Galectin-3抑制剂体外筛选方法的建立及评价

半乳糖凝集素-3 (galectin-3, Gal-3)属于β半乳糖苷酶结合凝集素家族,具有特异性结合半乳糖的特性。通过C端糖蛋白结合区(carbohydrate recognition domain, CRD), Gal-3可结合糖基化胰岛素受体(insulin receptor,IR)的半乳糖苷链,从而抑制IR信号通路,导致胰岛素抵抗,被视为治疗胰岛素抵抗和2型糖尿病的潜在药物作用靶点。本研究根据Gal-3结合糖基化蛋白半乳糖苷链的特性,设计了一种简便的Gal-3抑制剂筛选模型。在大肠杆菌表达Gal-3蛋白,经纯化后,用异硫氰酸荧光素(fluorescein isothiocyanate, FITC)修饰,获得Gal-3-FITC。Gal-3-FITC自发绿色荧光,与表面表达有大量糖蛋白的人胰腺癌细胞(PANC-1)孵育后, PANC-1细胞带有荧光信号。若待测化合物有Gal-3抑制活性,则该化合物可降低Gal-3-FITC与细胞的结合,从而降低PANC-1细胞荧光信号。通过荧光信号变化可评价Gal-3抑制剂的抑制强度。进一步研究表明,该筛选模型简易稳定,具有良好重复性, Z’因子...     

半乳糖凝集素-3和甲状腺过氧化物酶在甲状腺癌和良性结节中的表达

目的探讨半乳糖凝集素-3与甲状腺过氧化物酶(TPO)蛋白在甲状腺乳头状癌及良性甲状腺结节中的表达及其诊断价值。方法用免疫组化MaxVisionTM2法测定22例甲状腺乳头状癌(PTC组)及26例良性甲状腺结节(NC组,包括结节性甲状腺肿11例,乳头状增生7例,滤泡状腺瘤8例)组织中半乳糖凝集素-3和TPO的表达。结果半乳糖凝集素-3在PTC组呈中至强阳性表达,在NC组呈阴性或弱阳性表达。TPO在PTC组中呈阴性或弱阳性表达,在NC组呈中至强阳性表达。结论半乳糖凝集素-3和TPO联合检测对甲状腺良、恶性病变的诊断与鉴别诊断具有较好的临床实用价值。     

Expressions of galectin-3 and TPO in thyroid cancer and benign nodules

目的 探讨半乳糖凝集素-3与甲状腺过氧化物酶(TPO)蛋白在甲状腺乳头状癌及良性甲状腺结节中的表达及其诊断价值.方法 用免疫组化MaxVisionTM2法测定22例甲状腺乳头状癌(PTC组)及26例良性甲状腺结节(NC组,包括结节性甲状腺肿11例,乳头状增生7例,滤泡状腺瘤8例)组织中半乳糖凝集素-3和TPO的表达.结果 半乳糖凝集素-3在PTC组呈中至强阳性表达,在NC组呈阴性或弱阳性表达.TPO在PTC组中呈阴性或弱阳性表达,在NC组呈中至强阳性表达.结论 半乳糖凝集素-3和TPO联合检测对甲状腺良、恶性病变的诊断与鉴别诊断具有较好的临床实用价值.     

利普液基细胞学和肿瘤标记物检测在甲状腺细针穿刺中的临床应用

目的评价利普液基细胞学(LPT)技术联合肿瘤标志物检测在甲状腺细针穿刺(FNA)中的临床应用价值。方法 20例甲状腺FNA标本同时采用传统涂片和LPT技术制备细胞涂片。其中,14例用免疫细胞化学法测定穿刺标本中半乳糖凝集素3(Gal-3)与甲状腺过氧化物酶(TPO)的表达情况。结果与传统细胞涂片相比,LPT制片获得细胞数量较多,细胞呈单层均匀分布,结构清晰,涂片背景干净。Gal-3在高度怀疑恶性病变的甲状腺滤泡细胞中呈阳性表达,但在良性病变的甲状腺滤泡细胞中不表达或低表达。TPO在高度怀疑恶性病变的甲状腺滤泡细胞中不表达或低表达,但在良性病变的甲状腺滤泡细胞中呈中到强阳性表达。结论 LPT制片技术明显提高甲状腺FNA标本制片质量;LPT技术结合肿瘤标志物检测,可用于筛查和诊断甲状腺癌和甲状腺癌前病变。     

