生长抑素受体在人神经内分泌癌组织中的表达及受体激活对细胞生长的抑制

生长抑素（somatostatin,SST）通过与细胞膜上的G蛋白偶联的生长抑素受体（somatostatin receptors,SSTRs）结合而发挥其抑制细胞增殖的作用,因而生长抑素类似物（somatostatin analogue, SSA）常被用于肿瘤辅助治疗。然而,治疗效果存在相当大的个体差异,推测生长抑素类似物治疗效果不佳,与内源性生长抑素受体表达缺失或者表达量和亚型组合有关。为此,检测各亚型SSTR在几例罕见的神经内分泌肿瘤中的表达,并检测过表达SSTR2和SSTR5以及受体激活对细胞增殖的抑制效果,分析受体激活的可能机制,有助于临床筛选适合SSA肿瘤辅助治疗的病例,预估SSA的治疗效果。免疫组化检测肿瘤组织SSTR1-5的表达。在培养的293T细胞中过表达SSTR2和SSTR5,免疫共沉淀检测受体相互作用,免疫荧光和共聚焦显微镜检测受体细胞内定位。用MTT法检测受体过表达及激活对培养的人肺癌细胞NCI-H460细胞增殖的影响,用流式细胞技术检测细胞周期分布。SSTR1-5在10例神经内分泌肿瘤组织中均有不同程度的表达,表达亚型及表达量与肿瘤类型和年龄无关,SSTR5在所有肿瘤组织中均表达。SSTR2与SSTR5可形成受体相互作用。SSTR2与SSTR5活化后相互作用增加并定位于细胞质。共表达SSTR2和SSTR5显著抑制细胞增殖,并与受体激活剂呈现剂量相关性。SSTR2/SSTR5的共表达及激活显著减少S期的细胞而滞留于G₁期。     

乳腺癌荷瘤鼠99mTc-DOTANOC显像与生长抑素受体表达水平的相关性研究

目的:探讨乳腺癌荷瘤鼠模型中肿瘤组织中的生长押素受体(SSTR)的表达水平与99mTc-DOTANOC显像的相关性研究.方法:配体交换法标记99mTc-DOTANOC,通过尾静脉注射乳腺癌荷瘤鼠模型,行99mTc-DOTANOC显像,勾画ROI计算肿瘤与对侧正常组织(T/NT)的放射性比值并测定肿瘤及主要脏器单位组织的放射性摄取百分值(%ID/g),采用逆转录聚合酶反应(RT-PCR)检测肿瘤组织中各SSTR亚型mRNA的表达水平,对SSTR亚型表达水平与T/NT放射性摄取比值进行相关性研究.结果:99mTc-DOTANOC乳腺癌荷瘤鼠模型显像示肿瘤部位有较高的放射性浓聚,与对侧正常组织T/NT比值较高,4h达到2.41±0.21;99mTc-DOTANOC荷瘤鼠体内生物分布示药物在肿瘤部位有较高的摄取;RT-PCR示乳腺癌组织中有着丰富的SSTR表达,SSTR3和SSTR2亚型表达水平较高,两者mRNA的表达水平与荷瘤鼠显像T/NT比值呈正相关(两者分别r=0.94,r=81,P<0.05).结论:乳腺癌细胞株MDA-MB-435 高表达SSTR2和SSTR3,其中SSTR3和SSTR2 mRNA表达水平与肿瘤组织对99mTc-DOTANOC的摄取呈正相关.第三代生长抑素类似物99mTc-DOTANOC受体显像对乳腺癌有较好的影像诊断价值.     

鳜两种生长抑素受体cDNA全长克隆与组织表达

利用cDNA末端快速扩增(RACE)技术克隆了鳜(Siniperca chuatsi)脑中2种生长抑素受体(somatostatin receptor,SSTR2和SSTR3)cDNA全长序列。结果显示,鳜SSTR2 cDNA全长1 820 bp,含开放阅读框1 146 bp,编码382个氨基酸;SSTR3 cDNA全长1 874 bp,含开放阅读框1 458 bp,编码486个氨基酸。SSTR均由5个结构区域组成:N端、7个转膜区(TMD)、3个细胞外袢(ECLs)、4个细胞内袢(ICLs)和C末端。NJ系统进化树分析显示,鳜SSTR2和SSTR3分别形成相对独立的分支,两者间的氨基酸序列相似度为51.2%,表明它们是由不同基因编码而成。利用实时荧光定量RT-PCR技术检测了鳜SSTR2和SSTR3 mRNA的组织表达特征,它们均在多种组织中广泛表达,SSTR2 mRNA在肝中表达量最高,SSTR3 mRNA在胃中表达量最高。SSTR2、SSTR3表达差异反映它们可能参与不同生理调控作用。     

