HER2／neu抗体对SKBR3乳腺癌细胞株酪氨酸磷本化？…

研究抗ＨＥＲ２／ｎｅｕ胞外区的特异性抗体对ＳＫＢＲ３乳腺癌细胞株酪氨酸磷酸化及细胞增殖的影响。方法 Ｗｅｓｔｅｒｎ印迹法及抗体介导的特异性细胞增殖试验；筛选乳腺癌病人抗ＨＥＲ２／ｎｅｕ抗体阳恬血清ＩｇＧ的ＥＬＩＳＡ方法。结果 试验发现单抗ｃ－ｎｅｕ－５对     

原癌基因HER2／neu表达产物p185蛋白免疫原性研究

用表达ｐ１８５蛋白ＨＥＲ２／ｎｅｕ转基因３Ｔ３－ＨＥＲ２／ｎｅｕ细胞裂解物免疫Ｂａｌｂ／ｃ小鼠，经３次免疫后，免疫小鼠脾细胞对表达ｐ１８５蛋白的３Ｔ３－ＨＥＲ２／ｎｅｕ细胞呈现较强的增殖反应（平均刺激指数分别为ＳＩ＝３．１５±１．８８），而对未转染ＨＥＲ２／ｎｅｕ基因的３Ｔ３细胞的应答较弱（ＳＩ＝１．１４±０．９７）。并检测到１号免疫鼠脾细胞对３Ｔ３－ＨＥＲ２／ｎｅｕ细胞有特异杀伤作用。免疫鼠血清中出现高水平的抗ｐ１８５抗体（ＯＤ均值＝１．０２±０．１６），较正常对照组（ＯＤ均值＝０．３６±０．１０）有显著差异（Ｐ＜０．００１），表明ＨＥＲ２／ｎｅｕ表达产物ｐ１８５蛋白具有免疫原性，用于免疫小鼠可诱导出对ｐ１８５蛋白的特异免疫应答反应。     

肿瘤抗原p185蛋白的纯化及其鉴定

目的：从转染ＨＥＲ２／ｎｅｕ 基因的３Ｔ３／ｎｅｕ细胞中纯化ｐ１８５蛋白,研究其免疫原性和作为肿瘤疫苗的可能性。方法：采用溴化氰活化的Ｓｅｐｈａｒｏｓｅ４Ｂ为基质,通过交联５２０Ｃ９单克隆抗体（杂交瘤腹水中沉淀的γ－球蛋白）,用亲和层析的技术纯化ｐ１８５蛋白。经梯度盐溶液解离与抗体结合的ｐ１８５蛋白,收集到蛋白洗脱峰,用ＥＬＩＳＡ检测ｐ１８５的免疫反应性,并经ＳＤＳＰＡＧＥ和Ｗｅｓｔｅｒｎ Ｂｌｏ     

c-erbB-2基因工程抗体的构建及在哺乳动物细胞中的表达

原癌基因ＨＥＲ－２／ｎｅｕ／ｅｒｂＢ－２编码的１８５－ｋＤａ跨膜磷酸糖蛋白为受体酪氨酸激酶家族中的成员。已报道ｃ－ｅｒｂＢ－２在多种腺癌中过表达，其中包括１５％～４０％的早期及转移性乳腺癌和卵巢癌［１］。亦有人报道，约７３％的转移性乳腺癌和卵巢癌过表达 ＨＥＲ－２／ｎｅｕ［２］，表明其为乳腺癌靶向性免疫治疗的理想靶分子。我们采用基因工程技术，构建了ｃ－ｅｒｂＢ－２ ＳｃＦｖ基因，并获得了其在ＣＯＳ７细胞中的表达。ｌ 材料和方法１．ｌ材料ＣＯＳ７细胞为本室保存。载体ｐｃＤＮＡ３．ｌ购自Ｉｎ－ｖｉｔｒ…     

抗p185单克隆抗体的制备及初步应用

抗ｐ１８５单克隆抗体的制备及初步应用许金华杜红杨蔚孙素莲（北京市肿瘤防治研究所，北京１０００３４）ｐ１８５是癌基因ｃ－ｅｒｂＢ－２（或称ＨＥＲ－２／ｎｅｕ）的表达产物，其分子量为１８５ｋＤ，故称ｐ１８５。已知ｐ１８５在腺癌类如乳腺癌、卵巢癌、胃癌及...     

