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。     

妇科肿瘤的特异性主动免疫治疗进展

肿瘤的特异性主动免疫治疗(SAIT),即用肿瘤疫苗刺激机体产生针对肿瘤特异性抗原的免疫反应,作为癌症患者手术、化疗或放疗的一种辅助治疗,目的是克服因肿瘤产物造成的免疫抑制状态,增强肿瘤相关抗原(TAA)的免疫原性,以刺激特异性免疫来攻击肿瘤细胞.肿瘤疫苗包括细胞疫苗、分子疫苗和基因疫苗.     

光动力学治疗后的肿瘤细胞裂解物对卵巢上皮性癌大鼠的抗肿瘤作用

目的 探讨光动力学治疗(PDT)后的肿瘤细胞裂解物对卵巢上皮性癌(卵巢癌)大鼠的抗肿瘤作用.方法 选择6-8周龄Fischer344雌性大鼠,实验分为PDT组(腹腔注射PDT后的肿瘤细胞裂解物)、冻融组(腹腔注射冻融后的肿瘤细胞裂解物)、盐水组(腹腔注射生理盐水),间隔1周,3组均腹腔注射大鼠卵巢痛细胞系NuTu19细胞105个;设仅腹腔注射生理盐水者为空白对照组.采用酶联免疫斑点(ELISPOT)实验定量检测经PTD后的肿瘤细胞裂解物和冻融后的肿瘤细胞裂解物刺激后各组大鼠脾淋巴细胞中分泌肿瘤特异性γ干扰素(IFN-γ)的细胞数;乳酸脱氢酶(LDH)释放试验检测各组大鼠脾淋巴细胞中细胞毒T淋巴细胞(CTL)的杀伤活性;观察各组大鼠的生存时间以了解肿瘤细胞裂解物的抗肿瘤效果.结果 经PDT后的肿瘤细胞裂解物刺激后,PDT、冻融、盐水、空白对照组大鼠脾淋巴细胞中分泌肿瘤特异性IFN-γ的细胞数分别为(448.8+34.2)、(211.2±47.9)、(43.3±11.1)、(16.1±2.4)个,PDT组细胞数最多,分别与其他3组比较,差异均有统计学意义(P均<0.05);经冻融后的肿瘤细胞裂解物刺激后,上述4组大鼠脾淋巴细胞中分泌肿瘤特异性IFN-γ的细胞数分别为(151.7±22.6)、(188.7±53.0)、(18.2±12.2)、(8.8±7.7)个,PDT组与冻融组比较,差异无统计学意义(P>0.05).PDT组脾淋巴细胞中CTL的杀伤活性较其他3组明显增强(P<0.05).PDT、冻融、盐水组大鼠中位生存时间分别为234、171、168 d,空白对照组观察至截止时间全部存活,PDT组大鼠中位生存时间长于冻融和盐水组,差异有统计学意义(P<0.05).结论 PDT后的肿瘤细胞裂解物在大鼠体内诱发了特异性的抗肿瘤免疫反应,并可延长卵巢癌大鼠的生存时间,具有较好的抗肿瘤作用.     

化疗免疫“窗口期”对卵巢癌治疗影响的实验研究

目的:采用荷瘤动物模型经化疗联合被动免疫治疗,初步探讨化疗免疫"窗口期"对卵巢癌免疫应答的影响及其可能的免疫学机制。方法:以荷瘤大鼠为研究对象,根据紫杉醇联合卡铂对荷瘤鼠免疫功能改变的不同时期给予CTL(cytotoxic T-lymphocyte)主动免疫治疗。观察荷瘤体积及机体免疫微环境改变。结果:荷瘤鼠紫杉醇和卡铂化疗后不同时期给予CTL转输治疗,其中化疗后第6天即淋巴细胞数最少期,给予免疫治疗,肿瘤生长最缓慢;CT扫描结果示,化疗后6天+免疫治疗组的瘤体积最小,且与其余治疗组有统计学意义。荷瘤鼠免疫功能示,化疗后6天+免疫治疗组中淋巴细胞抗原特异性增殖及CD8+T细胞最明显,而Treg(T-Lymphocytes,Regulatory)细胞明显降低。结论:紫杉醇和卡铂联合CTL治疗肿瘤具有协同作用,其中化疗后6天(即淋巴细胞降到最低期)是CTL免疫治疗的最佳时间点。化疗造成的肿瘤个体免疫功能的低下或是免疫系统空间的释放(所谓的免疫"窗口期")为诱导特异性的抗肿瘤免疫应答提供了可能,免疫状态的检测是保证肿瘤患者化疗联合免疫治疗方案治疗有效性的重要前提之一。     

