增殖病毒对肿瘤的治疗作用

目的：探讨ＨＥＲ２／ｎｅｕ原癌基因胞外区片段ＤＮＡ免疫诱导的特异性细胞免疫应答及其在体内的抗瘤效应。方法：克隆并构建人ＨＥＲ２／ｎｅｕ原癌基因及其胞外区片段表达载体,分别转染ＳＰ２／０细胞以制备杀伤靶细胞。将质粒ＤＮＡ免疫小鼠,观察其诱导的细胞免疫应答,同时分析免疫动物体内抗瘤效应。结果：体外获得稳定表达ＨＥＲ２／ｎｅｕ基因的ＳＰ２／０靶细胞。目的基因ＤＮＡ免疫后,免疫鼠脾细胞在体外可检测到特异性杀伤作用,体内肿瘤细胞接种后可发现肿瘤结节出现时间延迟,肿瘤生长缓慢。结论： ＨＥＲ２／ｎｅｕ原癌基因胞外区片段ＤＮＡ免疫可诱导出特异细胞免疫应答,并具有一定抗瘤效应。     

分次小剂量放射免疫治疗预防和控制荷瘤小鼠肺转移的实验研究 *

目的：探讨ＨＥＲ２／ｎｅｎ的癌基因胞外区片段ＤＮＡ免疫诱导的特异性细胞免疫应答及其在体内的抗瘤效应。方法：克隆并构建人ＨＥＲ２／ｎｅｎ原癌基因及其胞外区片表达载体,分别转染ＳＰ２／０细胞以制备样伤靶细胞。将质粒ＤＮＡ免疫小鼠,观察其诱导的细胞免疫应答,同时分析免疫动物体内抗瘤效应。结果：体外获得稳定表达ＨＥＲ２／ｎｅｎ基因的ＳＰ２／０靶细胞。目的基因ＤＮＡ免疫后,免疫鼠脾细胞在体外可检测到特异性杀     

HER2/neu肽诱导BALB/c小鼠产生特异性CTL及其抑瘤作用的研究

目的：探讨来源于ＨＥＲ２／ｎｅｕ原癌蛋白的多肽诱导特异性细胞免疫应答及其在体内的抗癌效应。方法：利用合成的２个具有小鼠Ｈ－２Ｋ＾ｄ分子结合基序的ＨＥＲ２／ｎｅｕ肽作为肿瘤排斥抗原,在体外刺激小鼠淋巴细胞以及皮下免疫小鼠,观察淋巴细胞的增殖能力、ＣＴＬ的杀伤活性以及ＨＥＲ２／ｎｅｕ肽的抑瘤作用。结果：ＨＥＲ２／ｎｅｕ肽在体内和体外对ＢＡＬＢ／ｃ小鼠淋巴细胞的增殖都有显显的促进作用。ＨＥＲ２／ｎｅｕ肽免疫ＢＡＬＢ／ｃ小鼠后,可以从小鼠淋巴细胞中诱导出肽特异性ＣＴＬ,这些ＣＴＬ可以特异地杀伤ＨＥＲ２／ｎｅｕ阳性ＳＰ２／０细胞,而对ＨＥＲ２／ｎｅｕ阴性ＳＰ２／０细胞却无明显的杀伤活性。ＳＰ２／０ＨＥＲ２细胞在ＨＥＲ２／ｎｅｕ肽免疫小鼠体内的生长受到抑制。结论：研究结果表明ＨＥＲ２／ｎｅｕ肽可以起到一定的抑瘤保护作用,提     

ＨＥＲ-２／ｎｅｕ与胃癌临床病理及预后的相关性研究

目的　研究ＨＥＲ-２／ｎｅｕ基因蛋白表达与胃癌临床病理特征以及预后的相关性,探讨该基因在胃癌中作为预后因素的价值。方法　应用免疫组织化学染色法对２２９例胃癌组织芯片进行ＨＥＲ-２／ｎｅｕ基因蛋白表达的检测,并将检测结果与患者的临床病理特征以及生存随访结果进行统计分析。结果　２２９例胃癌组织中ＨＥＲ-２／ｎｅｕ基因蛋白表达阳性率为１４.８５％,其表达与患者Ｌａｕｒｅｎ分型、组织学分级、肿瘤大小、淋巴结转移和生存时间相关（Ｐ＜０.０５）;Ｌｏｇ-ｒａｎｋ单因素分析显示,ＨＥＲ-２／ｎｅｕ基因蛋白表达与预后相关（Ｐ＜０.０５）;Ｃｏｘ风险比例模型分析显示,ＨＥＲ-２／ｎｅｕ基因蛋白表达、Ｌａｕｒｅｎ分型、肿瘤大小、脉管浸润和ＴＮＭ分期均为独立预后因素（P＜０.０５）。结论　ＨＥＲ-２／ｎｅｕ基因蛋白表达与胃癌的浸润、转移相关,是胃癌独立的预后因素之一。     

