胸腔积液中肺腺癌细胞发生胶原化的形态观察与研究

目的胶原蛋白是细胞外基质的主要成分之一,我们在临床数千例恶性胸水转移的非小细胞肺癌细胞胞浆中常见到围绕细胞核或局灶状葱皮排列的纤维样结构,由此我们假设肺腺癌细胞在侵袭转移形成胸腔积液过程中发生与细胞外基质相同的结构变化,即肿瘤细胞胶原化形成“盔甲”式保护膜起到对肿瘤细胞结构及功能的支撑,达到抵御化疗药物、毒素的穿透和放射线的轰击,提高了自我保护能力,适应新的肿瘤细胞增生微环境。方法TCT方法选取224例肺腺癌细胞学标本,其中胸腔积液标本144例,肺泡灌洗液40例,痰液标本40例,24例为良性胸腔积液作为对照。常规制成细胞包埋块,切片、HE染色及CK7、TTF-l、vimintin的免疫细胞化学染色。透射电镜观察肿瘤细胞内部结构。Masson特殊染色,胶原蛋白工、Ⅱ、Ⅲ亚型染色,Western-blot蛋白检测。观察肺腺癌细胞是否存在胶原蛋白亚型及肺腺癌不同标本的表达情况。结果Masson细胞化学染色显示胶原纤维在恶性胸腔积液、支气管肺泡灌洗液及痰肺腺癌标本中的表达率分别为59．7％（sG／144）、0％（0／40）、o％（0／40）,在良性标本中不表达均为阴性（0／24）。免疫细胞化学结果显示：COLlAl、COL2Al和COL3Al在肺腺癌细胞中的阳性表达率分别为22．6％（38／1G8）,5．4％（9／1G8）,22．6％（38／168）,阳性表达主要定位于胞浆,而在增生的上皮细胞中不表达,统计学分析结果显示COLlAl、COL3Al的表达率要显著高于COL2Al的表达率（P=0．024）,并且我们发现COLlAl与COL3A1的表达成正相关性（r=0．886,P=0．000）。透射电镜可观察到在肿瘤细胞胞浆中可见多量纤维状物质。结论本研究从形态学上观察到在转移的肺腺癌细胞胞浆内有一些围绕胞核呈葱皮样改变的纤维样结构,结果表明肺腺癌细胞可以产生胶原蛋白,     

EGFR在晚期肺腺癌患者中的表达及临床意义

目的探讨表皮生长因子受体(EGFR)在晚期肺腺癌患者中的表达及其意义。方法收集60例晚期肺腺癌患者的胸腔积液及活检肺癌组织,采用免疫组化检测EGFR的表达,并探讨其表达与临床病理特征的关系。结果 EGFR在胸腔积液及肺癌组织中的阳性表达率分别为75.0%(45/60)、63.3%(38/60),两者差异无统计学意义(P0.05)。结论 EGFR均高表达于肺腺癌胸腔积液及腺癌组织,且与年龄、性别、吸烟史、分化程度及肿瘤的大小无明显相关,可指导肺腺癌患者的靶向治疗。     

EGFR信号通路在SiO2诱导肺上皮细胞间质转化中的作用机制

目的：在二氧化硅（SiO2）刺激下可引起肺部一系列的炎症反应及其伴随相关的成纤维细胞增殖,然而EGFR信号通路可维持细胞增殖、分化和凋亡的平衡,因此,我们可以设想EGFR信号通路是否在肺纤维化的发生发展中起到重要的作用。本实验探讨SiO2是否能诱导人肺上皮细胞（A549）发生上皮间质转化,并且研究EGFR信号通路在矽肺纤维化中的作用机制。方法：以A549为研究对象,用0（对照组）、50、100、200μg／mlSiO2孵育A549,作用48h后于倒置显微镜观察细胞形态学改变,并收集不同时段细胞,采用实时荧光定量PCR（RT-PCR）检测E-钙黏蛋白（E-cadherin）和α-平滑肌肌动蛋白（α-SMA）mRNA表达变化,细胞免疫荧光方法检测E-cadllerin、α-SMA及信号转导蛋白EGFR表达的变化。结果：倒置显微镜观察A549经SiO2处理后细胞形态由鹅卵石状转变为纺锤型或梭型,形态似成纤维细胞,随着SiO2浓度的升高,E-cadmRNA和蛋白表达逐渐下调,在200μg／ml组表达最低,α-SMAmRNA和蛋白表达逐渐上调,200μg／ml组α-SMA表达最高;EGFR蛋白表达上调;50、100、200μg／ml与对照组的差异具有统计学学意义（P〈0．05）。结论：SiO2可诱导肺上皮细胞向间质细胞转化,其机制可能与EGFR信号通路有关。     