乳腺癌病人抗HER2／neu阳性血清的筛选

ＨＥＲ2 /ｎｅｕ是一种原癌基因 ,编码的ｐ185跨膜糖蛋白具有受体酪氨酸激酶活性 ,属于表皮生长因子 (ＥＧＦ)受体家族中的成员。ＨＥＲ2 /ｎｅｕ基因的扩增和 /或ｐ185蛋白的过度表达可见于 2 0 %～ 4 0 %的乳腺癌病人[1] 。抗ＨＥＲ2 /ｎｅｕ胞外区的抗体可抑制肿瘤细胞的生长 ,而用于临床乳腺癌的治疗[2 ,3] 。本文旨在探索乳腺癌病人体内对ＨＥＲ2 /ｎｅｕ蛋白的体液免疫应答 ,分析其对细胞增殖的影响。1　材料与方法1 1　细胞株　见参考文献 [2 ]。1 2　研究对象　住院手术确诊为乳腺癌患者 14例 ,均为女性 ,其中 12例为浸润性导管癌 ,…     

抗人Ig轻链系列单抗的研制：Ⅲ抗人Ig游离λ型轻链共同抗原决定…

用多例提纯的ＢＪ－λ混合免疫ＢＡＬＢ／ｃ小鼠的脾细胞与骨髓瘤细胞Ｓｐ２／０融合，获得３株稳定分泌抗人Ｉｇ游离λ链ＭｃＡｂ的杂交瘤细胞株（ＡＨλＤ８、ＡＨλ１Ｃ１和ＡＨλ２Ｃｌ）。它们分泌的ＭｃＡｂ均能与我室所有１７例ＢＪ－λ起强反应，但不与结合的λ型轻链及Ｋ型轻链反应，表明这３株ＭｃＡｂ可能是抗人Ｉｇ游离λ链共同抗原决定簇的。３株ＭｃＡｂ均属小鼠ＩｇＧ１亚类。添加ＥＬＩＳＡ表明它们识别相同的表位。     

肿瘤抗原p185蛋白免疫鼠脾细胞的抗瘤效应研究

目的 研究肿瘤抗原ｐ１８５蛋白作为肿瘤疫苗所诱导的抗肿瘤效应。方法 亲和层析纯化的ｐ１８５蛋白皮下注射正常ＢＡＬＢ／ｃ小鼠,取脾细胞分别与ｐ１８５蛋白、３Ｔ３－ｍｅｕ细胞体外共培养,以单独佐剂注射组的小鼠脾细胞为对照,观察其培养上清中ＨＦＮ－γ和ＴＮＦ－α活性。将免疫鼠脾淋巴细胞经ｐ１８５抗原和低剂量的ＩＬ－２体外培养１周后,再用ＣＤ３抗体刺激扩增,用流式细胞仪分析其细胞表型。３Ｔｅ－ｎｅｕ和３Ｔ     

巨细胞病毒感染与可溶性白细胞介素2受体的关系

应用酶联免疫吸附试验（ＥＬＩＳＡ）对１０４例育龄妇女的血清进行了巨细胞病毒（ＨＣＭＶ）ＩｇＧ、ＩｇＭ抗体的检测，同时用ＥＬＩＳＡ双抗体夹心法测定了不同感染状态下血清中可溶性白细胞介素２受体（ｓＩＬ－２Ｒ）的水平，并将ｓＩＬ－２Ｒ水平与未感染ＨＣＭＶ的正常育龄妇女进行了比较。结果，育龄妇女中抗－ＨＣＭＶＩｇＧ的阳性率为８９．４％，ＩｇＭ的阳性率为９．６％，感染ＨＣＭＶ的妇女血清中ｓＩＬ－２Ｒ水平均大于未感染的对照组（１７８．１±５７．３Ｕ／ｍｌ），Ｐ＜０．０５，其中ＩｇＭ阳性者和ＩｇＭ、ＩｇＧ同时阳性者血清中ｓＩＬ－２Ｒ水平最高，分别为９１０±４６５．６Ｕ／ｍｌ和９０５±３４７．８Ｕ／ｍｌ，两者间的差异无显著性意义（Ｐ＞０．０５），但均大于仅抗－ＨＣＭＶＩｇＧ阳性者（４４６．８±１５８．９Ｕ／ｍｌ），Ｐ均＜０．０５。表明，ＨＣＭＶ感染可致ｓＩＬ－２Ｒ水平升高，并且活动性感染者上升明显。提示：ｓＩＬ－２Ｒ可能参与了ＨＣＭＶ的免疫致病机制。     

