小鼠肠癌RNA转染mIL-12修饰的树突细胞诱发特异性细胞毒性T淋巴细胞的研究

目的：研究小鼠结肠癌细胞CT-26RNA体外转染mIL-12基因修饰的树突细胞（DC），观察其诱导特异性细胞毒性T淋巴细胞（CTL）能力。方法：小鼠骨髓细胞体外经rmGM-CSF、rmIL-4诱导培养获取DC，流式细胞仪检测其纯度；293细胞扩增携带mIL-12基因的重组腺病毒，体外转染DC；Trizol法提取CT-26细胞总RNA，应用TransMessenger体外转染mIL-12基因修饰的DC；ELISA法检测细胞上清及小鼠血液中mIL-12，LDH释放法检测小鼠体内特异性CTL活性。结果：小鼠骨髓细胞经诱导培养后，获得大量高纯度的DC，流式细胞仪检测CD11c^＋的DC〉90％；携带mIL-12基因重组腺病毒转染的DC细胞高表达mIL-12；CT-26细胞总RNA体外转染mIL-12基因修饰的DC后，回输小鼠，能明显提高小鼠体内mIL-12的水平，并可诱导体内生成较高水平的CTL活性，而以该RNA转染Ad-LacZ修饰DC后的对照组及RNA转染DC的对照组，可诱导机体生成中等水平的特异性CTL活性，DC、PBS对照组则均无此作用。结论：小鼠肠癌CT-26细胞总RNA转染mIL-12基因修饰的DC后，免疫接种小鼠，可提高小鼠体内mIL-12的水平，并能有效诱导机体产生较高水平的特异性CTL活性。     

PADRE/MUC4 重组腺病毒转染树突状细胞诱导特异性CTL体外杀伤作用研究

目的:观察PADRE/MUC4重组腺病毒转染树突状细胞(DC)诱导特异性细胞毒性T细胞(CTL)及体外特异性杀伤作用.方法:pAd-CMV-PADRE/MUC4转染HLA-A2健康志愿者外周血单个核细胞(PBMC)来源的未成熟DC,TNF-α诱导成熟后与自体PBMC混合培养刺激3周,Cr51和Elispot实验检测CTL体外杀伤活性.结果:Cr51和Elispot检测结果显示PADRE/MUC4转染DC可以诱导产生特异性CTL,而pAd-CMV-GFP组和空白对照组不能产生有效特异性CTL.结论:腺病毒载体介导多表位嵌合基因(PADRE/MUC4)转染未成熟DC,在体外可以诱导产生特异性CTL,对表达MUC4肿瘤细胞具有杀伤效应.     

树突状细胞感染Her2/neu膜外及跨膜区蛋白基因重组腺病毒后的免疫功能变化

目的 观察体外树突状细胞（DC）感染编码Her2／neu基因膜外第一受体区（Her2-ECDs）、全长膜外区（Her2-ECD）和膜外跨膜区（Her2-TM）蛋白3种重组腺病毒（rAdrier2-ECDs、rAdrier2-ECD和rAdHer2-TM）后的免疫功能变化。方法 重组腺病毒感染未成熟DC后，Western blot法检测目的蛋白在DC中的表达。ELISA法检测DC感染3个重组腺病毒后的白介素-12（IL-12）分泌水平及与淋巴细胞共孵育后上清中干扰素-γ（IFN-γ）的含量。采用混合白细胞反应检测DC感染前后刺激同种异体淋巴细胞的增殖能力。MTT法检测细胞毒T淋巴细胞（CTL）活性。结果 Her2-ECDs、ECD、TM蛋白在DC中获得表达。转染DC培养第5天，上清中IL-12含量比未转染DC含量高（P〈0．05），但3种重组病毒之间无明显差异（P〉0．05）。DC刺激淋巴细胞增殖后培养上清中IFN-γ的含量显示随着时间的延长逐步增高，但病毒感染DC明显高于非感染DC。DC明显介导淋巴细胞增殖反应，除rAdHer2-TM感染DC外，另两种转染和非转染DC之间无差异（P〉0．05）。在DC诱导的CTL反应中，病毒感染DC诱导的杀伤率明显高于SK-OV-3修饰和非修饰DC，而SK-OV-3修饰又高于非修饰DC杀伤率（P〈0．05）。在病毒感染DC中．以rAdrier2-TM转染DC激发的CTL活性为最强。对高表达Her2／neu蛋白的乳腺癌细胞株的杀伤率明显高于对Her2／neu表达阴性的杀伤率（P〈0．05）。结论 编码Her2／neu膜外及跨膜区蛋白重组腺病毒转染DC后，明显增强DC的抗肿瘤免疫功能．诱导出Her2／neu特异性的CTL活性。     