半乳糖凝集素-3在心力衰竭临床应用中的研究进展

心力衰竭(HF）是心血管疾病发展到中晚期的一组复杂临床综合征,高发病率和病死率使其成为巨大的公共卫生负担。半乳糖凝集素-3(Gal-3)是近年来出现的新型生物标志物,大量研究证实其与HF的发生、发展密切相关,在HF患者临床管理应用中的价值日益突出。Gal-3参与心肌的纤维化和炎症反应,能直接反映心脏纤维化和心室重构的进程。Gal-3血清含量升高可促进心肌纤维化,降低心功能,增加HF患者的全因病死及再住院风险,因此监测该指标有助于HF的诊断及指导治疗,对HF患者的预后评估有着重要的临床意义。本文主要综述了Gal-3的结构、功能,Gal-3在HF临床诊断、预后评估中的作用,以及通过抑制Gal-3表达及中医药干预来辅助治疗HF。     

半乳糖凝集素3在乳腺癌患者中的表达及与上皮间质转化的关系

目的探讨半乳糖凝集素3(Gal-3)在乳腺癌患者中的表达水平及其与上皮间质转化(EMT)的关系。方法选择86例女性浸润性乳腺癌患者的癌组织和癌旁组织作为研究对象,其中发生肝转移者32例。采用免疫组化法检测Gal-3、波形蛋白(Vim)、E-钙黏附蛋白(E-cad)表达,采用Western blot法检测Gal-3的表达,分析Gal-3的表达与临床病理参数的关系。构建siRNA-Gal-3-vector和lentivirus-Gal-3-vector,转染SKBR-3细胞,用Western bolt法检测转染后细胞中Gal-3、E-cad及Vim的表达,并观察细胞的迁移及侵袭情况。结果86例患者癌旁组织Gal-3和Vim阳性表达率为0,E-cad表达阳性率为100%;86例患者乳腺癌组织Gal-3、Vim、E-cad阳性表达率分别为81%、57%和26%,与癌旁组织均有非常显著差异(均P<0.01)。发生肝转移者乳腺癌组织Gal-3、E-cad、Vim阳性表达率与未发生肝转移者有非常显著差异(分别为100%vs.70%,47%vs.13%,69%vs.48%,P<0.01)。Gal-3的阳性表达与TNM分期、腋窝淋巴结的转移及肝转移存在显著的相关性(P<0.05)。lentivirus-Gal-3-vector组乳腺癌细胞中Gal-3的表达显著增加,乳腺癌细胞的迁移与侵袭能力显著增加(P<0.01);EMT标志分子E-cad表达显著降低、Vim表达显著增加(P<0.01)。结论Gal-3在浸润性乳腺癌患者中呈现高表达,可能通过调控EMT促进乳腺癌细胞的浸润与转移。     

半乳糖凝集素-3和表皮生长因子受体蛋白表达与老年胃癌转移和预后的关系

目的探讨半乳糖凝集素-3（galectin-3,Gal-3）和表皮生长因子受体（epidemal growth factor receptor,EGFR）蛋白表达与老年胃癌临床病理特征和预后的关系。方法应用免疫组化技术检测60例老年胃癌、30例异型增生和20例癌旁正常胃黏膜组织中Gal-3和EGFR蛋白表达,并结合肿瘤的病理学行为和临床随访资料进行分析。结果在老年胃癌组织中Gal-3和EGFR阳性表达率分别为65.0%和46.7%,均显著高于异型增生和癌旁正常胃黏膜组织（P〈0.05）。Gal-3和EGFR表达与老年胃癌浆膜浸润、淋巴结转移和预后密切相关（P〈0.05）。Gal-3和EGFR蛋白表达呈显著正相关（r=0.30,P〈0.05）。结论 Gal-3和EGFR蛋白表达与老年胃癌发生、转移和患者生存期密切相关,同时检测Gal-3和EGFR蛋白表达可作为判断老年胃癌预后的参考指标。     