子宫内膜癌组织中生长抑素受体、VEGF的表达及其与血管形成的关系

目的探讨生长抑素受体（somatostatin receptor,SSTR）、血管内皮生长因子（vascular endothelial growth factor,VEGF）在子宫内膜癌组织中的表达及其与肿瘤血管形成的关系。方法应用免疫组织化学方法检测60例子宫内膜癌组织中SSTR各亚型、VEGF及CD34标记的微血管密度（microvessel denisity,MVD）的表达情况,探讨其与子宫内膜癌临床病理学特征及肿瘤血管形成的关系。结果在60例子宫内膜癌组织中,SSTR各亚型（SSTR1、SSTR2、SSTR3、SSTR4及SSTR5）的阳性表达率分别为70．0％,15．0％。21．7％,23．3％及18．3％;SSTR3、SSTR4在中高分化组表达阳性率明显高于低分化组（P〈0．05）。VEGF的阳性表达率为83．3％,VEGF在低分化组表达阳性率明显高于中高分化组、深肌层浸润组表达阳性率明显高于浅肌层浸润组、FIGO分期≥II期组表达阳性率明显高于I期组（P〈0．05）。子宫内膜癌组MVD（44．85±15．78）明显高于正常子宫内膜组MVD（18．96±4．30）（P〈0．01）。SSTR5的表达与VEGF呈负相关,VEGF阳性表达组子宫内膜癌组织MVD高于VEGF阴性组。结论联合检测SSTR和VEGF对子宫内膜癌预后的评估有一定临床意义。生长抑素类似物（somatostatin analogs,SSTA）可能为子宫内膜癌的诊治提供新的靶点。     

生长抑素受体2的小鼠时空组织表达谱

生长抑素(somatostatin, SST)作为一种抑制性多肽激素,在多种生物过程中发挥重要的功能。生长抑素受体2 (somatostatin receptor 2, SSTR2)作为生长抑素表达最广泛的受体在多种组织中表达,但其表达的具体细胞类型尚不清楚。本研究在小鼠不同发育阶段的多种组织中鉴定了SSTR2蛋白表达的细胞类型。通过多色免疫荧光在小鼠胚胎期15.5 d、出生后1 d、7 d、15 d、3个月和6个月的脑、骨、肺、肠道、皮肤、胃、脾和肾等组织中检测了Sstr2基因的表达。结果发现Sstr2在不同发育阶段的多种组织的特定细胞类型中表达,包括脑神经元和星形胶质细胞,骨的间充质基质细胞、造血细胞和B细胞,肺的巨噬细胞、Ⅱ型肺泡上皮细胞和气道纤毛细胞,肠道的上皮细胞和神经元,皮肤的毛囊细胞,胃体的上皮细胞,脾的造血干细胞、造血祖细胞和神经纤维,肾的肾小管上皮细胞等。本研究确定了小鼠多组织不同发育阶段Sstr2表达的细胞类型,为生长抑素与生长抑素受体2的生理功能研究提供了新的线索。     

应用组织芯片技术研究抑癌候选基因NDRG2在淋巴瘤组织中的表达及其意义

目的:应用组织芯片技术分析抑癌候选基因NDRG2在人类淋巴瘤组织、淋巴结正常组织及淋巴结良性病变组织中的表达情况.方法:采用小鼠抗NDRG2单克隆抗体的免疫组织化学ABC法,研究NDRG2在不同淋巴结组织的表达,并分析NDRG2在不同淋巴结组织中的表达差异.结果:免疫组化结果显示,淋巴瘤组织与淋巴结良性病变组织中DNRG2的表达相比呈显著性差异(P<0.05),淋巴瘤组织与正常淋巴结组织中DNRG2的表达相比呈显著性差异(P<0.05).淋巴结良性病变组织与正常淋巴结组织中DNRG2的表达相比呈显著性差异(P<0.05).正常淋巴结,淋巴结良性病变及淋巴瘤组织中NDRG2的阳性表达率分别为92.5%(37/40)、67.5%(27/40)和18.51%(15/81),3者间比较,差异有统计学意义.结论:NDRG2在淋巴瘤组织中呈低表达,提示其可能对淋巴瘤的发生或发展有重要作用,这不仅为研究淋巴瘤的发病机制进一步提供了线索,而且对淋巴瘤的诊断和治疗具有重要意义.     

