反义封闭NGAL基因表达对SHEEC食管癌细胞微丝骨架的影响

为了研究反义封闭NGAL基因表达对SHEEC食管癌细胞微丝骨架以及肿瘤细胞生物学行为的影响,以不同长度NGAL基因片段反义表达载体和硫代修饰反义寡核苷酸单链片段转染SHEEC食管癌细胞,通过G418筛选,建立一系列旨在封闭SHEEC食管癌细胞NGAL基因表达的亚细胞克隆.在细胞内F-肌动蛋白(F-actin)及DNA荧光双标记基础上,通过流式细胞术、激光共聚焦显微镜扫描术等技术手段检测封闭反义NGAL基因表达后, SHEEC食管癌细胞中F-actin和DNA含量、F-actin形态结构以及肿瘤细胞生物学行为的变化特征.结果显示,反义封闭NGAL基因表达后,SHEEC食管癌细胞F-actin的含量明显降低,与永生化食管上皮细胞SHEE相近,但细胞分裂增殖指数未见明显变化.表明反义封闭NGAL基因表达对SHEEC食管癌细胞的微丝骨架有明显影响,而对SHEEC食管癌细胞的分裂增殖影响不明显.激光共聚焦显微镜扫描观测显示,反义封闭NGAL基因表达可使SHEEC食管癌细胞F-actin分布均匀,F-actin小体减少,细胞间连接重新建立,结构较紧密,主要形态结构特征与SHEE细胞趋于一致.提示反义封闭NGAL基因表达可对SHEEC食管癌细胞的微丝骨架F-actin产生明显影响,推测癌细胞的微丝骨架F-actin可能是NGAL基因在SHEEC食管癌细胞中发挥功能的一种作用环节.     

SHEEC食管癌细胞中NGAL基因的功能

中性粒细胞明胶酶相关脂质运载蛋白 (neutrophilgelatinase associatedlipocalin ,NGAL)是脂质运载蛋白(lipocalin)家族的一个新成员 ,可能是人类的一种新的癌基因 ,但是在肿瘤中的功能不清楚。以往研究发现NGAL基因在SHEEC食管癌细胞中显著过表达 ,表明该细胞是一种用来揭示NGAL基因在肿瘤中功能的良好模型。采用反义封闭技术 ,同时结合裸鼠成瘤实验等研究了反义封闭NGAL基因转录对SHEEC食管癌细胞的浸润和分裂增殖等行为的影响。结果发现 ,反义封闭NGAL基因转录不但可以有效地降低SHEEC细胞分泌的基质金属蛋白酶 9和基质金属蛋白酶 2的活性 ,而与此同时裸鼠成瘤细胞的浸润行为也相应地受到了明显抑制 ,然而SHEEC细胞端粒的长度、拓扑异构酶II的含量以及细胞增殖指数等未发生明显变化。表明NGAL基因在食管癌细胞SHEEC中的功能可能主要是通过明胶酶在促进肿瘤细胞的浸润中发挥作用 ,而可能与肿瘤细胞分裂增殖的相关性不明显     

人食管癌cDNA酵母杂交文库的构建与鉴定及其应用

以往在研究由促癌物12-o-十四烷酰佛波醇-13-乙酯（12-o-tetradecanoylphorbol-13 -acetate,TPA）诱导的人永生化食管上皮细胞恶性变中基因的差异表达情况时,曾获得中性粒细胞明胶酶相关lipocalin（neutrophil gelatinase-associated lipocalin,NGAL）等新的食管癌癌变相关基因.为深入研究这些癌变相关基因在食管癌中以蛋白质-蛋白质或蛋白质-DNA相互作用为基础的功能网络调控关系,建立了一个食管癌cDNA酵母杂交文库.采用Trizol试剂从一种新的人食管癌细胞（SHEEC,由人永生化食管上皮细胞转化而来）中提取细胞总RNA,采用Oligotex mRNA Kit从细胞总RNA中制备PolyA⁺ mRNA,采用SuperScript^TM Choice System For cDNA Synthesis Kit合成cDNA,以PolyA⁺ mRNA为探针,化学发光法监测cDNA合成的质量,以pGADT₇为载体构建了SHEEC细胞的cDNA酵母杂交文库.文库的滴度为1.19×10⁹ cfu/ml,重组片段大小主要集中在0.5～6.0 kb,重组率为50％.在此基础上,应用酵母单杂交技术手段对该文库中的NF-κB元件结合因子进行了筛选,在SD/-his/-leu/［＋15 mmol/L 3-AT］缺陷培养基平板上共获得了约360个单克隆.按照平板上酵母单克隆直径的大小,选择直径大于2 mm者91个,提取质粒进行酶切鉴定和一对一酵母单杂交验证实验,结果获得了30个阳性重组子,并随机取其中的9个阳性重组子进行测序和GenBank/BLAST同源分析,发现某些基因编码的蛋白质产物在关键氨基酸残基位点上与p65和p50的NF-κB元件结合域公共序列具有高度一致性.这些实验结果表明,所构建的人食管癌cDNA酵母杂交文库是成功的.     

