肝癌细胞对重组人HSP70联合肝癌组织冻融抗原修饰树突状细胞的免疫应答

目的:研究重组人热休克蛋白70(rhHSP70)联合肝癌组织冻融抗原修饰的树突状细胞(dendritic cell,DC)诱导对肝癌细胞的免疫杀伤效应.方法:外周血单个核细胞经粒-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素4 (IL-4)诱导生成DC,负载冻融抗原的同时加入rhHSP70,不同分组致敏的DC激活淋巴细胞生成肿瘤抗原特异性细胞毒性T淋巴细胞(cytotoxic T cells,CTL),四甲基偶氮唑蓝(MTT)法及3H-TdR法检测DC刺激淋巴细胞增殖能力, MTT法检测CTL对肝癌细胞的体外杀伤活性,酶联免疫吸附试验(ELISA)测定细胞因子的分泌,流式细胞术(FCM)检测DC表型变化.结果:冻融抗原致敏的DC可明显促进淋巴细胞增殖,能有效呈递肝癌冻融抗原,诱导产生抗原特异性CTL,联合rhHSP70能进一步增强CTL对肝癌细胞的杀伤作用.结论:肝癌冻融抗原联合rhHSP70修饰的DC诱导CTL对肝癌细胞能产生高效杀伤作用.     

树突细胞激活的肿瘤浸润性淋巴细胞抗肝癌免疫活性的研究

目的　探讨树突细胞 (DC)激活的肿瘤浸润性淋巴细胞 (TIL)体外对肝癌细胞的杀伤活性。方法　从肝癌患者外周血获取DC ,应用粒 /巨噬细胞集落刺激因子 (GM -CSF)、白细胞介素 -4 (IL -4 )和肿瘤抗原激活DC ,然后用DC激活TIL ,观察TIL在体外对自体肝癌细胞和Hep3B细胞的杀伤活性。 结果　DC激活的TIL对自体肝癌细胞具有很强的杀伤活性 ,杀伤率为89 .39%± 3.0 5 %,明显高于未经DC激活的TIL、DC激活的T淋巴细胞和未经DC激活的T淋巴细胞对自体肝癌细胞的杀伤率(分别为 5 5 .2 3%± 1.5 3%、5 4.89%± 1.48%和 3.6 5 %± 0 .2 6 %)。而它们对Hep3B细胞的杀伤活性则相对较低。 结论　肝癌患者外周血DC能诱导TIL产生高效而特异的抗肝癌免疫活性。     

树突状细胞对肿瘤浸润性淋巴细胞杀伤自体肝癌细胞活性影响的研究

目的　探讨树突状细胞 (DC)激活的肿瘤浸润性淋巴细胞 (TIL )体外对自体肝癌细胞杀伤活性。方法　从肝癌患者外周血获取 DC,应用粒 /巨噬细胞集落刺激因子 (GM- CSF)、白细胞介素 - 4 (IL - 4 )和肿瘤抗原激活 DC,然后用 DC激活 TIL ,观察 TIL在体外对自体肝癌细胞的杀伤活性。结果　 DC激活的 TIL对自体肝癌细胞具有很高的杀伤活性 ,杀伤率为 (89.39± 3.0 5 ) % ,明显高于未经 DC激活的 TIL、DC激活的 T淋巴细胞和未经 DC激活的 T淋巴细胞对自体肝癌细胞的杀伤率。其杀伤率分别为 (5 5 .2 3± 1.5 3) %、(5 4 .89± 1.4 8) %和 (3.6 5± 0 .2 6 ) %。结论　肝癌患者外周血 DC能诱导 TIL产生高效而特异的抗肝癌免疫     