EGFR信号通路在SiO_2诱导肺上皮细胞间质转化中的作用机制(英文)

目的:在二氧化硅(SiO2)刺激下可引起肺部一系列的炎症反应及其伴随相关的成纤维细胞增殖,然而EGFR信号通路可维持细胞增殖、分化和凋亡的平衡,因此,我们可以设想EGFR信号通路是否在肺纤维化的发生发展中起到重要的作用。本实验探讨SiO2是否能诱导人肺上皮细胞(A549)发生上皮间质转化,并且研究EGFR信号通路在矽肺纤维化中的作用机制。方法:以A549为研究对象,用0(对照组)、50、100、200μg/ml SiO2孵育A549,作用48h后于倒置显微镜观察细胞形态学改变,并收集不同时段细胞,采用实时荧光定量PCR(RT-PCR)检测E-钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)mRNA表达变化,细胞免疫荧光方法检测E-cadherin、α-SMA及信号转导蛋白EGFR表达的变化。结果:倒置显微镜观察A549经SiO2处理后细胞形态由鹅卵石状转变为纺锤型或梭型,形态似成纤维细胞,随着SiO2浓度的升高,E-cad mRNA和蛋白表达逐渐下调,在200μg/ml组表达最低,α-SMA mRNA和蛋白表达逐渐上调,200μg/ml组α-SMA表达最高;EGFR蛋白表达上调;50、100、200μg/ml与对照组的差异具有统计学学意义(P0.05)。结论:SiO2可诱导肺上皮细胞向间质细胞转化,其机制可能与EGFR信号通路有关。关键词:表皮生长因子受体;矽尘;A549细胞;上皮间质转化     

活细胞中丝裂霉素诱导肺腺癌细胞凋亡机理的分析

为了分析丝裂霉素(mitomycin C,MMC)引起的肺腺癌细胞(ASTC-a-1)凋亡,实验通过CCK-8试剂盒检测不同浓度MMC对ASTC-a-1活性的影响,选择10μgS/mL的MMC处理ASTC-a-1.利用Hochest 33258染色观察MMC引起的ASTC-a-1凋亡,发现MMC可引起细胞缩小,核浓缩.为了进一步研究MMC引起凋亡的途径,通过脂质体转染pSCAT3质粒,利用光漂白技术和光谱技术观察Caspase-3是否活化;通过转染Bax和DsRed质粒观察Bax在凋亡过程中的位置变化及与线粒体的关系.结果显示:MMC可以诱导ASTC-a-1细胞内Caspase-3活化,并使Bax向线粒体转移聚集.在活细胞中证实Caspase-3和Bax参与了MMC引起的ASTC-a-1凋亡.     

乳腺癌细胞EGFR表达水平与长春瑞滨敏感度的相关性研究

乳腺癌是较早开始个体化治疗的肿瘤之一。表皮细胞生长因子受体（epidermalgrowthfactorreceptor,EGFR）6~表达与乳腺癌组织学分期、生长速度呈正相关,可作为乳腺癌患者预后的指标之一。临床上,长春瑞滨（Vinorelbine,NVB）~要作为耐药性晚期乳腺癌的挽救性化疗药物,单药治疗亦具有一定疗效。该研究结果发现,乳腺癌组织EGFR表达与NVB的敏感性相关（P=0．001）,而与紫杉醇、阿霉素及5．氟尿嘧啶无相关性。EGFRFH性乳腺癌细胞MDA-MB-435s对NVB耐药,而EGFR阳性细胞MCF-7则敏感．但是EGFR中和性抗体会降低敏感性。进一步研究发现,NVB会引起MCF-7表面EGFI滚达上调,以及胞内ERK1／2激酶的磷酸化,且这一效应会被抗EGFR抗体部分抑制。研究结果表明,乳腺癌细胞对NVB的敏感性与膜表面EGFR表达水平相关,提示EGFR可作为NVB治疗敏感性的预测分子。     