含hTERT片段的重组逆转录病毒感染对树突状细胞功能的影响

目的观察含人端粒酶逆转录酶（hTERT）片段的重组逆转录病毒感染对树突状细胞（DCs）功能的影响。方法ELISA试剂盒检测DCs培养液中IL-12水平;混合白细胞（MLR）反应检测含hTERT片段的重组逆转录病毒感染的DCs（hTERT-DCs）和未感染的DCs（N-DCs）刺激同种异体淋巴细胞增殖能力;流式细胞术检测DCs表面分子CD80、CD83、CD86和HLA-DR的变化;CytoTox96非放射性细胞毒性检测试剂盒检测细胞毒性T淋巴细胞（CTL）反应。结果hTERT-DCs和N-DCs在分泌IL-12的水平、刺激同种异体淋巴细胞增殖的能力方面无明显差异;hTERT-DCs的CD83表达水平低于N-DCs,同时,hTERT-DCs激发的CTL对端粒酶阳性的靶细胞杀伤率明显高于端粒酶阴性的靶细胞（P〈0．05）。结论hTERT-DCs尽管有可能阻止DCs自身的成熟,但在活化淋巴细胞、刺激淋巴细胞分化增殖的能力方面并没发生明显改变,并且还能激发hTERT特异性CTL。     

Her2/neu基因重组腺相关病毒转染人外周血来源树突状细胞的免疫功能

OBJECTIVE: To observe the immunostimulatory effect of human peripheral blood dendritic cells (DCs) transfected by Her2/neu gene delivered by adeno-associated virus. METHODS: The mononuclear cells in healthy donors were isolated by Ficol-Hypaque density gradient separation and divided into transfection group and control group without transfection by the recombinant virus. The cells were initially cultured in RPMI 1640 medium supplemented with 10% AB human serum, followed by addition of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4 and tumor necrosis factor (TNF)-alpha into the medium. The surface markers of DC were detected by flow cytometry, allogeneic mixed lymphocyte reaction assayed by 3H-TdR incorporation and the specific killing activity of T cells evaluated by MTT assay. RESULTS: The expression rates of CD1a, CD86 and CD83 of the transfected DCs were 98.10%, 99.42%, and 84.59%, and those of the non-transfected DCs were 92.69%, 98.07%, and 82.72%, respectively, showing no obvious differences between the two groups. The rates of CD4 and CD80 expression were 61.02% and 97.61%, respectively, in the transfected DCs, significantly higher than those in the non-transfected cells (36.19% and 55.5%). Both groups of DCs stimulated a strong T cell proferation response. The transfected DCs were capable of inducing specific killing of the target tumor cells, with the highest killing rate of (39.7+/-7.2)%. CONCLUSION: The immunostimulatory effect of human peripheral blood DCs can be enhanced by Her2/neu gene transfection mediated by adeno-associated virus.     

