树突细胞-食管癌融合细胞体外诱导特异性抗食管癌免疫反应

背景与目的:树突细胞(dendritic cells,DC)是人体专职的抗原呈递细胞,以DC为基础的DC/肿瘤细胞融合疫苗可以有效地诱导特异性抗肿瘤免疫应答.本研究旨在探讨成熟的DC与人食管癌细胞株ECl09细胞融合疫苗体外诱导特异性抗食管癌细胞的免疫反应.方法:从健康志愿者外周血中分离出单个核细胞(peripheral blood mononuclear cells,PBMC),在重组人粒/巨噬细胞集落刺激因子(recombinant human granulocyte-macrophage-colony-stimulating factor,rhGM-CSF)和白介素(interleukin-4,IL-4)作用下体外诱导DC,采用聚乙二醇(polyethylene glyco1,PEG)融合法使DC与EC109细胞融合制备DC/EC109细胞融合疫苗,四甲基偶氮唑盐(MTT)实验检测融合疫苗刺激T淋巴细胞增殖的活性,乳酸脱氢酶(LDH)实验检测融合疫苗活化的细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)在体外特异性杀伤EC109细胞的活性,并与对人胃癌细胞株SGC7901及人乳腺癌细胞株MCF7的杀伤作用进行比较.结果:DC与EC109细胞的融合效率最高达到22.25%.DC/EC109细胞融合疫苗能有效地刺激T淋巴细胞的增殖反应,其刺激T淋巴细胞增殖的能力显著高于DC或EC109细胞(P＜0.05).DC/EC109细胞融合疫苗活化的CTL对EC109细胞具有特异性的杀伤作用,对EC109细胞的杀伤作用显著强于对SGC7901细胞及MCF7细胞的杀伤作用(P＜0.05).结论:DC/EC109细胞融合疫苗能有效诱导抗EC109细胞的特异性免疫应答.     

兔肺VX2肿瘤经皮射频消融术前后HSP70表达的改变

目的　明确肺肿瘤经皮射频消融 (PRFA)术前后热休克蛋白 70 (HSP70 )表达的差异 ,探讨肺肿瘤PRFA治疗与机体免疫功能变化的关系。方法　采用VX 2肿瘤细胞悬液种植法建立兔肺肿瘤模型 ,进行PRFA后 ,观察原发灶及肿瘤的转移情况 ,并将肿瘤组织进行免疫组化染色 ,观察PRFA前后HSP70表达的变化。结果　PRFA后的VX 2肿瘤原发灶生长缓慢 ,腹腔淋巴结转移率低于对照组 ,7天时VX2肿瘤HSP70表达较对照组增加 ( 61.3 6%± 9.5 8%vs2 2 .13 %± 8.92 % ,P <0 .0 5 )。结论　PRFA后 ,肺VX 2肿瘤组织HSP70表达增加 ,可能增强递呈肿瘤特异性抗原而激活机体的抗肿瘤免疫     

靶向VEGF基因的siRNA体外抑制兔VX2细胞生长的研究

目的:探讨靶向VEGF基因的siRNA对兔VX2肿瘤细胞生长的影响。方法:采用化学合成法得到靶向兔VX2细胞VEGF基因的siRNA分子,应用脂质体将其转染体外培养的VX2细胞。转染48h后,RT-PCR法检测VEGF mRNA水平,ELISA法检测细胞分泌的VEGF蛋白,MTT法检测细胞生长活力。结果:VEGF-si1和VEGF-si3单独转染VX2细胞,细胞的生长抑制率分别为（38.5±7.3）%和（30.0±5.8）%,均显著高于scr-siRNA转染的对照组（P<0.01）;细胞VEGF mRNA表达水平分别为（0.37±0.06）和（0.45±0.07）,均显著低于对照组（P<0.01）;细胞分泌VEGF蛋白的抑制率分别为（58.1±7.3）%和（51.9±7.9）%,均显著高于对照组（P<0.01）。结论:VEGF siRNA分子在体外能特异地抑制兔VX2细胞的VEGF基因mRNA转录,VEGF蛋白分泌以及细胞增殖。     