Ret、粘蛋白-1和半乳糖凝集素-3蛋白表达与甲状腺滤泡源性肿瘤的相关性及预后分析

目的 判断与Ret、粘蛋白-1（Mucin-1）和半乳糖凝集素-3（Galectin-3）蛋白识别的相关因素及影响甲状腺肿块复发的可能因素。方法 观察Ret、Mucin-1（MUC1）和Galectin-3（Gal-3）表达与甲状腺滤泡源性肿瘤相关指标的关系,并与随访资料相结合。分析甲状腺肿瘤有关的生物学行为。结果 ①运用多因素逐步logistic回归分析法．分别得出Ret、MUC1、Gal-3表达的logistic回归方程。结果包膜存在、最大直径是Ret表达的抑制因素。包膜侵犯与双侧性对Ret表达有促进作用。合并病变、包膜存在是MUC1表达的抑制因素,包膜存在也是Gal-3表达的抑制因素,包膜侵犯对MUC1和Gal-3表达有促进作用。②预后分析的Cox比例风险函数模型可知：有包膜比无包膜或包膜不完整者在某时刻复发的相对危险度为0．126,即复发的可能性降低了87．4％;病程每增加-个等级,在某时刻复发的相对危险度为2．701,即复发的可能性增加了1．701倍。结论 ①包膜存在与甲状腺肿瘤不或低表达Ret、MUC1和Gal-3关联;恶性肿瘤的包膜侵犯与恶性标志物表达密切关联;囊性变与MUC1和Ret的阴性表达有关;双侧性同-病变者Ret表达的可能性增高;腺体外浸润、淋巴结转移者表达Ret、MUC1和Gal-3的机率增高．其关联性表明三种标志物能反映恶性肿瘤的生物学行为。②复发有关因素中有包膜者。不易复发;而及早诊治甲状腺病变。其术后复发可大大降低。     

半乳糖凝集素-3对心力衰竭的临床价值

半乳糖凝集素-3(galeetin-3,Gal-3)是一种糖结合蛋白,作为生物标志对心力衰竭(heart failure,HF)诊治价值的临床研究逐年增多.本文总结了近年有关临床试验,就Gal-3对HF的临床价值做一综述.这些研究显示Gal-3对于HF有一定的临床价值,有望成为继脑钠肽(brain natriuretic peptide,BNP)和N-末端脑钠肽前体(N-terminal pro-brain natriuretic peptide,NT-proBNP)后HF的又一个重要的生物标志.Gal-3在HF预后方面是一个很强的预测因子,对于HF的诊断也可以作为NT-proBNP的重要补充.在患者与治疗的选择上,没有足够的证据显示其可以作为某种特定治疗的选择标志.而在纵向研究及治疗效果方面,Gal-3没有显示什么重要价值.作为生物标志,Gal-3当前尚不能作为临床决策的常规而写入指南.在以后的临床研究中尚需扩大人群样本,深入研究Gal-3与HF的病理生理过程的关联机制,以明确Gal-3如何指导HF的临床决策,药物和治疗方法的研发.     

外周血Gal-3、NT-proBNP在慢性心力衰竭患者中表达意义的研究

目的 探讨慢性心力衰竭(CHF)患者外周血半乳糖凝集素(Gal)-3、N末端B型利钠肽原(NT-proBNP)表达水平的变化.方法 选择89例CHF患者作为研究对象,采用酶联免疫吸附法、电化学发光法分别检测入选患者外周血Gal-3、NT-proBNP表达水平,分析其在不同类型心力衰竭[左心射血分数保留的心力衰竭(HFPEF)、左心射血分数降低的心力衰竭(HFREF)]、心功能分级患者中的变化,并进行相关性分析.结果 HFPEF、HFREF患者Gal-3、NT-proBNP表达水平比较,差异均有统计学意义(P=0.000);心功能Ⅱ级、Ⅲ级、Ⅳ级患者Gal-3、NT-proBNP表达水平比较,差异均有统计学意义(P =0.000);Gal-3与NT-proBNP呈正相关(r=0.230,P=0.030),与LVEF呈负相关(r=-0.533,P=0.000);NT-proBNP与LVEF呈负相关(r=-0.372,P=0.000).结论 外周血Gal-3、NT-proB-NP可作为评判CHF患者病情危重程度的重要指标.     