多聚阳离子对非病毒载体转移效率的影响*

目的：制备Ｔ淋巴瘤ＴＣＲＶβ核酸疫苗。方法：利用ＲＴ－ＰＣＲ方法扩增出人Ｔ淋巴瘤细胞Ｊｕｒｋａｔ的ＴＣＲ　Ｖβ基因片段,并将其重组入真核表达载体ｐｃＤＮＡ３。用重组表达载体转染ＳＰ２／０细胞,在ｍＲＮＡ水平上检测其表达。再用ｐｃＤＮＡ３和重组载体分别免疫ＢＡＬＢ／ｃ小鼠,收集抗血清,用免疫组化技术检测抗体产生情况。结果：扩增出ＴＣＲ Ｖβ的基因片段,测序结果证实其序列与已发表的一致。并构建出重组表达载体ｐｃＤＮＡ３－ＴＣＲ Ｖβ。该质粒转染到 ＳＰ２／０细胞,可在ｍＲＮＡ水平上检测到其表达。在用ｐｃＤＮＡ３－ＴＣＲＶβ免疫的小鼠抗血清中检测到抗Ｊｕｒｋａｔ细胞 ＴＣＲ Ｖβ的抗体。免疫组化结果表明,该血清不与表达 ＴＣＲγδ的人Ｔ淋巴瘤细胞发生反应,而与转染该基因的ＳＰ２／０细胞产生强阳性反应。正常小鼠血清与ｐｃＤＮＡ３免疫的小鼠血清与Ｊｕｒｋａｔ细胞不产生显色反应。结论：所制备的Ｔ淋巴瘤ＴＣＶβ核酸疫苗能有效地诱导机体产生体液兔疫反应。     

HER2／neu基因介导的信号传递及其在肿瘤免疫中的作用

ＨＥＲ２／ｎｅｕ是一种跨膜生长因子受体基因，编码的１８５ｋＤ糖蛋白具有肥体酪氨酸激活性，ＨＥＲ２／ｎｅｕ基因的扩增或蛋白的过度表达可通过活化信呈传递系统而诱导的细胞恶变，针对ＨＥＲ２／ｎｅｕ蛋白的单克隆抗体可模拟或阻断配体的生物学功能而发挥促进或抑制肿瘤生长效应。对细胞生长的信号传递系统的深入了解不但有助于肿瘤发生机理的研究，而且为寻找有效的抗肿瘤方法提供了一条新的思路。     

正常子宫内膜、子宫内膜增殖症及子宫内膜癌ras、HER-2/neu癌基因表达的研究

为研究ｒａｓ、ＨＥＲ－２／ｎｅｕ癌基因的表达与子宫内膜癌发生、发展的关系，本文用免疫组化ＡＢＣ法检测了２３例正常子宫内膜、４４例子宫内膜增殖症及１０３例子宫内膜癌组织中ｒａｓ、ＨＥＲ－２／ｎｅｕ癌基因的表达情况。结果表明：在子宫内膜非典型增生中即存在ｒａｓ基因的过度表达，过度表达率为２０％，子宫内膜癌ｒａｓ的过度表达率为１８．４％，且ｒａｓ的过度表达与子宫内膜癌的病理分级、分期及患者的生存率无关，说明ｒａｓ癌基因的异常表达可能与癌形成的早期有关。子宫内膜癌中ＨＥＲ－２／ｎｅｕ的过度表达率为２７．２％，ＨＥＲ－２／ｎｅｕ的过度表达与子宫内膜癌的分期无关，与分级及患者的预后有关，存在ＨＥＲ－２／ｎｅｕ过度表达者的生存率低于无过度表达者，前者的５年生存率为５４．８％，后者为７９．６％，结果提示ＨＥＲ－２／ｎｅｕ的过度表达是判断子宫内膜癌预后的生物学指标。     

