体外负载冻融抗原的脐血树突状细胞诱导的抗肝癌效应

目的评价体外应用肝癌细胞冻融抗原负载的脐血树突状细胞（DC）所诱导的抗肝癌效应。方法采集健康足月剖宫产孕妇胎盘端脐血,分离脐血单个核细胞（CBMNC）及T淋巴细胞,用GM-CSF、IL-4及TNF-α联合诱导CBMNC分化为DC,观察形态学变化并以流式细胞术鉴定,选培养的第3天以肝癌细胞冻融抗原负载的CBMNC-DC,以负载抗原的DC刺激自体淋巴细胞活化为自体细胞毒性T淋巴细胞（CTL）,并用CTL对肝癌细胞进行杀伤,MTT法测定活化的自体淋巴细胞的相对数量和CTL对肝癌细胞的杀伤率。结果体外负载肿瘤冻融抗原的脐血DC可诱导显著的自体效应淋巴细胞增殖及抗肝癌效应。结论体外负载抗原的脐血DC可诱导显著的抗肝癌效应,是具有临床应用前景的肝癌疫苗。     

不同肝癌细胞抗原致敏树突状细胞体外诱导CTL活性的研究

目的比较不同方法提取的肝癌细胞(SMMC-7721)抗原致敏树突状细胞(Dendritic cell DC)后,对特异性细胞毒性T细胞(CTL)的诱导作用。方法分离脐带血单个核细胞,加入重组粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)诱导扩增出脐血DC;SMMC-7721细胞经反复冻融,直接超声破碎及热休克处理后再超声破碎细胞三种方法提取肿瘤抗原,然后分别致敏DC,以MTT法检测脐血DC诱导T细胞增殖及其特异性CTL杀伤活性。结果热休克处理后再超声破碎法提取的抗原致敏DC,能诱导更强的刺激T细胞增殖的能力,并且可以诱导更强的CTL活性。结论加热处理后再超声破碎细胞提取的肿瘤抗原致敏DC,在体外可诱导强大的抗肿瘤免疫保护效应。     

HepG2细胞抗原对脐血CD34^＋造血干细胞诱导分化的树突细胞免疫功能的影响

背景：树突细胞（DC）是体内功能最强的抗原呈递细胞,可激活初始型T细胞,生成辅助性T细胞和杀伤性T细胞。DC具有特异性呈递肿瘤抗原的能力,在肿瘤免疫中发挥重要作用。目的：探讨HepG2细胞抗原对脐血CD34^＋造血干细胞诱导分化的DC免疫功能的影响。方法：分离培养脐血CD34^＋造血干细胞后,加入细胞因子组合诱导生成DC并将其分成HepG2细胞抗原负载组和对照组．以流式细胞仪测定DC生成率和免疫表型,以酶联免疫吸附测定（ELISA）检测干扰素-γ（IFN-γ）含量,以MTT法检测细胞毒性T淋巴细胞（CTL细胞）对HepG2细胞的杀伤作用。结果：DC生成率为60．2％±9．4％。与对照组相比,HepG2细胞抗原负载组DC免疫表型CD1a^＋／CD40^＋、CD83^＋／CD86^＋、CD14^＋／HLA-DR^＋比例显著增高（57．6％±5．4％对33．2％±6．0％、32．5％±3．9％对26．0％±2．8％、38．1％±2．6％对29．1％±2．1％,P〈0．01）;IFN-γ含量呈时间依赖性增高;CTL细胞对HepG2细胞的杀伤作用显著增强（43.3％±11.3％对13．9％±4．6％,P〈0．01）。结论：应用HepG2细胞抗原孵育脐血CD34^＋造血干细胞可诱导分化成熟DC,DC可促进异基因淋巴细胞活化分泌IFN-γ,并产生特异性CTL细胞,杀伤肝癌HepG2细胞。     