尤文肉瘤融合基因修饰的重组腺病毒的构建及其转染的树突细胞体外抗尤文肉瘤免疫应答

目的:构建尤文肉瘤融合基因EWS-FLI1重组腺病毒,利用此腺病毒感染外周血单核细胞(PBMC),培养树突细胞(DC),检测感染后的DC致敏的淋巴细胞对尤文肉瘤细胞系的杀伤作用.方法:将质粒Pec1/ EWS-FLI1酶切,切出的EWS-FLI1 cDNA片段克隆至腺病毒穿梭质粒padtrack-cmv的hCMV启动子下游.将穿梭质粒和骨架质粒共同转化大肠杆菌BJ5183菌株,获得同源重组后的腺病毒质粒pADeasy-1/ EWS-FLI1.将此质粒转染293细胞,包装产生腺病毒Ad EWS-FLI1.扩增、纯化产生高滴度的Ad EWS-FLI1.转染PBMC并经过鉴定后致敏淋巴细胞,4 h 51Cr释放试验测定致敏后的淋巴细胞对尤文肉瘤细胞系的杀伤作用.结果: 同源重组后产生的pADeasy-1/ EWS-FLI1经PCR鉴定构建成功,纯化后的滴度为4×10¹⁰/mL.转染PBMC后,RT-PCR证实有EWS-FLI1 mRNA的转录.致敏后的淋巴细胞对尤文肉瘤细胞系有明显的杀伤作用,效靶比在40:1时,对尤文肉瘤673细胞系的杀伤率为35.1800%±0.0128%,和对照组相比差异有统计学意义. 结论 :重组腺病毒Ad EWS-FLI1构建成功,并能在PBMC中稳定有效地表达,Ad EWS-FLI1转染的DC可高效地诱发机体T细胞的特异性抗肿瘤免疫应答作用.     

In Vitro Anti-tumor Immune Response Induced by Dendritic Cells Transfected with Recombinant Adenovirus Carrying Mutant K-ras Genes

Summary： The specific anti-tumor immune response induced by mouse bone marrow dendritic cells （DCs） lransfected with recombinant adenovirus carrying mutant k-ras genes was investighted. DCs were generated from mouse bone marrow in the presence of rmGM-CSF （3.3 ng/mL） and rmIL-4 （1.3 ng/mL） and detected by FACS, and then transfecled with the recombinant adenovirus encoding mutant k ras gene. The efficacy of transfection and T cell stimulating activity of DCs were detected. CTL activity of the mice vaccinated with DCs was observed. The resuhs showed thai DCs had dendritic veiled morphology. BmDCs highly expressed B7-1（80%）, B7-2（77%）, MHC Ⅱ （70%）, CDllc （65%）, CD40 （70%） and CD54 （96%） with FACS, and no significant difference in the expression was observed before and after the transfection （P〈0.05）. The DCs transfeeled by mutant k-ras gene could significantly stimulate lymphoeytes proliferation as compared with those transfeeted by Ad e or non-modified DCs （P〈0.05）. DC vaccine transfected by mutant k-ras gene could induce CTL activity against Lewis lung cancer, but not against B16. The specific eytotoxicity against Lewis lung cancer in Ad-k-ras/12-transdueed DC group was signifieantly higher than those in the control, vector and non transfeeted DCs groups （P〈0.05）. It was concluded that special antitumor response could be induced by DCs transfected with recombinant adenovirus carrying mutant k-ras genes.     

多发性骨髓瘤抗原致敏树突状细胞介导的体外抗瘤活性

目的：探讨多发性骨髓瘤KM3细胞株冻融抗原和弱酸洗脱抗原肽负载树突状细胞（DC）后诱导的特异性细胞毒性T淋巴细胞（CTL）对KM3细胞的杀瘤作用。方法：联合GM-CSF,IL-4和TNF-α,体外诱导单核细胞生成DC,并使其致敏KM3特异性冻融抗原或酸洗脱抗原,然后与自体T淋巴细胞共同培养3d,生成特异性CTL,并采用MTT法检测其对KM3细胞的杀伤率。结果：在细胞因子作用下,体外培养的外周血单个核细胞可诱导分化为成熟的DC;应用KM3肿瘤抗原致敏DC,诱导生成的CTL对KM3肿瘤细胞具有较强的杀伤效应。结论：KM3冻融抗原和酸洗脱抗原激发的DC与自体T淋巴细胞孵育能诱生抗KM3细胞特异性CTL。     

In vitro induction of specific anti-tumoral immunity against laryngeal carcinoma by using human interleukin-12 gene-transfected dendritic cells