NGAL基因不同长度片段的克隆及其顺式作用元件定位分析

NGAL(neutrophilgelatinase-associatedlipocalin)是lipocalin家族的一个新成员,可能是人类的一种新癌基因,但其在肿瘤中的表达调控机制尚不清楚。应用生物信息学手段对NGAL(X99133,包括外显子和内含子)进行分析发现,此区域内含有许多潜在的顺式作用元件,其中主要为增强子元件。对此设计了以下实验:应用PCR技术从食管癌细胞SHEEC基因组DNA中扩增不同长度的NGAL基因片段,将其连接到PGEMT-eacy载体中进行扩增,并测序。NCBI/BLAST分析确认扩增片段为NGAL基因所有,与实验所设计相吻合。在此基础上并应用KEGG/MOTIF检索分析系统对NGAL基因的上述区段进行了顺式作用元件定位分析,结果共鉴定出5个Score值=100分的顺式作用元件位点。这为NGAL基因增强子的鉴定提供了新线索。     

食管癌细胞NGAL5′侧翼区存在TPA应答元件

在TPA诱导永生化食管上皮细胞癌变中,NGAL(neutrophil gelatinase-associated lipocalin)过表达,提示食管癌细胞NGAL转录调控区可能存在TPA应答元件.为了对食管癌细胞NGAL的这一TPA应答元件进行分段定位鉴定,首先将NGAL5′侧翼区不同长度片段-1 431~ 84-、1 137~ 84、-945~ 84、-657~ 84、-416~ 84和-152~ 84等依次插入质粒pGLP,构建NGAL/pGLP系列报告基因荧光素酶表达载体pGLP-1431、pGLP-1137、pGLP-945、pGLP-657、pGLP-416和pGLP-152;然后将上述质粒分别同pRL-TK共转染食管癌细胞EC109,最后用5 ng/ml的TPA刺激转染的EC109,检测报告基因荧光素酶活力,综合判定报告基因荧光素酶活力变化趋势,并以此对食管癌细胞NGAL5′侧翼区TPA应答元件进行分段定位.实验结果表明,食管癌细胞NGAL5′侧翼-657~-417区段内存在着较强的TPA应答元件.生物信息学分析显示,NGAL5′侧翼-657~-417区段至少存在3个潜在的TPA应答元件,表明有应答TPA刺激的结构基础.这些研究有助于从分子水平揭示TPA诱导永生化食管上皮细胞癌变中NGAL基因过表达机制.     

EZH2蛋白在食管上皮永生化细胞系和恶性转化细胞系中的表达

目的研究EZH2蛋白在食管上皮永生化细胞系(SHEE)和恶性转化细胞系(SHEEC)中的表达。方法采用免疫细胞化学染色、免疫印迹分析和流式细胞术检测两种细胞系EZH2蛋白的表达。结果两种细胞EZH2蛋白染色均呈阳性,阳性反应定位于细胞核,部分细胞胞浆也有阳性着色。免疫印迹分析表明SHEEC细胞和SHEE细胞的总蛋白、核蛋白在分子质量约90ku的位置均出现特异性印迹条带。SHEE细胞中EZH2蛋白的表达强于SHEEC细胞(P<0.05)。流式细胞术显示EZH2蛋白在两种细胞中均有表达,两者的平均荧光强度无明显差别;阳性细胞率均较高,其中SHEE细胞阳性率高于SHEEC细胞。结论EZH2蛋白在SHEE细胞和SHEEC细胞中高表达可能参与了它们的恶性改变;而EZH2蛋白在两种细胞系中的差异表达可能与细胞的分化程度及来源于胚胎食管上皮细胞相关。     