急性髓性白血病细胞体外诱导脐血细胞毒性T淋巴细胞增殖及抗白血病作用的研究

目的研究体外培养急性髓性白血病树突状细胞（AML-DC）,并利用该细胞联合细胞因子诱导脐血产生细胞毒性T淋巴细胞（CTL）,观察CTL对急性白血病细胞的杀伤效应,探讨从AML．DC体外诱导产生CTL的可行性。方法从急性髓性白血病细胞诱导AML—DC,联合细胞因子体外诱导脐血T细胞活化及增殖,流式细胞术检测培养前后的T淋巴细胞亚群变化,利用LDH试剂盒检测诱导后T细胞对相应急性白血病细胞的杀伤活性。结果可从人AML细胞中诱导出AML-DC,脐血T细胞在体外经过诱导培养后可获得增殖,T淋巴细胞比例较培养前明显增高,其中在AML—DC诱导组中,CD3^＋T淋巴细胞亚群比例达到（79．7±3．70）％,该T淋巴细胞对相应急性白血病细胞的杀伤效率最高达（48．35±12．75）％,与培养前及培养后无AML—DC刺激组相比,经过特异诱导培养的T细胞对相应白血病细胞杀伤作用大大加强（P〈0．05）。结论AML—DC联合细胞因子可以诱导活化脐血白血病细胞特异性细胞毒性T淋巴细胞（CTL）,该细胞能对相应白血病细胞产生特异性杀伤效应。     

Hep2-exo负载树突细胞诱导抗喉癌免疫效应的机制

目的探讨人喉癌细胞Hep2细胞来源外泌体(Hep2-exo)负载树突细胞(DC)后体外刺激细胞毒T细胞对Hep2细胞的杀伤作用。方法利用蔗糖密度梯度超速离心法从Hep2细胞培养上清液中分离Hep2-exo。透射电子显微镜鉴定Hep2-exo形态,Western印迹分析Hep2-exo表面CD9分子表达。分离培养喉癌患者外周血单个核细胞诱导的DC,流式细胞术鉴定细胞表型。用MTT法检测负载Hep2-exo的DC刺激T淋巴细胞增殖情况及细胞毒T细胞对Hep2细胞的杀伤作用。结果透射电镜下见Hep2-exo呈典型的杯状,大小相对均一,平均直径30~100 nm。Hep2-exo表面有CD9CD63分子表达。单个核细胞DC有CD1a分子表达说明诱导成功,边缘有绒毛样突起,呈典型的PC形态。负载Hep2-exo的DC刺激T细胞增殖能力及T细胞对靶细胞的杀伤效应明显强于未负载Hep2-exo的DC(P<0.05)。结论负载Hep2-exo的DC能促进T细胞增殖,可体外诱导细胞毒T细胞应答抑制喉癌细胞生长。     

人脐血和外周血树突状细胞诱导抗肝癌活性

目的　观察人脐血和外周血树突状细胞 (dendriticcells ,DC)的生物学特性及其对效应细胞体外杀伤人肝癌细胞株BEL 740 2 (B)的影响。方法　免疫组化和MTT法检测DC的生物学特性。抗肿瘤实验则分两大组 ,人外周血DC组为B +LAK +DC ,人脐血DC组为B +DC CTL ,检测效应细胞的细胞毒活性。结果　①人外周血和脐血DC均高表达HLA ABC、HLA DR、HLA DQ、CD54和S 10 0蛋白 ;刺激淋巴细胞增殖的效应均比对照组明显增高 (P <0 0 1) ,两种来源DC无显著性差异 (P >0 0 5)。②人外周血DC组和脐血DC组的细胞毒活性均为实验组 >对照组 (P <0 0 1) ,人脐血DC组 >人外周血DC组 (P <0 0 1)。结论　人外周血和脐血DC均为形态和功能成熟的DC ,均能明显增强效应细胞杀伤肝癌细胞的活性 ;而经肿瘤抗原致敏DC诱导的特异性CTL杀伤活性更强。提示临床上可根据患者的具体情况选择DC的来源     