不同转移表型肺腺癌细胞PI-PLC的比较研究

实验证明钙、磷脂酰乙醇胺(PE)、磷脂酸(PA)及脱氧胆酸钠(DOC)对两种不同转移表型癌细胞磷脂酰肌醇特异的磷脂酶C(PI-PLC)的活性影响有明显差异。高转移性人肺腺癌细胞亚系(Anip_(973))比低或无转移性人肺腺癌细胞系(AGZY_(83-a))腹水型癌细胞PI-PLC活性高2.25—7.1倍。Anip_(1211)细胞培养液PI-PLC活性也大于AGZY_(83-a)。说明不同表型肺腺癌细胞具有不同性质的PI-PLC,且活性变化与转移表型密切相关。     

雌激素受体在肺腺癌A549细胞系EMT过程中的作用

摘要 目的：探讨何种雌激素受体在肺腺癌A549细胞系上皮间质转化（EMT:Epithelial-mesenchymal transition）过程中发挥作用。方法：用雌激素受体拮抗剂分别抑制A549细胞系中的雌激素 和 受体,再用浓度为1×10-8 mol/L的雌激素刺激抑制前后的各组细胞系,用q-RT-PCR及Western blot实验检测各组细胞中EMT标志物的表达量,用Transwell实验检测各组细胞迁移量,计算各组间有无统计学差异。结果：雌激素组细胞中Vimentin的mRNA表达量为5.81±0.71,显著高于空白对照组,E-cadherin的mRNA表达量为0.43±0.07,显著低于空白对照组,细胞迁移数量为80.17±8.82,显著高于空白对照组,组间有统计学差异。用雌激素 受体拮抗剂MPP处理前后Vimentin的mRNA表达量分别为7.63±0.92和6.29±0.73,均显著高于空白对照组,组间有统计学差异;E-cadherin的mRNA表达量分别为0.39±0.05和0.42±0.06,均显著低于空白对照组,组间有统计学差异;细胞迁移数量分别为85.21±11.56和78.69±9.63,均显著高于空白对照组,组间有统计学差异。雌激素加雌激素 受体拮抗剂PHTPP组及雌激素加雌激素 和 受体拮抗剂组Vimentin的mRNA表达量分别为1.21±0.15和1.17±0.13,E-cadherin的mRNA表达量分别为0.86±0.12和0.93±0.11,细胞迁移数量分别为39.85±5.22和37.21±4.95,与对照组无统计学差异。各组细胞中EMT标志物蛋白表达量与mRNA表达量趋势相同。结论：雌激素?茁受体是雌激素促进A549细胞系发生EMT过程的重要靶点,在肺腺癌的肿瘤发生和发展过程中发挥了主要作用,可以作为进一步靶向治疗的研究方向。     

肺腺癌组织中表皮生长因子受体基因突变的检测及其临床意义

目的:通过检测肺腺癌组织中表皮生长因子受体(EGFR)基因的突变情况,研究EGFR突变与患者临床特征(性别、年龄和吸烟史)的相关性。方法:收集160例肺腺癌患者术前的石蜡组织标本,提取DNA后用实时定量PCR方法对EGFR基因18~21外显子进行突变检测;对基因突变结果与患者的性别、年龄和吸烟史分别做χ2检验。结果:160例肺腺癌组织标本中,有57例检测到EGFR基因突变,突变率为35.6%,且突变与患者性别、年龄和吸烟史均无显著相关性(P0.05);57例EGFR基因突变标本中,27例为19外显子缺失,22例为21外显子L858R点突变,这2种突变占总突变类型的85.96%,并且与患者性别、年龄及吸烟史无显著相关性(P0.05)。结论:EGFR基因在女性不吸烟肺腺癌中有较高的突变率,突变主要集中在19外显子缺失和21外显子L858R点突变,但突变率及突变类型与患者性别、年龄和吸烟史均不相关。     