食管癌细胞NGAL5′侧翼区存在TPA应答元件

在TPA诱导永生化食管上皮细胞癌变中,NGAL(neutrophil gelatinase associated lipocalin)过表达,提示食管癌细胞NGAL转录调控区可能存在TPA应答元件.为了对食管癌细胞NGAL的这一TPA应答元件进行分段定位鉴定,首先将NGAL 5′侧翼区不同 长度片段－1 431～+84、－1 137～+84、－945～+84、－657～+84、－416～+84和－152～+84等依次插入质粒pGLP,构建NGAL/pGLP系列报告基因荧光素酶表达载体pGLP 1431、pGLP 1137、pGLP 945、pGLP 657、pGLP 416和pGLP 152;然后将上述质粒分别同pRL TK共转染食管癌细胞EC109,最后用5 ng/ml的TPA刺激转染的EC109,检测报告基因荧光素酶活力,综合判定报告基因荧光素酶活力变化趋势,并以此对食管癌细胞NGAL 5′侧翼区TPA应答元件进行分段定位.实验结果表明,食管癌细胞NGAL 5′侧翼－657～－417区段内存在着较强的TPA应答元件.生物信息学分析显示,NGAL 5′侧翼－657～－417区段至少存在3个潜在的TPA应答元件,表明有应答TPA刺激的结构基础.这些研究有助于从分子水平揭示TPA诱导永生化食管上皮细胞癌变中NGAL基因过表达机制.     

NGAL的结构与功能研究进展

NGAL（neutrophil gelatinase-associated lipocalin)是一种新的lipocalin,其结构与功能的研究进展为人瞩目,目前认为NGAL具有运输疏水性分子,调节明胶酶B（gelatinaseB或matrix metalloproteinase-9,MMP-9)活性等功能,并可能参与免疫炎症反应以及肿瘤的发生发展,特别是肿瘤的浸润转移等过程,最近,NGAL蛋白的空间结构已被研究清楚,这对于促进NGAL生物学作用或功能的研究。特别是对其新的生物学作用或功能的探索,比如与肿瘤的关系（NGAL可能是一种新的癌基因或促癌基因）将具有十分重要的意义。对此作者进行了综合评述。     

利用cDNA微阵列-CGH筛选支气管上皮细胞恶性转化中的扩增基因

目的:基因组不稳定是导致肺癌发生与发展的重要分子机理之一。本研究旨在筛选支气管上皮细胞恶性转化过程中拷贝数扩增的基因。方法:利用业已建立的支气管上皮细胞体外恶性转化模型,通过cDNA微阵列-CGH技术对支气管上皮来源的永生化细胞和癌变细胞的基因拷贝数进行了检测,并对部分结果进行了实时PCR验证。结果:永生化BEP2D细胞染色体中的某些区域存在不同程度的扩增,包括5q31.3、9q32-33.1、14q22.2-23.1、19p13.12-13.13、20q13.12-13.31;恶性转化BERP35T2细胞染色体中的扩增区域集中在1p12-13.1、5q33.1、5q31.3、9q32、19p13.12-13.13;5q31.3、9q32、19q13.12-13.13是以上2种细胞系中的共同扩增区域。共检测到201个基因的拷贝数发生扩增,其中PCNA、TP53及GADD45A基因的异常扩增已经实时PCR进一步验证。结论:在支气管上皮细胞恶性转化过程中,病毒与低剂量辐射的双重作用使得某些重要基因的拷贝数发生扩增,因基因剂量增加而导致某些癌基因高表达可能是细胞恶性转化的重要机制之一。     

应用代表性差异分析法筛选人癌候选抑癌基因

运用cDNA代表性差异分析法(cDNA representational difference analysis,cDNA RDA),以正常人鼻咽上皮细胞及鼻咽癌HNE1细胞作为比较的样品来源,分离了四个在鼻咽癌中缺失的cDNA片段.以此四个片段作探针,分别进行DNA杂交、RNA杂交,结果显示,这些差异性的cDNA序列确实来自正常人鼻咽上皮且只在其中表达和/或在鼻咽癌HNE1中表达降低,并在鼻咽癌病人中存在不同程度的缺失.序列分析结果表明这些差异性表达的基因为具有相当抑癌基因功能的已知基因和可能与鼻咽癌相关的抑癌基因的新基因.从而说明cDNA RDA是一种高效、敏感、假阳性低的克隆抑癌基因的有效方法.     

