6811抗独特型微抗体负载树突状细胞对卵巢癌细胞系细胞杀伤作用的体外研究

目的 用6811抗独特型微抗体体外负载树突状细胞(DC)，以期诱导出抗原特异性的抗肿瘤免疫反应。方法 分离培养HLA-A2 健康人外周血树突状细胞，培养过程中用6811微抗体负载，无关抗原F(ab)2负载及未负载组为对照。光镜、电镜下观察树突的形态；流式细胞仪检测DC表面分子表达；^3H-TdR掺入法测DC刺激自体T细胞增殖；^51Cr6h释放试验测激活的T细胞对卵巢癌细胞系的杀伤。结果 培养的树突细胞有典型形态，CD80、CD86、HLA-DR等表面分子呈高表达(分别为65％、86．2％、93．7％)；6811微抗体负载的DC不仅能刺激自体T细胞的增殖，而且其诱导的CTL细胞对HLA-A2 、OC166-9 卵巢癌细胞系HOC1A有特异性的杀伤，结论 6811抗独特型微抗体模拟卵巢癌抗原OC166-9负载树突状细胞在体外可以诱导出抗原特异性的细胞毒T细胞，为进一步研究6811微抗体对卵巢癌免疫治疗作用提供了依据。     

应用卵巢癌抗独特型单克隆抗体6B11进行血清中卵巢癌抗体检测

探讨应用卵巢癌抗独型单克隆抗体６Ｂ１１进行血清中相关抗体检测的可行性。方法；采用间接免疫酶法测定妇科良性与恶性肿瘤患者各３０与８８例，子宫内膜异位症患者９例，正常女性献血员４５例共１７２份血清，１５例卵巢癌患者年内１３３份血清卵巢部抗体水平，并与病情作比较。     

卵巢癌单克隆抗-抗独特型抗体32F2的制备及其特性研究

目的6B11是可模拟卵巢癌抗原的鼠源性抗独特型单克隆抗体,为了深入探讨其作为肿瘤疫苗的主动免疫机制,制备抗6B11的IgG型抗-抗独特型单克隆抗体并对其特性进行研究。方法以卵巢癌抗独特型抗体6B11作为免疫原,与载体钥孔槭血兰蛋白(KLH)偶联并辅以福氏佐剂,多次免疫同系Balb/c小鼠,通过杂交瘤技术将免疫脾细胞与骨髓瘤细胞Sp2/0融合;采用酶联免疫吸附实验(EL1SA)筛选杂交瘤细胞,经克隆化后建立稳定分泌鼠源性抗-抗独特型单克隆抗体的杂交瘤细胞株。秋水仙素法确认杂交瘤细胞染色体数目及形态,ELISA法鉴定抗体类型,采用非竞争酶免疫结合实验测定抗体亲和力,EL1SA、免疫组织化学方法检测抗-抗独特型单克隆抗体特异性结合抗原的性质。结果制备出一株能稳定分泌抗-抗独特型单克隆抗体的杂交瘤细胞株,命名为32F2。其染色体数目平均为103条,并有端着丝点和亚中部着丝点染色体,符合小鼠杂交瘤细胞特点。抗体类型为IgG1型,抗体亲和力约为2.3119×107L/mol,能够竞争抑制卵巢癌单克隆抗体COC166-9与原始抗原OC166-9的结合,免疫组织化学染色证实32F2鼠腹水与84.6%卵巢浆液性乳头状囊腺癌呈阳性染色,与COC166-9类似,表明32F2可与OC166-9抗原产生特异性结合。结论通过杂交瘤技术建立了分泌IgG1亚型的卵巢癌抗-抗独特型单克隆抗体的杂交瘤细胞株。对抗体的初步研究显示其能特异性结合初始抗原OC166-9,并能竞争抑制COC166-9与OC166-9的结合,属Ab1样Ab3,间接证实6B11为抗原内影像型抗体,具有模拟抗原作用。32F2的制备成功为进一步研究卵巢癌抗独特型疫苗6B11的作用机制打下了基础,并且该抗体具有潜在的临床治疗卵巢癌的价值。     

卵巢癌抗独特型抗体的研究进展

卵巢癌抗独特型抗体的研究进展张香云崔恒钱和年抗独特型抗体（Ａｂ２）具有模拟抗原和免疫调节双重作用，同时能够克服机体免疫抑制或打破其免疫耐受状态。近年来，在国外已试用于结肠癌、恶性黑色素瘤、淋巴瘤等人类恶性肿瘤的治疗，并有成功的报道［１～４］。Ａｂ２在...     