妇科肿瘤的特异性主动免疫治疗进展

肿瘤的特异性主动免疫治疗(SAIT)，即用肿瘤疫苗刺激机体产生针对肿瘤特异性抗原的免疫反应，作为癌症患者手术、化疗或放疗的一种辅助治疗，目的是克服因肿瘤产物造成的免疫抑制状态，增强肿瘤相关抗原(TAA)的免疫原性，以刺激特异性免疫来攻击肿瘤细胞。肿瘤疫苗包括细胞疫苗、分子疫苗和基因疫苗。     

复发性卵巢癌的免疫治疗

近年来,生物学治疗作为肿瘤治疗的新模式日益受到重视。免疫治疗分为主动免疫治疗和被动(过继性)免疫治疗,免疫治疗作为卵巢癌生物学治疗的主要组成部分,许多制剂已进入临床试验阶段,其目的是提高机体免疫力,消灭微小病灶、延缓复发。1卵巢癌主动免疫治疗用于主动免疫的肿瘤细胞疫苗主要有两类,即完整细胞疫苗和以抗原为基础的疫苗,前者如经过各种方法处理或修饰的、灭活的自体肿瘤细胞,经各种抗原修饰后的自体树突状细胞(DC),后者如肿瘤特异性抗原疫苗和DNA疫苗等。由于肿瘤特异性抗原克隆鉴别困难,而抗独特型抗体(anti Id或Ab2)中的Ab…     

紫杉醇联合卡铂对荷瘤鼠细胞免疫功能影响的研究

目的:揭示紫杉醇联合卡铂对荷瘤大鼠免疫系统的影响,探讨化疗后是否存在免疫功能逆转的"窗口期"。方法:取25只Fischer 344大鼠,随机分为5组,取4组于右腋下种植NuTu-19卵巢癌细胞株,待荷瘤体积生长至0.5cm3后,以牺牲大鼠的时间为第0天,取3组分别于-15天(化疗晚期)、-10天(化疗中期)、-6天(化疗早期)在荷瘤的大鼠腹腔内注射紫杉醇、卡铂。流式细胞技术检测化疗后各时期淋巴细胞数量及刺激活化后外周血Tc1淋巴细胞的功能亚群,H3-TdR掺入法检测化疗后淋巴细胞的增殖。结果:荷瘤鼠与正常大鼠相比,淋巴细胞数量无显著差异,荷瘤组淋巴细胞亚群CD3+T、CD4+T、CD8+T、NK细胞下降,但仅CD3+T细胞较正常组有差异(P<0.05)。荷瘤组肿瘤抑制性Treg细胞明显增高(P<0.05),而具有杀伤功能的Tc1细胞在荷瘤组明显下降(P<0.05),NK的杀伤功能在两组间无显著差异。移植瘤大鼠化疗后免疫功能变化:(1)在用药后不同阶段淋巴细胞数呈先下降后逐渐恢复至正常的变化规律,在化疗后6天降到最低点,15天恢复至化疗前水平;(2)化疗后6天淋巴细胞降到最低,其增殖力最强;(3)化疗后机体外周血中CD3+T、CD4+T、CD8+T细胞、肿瘤抑制性Treg细胞绝对数均先下降后恢复,化疗后6天降到最低,较未化疗组均有统计学意义,化疗后10天开始恢复,化疗后15天均恢复到化疗前水平;(4)化疗后CD8+T细胞中Tc1细胞比例逐渐增高,化疗后6天较未化疗组增高且有统计学意义,化疗后15天达到最高,但较化疗后6天无显著性差异,表明化疗促进具有杀伤功能Tc1的表达。结论:荷瘤鼠的机体呈负调节免疫状态,肿瘤的发生引起了免疫功能的逃逸,肿瘤的免疫治疗应该具有针对性;紫杉醇联合卡铂打破机体内原有的抗肿瘤免疫抑制状态,机体有可能通过免疫重建诱生了增强的抗肿瘤免疫应答,从而使抗肿瘤免疫抑制状态得到暂时逆转,表明机体经化疗后存在免疫"窗口期"。     

