CARD In Situ Hybridization: Sights and Signals

During the last decade, several strategies have been developed to improve the detection sensitivity ofin situ hybridization (ISH) by amplification of either target nucleic acid sequences prior to ISH (e.g.,in situ PCR), or the detection signals after the hybridization procedures (signal amplification). Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, summarize applications as well as possible limitations of CARD ISH, and discuss some future directions ofin situ nucleic acid detection using this amplification strategy.     

非霍奇金淋巴瘤中p53基因mRNA的表达

目的　探索研究非霍奇金淋巴瘤中 p5 3mRNA的表达与突变型p5 3蛋白和临床病理的相关性。 方法　采用原位杂交和ABC法。结果　在 4 8例各型非霍奇金淋巴瘤中 p5 3mRNA有不同程度的表达 ,检出率为5 8.3 % ,突变型 p5 3蛋白染色阳性率 4 1.7% ;在突变型p5 3蛋白阳性组中 p5 3mRNA的检出率为 80 .0 % ,显著高于突变型 p5 3蛋白阴性组 ( 4 2 .8% ) (P <0 .0 5 )。 结论　在非霍奇金淋巴瘤中存在 p5 3基因突变 ,产生了突变型p5 3mRNA ,提示后者的表达增强可能是导致突变型 p5 3蛋白表达增强的主要原因之一。     

Influence of α-tocopherol over the time course of experimental IgA nephropathy

 The present study investigated the pathogenesis and the time course of kidney injury in experimental IgA nephropathy. In order to determine an appropriate period in the course of experimental IgA nephropathy to study renal injury and repair, we examined proteinuria and IgA deposition in the renal mesangium after 4, 8, and 16 weeks of mucosal challenge by bovine gamma globulins (BGG) provided in the drinking water. The hallmark of IgA deposition in the mesangium was present after 4 weeks and 8 weeks of BGG inoculation, but by 16 weeks, the mesangial IgA deposition had resolved. In addition, we confirmed our previous report on the beneficial effects of α-tocopherol in reducing proteinuria in IgA nephropathy at 8 weeks, and extended this observation to investigate the effects of dietary supplementation of α-tocopherol at both 4 weeks and 16 weeks. Proteinuria resolved spontaneously at 16 weeks. There is oxidative stress, as suggested by the elevation in plasma and renal malondialdehyde content, and increased fibrogenic cytokine message, as suggested by elevated transforming growth factor β1 mRNA. These increases were clearly blunted by α-tocopherol at both 4 weeks and 8 weeks. Treatment with α-tocopherol was associated with a significant reduction in the severity of proteinuria. Thus, our data suggest that the period between 4 and 8 weeks of BGG vaccination could be relevant in designing an appropriate model to study the molecular biology of the pathogenesis of renal injury and the effects of treatment. The 16-week model may be useful in exploring gene expression involved with spontaneous resolution. Received: 17 February 1998 / Revised: 2 June 1998 / Accepted: 3 June 1998     

Sexually dimorphic expression of sst1 and sst2 somatostatin receptor subtypes in the arcuate nucleus and anterior pituitary of adult rats

The pattern of growth hormone (GH) secretion and rate of somatic growth are markedly sexually dimorphic, but the underlying neuroendocrine mechanisms are far from clear. In the present study, we tested the hypothesis that the sexual dimorphism of GH secretion may be due to gender-related differences in the transduction of somatostatin's actions in brain and/or pituitary. To accomplish this, we compared the distributional pattern and level of expression of two somatostatin receptor subtypes, sst1 and sst2, in the brain and pituitary of adult male and female rats by in-situ hybridization using 35S-labelled antisense riboprobes. In the brain, the hybridization pattern and labelling density of sst1 and sst2 mRNA-expressing cells, as revealed by computer-assisted image analysis, in areas including the cerebral cortex, medial habenula (MHb) and ventromedial hypothalamic nucleus (VMN), were similar in male and female rats. In contrast, there was a marked sex-related difference in sst1 expression in the arcuate nucleus of the hypothalamus; both the number and labelling density of sst1 mRNA-expressing cells were two- to threefold greater in males than in females and this significant increase was homogenous throughout the rostrocaudal extent of the nucleus. No gender-related differences in arcuate sst2 mRNA levels were found. At the level of the anterior pituitary, the labelling density of sst2 mRNA in males was significantly higher than that of females. No sex-related difference in pituitary sst1 mRNA was observed. These results demonstrate a sexual dimorphism in the expression of two somatostatin receptor subtypes, sst1 and sst2, at the level of the arcuate nucleus and anterior pituitary, respectively. Such dimorphism suggests a differential involvement of sst1 and sst2 in GH regulation with respect to gender, and may imply roles for sst2 and sst1 in transducing somatostatin's actions on pituitary somatotrophs and GH-releasing hormone-containing arcuate neurones, respectively, to generate the lower basal and higher GH pulse levels characteristic of the male rat.     