抗独特型抗体及其在卵巢癌研究中的现状

随着近年来对独特型-抗独特型免疫调节网络的深入研究,抗独特型抗体作为肿瘤免疫治疗的新途径越来越受到人们重视。本文简述近年来抗独特型抗体在肿瘤研究中的进展,重点介绍有关卵巢癌方面抗独特型抗体研究现状,为妇科肿瘤免疫学研究提供新信息。     

6B11ScFv-mHSP70融合蛋白诱导抗卵巢癌免疫应答的体外实验研究

目的 既往研究证实,6B11抗独特型单链抗体（6B11ScFv）具有模拟卵巢癌相关抗原OC166-9的作用。本文研究6B11ScFv与小鼠热休克蛋白70（mHSP70）构建而成的融合蛋白（6B11ScFv-mHSP70）在体外抗卵巢癌免疫应答,探讨其作为卵巢癌疫苗的可能性。方法 以大肠杆菌表达6B11ScFv-mHSP70融合蛋白,经变性复性后纯度达95%。采用ELISA检测其免疫学活性。采集HLA-A2阳性的女性健康志愿者的外周血单个核细胞,用6B11ScFv-mHSP70刺激并培养。采用CCK-8法、LDH释放法分别观察淋巴细胞增殖情况和对卵巢癌细胞系的细胞毒作用,流式细胞术检测6B11ScFv-mHSP70刺激前后淋巴细胞表型的改变。结果 6B11ScFv-mHSP70具有特异性结合卵巢癌单抗COC166-9及抗小鼠-HSP70抗体的双重免疫学活性;可以刺激人外周血淋巴细胞的增殖;并对HLA-A2阳性、OC166-9表达阳性的卵巢癌细胞系有细胞毒作用;经6B11ScFv-mHSP70刺激后,实验组的淋巴细胞表型与未经蛋白刺激的对照组相比：CD4＋T细胞明显增加,CD8＋T细胞略有增加。CD4＋/CD8＋的比率明显增加,CD4＋CD25＋T细胞占CD4＋T细胞的比率明显下降,差异有统计学意义。结论 6B11ScFv-mHSP70融合蛋白在体外可诱导特异性抗卵巢癌的细胞免疫反应,有可能作为抗独特型疫苗用于卵巢癌的主动免疫。     

抗独特型抗体免疫治疗卵巢癌的动物实验研究

目的：研究抗独特型抗体对卵巢癌的免疫治疗作用，为临床试验提供动物实验依据。方法：用人卵巢癌抗独特型抗体（Ａｂ２）免疫杂交一代小鼠ＢＣＦ１，为实验组；用正常小鼠ＩｇＧ免疫小鼠ＢＣＦ１，为对照组。两组均免疫３次，于末次免疫后１周，将经裸鼠传代的人卵巢癌细胞系移植于ＢＣＦ１小鼠肾脏包膜下，分别于移植后第２、４、６、８和１０天处死小鼠，取血清进行抗独特型抗体（Ａｂ３）分析。移植瘤行组织学检查，观察宿主淋巴细胞浸润情况及瘤细胞可见率。结果：实验组小鼠肾脏包膜下人卵巢癌细胞系很快受到排斥，在移植后第６天，淋巴细胞浸润即达高峰，而对照组在第１０天才达高峰；瘤细胞可见率，在移植第６天后即明显降低，而对照组呈逐渐下降。结论：Ａｂ２作为抗原免疫小鼠后，能够排斥瘤细胞的生长。     

抗独特型抗体免疫治疗卵巢癌的动物实验动物实验研究

目的：研究抗独特型抗体对卵巢癌的免疫治疗作用，为临床试验提供动物实验依据。方法用人卵巢癌抗独特型抗体免疫杂交一代小鼠ＢＣＦ１，为实验组；用正常小鼠ＩｇＧ免疫小鼠ＢＣＦ１，为对照组。     

卵巢癌抗独特型抗体诱导特异性迟发过敏反应的实验研究

本研究应用卵巢癌抗原内影像型单克隆抗独特型抗体６Ｂ１１和１Ｈ１２代替肿瘤抗原，诱导同系小鼠产生卵巢癌特异性细胞免疫反应－迟发性过敏反应（ＤＴＨ），从而证明了６Ｂ１１和１Ｈ１２具有抗原模拟作用。经过６Ｂ１１或１Ｈ１２免疫的小鼠，用卵巢癌细胞系ＳＫＯＶ３进行脚掌注射攻击。而后，测量脚掌水肿厚度作为ＤＴＨ反应强度。６Ｂ１１诱导的ＤＴＨ平均水肿厚度为０．９３ｍｍ，卵巢癌组织抗原ＯＣ１６６－９诱导的阳性对照     