将抗体库所获抗-HBs Fab转换为全长人IgG

目的 将大肠杆菌表达的抗－乙肝表面抗原（ＨＢｓ）人Ｆａｂ转换为真核表达的全长人ＩｇＧ１。方法 用重叠ＰＣＲ法将人工合成的人Ｖｋ前导序列拼到抗－ＨＢｓ的ＶＨ和ＶＫ上,构建人ＩｇＧ１真核表达载体。转染真核细胞,通过ＥＬＩＳＡ、ＲＴ－ＰＣＲ、免疫印迹检测抗－ＨＢｓ人ＩｇＧ的表达。结果 用轻链和重链同时表达的单一载体在ＣＨＯ细胞中获得了抗－ＨＢｓ人ＩｇＧ的表达。结论 通过噬菌体抗体库所获Ｆａｂ段可经拼接人     

CD98 regulates the phosphorylation of HER2 and a bispecific anti-HER2/CD98 antibody inhibits the growth signal of human breast cancer cells

Human epidermal growth factor receptor (HER) family proteins are currently major targets of therapeutic monoclonal antibodies against various epithelial cancers. However, the resistance of cancer cells to HER family-targeted therapies, which may be caused by cancer heterogeneity and persistent HER phosphorylation, often reduces overall therapeutic effects. We herein showed that a newly discovered molecular complex between CD98 and HER2 affected HER function and cancer cell growth. The immunoprecipitation of the HER2 or HER3 protein from lysates of SKBR3 breast cancer (BrCa) cells revealed the HER2-CD98 or HER3-CD98 complex. The knockdown of CD98 by small interfering RNAs inhibited the phosphorylation of HER2 in SKBR3 cells. A bispecific antibody (BsAb) that recognized the HER2 and CD98 proteins was constructed from a humanized anti-HER2 (SER4) IgG and an anti-CD98 (HBJ127) single chain variable fragment, and this BsAb significantly inhibited the cell growth of SKBR3 cells. Prior to the inhibition of AKT phosphorylation, BsAb inhibited the phosphorylation of HER2, however, significant inhibition of HER2 phosphorylation was not observed in anti-HER2 pertuzumab, trastuzumab, SER4 or anti-CD98 HBJ127 in SKBR3 cells. The dual targeting of HER2 and CD98 has potential as a new therapeutic strategy for BrCa.     

抗人P185~(erbB_2)单克隆抗体的制备及特性分析

目的制备可特异识别 P185 erb B2 胞外区的单抗 ,选出亲和力较高并具有抑瘤作用的克隆。方法以高表达 erb B2 的人乳腺癌细胞系 SKBR3免疫 BABL / c小鼠 ,用纯化的重组 DNA表达的 P185胞外区蛋白作为抗原 ,以间接 EL ISA方法筛选阳性克隆。结果获得 10株稳定分泌抗 P185胞外区单抗的杂交瘤细胞株。 4株为 Ig G1、1株为 Ig G3、5株为 Ig M,分别可识别 P185胞外区 3个不同表位 ,且只与天然形式的 P185胞外区特异结合。其中 4株 Ig G1类抗体可明显抑制 SKBR3及 SKOV3细胞增殖。免疫组化实验可使 SKBR3及 SKOV3细胞膜特异性染色 ,且可检出乳腺癌、卵巢癌等石蜡切片标本中 erb B2 高表达的阳性细胞。结论所获单抗可特异识别 P185胞外区抗原 ,具有肿瘤诊断、判断预后及人源化改造后用于治疗的应用潜力。     