生长抑素受体的生理功能及动力学特征

生长抑素受体家族(somatostatin receptors,SSTRs)是一类介导生长抑素及其类似物,具有多种生物学效应的G蛋白偶联受体家族,其生理功能和作用机制长期以来倍受关注.研究表明,这些细胞膜上存在的特定膜受体包括SSTR1、SSTR2、SSTR3、SSTR4以及SSTR5,可以通过cAMP、PTP和MAPK信号通路,在调控GH分泌、诱导细胞凋亡、抑制肿瘤细胞增生、抑制胰岛素作用和抑制细胞生长等生物学过程发挥重要的作用,同时表现出与其它G蛋白偶联受体性质相似的动力学特征.本文将SSTRs的结构、分布和生理功能、配体选择性、下游信号通路,以及该受体家族的动力学特征最新研究进展作一综述.     

南海柳珊瑚鳞海底柏化学成分的研究

本文对南海柳珊瑚鳞海底柏Melitodes squarnata Nutting进行了化学成分的研究.通过用工业乙醇:二氯甲烷(2∶1)进行了提取,采用硅胶柱色谱、Sephadex LH-20凝胶色谱、高效液相半制备色谱和薄层制备等方法对鳞海底柏的化学成分进行了分离,并利用波谱分析技术和文献对照鉴定其结构.从鳞海底柏中分离得到了17个化合物,分别鉴定为:malonganenone E(1),malonganenone D(2),nuttingin A(3),腺嘌呤核苷(4),1,3,7-三甲基黄嘌呤(5),2'-脱氧胸腺嘧啶核苷(6),3-甲基-6-次黄嘌呤(7),2'-脱氧尿苷(8),(22E,24R)-ergosta-8 (14),22-dien-3β,5α,6β,7α-tetrol(9),(22E)-胆甾-5,22-二烯-3β-醇(10),胆甾醇(11),(2S,3S,4R)-N-hexadecanoyl-2-amino-1,3,4-eicosanetriol( 12),(2S,2'R,3R,4E,8E)-N-2'- Hydroxytetradecanoyl-2-amino-9-methyl-4,8-octadecaa-diene-1,3-diol(13),鲨肝醇(14),十六烷基甘油醚(15),三油酸甘油酯(16)和4-hydroxy-2,3-dimetlhy1-2-nonen-4-olide(17).     

猕猴发育过程中肠肝组织生长抑素及其受体表达演变规律

探讨在猕猴发育过程中生长抑素(somatostatin,SST)及其受体在肠肝组织的演变规律。通过手术途径获得胚胎6月、新生2 d、新生45 d和成年猕猴的回肠、肝脏、门静脉和外周血等标本。应用放射免疫分析法测定各标本中的SST含量; 通过免疫组化方法观察SST在肠、肝组织内的分布;利用原位杂交检测SST受体2(somatostatin receptor 2,SSTR2)的表达。结果显示:(1)胚胎6月的猕猴,小肠内SST含量为(27．3±16．6)ng／mg蛋白;黏膜隐窝处SST呈弱阳性染色,肌层 SST染色阴性。在发育过程中,小肠内SST含量逐渐增加,成年期时达最高水平(120．1±35．3)ng／mg蛋白,较胚胎6月显著增加(P<0．01)。(2)成年小肠黏膜隐窝处及肌间神经丛SST呈强阳性染色。(3)胚胎6月,小肠粘膜上皮可见大量SSTR2 表达,成年时SSTR2表达下调,且主要定位于腺上皮隐窝处;胚胎及新生期肌层SSTR2染色阴性,成年时小肠肌间神经丛则可见阳性SSTR2染色。(4)肝脏在发育过程中SST及SSTR2含量逐渐降低;发育的各个时期,小肠组织的SST含量均明显高于肝脏组织含量,门静脉SST水平也始终高于外周血。总之,位于小肠黏膜隐窝处的SST和SSTR2随着发育逐渐增加,来自肠道的SST进入门静脉后迅速被降解。SST阳性的肠肌间神经丛仅在发育成熟后才出现。     

SSTR-2、E-cad与不明原因的反复自然流产相关性研究

目的:研究生长抑素2型受体(SSTR2)、E-钙粘附素(E-cad)与不明原因的反复自然流产相关性。方法:选取2011年1月到2014年12月我院收治的不明原因反复自然流产患者60例(研究组),另选同期早期正常孕妇且准备流产者60例(对照组),应用免疫组化技术检测两组蜕膜组织中的SSTR2和E-cad表达,并进行比较。结果:研究组蜕膜组织中SSTR-2阳性细胞平均光密度显著高于对照组,而E-cad阳性细胞平均光密度显著低于对照组,两组比较差异具有统计学意义(P0.05);研究组蜕膜组织中SSTR-2与E-cad水平呈负相关关系,对照组蜕膜组织中SSTR-2与E-cad水平呈正相关关系(P0.05)。结论:蜕膜组织中SSTR-2高表达与E-cad低表达可能与不明原因的反复自然流产有关。     