肝癌细胞抗原致敏树突状细胞诱导的体外抗肿瘤作用

目的　探讨原发性肝癌患者外周血树突状细胞 (DC)经自体肝癌细胞抗原致敏后诱导的体外抗肿瘤作用。方法　对肝癌患者外周血采用密度梯度离心法分离 ,获得DC前体细胞 ,用重组人粒细胞 巨噬细胞集落刺激因子 (rhGM CSF)和重组人白细胞介素 4(rhIL 4)联合培养 ,诱导扩增DC。制备自体肝癌细胞抗原 ,体外脉冲DC ,检测DC诱导自体T细胞增殖能力及细胞毒性T细胞 (CTL )在体外对自体肝癌细胞的杀伤活性 ,并检测肿瘤抗原致敏DC分泌的IL 12水平。结果　经自体肝癌细胞抗原致敏的DC能分泌IL 12和诱导较强的自体T细胞增殖 ,且能诱导特异性CTL ,该CTL对自体肝癌细胞具有很强的杀伤活性 ,杀伤率显著高于DC、未经肝癌细胞抗原致敏的DC激活的CTL及T淋巴细胞的杀伤率 ,而对CT 2 6细胞、BEL 740 2细胞无明显的杀伤作用。结论　肝癌患者外周血DC经自体肝癌细胞抗原致敏后能诱导高效而特异的抗肝癌免疫 ,其机制可能与增强T细胞应答和诱导机体产生肿瘤特异CTL从而发挥特异性的抗肿瘤作用有关。     

NY-ESO-1致敏树突状细胞诱导的CTL对肝癌细胞株的特异性杀伤作用

目的:探讨肿瘤睾丸抗原NY-ESO-1(New York-esophageal-1)致敏树突状细胞体外诱导特异性CTL对肝癌细胞株的杀伤作用.方法:重组质粒pGEX-ESO1经原核诱导表达并纯化GST-ESO1融合蛋白肽.重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素4(rhIL-4)诱导培养人外周血来源的树突状细胞(dendritic cells, DCs),经GST-ESO1融合蛋白肽致敏后诱导特异性CTL增殖.以此CTL为效应细胞,分别以NY-ESO-1阳性表达的肝癌细胞株HepG2和不表达NY-ESO-1的肝癌细胞株H2P为靶细胞,MTT法检测CTL对肝癌细胞株的杀伤作用.结果:重组质粒pGEX-ESO1经IPTG诱导,在大肠杆菌中表达相对分子质量约36 000的GST-ESO1融合蛋白肽,纯化后的质量浓度为50 μg/ml;经rhGM-CSF和rhIL-4联合诱导成功培养人外周血DCs,其表型分子HLA-DR为91.4%、CD86为70.5%、CD83为71.2%、CD80为55.3%.NY-ESO-1致敏的DCs能明显诱导CTL增殖,此CTL对肝癌细胞株HepG2的杀伤率显著高于GST刺激组、未致敏DC组和无DC刺激组(均P<0.05),效靶比为50 ∶1时杀伤效应达到最高峰[(53.23±3.78)%,P<0.01];相同条件下CTL对H2P细胞无特异性杀伤作用.结论: NY-ESO-1抗原致敏的DCs在体外可诱导同种CTL产生和增殖,后者对NY-ESO-1阳性肝癌细胞株具有特异性杀伤效应,该方法为肝癌免疫治疗提供了一条新思路.     

H22细胞全细胞抗原负载的树突状细胞激活肿瘤浸润性淋巴细胞抗小鼠肝癌的实验

目的探讨H22小鼠肝癌细胞（H22细胞）全细胞抗原致敏的树突状细胞激活肿瘤浸润淋巴细胞抗小鼠肝癌细胞活性。方法取得小鼠骨髓细胞并诱导生成树突状细胞,由冻融法制备的H22细胞全细胞抗原致敏,然后用已致敏的树突状细胞激活肿瘤浸润性淋巴细胞,测定致敏前后的DC表面抗原CD11c、CD80、CD86、CD40、MHCⅡ,并评估激活前后的TIL对H22细胞的杀伤活性,同时脾淋巴细胞作为杀伤对照。结果CD11c阳性细胞中CD80、CD86、CD40、MHCⅡ阳性细胞所占比例在致敏后的DC表现为明显上调。经致敏后成熟DC激活的TIL对H22细胞杀伤活性明显高于未激活的TIL,并高于激活或未激活的小鼠脾脏淋巴细胞。结论在H22细胞全抗原致敏后,小鼠成熟DC中CD80、CD86、CD40、MHCⅡ的表达率明显高于未成熟DC。经H22细胞全细胞抗原致敏的DC能诱导活化TIL,明显提高其在体外对H22细胞的杀伤活性。     

