中国人胶质瘤中表皮生长因子受体基因的扩增及对患者预后的影响

目的：了解国人胶质瘤中表皮生长因子受体基因（EGFR）的扩增情况及对患者预后的影响。方法：使用半定量的Differential PCR方法对113例福尔马林固定-石蜡包埋（FFEP）胶质瘤标本内的EGFR基因扩增情况进行了检测；对其中的83例进行了生存分析研究。结果：113例胶质瘤中存在EGFR基因扩增的有26例，其中低度恶性组中仅占3.5％，而在高度恶性组中占42.1％。生存分析显示：EGFR基因扩增对患者预后并无显著影响，而起病年龄、肿瘤病理分级、肿瘤生长部位及起病症状为失语对患者术后生存期有显著影响。结论：恶性胶质瘤中存在着较为广泛的EGFR基因扩增，但EGFR基因的扩增与胶质瘤患者预后之间并不存在显著关联。     

神经上皮组织起源的脑肿瘤增殖活性与表皮生长因子受体基因表达的相关性研究

目的　探讨表皮生长因子受体 (EGFR)基因表达与神经上皮组织起源的脑肿瘤增殖活性的关系。　　方法　利用EGFR和Ki-67单抗免疫组化染色及核仁组成区嗜银蛋白 (AgNOR)染色对8例正常脑组织、50例神经上皮组织起源的脑肿瘤和 4个恶性脑肿瘤体外细胞系进行检测。　　结果　神经上皮组织起源的脑肿瘤中EGFR蛋白表达阳性率为 54% ,WHO分级与EGFR蛋白表达呈正相关 (r =0 5597,P <0 0 1 )。Ki-67标记指数 (Ki-67LI)及AgNOR平均数均与神经上皮组织起源的脑肿瘤的分级有明显关系 (r分别为 0 8971和 0 880 1 ,P <0 0 1 ) ,且Ki-67LI与Ag NOR平均数有显著相关性 (r=0 9896,P <0 0 1 )。EGFR蛋白表达阳性的肿瘤组织及细胞系中高Ki-67LI及高AgNOR平均数者占 64 5% ,而无EGFR蛋白表达的肿瘤组织和正常脑组织中高Ki-67LI及高AgNOR平均数者仅为 2 2 6% ,二者间差别有统计学意义 (P <0 0 1 )。　　 结论　神经上皮组织起源的脑肿瘤中EGFR表达与细胞的分裂增殖活动密切相关     

Epidermal growth factor receptor expression and activation in neuroendocrine tumours

Epidermal growth factor receptor (EGFR) is expressed in many cancers and is associated with poor prognosis. EGFR activation pathways have been well characterised using tumour cell lines and are known to involve EGFR activation through autophosphorylation. Phosphorylation of downstream signalling molecules, such as ERK1/2 (extra-cellular regulated kinase 1 and 2) and PKB/Akt (protein kinase B), leads to enhanced tumour cell survival and proliferation. Although EGFR expression has been determined in neuroendocrine tumour tissue, its activation and subsequent effects on the downstream signalling molecules, ERK1/2 and Akt, have not been studied. We therefore planned to determine the role of EGFR in neuroendocrine tumours (NETs) by determining its pattern of expression and activation, and the subsequent activation of downstream signalling molecules ERK1/2 and Akt. Paraffin-embedded tumour tissue was available from 98 patients with NETs (39 foregut, 42 midgut, four hindgut, five paragangliomas, and four of unknown origin). Immunohistochemical evaluation was performed for the expression of EGFR, p-EGFR, p-Akt, and p-ERK1/2. Ninety-six percent of tumour samples were positive for EGFR expression; 63% were positive for activated EGFR; 76% were positive for activated Akt; and 96% were positive for activated ERK1/2. Importantly, the histological score for the activation of Akt and ERK1/2 correlated with the histological score for activated EGFR. These data provide a rationale for considering EGFR inhibitors in the treatment of NETs. Additionally, direct inhibition of Akt and ERK1/2 may provide further therapeutic options in the treatment of NETs in the future.     

