负载膀胱癌酸洗抗原肽对树突状细胞分泌IL-12的影响及意义

目的：研究负载膀胱癌酸洗抗原肽对树突状细胞（DCs）分泌IL-12的影响和意义.方法：用弱酸洗脱法获得膀胱癌细胞株BIU-87肿瘤抗原肽;联合应用重组人GM-CSF、IL-4和TNF-α体外诱导健康志愿者DCs并负载膀胱癌肿瘤抗原肽;DC诱导膀胱癌抗原特异性CTLs;用MTT法检测其对BIU-87体外杀伤效应;用ELISA法检测第3、6、9天培养细胞的上清液,评价DCs分泌IL-12 p70的能力.结果：联合应用重组人GMCSF、IL-4和TNF-α可在体外诱导出DCs;负载膀胱癌抗原肽的DC诱导的特异性CTLs可杀伤高达（78.5±7.0）%的BIU-87细胞,显著高于各对照组（P〈0.01）;经ELISA方法检测不同时期的DCs分泌IL-12的量不同,而且负载抗原肽DCs较未负载抗原DCs有更强的分泌能力（P〈0.05）.结论：IL-12 p70在DCs刺激特异性CTLs的过程中发挥重要作用,其分泌量受DCs的成熟状态及某些刺激信号的影响,膀胱癌酸洗抗原肽是其刺激信号之一.     

超抗原SEA联合树突状细胞诱导特异性抗膀胱肿瘤研究

目的 观察超抗原金黄色葡萄球菌肠毒素A(SEA)联合树突状细胞(Dc)诱导CTL对膀胱肿瘤高效特异性的免疫杀伤作用和对肿瘤局部免疫提呈功能的改善情况.方法 用GM-CSF和IL-4联合刺激诱导外周血单个核细胞分化为DC.将Dc与同源淋巴细胞(DC-L组)、超抗原SEA淋巴细胞(DC-SEA-L组)共培养,用FCS分析DC供刺激分子表型,酶联免疫吸附测定法检测细胞因子IL-2,MTT法测定激活的CTL对膀胱癌E-J细胞的体外杀伤效应.结果 在体外,DCSEA-L对膀胱癌细胞具有相对特异的杀伤作用,SEA-L和Dc-L则无明显特异性.结论 SEA和Dc联合应用可诱导产生高效并具有相对特异性的抗膀胱癌效应,并改善膀胱癌组织局部的DC抗原提呈功能.     

负载抗原的树突状细胞诱导特异性抗膀胱癌细胞的效应

树突状细胞(DCs)是体内“专职的”抗原提呈细胞，与机体抗瘤免疫关系密切。本研究旨在用DCs负载膀胱癌细胞冻融抗原，探讨其在体外对膀胱癌细胞的杀伤效应。     

乳腺癌细胞抗原负载的自体树突状细胞体内诱导特异性T细胞应答

目的 以乳腺癌细胞抗原体外负载自体树突状细胞(DCs)对24例乳腺癌患者进行自身免疫,探讨其体内诱导特异性T细胞免疫应答的能力.方法 新鲜癌组织制备成热休克凋亡细胞抗原负载外周单核细胞来源DC,分别在采血后第1、2、4、6周于患者腹股沟淋巴结富集区进行皮内注射,细胞剂量为4×10+～6×106/次.结果 DC免疫治疗后患者血清抗瘤免疫因子水平明显上升:白细胞介素(IL)-2治疗前为(33.8±7.2)ng/L,治疗后为(68.5±12.4)ng/L;IL-12治疗前为(48.5±10.9)ng/L,治疗后为(118.2±31.5)ng/L;肿瘤坏死因子(TNF)-α治疗前为(18.7±5.3)ng/L,治疗后为(78.3±11.5)ng/L;干扰素(IFN)-γ治疗前为(20.5±6.3)ng/L,治疗后为(92.6±14.9)ng/L,治疗前后比较差异有统计学意义(P<0.01);DTH试验阳性率为7/10;4/10例IFN-γ+CD8+T细胞频率较治疗前明显增加.随访发现:除1例患者在治疗后3个月内疾病进展(PD),其余患者病情稳定无复发与转移症状(SD).结论 以乳腺癌细胞为抗原负载自体DC免疫患者,能够有效提高患者非特异免疫水平,激发体内特异性T细胞免疫应答,是一种安全、副反应较小、有效的乳腺癌辅助治疗手段.     