ZAC1 and SSTR2 Are Downregulated in Non-Functioning Pituitary Adenomas but Not in somatotropinomas

Introduction

There are few data regarding ZAC1 expression in clinically non-functioning pituitary adenomas (NFPA). Because somatotropinomas and NFPA behave differently with respect to tumor shrinkage during somatostatin analogs (SA) therapy, we sought to compare the ZAC1 and somatostatin receptor (sstr) types 1, 2, 3 and 5 mRNA expression in these two pituitary adenoma subtypes and in normal human pituitaries.

Methods

ZAC1 and SSTR mRNA expression levels were evaluated using real-time RT-PCR (TaqMan) in 20 NFPA and compared with the expression levels in 23 somatotropinomas and five normal pituitaries. The NFPA invasiveness was evaluated using magnetic resonance imaging with Hardy’s modified criteria. Ki-67 and p53 were evaluated using immunohistochemistry.

Results

A total of 20 patients with NFPA [6 males, median age 56 years (range: 30-78)], 23 with acromegaly [12 males, median age 43 years (range: 24–57)] and five normal pituitaries [4 males, median age 48 years (range: 36–54)] were included. Four of the patients (20%) had Hardy’s grade 2 tumors; all of the others had Hardy’s grade 3 tumors. The Ki-67 median expression was 2.35 (range: 0.2–9.23), and only four of the tumors (20%) were positive for p53. The ZAC1 mRNA expression was significantly lower in NFPA than in somatotropinomas and in normal pituitaries (p<0.001 for both), as well as the SSTR2 (p=0.001 and 0.01, respectively). The SSTR3 expression was higher in the NFPA than in the somatotropinomas and in the normal pituitaries (p=0.03 and 0.02, respectively). No correlation was found between the ZAC1 mRNA expression and the tumor invasiveness, Ki-67 and p53.

Conclusion

ZAC1 and SSTR2 are underexpressed and SSTR3 is overexpressed in NFPA compared to those in somatotropinomas and in normal pituitaries, which might explain the lack of tumor shrinkage that is observed in response to commercially available SA therapy in patients with NFPA.     

Synthesis,radiolabeling, and preclinical evaluation of a new octreotide analog for somatostatin receptor‐positive tumor scintigraphy

Radiolabeled somatostatin analogs have become powerful tools in the diagnosis and staging of neuroendocrine tumors, which express somatostatin receptors. The aim of this study was to evaluate a new somatostatin analog, 6‐hydrazinopyridine‐3‐carboxylic acid‐Ser³‐octreotate (HYNIC‐SATE) radiolabeled with ^99mTc, using ethylenediamine‐N,N′‐diacetic acid and tricine as coligands, to be used as a radiopharmaceutical for the in vivo imaging of somatostatin receptor subtype 2 (SSTR2)‐positive tumor. Synthesis of the peptide was carried out on a solid phase using a standard Fmoc strategy. Peptide conjugate affinities for SSTR2 were determined by receptor binding affinity on rat brain cortex and C6 cell membranes. Internalization rate of ^99mTc‐HYNIC‐SATE was studied in SSTR2‐expressing C6 cells that were used for intracranial tumor studies in rat brain. A reproducible in vivo C6 glioma model was developed in Sprague–Dawley rat and confirmed by histopathology and immunohistochemical analysis. Biodistribution and imaging properties of this new radiopeptide were also studied in C6 tumor‐bearing rats. Radiolabeling was performed at high specific activities, with a radiochemical purity of >96%. Peptide conjugate showed high affinity binding for SSTR2 (HYNIC‐SATE IC₅₀ = 1.60 ± 0.05 n m ) and specific internalization into rat C6 cells. After administration of ^99mTc‐HYNIC‐SATE in C6 glioma‐bearing rats, a receptor specific uptake of radioactivity was observed in SSTR‐positive organs and in the implanted intracranial tumor and rapid excretion from nontarget tissues via kidneys. ^99mTc‐HYNIC‐SATE is a new receptor‐specific radiopeptide for targeting SSTR2‐positive brain tumor and might be of great promise in the scintigraphy of SSTR2‐positive tumors. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of somatostatin receptors and associated signaling pathways in pancreas of R6/2 transgenic mice