含甲胎蛋白肽段的重组腺病毒转染树突状细胞诱导细胞毒性T细胞对肝癌细胞的体外杀伤效应

目的 研究人外周血单核细胞来源的树突状细胞(DC)转染含甲胎蛋白(AFP,137-145)片段的重组腺相关病毒后所诱导的特异性T细胞对肝癌细胞株HepG2和SMMC-7721的体外杀伤作用.方法 抽取健康志愿者外周血,分离单核细胞,体外培养,使用含AFP片断的重组腺相关病毒转染未成熟DC,诱导特异性T细胞.检测体外培养的DC和细胞毒性T细胞(CTL)活性,应用四甲基偶氮唑蓝(MTT)法检测CTL对HepG2和SMMC-7721细胞的杀伤作用.结果 转染或未转染的体外培养的成熟DC高表达CD40、CD86和IL12,成熟DC诱导的CTL高表达IFNγ;修饰成熟后的DC后体外能诱导特异性CTL,该CTL在休外对肝癌细胞株HepG2和SMMC-7721均有杀伤作用.结论 重组腺相关病毒转染DC,不明显改变DC表型和刺激淋巴细胞增殖和分化功能,可诱导自体CTL增殖,含AFP(137～145)片断的腺相关病毒转染DC诱导自体CTL对肝癌细胞株HepG2和SMMC-7721细胞有明显杀伤作用,DC疫苗可以作为肝癌患者免疫治疗的有效补充.     

肝癌树突状细胞瘤苗诱导抗肿瘤作用的研究

目的：研究负载肝癌抗原的肝癌树突状细胞（dendritic cell,DC）瘤苗诱导的抗肿瘤作用。方法：于体外用rhGM-CSF和rhIL-4从肝癌患者外周血诱导DC,并用肝癌细胞抗原冲击致敏,制成负载肝癌抗原的肝癌DC瘤苗。肝癌DC瘤苗活化自体T淋巴细胞分化为细胞毒性T淋巴细胞（cytotoxic of T-lymphocytes,CTL）,采用流式细胞术检测CTL分型;乳酸脱氢酸（LDH）4 h释放法检测CTL对肝癌细胞的特异性杀伤作用。MTT法检测肝癌DC瘤苗及其活化CTL培养上清对肿瘤细胞的抑制作用;ELISA法检测肝癌DC瘤苗活化的CTL培养上清中IL-12、IFN-γ和TNF-α水平。结果：经肝癌DC瘤苗活化后的T淋巴细胞群中,CD56^＋细胞数量显著减少,CD4^＋T和CD8^＋T细胞数量增加,其中以CD8^＋T细胞增加较明显;细胞毒实验显示,该CTL对自体肝癌细胞具有较强的杀伤作用,而对与致敏DC的肝癌抗原无关的CT26结肠腺癌细胞无明显杀伤作用;单纯肝癌DC瘤苗或其激活的CTL培养上清也表现出较强的抑瘤作用,而且这种抑瘤作用具有广谱性,可抑制多种肿瘤细胞的生长;在CTL培养上清中可检测到IL-12、TNF-α和IFN-γ的含量。结论：肝癌DC瘤苗不仅诱导了对肝癌细胞的特异性CTL杀伤作用,而且可激活CD4^＋T和CD8^＋T细胞分泌IL-12、TNF-α和IFN-γ等细胞因子,以非杀伤方式直接或间接地抑制肿瘤细胞生长,发挥非特异性的抗肿瘤作用。     