树突状细胞与膀胱癌的治疗进展

树突状细胞（DC）是人体内抗原递呈能力最强的细胞，由DC激活的T细胞介导的免疫应答在机体抗肿瘤过程中起着主导作用，本文订对DC的生物学特性，DC参与抗肿瘤的机制，膀胱癌病人DC的变化及目前DC对膀胱癌免疫治疗的研究进展作一综述。     

负载肾癌肿瘤裂解物的树突状细胞诱导产生抗原特异性细胞毒T淋巴细胞

目的　利用树突状细胞呈递肿瘤抗原的特性提高细胞毒T淋巴细胞 (CTLs)对肾癌细胞的杀伤活性。　方法　肾细胞癌患者骨髓来源的有核细胞体外经GM CSF和IL 4诱导产生树突状细胞 ,负载肿瘤裂解物后诱导自体CTLs产生。用细胞毒试验和ELISA测定CTLs杀伤活性和细胞因子的分泌。　结果　肾癌患者自体来源的DC Tuly能 (1)增加CTLs增殖 ,16d时使T细胞增殖达 4 3倍 ;(2 )上调CTLs中CD3 和CD8 T细胞群 ;(3)诱导产生的CTLs对自体肾癌细胞具有高杀伤率 ,显著高于异体肾癌和异种肿瘤细胞 (P <0 .0 5 ) ;(4)上调TNF α分泌。　结论　DCs能呈递Tuly抗原。诱导产生抗原特异性CTLs,提示DC Tuly具有为肾癌患者制作疫苗和进行特异性CTLs过继免疫治疗的临床应用前景     

自体负载肿瘤抗原树突状细胞治疗大鼠甲状腺癌

目的 观察自体负载肿瘤抗原树突状细胞(DC)对大鼠甲状腺癌的治疗作用.方法 建立Wistar荷瘤大鼠动物模型,随机分为治疗组和对照组,治疗组静脉输入负载肿瘤抗原DC、对照组输入生理盐水,检测分析2组血清中自细胞介素(IL)-12、IL-8表达水平,以及瘤重、瘤体体积及大鼠存活.结果 治疗组与对照组瘤重、瘤体体积、存活期、IL-12及IL-8表达水平分别为:(3.56±0.79)g比(4.97±0.56)g、(3.98±0.84)cm3比(5.06±0.58)cm3、(36.0±3.0)d比(28.0±3.2)d、(368.9±21.7)ng/L比(227.7±13.4)ng/L和(218.9±19.3)ng/L比(371.2±24.1)ng/L(P值分别<0.01、<0.01、<0.05、<0.01、<0.01).结论 自体负载肿瘤抗原DC对甲状腺癌具有较好的抑瘤效应,可延长荷瘤鼠的生存期.     

乳腺癌细胞抗原负载的自体树突状细胞体内诱导特异性T细胞应答

Objective To explore the potential of autologous dendritic cells (DCs) loaded with breast tumor cells antigen in inducing tumor-specific T cells response in vivo. Methods Fresh tumor samples from patients with breast cancer were collected and made into apoptotic heat shock tumor cells. Peripheral blood mononuclear cells (PBMCs) were isolated. DCs were induced from monocytes with granulocytemacrophage colony-stimulating factor (rhGM-CSF) and interleukin-4 (rhIL-4) combination for 5 days. DCs were co-cultured with autologous apoptotic heat shock tumor cells for 48 h. Twenty-four patients with breast cancers received four vaccinations of auto-DCs at 1 st, 2nd, 4th, and 6th week with cell number from 4×106 to 6×106. Results The vaccinations were well tolerated, and most patients experienced improved quality of life after DC immunization. Cytokine levels including IL-2, IL-12, TNF-α and IFN-γ in sera were obviously increased after DC vaccination [(68. 5±12. 4), (118. 2±31. 5), (78. 3±11.5) and (92. 6±14. 9) ng/L respectively] as compared with those before vaccinations [(33. 8±7. 2), (48. 5±10. 9), (18. 7±5. 3 ) and (20. 5±6. 3) ng/L respectively]. Both antigen specific delayed type hypersensitivity (DTH) reaction and the frequency of antigen specific IFN-gamma+CD8+T lymphocytes were examined after 4 times of DC vaccinations. The DTH tests were positive in 7 out of 10 patients. The frequency of IFN-γ+CD8+T cells was enhanced in 4 out of 10 patients. One out of 24 patients experienced a progressive disease, while the others showed no progress during a follow-up period of 34 months. Conclusion Autologous DCs loaded with heat-shocked apoptotic breast cancer cells antigen elicit specific anti-tumor T cell immune responses companying with high levels of Thl type cytokine secretion. DCs vaccination after surgery may be a novel and effective combination tool for tumor treatment.     