The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition

First identified as a neutrophil granule component, neutrophil gelatinase-associated lipocalin (NGAL; also called human neutrophil lipocalin, 24p3, uterocalin, or neu-related lipocalin) is a member of the lipocalin family of binding proteins. Putative NGAL ligands, including neutrophil chemotactic agents such as N-formylated tripeptides, have all been refuted by recent biochemical and structural results. NGAL has subsequently been implicated in diverse cellular processes, but without a characterized ligand, the molecular basis of these functions remained mysterious. Here we report that NGAL tightly binds bacterial catecholate-type ferric siderophores through a cyclically permuted, hybrid electrostatic/cation-pi interaction and is a potent bacteriostatic agent in iron-limiting conditions. We therefore propose that NGAL participates in the antibacterial iron depletion strategy of the innate immune system.     

Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa lipocalin originally purified from human neutrophils. It exists in monomeric and homo- and heterodimeric forms, the latter as a dimer with human neutrophil gelatinase. It is secreted from specific granules of activated human neutrophils. Homologous proteins have been identified in mouse (24p3/uterocalin) and rat (alpha(2)-microglobulin-related protein/neu-related lipocalin). Structural data have confirmed a typical lipocalin fold of NGAL with an eight-stranded beta-barrel, but with an unusually large cavity lined with more polar and positively charged amino acid residues than normally seen in lipocalins. Chemotactic formyl-peptides from bacteria have been proposed as ligands of NGAL, but binding experiments and the structure of NGAL do not support this hypothesis. Besides neutrophils, NGAL is expressed in most tissues normally exposed to microorganisms, and its synthesis is induced in epithelial cells during inflammation. This may indicate either a microbicidal activity of NGAL or a role in regulation of inflammation or cellular growth, putative functions yet to be demonstrated.     

Neutrophil Gelatinase-associated Lipocalin in Normal and Neoplastic Human Tissues. Cell Type-specific Pattern of Expression

Neutrophil gelatinase-associated lipocalin (NGAL) has recently been identified in myeloperoxidase-negative neutrophil granules. Members of the lipocalin family are thought to bind and transport small lipophilic molecules such as retinoids and roles in cell regulation have been proposed. Recently, NGAL has also been demonstrated in the colonic mucosa in certain pathologic conditions.The aim of this study was to examine the distribution of NGAL in normal and neoplastic tissues by immunohistochemistry. Interestingly, NGAL was found in a variety of normal and pathological human tissues. A cell type-specific pattern of expression was seen in bronchus, stomach, small intestine, pancreas, kidney, prostate gland, and thymus. The comparative analysis of the putative rat homologue neu-related lipocalin showed a very similar pattern of expression with the exception of pancreas and kidney. Neoplastic human tissues showed a very heterogeneous expression of NGAL protein. High NGAL levels were found in adenocarcinomas of lung, colon and pancreas. In contrast, renal cell carcinomas of various subtypes and prostate cancers contained low NGAL levels. Lymphomas and thymic tumours were negative for NGAL immuno-labeling. Knowledge about the location of NGAL in normal cells and in disease states provides the first clues towards understanding its biological function.     

Ex-FABP: a fatty acid binding lipocalin developmentally regulated in chicken endochondral bone formation and myogenesis

Extracellular fatty acid binding protein (Ex-FABP) is a 21 kDa lipocalin specifically binding fatty acids, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibers and in blood granulocytes. In chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory agents and repressed by anti-inflammatory agents. In adult cartilage Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chickens. The possible mammalian counterpart is the Neu-related lipocalin (NRL), a lipocalin overexpressed in rat mammary cancer; NRL is homologous to the human neutrophil gelatinase associated lipocalin (NGAL) expressed in granulocytes and in epithelial cells in inflammation and malignancy and to the Sip24 (super-inducible protein 24), an acute phase lipocalin expressed in mouse after turpentine injection. Immunolocalization and in situ hybridization showed that NRL/NGAL is expressed in hypertrophic cartilage, in forming skeletal muscle fibers and in developing heart. In adult cartilage NRL/NGAL was expressed in articular cartilage from osteoarthritic patients and in chondrosarcoma. Moreover, NRL was induced in chondrocyte and myoblast cultures by an inflammatory agent. We propose that these lipocalins (Ex-FABP, NRL/NGAL, Sip24) represent stress proteins physiologically expressed in tissues where active remodeling is taking place during development and also present in tissues characterized by an acute phase response due to pathological conditions.