K562细胞可溶性抗原负载树突状细胞体外诱导CTL作用的研究

目的探讨经K562细胞裂解物冲击致敏的外周血单个核细胞衍生的树突状细胞（DC）的生物特性及体外诱导抗原特异性CTL应答的能力。方法采集健康人抗凝外周血分离单个核细胞，贴壁细胞用含rhGM—CSF、rhIL-4、TNF—α的RPM1640＋10％FBS培养基体外诱导培养产生DC，5天收获细胞并将细胞分组：A组：未负载抗原DC；B组：加入K562细胞裂解液脉冲DC。7天后用流式细胞仪检测成熟DC免疫表型，并将非贴壁细胞（淋巴细胞）作为效应细胞与各组DE共育，以产生细胞毒性T淋巴细胞（CTL）。12天用LDH释放试验测定对K562细胞的杀伤活性。并用ELISA方法测定细胞上清液中IL-12的含量。结果（1）经细胞因子联合体外诱导的各组DC较培养前在数量，形态及免疫表型上差异有统计学意义，CD86、CD83、CD40、CD1a表达增加，其中经K562细胞裂解液冲击的DC的CD83CD86表达率明显升高。（2）效应细胞与K562细胞混合培养时，负载K562细胞裂解液的DC刺激后的T细胞比单独DC刺激后的T细胞对K562细胞的杀伤作用更明显。（3）负载K562细胞裂解液的DC细胞培养上清液中产生IL-12含量较未负载抗原的DC明显增加。结论用GM—CSF、IL-4以及TNF—α诱导培养健康人外周血单个核细胞可以得到成熟的DC，且经K562细胞裂解液致敏可以进一步促进DC的成熟并体外诱导特异性杀伤靶细胞的CTL。     

人脐血来源树突细胞抗乙型肝炎病毒细胞免疫的体外实验研究

目的借助DC的强大抗原呈递能力,使其负载HBsAg后以激活HBsAg特异性的CTL,从而探索一种可有效活化机体抗HBV细胞免疫的免疫方式。方法从健康产妇分娩的胎儿脐血中分离出单个核细胞,在细胞因子组合的作用下诱导出DC,于体外负载HBsAg后,激活自体T细胞成CTL,于体外培养环境下检测其对表达HBsAg的靶细胞HepG2-S的杀伤效应。结果人脐血来源的单个核细胞可在多种细胞因子的作用下,被诱导为DC。DC在负载HBsAg后可于体外活化HBsAg特异性的CTL。效应细胞为负载HBsAg的DC活化脐血单个核细胞,靶细胞分别为不表达和表达HBsAg的HepG2,当效应细胞和靶细胞比为1:1时出现最大的杀伤效率,CTL对靶细胞的杀伤率分别为(16.36±6.42)%和(78.09±9.66)%,差别有统计学意义(P<0.01)。结论健康人脐血来源的DC,具有较强的抗原呈递功能,可在体外激活产生HBsAg特异性CTL,在表达HBsAg的靶细胞模型上可观察到一定杀伤效果,为人脐血来源DC用于抗HBV治疗进行了有益的探索。     

rAAV／HCV核心抗原基因转染树突状细胞的免疫功能研究

目的 研究通过rAAV／HCV（hepatitis Cvirus,HCV）核心抗原基因（Coregene）转染树突状细胞（dendritic cell,DC）制备DC疫苗的免疫功能。方法分离外周血单个核细胞（DC前体细胞）,以rAAV／Core／Neo病毒转染DC前体细胞（基因转染组）,同时设293细胞裂解物刺激为对照组,转染12h后,均采用GM-CSF、IL-4、TNF-α诱导成熟。7天后收集细胞,流式细胞仪检测rAAV／Core／Neo病毒转染效率（即HCV核心抗原表达率）及DC表面标志CDl4、CD40、CD80、CD83、CD86的表达情况,混合淋巴细胞实验检测DC刺激自体T细胞增殖的能力,^51Cr释放法检测CTL对抗原阳性靶细胞的杀伤效率及特异性。结果rAAV／Core／Neo转染DC的效率超过90％,成熟DC高表达CD40、CD80、CD83、CD86,转染后的DC具有较强的刺激自体T细胞增殖能力,其CTL对HCV核心抗原阳性靶细胞具有很高的杀伤率及抗原特异性。结论rAAV／Core／Neo能够高效转染DC,转染后的DC能刺激自体T细胞增殖,使CTL对HCV核心抗原阳性靶细胞具有杀伤活性。     

结核分枝杆菌MPT64抗原DNA疫苗在小鼠体内诱导的免疫应答

目的研究结核分枝杆菌MPT64抗原DNA疫苗在小鼠体内诱导的免疫应答。方法用表达MPT64的真核表达质粒pcDNA-M免疫BALB/c小鼠,ELISA法检测免疫小鼠的特异性抗体滴度和抗体亚类。分离免疫小鼠的脾淋巴细胞,检测淋巴细胞增殖、IFN-γ和IL-12产生水平、流式细胞仪计CD4+细胞和CD8+细胞数、脾淋巴细胞特异性CTL杀伤效应。结果MPT64基因免疫可诱导小鼠高水平的体液免疫应答,免疫小鼠脾淋巴增殖显著,IFN-γ和IL-12含量增加,CD4+细胞和CD8+细胞百分比明显增加,CTL杀伤效应明显。结论MPT64 DNA疫苗可诱导小鼠有效的体液和细胞免疫应答,有可能作为新型TB疫苗的组分。     

