RET原癌基因在甲状腺癌中的研究进展

原癌基因RET(rearranged during transfection)在1985年被鉴定,随后,在甲状腺乳头状癌（papillary thyroid carcinoma,PTC）中发现了不同类型的RET/PTC染色体重排。在遗传性和散发性的甲状腺髓样癌（medullary thyroid carcinoma, MTC）中发现RET点突变基因。与PTC中RET的变化存在不同,MTC中RET的活化主要通过激活点突变。RET不同类型的重排活化与甲状腺癌的高发密切相关,RET不同的活化机制需对应不同的治疗对策。讨论了RET原癌基因在甲状腺乳头状癌和甲状腺髓样癌中的致病、诊断和预后的作用,以期为RET活化引起的甲状腺癌的治疗提供理论依据。     

中国人先天性巨结肠RET基因突变特征研究

为了研究中国人先天性巨结肠(HD)RET基因突变特征,从分子水平探讨先天性巨结肠症(HD)的发病机理,揭示中国人群HD与RET基因突变的关系。我们应用聚合酶链反应-单链构象多态性(PCR—SSCP)-DNA测序方法,对50例HD患儿和30例无便秘正常对照者,进行RET基因第13外显子突变筛查,并对第13外显子突变阳性患儿家系作研究。结果发现在50例HD患儿中,有7例第13外显子扩增片段在SSCP分析时出现泳动变位。经DNA测序证实,有3种点突变类型(错义、同义和移码),第13外显子突变率为14％(7／50)。在7例第13外显子突变的患儿中,发现2例患儿的父亲存在与其子相同的突变。实验表明中国先天性巨结肠人群存在着RET基因突变,且以杂和性点突变为主,HD具有一定的遗传性。     

中国人先天性巨结肠RET基因突变特征研究

为了研究中国人先天性巨结肠(HD)RET基因突变特征,从分子水平探讨先天性巨结肠症(HD)的发病机理,揭示中国人群HD与RET基因突变的关系。我们应用聚合酶链反应-单链构象多态性(PCR-SSCP)-DNA测序方法,对50例HD患儿和30例无便秘正常对照者,进行RET基因第13外显子突变筛查,并对第13外显子突变阳性患儿家系作研究。结果发现在50例HD患儿中,有7例第13外显子扩增片段在SSCP分析时出现泳动变位。经DNA测序证实,有3种点突变类型(错义、同义和移码),第13外显子突变率为14%(7/50)。在7例第13外显子突变的患儿中,发现2例患儿的父亲存在与其子相同的突变。实验表明中国先天性巨结肠人群存在着RET基因突变,且以杂和性点突变为主,HD具有一定的遗传性。     

RET原癌基因与肿瘤相关性研究的进展现状

RET原癌基因是一种重要的癌症驱动基因,与人类多种肿瘤的发生、发展密切相关。RET相关肿瘤的发病机制包括RET基因激活性点突变与RET基因融合突变。近年来,针对致癌性RET基因融合突变开发的精准靶向药物取得了突破性进展。综述了RET原癌基因与肿瘤发生、发展相关性研究及近年来临床诊疗方面的研究进展,旨在为RET基因突变癌症患者的精准化诊疗提供参考,并通过精准高效地抑制RET基因突变,提高疾病缓解率和控制率。     

多巴胺能神经细胞慢性损伤过程中RET的表达变化

目的:为了检测RET在多巴胺能神经细胞损伤过程中中脑黑质部位的表达变化,并探索其在多巴胺能神经细胞损伤中的可能作用.方法:本实验将C57BL/6 d、鼠分为三组:MPTP组、NS组和空白对照组,采用MPTP腹腔注射建立小鼠中脑黑质多巴胺能神经细胞慢性损伤模型,设立第一周到第六周六个时间点(1w-6w),检测小鼠行为学变化,并采用免疫荧光染色和western blotting等方法检测中脑黑质部酪氨酸羟化酶(TH)和PET的表达情况.结果:行为学结果显示,随给药次数及时间延长,悬挂实验中,MPTP组悬挂评分逐渐下降,MPTP组第三周给药以后与NS组比较差异有统计学意义(P<0.05);跳台实验中,小鼠受电击后跳上跳台的时间逐渐延长,错误次数逐渐增多.免疫荧光双标结果显示,在检测的各时间点,在小鼠中脑黑质一直有RET阳性细胞存在,且与TH阳性细胞共表达;western blotting结果显示,TH在给予MPTP后第三周表达开始降低,RET在给予MPTP后第一周和第二周持续高表达,并且也从第三周开始,其表达量明显降低.结论:这种表达变化提示RET的表达与多巴胺能神经细胞损伤有关.     