腺病毒介导的RNA干涉对乳腺癌SKBR3细胞HER2的下调及生长抑制效应

目的:探讨采用腺病毒做为载体介导基于RNA干涉的针对高HER2肿瘤的基因治疗的可能性。方法:构建HER2-绿色荧光蛋白融合蛋白表达质粒pHER2-GFP,并与9种针对HER2不同靶序列的siRNA表达质粒分别共转染CHO-K1细胞,根据荧光蛋白表达量从中筛选出沉默效率最高的质粒。将筛选出的质粒转染HER2高表达乳腺癌SKBR3细胞,检测它们对HER2表达的影响。随后,将siRNA转录单元克隆入腺病毒载体,成功包装病毒后感染SKBR3细胞,再次测定其下调HER2的效应及其对细胞生长的影响。结果:2种有效下调HER2表达的质粒被筛选出来。将此2种质粒所含siRNA转录单元包装入腺病毒后仍然保持了原有的基因沉默效应。HER2下调增加了SKBR3细胞中G1期细胞的比例,并且诱导部分细胞凋亡。MTT和细胞长期增殖抑制实验表明腺病毒介导的RNA干涉抑制了SKBR3细胞生长。结论:重组腺病毒介导的RNA干涉能够下调HER2的表达并且对高表达HER2的乳腺癌细胞有生长抑制作用。     

去甲斑蝥素抑制乳腺癌SKBR3细胞增殖及侵袭转移的体外研究

目的： 探讨去甲斑蝥素（norcantharidin,NCTD）抑制乳腺癌细胞SKBR3增殖及侵袭转移的机制。方法： 体外培养人乳腺癌SKBR3细胞株，采用MTT法检测NCTD对乳腺癌细胞SKBR3增殖抑制能力；流式细胞术测NCTD对SKBR3细胞周期和凋亡的影响；应用肿瘤细胞体外黏附实验、迁移实验和Transwell体外侵袭转移模型检测NCTD对SKBR3细胞黏附、运动、侵袭转移的抑制作用。结果： NCTD可抑制人乳腺癌SKBR3细胞增殖，具有明显量效和时间依赖性，24 h的IC₅₀为12.5 mg/L；10 mg/L NCTD 作用SKBR3细胞24 h、48 h后，特异地使SKBR3细胞发生G₂/M期阻滞，G2/M期细胞百分比分别为35.82%、38.70%，G₁期和S期细胞较对照组明显减少，亚G₁和G₀峰DNA含量逐渐增加, 24 h、48 h的凋亡率分别为3.44%和6.17%；SKBR3细胞的体外黏附、运动、侵袭转移能力随NCTD药物浓度提高而下降，表现在黏附抑制率增加，过河时间延长，过膜细胞数减少(P<0.05)。结论： NCTD可通过抑制乳腺癌细胞增殖，阻滞细胞周期，诱导细胞凋亡，抑制肿瘤细胞对基质黏附、迁移运动、侵袭转移等途径抑制癌症的发展。     

Interleukin-2 enhances the natural killer cell response to Herceptin-coated Her2/neu-positive breast cancer cells

