HER2/neu原癌基因胞外区片段DNA克隆及其在小鼠中诱导的抗体应答 *

目的：探讨维甲酸在预防和治疗肿瘤中的作用机制,分离受维甲酸调控的靶基因。方法：采用ＡＰ－ＰＣＲ测序、Ｎｏｒｔｈｅｒｎ杂交及序列２同源分析等方法。结果：首次发现ＡＴＲＡ可上调线粒体基因ＡＴＰ酶第六亚单位表达,这种表达上调开始于Ａ－ＴＲＡ诱导早期并持续整个诱导过程（７ｄ）。Ｎｏｒｔｈｅｒｎ杂交结果证实了ＡＴＲＡ对线粒体基因ＡＴＰ酶第六亚单位的上调作用。夺线粒体基因组的转录调控区Ｄ－ｌｏｏｐ序列进行了初     

腺病毒E1A下调HER2/neu原癌基因表达及其抗瘤效应研究

目的　研究腺病毒E1A基因对HER2 /neu高表达肿瘤细胞生长的抑制作用及增强其化疗敏感性的作用。方法　以腺病毒Ⅴ型为载体 ,经体、内外对HER2 /neu高表达及低表达的肿瘤细胞株转染腺病毒E1A基因后 ,观察E1A基因对肿瘤细胞生长抑制作用 ;用MTT法检测E1A增强肿瘤细胞化疗敏感性作用。结果　E1A能显著抑制HER2 /neu高表达的肿瘤细胞在体内外的生长 ,延长荷瘤裸鼠的生存期。免疫印迹及免疫组织化学分析显示 ,转染E1A的HER2 /neu高表达肿瘤细胞 ,其HER2 /neu基因产物p185蛋白表达明显降低 ;经AdE1A 处理的HER2 /neu高表达的乳腺癌细胞株能明显增强对TaxotereTM的化疗敏感性。结论　E1A通过下调HER2 /neu原癌基因的表达 ,抑制HER2 /neu高表达的肿瘤细胞生长 ,增强肿瘤细胞对化疗药的敏感性。     

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。     

尿激酶氨基末端基因抗乳腺癌细胞转移的实验研究

目的：探讨尿激酶氨基末端（ATF）基因转移对肿瘤转移的抑制作用。方法：构建重组ATF基因真核表达载体pcDNA3-ATF,用脂质体Lipofectin介导,将其导入其证明尿激酶（uPA)和尿激酶受体（uPAR）均有表达的人乳腺癌细胞系MCF－7,用Western blot检测转染细胞ATF基因的表达,体外观察并比较了野生型和转基因MCF－7细胞侵袭人工基底膜能力;裸鼠皮下接种癌细胞,复制自发性肿瘤转移模型,观察成瘤性和转移性。结果uPA／uPAR在MCF－7中有较高水平的表达;转ATF基因后,可检测到ATF的明显表达,体外侵袭穿透人工基底膜的能力明显降低,体内原位成瘤性不受影响,但自主性体肺转移能力有一定程度的下降,结论ATF基因转移后,ATF的表达可能竞争 抑制内源性uPA与uPAR的结合,在一定程度上抑制癌细胞的侵袭和转移。     

mB7－1基因转染小鼠黑色素瘤细胞诱导的抗瘤效应研究

目的：研究共刺激Ｂ７－１（ＣＤ６０）在诱导有效的抗肿瘤免疫反应反应中所起的作用。方法：在体外,三种肿瘤细胞与同上鼠脾淋巴细胞混合培养（ＭＬＴＣＳ）后,测定淋巴细胞增殖指数（ＭＴＴ法）和特异杀伤活性（ＬＤＨ释放改良法）;ＭＴＴＩ地测定肿瘤细胞体外增殖能力后,将Ｂ１６－ｎｅｏ和Ｂ１６－ｍＢ７－１妆种于Ｃ５７ＢＬ／６小鼠皮下后观察成瘤期、荷瘤小鼠存活以及肿瘤结节生长速度。结果：与野生型Ｂ１６细胞和模拟转     