RET原癌基因与先天性巨结肠相关性的分子遗传学研究进展

先天性巨结肠(hirschsprung disease,HSCR),又称肠无神经节细胞症,是典型的肠神经系统发育异常疾病.目前已经发现11种基因和5个易感位点与HSCR发病相关.其中RET原癌基因(RET proto-oncogene,RET)是主要的易感基因.虽然大部分HSCR发病风险都与RET基因相关,但只有不到15%的散发性HSCR患者发生RET基因编码区的突变.近期研究发现,RET基因非编码区的调节性突变可能在HSCR发生中起重要作用.现着重对RET基因与HSCR相关性的最新研究进展进行综述.     

RET PLCγ phosphotyrosine binding domain regulates Ca2+ signaling and neocortical neuronal migration

The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca(2+)) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca(2+) from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca(2+) and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015.     

Distribution of GDNF family receptor α3 and RET in rat and human non-neural tissues

The neurotrophic growth factor artemin binds selectively to GDNF family receptor α3 (GFRα3), forming a molecular complex with the co-receptor RET which mediates downstream signaling. This signaling pathway has been demonstrated to play an important role in the survival and maintenance of nociceptive sensory neurons and in the development of sympathetic neurons. However, the presence and potential role of this artemin-responsive pathway in non-neural tissues has not been fully explored to-date. To study the distribution of GFRα3 and RET in adult rat and human non-neural tissues, we carried out a comprehensive immunohistochemical study. We stained major organs from the digestive, urinary, reproductive, immune, respiratory and endocrine systems, and from other systems (cardiovascular, skeletal muscle), as well as regions of the nervous system for comparison. In both rat and human, the majority of non-neural cells did not exhibit detectable GFRα3-like immunoreactivity. In the rat, GFRα3- and RET-like staining were found in the same non-neural cell type only in kidney. In the human digestive and reproductive systems, a subset of epithelial cells exhibited GFRα3- and RET-like staining, suggesting co-localization. In other tissues, sub-populations of cells expressed either GFRα3- or RET-like immunoreactivity. The functional consequences of GFRα3 expression in non-neural cells remain to be determined.     

SorLA Controls Neurotrophic Activity by Sorting of GDNF and Its Receptors GFRα1 and RET

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Highlights? SorLA is a sorting receptor for GDNF and its signaling receptors GFRa1 and RET ? The SorLA/GFRa1 complex targets GDNF for lysosomal degradation, while GFRa1 is recycled ? SorLA/GFRa1 targets RET for endocytosis and influences GDNF-induced neurotrophic effects ? SorLA knockout mice display altered dopaminergic function and an ADHD-like phenotype     