脐血来源的细胞毒性T淋巴细胞对人卵巢癌裸鼠移植瘤治疗的研究

目的研究脐血来源的细胞毒性T淋巴细胞用于卵巢癌过继免疫治疗的可行性。方法采用肿瘤细胞和淋巴细胞混合培养法获得脐血来源的细胞毒性T淋巴细胞对人卵巢癌裸鼠移植瘤进行治疗并与外周血来源淋巴因子激活的杀伤细胞相对照。结果脐血来源的细胞毒性T淋巴细胞有明显的抑瘤作用,移植物抗宿主病的发生不明显。结论脐血可作为过继免疫治疗中效应细胞来源试用于卵巢癌的治疗。     

卵巢癌免疫治疗的现状与进展

卵巢癌是最致命的妇科恶性肿瘤之一.传统的治疗方法包括手术和化疗,但是,约70％的患者在初始治疗后出现疾病复发.在过去的20年中,免疫疗法迅速发展,并彻底改变了各种癌症的治疗方法.尽管卵巢癌患者对免疫治疗的反应率仍然不高,但免疫检查点抑制剂、治疗性肿瘤疫苗、过继性细胞免疫治疗正在迅速发展.同时不同的联合治疗策略也在研究和...     

树突细胞疫苗与卵巢癌免疫治疗的研究进展

以免疫治疗为代表的生物治疗日益受到重视.细胞能否进行有效的抗原提呈直接关系到免疫激活和免疫耐受的诱导.树突细胞(DC)是体内抗原提呈功能最强的细胞,在肿瘤免疫治疗中占重要地位,虽然其在体内含量甚微(占外周血1%以下),但分布广泛,具有吞噬、加工、提呈抗原及启动并刺激T细胞介导的特异性肿瘤免疫反应等多种功能.通过不同形式的抗原修饰的DC可增强其抗肿瘤的免疫原性.就DC疫苗与卵巢癌的免疫治疗研究进展做一综述.     

IL-2 enhances standard IFNgamma/LPS activation of macrophage cytotoxicity to human ovarian carcinoma in vitro: a potential for adoptive cellular immunotherapy.

OBJECTIVE: The objective was to evaluate the enhancement of human peritoneal macrophage cytotoxic in vitro activity by the addition of interleukin-2 (IL-2) to the standard interferon gama (IFNgamma) and lipopolysaccharide (LPS) activation procedure used for cellular adoptive immunotherapy in a human ovarian cancer system. This cytotoxic effect of these activated macrophages was tested on cells from ovarian cancers of various stages, histology type, and grade, both prior to chemotherapy and at recurrence, in ovarian carcinoma cells lines and normal cells. Increased activation of the macrophage may make it a better candidate for intraperitoneal cellular adoptive immunotherapy as a component of ovarian cancer therapy. This was not a study of the mechanism of macrophage killing. METHODS: Ascites specimens were collected from 24 ovarian cancer patients at the time of surgery or by paracentesis. The mononuclear cell fraction was isolated by discontinuous density gradient centrifugation and used as a cellular source of peritoneal macrophages (PMs) and primary cultured ovarian cancer cells. PMs were separated by 1-h adhesion followed by intensive washing to remove floating cells. The floating cells were cultured for 24 h which left the cancer cells attached after unattached cells were removed by washing. These cells formed a monolayer of cancer cells, which could be subcultured in 22 patients. The cells from the third to fifth passages were used as target cells without coculture with other cells. PMs were identified by latex ingestion, and their purity after isolation by adhesion culture was tested by flow cytometry and immunofluorescence. PMs were activated by culturing in the presence of IFNgamma, with or without IL-2, for 18 h followed by the addition of LPS 6 h prior to use as effector cells in cytotoxicity assays. Ovarian cancer cells of both established cell lines and primary cultures were labeled with (51)Cr and utilized as target cells to quantitatively measure PM-mediated cytotoxicity. Ovarian cancer cells were also cocultured with PMs for morphologic observations to provide supporting evidence to the cytotoxicity assays. RESULTS: IL-2 enhances the cytotoxicity of the standard IFNgamma/LPS macrophage activation in this system. Peritoneal macrophages so activated are cytotoxic to autologous and allogenic primary cultured ovarian tumors and to ovarian carcinoma cell lines. The macrophages are cytotoxic to cells both prior to treatment and at recurrence, but the data from the few recurrent patients did reach statistical significance. This cytotoxicity is not MHC associated. Normal cells are minimally affected. CONCLUSIONS: IL-2 augmented the standard IFNgamma/LPS method of activating peritoneal macrophage cell killing of human ovarian cancer cells in this in vitro system. The cell killing occurred with autologous and allogenic tumor cells from patients with primary and possibly recurrent tumors. Activated PMs minimally affected the normal cells tested. This enhanced activation may improve the disappointing results of previous adoptive cellular immunotherapy human trials and should be considered for ovarian cancer clinical trials.     