脑膜瘤中EGFR和PTEN的表达及意义

目的探讨脑膜瘤组织中表皮生长因子受体（EGFR）和人10号染色体上缺失的磷酸酶和张力蛋白类似物（PTEN）表达的相互关系及意义。方法应用免疫组织化学SP法对60例脑膜瘤标本的EGFR和PTEN表达进行检测与分析。结果在90％（54／60）的脑膜瘤中EGFR过表达，非典型脑膜瘤及间变性脑膜瘤中EGFR的染色强度明显高于典型脑膜瘤（P〈0．05）；PTEN在各病理级别脑膜瘤中的阳性率为100％，而且各病理级别脑膜瘤中PTEN的染色强度无统计学差别（P〉0．05）。结论EGFR的表达强度与脑膜瘤的恶性程度有关，抑癌基因PTEN在脑膜瘤中未见缺失，可能与脑膜瘤的发生、发展无关。     

miR-7沉默表皮生长因子受体对胶质瘤细胞周期的影响

目的 研究miR-7阻遏脑胶质瘤细胞增殖周期由G1期向S期转化的内在机制.方法 脂质体转染miR-7表达质粒进入人脑胶质瘤细胞株U251,原位杂交和RT-PCR方法检测外源miR-7基因的整合效果;流式细胞术检测细胞周期变化,Western blot方法检测EGFR、CyclinD1、CyclinE、CDK2、CDK4、CDK6、P16、P21和P27的蛋白表达;SPSS13.0软件进行统计学分析.结果 RT-PCR结果显示miR-7基因被成功整合入U251胶质瘤细胞,原位杂交结果示miR-7主要定位于细胞核和细胞质;转染后瘤细胞增殖速度减慢,且大部分阻滞于G1期,处于分裂期S期的细胞比例明显减少(P＜0.05);miR-7转染组EGFR、CyclinD1、CyclinE、CDK2、CDK4和CDK6蛋白表达水平均有不同程度下降(P＜0.05),其中EGFR、CyclinD1、CDK4和CDK6的降低程度更为显著(P＜0.01),P16水平显著升高(P＜0.01),P21和P27的表达没有明显变化(P＞0.05).结论 miR-7可能通过有效沉默癌基因EGFR的表达途径,抑制细胞周期G1期调节蛋白复合体CyclinD1/CDK4/6的活性,并接受P16的协同作用,共同阻遏胶质瘤由G1期向S期转化,进而抑制肿瘤增殖;miR-7有望成为一种新的胶质瘤治疗候选药物.     

垂体生长激素腺瘤细胞表皮生长因子受体的表达

目的了解大鼠垂体生长激素腺瘤(GH3)细胞的表皮生长因子受体(EGFR)的表达情况.方法应用免疫组化的方法检测GH3细胞及阴性对照U-2OS细胞和阳性对照HO8910PM细胞的EGFR表达情况.结果 GH3细胞和HO8910PM细胞明显黄染,U-2OS细胞无明显染色;GH3、U-2OS和HO8910细胞表达EGFR的阳性细胞率分别为90.57%、4.84%和93.33%.结论垂体生长激素腺瘤细胞中存在着广泛的EGFR表达,而且具有较高的表达强度.     

人脑胶质瘤的表皮生长因子基因表达

采用Ｎｏｒｔｈｅｒｎ杂交和斑点杂交的方法检测了５０例胶质瘤、４个恶性胶质瘤体外细胞系和７例正常脑组织表皮生长因子（ＥＧＦ）ｍＲＮＡ表达水平。结果显示：上述组织或细胞都可表达５．０ｋｂ的ＥＧＦｍＲＮＡ片断，高恶度胶质瘤较低恶度胶质瘤和正常脑组织ＥＧＦｍＲＮＡ表达水平增高，低恶度胶质瘤与正常脑组织的表达无显著差异。在５０例胶质瘤中２９例（５８％）ＥＧＦｍＲＮＡ过表达，其中多见于恶性胶质瘤，ＥＧＦｍＲＮＡ表达与肿瘤的分级呈正相关。提示胶质瘤可以合成ＥＧＦ，在胶质瘤的发生发展过程中，ＥＧＦ参与或促进了其恶性进展。     