人肝癌细胞株中EGFR的表达和EGF对肝癌细胞生长的促进

本文分析了人肝癌细胞株7404,7721细胞中表皮生长因子受体(EGFR)基因表达和EGF对肝癌细胞生长的促进作用。~(125)Ⅰ-EGF对7404细胞的结合试验表明结合是可饱和的和专一的,从~(125)Ⅰ-EGF对7404、7721细胞结合浓度曲线作Scatchard作图和计算,提示每个7404和7721细胞表面分别有1.1×10~5和0.7×10~5的EGFR分子。Northern杂交分析证明EGFR基因在7404,7721细胞中的转录产物主要是5.6 kb EGFR mRNA,免疫印迹分析证明7404细胞和7721细胞的EGFR为170kd的蛋白。EGF对培养于含10%或0.5%小牛血清的RPMI-1640培液中的7404、7721细胞的贴壁依赖性生长有促进作用,促进作用的程度与培液中CS含量有一定关系,提示EGF的促生长作用可能是EGF与血清中其他成分协同作用的结果。EGF对培养于软琼脂中的7404,7721细胞的贴壁不依赖性生长也有明显促进作用。综合上述实验结果说明EGFR基因在人肝癌细胞中是活跃表达的,EGF可能是肝癌细胞生长依赖的一个重要有丝分裂原。     

TP53 mutations in circulating tumor DNA in advanced epidermal growth factor receptor-mutant lung adenocarcinoma patients treated with gefitinib

Tumor protein p53 (TP53) is a tumor suppressor gene and TP53 mutations are associated with poor prognosis in non-small cell lung cancer. However, the in-depth classification of TP53 and its relationship with treatment response and prognosis in epidermal growth factor receptor (EGFR)-mutant tumors treated with EGFR tyrosine kinase inhibitors are unclear. Circulating tumor DNA was prospectively collected at baseline in advanced treatment-naïve EGFR-mutant lung adenocarcinoma patients treated with gefitinib in an open-label, single-arm, prospective, multicenter, phase 2 clinical trial (BENEFIT trial) and analyzed using next-generation sequencing. Survival was estimated using the Kaplan–Meier method. Of the 180 enrolled patients, 115 (63.9%) harbored TP53 mutations. The median progression-free survival (PFS) and overall survival (OS) of patients with TP53-wild type tumors were significantly longer than those of patients with TP53-mutant tumors. Mutations in exons 5–8 accounted for 80.9% of TP53 mutations. Mutations in TP53 exons 6 and 7 were significantly associated with inferior PFS and OS compared to wild-type TP53. TP53 mutation also influenced the prognosis of patients with different EGFR mutations. Patients with TP53 and EGFR exon 19 mutations had significantly longer PFS and OS than patients with TP53 and EGFR L858R mutations, and both groups had worse survival than patients with only EGFR mutations. Patients with TP53 mutations, especially in exons 6 and 7, had a lower response rate and shorter PFS and OS when treated with gefitinib. Moreover, TP53 exon 5 mutation divided TP53 mutations in disruptive and non-disruptive types.     

Specific epidermal growth factor receptors on porcine thyroid cell membranes

The influence of islet-activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTP gamma S, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid-induced stimulation of a high affinity GTPase. The data suggest that a GTP-hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.     

Targeting epidermal growth factor receptors inhibition in non-small-cell lung cancer: a computational approach

Epidermal growth factor receptors (EGFRs) are transmembrane receptors present on cell membranes, play an important role in controlling cell growth, apoptosis and other cellular functions. They have an extracellular binding moiety, a transmembrane component and an intracellular tyrosine kinase unit. Mutations of EGFRs can lead to continual or abnormal activation of the receptors causing unregulated cell division, causing cancer such as non-small-cell lung cancer (NSCLC). Hence, the objective is to recognise the potential drug targets through generating pharmacophoric pattern, identifying and building suitable ligands and docking studies with dynamics applications. The pharmacophore of these compounds explains about physicochemical properties required for designing new compounds which provides the design to develop desired targeted drug therapy. The simulation uncovers changes in the width of the essential channel to the active site, extensive to concede substrates. This study concludes the interaction of EGFRs with its inhibitors through computational modelling which can be important initial steps toward the development of novel pharmaceuticals in the fight against NSCLC.     