弥散成像评价兔软组织VX2肿瘤细胞增殖的价值

目的:分析反映兔软组织VX2肿瘤细胞增殖情况Ki-67标记指数与由弥散加权影像(DWI)计算出表观弥散系数(ADC)的相关性.方法:将VX2 肿瘤接种于18只健康新西兰大白兔右侧后腿肌肉内,3～4周后对实验用兔行DWI检查.利用MR扫描仪上的软件对所获DWI图像进行处理生成ADC图,测量在肿瘤内所选2～4个感兴趣区(ROI)的ADC值.随后处死实验用兔,获取肿瘤标本,在与ADC图的ROI相对应非坏死区进行取材,对所获肿瘤组织行Ki-67免疫组化染色后测量Ki-67标记指数.利用Spearman相关分析法评价VX2肿瘤组织Ki-67标记指数与ADC值的相关性.结果:所有实验用兔的VX2肿瘤种植成功率为100%.在进行DWI检查前,1只实验用兔因麻醉意外死亡,另1只于饲养过程中不明原因死亡.总共分析了39块VX2肿瘤组织,其Ki-67标记指数与ADC值呈显著负相关,r=-0.873,P<0.001.结论:ADC值通过反映Ki-67标记指数可用于评价软组织肿瘤的细胞增殖情况,从而帮助判断预后、指导治疗并监测疗效.     

双歧杆菌对兔VX2瘤HIF 1α和VEGF表达的影响

目的: 探讨青春型双歧杆菌对兔VX2瘤组织HIF1α、VEGF表达的影响。方法: 建立兔荷VX2瘤模型20只，随机分成2组，每组10只。对照组经耳缘静脉注射生理盐水，实验组经耳缘静脉注射青春型双歧杆菌。处死动物，取出脏器和肿瘤组织粉碎后进行厌氧培养和革兰染色，观察双歧杆菌对肿瘤的靶向性；另取部分肿瘤组织HE染色观察肿瘤组织的形态学变化；应用免疫组织化学方法分别检测肿瘤组织中HIF1α、VEGF的表达。结果: 青春型双歧杆菌能够靶向定植于肿瘤组织中；经双歧杆菌治疗后实验兔肿瘤组织坏死面积明显增加。免疫组化显示实验兔VX2瘤组织中HIF1α、VEGF阳性表达细胞密度显著低于对照组（P<0.05），且两组中HIF1α与VEGF的表达呈显著正相关（r=0.86）。结论: 青春型双歧杆菌能够在兔VX2瘤组织中靶向定植，且能下调兔VX2瘤中HIF1α和VEGF的表达     

兔VX2骨肿瘤模型研究进展

兔VX2骨肿瘤模型是一种非骨源性的同种异体移植性骨肿瘤模型,具有制备方法简单,生物学性质稳定,传代迅速并且容易复制等特点。该模型在对恶性骨肿瘤的诊断及治疗方法的研究中广泛应用。兔VX2骨肿瘤模型建立方法包括细胞悬液种植法和组织包块种植法,种植点也多种多样,研究人员可以根据其研究目的选用适当的方案。随着医学技术的快速发展,兔VX2骨肿瘤模型的制作和应用日趋多样。本文对兔VX2骨肿瘤的来源、生物学特性、模型制作方法及研究应用等方面的研究进展进行综述。     

兔VX2移植瘤PET-CT的显像研究

目的:研究兔VX2移植瘤的PET/CT显像及其应用前景.方法:48只日本大白兔种植VX2肿瘤,随机分为4组,分别于2、4、6和8周进行PET/CT显像,获得注射18F-FDG后不同时间(20、40、60、80、100和120 min)PET/CT图像,以及肿瘤组织和正常组织的SUV值.显像后处死大白兔,进行病理检查,观察肿瘤细胞的核分裂像及平均肿瘤微血管密度.结果:VX2移植瘤总成活率为80%;注射18F-FDG后40～100 min的SUV值与20和120 min的SUV值相比,差异有统计学意义,P≤0.001.种植VX2肿瘤细胞后2、4、6和8周的肿瘤组织和正常组织的SUV值差异均有统计学意义,P均<0.05.肿瘤大小的增殖、病理性核分裂像和平均肿瘤微血管密度与注射18F-FDG后60 min 的SUV值密切相关.结论:PET/CT还可以清楚地显示肿瘤的解剖部位、形态、大小和肿瘤细胞的代谢状况,及时发现转移,是一项可靠和有价值的检查手段.     