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     

IL—2基因转染的肿瘤细胞增强机体免疫功能的实验研究

目的：探讨小鼠接种腺病毒介导的IL－2基因转移的CT26小鼠结肠腺癌细胞后机体免疫功能的变化（包括CTL、LAK细胞、NK细胞、巨噬细胞的杀伤活性变化）。方法：体外将腺病毒介导的小鼠IL－2基因转染CT26细胞（CT26-mIL-2)，然后将CT26－mIL2细胞接种于小鼠皮下。采用4h乳酸脱氢酸释放法检测荷瘤小鼠CTL、LAK、NK细胞的杀伤活性，MTT法检测巨噬细胞的杀伤活性。结果：接种CT26－mIL－2后第14d，小鼠脾细胞NK活性和诱导后的LAK活性和CTL活性均显著高于对照组（P＜0．01）；小鼠腹腔巨噬细胞数量显著增加，杀伤活性显著增强（P＜0．01）。结论：IL－2基因转染的肿瘤细胞能诱导机体的特异性和非特异性免疫功能参与机体的抗肿瘤作用。     

腺病毒介导p21WAF1/CIP1基因转移及诱导胃癌细胞凋亡

目的：探讨p21^WAF1/CIP1基因（p21基因）过表达对人胃癌细胞恶性表型和细胞凋亡的影响，进一步了解p21基因对肿瘤细胞的治疗作用。方法：通过腺病毒介导外源性p21基因转移到有p53基因突变的人胃癌细胞系SGC－7901，对p21基因的表达、细胞生长的抑制与投机和对肿瘤模型的治疗效果进行分析。结果：外源性p21基因在靶细胞高水平表达显著抑制了胃癌细胞的生长和集落形成，流式细胞仪检测显示G1期阻滞并发生了凋亡。瘤内注射Ad-p21对裸鼠体内移植瘤有一定抑瘤作用。结论；p21基因可能参与肿瘤细胞凋亡的诱导，使p21基因在肿瘤的基因治疗，特别是在与其他凋亡诱导因子联合作用的方案中有更好的应用前景。     

hMIP-1β基因修饰瘤苗体内诱导抗肿瘤免疫效应的研究

目的：探讨人巨噬细胞炎性蛋白-1β（hMIP-1β）基因修饰瘤苗体内诱导的抗肿瘤免疫效应。方法：通过重组腺病毒载体介导将hMIP-1β基因导入CT26细胞中,X-gal染色法检测基因转染效率;ELISA法检测hMIP-1β基因修饰CT26细胞培养上清中hMIP-1β的含量;Boyden趋化小室法检测培养上清对CD4^＋T细胞、CD8^＋T细胞、NK细胞及未成熟树突状细胞（imDCs）的趋化作用;制备hMIP-1β基因修饰的CT26细胞瘤苗并免疫BALB／c小鼠,观察其诱导免疫细胞的杀伤活性和保护性免疫反应。结果：腺病毒载体可介导hMIP-1β基因转染CT26细胞和表达（X—gal染色的阳性率〉95％）,培养上清中hMIP-1β水平为980pg／mL,并对CD4^＋T细胞、CD8^＋T细胞、NK细胞及imDCs有显著的趋化作用,与转染对照基因LacZ的CT26细胞及野生型CT26细胞比较差异均有统计学意义,P〈0．01。hMIP-1β基因修饰的CT26细胞瘤苗免疫小鼠能有效诱导肿瘤特异性CTL活性和非特异性NK活性,产生明显的免疫保护作用,可抵抗肿瘤细胞的再攻击,成瘤率降低,肿瘤生长速度减慢,小鼠生存期延长。结论：hMIP-1β基因修饰瘤苗可诱导产生有效的抗肿瘤免疫保护作用,有希望成为预防肿瘤转移与复发的有效手段。     