Galectin-3 inhibitors: a patent review (2008–present)

Introduction: Galectin-3 (Gal-3), a lectin with preference for β-galactoside-containing carbohydrates, is a structurally unique member of the galectin family. It is ubiquitously expressed in various mammalian tissues with a wide distribution from the intracellular environment to the extracellular space. Gal-3 is a well-established player in numerous diseases, from infections to heart failure. Notably, as Gal-3 overexpression is associated with cancer drug resistance, it has been identified as a valuable therapeutic target in the fight against cancers.

Areas covered: This review discusses the recent progress of patent applications (2008–present) and the current knowledge of pertinent Gal-3-inhibitor interactions in an effort to progress the development of selective and high affinity carbohydrate-based inhibitors targeting Gal-3, with an emphasis on engaging a structure-based drug design rationale.

Expert opinion: The lack of commercially available anti-Gal-3 therapeutic reagents and its clear involvement in serious disease, notably cancer, leads to an urgent need for development of inhibitors that specifically target Gal-3. Design of potent and selective carbohydrate inhibitors targeting Gal-3 is challenging due to relative weak protein-carbohydrate interactions along with the high sequence homology in the carbohydrate binding site region among galectins. To date, some chemical scaffolds have been exploited for design of promising effective Gal-3 inhibitors for cancer therapy.     

Obestatin、Gal-3和AGEs水平检测在Ⅱ型心肾综合征中的临床意义

目的 观察肥胖抑制素(Obestatin),乳糖凝集素-3(Gal-3)和晚期糖基化终产物(AGEs)在Ⅱ型心肾综合征疾病发生发展中的临床意义.方法 慢性心力衰竭患者95例,根据肾小球滤过率(eGFR)水平分为单纯心衰组(n=30)和心肾综合征组(n=65).选择同期行健康体检者20例,为健康对照组.观察3组机体Obestatin,Gal-3和AGEs水平的变化,心肾综合征患者血清Obestatin,Gal-3和AGEs水平与心功能分级和肾功能分期的关系及其各指标之间的相关性分析.结果 心肾综合征组的Obestatin,Gal-3和AGEs水平明显高于单纯心衰组和健康对照组(P<0.01),而单纯心衰组明显高于健康对照组(P<0.01).Ⅱ型心肾综合征患者机体的Obestatin,Gal-3和AGEs水平随着心功能和肾功能分期升高而升高(P<0.01).Ⅱ型心肾综合征患者机体的Obestatin水平与Gal-3和AGEs水平呈正相关(r值分别为0.572、0.726,P<0.05),Gal-3与AGEs水平呈正相关(r=0.587,P<0.05).结论 Obestatin,Gal-3和AGEs参与了Ⅱ型心肾综合征疾病的发生发展过程,联合检测有助于疾病的早期诊断,对病情严重程度的判断具有重要临床意义.     

Increased Galectin-9 expression,a prognostic biomarker of aGVHD,regulates the immune response through the Galectin-9 induced MDSC pathway after allogeneic hematopoietic stem cell transplantation

Galectin-9 (Gal-9) is a β-galactoside-binding soluble lectin family member that exerts its primary biological functions via specific glycoconjugate interactions. Gal-9 expression is closely related to tumor occurrence, development, metastasis and prognosis. In transplant immunology, a high level of Gal-9 expression has been shown to markedly reduce the severity of acute graft rejection and effectively prolong survival time in organ and bone marrow transplantation (BMT) models. The main mechanism of Gal-9-mediated immunoregulation involves the Tim-3/Gal-9 axis in T cells. However, myeloid-derived suppressor cell (MDSC) accumulation in transgenic mice with persistently high Gal-9 expression was observed in a model of lung inflammation, indicating that a potential immunosuppressive mechanism distinct from the Gal-9/Tim-3 axis might exist. In the present study, increased Gal-9 expression and MDSC frequencies before acute graft-versus-host disease (aGVHD) onset were observed in patients who developed aGVHD. Patients with higher Gal-9 expression (≥14.8417 ng/ml) exhibited reduced overall survival and increased cumulative incidences of GVHD at +100 day. We considered the elevated Gal-9 expression before aGVHD onset a secondary inflammatory response. This increase might be part of a negative feedback pathway corresponding to aGVHD pathogenesis. Additionally, a high Gal-9 concentration induced MDSC proliferation in vivo and in vitro. Gal-9-induced MDSCs (G9-MDSCs) suppressed T cell proliferation and activation. An infusion of G9-MDSCs into a graft contributed to the successful control of severe aGVHD and long-term survival in an allogeneic (allo)-BMT mouse model. Thus, we speculated that increased Gal-9 expression after allo-hematopoietic stem cell transplantation is a potential prognostic biomarker of aGVHD. The Gal-9-associated immunosuppressive effects on aGVHD development might occurr through G9-MDSCs and were independent of the Gal-9/Tim-3 axis.     