乳腺癌细胞抗原负载的自体树突状细胞体内诱导特异性T细胞应答

Objective To explore the potential of autologous dendritic cells (DCs) loaded with breast tumor cells antigen in inducing tumor-specific T cells response in vivo. Methods Fresh tumor samples from patients with breast cancer were collected and made into apoptotic heat shock tumor cells. Peripheral blood mononuclear cells (PBMCs) were isolated. DCs were induced from monocytes with granulocytemacrophage colony-stimulating factor (rhGM-CSF) and interleukin-4 (rhIL-4) combination for 5 days. DCs were co-cultured with autologous apoptotic heat shock tumor cells for 48 h. Twenty-four patients with breast cancers received four vaccinations of auto-DCs at 1 st, 2nd, 4th, and 6th week with cell number from 4×106 to 6×106. Results The vaccinations were well tolerated, and most patients experienced improved quality of life after DC immunization. Cytokine levels including IL-2, IL-12, TNF-α and IFN-γ in sera were obviously increased after DC vaccination [(68. 5±12. 4), (118. 2±31. 5), (78. 3±11.5) and (92. 6±14. 9) ng/L respectively] as compared with those before vaccinations [(33. 8±7. 2), (48. 5±10. 9), (18. 7±5. 3 ) and (20. 5±6. 3) ng/L respectively]. Both antigen specific delayed type hypersensitivity (DTH) reaction and the frequency of antigen specific IFN-gamma+CD8+T lymphocytes were examined after 4 times of DC vaccinations. The DTH tests were positive in 7 out of 10 patients. The frequency of IFN-γ+CD8+T cells was enhanced in 4 out of 10 patients. One out of 24 patients experienced a progressive disease, while the others showed no progress during a follow-up period of 34 months. Conclusion Autologous DCs loaded with heat-shocked apoptotic breast cancer cells antigen elicit specific anti-tumor T cell immune responses companying with high levels of Thl type cytokine secretion. DCs vaccination after surgery may be a novel and effective combination tool for tumor treatment.     

自体负载肿瘤抗原树突状细胞治疗大鼠甲状腺癌

Objective To investigate the immunotherapeutic effects of auto dendritic cells (DC) loaded with tumor lysates on rat transitional thyroid carcinoma. Methods After the thyroid tumor models in Wistar rats were successfully established, all rats were divided randomly into the treatment group and the control group. DC loaded with tumor lysates were injected into the tail vein of the rats in the treatment group, and the rats in control group, normal saline was injected into the tail vein. The levels of interleukin (IL)-12 and IL-8 in serum, the tumor weight and volume, and the life span of the rats were measured. Results The average tumor weight and volume, the life span, the levels of IL-12 and IL-8 in the treatment group and the control group were respectively (3. 56±0. 79) g vs (4. 97±0. 56) g, (3. 98±0. 84) cm3 vs (5.06±0.58) cm3, (36.0±3.0) days vs (28.0±3.2) days, (368.9±21.7) ng/L vs (227.7±13.4) ng/L, (218.9±19.3) ng/L vs (371.2±24. 1) ng/L. P values <0.01, <0.01, <0.05,<0.01, <0.01, respectively. Conclusion Auto DC loaded with tumor lysates can inhibit tumor growth and prolong the survival time of Wistar rats with thyroid carcinoma.     

Effector cells in natural cytotoxicity against human bladder cancer cell lines

Summary Human peripheral blood mononuclear cells obtained by ficoll-hypaque sedimentation were depleted of Fc-receptor-bearing (FcR+) cells. Cytotoxicity (direct killing of target cells by effector cells), tested in a 40 h assay, was significantly decreased against a variety of target cells. Tests in which no FcR+ cells could be detected were also positive for natural killing (NK) against a spectrum of target cells from normal donors. NK in this system was mediated by more than one subpopulation of lymphocytes. Monocytes probably did not play a significant role.Decreasing the FcR+ cells in peripheral blood mononuclear cells in patients with bladder cancer and in controls did not reveal specific antitumour activity.This work was supported by NTH grant CA16880 through the National Bladder Cancer Project, and grant CA12800 from the National Cancer Institute     