Background  Objective evaluation of the antitumor effect of interleukin-12 (IL-12) gene-transfected dendritic cell (DC) vaccine on laryngeal carcinoma requires in vivo and in vitro tests. The aim of this study was to investigate the function of IL-12 gene transfected DC at initiating specific immune response to laryngeal carcinoma in vitro．

Methods  Recombinant adenovirus with IL-12 gene was constructed. DCs were isolated from the peripheral blood of patients with laryngeal carcinoma, pulsed with tumor lysate of laryngeal carcinoma cells (DC⁺Ag), and transfected with IL-12 (DC-IL-12⁺Ag). The cells pheotypes including CD83, CD86 and HLA-DR on surface of DCs were assayed by flow cytometry (FCM). The concentration of IL-12 in culture supernatant of DCs and interferon γ (IFN-γ) in culture supernatant of T cells cocultured with DCs were quantified by ELISA. Methyl thiazolys tetrazolium (MTT) was used to evaluate proliferation of autologous T lymphocytes and activation of cytotoxic T lymphocytes (CTL) stimulated by IL-12-transfected DCs pulsed with tumor lysate against laryngeal carcinoma cells.

Results  The recombinant adenovirus expressing IL-12 gene was constructed successfully. Gene-transfected DC plused with tumor lysate with IL-12 (DC-IL-12⁺Ag) expressed higher level of CD83, CD86 and produced higher level of IL-12 than untransfected DCs (DC⁺Ag) (CD83: (60.2±1.8)% vs. (50.7±1.2)%, P <0.05; CD86: (88.9±2.1)% vs. (78.2±3.9)%, P <0.05; IL-12: (262.5±3.0) ng/L vs. (103.8±5.1) ng/L, P <0.05). The proliferation of autologous T lymphocytes and production of IFN-γ stimulated by DC transfected with IL-12 were more obviously than untransfected DCs. Cytotoxicity of CTL stimulated by IL-12-transfected DC pulsed with tumor lysate against laryngeal carcinoma cells were significantly stronger than stimulated by untransfected DC.

Conclusion  It is a promising approach for IL-12-transfected DC pulsed with tumor lysate to increase the antitumoral effect.

     

TNF-α gene-modified dendritic cells act as more potent adjuvants for peptide delivery to induce specific antitumor immunity in mice

Objective To investigate the antitumor immune efficiency of mouse dendritic cells (mDCs) by using adenovirus-mediated tumor necrosis factor-alpha (AdV-TNF-α) gene transfer.Methods MDCs infected with AdV-TNF-α and AdV-pLpA (no gene insert) at 100 multiplicity of infection (MOI) were analyzed by RNase protection assay for their cytokine secretion. Mixed lymphocyte reactions were also performed to analyze their capacity for alloantigen-presentation. C57BL/6 mice were challenged with R3LL tumor cells (Lewis lung carcinoma line) 10 days after vaccination with different engineered DCs and regular DCs as well.Results Compared to AdV-pLpA and mock-infected DCs, AdV-TNF-α-infected DCs displayed up-regulated expression of alpha tumor necrosis factor, interleukin-12 (IL-12), interleukin-18 (IL-18) and granulocyte macrophage colony stimulation factor (GM-CSF), and indicated stronger allogeneic T cell proliferative responses. Furthermore, vaccination of mice with dendritic cell tumor necrosis factor-alpha (DCTNF-α) pulsed with Mut1 peptide induced more efficient tumor-specific cytotoxic T lymphocyte (CTL) cytotoxicity against R3LL tumor cells in vitro and with efficient antitumor immunity in vivo. Conclusion This type of engineered DCs could be applied in clinical settings of DC-based cancer vaccines     

mIL-12基因转染促进小鼠树突状细胞混合淋巴细胞反应

目的 观察ｍＩＬ－１２基因转染的小鼠树突状细胞（ＤＣ）功能的改变。方法 分离小鼠骨髓单个核细胞与ｒｍＧＭ－ＣＳＦ和ｒｍＩＬ－４培养１周,对培养的细胞进行形态学观察,ＦＡＣＳ检测细胞表面ＤＥＣ２０５、ＣＤ８６表达。以ｍＩＬ－１２重组腺病志染培养的ＤＣ,ＥＬＩＳＡ测定培养上清中ｍＩＬ－１２的水平。混合淋巴细胞反应检测转染细胞的功能。结果培养１周后,得到具有典型ＤＣ形态的细胞,以ｍＩＬ－１２重组腺病毒为     