The Her2/neu (c-erbB-2) oncogene encodes a 185-kDa protein tyrosine kinase which is overexpressed in 20% of breast adenocarcinomas and is recognized by a humanized anti-Her2/neu monoclonal antibody (mAb) (rhu4D5 or Herceptin). Natural killer (NK) cells are capable of mediating antibody-dependent cell cytotoxicity (ADCC) against antibody-coated targets via their expression of a low-affinity receptor for IgG (FcgammaRIII or CD16). NK cells can be expanded in cancer patients via the administration of low-dose interleukin-2 (IL-2) and become potent cytotoxic effectors following exposure to high doses of IL-2. We tested IL-2-activated NK cells against Her2/neu+ (MCF-7Her2/neu) and Her2/neu- (MDA-468) breast cancer cell lines in a 4-h 51Cr-release cytotoxicity assay in the presence or absence of rhu4D5 mAb (effector : target ratio = 10 : 1). Specific lysis of rhu4D5-coated MCF-7Her2/neu and MDA-468 target cells by IL-2-activated NK cells was 35% and 3%, respectively (p < 0.05). Lysis was less than 5% when targets were treated with either the non-humanized mu4D5 mAb or control huIgG. Lysis of rhu4D5-coated MCF-7Her2/neu cells was inhibited by 80 % when NK cells were pre-treated with an anti-Fc receptor antibody prior to use in the cytotoxicity assay. Enhanced ADCC of MCF-7Her2/neu target cells was seen when the effector cells consisted of mononuclear cells obtained from a patient demonstrating significant expansion of NK cells secondary to therapy with low-dose IL-2. Serum from patients receiving infusions of rhu4D5 mAb could substitute for exogenous antibody in the ADCC assay. NK cells activated by rhu4D5-coated tumor cells in the presence of IL-2 also produced large amounts of IFN-gamma with concomitant up-regulation of cell-surface activation markers CD25 and CD69. These results lend support to the concurrent use of rhu4D5 mAb and IL-2 therapy in patients with cancers that express the Her2/neu oncogene.     

Trastuzumab enhances the anti-tumor effects of the histone deacetylase inhibitor sodium butyrate on a HER2-overexpressing breast cancer cell line

Trastuzumab has efficacy to improve the effect of cytotoxic drugs, such as paclitaxel and anthracyclin, against HER2-overexpressing breast cancer cells. Sodium butyrate (NaB), a histone deacetylase inhibitor, is known to have antitumoral properties. However, whether and how trastuzumab possesses the potential to synergize the anti-tumor effect of NaB on breast cancer cells is still equivocal. To elucidate whether combined treatment with NaB and trastuzumab exerts anti-tumor effects on a HER2-overexpressing breast cancer cell line, SKBR3 cells were treated with NaB alone or in combination with trastuzumab, and the effects on proliferation and cell cycle progression were analyzed. Combinatory treatment with NaB (4 mmol/l) and trastuzumab (20 μg/ml) significantly increased the growth-inhibitory effect on SKBR3 breast cancer cells, in comparison to NaB or trastuzumab treatment alone. The growth-inhibitory effect of the combination of NaB and trastuzumab was accompanied by elevated mRNA and protein levels of the cyclin-dependent kinase inhibitor p27Kip1. In contrast, this effect was absent in HER2-negative HCC1937 cells. In conclusion, trastuzumab significantly improved the antitumor effect of NaB on HER2-overexpressing breast cancer cell line in vitro.     

抗人IL-11单克隆抗体的研制及生物学特性研究

以rhIL 11作为免疫原常规免疫BALB/c小鼠 ,采用细胞融合、ELISA检测和杂交瘤细胞克隆筛选等方法获得了 2株鼠抗人IL 11的单克隆抗体 (5A4、 1G12 ) ,分别属于IgG1亚类和IgG2a亚类。MTT法和细胞增殖试验分析表明 ,5A4、 1G12均不同程度地抑制IL 11对其依赖株细胞B9 11的增殖效应。表明成功地研制成了 2株鼠抗人IL 11的中和功能性单克隆抗体     

Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference

Gene silencing using short interfering RNA (siRNA) is fast becoming an attractive approach to probe gene function in mammalian cells. Although there have been some success in the delivery of siRNA using various methods, tracking their delivery and monitoring their transfection efficiency prove to be hard without a suitable tracking agent. Therefore, a challenge lies with the design of an efficient and at the same time, self-tracking, transfection agent for RNA interference. In this paper, chitosan nanoparticles (NPs) with encapsulated quantum dots (QDs) were synthesized and used to deliver HER2/neu siRNA. Using such a construct, the delivery and transfection of the siRNA can be monitored by the presence of fluorescent QDs in the chitosan NPs. Targeted delivery of HER2 siRNA to HER2-overexpressing SKBR3 breast cancer cells was shown to be specific with chitosan/QD NP surface labeled with HER2 antibody targeting the HER2 receptors on SKBR3 cells. Gene-silencing effects of the conjugated siRNA was also established using the luciferase and HER2 ELISA assays. These self-tracking siRNA delivery NPs will also aid in the monitoring of future gene silencing studies in vivo.