应用基因工程方法制备卵巢癌抗OC125独特型抗体

目的；扩增和表达卵巢癌抗独特型鼠单克隆抗体变区基因，用于制备新型卵果癌疫苗。方法：自产生卵巢抗原内影象型抗ＯＣ１２５特特型抗体的小鼠杂交瘤细胞中提取ｍＲＮＡ，采用反转录聚合酶链，扩增抗体轻、重链可变区基因，连接成单链后重组至表面表达载体ｐＣＡＮＴＡＢ５Ｅ，转化大肠杆菌ＴＧ１，用辅助噬菌体拯救包装成完整噬菌体，用酶联免疫吸附法进行筛选鉴定。结果：获得抗ＯＣ１２５独特型抗体轻、重链可变区基因，长度约３     

The anti-tumor immune responses induced by a fusion protein of ovarian carcinoma anti-idiotypic antibody 6B11ScFv and murine GM-CSF in BALB/c mice

Ovarian carcinoma anti-idiotypic antibody 6B11 was murine derived; we previously have cloned 6B11 single-chain Fv antibody (6B11ScFv) and constructed the 6B11ScFv/human granulocyte-macrophage colony stimulating factor (GM-CSF) fusion protein (designated as 6B11GM) to enhance the immunogenecity of the single-chain Ab(2). Because of the difference in species specificity between human GM-CSF and murine GM-CSF, there is no immune competent animal model on which the effect and metabolism of 6B11GM as a vaccine could be observed. In this study, 6B11mGM fusion gene was constructed by the fusing murine GM-CSF cDNA gene with 6B11ScFv. The fusion gene was cloned and expressed. The product of this gene is a fusion protein. It could specifically interact with the primary anti-ovarian carcinoma monoclonal antibody (COC166-9) and rat anti-mouse GM-CSF monoclonal antibody, respectively, and stimulate the growth of NFS-60 cells (a murine GM-CSF-dependent cell line). The specific anti-tumor immune response could be induced in BALB/c mice after immunized with anti-idiotypic fusion protein instead of ovarian carcinoma antigen without carrier proteins and adjuvant. Ab(3) could be detected in the sera of immunized mice with 6B11mGM by enzyme-linked immunoadsorbent assay test. Moreover, the fusion protein stimulated proliferation of CD4+ T cell from the spleen of BALB/c mice and proliferation of CD8+ T cell to a lesser degree. Therefore, 6B11mGM probably induces both humoral and cellular immunity against ovarian carcinoma in vivo.     

胆酸钠对人卵巢癌细胞系作用的研究

目的 探讨胆酸钠单独应用及联合全反式维甲酸对2株卵巢癌细胞系(COC2、CAOV3)的体外抑制作用。方法 2003年5～8月对经不同浓度的胆酸钠及胆酸钠联合全反式维甲酸作用后的两株细胞采用MTT比色法测定细胞生长抑制率，流式细胞仪测定细胞周期变化，并行培养细胞的光镜、电镜观察。结果胆酸钠对两株细胞的抗增殖活性在50～150mg／L范围内呈现剂量依赖效应，并且在两药合用时此作用更明显；单用胆酸钠及两药联合应用均能随用药浓度而明显增加G0／G1期细胞比例，降低S、G2／M期比例，并以两药合用更为明显；光镜、电镜结果显示：胆酸钠100mg／L组与全反式维甲酸10μmol／L组细胞形态变化相似，但两者合用时细胞凋亡明显。结论 在两株卵巢癌细胞中胆酸钠不但具有单独而且有协同全反式维甲酸抑制肿瘤的作用，胆酸钠能明显降低全反式维甲酸的用量从而减少其副反应，并且不降低其抗肿瘤作用。     