自体宫颈癌-树突细胞疫苗激活的CTL杀伤效应

背景与目的:树突细胞(dendriticcells,DC)是目前已知的功能最强的抗原递呈细胞(antigen-presentingcell,APC),它可以在体内、外向T淋巴细胞递呈抗原,并诱发细胞毒T淋巴细胞(cytotoxicTlymphocyte,CTL)反应。本研究旨在探讨负载自体宫颈癌抗原的DC体外激发的CTL对自体宫颈癌细胞的杀伤效应。方法:先冻融宫颈癌细胞制备抗原,然后以GM-CSF、IL-4诱导自体外周血单个核细胞(peripheralbloodmononuclearcell,PBMC)获得DC并负载抗原,刺激自体T淋巴细胞制备宫颈癌抗原特异性CTL,观察CTL对宫颈癌细胞的杀伤活性。结果:负载自体宫颈癌抗原DC诱导的特异性CTL对自体宫颈癌细胞的体外杀伤率高达79.32%～89.27%,显著高于淋巴因子激活的杀伤细胞(lymphokine-activatedkillingcells,LAK)的杀伤率(t≥2.89,P<0.05);且对宫颈癌HeLa细胞株具有一定杀伤效应(40.35%～58.09%),但低于自体癌细胞组(t≥2.97,P<0.05);特异性CTL对HepG2、MCF7、A549、MGC803细胞无明显杀伤效应。结论:自体宫颈癌-树突细胞疫苗体外诱导的CTL具有高效而特异的抗自体宫颈癌细胞免疫活性,可望成为宫颈癌生物治疗的一个有力手段。     

术中失血来源的树突状细胞用于肝癌治疗的体外实验研究

目的 探讨从肝癌患者术中失血来源的单个核细胞中培养树突状细胞(DC)的可行性,为个体化的DC介导免疫治疗提供新的细胞来源.方法 采集9例原发性肝细胞癌患者术中失血及8例脐血,分离其单个核细胞,其中贴壁的单个核细胞经重组人粒细胞巨噬细胞集落刺激因子(RHGM-CSF),重组人白细胞介素4(rhIL-4)诱导和负载癌细胞抗原,制成不同的DC瘤苗,悬浮的单个核细胞经细胞因子处理成为细胞因子诱导的杀伤细胞(CIK).采用二苯基溴化四氮唑蓝(MTT)法测定DC激活同源CIK的相对增殖率和CIK对肝癌细胞的杀伤效果.结果 肝癌患者术中失血来源的单个核细胞在体外能够诱导分化为具有典型形态和表型的DC.术中失血来源的DC表面标志物表达水平低于脐血来源的DC,但二者均能有效地激活CIK,并增强其对肝癌细胞的杀伤活性.负载患者自身癌细胞抗原的术中失血IX:和脐血DC,其激活的CIK增殖率分别为(388.9±137.3)%和(315.1±44.5)%,对肿瘤细胞的杀伤率分别为(87.1±8.0)%和(90.0±5.1)%;而负载SMMC-7721抗原的术中失血DC和脐血DC,其激活的CIK增殖率分别为(239.9±48.7)%和(226.3±32.3)%,对肿瘤细胞的杀伤率分别为(76.4±7.9)%和(81.1 ±4.3)%.在激活CIK和增强对肝癌细胞杀伤能力方面,相同抗原负载的两种Dc差异无统计学意义,但负载自身抗原优于负载SMMC-7721抗原.结论 肝癌患者术中失血来源的DC可有效激活CIK,并增强其对肝癌细胞的杀伤效应,为临床研究和应用DC瘤苗提供了一个新的来源.     

负载腮腺腺样囊性癌抗原的树突状细胞诱导的抗肿瘤效应

目的:探讨腺样囊性癌肿瘤抗原负载的树突状细胞通过淋巴细胞介导的免疫反应,体外杀伤腺样囊性癌细胞的细胞毒性效应.方法:外周血单核细胞在GM-CSF + IL-4 的诱导下体外培养,用肿瘤细胞抗原冲击后,流式细胞仪检测树突状细胞抗原负载前后CD1a、CD83表达量的变化.MTT比色法检测同种异体的混合淋巴细胞反应和诱导细胞毒淋巴细胞CTL杀伤肿瘤细胞.结果:凋亡肿瘤抗原刺激后,CD83 表达增加﹙P<0.01﹚,而CD1a表达量下调﹙P<0.05).负荷肿瘤抗原树突状细胞体外诱导出明显的细胞不良反应,并刺激同种T淋巴细胞增殖.结论:GM-CSF + IL-4 诱导的单核细胞来源的树突状细胞,能在体外摄取肿瘤抗原而进一步成熟,通过激活淋巴细胞杀伤癌细胞.     

