p185蛋白及其抗体的研究进展

描述了p185在信号转导过程中的功能,讨论了抗p185抗体的蛋白质工程及它们在肿瘤治疗中的应用.     

基质金属蛋白酶-2在酵母系统中表达的初步研究

基质金属蛋白酶-2（明胶酶A）在肿瘤侵袭和转移中具有重要作用,为进一步研究其作用机制,在酵母系统中表达了金属蛋白酶-2通过PCR方法获得明胶酶A的表达序列,酶切和测序结果证明序列正确后,构建明胶酶A的表达载体pPIC9/GelA,电击法转化酵母得到阳性克隆.明胶酶谱分析说明表达产物具有明胶底物特异性,SDS-聚丙烯酰胺凝胶电泳表明,表达产物的分子质量为66 ku.     

血清基质金属蛋白酶-2与冠心病的相关性分析

目的:探讨冠心病患者血清基质金属蛋白酶-2(MMP-2)的表达水平及与冠状动脉内径狭窄程度的相关关系.方法:通过ELISA法检测冠心病患者(n=24)和正常对照组(n=25)血清MMP-2水平,并分析冠心病患者MMP-2水平与冠状动脉内径狭窄程度的相关关系.结果:冠心病患者和健康对照组血清MMP-2表达水平依次为(48.92±14.86)ng/mL和(25.77±8.82)ng/mL,差异具有显著性(t=6.663,P=0.001);冠心病患者的MMP-2水平与冠状动脉内径狭窄程度之间存在明显相关关系(P＜0.01).结论:冠心病患者血清的MMP-2水平有明显的升高,并且MMP-2水平与冠状动脉内径狭窄程度关系密切,可能参与了冠心病的发生发展过程.     

人静脉移植物基质金属蛋白酶-2和波型蛋白的表达

目的:探讨人静脉移植物基质金属蛋白酶-2(Matrix Metalloproteinase-2,MMP-2)和波型蛋白(Vimentin)的表达情况。方法:应用免疫荧光组织化学技术对30个静脉移植物再塞标本中MMP-2、Vimentin的表达进行了检测,用激光共聚焦显微镜拍片,图片用Silicon Graphics Octane进行处理。结果:在正常静脉血管,有少量MMP-2的表达;Vimentin的表达较弱。在静脉移植的血管, MMP-2的表达显著增加,是正常血管的3．5倍;Vimentin(波形蛋白)在中膜和内膜的表达明显增多。另外,统计分析表明MMP-2在新内膜的表达均显著高于在中膜的表达(P＜0．01)。结论:在静脉移植物的新内膜,MMP-2和Vimentin的表达均上调,提示这些重要分子在新内膜的形成和静脉再狭窄的病理过程中具有重要的作用。     

人基质金属蛋白酶-2的表达、纯化和性质鉴定

PCR方法扩增人基质金属蛋白酶 2 (MMP 2 )不含信号肽的表达序列 ,酶切和测序鉴定正确后 ,构建酵母重组表达质粒pPIC9 MMP 2 ,电击法转化毕赤酵母 (Pichiapastoris)细胞得到阳性克隆 ,甲醇诱导获得含大量基质金属蛋白酶 2的培养上清 ,经SephacrylS 2 0 0纯化后 ,纯度达到电泳纯。明胶酶谱和SDS PAGE分析说明重组MMP 2能够降解明胶和IV型胶原 ,表明重组蛋白具有与天然MMP 2相似的底物特异性。糖基化分析和SDS PAGE表明 ,表达产物的分子量约为 5 0kD ,重组MMP 2的C 末段可能发生了降解。     

基质金属蛋白酶2抑制剂的筛选及鉴定

以原核表达的具有明胶水解活性的人基质金属蛋白酶 2的催化区 (MCD)为靶标 ,筛选噬菌体随机环七肽库和十二肽库 .找到 6种与MCD特异结合的小肽 ,将 6种小肽基因分别与GST表达质粒重组 ,进行GST融合表达 ,制备融合蛋白 .采用Glutathione Sepharose 4B亲和层析法纯化融合蛋白 ,通过酶抑制实验、体外侵袭实验检测融合蛋白的活性 .结果表明 ,GST C71能够抑制MCD水解 β酪蛋白的活性 ,并且对人纤维肉瘤细胞HT10 80的体外侵袭有明显的抑制作用     