In vivo anti-tumor effect of hybrid vaccine of dendritic cells and esophageal carcinoma cells on esophageal carcinoma cell line 109 in mice with severe combined immune deficiency

AIM： To develop a fusion vaccine of esophageal carcinoma cells and dendritic cells （DC） and observe its protective and therapeutic effect against esophageal carcinoma cell line 109 （EC109）.
METHODS： The fusion vaccine was produced by fusing traditional polyethyleneglycol （PEG）, inducing cytokine, sorting CD34＋ magnetic microbead marker and magnetic cell system （MACS）. The liver, spleen and lung were pathologically tested after injection of the fusion vaccine. To study the therapeutic and protective effect of the fusion vaccine against tumor EC109, mice were divided immune group and therapeutic group. The immune group was divided into P, E, D and ED subgroups, immunized by phosphate buffered solution （PBS）, inactivated EC109, DC and the fusion vaccine respectively, and attacked by EC109 cells. The tumor size, weight, latent period and mouse survival period were recorded and statistically analyzed. The therapeutic group was divided into four subgroups： P, inactivated EC109, D and ED subgroups, which were attacked by EC109 and then treated with PBS, inactivated EC109, DC, and EC109-DC respectively. Pathology and flow cytometry were also used to study the therapeutic effect of the fusion vaccine against EC109 cells.
RESULTS： Flow cytometry showed that the expression of folate receptor （FR）, EC109 （C）, Des （D） in human nasopharyngeal carcinoma cell line （HNE1） （B） was 78.21%, 89.50%, and 0.18%, respectively. The fusion cells （C） were highly expressed. No tumor was found in the spleen, lung and liver after injection of the fusion vaccine. Human IgG was tested in peripheral blood lymphocytes （PBL）. In the immune group, the latent period was longer in EC109-DC subgroup than in other subgroups, while the tumor size and weight were also smaller than those in ED subgroup. In the therapeutic group, the tumor size and weight were smaller in ED subgroup than in P, inactivated EC109 and DC subgroups.
CONCLUSION： Fusion cells are highly expressed not only in FR but     

Effect of a cancer vaccine prepared by fusions of hepatocarcinoma cells with dendritic cells

AIM To prepare a cancer vaccine(H-(22)-OC)expressinghigh levels of costimulatory molecules based on fusions ofhepatocarcinoma cells(H_(22))with dendritic cells(DC)ofmice and to analyze the biological characteristics andinduction of specific CTL activity of H_(22)-DC.METHODS DCs were isolated from murine spleen bymetrizamide density gradient centrifugation,purifiedbased on its characteristics of semi-adhesion to cultureplates and FcR,and were cultured in the mediumcontaining GM-CSF and IL-4.A large number of DC wereharvested.DCs were then fused with H_(22) cells by PEG andthe fusion cells were marked with CO11c MicroBeads.TheH_(22)-DC was sorted with Mimi MACS sorter.The techniquesof cell culture,immunocytochemistry and lightmicroscopy were also used to test the characteristics ofgrowth and morphology of H_(22)-DC in vitro.As theimmunogen,H_(22)-DC was inoculated subcutaneously intothe right armpit of BALB/C mice,and their tumorigenicityin vivo was observed.MTT was used to test the CTLactivity of murine spleen in vitro,RESULTS DC cells isolated and generated were CO11c cells with irregular shape,and highly expressed CD80.CD86 and CD54 molecules. H_(22) cells were CO11c~- cellswith spherical shape and bigger volume,and did notexpress CD80, CD86 and CD54 molecules.H_(22)-DC wasCO11c~ cells with bigger volume,being spherical,flat orIrregular in shape,and highly expressed CD80,CD86 andCD54 molecules,too.H_(22)-DC was able to divide andproliferate in vitro,but its activity of proliferation wassignificantly decreased as compared with H_(22) cells and itsgrowth curve was flatter than H_(22) cells,Aftersubcutaneous inoculation over 60 days,H_(22)-DC showed notumorigenecity in mice,which was significantly differentfrom control groups(P<0.01).The spleen CTL activityagainst H_(22) cells in mice implanted with fresh H_(22)-DC wassignificantly higher than control groups(P<0.01).CONCLUSION H_(22)-DC could significantly stimulate thespecific CTL activity of murine spleen,which suggests that the fusion cells have already obtained the function ofantigen presenting of parental DC and could present H_(22)specific antigen which has not been identified yet,andH_(22)-DC could induce antitumor immune response;althoughsimply mixed H_(22) cells with DC could stimulate the specificCTL activity which could inhibit the growth of tumor insome degree,it could not prevent the generation oftumor.It shows that the DC vaccine is likely to become ahelpful approach in immunotherapy of hepatocarcinoma.     