Growth effects of epidermal growth factor (EGF) and a monoclonal antibody against the EGF receptor on four glioma cell lines

Summary Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis and cell division in normal glia. At least half of malignant human gliomas (MHG) express high levels of the EGF receptor (EGFR), which are above those detected in normal brain. The demonstration that antibodies against the EGFR inhibit the growth of squamous cell carcinoma line A-431, with large numbers of EGFR, in vitro and in vivo raises the possibility that these agents could be used therapeutically against malignant human gliomas either alone or conjugated to other agents. We have measured the growth effects of EGF and an anti-EGFR monoclonal antibody, 528 (Ab-528), on four well-characterized human malignant glioma cell lines, D-263 MG, D-247 MG, U-343 MGa Cl 26, and D-37 MG, with 2.9×10⁴, 1.5×10⁵, 8.6×10⁵ and 1.59×10⁶ EGFRs per cell, respectively. EGF significantly increased cell number in D-263 MG and D-37 MG by 65% and 74%, respectively, had no effect on D-247 MG, and significantly decreased cell number in U-343 MGa Cl 26 by 39%. U-343 MGa Cl 26 growth was inhibited 19% by Ab-528, but Ab-528 had no effect on growth of the other MHG lines. Ab-528 significantly inhibited all EGF-mediated growth effects. These studies demonstrate that, although Ab-528 alone has little antiproliferative activity on MGH, it successfully competes with EGF to reduce the biological effects of EGF-EGFR binding. Therefore, this antibody could potentially be used to target radioisotopes to MHG via the EGFR for diagnosis and therapy.Supported by Grants CA-11898, NS-20023, CA-43722, and the Association for Brain Tumor Research (MHW, PAH)     

髓母细胞瘤中表皮生长因子受体的表达及基因扩增分析

目的：研究表皮生长因子受体在髓母细胞瘤中的蛋白表达及基因水平的扩增情况，从而分析二关系。方法：对48例髓母细胞瘤标本进行免疫组化染色，同时进行差异多聚酶链反应，放射自显影后根据其信号强弱来判断基因的扩增情况。结果：48例髓母细胞瘤中有7例表皮生长因子受体呈阳性表达（14．58％），其中成人6例，儿童1例；差异多聚酶链反应未显示表皮生长因子受体有基因水平的异常扩增。结论：表皮生长因子受体蛋白的过度表达存在于髓母细胞瘤，尤以成人为主，但其机制可能与基因的异常扩增无关。     

Amplification of the epidermal growth factor receptor in astrocytic tumours by chromogenic in situ hybridization: association with clinicopathological features and patient survival

Chromogenic in situ hybridization (CISH) was used to detect amplification of the epidermal growth factor receptor (EGFR) gene in tissue microarrays of tumours derived from 287 patients with grade II-IV diffuse astrocytomas. Amplification was found in 32% of the tumours with a highly significant association with histological grade (4% in grade II, 21% in grade III and 39% in grade IV; P < 0.001). Amplification of the EGFR gene was more common in primary than in secondary glioblastomas (41%vs. 16%, P = 0.033). Overexpression of EGFR mRNA and protein (wild-type and vIII variant) was found to correlate with EGFR gene amplification (P = 0.028, P = 0.035 and P = 0.014 respectively), but wild-type EGFR protein was also frequently overexpressed in tumours without EGFR gene amplification. Patients with older age (P < 0.001) and tumours with lack of p53 overexpression (P = 0.03) and higher apoptosis rate (P < 0.001) had significantly more EGFR gene amplifications than their counterparts. No such correlation with apoptosis was found in glioblastomas. The survival of patients with EGFR gene-amplified grade III tumours was significantly shorter than in those with grade III non-amplified tumours (P = 0.03). No such difference was noted in glioblastomas (grade IV tumours). Our data verify the central role of EGFR in the pathobiology of astrocytic tumours, and highlight the advantages of CISH as a simple and practical assay to screen for EGFR gene amplification in astrocytic tumours.     

