met-enkephalin. Leu-enkephalinArg⁶ levels were reduced in the ventral tegmental area (VTA). Morphine-treated rats had very low Leu-enkephalinArg⁶ levels in the hippocampus as compared to saline control rats. Comparison of the relative amounts of dynorphin peptides and the shorter prodynorphin-derived peptides, Leu-enkephalinArg⁶ and Leu-enkephalin, revealed a relative increase in dynorphin peptides versus shorter fragments in the nucleus accumbens, VTA and hippocampus. Morphine-tolerant rats had lower levels of dynorphin A in both lobes of the pituitary gland, whereas hypothalamic dynorphin levels were unaffected by morphine. Leu-enkephalinArg⁶ levels were reduced in the hypothalamus, but not changed in the pituitary gland. Naloxone-precipitated withdrawal accentuated the increase in dynorphin A and B levels in the accumbens and dynorphin A levels in the striatum, while inducing an increase in enkephalin levels in the accumbens and Met-enkephalin in the VTA. In the hippocampus, Leu-enkephalinArg⁶ levels remained low in the withdrawal state. The low dynorphin levels in the anterior part of the pituitary gland were reversed by naloxone, whereas the low dynorphin A levels in the neurointer-mediate lobe were even lower in the withdrawal state. In conclusion, morphine tolerance and withdrawal affected prodynorphin-derived peptides in areas related to central reward mechanisms, and in the pituitary gland. The dynorphin peptides and the LeuenkephalinArg⁶ fragment were not affected similarly, indicating an effect also on metabolic interconversion.     

Role of dynorphin and enkephalin in the regulation of striatal output pathways and behavior

Projection neurons in the striatum give rise to two output systems, the “direct” and “indirect” pathways, which antagonistically regulate basal ganglia output. While all striatal projection neurons utilize GABA as their principal neurotransmitter, they express different opioid peptide co-transmitters and also different dopamine receptor subtypes. Neurons of the direct pathway express the peptide dynorphin and the D1 dopamine receptor, whereas indirect pathway neurons express the peptide enkephalin and the D2 receptor. In the present review, we summarize our findings on the function of dynorphin and enkephalin in these striatal output pathways. In these studies, we used D1- or D2-receptor-mediated induction of immediate-early genes as a cellular response in direct or indirect projection neurons, respectively, to investigate the role of these opioid peptides. Our results suggest that the specific function of dynorphin and enkephalin is to dampen excessive activation of these neurons by dopamine and other neurotransmitters. Levels of these opioid peptides are elevated by repeated, excessive activation of these pathways, which appears to be an adaptive or compensatory response. Behavioral consequences of increased opioid peptide function in striatal output pathways may include behavioral sensitization (dynorphin) and recovery of motor function (enkephalin).