DC融合瘤苗治疗大鼠腹腔播散性卵巢癌

目的:研究树突状细胞(DC)与卵巢癌融合的融合瘤苗对大鼠腹腔播散性卵巢癌的治疗作用,并观察其体内应用的安全性。方法:用聚乙二醇法将Fischer344(F344)大鼠骨髓来源的DC与卵巢癌上皮细胞株NuTu19细胞在体外融合制备融合瘤苗(DCNuTu19)。流式细胞术检测DCNuTu19的免疫相关表型。将DCNuTu19接种于F344大鼠皮下,观察它在体内的成瘤性。用乳酸脱氢酶释放法检测经过DCNuTu19免疫的大鼠脾细胞毒性T淋巴细胞(CTL)对NuTu19细胞的杀伤作用。复制腹腔播散性卵巢癌的F344大鼠模型,观察DCNuTu19对它的治疗作用。结果:DCNuTu19高表达主要组织相容性复合物II(MHCII)分子OX6、共刺激分子B72、细胞间粘附分子ICAM1和大鼠DC的标志性抗原整合素OX62。与NuTu19相比,DCNuTu19在大鼠体内成瘤率降低,成瘤时间延迟,两组平均瘤重具有统计学差异(P<0.05)。经DCNuTu19免疫的大鼠脾CTL对NuTu19细胞的杀伤活性显著增高(P<0.01)。与对照组相比,经DCNuTu19治疗后大鼠的病情发展速度减缓,生存期明显延长(P<0.01)。结论:卵巢癌DC融合瘤苗能显著提高大鼠CTL杀伤活性,诱导高效而特异的抗卵巢癌免疫效应,延长患有腹腔播散性卵巢癌大鼠的生存时间。     

冻融抗原致敏的树突细胞对裸鼠人卵巢癌移植瘤治疗作用的研究

目的 :研究冻融抗原致敏的树突细胞 (DC)对裸鼠人卵巢癌移植瘤的治疗作用。方法 :联合应用粒性白细胞与巨噬细胞集落刺激因子 (GM CSF)及白介素 4 (IL 4 )从正常足月产妇分娩后新生儿脐血中培养出DC ,以人卵巢癌细胞系 3AO细胞冻融抗原激活DC ,测定其诱导的细胞毒性T淋巴细胞 (CTL)对 3AO的杀伤活性 ;CTL预防性接种于裸鼠皮下 ,观察裸鼠人卵巢癌移植瘤的发生率 ,以DC激活的CTL治疗裸鼠人卵巢癌移植瘤并观察治疗效果。结果 :体外抗原冲击致敏的DC能显著刺激T淋巴细胞增殖 ,其诱导的CTL对细胞系 3AO具有显著的杀伤作用 ,在效靶比为 4 0 :1、2 0 :1、10 :1、5 :1时 72h杀伤率平均分别为 90 .1%、67.4 %、4 0 .4 %、17.8%。DC激活的CTL能预防裸鼠人卵巢癌移植瘤的发生 (预防组 16.6% ,对照组 10 0 % ,P <0 .0 0 1) ,并能抑制移植瘤生长 ,对照组、治疗组移植瘤的大小分别为 (5 .6± 1.1)cm3 、(2 .7± 0 .78)cm3 (P <0 .0 1)。结论 :人卵巢癌细胞冻融抗原体外冲击致敏的DC可作为一种抗癌疫苗在免疫治疗卵巢癌患者中发挥重要作用     