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。     

SUCI02选择性抑制HER-2/neu受体酪氨酸激酶磷酸化及其对HER-2/neu过表达乳腺癌细胞生长的影响

背景与目的：HER-2／neu受体过度表达与肿瘤的发生发展、对化疗的敏感性以及患者预后有关;我们通过大量筛选,发现SUCI02[N-(4-乙氧苯基)-2-羟基酸胺]能抑制HER2／neu受体酪氨酸激酶磷酸化。本研究拟探讨SUCI02对HER-2／nell过度表达的肿瘤细胞生长的影响。方法：用免疫沉淀、免疫印迹法检测：HER-2／neu受体酪氨酸激酶磷酸化、酪氨酸激酶蛋白水平的变化;MTT法检测SUCI02对乳腺癌细胞的抑制作用。结果：SUCI02能抑制HER-2／neu受体的自身磷酸化,在乳腺癌MDA—MB．453m1细胞中,SUCI02对HER-2／neu受体自身磷酸化的IC50为4．34μg／ml,对HER-2／neu的表达没有任何影响。在用SUCI02处理MDA-MB-453ml细胞30min后,用培养液洗掉药物,继续培养不同时间,结果可见洗掉药物后30min,SUCI02对HER-2／neu受体磷酸化的抑制就开始恢复。SUCI02处理MDA-MB-453ml细胞后其下游靶分子MAPK和AKT激活明显受抑制,呈现剂量依赖性。SUCI02对EGFR受体酪氨酸磷酸化在最高剂量用到40μg／ml下没有明显的抑制作用。应用MTT法检测了SUCI02对过度表达HER-2／neu的MDA-MB-453ml细胞的生长抑制作用,同时应用过表达EGFR的乳腺癌MDA—MB-468细胞作为对照,结果可见SUCI02对HER-2／neu过表达的肿瘤细胞相对于过表达EGFR的肿瘤细胞有更明显的抑制作用。结论：SUCI02选择性抑制HER-2／neu受体酪氨酸激酶磷酸化,阻断其下游信号途径,对过度表达HER-2／neu的肿瘤细胞具有更强的生长抑制作用。     

HER-2/neu基因在鼻咽癌中的表达及其临床意义

目的：探讨HER－2／neu基因在鼻咽癌中的表达及其临床意义。方法：采用免疫组化法测定HER－2／neu基因蛋白的表达。结果：69例鼻咽癌病人中52％（36／69）有HER－2／neu基因蛋白表达。这种过表达与肿瘤分期、局部淋巴结和远处脏器转移以及5年总生存期、3年无病生存期均无相关性。同一病例原发病灶和复发或转移灶HER－2/neu基因蛋白的表达较一致。HER－2／neu基因蛋白过表达对鼻咽癌化疗的反应性不产生影响。结论：在鼻咽癌,HER－2／neu基因蛋白过表达的临床意义不前,HER-2/neu基因蛋白能否成为治疗新的靶点还有待进一步研究。     

Pre-existent immunity to the HER-2/neu oncogenic protein in patients with HER-2/neu overexpressing breast and ovarian cancer

Immunomodulatory strategies, such as antibody therapy and cancer vaccines, are increasingly being considered as potential adjuvant therapies in patients with advanced stage breast cancer to either treat minimal residual disease or prevent relapse. However, little is known concerning the incidence and magnitude of the pre-existent breast cancer specific immune response in this patient population. Using the HER-2/neu oncogenic protein as a model, a well-defined tumor antigen in breast cancer, we questioned whether patients with advanced stage HER-2/neu overexpressing breast and ovarian cancers (III/IV) had evidence of pre-existent immunity to HER-2/neu. Forty-five patients with stage III or IV HER-2/neu overexpressing breast or ovarian cancer were evaluated for HER-2/neu specific T cell and antibody immunity. Patients enrolled had not received immunosuppressive chemotherapy for at least 30 days (median 5 months, range 1–75 months). All patients were documented to be immune competent prior to entry by DTH testing using a skin test anergy battery. Five of 45 patients (11%) were found to have a significant HER-2/neu specific T cell response as defined by a stimulation index 2.0 (range 2.0–7.9). None of eight patients who were HLA-A2 had a detectable IFN secreting T-cell precursor frequency to a well-defined HER-2/neu HLA-A2 T cell epitope, p369-377. Three of 45 patients (7%) had detectable HER-2/neu specific IgG antibodies, range 1.2–8.9g/ml. These findings suggest that patients with advanced stage HER-2/neu overexpressing breast and ovarian cancer can mount a T cell and/or antibody immune response to their tumor. However, in the case of the HER-2/neu antigen, the pre-existent tumor specific immune response is found only in a minority of patients.     

重组人白介素2-卡介苗对荷瘤小鼠腹腔巨噬细胞体外杀伤活性的影响 *

目的：研究ＨＥＲ２／ｎｅｕ原癌基因胞外区片段（ＥＣＤ）ＤＮＡ直接肌肉注射在小鼠体内表达,体液免疫状况,利用小鼠流产模型观察其体内效应。方法：克隆并构建了人及大鼠ＨＥＲ２／ｎｅｕ原癌基因胞外区片段真核表达载体ｐＣＤＮＡ３－Ｘ,采用肌肉直接注射的方法免疫小鼠,分析目的基因在小鼠体内的表达及其诱导的兔疫应答。结果：在质粒ＤＮＡ直接注射部位可检测到目的基因的表达,并诱导了针对ＨＥＲ２／ｎｅｕ的抗体反应;同时诱发小鼠流产,使免疫鼠产仔数减少,可观察到流产结节形成。结论：通过基因免疫,可诱导针对ＨＥＲ２／ｎｅｕ原癌基因产物的有效免疫应答。     