靶向表皮生长因子受体的shRNA抑制U251胶质瘤生长的实验研究

目的探讨靶向表皮生长因子受体(epidermal growth factor receptor,EGFR)的RNA干涉(RNAi)技术抑制恶性胶质瘤体外细胞系U251的EGFR表达后,在体内外对U251细胞生长抑制作用。方法在人EGFR开放阅读框的细胞内区酪氨酸激酶结构域选择1个siRNA靶序列、进行短发夹RNA(shRNA)表达载体psiRNA-2400的构建,并以含有随机序列的psiRNA-scr表达质粒为对照进行了脂质体介导的U251人脑恶性胶质瘤细胞系表达。应用RT-PCR检测EGFR表达水平,应用MTT法、流式细胞术和Matrigel基质生长实验评价肿瘤细胞转染前后的生物学行为。进一步应用裸鼠皮下荷瘤模型观察脂质体介导shRNA基因治疗对U251细胞生长抑制作用。对肿瘤组织应用免疫组化的方法进行EGFR、PCNA和GFAP表达比较。结果脂质体介导、靶向EGFR的shRNA可显著抑制U251细胞ECFR表达,MTT法分析显示psiRNA-2400转染组细胞生长抑制率为68%;与对照组和psiRNA-scr转染组比较,细胞周期分析结果表明psiRNA-2400转染组G_0～G_1期细胞数没有明显变化,进入S期的细胞数较对照组减少了12.7%～13%,而进入G_2期细胞则增加了10.5%～11.7%。Matrigel基质生长实验显示对照组和psiRNA-scr转染组细胞呈正常形态贴壁生长,而psiRNA-2400转染组细胞不能贴壁生长,呈团块状簇集生长。裸鼠皮下荷瘤模型实验显示psiRNA-2400显著抑制皮下肿瘤生长,组织病理学分析显示治疗组EGFR表达下降、PCNA标记指数降低而GFAP表达上调。结论靶向EGFR的RNAi技术可以显著抑制U251细胞的EGFR表达,在体内外对U251细胞生长均产生明显抑制作用。因此,shRNA表达质粒介导的基因治疗可以成为胶质瘤靶向性EGFR治疗的新策略。     

Amplification of the epidermal growth factor receptor gene in glioblastoma: An analysis of the relationship between genotype and phenotype by CISH method

We examined epidermal growth factor receptor (EGFR) overexpression and EGFR gene amplification using immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) in 109 glioblastomas, including 98 primary glioblastomas and 11 secondary glioblastomas. EGFR overexpression and EGFR gene amplification were found in 33% and 24% of glioblastoma, respectively, and all of those cases were primary glioblastoma. Large ischemic necrosis was significantly more frequent in primary glioblastomas than in secondary glioblastomas (54% vs. 18%), but pseudopalisading necrosis was not (65% vs. 54%). EGFR gene amplification was detected significantly more frequently in cases with both types of necrosis. Although glioblastomas with EGFR gene amplification invariably exhibited EGFR overexpression at the level of the whole tumor, tumor cells with EGFR gene amplification did not always show EGFR overexpression at the level of individual tumor cells. Cases of “strong” EGFR overexpression on IHC could be regarded as having EGFR gene amplification, and cases without EGFR overexpression could not. Cases of “weak” EGFR overexpression should be tested with CISH to confirm the presence of EGFR gene amplification. We found that 54% of glioblastomas with EGFR gene amplification were composed of areas with and without EGFR gene amplification; however, there were no obvious differences in morphology between tumor cells with and without EGFR gene amplification. Although small cell architecture might be associated with EGFR gene amplification at the level of the whole tumor, it did not always suggest amplification of the EGFR gene at the level of individual tumor cells. In one case, it seemed to suggest that a clone with EGFR gene amplification may arise in pre‐existing tumor tissue and extend into the surrounding area. In cases of overall EGFR amplification, CISH would be a useful tool to decide the tumor border in areas infiltrated by tumor cells.     