Role of galectin-3 in acetaminophen-induced hepatotoxicity and inflammatory mediator production

Galectin-3 (Gal-3) is a β-galactoside-binding lectin implicated in the regulation of macrophage activation and inflammatory mediator production. In the present studies, we analyzed the role of Gal-3 in liver inflammation and injury induced by acetaminophen (APAP). Treatment of wild-type (WT) mice with APAP (300 mg/kg, ip) resulted in centrilobular hepatic necrosis and increases in serum transaminases. This was associated with increased hepatic expression of Gal-3 messenger RNA and protein. Immunohistochemical analysis showed that Gal-3 was predominantly expressed by mononuclear cells infiltrating into necrotic areas. APAP-induced hepatotoxicity was reduced in Gal-3-deficient mice. This was most pronounced at 48-72 h post-APAP and correlated with decreases in APAP-induced expression of 24p3, a marker of inflammation and oxidative stress. These effects were not due to alterations in APAP metabolism or hepatic glutathione levels. The proinflammatory proteins, inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, and MIP-3α, as well as the Gal-3 receptor (CD98), were upregulated in livers of WT mice after APAP intoxication. Loss of Gal-3 resulted in a significant reduction in expression of iNOS, MMP-9, MIP-3α, and CD98, with no effects on IL-1β. Whereas APAP-induced increases in MIP-2 were augmented at 6 h in Gal-3(-/-) mice when compared with WT mice, at 48 and 72 h, they were suppressed. Tumor necrosis factor receptor-1 (TNFR1) was also upregulated after APAP, a response dependent on Gal-3. Moreover, exaggerated APAP hepatotoxicity in mice lacking TNFR1 was associated with increased Gal-3 expression. These data demonstrate that Gal-3 is important in promoting inflammation and injury in the liver following APAP intoxication.     

Galectin-3 gene silencing inhibits migration and invasion of human tongue cancer cells in vitro via downregulating β-catenin

Aim:

Galectin-3 (Gal-3) is a member of the carbohydrate-binding protein family that contributes to neoplastic transformation, tumor survival, angiogenesis, and metastasis. The aim of this study is to investigate the role of Gal-3 in human tongue cancer progression.

Methods:

Human tongue cancer cell lines (SCC-4 and CAL27) were transfected with a small-interfering RNA against Gal-3 (Gal-3-siRNA). The migration and invasion of the cells were examined using a scratch assay and BD BioCoat Matrigel Invasion Chamber, respectively. The mRNA and protein levels of β-catenin, Akt/pAkt, GSK-3β/pGSK-3β, MMP-9 in the cells were measured using RT-PCR and Western blotting, respectively.

Results:

Transient silencing of Gal-3 gene for 48 h significantly suppressed the migration and invasion of both SCC-4 and CAL27 cells. Silencing of Gal-3 gene significantly decreased the protein level of β-catenin, leaving the mRNA level of β-catenin unaffected. Furthermore, silencing Gal-3 gene significantly decreased the levels of phosphorylated Akt and GSK-3β, and suppressed the mRNA and protein levels of MMP-9 in the cells.

Conclusion:

Our data suggest that Gal-3 mediates the migration and invasion of tongue cancer cells in vitro via regulating the Wnt/β-catenin signaling pathway and Akt phosphorylation.     