甲状腺癌HER—2／neu癌蛋白和mRNA表达的研究

「目的」观察人类甲状腺癌中ＨＥＲ－２／ｎｅｕ癌蛋白和ｍＲＮＡ的表达及其关系。「方法」应用免疫组化和同位素点杂交技术对新鲜冰冻甲状腺组织ＨＥＲ－２／ｎｅｕ癌蛋白和ｍＲＮＡ的表达进行研究。「结果」２１例甲状腺乳头状癌中１４例ＨＥＲ－２ｅｎｕ癌蛋白表达阳性,而其他类型肿瘤和非肿瘤组织均未见ＨＥＲ－２／ｎｅｕ ｍＲＮＡ表达研究显示,乳头状癌和淋巴结转移灶中,ｍＲＮＡ的表达水平明显高于其他肿瘤和非肿瘤组织。本研究还证明石蜡包埋的乳头状癌标本中ＨＥＲ－２／ｎｅｕ癌蛋白表达率明显低于新鲜标本。「结论」ＨＥＲ－２／ｎｅｕ蛋白的表达是甲状腺乳头状癌的一个特征性改变,ＨＥＲ－２ｎｅｕ蛋白的表达与ｍＲＮＡ的表达相一致,ＨＥＲ－２ｎｅｕ蛋白检测的稳定性取决于组织标本的质量。     

抗体导向酶——前药疗法的研究和应用进展

目的：制备Ｔ淋巴瘤ＴＣＲ－Ｖβ核酸疫苗。方法：利用ＲＴ－ＰＣＲ方法扩增出人Ｔ淋巴瘤细胞Ｊｕｒｋａｔ的ＴＣＲ Ｖｐ基因片段,并将其重组人真核表达载体ｐｃＤＮＡ３。用重组表达载体转染ＳＰ２／０细胞,在ｍＲＮＡ水平上检测其表达。得用ｐｃＤＮＡ３和重组载体分别免疫ＢＡＬＢ／ｃ小鼠,收集抗血甭,用免疫组化技术检测抗体产生情况。结果：扩增出ＴＣＲ Ｖβ的基因片段,测证实其序列与已发表的一致。并构建出重组表达载     

乳腺癌中C－erbB－2扩增及过度表达的临床意义

乳腺癌中Ｃ－ｅｒｂＢ－２扩增及过度表达的临床意义北京医科大学附属人民医院（北京市１０００４４）于虹，阚秀综述Ｃ－ｅｒｂＢ－２又称ｎｅｕ、ＨＥＲ－２、ＮＧＬ，是一种原癌基因。最初在化学制剂乙基硝基酸诱导的大鼠神经母细胞瘤的转染研究中，作为一种转化的肿瘤...     

HER2/neu胞外区片段基因免疫诱导特异细胞免疫及其抗瘤效应研究 *

目的：了解鱼精蛋白应用于血管内皮生长因子受体介导的靶向性非病毒载体的可行性。方法：ＣＶ１,ＣＶ２靶向性非病毒载体来比较多聚赖氨酸与鱼精蛋白对靶向性基因转移复合体携带ＤＮＡ的能力及体外基因转移效率的影响。结果：在Ａ３７５细胞中,鱼业 白与多聚赖氨酸参与形成的复合基因导入率都为５０％左右。在ＡＢＡＥ细胞中,鱼精蛋白参与形成的复合体基因导入率只有２０％左右,而多聚赖酸可达７０％左右。鱼精蛋白参与形成的复     

Antibody and CD8+ T cell responses against HER2/neu required for tumor eradication after DNA immunization with a Flt-3 ligand fusion vaccine.

PURPOSE: HER2/neu is frequently overexpressed in breast cancer. In a mouse model, vaccination with HER2/neu DNA elicits antibodies that confer partial protection against tumor challenge. EXPERIMENTAL DESIGN: To enhance antitumor immunity, we fused cDNA encoding Flt-3 ligand (FL) to the rat HER2/neu extracellular domain (neu), generating a chimeric FLneu molecule. FLneu and neu DNA vaccines were compared for immunogenicity and their ability to protect mice from tumor challenge. RESULTS: The neu vaccine generated a HER2/neu-specific antibody response. In contrast, vaccination with FLneu induced CD8+ T cells specific for HER2/neu but a negligible anti-HER2/neu antibody response. The switch from an antibody-mediated to T cell-mediated response was due to different intracellular localization of neu and FLneu. Although the neu protein was secreted, the FLneu protein was retained inside the cell, co-localizing with the endoplasmic reticulum, facilitating processing and presentation to T cells. The neu and FLneu vaccines individually conferred only weak tumor immunity. However, efficient tumor rejection was seen when neu and FLneu were combined, inducing both strong anti-HER2/neu-specific antibody and T cell responses. Adoptive transfer of both immune CD8+ T cells and immune sera from immunized mice was required to confer tumor immunity in na?ve hosts. CONCLUSIONS: These results show that active induction of both humoral and cellular immunity to HER2/neu is required for efficient tumor protection, and that neither response alone is sufficient.     