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     

树突细胞疫苗诱发抗肿瘤免疫特异性的体内研究

目的:探讨卵巢肿瘤提取物负载的树突细胞(DC)体内诱发抗肿瘤免疫效应的特异性。方法:应用rrGM-CSF(终浓度40ng/ml)、rrIL-4(终浓度40ng/ml)及rrTNF-α(终浓度40ng/ml)体外培养Fischer344大鼠骨髓来源的单个核细胞,于培养第12天通过流式细胞仪检测细胞标志CD86、OX62抗原,用电镜观察细胞形态,证实获得树突细胞。分别利用NuTu-19卵巢肿瘤细胞和CBRH-7919大鼠肝癌细胞肿瘤提取物制备肿瘤抗原致敏Fischer344大鼠骨髓来源的树突细胞,得到两种肿瘤DC疫苗,即NuTu-DC和CBRH-DC。体内试验:Fischer344大鼠随机分为3组(1)对照组;(2)NuTu-DC治疗组;(3)CBRH-DC治疗组。于大鼠皮下接种NuTu-19细胞,5天后出瘤,开始于肿瘤接种对侧接种DC疫苗,每隔1周给予疫苗1次,共治疗2次。于第0天(未荷瘤时),第5天(出瘤后),第26天(治疗后1周),第40天(治疗后3周)天眼球取血分析各组动物外周血CD3/4/8表达水平;于荷瘤30,32,34,36,38,40天测量肿瘤体积,观察大鼠皮下肿瘤生长情况。于荷瘤40天处死各组实验动物,取肿瘤组织称重,评价治疗效果。结果:大鼠骨髓来源的骨髓单个核细胞(bone marrow mononuclearcells,BMMNC)在相关细胞因子的作用下可以培养出成熟的树突细胞,负载肿瘤提取物后,高表达表面抗原OX-62,CD-86。流式细胞仪检测外周血CD3/4/8水平显示:各组大鼠CD4/CD8比值升高,与未荷瘤时(第0天)相比,差异有统计学意义(P<0.05);CD3+CD4+T细胞表达百分率及CD4/CD8在治疗后3周时(第40天)NuTu-DC治疗组明显高于另两组(P<0.05),而对照组与CBRH-DC比较无差异。通过测量肿瘤生长曲线可见NuTu-DC治疗组各大鼠肿瘤体积较其它两组小,荷瘤第40天时,NuTu-DC治疗组为106.57±56.65mm3,对照组为299.78±118.17mm3,CBRH-DC治疗组为299.12±85.61mm3,与对照组和CBRH-DC组比较差异有统计学意义(P<0.01);而且NuTu-DC治疗组肿瘤平均瘤重为0.197±0.039g,与对照组和CBRH-DC治疗组有显著差异(P<0.01),其抑瘤率为64.4%;而CBRH-DC治疗组大鼠平均肿瘤体积、生长速度及瘤重与对照组无明显差异(P>0.05),其抑瘤率仅为0.22%。结论:应用大鼠卵巢癌细胞肿瘤提取物负载的树突细胞体内治疗卵巢癌荷瘤大鼠,能有效抑制肿瘤生长,且所诱发的抗肿瘤免疫具有组织特异性。     