组织块悬液注射法制作兔VX2乳腺癌模型

目的：建立稳定的兔VX2乳腺癌模型，选择最佳的模型制作方法。方法：30只兔随机分为3组，每组10只，分别采用乳腺内瘤细胞液注射法、组织块悬液注射法、组织块种植法制作兔VX2乳腺癌模型，比较各组的种植成活率、肿瘤生长率、荷瘤兔存活时间，并观察肿瘤的生物学形态。结果：组织块悬液注射法操作简便，成瘤率高，肿瘤增殖旺盛，生物学特征符合乳腺鳞状细胞癌。结论：成功建立了兔VX2乳腺癌模型，组织块悬液注射法是最佳模型制作方法。     

高强度聚焦超声消融兔VX2种植性乳腺肿瘤的病理学变化

目的:观察高强度聚焦超声(high intensity focused ultrasound,HIFU ) 消融兔VX2种植性乳腺肿瘤后,"即刻"这一时间点的肿瘤组织病理学变化.方法:36只新西兰兔接受VX2肿瘤种植后2周,随机分为治疗组24只和对照组12只.治疗组在B型超声监控下接受HIFU消融治疗后即刻取材,HE染色后在光学显微镜下观察病理组织学变化,然后进行电子显微镜观察、琥珀酸脱氢酶(succinate dehydrogenase,SDH)活性检测和肿瘤抗原性免疫组织化学检测.结果:组织学观察发现,HIFU消融后的靶区内肿瘤细胞形态和细胞核型无明显改变,细胞质出现空泡样变;电子显微镜观察显示,肿瘤细胞呈凝固性坏死;SDH活性检测显示肿瘤细胞失活;增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)在对照组的肿瘤细胞内呈阳性表达,而在治疗组的肿瘤细胞中不表达. 结论:VX2种植性乳腺肿瘤经HIFU消融后, 组织学观察到看似正常的肿瘤细胞经电子显微镜、酶学和免疫组织化学检测证实已经死亡.     

双歧杆菌对兔VX2瘤HIF-1α和VEGF表达的影响

目的：探讨青春型双歧杆菌对兔VX2瘤组织HIF-1α、VEGF表达的影响。方法：建立兔荷VX2瘤模型20只，随机分成2组，每组10只。对照组经耳缘静脉注射生理盐水，实验组经耳缘静脉注射青春型双歧杆菌。处死动物，取出脏器和肿瘤组织粉碎后进行厌氧培养和革兰染色，观察双歧杆菌对肿瘤的靶向性；另取部分肿瘤组织H—E染色观察肿瘤组织的形态学变化；应用免疫组织化学方法分别检测肿瘤组织中HIF-1α、VEGF的表达。结果：青春型双歧杆菌能够靶向定植于肿瘤组织中；经双歧杆菌治疗后实验兔肿瘤组织坏死面积明显增加。免疫组化显示实验兔VX2瘤组织中HIF-1α、VEGF阳性表达细胞密度显著低于对照组（P〈0．05），且两组中HIF-1α与VEGF的表达呈显著正相关（r=0．86）。结论：青春型双歧杆菌能够在兔VX2瘤组织中靶向定植，且能下调兔VX2瘤中HIF-1α和VEGF的表达。     

红细胞肿瘤疫苗的制备及对肿瘤复发转移作用的观察

目的：制备一种新型肿瘤疫苗，观察其对肿瘤复发的转移的作用。方法：用PEG将血红细胞（RBC）与小鼠于宫颈癌（U14）细胞融合，制图瘤苗。MTT法检测新型瘤苗诱发的CTL活性。用复发瘤抑制率及肿瘤转移凶制率来评价瘤苗的疗效。结果：融合细胞携带了RBC的血型抗原。在小鼠体内瘤苗可激发特异的CTL活性，对U14细胞的杀伤率为37．8％；而对照小鼠前胃癌（MFC）细胞的杀伤率为22．0％。瘤苗治疗组复发瘤抑制率为39．9％，肺转移的抑制率为69．9％。结论：RBC与肿瘤细胞融合制备的细胞可作为瘤苗。该瘤苗可有效抑制肿瘤复发和转移。     