Coexpression of Flt3 ligand and GM-CSF genes modulates immune responses induced by HER2/neu DNA vaccine

DNA vaccine and dendritic cells (DCs)-based vaccine have emerged as promising strategies for cancer immunotherapy. Fms-like tyrosine kinase 3-ligand (Flt3L) and granulocyte-macrophage-colony-stimulating factor (GM-CSF) have been exploited for the expansion of DC. It was reported previously that combination of plasmid encoding GM-CSF with HER2/neu DNA vaccine induced predominantly CD4(+) T-cell-mediated antitumor immune response. In this study, we investigated the modulation of immune responses by murine Flt3L and GM-CSF, which acted as genetic adjuvants in the forms of bicistronic (pFLAG) and monocistronic (pFL and pGM) plasmids for HER2/neu DNA vaccine (pN-neu). Coexpression of Flt3L and GM-CSF significantly enhanced maturation and antigen-presentation abilities of splenic DC. Increased numbers of infiltrating DC at the immunization site, higher interferon-gamma production, and enhanced cytolytic activities by splenocytes were prominent in mice vaccinated with pN-neu in conjunction with pFLAG. Importantly, a potent CD8(+) T-cell-mediated antitumor immunity against bladder tumors naturally overexpressing HER2/neu was induced in the vaccinated mice. Collectively, our results indicate that murine Flt3L and GM-CSF genes coexpressed by a bicistronic plasmid modulate the class of immune responses and may be superior to those codelivered by two separate monocistronic plasmids as the genetic adjuvants for HER2/neu DNA vaccine.     

Adenovirus vaccination against neu oncogene exerts long-term protection from tumorigenesis in BALB/neuT transgenic mice

The transforming rat HER2/neu oncogene (neu), when embedded in the genome of transgenic BALB/c (neuT) mice, provokes the development of an invasive carcinoma in each of their 10 mammary glands. We used the neuT mice model system to evaluate the immunization efficiency and the protective effect of intramuscular injection of adenovirus (Ad) and/or of DNA with electrostimulation (DNA+ES), both expressing the rat p185(neu) protein. A neu cDNA sequence, which exclusively contains codons preferred by highly expressed mammalian genes, was used in this study. This "optimized" cDNA displayed higher expression in cultured cells and greater cell-mediated response than the original gene when injected as DNA+ES. Ad expressing the optimized sequence (Ad5-neu.opt) induced a higher immune response, as measured by the frequency of IFN-gamma-secreting spleen cells and antibody titers. Different Ad/DNA combinations and immunization schedules confirmed the superiority of Ad5-neu.opt in inducing a strong Th1-skewed humoral and CD8(+) cell-mediated response. Two Ad5-neu.opt injections of 10(9) viral particles at week 10 and 12 were sufficient to induce the highest response, which persisted at detectable levels up to 33 weeks of age. Anti-Ad5 antibodies elicited by previous injections neutralized the effect of an additional Ad5-neu.opt immunization at week 19. A group, which received 3 injections of DNA+ES at week 23, 27 and 31, in addition to the 3 Ad injections at week 10, 12 and 19 showed an increased frequency of IFN-gamma(+), CD8(+) PBMC at week 25, which persisted at detectable levels till week 38. Ad5-neu.opt administration at 10 and 12 weeks of age had a significant impact on tumor progression. At 44 weeks, 40% of the mice were completely protected from tumors with a mean tumor of 3.8. In contrast, control mice developed 10 tumors and died by week 27. Vaccination blocked the tumor development at the atypical hyperplasia stage present at the time of treatment. Tumors developing at later times express reduced levels of rat p185(neu) protein.     

Her2／ECD －sf162／TM嵌合 VLPs 的制备及抗肿瘤免疫效果研究

目的：成功制备 Her2胞外段基因与猴免疫缺陷病毒（simian immunodeficiency virus,SIV）包膜蛋白sf162跨膜区基因融合的 Her2／ECD －sf162／TM病毒样颗粒（VLPs）,并在小鼠体内进行初步的抗肿瘤免疫效果研究。方法：利用前期构建好的 Her2／neu 与 SIV －gag 嵌合的表达载体 Her2／ECD －sf162／TM,制备 Her2／neu 与 SIV －gag 嵌合型 VLPs 疫苗,并用该疫苗免疫小鼠。结果：VLPs 可成功激发小鼠体内免疫应答反应,产生血清抗 VLPs 的抗体;VLP 免疫后接种 Her2／neu ＋小鼠乳腺癌细胞 EMT6,结果显示该疫苗可有效抑制肿瘤生长;同时,VLP 对荷瘤鼠治疗结果也显示,该疫苗可在一定程度上抑制肿瘤生长。结论：VLPs 疫苗具有良好的免疫原性,且免疫后对肿瘤攻击具有保护作用。     

Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine.