胆管癌肿瘤裂解物修饰和P53基因导入对树突状细胞免疫效应的影响

目的 :研究胆管癌裂解物对转染全长野生型P5 3的树突状细胞 (DC)免疫功能的影响。方法 :用腺病毒作为介质 ,将全长野生型P5 3转染入DC ,然后用胆管癌裂解物修饰已转染全长野生型P5 3的DC(LywtP5 3DC) ,检测这种DC表面分子B7 1、B7 2、MHC Ⅰ、MHC Ⅱ表达的高低 ,诱导特异的细胞毒性T淋巴细胞 (CTLs)的能力 ,对小鼠的免疫保护和对动物模型治疗作用。结果 :经流式细胞仪检测胆管癌裂解物刺激的野生型P5 3DC表面高表达B7 1(86 .70 %± 0 .0 7% )、B7 2 (18.77%± 0 .0 8% )、MHC Ⅰ (87.2 0 %± 0 .0 5 % )、MHC Ⅱ (5 6 .70 %± 0 .0 7% ) ,单纯DC低表达 ;LywtP5 3DC能够特异性地杀伤胆管癌细胞 ,杀伤率为 81%。LywtP5 3DC治疗组和其它组在肿瘤生长的直径大小之间有显著差异 (P <0 .0 5 ) ,在LywtP5 3DC治疗组中 ,肿瘤生长明显减慢。结论 :全长野生型P5 3基因转染 +胆管癌裂解物联合修饰的DC能诱导细胞毒性T淋巴细胞的特异性 ,显著地提高DC的抗原提呈功能 ,体外能诱导高效而特异的抗胆管癌免疫效应。     

未成熟DCs诱导同种免疫低应答及 CD14分子在DCs上的表达

目的：从健康人外周血单核细胞诱导树突状细胞（DCs）,并且证实未成熟DCs在体外可以诱 导同种T细胞的低应答。 方法：人外周血单核细胞经GM-CSF,IL-4及TNF-α联合诱导,分化出不同发育阶段的DCs。采用再次混合淋巴细胞培养的方法观察未成熟DCs处理过的T细胞对与未成熟DCs同一来源的T细胞的应答能力。 结果：在DCs的未成熟期与成熟期均中度以上表达CD14分子;随着培养时间的不同DCs内吞能 力不断变化,在第7天达高峰,且内吞量高于相同培养时间的单核细胞（P＜001）;在再次混合淋巴细胞反应中,供者未成熟DCs预处理的受者T细胞当再次被供者的T细胞刺激时应答降低,而对第三者的T细胞刺激则表现强反应性。 结论：诱导人外周血单核细胞获得不同发育阶段的DCs,经过再次混合淋巴细胞反应发现具 有中度表达共刺激分子的未成熟DCs在体外可以诱导T细胞免疫低应答。     

抗原致敏树突状细胞诱导CTL对MCF-7细胞的体外杀伤效应

目的:采用MCF-7乳腺癌细胞裂解物致敏树突状细胞(DC),观察其体外诱导人T淋巴细胞产生特异性抗乳腺癌免疫的效能。方法:联合应用细胞因子从外周血单个核细胞(PBMC)中诱导出DC,体外负载MCF-7细胞冻融抗原,活化自身T淋巴细胞,采用ELISA法测定γ-干扰素(IFN-γ)、白介素-12(IL-12)水平,乳酸脱氢酶释放法检测细胞毒性T淋巴细胞(CTL)对MCF-7细胞的杀伤效应。结果:负载抗原DC与T淋巴细胞共培养产生IFN-γI、L-12的水平,显著高于未负载抗原DC组(P<0.05)、单纯抗原组(P<0.01)和对照组(P<0.01);负载抗原DC诱导CTL对MCF-7的杀伤效应,显著高于其他3组(P<0.01)。结论:MCF-7细胞裂解物致敏DC诱导的人CTL,对MCF-7细胞具有明显的特异性杀伤效应。     