Effects of dendritic cells from cord blood CD34^＋ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in sever combined immunodeficiency (SCID) mice. METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocartinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect. RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%, 47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80% vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11d vs 7 d,P<0.01). CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.     

肝癌细胞裂解物致敏的树突状细胞瘤苗体外诱导特异性抗肝癌免疫

目的 探讨肝癌患者肿瘤细胞裂解物致敏的树突状细胞(DC)瘤苗体外诱导自体T淋巴细胞特异性抗肝癌免疫效应。 方法 从肝癌患者外周血单个核细胞中诱导D C,用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素-4(rhIL-4)刺激活化,经自体肝癌细胞裂解物致敏。用流式细胞仪检测D C细胞表面分子的表达,酶联免疫吸附法检测T淋巴细胞培养上清液中干扰素(I F N)γ和白细胞介索-12(IL-12)的含量,液体闪烁计数仪测定肝癌细胞裂解物致敏的D C刺激自体T淋巴细胞增殖效应,四甲基偶氮唑盐法检测肝癌细胞裂解物致敏D C诱导的细胞毒T淋巴细胞对自体肝癌细胞的特异性杀伤作用。 结果 肝癌细胞裂解物致敏的DC瘤苗可上调DC表面CD1 a、CD40、CD86和人类白细胞抗原-DR分子表达水平,其与T淋巴细胞共培养产生的IFN γ、IL-12的浓度明显高于未致敏的D C组(t值分别为2.30、2.14,P<0.05),肝癌细胞裂解物组(t值分别为14.01、15.40,P<0.01)和对照组(t值分别为14.85、16.87,P<0.01)。同时肝癌细胞裂解物致敏的瘤苗可明显诱导T淋巴细胞的增殖,其诱导的细胞毒性T淋巴细胞对自体肝癌细胞的杀伤率(81.72％±9.49％)显著高于对HepG2的杀伤率(49.37％±11.21％)和人鼻咽癌肿瘤细胞的杀伤率(17.14％±5.65％),P<0.01。 结论 肝癌细胞     

Human umbilical cord blood-derived stromal cell, a new resource of feeder layer to expand human umbilical cord blood CD34+ cells in vitro

Allogeneic transplantation with human umbilical cord blood (hUCB) in adult recipients is mainly limited by a low CD34+ cell dose. To break the limit, hUCB as a novel source of hUCB-derived stromal cells was incorporated in an attempt to expand CD34+ cells from hUCB in vitro. Cord blood CD34 cells were separated by MACS system. HUCB-derived stromal cells were cultured by the Dexter system and characterized by morphologic, immunophenotypical, and functional analysis. We studied the effects of hUCB-derived stromal cells, cytokines, and hUCB-derived stromal cells combined with cytokines on expansion of hUCB CD34 cells. The CD34+ cells were assessed for the degree of expansion and the number of colony-forming units in semisolid culture. Our research found that hUCB-derived stromal cells were mainly composed of three kinds of cell components, with CD106, CD29, CD44, CD45, CD50, CD68, CD31, Fn, Lm, and collagen IV positive, but CD34 negative immunophenotype. Functionally, it was discovered by cell cycle and growth curve analyses that the capability of colony and parietal layer formation of hUCB-derived stromal cells was poorer than that of BM stromal cells, and the doubling time of hUCB-derived stromal cells was longer than that of BM stromal cells. It was indicated by ELISA and RT-PCR that hUCB-derived stromal cells express higher level of TPO and less GM-CSF and SCF than BM stromal cell. Adherent layer of hUCB-derived stromal cells alone or combining with cytokines, increased CD34+ cell expansion. In vitro formation of CFUs by expanded CCD34 cells was significantly higher than that of unexpanded CD34+ cells (P < 0.05). When cocultured with hUCB-derived stromal cells in the presence of cytokines, cell growth was significantly enhanced: CD34 cells by 8.02 +/- 0.96-fold, CFU-GM by 217.60 +/- 6.72-fold, CFU-E by 1940.80 +/- 52.78-fold, and CFU-Mg by 142.60 +/- 4.39-fold. HUCB-derived stromal cells have significant superiority on the expansion of CFU-Mg (P < 0.05). The results indicate that human umbilical cord blood-derived stromal cells may be a suitable feeder layer for expansion of hematopoietic progenitors from hUCB in vitro.     