阻断B7-H1通路增强膀胱癌患者外周血树突状细胞免疫功能的实验研究

目的:探讨膀胱癌患者外周血与正常人外周血树突状细胞(DCs)免疫功能的差异以及B7-H1阻断对膀胱癌患者外周血DCs免疫功能的影响。方法:采集膀胱癌患者及正常人的外周血,用rhGM-CSF、rhIL-4和rhTNF-α自外周血单个核细胞(PBMC)诱导分离DCs,混合淋巴细胞反应检测DCs刺激淋巴细胞增殖的能力。然后于膀胱癌DCs组加入B7-H1的阻断型抗体,混合淋巴细胞反应检测B7-H1阻断后DCs刺激淋巴细胞增殖的能力,ELISA法检测阻断后DCs分泌IL-10和IL-12的水平。结果:膀胱癌患者外周血DCs刺激淋巴细胞增殖的能力低于正常人外周血DCs,而加入B7-H1阻断型抗体阻断B7-H1后,膀胱癌患者外周血DCs刺激淋巴细胞增殖的能力得到明显提高,同时其分泌IL-10水平明显降低,而分泌IL-12水平明显增加。结论:膀胱癌患者外周血DCs存在免疫功能缺陷,其机制可能与DCs表面B7-H1的诱导高表达有关,而B7-H1阻断能够增强膀胱癌患者外周血DCs的免疫功能。     

Antitumor effect of simultaneous transfer of interleukin-12 and interleukin-18 genes and its mechanism in a mouse bladder cancer model

BACKGROUND: The objectives of this study were to evaluate the antitumor effects of the simultaneous introduction of interleukin 12 (IL-12) and IL-18 genes into a mouse bladder cancer cell line (MBT2). We intended to compare these with those of either gene alone and to investigate the mechanism of the effects induced by the transfer of IL-12 and/or IL-18 genes in this model system. METHODS: We transfected the IL-12 and/or IL-18 genes into MBT2 cells by the liposome-mediated gene transfer method. We confirmed the secretion of IL-12 and/or IL-18 by enzyme-linked immunosorbent assay. Parental (MBT2/P), IL-12-transfected (MBT2/IL-12), IL-18-transfected (MBT2/IL-18) or both IL-12- and IL-18-transfected (MBT2/Both) cells were subcutaneously or intravenously injected into syngeneic C3H mice. To analyze the mechanism of tumor rejection, these clones were subcutaneously injected into naive nude mice and those depleted with natural killer (NK) cells by antibody. RESULTS: MBT2/IL-12, MBT2/IL-18 and MBT2/Both were completely rejected when they were injected subcutaneously or intravenously into syngeneic mice. However, MBT2/IL-12, but not MBT2/IL-18, could grow in nude mice. Moreover, the antitumor effect of MBT2/IL-18 was partially abrogated when injected into nude mice of which NK cells were depleted by antibody treatment. MBT2/Both was completely rejected in both nude mice with and without NK cells. CONCLUSION: The results of the present study indicate that T cells and NK cells seem to play important roles in the antitumor effects by the secretion of IL-12 and IL-18, respectively, and MBT2/Both possesses both mechanisms.     

二次TURBt联合膀胱灌注化疗及肿瘤细胞抗原负载的树突状细胞治疗非肌层浸润性膀胱癌的临床研究

目的 观察二次TURBt联合膀胱灌注化疗及肿瘤细胞抗原负载的树突状细胞(DC)治疗非肌层浸润性膀胱癌的安全性及疗效. 方法 T1期膀胱尿路上皮癌患者80例.男59例,女21例.年龄30～ 85岁,平均65岁.入组患者均在第一次TURBt术后4～6周行二次TURBt,术后常规膀胱灌注化疗.分为2组:DC组40例,对照组40例.DC组自外周血分离出单核细胞,体外诱导分化为DC,加入该患者的肿瘤抗原共培养,获取负载肿瘤细胞抗原的DC;在二次TURBt术后6～8周将肿瘤细胞抗原负载的DC回输,每周1次,共4次,每次腹股沟皮下注射细胞数不低于1×106个,每疗程回输细胞总数＞4×106个.观察DC组免疫指标改变及不良反应,比较2组患者肿瘤复发比例.结果 80例患者第一次TURBt病理分级G117例(21.3％)、G254例(67.5％)、G39例(11.2％);二次TURBt病理检查发现残存肿瘤27例,总阳性率33.7％;Ta期8例(29.6％)、T1期19例(70.4％);G13例(11.1％)、G2 19例(70.4％)、G3 5例(18.5％).二次TURBt时Ta期8例中分级同第一次TURBt 6例,分级升高2例;T1期19例中分级同第一次TURBt 12例,分级升高5例,降级2例.单发16例,均位于原电切处;多发11例,其中原电切处可见菜花样肿瘤7例.DC回输治疗时出现寒战、发热5例,给予地塞米松10 mg静脉推注治疗后缓解.治疗前、治疗后1年及2年患者血中白细胞、SCr、ALT值比较差异无统计学意义(P＞0.05).与治疗前比较,治疗后1年及2年CD4、CD8、CD4/CD8等指标比较差异均有统计学意义(P＜0.05),而治疗后1年后及2年各指标比较差异无统计学意义.DC组1年内复发1例(2％),2年内复发3例(6％);对照组中1年内复发6例(20％);2年内复发9例(30％),2组复发率比较差异有统计学意义(P＜0.05). 结论 二次TURBt联合膀胱灌注及肿瘤细胞抗原负载的DC回输治疗是降低非肌层浸润性膀胱癌复发率较有效的方法.     