Targeting of IL-2 and GM-CSF immunocytokines to a tumor vaccine leads to increased anti-tumor activity

Fusion proteins combining antibodies with cytokines such as IL-2 and GM-CSF appear to be promising reagents for tumor therapy. In this study, we combined such immunocytokines with the tumor vaccine ATV-NDV consisting of irradiated tumor cells infected with Newcastle disease virus (NDV). The two fusion proteins bsF-GMCSF and tsHN-IL2-GM-CSF, binding, respectively, to the viral fusion protein (F) or to hemagglutinin-neuraminidase (HN) expressed on the surface of the vaccine cells and containing GM-CSF or GM-CSF and IL-2-activities were produced by recombinant antibody technology. The purified molecules showed the expected binding specificity and biological activity inherent to the respective cytokine. Using a newly established in?vitro tumor neutralisation assay (TNA), we showed improved antitumoral effect through tumor growth inhibition when human peripheral blood mononuclear cells from healthy donors were stimulated with immunocytokine modified versus non-modified tumor vaccine cells. These effects induced by the fusion proteins, in the presence of a suboptimal T cell activation signal 1 provided by bsHN-CD3, occured only when these were bound to the tumor vaccine. Furthermore, it was shown that CD14+ monocytes could be activated by the GM-CSF cytokine fused within the recombinant proteins and that they contributed essentially to the antitumor effect in the TNA. The data presented here suggest an easy way for a broad clinical development and application of tumor-targeted IL-2- and GM-CSF-based immunocytokines based on the associated increase of anti-tumor activity mediated by T cells and monocytes.     

Enhanced anti-tumor immunity generated by Rituximab-coated tumor cell vaccine

Rituximab is a chimeric monoclonal antibody against CD20, and has been used to treat malignant tumors derived from B cell. We designed a tumor cell vaccine modified by Rituximab, and evaluated anti-tumor effect in human CD20 gene transfected mice tumor model in vivo, and in human CTLs induction by mixed lymphocyte tumor cell culture in vitro. The results demonstrated that the Rituximab-coated tumor cell vaccine had a significant therapeutic effect against tumor pulmonary metastasis formation. Antibody depletion experiments showed CD8+ T Cells were essential for the anti-tumor effect but not NK cells. Capture rate of tumor cells by DCs, which were detected by flow cytometry, was increased by adding Rituximab. The tumor specific cytolysis could be induced by Rituximab-coated tumor cell in human in vitro assay. This therapeutic strategy provides a simple way to potentialize CTLs function to combat cancer and may promote more clinical consideration in immunotherapy for tumors. Rituximab-coated tumor cell vaccine also expanded the clinical Rituximab applications.     

人LAK细胞在体外和裸鼠体内抗人肺巨细胞癌（PG）的研究

Human LAK cells were prepared by culturing normal human peripheral blood mononuclear cells (PBMC) with or without rIL-2 and assayed for T cell surface markers as well as anti-tumor activity against PG in vitro and in nude mice. Although the percentages of T3, T4 and T8 positive cells in rIL-2-activated cells did not differ significantly from those of control cells in vitro, the former showed stronger cytotoxicity than control cells to PG tumor cells in vitro. In vivo, LAK cells completely inhibited the growth of PG tumor in nude mice, whereas PBMC control cells were of no effect. The anti-tumor effect of human LAK cells in nude mice may offer a useful model to study the role of human LAK cells against human tumor in vivo.     