负载腮腺腺样囊性癌抗原的树突状细胞诱导的抗肿瘤效应

目的：探讨腺样囊性癌肿瘤抗原负载的树突状细胞通过淋巴细胞介导的免疫反应,体外杀伤腺样囊性癌细胞的细胞毒性效应。方法：外周血单核细胞在GM—CSF＋IL-4的诱导下体外培养,用肿瘤细胞抗原冲击后,流式细胞仪检测树突状细胞抗原负载前后CD1a、CD83表达量的变化。MTT比色法检测同种异体的混合淋巴细胞反应和诱导细胞毒淋巴细胞CTL杀伤肿瘤细胞。结果：凋亡肿瘤抗原刺激后,CD83表达增加（P〈0．01）,而CD1a表达量下调（P〈0．05）。负荷肿瘤抗原树突状细胞体外诱导出明显的细胞不良反应,并刺激同种T淋巴细胞增殖。结论：GM—CSF＋IL-4诱导的单核细胞来源的树突状细胞,能在体外摄取肿瘤抗原而进一步成熟,通过激活淋巴细胞杀伤癌细胞。     

Restoration of Tumor-Specific HLA Class I Restricted Cytotoxicity in Tumor Infiltrating Lymphocytes of Advanced Breast Cancer Patients by in vitro Stimulation with Tumor Antigen-Pulsed Autologous Dendritic Cells

Breast tumor infiltrating lymphocytes (TIL) are enriched in tumor-specific cytotoxic T lymphocytes (CTL), and may represent a superior source of CTL compare to peripheral blood lymphocytes (PBL), for adoptive T cell immunotherapy of breast cancer. However, the immunocompetence of TIL and the possibility to consistently restore their tumor-specific lytic activity in vitro remains an open issue. In this study we evaluated the potential of tumor antigen-pulsed fully mature dendritic cell (DC) stimulation in restoring tumor-specific cytotoxicity in anergic TIL populations from advanced breast cancer patients. In addition we have compared tumor-specific T cell responses induced by tumor antigen-loaded DC stimulation of TIL to responses induced from PBL. Although TIL were consistently non-cytotoxic after isolation or culture in the presence of interleukin-2 (IL-2), in matched experiments from three consecutive patients, tumor-lysate-pulsed DC-stimulated CD8+ T cell derived from TIL were found to be significantly more cytotoxic than PBL (p < 0.05). In addition, cytotoxicity against autologous tumor cells was more significantly inhibited by an anti-HLA class I (W6/32) MAb in TIL compared to PBL (p < 0.05). CTL populations derived from TIL and PBL did not lyse autologous EBV-transformed lymphoblastoid cell lines, and showed negligible cytotoxicity against the NK-sensitive cell line K562. Furthermore, in both CD8+ T cell populations the majority of the tumor-specific CTL exhibited a Th1 cytokine bias (IFN-^high/IL-4^low). Taken together, these data show that tumor lysate-pulsed mature DC can consistently restore tumor-specific lytic activity in non-cytotoxic breast cancer TIL. These results may have important implications for the treatment of chemotherapy resistant breast cancer with active or adoptive immunotherapy.     

Immunologic character of tumor infiltrating lymphocytes in ovarian carcinoma

Tumor-infiltratinglymphocytes(TIL)wasdirectlyisolatedfrompatient'stumortissues.ByrIL2activationandexpansioninvitro,TILwasagainimportedintothesamepatient'sbodytotreattumor,withapparenteffectsofanti-tumorandcomparativelessside-effect,withoutkillingothertumorcellsandnormalcells,allofwhichhavemadeitaeffectivewayoftreatingtumoratanadvancedstage.Inrecentyears,someresearchersfoundthattheeffectsofTIL'treatmentwerenotsofarapparent,withcomparativelyapparentdifference,whichmightbecloselyrelatedtothef…     