褪黑素对新生儿脐带血单个核细胞产生细胞因子的影响

目的 探讨褪黑素(melatonin,MLT)对新生儿脐带血单个核细胞(cord blood mononuclear cells,CBMC)产生细胞因子的影响. 方法 体外培养10例健康足月新生儿CBMC,取10例健康成人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)作正常对照,不同浓度MLT(50 pg/ml～50 ng/ml)作为刺激物与CBMC或PBMC共培养,测定培养上清中IL-β、IL-2、IL-4、IL-6、IL-8、IL-10、IL-12、IFN-γ及TNF-α水平. 结果 500 pg/ml MLT可刺激CBMC产生较高水平的IL-2[(12.18±7.31)pg/ml]、IL-6[(2091.08±968.58)pg/ml]、IL-8[(2779.04±1168.49)pg/ml]、IL-12[(15.29±6.88)pg/ml]及IFN-γ[(52.22 17.77)pg/ml],与对照组[分别为(6.99±4.94)pg/ml、(1017.06±452.60)pg/ml、(1293.33±791.34)pg/ml、(6.84±2.26)pg/ml、(22.24±10.38)pg/ml]相比,差异有统计学意义(P<0.05);与对照组[(175.63±62.10)pg/ml]相比,不同浓度MLT(50 ng/ml、5 ng/ml、500 pg/ml、50 pg/ml)均可降低CBMC培养上清中TNF-α水平,分别为(24.87±11.21)pg/ml、(18.14±9.44)pg/ml、(31.38±15.28)pg/ml和(28.18±17.35)pg/ml,差异均有统计学意义(P<0.05).5 ng/ml MLT可诱导PBMC产生IL-2、IL-12及IFN-γ,抑制TNF-α的分泌,与对照组比较,差异有统计学意义(P<0.05).比较各自最佳效应浓度的MLT对CBMC(MLT最佳效应浓度为500 pg/ml)和PBMC(MLT最佳效应浓度为5 ng/ml)释放细胞因子IL-2、IL-12、IFN-γ的诱导作用及对TNF-α的抑制作用,除TNF-α外,差异均有统计学意义(P<0.01). 结论 MLT不但可调节PBMC细胞因子的释放,也可调节CBMC细胞因子的释放,且MLT对CBMC分泌细胞因子IL-12、IFN-γ等的调节作用强于其对PBMC的作用.因此,MLT有可能提高新生儿的免疫功能,为临床应用提供了实验依据.     

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.     

卵巢上皮性癌肿瘤浸润淋巴细胞的增殖、表型及细胞毒活性研究

目的:探讨卵巢上皮性癌肿瘤浸润淋巴细胞TIL的生物学特性。方法:用机械法和酶法消化实体瘤后,经分步低速离心行连续密度梯度法分离卵巢癌TIL,再在IL-2、PHA或OKT_3条件下培养,表型分析采用间接免疫荧光染色法,细胞毒性检测采用MTT法。结果:15份卵巢癌TIL中,得率范围106-107/g,13份TIL活化（另2份污染）,其中7份TIL经培养达治疗量（109细胞）;初分离的卵巢癌TIL处于功能抑制状态,抑制期5-15d,随着活化后体外培养,CD3、CD8、CD25（IL-2R）和 HLA-DR表达显著增多;初分离的卵巢癌TIL对K562和自体瘤细胞杀伤力均低下,随着体外培养,细胞毒性增加,对自体瘤的特异性杀伤力增加尤为显著。结论:卵巢癌TIL体外易分离及活化,特异性杀伤力较强,适于肿瘤的生物学治疗。     