VLA-2 and VLA-5 cell adhesion molecules expression in CD34+ cells from umbilical cord blood and from bone marrow

BACKGROUND/AIMS: Umbilical cord blood contains a large number of early hematopoietic cells with high proliferating capacity, that has been used as an alternative to bone marrow transplantation. The aim of this study is to investigate the number of two cell adhesion molecules in cord blood and in bone marrow. METHODS: We investigated two integrins, named VLA-2 and VLA-5 (Very Late Appearing Antigen), expressed in the surface of CD34+ cells. The CD34+ cells, isolated with MACS CD34+ isolation kit, were labelled with the appropriate monoclonal antibodies. RESULTS: Cell adhesion molecules showed highly expressed in both cord blood and bone marrow CD34+ cells. CONCLUSION: There are no significant differences between the two sources of CD34+ populations.     

负载食管癌抗原的DC诱导特异性CTL对食管癌细胞体外杀伤作用观察

目的观察负载食管癌抗原的树突状细胞（DC）活化的特异性细胞毒性T淋巴细胞（CTLs）对食管癌细胞的体外杀伤作用。方法冻融法获取食管癌细胞抗原,联合应用粒细胞—巨噬细胞集落刺激因子（rhGM-CSF）、白细胞介素4（IL-4）和肿瘤坏死因子α（TNF-α）诱导培养外周血DC并负载肿瘤抗原,激活自体T淋巴细胞,制备特异性CTLs。将其加入食管癌细胞中培养48 h,用MTT法检测食管癌细胞裂解率,ELISA法检测γ干扰素（γ-IFN）水平。结果负载食管癌抗原的DC激活的CTLs表现出对食管癌Eca109细胞的特异性杀伤作用,γ-IFN水平为（1 625±37.55）pg/ml;而对A549细胞仅有微弱的杀伤作用,γ-IFN水平为（169.04±13.81）pg/ml。未负载食管癌抗原DC刺激的CTLs对食管癌细胞几无杀伤作用。结论负载食管癌抗原的DC激活的CTLs在体外对食管癌细胞能产生高效而特异性的杀伤作用。     

Different behaviour of fresh and cultured CD34+ cells during immunomagnetic separation

In-vitro expansion of human cord blood (CB) cells could enhance peripheral blood recovery and ensure long-term engraftment of larger recipients in the clinical transplant setting. Enrichment of CD34+ cells using the MiniMACS column has been evaluated for the preparation of CB CD34+ cells before and after expansion culture. Repurification of CD34+ cells after culture would assist accurate phenotypic and functional analysis. When fresh CB mononuclear cells (MNC) were separated, the MACS positive (CD34+) fraction (90.1% pure) contained a mean (+/- SD, n = 5) of 93.0 +/- 8.0% of the eluted CD34+ cells, 99.6 +/- 0.7% of the CFU-GM and all of the eluted long-term culture-initiating cells (LTC-IC). Cord blood CD34+ cells were then cultured for 14 d with IL-3, IL-6, SCF, G-CSF and GM-CSF, each at 10 ng/ml. The total cell expansion was 2490 +/- 200-fold and the CD34+ cell expansion was 49 +/- 17-fold. The percentage of CD34+ cells present after expansion culture was 1.2 +/- 0.85%. When these cells were repurified on the MiniMACS column, the MACS positive fraction only contained 40.3 +/- 13.4% of the eluted CD34+ cells which was enriched for the mature CD34+ CD38+ subset, 24.4 +/- 8.8% of the eluted CFU-GM and 79.5 +/- 11.0% of the LTC-IC. The remaining cells were eluted in the MACS negative fraction. In conclusion, repurification of cultured CD34+ cells does not yield a representative population and many progenitors are lost in the MACS negative fraction. This can give misleading phenotypic and functional data. Cell losses may be important in the clinical setting if cultured cells were repurified for purging.