转染IL-18基因慢性粒细胞白血病树突状细胞的抗白血病作用

目的：探讨转染IL-18基因慢性粒细胞白血病（CML）树突状细胞（DCs）抗白血病免疫作用。方法：以脂质体介导法将pVAX1-IL-18真核表达质粒转染CML DCs,检测转染IL-18 DCs的IL-18表达,通过流式细胞仪检测转染IL-18 DCs的CD80⁺、CD86⁺细胞百分率,并检测IL-18基因修饰DCs刺激T细胞增殖能力、特异性CTL杀伤作用及诱导的NK细胞的杀伤活性。结果：转染IL-18 DCs上清中IL-18的含量为（596±34.1）pg/2×10⁶cells/48 h,而在转染空质粒DCs和DCs上清中未检测到IL-18,且转染IL-18 DCs的CD80⁺、CD86⁺细胞百分率高于转染空质粒的DCs（P<0.05）。转染IL-18的DCs刺激T细胞增殖能力、特异性CTL杀伤作用及诱导的NK细胞的杀伤活性明显高于转染空质粒的DCs（P<0.05）,且随着S/R或R/T增加,其多种免疫反应逐渐增强。结论：IL-18与DCs抗肿瘤作用具有协同性。     

抗结核DC疫苗的初步研究

目的:研制抗结核的新型疫苗(DC疫苗)。方法:无菌分离C57BL/6小鼠骨髓细胞,以rGM-CSF及rIL-4诱导分化获得树突状细胞(DC),以pEGHsp65融合质粒转染DC,共聚焦激光扫描显微镜检测转染率,继以电镜鉴定细胞形态、流式细胞术分析组合性表面分子表达、混合淋巴细胞反应(MLR)检测体外刺激T细胞增殖的功能。将制备的DC疫苗静脉接种正常同系小鼠,分别取各脏器冰冻切片后荧光显微镜观察;取小鼠静脉血用ELISA法检测IL-12及IFN-γ的表达量。结果:24 h DC转染率约20%,电镜可见细胞呈典型毛刺状突起,流式细胞分析表明转染后的DC表面MHCⅡ、33D1的表达量均呈显著增高。MLR显示,pEGHsp65转染DC组与pEG-FP-C1转染DC组及空白DC组比较呈统计学增高。脏器冰冻切片显示,接种pEGHsp65转染DC小鼠组及pEG-FP-C1转染DC小鼠组与未转染DC组比较,在各脏器分布中,以脾脏分布显著增高。其余脏器分布无显著差别。pEGHsp65转染DC小鼠组IL-12及IFN-γ表达量显著高于pEGFP-C1转染DC小鼠组及未转染DC小鼠组,P<0.05,未转染DC组与阴性对照组相比,IL-12及IFN-γ表达量也有统计学增高,P<0.05。结论:pEGHsp65制备的DC疫苗具有成熟表型及功能,能够刺激小鼠IFN-γ及IL-12分泌增高。     

用脂质体包裹重组survivin腺病毒治疗肿瘤的实验研究

目的 本实验拟用脂质体包裹重组survivin腺病毒治疗肿瘤,观察其抗肿瘤效应。方法 在BALB／c小鼠建立CT26结肠癌模型,将小鼠随机分成用脂质体包裹重组survivin腺病毒组、重组survivin腺病毒组、用脂质体包裹空病毒组、脂质体组、PBS对照组5组,观察肿瘤的生长以及小鼠的存活情况和不良反应,并做细胞毒T淋巴细胞（CTL）分析。HE染色观察肿瘤组织及肿大淋巴结的病理改变。结果 ①用脂质体包裹重组survivin腺病毒对小鼠有免疫保护和治疗作用,小鼠肿瘤生长受到明显抑制,肿瘤大小和存活率与其它组比较,差异均有统计学意义（P〈0．05）。②肿瘤病理切片显示用脂质体包裹重组survivin腺病毒组肿瘤组织内有较多的淋巴细胞浸润,有大量的坏死灶。③CTL分析显示用脂质体包裹重组survivin腺病毒组免疫小鼠的T细胞具有较高的CT26细胞杀伤活性,并具有特异性及不依赖NK细胞活性。结论 用脂质体包裹重组survivin腺病毒对CT26移植瘤有治疗作用。     