细胞融合大鼠肝癌疫苗体内治疗和预防作用研究

目的 :观察大鼠 BERH - 2肝癌细胞与激活 B细胞的细胞融合大鼠肝癌疫苗对肿瘤的预防和治疗作用。方法 :应用 PEG将大鼠 BERH- 2肝癌细胞与激活 B细胞融合 ,制备细胞融合大鼠肝癌疫苗 ;部分肝癌疫苗免疫动物前经 6 0 Co照射 ;大鼠被肝癌疫苗免疫之前或之后 ,接种肝癌 BERH- 2细胞或组织块 ,观察疫苗对肝癌的预防和治疗作用。结果 :经细胞融合疫苗保护的大鼠可获 10 0 %无瘤生存 (8/8) ,而 HERH- 2或照射后 BERH- 2对照组大鼠均生长肿瘤死亡 (0 /8)。经皮下接种细胞融合肝癌疫苗治疗大鼠 ,肝内注射肝癌细胞的大鼠可获 85 %以上无瘤存活。照射后细胞融合肝癌疫苗与未照射的获得同样治疗效果。不经筛选的细胞融合疫苗也获相同治疗效果 ,而对照组大鼠均生长肿瘤后死亡。结论 :细胞融合肝癌疫苗对大鼠有预防和治疗作用 ,照射的细胞融合大鼠肝癌疫苗和不经筛选的融合细胞同样具有较好的治疗作用。     

The p38 MAPK inhibitor BIRB796 enhances the antitumor effects of VX680 in cervical cancer

VX680 is a potent and selective inhibitor that targets the Aurora kinase family. The p38 mitogen-activated protein kinase (MAPK) regulates a large number of cellular pathways and plays an important role in the regulation of cell survival and apoptosis. This study aimed to evaluate the effect of VX680 on cervical cancer cells and investigate whether the effects on apoptosis are enhanced by the ablation of p38 MAPK activation. The results suggested that VX680 inhibited the proliferation of cervical cancer cells by causing G2/M phase arrest and endoreduplication and that the apoptotic effect was attenuated by the activation of p38 MAPK. However, the addition of BIRB796, which is an important p38 MAPK inhibitor, effectively eliminated the expression of p-p38 and hence significantly enhanced the cell death induced by VX680 in vitro. Further study demonstrated that BIRB796 cooperated with VX680 to suppress cervical cancer cell growth in a mouse xenograft model. Taken together, our results demonstrated that VX680 induced cell cycle arrest and endoreduplication in human cervical cancer cells. Combined treatment with VX680 and BIRB796 synergistically inhibited tumor growth both in vitro and in vivo. Dual blockade of Aurora kinases and p38 MAPK is therefore a promising strategy for cervical cancer treatment.     

Biochemical modulation of 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine incorporation into DNA in VX2 tumor-bearing rabbits.

The thymidine analogues 5-bromo-2'-deoxyuridine (Brd-Urd) and 5-iodo-2'-deoxyuridine (IdUrd) compete with thymidine for incorporation into the DNA of replicating cells. This incorporation results in radiosensitizing effects which are directly related to the degree of analogue substitution. In vitro and in vivo evidence suggests that preadministration or coadministration of the thymidylate synthetase inhibitors fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd) can modulate analogue incorporation into DNA. We have evaluated in the rabbit VX2 tumor model the effects of thymidylate synthetase inhibitor (fluorouracil or FdUrd) coadministration (as 24-hour, intravenous infusions) on the incorporation of BrdUrd or IdUrd into the DNA of relevant normal tissues (bone marrow, gut mucosa) and intrahepatic VX2 tumor. Tissues were harvested and processed for gas chromatography-mass spectrometry analysis of the thymine, 5-bromouracil, and 5-iodouracil contents in hydrolyzed DNA. Coadministration of FdUrd resulted in statistically significant (P less than .01) enhancement of IdUrd incorporation into the DNA of intrahepatic VX2 tumor and normal (bone marrow and duodenal mucosa) rabbit tissues. Coadministered fluorouracil, on the other hand, significantly enhanced IdUrd incorporation only into DNA of intrahepatic VX2 tumor. Statistically significant enhancement of BrdUrd incorporation was achieved only with FdUrd coadministration and then only into the DNA of intrahepatic VX2 tumor. The percent of thymine replaced by analogue (I) is related to the steady-state arterial plasma drug concentration (C) by the Michaelis-Menten equation: I = I(MAX.) C/(C50 + C). The primary effect of FdUrd coadministration on BrdUrd incorporation into VX2 tumor DNA was a reduction of the C50 parameter (plasma BrdUrd concentration eliciting I = I(MAX)/2) from 8.17 microM to 1.78 microM. On the other hand, the I(MAX) parameter (I as C approaches infinity) was only slightly affected (29.7% to 25.2%). Thus, the degree to which the modulator enhanced analogue incorporation varied inversely with the analogue's steady-state plasma concentration. These results, which describe potential tissue specificity of modulator efficacy and characterize the effects of thymidylate synthetase inhibitor modulation on thymidine analogue incorporation pharmacodynamics, should provide guidance as to dose scheduling of BrdUrd and IdUrd in clinical trials for improved tumor specificity of uptake.     