HER-2/neu is a "self" tumor antigen that is overexpressed in 15-30% of human adenocarcinomas. Vaccine strategies directed against HER-2/neu and other self tumor antigens require development of methods to overcome immune tolerance to self-proteins. In rats, rat neu peptide vaccines have been shown to be an effective way of circumventing tolerance to rat neu protein and generating rat neu-specific immunity. The present report validates that a similar peptide-based vaccine formulation is effective for inducing T-cell immunity to HER-2/neu protein in humans with breast and ovarian cancer. The vaccine formulation included groups of peptides derived from the HER-2/neu extracellular domain (ECD) or intracellular domain (ICD) mixed with granulocyte macrophage colony stimulating factor as an adjuvant. These peptides were 15-18 amino acids in length and designed to elicit a CD4 T helper-specific immune response. Patients underwent intradermal immunization once a month for a total of two to six immunizations. To date, all of the patients immunized with HER-2/neu peptides developed HER-2/neu peptide-specific T-cell responses. The majority of patients (six of eight) also developed HER-2/neu protein-specific responses. Responses to HER-2/neu protein occurred with epitope spreading. Immune T cells elicited by vaccination were shown to migrate outside the peripheral circulation by virtue of generating delayed type hypersensitivity responses distant from the vaccine site, which indicated the potential ability to traffic to the site of tumor. The use of peptide-based vaccines may be a simple, yet effective, vaccine strategy for immunizing humans to oncogenic self-proteins.     

Production of antibodies by single DNA immunization: comparison of various immunization routes

DNA immunization is a recent vaccination method that induces humoral and cellular immune responses in a range of hosts. Different immunization routes induce a different degree of the immune response. In the present report, we demonstrate that multiple intramuscular immunizations of plasmid DNA encoding various leukocyte surface molecules induced a specific antibody response. In contrast, a single intramuscular immunization could not induce antibody production. To study the induction of antibody response after a single immunization of plasmid DNA, mice were single-dose intramuscularly, intraperitoneally, intravenously and intrasplenically immunized, simultaneously, with the same preparation of plasmid DNA encoding CD147 membrane protein. We observed that only the intrasplenic route induced specific antibody production. The induction of antibody by intrasplenic immunization was confirmed by using plasmid DNA encoding CD54 molecule. By this single-dose DNA intrasplenic immunization, the generated antibodies could be detected in mice up to 6 months. These results suggest that the injected DNA is expressing the relevant protein antigen in the spleen for several months after injection. Our results demonstrate that direct immunization of antigen-encoding DNA into the spleen is a more effective method for induction of antibody production. This finding may support future investigations of DNA vaccination strategies that specifically promote the uptake of plasmid by splenocytes. Intrasplenic immunization may also be helpful in the understanding of the early events of the immune response to DNA vaccine and be useful as an effective route for the induction of immune responses.     

DNA vaccination against neu reduces breast cancer incidence and metastasis in mice

The gene for HER2/neu is overexpressed in 30-40% of breast and ovarian cancers, and this overexpression correlates with increased metastasis and poor prognosis. The HER2/neu gene product, a transmembrane protein kinase member of the EGF receptor family, has significant potential as a tumor antigen for vaccination. We inserted the sequence for neu into a novel plasmid called ELVIS containing a Sindbis virus replicon that reproduces multiple copies of mRNA. Mice vaccinated one time intramuscularly demonstrated a strong antibody response against A2L2, a murine breast cancer cell line transfected to express neu. Vaccinated mice challenged in the mammary fatpad with A2L2 had reduced tumor incidence and reduced tumor mass compared to mice challenged with tumor cells lacking the neu insert. Intradermal vaccination was also protective and required 80% less plasmid for a similar level of protection. Vaccination reduced the incidence of lung metastasis from mammary fatpad tumors and reduced the number of lung metastases resulting from intravenous injection of A2L2 cells. Cytotoxic T lymphocytes cultures of immune spleen cells with P815-neu cells produced high levels of interferon-gamma indicating an antigen-specific Th1-type immune response resulting from the vaccination. In a spontaneous breast tumor model using neu transgenic mice, vaccination with ELVIS-neu protected against development of spontaneous breast tumors. Our preclinical data indicate that therapeutic vaccination of patients with ELVIS-neu may reduce metastasis from HER2/neu-expressing breast and ovarian tumors.