rhHSP70对食管癌细胞RNA转染的脐血树突状细胞诱导CTL抗肿瘤功能的影响

目的研究重组人热休克蛋白70(rhHSP70)联合食管癌细胞RNA转染脐血树突状细胞(DCs)对细胞毒性T淋巴细胞(CTL)特异性抗肿瘤作用的影响。方法分离脐血单个核细胞,经rhSCF、rhGM-CSF和IL-4诱导和食管癌RNA转染形成成熟DCs,加入rhHSP70,检测DCs表面标志及DCs诱导的T细胞增殖、CTL杀伤活性。结果食管癌RNA转染后DCs高表达抗原提呈分子、协同刺激分子和黏附分子,对T细胞的促增殖作用和CTL杀伤活性显著增强(P<0.01),rhHSP70能显著促进DCs功能。结论 rhHSP70具有增强食管癌细胞RNA转染的DCs对T细胞的促增殖和CTL杀伤活性的作用。     

Her2/neu基因重组腺相关病毒转染人外周血来源树突状细胞的免疫功能

目的 Her2/neu基因重组腺相关病毒(rAAV-Her2/neu)转染人外周血树突状细胞(DC),并检测其免疫功能。方法 采用Ficoll分离健康人外周血中的单个核细胞。将其分成两组,给其中一组加入病毒。应用含10%人AB血清的RPMI-1640培养基及粒细胞-巨噬细胞集落刺激因子,白细胞介-4、肿瘤坏死因子-α培养。第7天收获成熟DC。流式细胞仪检测DC表型。³H-TdR检测同种混合淋巴细胞反应。MTT法检测DC诱导T细胞的杀伤活性。结果 加病毒组CD1a、CD86和CD83分别为:98.10%、99.42%、84.59%;无病毒组为:92.69%、98.07%、82.72%,组差别不明显。加病毒组CD40、CD80分别为:61.02%、97.61%;无病毒组为:36.19%、55.5%,加病毒组高于无病毒组。两组均能刺激T淋巴细胞增殖。加病毒组DC可诱导特异性杀伤,最高杀伤率(39.7±7.2)%。结论 rAAV-Her2/neu转染的DC有更强的免疫功能。     

Functional change of human peripheral blood monocyte-derived dendritic cells after recombinant adeno-associated virus type 2-mediated HBsAg gene infection.

OBJECTIVE: To observe the changes in the functions of human peripheral blood monocyte-derived dendritic cells (DCs) after hepatitis B virus surface antigen (HBsAg) gene infection mediated by recombinant adeno-associated virus type 2 (rAAV). METHODS: The levels of both interleukin (IL)-12 in the supernatant of in vitro cultured DCs infected with rAAV-HbsAg and interferon gamma (IFN-gamma) in the supernatant of the lymphocyte populations co-cultured with DCs were determined by ELISA. The functions of the rAAV-HbsAg-infected DCs were assessed by mixed leukocyte reaction (MLR), and the changes of the surface markers (including CD80, CD83, CD86 and HLA-DR) in response to the infection were detected by flow cytometry. RESULTS: After rAAV-HBsAg infection, the IL-12 secretion of DCs was significantly enhanced (P <0.05), while IFN-gamma production by the lymphocyte populations co-cultured with rAAV-HbsAg-infected DCs was reduced (P <0.01). rAAV-HBsAg infection of DCs did not affect the surface marker expressions and stimulation ability of the DCs in allogeneic lymphocytes reaction. CONCLUSION: DCs infected by rAAV-HBsAg are more efficient than naive DCs in eliciting the differentiation of the lymphocytes toward T helper type I cells, but the functions and the surface markers of DCs remain unaffected after the infection, suggesting the applicability of rAAV as a potentially useful vector for HBsAg gene transfer into the DCs for HBV immunotherapy.