Effect of tunicamycin on the turnover of epidermal growth factor receptors in cultured calf aorta smooth muscle cells: Comparison with IMR-90 human lung fibroblasts

Treatment of cultured calf aorta smooth muscle cells with tunicamycin, a potent inhibitor of dolichol-mediated glycosylation, resulted in progressive loss of receptors for epidermal growth factor with 50% of receptors lost after 6 h. Receptor half-life was also 6 h with cycloheximide treatment but was 12 h with either actinomycin D or camptothesin treatment. The epidermal growth factor-induced processing (internalization and/or degradation) of residual receptors remaining after tunicamycin treatment appeared to be unaltered.50% decrease in ¹²⁵I-labeled epidermal growth factor binding was observed also with IMR-90 fibroblasts upon 6 h treatment with tunicamycin, although these cells were less sensitive to inhibition by tunicamycin of glycosylation and protein synthesis.     

The extracellular matrix protein Del1 induces apoptosis via its epidermal growth factor motif

Mouse Del1 is an extracellular matrix protein mainly expressed in the developing embryo. Del1 has three EGF motifs and two discoidin domains. The second EGF motif reportedly contains an RGD sequence that binds to integrin receptors. Here, we provide evidence that Del1 protein induces cell death in vitro. Chromatin condensation and DNA laddering were observed, suggestive of apoptosis. The results of analysis using the TUNEL method and annexin V staining were also consistent with apoptosis. The apoptosis-inducing activity of Del1 could be mapped to the third EGF motif, which fitted the consensus sequence CX(D/N)XXXX(F/Y)XCXC, wherein the aspartic acid residue (D) could be β-hydroxylated. As little as twenty-five picomolar of recombinant E3 could induce apoptosis.     

Effects of gangliosides on the differentiation of human mesenchymal stem cells into osteoblasts by modulating epidermal growth factor receptors

Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation as well as the signals of several signal molecules, including epidermal growth factor receptors (EGFR). These compounds are localized in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role that gangliosides play in osteoblastogenesis is not yet clearly understood, therefore, in this study, the relationship between gangliosides and EGFR activation was investigated during osteoblast differentiation in human mesenchymal stem cells (hMSCs). The results of high-performance thin-layer chromatography (HPTLC) showed that ganglioside GM3 expression was decreased, whereas ganglioside GD1a expression was increased during the differentiation of hMSCs into osteoblasts. In addition, an increase in the activation of alkaline phosphatase (ALP) was observed in response to treatment with EGF (5 ng/ml) and GD1a (1 μM) (p < 0.05). The activation of ALP was significantly elevated in response to treatment of ganglioside GD1a with EGF when compared to control cells (p < 0.01). However, treatment with GM3 (1 μM) resulted in decreased ALP activation (p < 0.01), and treatment of hMSCs with a chemical inhibitor of EGFR, AG1478, removed the differential effect of the two gangliosides. Moreover, incubation of the differentiating cells with GD1a enhanced the phosphorylation of EGFR, whereas treatment with GM3 reduced the EGFR phosphorylation. However, AG1478 treatment inhibited the effect of ganglioside GD1a elicitation on EGFR phosphorylation. Taken together, these results indicate that GD1a promotes osteoblast differentiation through the enhancement of EGFR phosphorylation, but that GM3 inhibits osteoblast differentiation through reduced EGFR phosphorylation, suggesting that GM3 and GD1a are essential molecules for regulating osteoblast differentiation in hMSCs.     

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 10⁴ receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.     

Click chemistry for improvement in selectivity of quinazoline-based kinase inhibitors for mutant epidermal growth factor receptors

Discovery of mutant-selective kinase inhibitors is one of the challenges in medicinal chemistry and is a main issue for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. We tried to improve the selectivity of pan-HER inhibitors for mutant EGFRs. Utilizing click chemistry, triazole-tethered quinazoline derivatives were synthesized, based on a quinazoline scaffold showing pan-HER inhibition. The representative compound 5j exhibited 17- and 52-fold improved selectivity for EGFR L858R/T790M over wild-type EGFR and HER2, respectively, and demonstrated 6.7-fold more potent antiproliferative activity against PC9 cells harboring EGFR-activating mutation than gefitinib. Although the described quinazolines did not surpass pyrimidines as 3rd generation EGFR inhibitors in terms of selectivity for mutant EGFRs, our approach might provide information that would help in the identification of mutant-selective compounds among pan-HER inhibitors using the quinazoline scaffold.