HSP70-Id复合物修饰的树突状细胞体外诱导特异性抗肿瘤作用

目的观察人慢性B淋巴细胞性白血病（B-CLL）细胞的独特型抗原Id-ScFv与热休克蛋白70（HSP70）形成复合物修饰的树突状细胞（DC）体外诱导特异性抗肿瘤作用,并初步探讨其机制。方法将HSP70与Id-ScFv体外结合形成复合物HSP70-Id,修饰自人外周血单核细胞获取的DC。倒置相差显微镜观察DC的形态特征;流式细胞仪检测修饰前后DC的表型变化,酶联免疫吸咐试验（ELISA）检测DC分泌的白细胞介素12（IL-12）和肿瘤坏死因子-α（TNF-α）,四甲基偶氮唑蓝（MTT）法检测修饰的DC对自身淋巴细胞的激活和增殖作用,流式细胞仪检测激活的自身淋巴细胞T细胞亚群的变化,台盼蓝染色法检测其对Daudi、K562和HepG2等细胞的杀伤作用。结果DC体外诱导培养成功,HSP70-Id复合物可使DC成熟,镜下可见典型的DC形态,其CD1a表达率为20％-30％,CD83表达率〉72％,CD86和HLA-DR表达显著增加（P〈0．05）,上清中IL-12、TNF-α亦显著高于DC对照组（P〈0．01）。HSP70-Id复合物修饰的DC激活自身淋巴细胞,对Daudi细胞的杀伤率为71．24％,而对K562细胞杀伤作用较弱,对HepG2细胞无明显作用。其淋巴细胞亚群中,CD4^＋T细胞、CD8^＋T细胞的比例均显著增加,分别为56．51％和70．21％,CD4^＋T细胞／CD8^＋T细胞比值由空白对照组的1．49倒置为0．81。结论HSP70-Id复合物修饰的DC生物学活性增强,经其刺激后,传代培养的淋巴细胞可产生高效而特异性的抗肿瘤免疫效应,可能是CD4^＋T细胞、CD8^＋T细胞及DC协同作用的结果。     

A Correlation between the Expression of CD 8 Antigen and Specific Cytotoxicity of Tumor-infiltrating Lymphocytes

Tumor-infiltrating lymphocytes (TIL) from six gynecologic malignant tumors (two uterine cervical cancers, two ovarian serous cystadenocarcinomas, and two uterine corpus cancers), cultured in the presence of recombinant interleukin 2, were assayed for their cytotoxic activities against various fresh tumor cells including autologous tumors. A clear correlation between phenotype and cytotoxic activity of TIL was observed. Four of six TIL preparations exhibited strong cytotoxic activity against autologous fresh tumor target cells, and were all CD8^±. In contrast, cytotoxic activity was not detected in any of the CD4^± TIL preparations. The cytotoxic activities of the CD8^± TIL preparations were highly specific; only autologous fresh tumor cells were lysed. This result is consistent with the notion that TIL are of a different cell lineage from lympholdne-activated killer cells which are antigen-nonspecific and CD8^-. Instead, TIL appear to be of cytotoxic T cell lineage that is highly antigen-specific and CD8^±. To explore the potential for clinical use, we have attempted to augment the cytotoxic activities of these CD8^± TIL by treatment of the target tumor cells with gamma interferon (IFN) in vitro , hoping that elevated expression of MHC class I gene products on the cell surface would enhance their recognition. It was observed that brief treatment of freshly prepared tumor cells in vitro with gamma-IFN resulted in augmentation of the expression of MHC class I gene products, and the treated tumor cells were more susceptible to lysis by TIL than untreated cells.     

抗原致敏DC联合CIK在原发性肝癌治疗中的应用

目的:探讨负载自身肝癌裂解物的树突状细胞(DC)与细胞因子诱导杀伤细胞(CIK)共培养对CIK体外杀伤活性的影响,并观察抗原致敏DC(Ag-DC)联合CIK治疗原发性肝癌后患者的免疫状态、临床疗效及毒副反应.方法:选择24例原发性肝癌患者,分离外周血单个核细胞,其中贴壁细胞经GM-CSF和IL-4诱导产生DC,并负载自体肿瘤裂解物;悬浮细胞经IFN-γ、IL-2、抗CD3单抗、IL-1α体外诱导产生CIK.将Ag-Dc与CIK共培养,观察CIK在体外对肝癌细胞株SMMC-7721的杀伤活性;24例患者接受Ag-DC+CIK的过继免疫治疗,观察疗效.结果:1)Ag-DC与CIK共培养后,CIK体外肿瘤杀伤活性明显提高;2)Ag-Dc联合C1K治疗原发性肝癌可明显改善患者细胞免疫功能,提高临床疗效;3)除一过性发热、畏寒外未见其它不良反应.结论:负载自体肿瘤细胞裂解物的DC疫苗联合CIK可作为原发性肝癌常规治疗的有效辅助手段.     