中药香加皮提取物的体内外抑瘤效果研究

背景与目的:研究中药香加皮水提取物(cortex periplocae extract,CPE)的体内、外抗肿瘤效果.材料与方法:采用MTT法和克隆形成实验,检测CPE对6种不同组织来源肿瘤细胞(K562、BGC-823、TE-13、SMMC-7721、MCF-7和HeLa)增殖反应的影响;将CPE灌胃给予S180和colon 26荷瘤小鼠,观察其对肿瘤形成及小鼠生存期的影响.结果:CPE对6种不同肿瘤细胞株均有明显的抑制作用(P＜0.05),其中对K562,BGG-823,TE-13的抑制作用量效关系明显(r分别为0.89、0.71和0.72,P值均＜0.05).将CPE灌胃S180或colon26荷瘤小鼠,可明显抑制肿瘤在体内的生长,并可明显延长荷瘤小鼠的生存期(P＜0.05).结论:CPE对6种不同组织来源的肿瘤细胞均具有抑制作用,可明显抑制荷瘤小鼠肿瘤的生长,延长荷瘤小鼠寿命.香加皮作为抗肿瘤药物有待进一步开发研究.     

Tumor antigen-specific T-cell expansion is greatly facilitated by in vivo priming.

PURPOSE: Adoptive T-cell therapy is a promising strategy for the treatment of patients with established tumors but is often limited to specific cancers where tumor-infiltrating lymphocytes, the source of T cells for ex vivo culture, can be obtained. In this study, we evaluated the feasibility of expanding HER-2/neu-specific T cells derived from peripheral blood ex vivo following in vivo priming with a HER-2/neu peptide vaccine. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells from cytomegalovirus (CMV)-seronegative and CMV-seropositive donors as well as HER-2/neu-positive cancer patients who had or had not been vaccinated with a HER-2/neu peptide-based vaccine was used as a source of T lymphocytes. Antigen-specific T-cell lines were generated by in vitro stimulation with antigen followed by nonspecific expansion on CD3/CD28 beads. The ability to expand antigen-specific T cells was assessed using IFN-gamma and granzyme B enzyme-linked immunosorbent spot. The phenotype of the resultant T-cell lines was evaluated by flow cytometry, including the presence of FOXP3-expressing CD4(+) T cells. RESULTS: The frequencies of CMV-specific T cells generated from CMV(+) donors were >11-fold higher than the frequencies from CMV(-) donors (P = 0.001), with 22-fold increase of total number of CD3(+) T cells. The frequencies of HER-2/neu-specific T cells generated from the primed patients were >25-fold higher than the frequencies from unvaccinated patients (P = 0.006), with an average of a 19-fold increase of total number of CD3(+) T cells. Using peripheral blood as the source of T cells did not result in concurrent expansion of FOXP3(+)CD4(+) regulatory T cells despite the use of interleukin-2 in in vitro culture. Both CD4(+) and CD8(+) HER-2/neu-specific T cells could be expanded. The extent of ex vivo expansion correlated with the magnitude of immunity achieved during immunization (P = 0.008). CONCLUSION: Tumor-specific T cells can be efficiently expanded from the peripheral blood ex vivo following in vivo priming with a vaccine. This approach provides an effective method to generate tumor-specific polyclonal T cells for therapeutic use that could be applied to cancer patients with any tumor type.