窖蛋白-1、基质金属蛋白酶-2与乳腺肿瘤的侵袭和转移

窖蛋白(caveolin)是分子量为21～24 kD的整合膜蛋白,是胞膜窖(caveolae)的标志性结构分子,其家族成员窖蛋白-1(caveolin-1,Cav-1)参与细胞内许多重要的生命活动.近来研究发现,窖蛋白-1与乳腺上皮细胞转化及乳腺癌的发生密切相关.基质金属蛋白酶(matrix metalloproteinases,MMPs)是基质降解代谢的主要酶类,几乎能降解细胞外基质和基底膜的所有成分,其家族成员明胶酶A(MMP-2)在乳腺癌的浸润和转移过程中起重要作用.新近发现,窖蛋白-1与基质金属蛋白酶-2在胞膜窖中共定位,窖蛋白-1通过抑制基质金属蛋白酶-2的激活来抑制乳腺癌的侵袭和转移,起到肿瘤抑制因子的作用.本文对窖蛋白-1与基质金属蛋白酶-2各自在乳腺肿瘤侵袭转移中的作用及两者关系的研究进行综述.     

层粘连蛋白及基质金属蛋白酶-2在人脑胶质瘤侵袭过程中的相关研究

目的研究层粘连蛋白(Laminin, LN)及基质金属蛋白酶-2(Matrix metalloproteinases-2,MMP-2)在人脑胶质瘤侵袭中的相互关系,探索人脑胶质瘤侵袭转移的机制.方法用免疫组织化学SP法检测LN和MMP-2在27例人脑胶质瘤、6例颅内转移瘤及5例正常脑组织的表达情况.结果 1)LN在高度恶性的胶质瘤中的表达显著高于颅内转移瘤及其它各组(9.584±6.448,P<0.05), MMP-2的表达随着胶质瘤恶性程度的增高而增加(P<0.05),其在颅内转移瘤中的表达与高度恶性组无显著差异性(P>0.05);2)MMP-2/LN比值在颅内转移瘤中显著高于其它各组(0.120±0.069, P<0.05),其它各组间无统计学差异,该值相对恒定;3)LN与MMP-2在上述组织中的表达呈正相关( r＝0.605,P=0.0002<0.01).结论在人脑胶质瘤中,MMP-2表达增加使得胶质瘤细胞呈现颅内浸润,而LN的表达随MMP-2表达的增加而增加却阻遏了胶质瘤细胞向颅外转移,两者的相互作用使得胶质瘤既表现出颅内广泛的侵袭性,又不转移到颅外.     

组织基质金属蛋白酶抑制剂-2的表达纯化及活性鉴定

组织基质金属蛋白酶抑制剂-2(TIMP-2)抑制肿瘤迁移及侵袭。文中以人TIMP-2为研究对象,探索人TIMP-2蛋白的原核表达特征,并进行纯化及活性鉴定。以人肺癌A549细胞的总RNA反转录得到的cDNA为模板,克隆人TIMP-2基因,构建pET28a重组表达载体;经酶切检测和测序分析的重组表达载体pET28a-TIMP-2转入大肠杆菌Escherichia coli BL21(DE3)中,利用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,并对表达条件进行优化。经镍亲和柱纯化后,用Westernblotting法鉴定融合蛋白His-TIMP-2,并用明胶酶谱法检测融合蛋白的活性。研究发现融合蛋白His-TIMP-2在E.coliBL21(DE3)中以包涵体的形式存在;在一定范围内,IPTG浓度对His-TIMP-2的表达量没有显著影响;而在该表达系统中,诱导温度和时间是关键参数,His-TIMP-2的表达量随诱导温度升高而增加;纯化并复性后的融合蛋白His-TIMP-2能有效抑制人肺癌A549细胞表达的基质金属蛋白酶的活性。具有活性的融合蛋白的获得为后续深入研究人TIMP-2的功...     