B、T淋巴细胞弱化因子胞外段联合热休克蛋白70-子宫颈癌TC-1细胞抗原肽复合物治疗小鼠子宫颈癌模型的效果

目的 探讨B、T淋巴细胞弱化因子(BTLA)胞外段联合热休克蛋白(HSP)70-宫颈癌TC-1细胞(HSP70-TC-1)抗原肽复合物治疗小鼠宫颈癌模型的效果.方法 (1)将TC-1细胞接种于C57BL/6小鼠成瘤后,实时荧光定量PCR技术检测小鼠肿瘤组织中BTLA、疱疹病毒侵入介质(HVEM)mRNA的表达,流式细胞仪检测肿瘤组织中浸润的淋巴细胞细胞膜表面BTLA、HVEM的表达(以荧光强度表示).(2)小鼠接种TC-1细胞后,根据处理方法的不同分为5组,即pcDNA3.1(注射空载体pcDNA3.1质粒,作为对照)、psBTLA(注射表达BTLA胞外段的真核表达载体psBTLA质粒)、HSP70(注射HSP70-TC-1抗原肽复合物)、HSP70+pcDNA3.1(注射HSP70-TC-1抗原肽复合物和pcDNA3.1质粒)、HSP70+psBTLA(注射HSP70-TC-1抗原肽复合物和psBTLA质粒)组,观察各组小鼠体内肿瘤的生长情况;并采用实时荧光定量PCR技术检测各组肿瘤组织中相关免疫基因包括γ干扰素(IFN-γ)、白细胞介素(IL)2、IL-10、转化生长因子β(TGF-β)和人叉头型基因P3(FoxP3)mRNA的表达;免疫组化法计数各组瘤旁组织中CD8+T淋巴细胞;7-氨基放线菌素D/羧基荧光素乙酰乙酸琥珀酰亚胺酯双标法检测各组脾淋巴细胞的杀伤效应(以杀伤率表示);氚标记胸腺嘧啶核苷掺入法测定各组脾淋巴细胞的增殖活性,酶联免疫吸附试验测定各组脾淋巴细胞培养上清液中IL-2和IFN-γ的浓度.结果 (1)小鼠接种TC-1细胞后第7、14、21、28天,其肿瘤组织中BTLA mRNA的表达水平逐渐上调,第14天达最高,为2.83±0.35,与第7天的1.66±0.25比较,差异有统计学意义(P<0.05);而HVEM mRNA的表达水平无明显变化(P>0.05).接种TC-1细胞后第7、14天,肿瘤组织中浸润的淋巴细胞细胞膜表面BTLA的平均荧光强度分别为33.5和51. 8,两者比较,差异有统计学意义(P<0.05);而HVEM的平均荧光强度分别为57.2和49.3,两者比较,差异无统计学意义(P>0.05).(2)接种后第14、28天,HSP70+psBTLA组抑瘤率分别为65%、88%,均明显高于其他组(P<0.05).接种后第28天,HSP70+psBTLA组IFN-γ、IL-2 mRNA的相对表达水平(分别为3.12±0.71、3.20±0.62)高于其他组,TGF-β、IL-10和Foxp3 mRNA的相对表达水平(分别为0.25±0.03、0.31±0.04、0.19±0.03)低于其他组,分别比较,差异均有统计学意义(P<0.05).HSP70+psBTLA组瘤旁组织中CD8+T淋巴细胞数为(52±6)个/高倍视野,脾淋巴细胞对TC-1细胞的杀伤率为(65.5±2.4)%,脾淋巴细胞的增殖活性为15.0×103 cpm,IL-2和IFN-γ的浓度分别为(824±51)、(1096±112)pg/ml,均明显高于其他组(P<0.05).结论 真核表达载体psBTLA表达的BTLA胞外段联合HSP70-TC-1抗原肽复合物可使肿瘤微环境中的免疫相关基因表达更有利于抗肿瘤免疫应答,对TC-1细胞构建的小鼠宫颈癌模型具有很好的治疗效果.     

Induction of ovarian tumor-specific CD8+ cytotoxic T lymphocytes by acid-eluted peptide-pulsed autologous dendritic cells

OBJECTIVE: To evaluate the potential of dendritic cells pulsed with acid-eluted peptides derived from autologous ovarian cancer cells for eliciting a tumor-specific cytotoxic T cell response in women with advanced ovarian cancer. METHODS: CD8+ T lymphocytes derived from peripheral blood mononuclear cells stimulated in vitro with autologous ovarian tumor peptide-pulsed dendritic cells were tested for their ability to induce an HLA class I-restricted cytotoxic T lymphocyte response against autologous tumor cells. To correlate cytotoxic activity by cytotoxic T lymphocytes with T cell phenotype, we used two-color flow cytometric analysis of surface markers and intracellular cytokine expression (interferon-gamma versus interleukin-4). RESULTS: CD8+ cytotoxic T lymphocyte responses against autologous ovarian tumor cells were elicited in three consecutive women who had advanced ovarian cancer. Although cytotoxic T lymphocyte populations from all women expressed strong cytolytic activity against autologous tumor cells, they did not lyse autologous lymphoblasts or Epstein-Barr virus-transformed cell lines, and they showed negligible cytotoxicity against the natural killer-sensitive cell line K-562. Cytotoxicity against the autologous tumor cells was significantly inhibited by anti-HLA class I (W6/32) and anti-HLA-A2 (BB7-2) monoclonal antibodies. CD8+ cytotoxic T lymphocytes expressed variable levels of CD56 and preferentially expressed interferon-gamma rather than interleukin-4. CONCLUSIONS: Peptide-pulsed dendritic cells induced specific CD8+ cytotoxic T lymphocytes that killed autologous tumor cells from women with advanced ovarian cancer. This finding might contribute to the development of active or adoptive immunotherapy for residual or resistant ovarian cancer after standard surgery and cytotoxic treatment.