调控RET依赖性GDNF信号通路对大鼠癫痫模型的影响

目的:探究双侧海马CA1区立体定向注射anti-GDNF抗体对匹鲁卡品诱导的大鼠癫痫模型的影响。方法:选择成年雄性SD大鼠60只,并随机分为3组,即假手术组(sham组,n=20)、癫痫模型组(model组,n=20)和GDNF抑制剂组(anti-GDNF组,n=20)。使用氯化锂-匹鲁卡品腹腔注射诱导癫痫模型,sham组只给予氯化锂,anti-GDNF组在造模前2 h给予大鼠双侧海马CA1区立体定向注射anti-GDNF抗体。在造模后1、3、7 d观察大鼠癫痫的发作频率,7 d后采用脑电图监测(EEG)测定脑电波的变化情况,通过免疫组化方法测定海马CA1区域神经元数量变化(Neu N表达水平),造模后1 d时使用western blot方法测定海马CA1区GDNF、RET和P53蛋白的表达。结果:Model组大鼠棘-慢波数量明显高于Sham组,anti-GDNF组以上指标较model组显著减少(P0.05);Model组海马CA1区神经元大量凋亡,但anti-GDNF组凋亡较model组显著减少(P0.05)。与Sham组比较,在癫痫发作后1 d,model组的GDNF、RET表达水平上调,P53表达水平下降(P0.05),而anti-GDNF组大鼠海马CA1区GDNF、RET表达较model组明显下调,P53表达水平显著上降(P0.05)。结论:双侧海马CA1区立体定向注射anti-GDNF抗体能够减少癫痫发作,并对海马神经元起到保护作用,可能与其抑制GDNF/RET/P53信号通路有关。     

Synthesis,structure–activity relationship and crystallographic studies of 3-substituted indolin-2-one RET inhibitors

The synthesis, structure–activity relationships (SAR) and structural data of a series of indolin-2-one inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the available space around the indolinone scaffold within RET active site. Several substitutions at different positions were tested and biochemical data were used to draw a molecular model of steric and electrostatic interactions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results. These data provide important information for the development of indolinone inhibitors for the treatment of RET-driven cancers.     

The RET–Glial Cell-derived Neurotrophic Factor (GDNF) Pathway Stimulates Migration and Chemoattraction of Epithelial Cells

Embryonic development requires cell migration in response to positional cues. Yet, how groups of cells recognize and translate positional information into morphogenetic movement remains poorly understood. In the developing kidney, the ureteric bud epithelium grows from the nephric duct towards a group of posterior intermediate mesodermal cells, the metanephric mesenchyme, and induces the formation of the adult kidney. The secreted protein GDNF and its receptor RET are required for ureteric bud outgrowth and subsequent branching. However, it is unclear whether the GDNF–RET pathway regulates cell migration, proliferation, survival, or chemotaxis. In this report, we have used the MDCK renal epithelial cell line to show that activation of the RET pathway results in increased cell motility, dissociation of cell adhesion, and the migration towards a localized source of GDNF. Cellular responses to RET activation include the formation of lamellipodia, filopodia, and reorganization of the actin cytoskeleton. These data demonstrate that GDNF is a chemoattractant for RET-expressing epithelial cells and thus account for the developmental defects observed in RET and GDNF mutant mice. Furthermore, the RET-transfected MDCK cells described in this report are a promising model for delineating RET signaling pathways in the renal epithelial cell lineage.     

Synthesis and structure–activity relationship study of pyrazolo[3,4-d]pyrimidines as tyrosine kinase RET inhibitors

Three series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized and evaluated as RET kinase inhibitors. Compounds 23a and 23c were identified to show significant activity both in the biochemical and the BaF3/CCDC6-RET cell assays. Compound 23c was found to significantly inhibit RET phosphorylation and down-stream signaling in BaF3/CCDC6-RET cells, confirming its potent cellular RET-targeting profile. Different from other RET inhibitors with equal potency against KDR that associated with severe toxicity, 23c did not show significant KDR-inhibition even at the concentration of 1 μM. These results demonstrated that 23c is a potent and selective RET inhibitor.     