糖尿病肾病患者血浆基质金属蛋白酶-2和同型半胱氨酸水平及其临床意义

目的:探讨糖尿病肾病患者血浆基质金属蛋白酶-2和同型半胱氨酸水平及其临床意义。方法:选取糖尿病患者71例,其中并发糖尿病肾病组(n=35)和未并发肾病组(n=36),同时选取健康人群40例为对照组。检测患者MMP-2、Hcy、MAU及Scr水平。结果:糖尿病肾病组、糖尿病无肾病组血浆MMP-2、Hcy及MAU水平均显著高于健康对照组(P0.01);与糖尿病无肾病组比较,糖尿病肾病组患者MMP-2、Hcy及MAU水平较高(P0.05);糖尿病肾病组患者的血浆SCr水平明显高于其他两组(P0.05)。糖尿病肾病患者血浆MMP-2与MAU呈显著正相关(r=0.522,P0.05),与Hcy呈显著负相关(r=-0.007,P0.05),与Scr不相关(P0.05);Hcy与MAU(r=0.012,P0.05)、Scr均(r=0.031,P0.05)呈正相关。结论:糖尿病肾病患者MMP-2水平降低,Hcy水平升高,有望作为糖尿病肾病患者的诊断和预后预测参考指标。     

HER-2/neu基因高表达及靶向治疗

HER-2/neu癌基因在许多肿瘤,如乳腺癌、卵巢癌、非小细胞肺癌等肿瘤中高表达,在肿瘤的发生与发展中起重要作用,与肿瘤的转化、转移、复发、预后差、患者生存期缩短有关。HER-2/neu在乳腺癌过度表达率约为20%～30%,编码蛋白P185HER2属生长因子受体家族,抗P185HER2单克隆抗体(Herceptin)作为靶向药物已临床应用治疗HER2/neu高表达乳腺癌。     

Autoantibody to p185 erbB2/neu oncoprotein by vaccination with xenogenic DNA

 The passive transfer of antibodies and vaccination procedures against p185, the erbB2/neu oncoprotein, are approaches being explored for treatment of human breast cancer. We now report the possibility of using the erbB2/neu gene as an immunogen. This study demonstrates that intramuscular or intradermal injections of rat neuNT full-length DNA into mice generate anti-p185 autoantibodies. Anti-p185 polyclonals were also shown to bind the homologous human receptor ErbB2 and to stain specimens of breast adenocarcinoma from both neu-transgenic mice and humans. Further, in vitro assays demonstrated that anti-p185 IgG (probably dependent on CD4⁺ Th1) were able to inhibit human SKBR3 tumour cell growth and to mediate their lysis by natural killer cells. The continuous presence of circulating neu autoantibodies in mice did not cause any discernible toxic effects on normal tissues expressing low levels of self-antigen, even after 1 year. Received: 29 August 1996 / Accepted: 31 October 1996     

HER-2/neu raises SHP-2, stops IFN-gamma anti-proliferation in bladder cancer

Gene amplification or HER-2/neu protein overexpression signals a poor outcome for bladder cancer patients. We investigated the anti-proliferative effect of IFN-gamma in HER-2/neu-transfected human bladder cancer cells (TCC-N5 and TCC-N10). The cells continued growing after IFN-gamma stimulation but did not activate the Janus kinase (Jak)/Stat pathway. We found Jak/Stat protein phosphatase in TCC-N5 and TCC-N10 cells with upregulated Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2). After the cells had been treated with AG825, a HER-2/neu-specific inhibitor, SHP-2 expression declined, and Jak2/Stat1 reactivated. Similar results were reported in a mouse bladder cancer cell line, MBT2, with constitutive HER-2/neu overexpression. Further, AG825 pretreatment restored the anti-proliferation activity of IFN-gamma in TCC-N5 and TCC-N10 cells. Therefore, the suppression of IFN-gamma signaling in HER-2/neu-overexpressing bladder cancer cells might be due to SHP-2 upregulation. The regulation of SHP-2 by HER-2/neu provides a new target for blocking the HER-2/neu oncogenic pathway.     

Interaction of the neu/p185 and EGF receptor tyrosine kinases: Implications for cellular transformation and tumor therapy

Growth factor receptors such as the epidermal growth factor receptor (EGFR) and the p185^c-neu protein serve vital roles in the transduction of differentiation, developmental, or mitogenic signaling within normal cells. Two methods of analysis suggest that the inappropriately high expression of either protein tyrosine kinase promotes malignant transformation. First, data from in vitro experiments indicate that overexpression of either EGFR or p185^c-neu (or the human homolog c-erbB-2) transforms cell-lines. Second, analysis of primary tumors and tumor cell-lines derived from many epithelial tissues (breast, stomach, ovary, and pancreas) show growth factor receptor gene amplification and elevated protein levels. The physical and functional interaction of p185^c-neu and EGFR leads to the formation of a highly active, heterodimeric tyrosine kinase complex which synergistically activates cellular transformation. Anti-receptor antibodies have shown potential utility for the down modulation of these cell-surface proteins and suppression of the malignant phenotype. Design of organic antibody “mimetics” based on the structure of antireceptor antibodies may provide useful therapies and biological reagents to affect growth factor receptor function.     