Prevention of HER-2/neu transgenic mammary carcinoma by tamoxifen plus interleukin 12

FVB-NeuN (N#202) female mice transgenic for the HER-2/neu protooncogene driven by the murine mammary tumor virus (MMTV) promoter develop mammary carcinomas with a progression from focal atypical hyperplasia to in situ carcinoma and to invasive carcinoma that closely resembles that of human neoplasia. Here we report that the combination of tamoxifen plus interleukin 12 (IL-12) results in a very effective prevention of mammary carcinogenesis, significantly higher than those obtained with either tamoxifen or IL-12 alone. At 1 year of age, 20% of control mice resulted tumor-free, whereas 80% of mice receiving the combined treatment were tumor-free. At 2 years of age, less than 5% of control mice were tumor-free, as opposed to 70% of mice treated with tamoxifen plus IL-12. The combined treatment inhibited mammary carcinogenesis mainly through a reduction in the number of mammary cells at risk of progression, a reduction in estrogen receptors (ERs) expression and a reduction in the angiogenic support to mammary development, likely due to cross-talk between tamoxifen and interferon-gamma (IFN-gamma) (the main downstream mediator elicited in vivo by IL-12). The addition of IL-12 to the tamoxifen treatment more than doubled mouse lifetime and did not exacerbate known side effects of tamoxifen.     

Frequencies of HER-2/neu overexpression relating to HLA haplotype in patients with gastric cancer

We have identified that HER-2/neu-derived peptides are naturally processed as tumor rejection antigens recognized by tumor-specific, HLA-A2-restricted cytotoxic T lymphocytes in gastric cancer. To evaluate candidates for immunotherapy using HER-2/neu-derived, HLA-A2-restricted peptides, we examined the frequency of HLA-A2 relating to HER-2/neu overexpression or the infiltrating grade of tumor-infiltrating lymphocytes (TILs) in Japanese patients with gastric cancer. HER-2/neu-overexpressing tumors detected by immunohistochemistry amounted to 19% of primary gastric cancers and HLA-A2-positive patients with gastric cancer were 31% of primary gastric-cancer cases. Finally, gastric-cancer patients with both HLA-A2-positive and HER-2/neu-overexpressing tumors amounted to 6.6% of these cases. There was no significant difference in the infiltrating grade of TILs between gastric cancers overexpressing HER-2/neu and those that did not. The candidate for HER-2/neu-based immunotherapy with HLA-A2-restricted peptides represent a very limited population of Japanese patients.     

非小细胞肺癌HER-2/neu基因表达及曲妥珠单抗治疗研究进展

HER-2/neu是一种原癌基因,其高度表达与人肿瘤发生、预后不佳有一定关系。该文就HER-2/neu高度表达与非小细胞肺癌(NSCLC)发生、发展的关系以及单克隆抗体——曲妥珠单抗(Trastuzumab)在临床中对NSCLC的应用价值作一综述。     

Effect of resveratrol on the development of spontaneous mammary tumors in HER-2/neu transgenic mice

Resveratrol (Res) has been reported to possess cancer chemopreventive activity on the basis of its in vitro effects on tumor cells and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of Res on the development of mammary tumors appearing spontaneously in HER-2/neu transgenic mice at an early age. The mechanisms involved in the Res antitumor effect were evaluated by studying the immune effectiveness, tumor apoptosis and expression of mRNA and protein for HER-2/neu in tumoral mammary glands from Res-treated mice and in tumor cell lines. Res supplementation delayed the development of spontaneous mammary tumors (p < 0.001), reduced the mean number and size of mammary tumors (p < 0.0001) and diminished the number of lung metastases in HER-2/neu transgenic mice. The effects of Res were associated with downregulation of HER-2/neu gene expression and increased apoptosis both in tumoral mammary glands and in murine (N202) and human (SKBr3) tumor cell lines. Neither the basal, the IL-2-induced NK activity nor the lymphocyte number and proliferation was modified in Res-supplemented compared to control mice. Our results demonstrate that Res supplementation delays the development and reduces the metastasizing capacity of spontaneous mammary tumors in HER-2/neu transgenic mice. The antitumor effect of Res might be related to the downregulation of HER-2/neu expression and the induction of apoptosis in tumor cells.