Distribution of epidermal growth factor receptor gene amplification in brain tumours and correlation to prognosis

In 75 gliomas and 31 meningiomas, mutations at the epidermal growth factor receptor (EGFR) gene locus were restricted to gliomas. The ligands of this receptor, epidermal growth factor and transforming growth factor alpha, lacked quantitative changes at their loci in gliomas and meningiomas. EGFR gene amplification occurred in astrocytomas, oligodendrogliomas, ependymomas and glioblastomas. The frequency of this mutation significantly increased with the malignancy grade and the patient's age. Especially in glioblastomas of individuals aged over 64 years, EGFR gene mutations were observed without chromosome-10-specific allele losses. This finding contradicts the hypothesis that deletion of one entire chromosome 10 regularly precedes EGFR gene amplification in primary glioblastomas of patients aged over 50 years. It was found that most individuals whose gliomas carry an EGFR gene mutation have a poor prognosis, comparable to that of glioblastoma patients even when the tumour is graded as benign.     

Low serum levels of brain-derived neurotrophic factor and epidermal growth factor in patients with chronic schizophrenia

Neurotrophic factors (NFs) play a pivotal role in the development of the central nervous system. They are thus also suspected of being involved in the etiology of schizophrenia. Previous studies reported a decreased level of serum brain-derived neurotrophic factor (BDNF) in schizophrenia, whereas the association of epidermal growth factor (EGF) with this illness remains controversial. Using a two-site enzyme immunoassay, we conducted the simultaneous measurement of serum BDNF and EGF levels in a group of patients with chronic schizophrenia (N = 74) and a group of normal controls matched in age, body mass index, smoking habit and sex (N = 87). We found that, compared to normal controls, patients with chronic schizophrenia exhibited lower serum levels of both BDNF and EGF across all ages examined (21–59 years). The serum levels of BDNF and EGF were negatively correlated in the controls (r = − 0.387, P = 0.0002) but not in the patients. Clinical parameters such as duration of illness and psychiatric rating scale also showed no robust correlations with the NF levels. Collectively, these results suggest that pervasive, abnormal signaling of NFs underlies the pathophysiology of chronic schizophrenia.     

Visualization of growth factor receptor sites in rat forebrain

It is now known that various growth factors may also act in the central nervous system. Among them, it has recently been shown that epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) may possess trophic effects in the mammalian brain. We report here on the respective autoradiographic distribution of [125I]EGF and [125I]IGF-I receptor binding sites in the rat brain, both during ontogeny and in adulthood. It appears that [125I]EGF sites are mostly found in the rat forebrain during brain development. On the other hand, [125I]IGF-I sites are more widely distributed both during ontogeny and in adulthood. These results reveal the plasticity of the expression of EGF and IGF-I receptor sites in the mammalian brain. This could be relevant for the respective role of these two growth factors in the development and maintenance of neuronal function.     

Influence of epidermal growth factor on long-term potentiation in the hippocampal slice

Rat hippocampal slices were perfused with epidermal growth factor (EGF) at a concentration of 10(-8) M. EGF is a well known mitogen which exhibits neurotrophic action on neurons of the CNS. During extra- and intracellular recordings in the pyramidal cell body layer of the CA1-region no influence of EGF on evoked potentials was seen if single-pulse or paired-pulse stimulation was used. Furthermore EGF has no influence on the resting membrane potential of the cells investigated. However after tetanic stimulation a significant increase in the magnitude of long-term potentiation was observed. Therefore it is concluded, that EGF might be involved in modulation of neuronal plasticity.