T cell immunoglobulin-3 as a new therapeutic target for rheumatoid arthritis

T cell immunoglobulin-3 (Tim-3) is a surface molecule expressed on various cell types of the immune system which plays a central role in immune regulation. Recently, identi?cation of galectin-9 (Gal-9) as a ligand for Tim-3 has established the Tim-3–Gal-9 pathway as an important regulator of Th1 immunity and induction of tolerance. The interaction of Tim-3 with Gal-9 induces cell death; the in vivo blockade of this interaction results in exacerbated autoimmunity and abrogation of tolerance in experimental models, thus establishing Tim-3 as a negative regulatory molecule. A number of previous studies have demonstrated that Tim-3 in?uences chronic autoimmune diseases, such as multiple sclerosis and systemic lupus erythematosus. In addition, an association between Tim-3 polymorphisms and susceptibility to several autoimmune diseases has been identi?ed in various autoimmune diseases, including rheumatoid arthritis (RA). Recent work has focused on the role of Tim-3 in RA, and the results indicate that Tim-3 may represent a novel target for the treatment of RA. In this article we will discuss the Tim-3 pathway and the therapeutic potential of modulating the Tim-3 pathway in RA.     

Structural requirement for galanin action in the pancreatic beta cell line Rin m 5F

Galanin fragments were tested for their ability to alter forskolin-stimulated cyclic AMP production and insulin release from Rin m 5F cells. Galanin and its fragments inhibited both events with the following potencies: Gal-(1-29) greater than N-AcGal-(2-29) greater than Gal-(2-29) greater than Gal-(1-15). In contrast, Gal-(3-29), Gal-(10-29) and [Ile2]Gal were inactive. [Phe2]- and [Tyr2]Gal were moderately effective. We conclude that the N-terminal portion of galanin (in particular the aromatic amino acid in position 2) is crucial for activity.     

Regulated expression of galectin-1 after in vitro productive infection with herpes simplex virus type 1: implications for T cell apoptosis

Apoptosis of cytotoxic T lymphocytes by herpes simplex virus type-1 (HSV-1) has been reported to be a relevant mechanism of viral immune evasion. Galectin-1 (Gal-1), an endogenous lectin involved in T-cell apoptosis, has recently gained considerable attention as a novel mechanism of tumor-immune evasion. Here we investigated whether infection of cells with HSV-1 can modulate the expression of Gal-1. Results show that pro-apoptotic Gal-1, but not Gal-3, is remarkably up-regulated in cell cultures infected with HSV-1. In addition, this protein is secreted to the extracellular milieu, where it contributes to apoptosis of activated T cells in a carbohydrate-dependent manner. Since many viruses have evolved mechanisms to counteract the antiviral response raised by the infected host, our results suggest that HSV-1 may use galectin-1 as a weapon to kill activated T cells and evade specific immune responses.     

Association between heart rate variability indices and features of spontaneous ventricular tachyarrhythmias in mice

Direct evidence is limited for the association between heart rate variability (HRV) indices and ventricular tachyarrhythmias (VTAs). While galectin-3 (Gal-3) is regarded as a causal factor for cardiac remodelling and a biomarker for arrhythmias, its regulation on VTAs and HVR is unknown. Using aged transgenic (TG) mice with cardiac overexpression of β₂-adrenoceptors and spontaneous VTAs, we studied whether changes in HRV indices correlated with the severity of VTAs, and whether Gal-3 gene knockout (KO) in TG mice might limit VTA. Body-surface ECG was recorded (10-minute period) in 9- to 10-month-old mice of non-transgenic (nTG), TG and TG × Gal-3 knockout (TG/KO). Time-domain, frequency-domain and nonlinear-domain HRV indices were calculated using the R-R intervals extracted from ECG signals and compared with frequency of VTAs. TG and TG/KO mice developed frequent VTAs and showed significant changes in certain time-domain and nonlinear-domain HRV indices relative to nTG mice. The severity of VTAs in TG and TG/KO mice in combination, estimated by VTA counts and arrhythmia score, was significantly correlated with certain time-domain and nonlinear-domain HRV indices. In conclusion, significant changes in HRV indices were evident and correlated with the severity of spontaneous VTAs in TG mice. The frequency of VTA and HRV indices were largely comparable between TG and TG/KO mice. Deletion of Gal-3 in TG mice altered certain HRV indices implying influence by neuronally localized Gal-3 on autonomic nervous activity.