Interferon-induced changes in human ovarian carcinoma cell lines

Metcalf KS, Selby PJ, Trejdosiewicz LK, Southgate J. Interferon-inducedchanges in human ovarian carcinoma cell lines. Int J Gynecol Cancer 1997; 7 : 355–363.
Interferon α (IFNα) may have a clinical role in maintenance therapyof advanced epithelial ovarian cancer following chemotherapy. To investigatethe action of IFN, we havestudied the direct effects of IFNα and IFNγ on cell surface antigenexpression in four human ovarian cancer cell lines (Caov-3, SK-OV-3, SW626 andNIH OVCAR 3). All celllines demonstrated responses to the IFNs, indicating that IFN response elementshad been retained. The effects of IFNα and IFNγ on cell surfacephenotype were investigatedby analytical flow cytometry for a panel of 15 cell surface antigens, whichincluded tumor-associated antigens, growth factor receptors, MHC antigens,adhesion molecules andepithelial mucins. As single agents or in combination, the effects of theinterferons on cell surface phenotype were heterogeneous, with up-regulation ofMHC class I being the mostconsistent observation. IFNγ, alone or with IFNα, also up-regulatedexpression of MHC class II and ICAM-1. IFNα caused down-regulation of cellsurface expression of theCA125 antigen in NIH OVCAR 3 cells by inducing antigen shedding. This haspotential clinical implications for tumor monitoring during IFN therapy inovarian cancer.     

Cytotoxic effects of T cells induced by fusion protein 6B11-pulsed dendritic cells on ovarian carcinoma cells

INTRODUCTION: 6B11 anti-idiotype minibody, a fusion protein, has been shown to mimic ovarian carcinoma associated antigen OC166-9. This study was designed to determine whether 6B11 anti-idiotype minibody-pulsed dendritic cells (DCs) can induce cytotoxic T cells against ovarian cancer cells. METHODS: Monocytes were isolated from peripheral blood mononuclear cells collected from patients with epithelial ovarian carcinoma (n=10). The monocytes-derived immature DCs were stimulated by cytokines, and mature DCs were pulsed with 6B11 anti-idiotype-minibody or murine F(ab)'2 fragments. The proliferation of autologous T cells induced by DCs was determined by 3H-thymidine uptake. The cytotoxicity of DC-activated T cells against autologous carcinoma cells was determined by 51Cr-release assay. RESULTS: Purified T cells demonstrated strong proliferation following incubation with 6B11 anti-idiotype minibody-pulsed DCs in 4 of 10 patients. The specific cytotoxicity of purified T cells against autologous carcinoma cells was induced after stimulation with 6B11 anti-idiotype minibody-pulsed DCs in 5 of 10 patients with cytotoxic effects ranging from 25 to 95%. In contrast, isotype murine F(ab)'2 fragments-pulsed DCs did not induce T cell proliferation and cytotoxicity against the targets. Additionally, the cytotoxic effect was partially inhibited by anti-MHC class-I antibody indicating that the cytotoxic effects are antigen-specific. CONCLUSION: 6B11 anti-idiotype-antibody-pulsed DCs can induce T cell proliferation and T cell-mediated cytotoxicity against autologous ovarian tumor cells in vitro. The cytotoxic effects of T cells against autologous tumor cells are antigen-specific. These data implicate the rationale for the use of 6B11 anti-idiotype minibody as immunotherapy against ovarian carcinoma.     

树突状细胞体外诱导抗卵巢癌免疫的实验研究

目的 观察人外周血树突状细胞(Dendritic cells,DC),体外能否诱导抗卵巢癌免疫应答。方法 用粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)和肿瘤坏死因子α(TNF-α)从健康女性外周血分化诱导DC,以源于人卵巢癌细胞系HO-8910的肿瘤抗原粗提物冲击致敏DC,将致敏DC、同源淋巴细胞和卵巢癌细胞共育,观察负载抗原DC体外诱导淋巴细胞对HO-8910细胞的杀伤作用,同时设不同类型肿瘤细胞(Eca-109和PC-12)作为对照。MTT法测定细胞杀伤活性。结果 经卵巢癌细胞HO-8910肿瘤抗原脉冲致敏的DC能诱导淋巴细胞特异性地杀伤卵巢癌细胞。结论 用GM-CSF、IL-4和TNF-α从人外周血诱生的DC能从卵巢癌细胞HO-8910冻融物有效递呈抗原并诱导出高效而特异的抗卵巢癌免疫反应。     