肿瘤坏死因子的调节性T细胞与肝癌发生的相关性

目的探讨肿瘤坏死因子(tumor necrosis factor,TNF)的调节性T细胞与肝癌发生的相关性。方法选取124例肝癌患者作为肝癌组,选择同期健康查体者124例作为对照组。检测2组血清TNF-α含量与肝功能,采用流式细胞仪检测外周血CD4⁺调节性T细胞比例并进行相关性分析。结果肝癌组的血清TNF-α含量高于对照组,血清AFP、ALT、AST、TBil含量也高于对照组,对比差异都有统计学意义(P>0.05)。肝癌组的CD4⁺调节性T细胞比例显著低于对照组(P<0.05)。相关分析显示肝癌患者CD4⁺调节性T细胞比例与TNF-α、AFP、ALT、AST、TBil都存在相关性(P<0.05)。多元线性回归分析显示TNF-α、AFP、ALT为影响CD4⁺调节性T细胞比例的主要因素(P<0.05)。结论肝癌患者的外周血CD4⁺调节性T细胞比例呈现下降趋势,与患者的TNF-α含量与肝功能状态有相关性。     

Clonal analysis of tumor-infiltrating lymphocytes from human primary and metastatic liver tumors

Phenotypic and functional characteristics of tumor-infiltrating lymphocytes (TIL) obtained from human primary and metastatic liver tumors were studied. Lymphocytes isolated from 18 tumors and autologous (A) peripheral blood (6 cases) were phenotyped by 2-color flow cytometry and cloned in a limiting dilution system, which allows virtually all normal T lymphocytes to proliferate; 70-80% of fresh TIL were T cells (i.e., CD3+), and the ratio of CD4+/CD8+ cells was 1.2 in both primary and metastatic liver tumors. TIL contained significantly more CD56+ (NKHI+) cells, half of which were CD3+CD56+, CD3+CD25+ cells and CD3+HLA-DR+ cells, than A-PBL. The frequencies of proliferating T-cell precursors (PTL-p) and cytolytic T-lymphocyte precursors (CTL-p) reactive with K562, allogeneic tumor cells and autologous tumor cells, were determined. Mean PTL-p frequencies for TIL from hepatocellular carcinomas, cholangiocarcinomas and metastatic liver tumors were 0.52 (0.22-0.83), 0.10 (0.05-0.16) and 0.16 (0.01-0.30), respectively. The frequency of CTL-p with natural-killer-like activity was lower in TIL than in A-PBL. The frequency of CTL-p for autologous tumor cells in fresh TIL isolated from primary liver tumors was 0.02-0.13 and 12/81 clones were reactive against autologous tumor. In contrast, only 1/66 TIL clones obtained from colon carcinomas metastatic to liver showed autotumor reactivity. No clones reactive with autologous tumor were obtained from peripheral blood of patients with liver cancer. These data indicate that substantial differences in anti-tumor functions of TIL between primary and metastatic liver tumors exist, which can be detected at a clonal level.     

酸性环境抑制CIK细胞对肝癌HepG2细胞的杀伤活性

目的 研究酸性环境对CIK（cytokine-induced killer, CIK）细胞杀伤肝癌HepG2细胞的影响。方法 利用IFN-γ、IL-2及CD3抗体诱导外周血单个核细胞获得CIK细胞。在pH6.5及pH7.4条件下将CIK细胞和荧光素酶标记的HepG2细胞（HepG2-luc）按不同的效靶比混合培养,用小动物活体成像系统检测HepG2-luc荧光强度并计算杀伤活性,用MTT法检测并计算杀伤活性。在pH6.5及pH7.4条件下,在含CIK条件培养液0、50%和100%的情况下培养HepG2细胞,流式细胞仪检测凋亡坏死的细胞比例。结果 pH7.4时CIK细胞对HepG2细胞的杀伤率明显高于pH6.5时。CIK条件培养液作用下,pH7.4时HepG2细胞的凋亡坏死比例明显高于pH6.5。结论 酸性环境明显抑制了CIK细胞对肝癌细胞HepG2的杀伤活性。