The present study describes the status of somatostatin receptors (SSTRs) and their colocalization with insulin (β), glucagon (α) and somatostatin (δ) producing cells in the pancreatic islets of 11 weeks old R6/2 Huntington's Disease transgenic (HD tg) and age-matched wild type (wt) mice. We also determined expression of tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and presynaptic marker synaptophysin (SYP) in addition to signal transduction pathways associated with diabetes. In R6/2 mice, islets are relatively smaller in size, exhibit enhanced expression and nuclear inclusion of mHtt along with the loss of insulin, glucagon and somatostatin expression. In comparison to wt, R6/2 mice display enhanced mRNA for all SSTRs except SSTR2. In the pancreatic lysate, SSTR1, 4 and 5 immunoreactivity decreases whereas SSTR3 immunoreactivity increases with no discernible changes in SSTR2 immunoreactivity. Furthermore, at the cellular level, R6/2 mice exhibit a receptor specific distributional pattern of SSTRs like immunoreactivity and colocalization with β, α and δ cells. While GAD expression is increased, TH and SYP immunoreactivity was decreased in R6/2 mice, anticipating a cross-talk between the CNS and pancreas in diabetes pathophysiology. We also dissected out the changes in signaling pathway and found decreased activation and expression of PKA, AKT, ERK1/2 and STAT3 in R6/2 mice pancreas. These findings suggest that the impaired organization of SSTRs within islets may lead to perturbed hormonal regulation and signaling. These interconnected complex events might shed new light on the pathogenesis of diabetes in neurodegenerative diseases and the role of SSTRs in potential therapeutic intervention.     

Cytoplasmic expression of SSTR2 and 5 by immunohistochemistry and by RT/PCR is not associated with the pharmacological response to octreotide

ObjectiveTo evaluate expression of somatostatin receptor subtypes 2 and 5 (SSTR 2 and 5) by RT/PCR and immunohistochemistry (IHC) in GH-secreting adenomas, seeking correlations with response to octreotide.MethodsSSTR2 and 5 expression was tested by IHC (n = 37), RT/PCR (n = 36) or both (n = 13) in GH-secreting adenomas from 60 patients with acromegaly who had undergone pituitary surgery; 36 had been treated preoperatively with octreotide LAR for 3–6 months, and were categorized as responders (achievement of GH <2.5 ng/mL and a normal age-adjusted IGF-1), partial responders (GH and IGF-1 reduction >50% and >30%, respectively) or non-responders. IHC was performed on a tissue microarray using specific antibodies directed to the carboxyl terminus of SSTR2 and 5.ResultsSSTR5 was the predominantly expressed receptor subtype by both IHC and RT/PCR in all tumors tested, regardless of whether they came from octreotide-naïve, octreotide-responsive, or octreotide-resistant patients. Immunostaining was concentrated in the cytoplasm. Neither SSTR2 nor SSTR5 expression correlated with baseline or post-octreotide GH or IGF-1 levels or tumor volume by either method. The agreement rate between RT/PCR and IHC was 77% in all 13 adenomas in which both methods were used.ConclusionExpression of these receptors does not guarantee an adequate response to somatostatin analogs; other functional aspects of this interaction, such as receptor homo- and heterodimerization, and the resulting signaling cascade, probably play a role in determining whether a patient will respond or not to these agents.     

Somatostatin analogues, a series of tissue transglutaminase inducers, as a new tool for therapy of mesenchimal tumors of the gastrointestinal tract

Summary. Imatinib, a tyrosine kinase inhibitor directed against the enzymatic domain of KIT protein, was found to produce dramatic clinical responses in metastatic gastrointestinal stromal tumors (GISTs). However, resistance usually develops thus determining treatment failure. The present study was performed to analyse the expression of somatostatin receptor (SSTR) subtypes, modulators of tissue transglutaminase, in a series of GISTs and leiomyosarcomas by immunohistochemistry to identify a new potential therapeutic target. Sixteen cases (8 males and 8 females, age range: 38–73; 11 GISTs, 4 leiomyosarcomas, 1 leiomyoma) were studied. Immunohistochemical detection of the relevant SSTRs was performed on paraffin-embedded tissue sections, stained with polyclonal antibodies directed against the five somatostatin receptor subtypes. We found 7 out of 16 (44%) tumors expressing all SSTRs and 14 out of 16 (87%) tumors positive for at least 3 subtypes. SSTR2_A was the most represented subtype in the tumors studied, being expressed in approximately 70% of cases exhibiting an intense labeling in most of these cases. The significant expression of SSTRs shown in this series of GISTs and gastrointestinal leiomyosarcomas suggests a potential therapeutic target to be explored alone and/or in combination with other therapeutic agents in the setting of refractory GI stromal tumors.