锰对大鼠子代新生鼠黑质酪氨酸羟化酶的影响

为了研究锰对子代新生鼠黑质中酪氨酸羟化酶（ｔｙｒｏｓｉｎｅｈｙｄｒｏｘｙｌａｓｅ,ＴＨ）的影响,对雌性大鼠腹腔注射氯化锰（ＭｎＣｌ２·４Ｈ２Ｏ）,然后采用原位杂交组织化学技术检测其子代新生鼠黑质中ＴＨ．ｍＲＮＡ神经元。结果：对照组新生鼠黑质中观察到ＴＨ．ｍＲＮＡ神经元,各实验组均未观察到ＴＨ．ｍＲＮＡ神经元。提示：锰在基因水平上抑制了染锰者子代新生鼠黑质中ＴＨ活性,进而干扰和抑制了胺能神经递质的正常代谢     

Effect of repeated administration of dopamine agonists on striatal neuropeptide mRNA expression in rats with a unilateral nigral 6-hydroxydopamine lesion

Summary Striatal mRNA expression for preproenkephalin (PPE) and preprotachykinin (PPT) was studied in unilateral 6-OHDA lesioned rats treated subchronically with a range of selective and non-selective D-1 or D-2 dopamine (DA) agonists. Apomorphine (5mg/kg sc), pergolide (0.5mg/kg sc), SKF 38393 (5mg/kg sc), SKF 80723 (1.5mg/kg sc), and quinpirole (5mg/ kg sc), or 0.9% saline (150l sc) were all given twice daily (except pergolide: once daily) for 7 days. The abundance of PPE mRNA was not altered by any of these DA agonists in the intact striatum contralateral to the 6-OHDA lesion. Only apomorphine and quinpirole increased the abundance of PPT mRNA in the intact striatum. In saline treated 6-OHDA lesioned animals PPE mRNA was elevated (+160%, p < 0.005) and PPT mRNA decreased (–36%, p < 0.005) in the denervated striatum. The up-regulation of striatal PPE mRNA in the lesioned striatum was reversed only by pergolide. The downregulation of striatal PPT mRNA in the lesioned striatum was reversed only by apomorphine. The differential sensitivity of the striatal PPE message to the long-acting DA agonist pergolide, and of the striatal PPT message to the mixed D-1/D-2 DA agonist apomorphine suggests that the striatopallidal enkephalinergic pathways are mainly regulated by prolonged DA receptor stimulation, whereas the striatonigral substance P pathways are mainly regulated by mixed D-1/D-2 DA receptor stimulation.     

胆囊癌P~（53）、C－mycmRNA异常表达与TNM分期相关研究

应用地高辛标记的Ｐ￣(５３)、ｃ－ｍｙｃｃＤＮＡ探针对２１例胆囊癌石腊切片上的ｍＲＮＡ进行原泣杂交。结果表明ｃ－ｍｙｃ和Ｐ￣(５３)ｍＲＮＡ的表达均位于细胞核内。ｃ－ｍｙｃ和Ｐ￣(５３)ｍＲＮＡ在胆囊癌的表达率分别为６６．６％和５７．１％,在癌周正常组织中没有ｃ－ｍｙｃ、Ｐ￣(５３)ｍＲＮＡ的表达。ｃ－ｍｙｃ、Ｐ￣(５３)ｍＲＮＡ表达的强度和胆囊癌的原发肿瘤大小,有无淋巴结转移及远处转移有关。分期越晚,表达率越高。有淋巴结转移者高于无转移者。Ｐ￣(５３)ｍＲＮＡ表达的阳性率和胆囊癌的分化程度无关;而ｃ－ｍｙｃｍＲＮＡ的表达和分化程度有关,分化越差,表达率越高。     

大鼠心脏和肾脏多巴胺D_1A受体mRNA水平与大鼠年龄的关系

目的　观察了幼年、成年及老年大鼠心、肾组织中的多巴胺 D1 A受体 m RNA水平的变化 ,借以阐明外周多巴胺 D1 A受体与年龄变化的关系。方法　采用了反转录 -多聚酶链反应 (RT- PCR)方法。结果　以D1 A受体与 β- actin吸光度值之比表示组织中 D1 A受体 m RNA水平 ,以 3个月龄大鼠 m RN A水平为 10 0 % ,10 d龄大鼠 m RNA水平为 (6 1± 6 ) % (P<0 .0 5 ) ,18个月龄为 (6 9± 11) % (P<0 .0 5 ) ,10 d龄大鼠肾脏 m RNA为 (49± 8) % (P<0 .0 5 ) ,18个月龄大鼠为 (5 2± 7) % (P<0 .0 5 )。提示 10 d龄及 18个月龄大鼠心脏及肾脏 D1 A受体m RNA水平较 3个月龄大鼠明显下降 (P<0 .0 5 )。结论　 1外周 D1 A受体与中枢多巴胺受体相似 ,也有明显的年龄依赖性特征。2外周多巴胺受体的年龄相关性影响 ,提示多巴胺类药物对不同年龄段患者的效果可能有所不同 ,应引起临床用药的注意     

针刺对吗啡戒断大鼠脑组织一氧化氮合酶基因表达的影响

目的 :观察针刺对吗啡戒断大鼠脑组织神经元一氧化氮合酶基因 (nNOSmRNA)表达的影响 ,探讨针刺改善戒断症状的分子生物学机理。方法 :建立大鼠吗啡自然戒断模型 ,运用逆转录聚合酶链反应 (RT PCR)测定戒断大鼠脑组织nNOSmRNA的表达。观察戒断后 ,针刺“足三里”和一氧化氮合酶 (NOS)抑制剂L N 硝基精氨酸 (L NAME)对吗啡戒断大鼠戒断症状和脑组织nNOSmRNA表达的影响。结果 :戒断组nNOSmRNA表达增加 ,针刺组和L NAME组nNOSmRNA表达比戒断组减少。针刺组戒断积分比戒断组少 ,组间差异显著 (P <0 .0 1 )。结论 :针刺“足三里”穴具有抑制吗啡戒断大鼠脑组织nNOSmRNA表达的作用 ,提示这可能是针刺改善吗啡戒断症状的一个重要机制。