BRAF~(T1799A)突变、RET/PTC重排对术前诊断甲状腺乳头状癌的作用

目的:探讨术前超声引导下细针抽吸细胞学检查(US-FNAB)联合BRAFT1799A突变、RET/PTC1及RET/PTC3重排对诊断甲状腺乳头状癌(PTC)的特异性及敏感性,为临床术前准确诊断PTC选择适用的分子标记物。方法:收集346个超声怀疑为甲状腺恶性结节的US-FNAB细胞标本及对应结节(152个)的术后新鲜组织,采用PCR分别扩增BRAFT1799A、RET/PTC1及RET/PTC3基因,产物经基因测序证实。结果:346个甲状腺术前穿刺的结节中,选择观察而未手术的结节192个,手术治疗152个,未接受手术建议的结节2个。术前US-FNAB的细胞标本中共检测到51个结节发生BRAFT1799A突变,其细胞学分类为36个恶性,11个可疑恶性,4个良性。该51个结节术后病理证实均为PTC。20个发生RET/PTC1重排,其术前细胞学结果为17个恶性,2个可疑恶性,1个滤泡性肿瘤或可疑滤泡性肿瘤,术后病理证实均为PTC。3个结节发生RET/PTC3重排,其术前细胞学结果为恶性,术后病理证实均为PTC。对45个术前US-FNAB标本检测BRAFT1799A突变阴性而术后病理证实为PTC的结节,将其对应结节的术中组织行该基因的检测,仅有1个结节的术后组织中检测到该突变。本研究中,术前US-FNAB联合多分子标志物的检测,将细胞学诊断PTC的敏感度由73.96%提高到92.71%。结论:术前US-FNAB联合多分子标志物的检测可提高其诊断PTC的敏感性及特异性,并有助于患者的个体化诊治。     

EIGHT NEW SPECIES OF THE GENUS COPTOTERMES AND RET1CULITERMES FROM GUANGDONG PROVINCE, CHINA(Isoptera: Rhinotermitidae)

1. Coptotermes guangzhouensis, sp. n. This new sepcies can be pesarated from C.formosanus Shiraki, by thelength of left mandble<0.95, by the posterior margin of pronotum almoststraight, and by the width of head 0.99-1.12 (1.06).     

Site-specific gene expression and localization of growth factor ligand receptors RET, GFRα1 and GFRα2 in human adult colon

Two of the glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), namely GDNF and neurturin (NRTN), are essential neurotropic factors for enteric nerve cells. Signal transduction is mediated by a receptor complex composed of GDNF family receptor alpha 1 (GFRα1) for GDNF or GFRα2 for NRTN, together with the tyrosine kinase receptor RET (rearranged during transfection). As both factors and their receptors are crucial for enteric neuron survival, we assess the site-specific gene expression of these GFLs and their corresponding receptors in human adult colon. Full-thickness colonic specimens were obtained after partial colectomy for non-obstructing colorectal carcinoma. Samples were processed for immunohistochemistry and co-localization studies. Site-specific gene expression was determined by real-time quantitative polymerase chain reaction in enteric ganglia and in circular and longitudinal muscle harvested by microdissection. Protein expression of the receptors was mainly localized in the myenteric and submucosal plexus. Dual-label immunohistochemistry with PGP 9.5 as a pan-neuronal marker detected immunoreactivity of the receptors in neuronal somata and ganglionic neuropil. RET immunoreactivity co-localized with neuronal GFRα1 and GFRα2 signals. The dominant source of receptor mRNA expression was in myenteric ganglia, whereas both GFLs showed higher expression in smooth muscle layers. The distribution and expression pattern of GDNF and NRTN and their corresponding receptors in the human adult enteric nervous system indicate a role of both GFLs not only in development but also in the maintenance of neurons in adulthood. The data also provide a basis for the assessment of disturbed signaling components of the GDNF and NRTN system in enteric neuropathies underlying disorders of gastrointestinal motility.     

Neurturin, RET, GFRα-1 and GFRα-2, but not GFRα-3, mRNA are expressed in mice gonads

The gonads are known to produce numerous hormones and also neurotrophins and their receptors. Here we demonstrate expression of glial-cell-line-derived neurotrophic factor (GDNF) family ligands and related receptors in adult mice gonads by in situ hybridization. GDNF mRNA was expressed in the ovary, but was not detectable in testis. Neurturin (NTN), another ligand in this family, gave rise to strong mRNA hybridization signals in a mosaic pattern in the seminiferous tubules of the testis at stages IX-XII and I-II of the cycle. NTN mRNA signals were also found in uterus and the oviduct. In testis, the transducing receptor RET as well as GDNF receptor alpha-1 (GFR)alpha-1 and GFRalpha-2 were distributed in complementary and overlapping patterns, the former at stages XI-XII-I and the latter at stages VII and VIII. GFRalpha-3 could not be detected. Expression of these trophic molecules suggests involvement of GDNF family ligands and related receptor components in reproduction.