卵巢癌抗独特型抗体6B11特异性表位肽在体内诱导抗原特异性体液免疫应答

目的探讨卵巢癌抗独特型抗体6B11表位多肽与诱导免疫应答之间的关系。方法采用化学合成的方法，合成抗独特型抗体6B11轻重链可变区的六条CDR区，分别以三种不同浓度免疫BALB／c小鼠。采用ELISA的方法检测免疫鼠血清，进行体内免疫学功能研究。结果6B11抗独特型抗体的六条CDR肽分别加入佐剂免疫小鼠后，其中有两条肽可以诱导出Ab3，分别为重链的CDR2区和轻链的CDR2区。结论这两条肽作为后选肽有可能是6B11抗独特型抗体特异性的CTL表位。     

Genistein对人卵巢癌细胞系TC-1增殖抑制和诱导凋亡作用的研究

目的:研究植物雌激素染料木黄酮(genistein)对人卵巢癌细胞系TC-1细胞增殖的抑制作用和凋亡的诱导作用,探讨其抗癌作用的机制。方法:应用MTT法检测不同浓度genistein对TC-1细胞的生长抑制作用,电镜观察药物作用后细胞超微结构的改变,流式细胞仪检测细胞周期、定量分析凋亡,DNA琼脂糖凝胶电泳法确定凋亡。结果:TC-1细胞经不同浓度genistein处理后,细胞生长明显受到抑制,且这种抑制作用呈时间及浓度依赖性,5mg/L和10mg/L genistein作用72h后,抑制率分别达到89·84%和97·99%。genistein阻断TC-1细胞生长于G2/M期,在G0/G1前出现典型的亚二倍体凋亡峰,且凋亡呈时间依赖性。电镜观察到用药后凋亡细胞典型的形态学特征。DNA凝胶电泳可观察到细胞凋亡特异的DNA梯形带。结论:genistein对TC-1细胞生长的抑制作用呈明显的时间及浓度依赖性,并诱导其发生凋亡。     

卵巢癌抗独特型抗体融合蛋白细胞免疫功能的体外实验

目的 探讨卵巢癌抗独特型单链抗体与人粒细胞－巨噬细胞集落刺激因子形成的融合蛋白作为肿瘤疫苗的可能性。方法 从卵巢癌病人外周血中分离得到单个核细胞、Ｔ淋巴细胞和作为抗原呈递细胞的单核细胞,同时从病人腹水中分离得到癌细胞,分别以卵巢癌抗独特型抗体的全抗体、６Ｂ１１ＧＭ、６Ｂ１１＋ｈＧＭ－ＣＳＦ、ｈＧＭ－ＣＳＦ进行体外Ｔ淋巴细胞增殖实验和自身肿瘤细胞杀伤实验。并对６Ｂ１１和６Ｂ１１ＧＭ的作用进行比较。结     

Profiling of proteins associated with cisplatin resistance in ovarian cancer cells

Resistance to cisplatin is a major impediment to the successful treatment of ovarian cancer, but the precise nature of the resistance is still unclear. In the current study, we aimed to investigate and compare the protein expression profiles in cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines. We employed the recent development of surface-enhanced laser desorption/ionization ProteinChip technology to measure protein expression in three human ovarian cancer cell lines (KF-1, MN-1, and A2780) and their sublines (KF-r, MN-r, and A2780cp) resistant to cisplatin. The ProteinChip Arrays were analyzed using the ProteinChip Reader. We did not find any regularity in protein expressions in secretions of cisplatin-sensitive and cisplatin-resistant cells. But on the IMAC3 array, we captured 12 identical expressions which represent a subset of proteins whose expression levels are different between parent ovarian cancer cells and their cisplatin-resistant cells. In particular, at the molecular weight of 7829 d, three kinds of parent cell lines exhibited an elevated expression and their cisplatin-resistant sublines revealed a lowered expression. At the molecular weight of 6881 d, for KF and MN cell lines, opposite protein expressions were seen in the parent cell line and its cisplatin-resistant subline. We think the interesting protein expressions perhaps suggest some mechanisms involved in cisplatin resistance.     

RelA与uPA在卵巢癌细胞系中表达的相关性研究

目的 探讨RelA对人卵巢癌细胞株A2780、OVCAR-3 uPA合成的影响。方法用RelA、IкBα反义寡核苷酸(ASODN)处理A2780、OVCAR-3细胞株,然后以Western Blot、RT—PCR检测RelA、uPA的表达以及RelA在蛋白、mRNA水平对uPA的调节作用。结果 RelA表达与uPA蛋白及mRNA表达水平均呈明显正相关。RelAASODN下调uPA的表达与其作用时间相俄。结论用RelA ASODN抑制RelA表达可明显下调uPA的表达。