锌指E-盒结合同源异形盒-1在膀胱癌细胞中的表达及对肿瘤细胞侵袭的影响

目的:锌指E-盒结合同源异形盒-1(ZEB1)是上皮-间质转换的重要调控因子。本研究探讨ZEB1在膀胱癌细胞系中的表达情况,以及对膀胱癌发展与转移的影响。方法:采用RT-PCR检测膀胱癌细胞系ZEB1的表达,免疫荧光检测ZEB1蛋白表达定位;转染ZEB1siRNA后通过RT-PCR与Western Blot检测ZEB1的mRNA与蛋白表达变化;以及通过细胞侵袭实验观察ZEB1影响细胞侵袭能力的变化。结果:膀胱癌细胞系UMUC3和5637均表达ZEB1,SV-HUC-1不表达ZEB1;ZEB1蛋白定位于胞核;ZEB1敲低后,其mRNA与蛋白表达降低,膀胱癌细胞系侵袭能力降低。结论:膀胱癌细胞系UM-UC3和5637可用于ZEB1与肿瘤相关的机制研究;ZEB1促进膀胱癌细胞侵袭。     

吡柔比星与丝裂霉素C对膀胱癌T24细胞株抑制作用的实验研究

目的探讨吡柔比星（THP）与丝裂霉素C（MMC）对膀胱癌T24细胞株的抑制作用。方法将膀胱癌T24细胞株分为4组,分别给予THP、MMC、THP＋MMC和MMC＋THP。用MTT法测定T24细胞的增殖抑制率,用TuNEL法检测T24细胞的凋亡情况。结果THP、MMC、THP＋MMC和MMC＋THP四组的增殖抑制率（％）分别为54．3±6．4,48．9±7．2,62．3±5．9,69．1±6．2;凋亡指数（％）分别为6．2±2．5,4．4±2．7,6．7±3．0,7．4±3．2。联合用药组对T24细胞的抑制作用优于单药THP和MMC对肿瘤细胞的抑制作用（P〈0．05）。在联合用药组中,MMC＋THP组对T24细胞的抑制作用优于THP＋MMC组对肿瘤细胞的抑制作用（P〈O．05）。结论THP和MMC均可抑制膀胱癌T24细胞株的增殖并诱导凋亡,两药序贯联合用于膀胱灌注化疗可能具有合理性。     

大鼠诱发肝癌模型中脾内转染IL-2和IL-12基因激活NK细胞

目的研究大鼠诱发肝癌模型中脾内直接注射携带白介素2（IL-2）和（或）白介素12（IL-12）基因的逆转录病毒包装细胞株对血IL-2和IL-12,以及NK细胞活性的影响.方法大鼠随即分为生理盐水对照组、空载体对照组、IL-12基因治疗组、IL-2基因治疗组及IL-2/IL-12联合基因治疗组.构建携带IL-2和（或）IL-12基因的逆转录病毒载体.口服二乙基亚硝胺诱癌后,含IL-2和（或）IL-12基因的包装细胞转染脾细胞.比较大鼠血IL-2和IL-12浓度、NK细胞活性、病理变化和毒性反应.结果IL基因治疗后血IL-2和IL-12明显增加.联合基因组同时表达IL-2和IL-12,总水平高于单基因治疗组.病理示治疗后肝癌组织中淋巴细胞浸润明显增多.IL治疗组NK细胞活性较对照组显著增高（P＜0.01）.联合基因组较IL单基因增高（P＜0.05）.治疗后3 d血清IL达高峰,以后逐步下降.结论脾内直接注射携带IL-2和（或）IL-12基因的逆转录包装细胞株可明显增强NK细胞活性,IL联合基因治疗优于IL单基因.