Immunogenic HER-2/neu peptides as tumor vaccines

During the last decade, a large number of tumor-associated antigens (TAA) have been identified, which can be recognized by T cells. This has led to renewed interest in the use of active immunization as a modality for the treatment of cancer. HER-2/neu is a 185-KDa receptor-like glycoprotein that is overexpressed by a variety of tumors including breast, ovarian, lung, prostate and colorectal carcinomata. Several immunogenic HER-2/neu peptides recognized by cytotoxic T lymphocytes (CTL) or helper T lymphocytes (TH) have been identified thus far. Patients with HER-2/neu over-expressing cancers exhibit increased frequencies of peripheral blood T cells recognizing immunogenic HER-2/neu peptides. Various protocols for generating T cell-mediated immune responses specific for HER-2/neu peptides have been examined in pre-clinical models or in clinical trials. Vaccination studies in animals utilizing HER-2/neu peptides have been successful in eliminating tumor growth. In humans, however, although immunological responses have been detected against the peptides used for vaccination, no clinical responses have been described. Because HER-2/neu is a self-antigen, functional immune responses against it may be limited through tolerance mechanisms. Therefore, it would be interesting to determine whether abrogation of tolerance to HER-2/neu using appropriate adjuvants and/or peptide analogs may lead to the development of immune responses to HER-2/neu epitopes that can be of relevance to cancer immunotherapy. Vaccine preparations containing mixtures of HER-2/neu peptides and peptide from other tumor-related antigens might also enhance efficacy of therapeutic vaccination. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004     

Downregulation of wild-type p53 protein by HER-2／neu mediated PI3K pathway activation in human breast cancer cells： its effect on cell proliferation and implication for therapy

Overexpression and activation of HER-2/neu (also known as c-erbB-2), a proto-oncogene, was found in about 30% of human breast cancers, promoting cancer growth and making cancer cells resistant to chemo- and radio-therapy.Wild-type p53 is crucial in regulating cell growth and apoptosis and is found to be mutated or deleted in 60-70% of human cancers. And some cancers with a wild-type p53 do not have normal p53 function, suggesting that it is implicated in a complex process regulated by many factors. In the present study, we showed that the overexpression of HER-2/neu could decrease the amount of wild-type p53 protein via activating PI3K pathway, as well as inducing MDM2 nuclear translocation in MCF7 human breast cancer cells. Blockage of PI3K pathway with its specific inhibitor LY294002 caused G1-S phase arrest, decreased cell growth rate and increased chemo- and radio-therapeutic sensitivity in MCF7 cells expressing wild-type p53. However, it did not increase the sensitivity to adriamycin in MDA-MB-453 breast cancer cells containing mutant p53. Our study indicates that blocking PI3K pathway activation mediated by HER-2/neu overexpression may be useful in the treatment of breast tumors with HER-2/neu overexpression and wild-type p53.     

HER-2/neu peptide specificity in the recognition of HLA-A2 by natural killer cells

Although natural killer (NK) cells have been described as non-MHC-restricted, new evidence suggests that NK activity can be either up- or down-regulated after interaction with the peptide–MHC-class-I complex expressed on target cells. However, the epitope(s) recognized by NK cells have remained ill-defined. We investigated NK cell recognition of synthetic peptides representing a portion of a self-protein encoded by the HER-2/neu (HER-2) proto-oncogene and presented by HLA-A2. HER-2 nonapeptides C85, E89, and E75 were found partially to protect T2 targets from lysis by freshly isolated and interleukin-2(IL-2)-activated NK cells (either HLA-A2⁺ or A2⁻). This inhibition was not solely due to changes in the level of HLA-A2 expression or conformation of serological HLA-A2 epitopes. Using single-amino-acid variants at position 1 (P1) of two HER-2 peptides, we observed that protection of targets was dependent on the sequence and the side-chain. These results suggest similarities in the mechanism of target recognition by NK and T cells. This information may be important for understanding the mechanisms of tumor escape from immunosurveillance and could help explain the aggressiveness of HER-2-overexpressing tumor cells. Received: 16 March 1999 / Accepted: 3 June 1999     

Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer

The HER-2/neu transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target for breast cancer. Accurate determination of HER-2/neu status is a prerequisite for selecting breast tumors for HER-2/neu immunotherapy or for taxan based chemotherapy. Unfortunately, there is no consensus concerning how this determination should be reached. We compared assessment of HER-2/neu status using Multiplex ligation-dependent probe amplification (MLPA) and immunohistochemistry (IHC). The patient group comprised 60 Indonesian breast cancers patients. IHC was performed on paraffin sections using the CB11 antibody from Novocastra. Results were scored according to the Hercept test. For MLPA, DNA was extracted from frozen samples, PCR amplified with a probe set containing three hemi-primer sets for the HER-2 locus and another nine control probes spread over chromosome 17 and other chromosomes, and analyzed on a gene scanner. A ratio above two for at least two HER-2 locus probes compared to the control probes was regarded as amplification. IHC for HER-2/neu was negative in 36 cases, and 24 cases (40%) showed expression. Seven, eight and nine of the latter cases were 1+, 2+ and 3+ positive, respectively. Forty-seven cases showed no amplification by MLPA, and 13 cases (22%) were amplified. Comparison of IHC and MPLA showed that none of the 36 IHC-negative or seven IHC 1+ cases was amplified. Five of the eight (63%) 2+ cases were amplified, and eight of nine (89%) of the IHC 3+ tumors showed gene amplification by MLPA assay. For HER-2/neu, there is a good correlation between gene amplification detected by MLPA and overexpression by IHC in invasive breast cancer. It appears that MLPA can detect the HER-2 amplified cases in the IHC 2+ class. Because MLPA is quick and inexpensive, it is an attractive method for detecting HER-2/neu amplification in daily laboratory practice.     

Chromogenic in situ hybridization analysis of HER-2/neu status in cytological samples of breast carcinoma

The current use of humanized monoclonal antibody trastuzumab for the treatment of patients with metastatic breast cancer has made evaluation of HER-2/neu status an important clinical issue. Chromogenic in situ hybridization (CISH), in which the DNA probe is detected with an immunohistochemistry (IHC)-like peroxidase reaction, has been recently developed for the assessment of HER-2/neu status in formalin-fixed breast cancer specimens. We have applied the technique of dual-colour CISH using HER-2/neu and chromosome 17 centromere probes in 27 cytological smears, and these cytological samples were obtained from scrapings of fresh breast tumours. We also investigated HER-2/neu amplification and protein overexpression in the corresponding surgical tissues by CISH and IHC using the monoclonal antibody CB11. Of the 27 cytological cases, HER-2/neu gene amplification was observed in nine cases that were positive cases (2+ and 3+) for IHC. Among the 13 IHC positive cases (2+ and 3+), four of them showed no gene amplification. Identical results for the CISH technique were obtained in the matched surgical samples. The scrape samples from fresh breast tumour offer a monolayer cell population that is especially suitable for CISH. This study has shown that the cytological smear might be a good alternative for the CISH test.     

Danger signals and nonself entity of tumor antigen are both required for eliciting effective immune responses against HER-2/neu positive mammary carcinoma: implications for vaccine design

Using parental FVB mice and their neu transgenic counterparts, FVBN202, we showed for the first time that dangerous hyperplasia of mammary epithelial cells coincided with breaking immunological tolerance to the neu "self" tumor antigen, though such immune responses failed to prevent formation of spontaneous neu-overexpressing mammary carcinoma (MMC) or reject transplanted MMC in FVBN202 mice. On the other hand, neu-specific immune responses appeared to be effective against MMC in parental FVB mice because of the fact that rat neu protein was seen as "nonself" antigen in these animals and the protein was dangerously overexpressed in MMC. Interestingly, low/intermediate expression of the neu "nonself" protein in tumors induced immune responses but such immune responses failed to reject the tumor in FVB mice. Our results showed that self-nonself (SNS) entity of a tumor antigen or danger signal alone, while may equally induce an antigen-specific immune response, will not warrant the efficacy of immune responses against tumors. On the other hand, entity of antigen in the context of dangerous conditions, i.e. abnormal/dangerous overexpression of the neu nonself protein, will warrant effective anti-tumor immune responses in FVB mice. This unified "danger-SNS" model suggests focusing on identification of naturally processed cryptic or mutated epitopes, which are considered semi-nonself by the host immune system, along with novel dangerous adjuvant in vaccine design.