IL-27基因转染对树突状细胞表型和功能的影响

目的 探讨转染IL-27基因体外对人外周血单个核细胞来源树突状细胞表型和功能的影响。方法 采集健康成人外周血,密度梯度离心法获得外周血单个核细胞,用GM-CSF、IL-4诱导树突状细胞（Dendritic cell, DC）,第5天后将DC分为3组：IL-27基因转染组、TNF-α组和阴性对照组。倒置显微镜观察DC形态;流式细胞术检测DC表面分子CD1a、CD80、CD83和CD86的表达情况;MTT法检测DC刺激T细胞增殖的能力;ELISA法检测细胞培养上清中IL-12和IFN-γ的含量。结果 转染IL-27基因后外周血单个核细胞来源DC呈现典型的成熟DC形态学特征。IL 27基因转染组DC表面CD1a、CD80、CD83和CD86表达水平较阴性对照组均明显上调（P<0.05）。IL-27基因转染组DC诱导T细胞增殖的能力较对照组DC明显增高（P<0.05）。IL-27基因转染组DC培养上清中IL-12和IFN-γ的含量均明显高于对照组DC（P<0.01）。结论 转染IL-27基因可以上调成熟DC的细胞表型,增强DC的免疫学活性。     

自体恶性黑色素瘤抗原激活的树突状细胞体外诱导免疫试验

树突状细胞(dendritic cells,DC)是迄今为止所知的抗原呈递能力最强的抗原呈递细胞(antigen presenting cells,APC).肿瘤患者因DC免疫功能低下致宿主抗肿瘤免疫无能.研究表明,人外周血单核细胞在体外用粒/巨噬细胞集落刺激因子(granulocyte/macrophage colony-stimulating factor,GM-CSF)及白介素4(IL-4)诱导后,可培养出表达高水平MHCI、MHCII类抗原和CD80、CD86等因子的DC.本实验拟应用黑色素瘤患者外周血单核细胞,经GM-CSF及IL-4诱导后获得DC,再经自体肿瘤细胞冻融物冲击,在体外激活自体T淋巴细胞产生细胞毒性T淋巴细胞(CTL),观察其体外抗肿瘤作用.     

粒细胞-巨噬细胞集落刺激因子基因修饰的树突状细胞疫苗增强体外抗肿瘤免疫效应

目的 研究粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因修饰树突状细胞(DC)后形态、表型及功能的变化,以及增强DC疫苗对肿瘤细胞的体外杀伤作用.方法 小鼠尾静脉注射趋化因子配体3(CCL3),分选得到B220- CDllc+细胞,经细胞因子培养诱导分化DC.在体外用含GM-CSF基因的重组腺病毒(AdGM-CSF)转染DC,酶联免疫吸附试验(ELJSA)检测转染后GM-CSF的水平.通过细胞形态学观察、表型分析及混合淋巴细胞反应(MLR),检测GM-CSF基因修饰前后DC的变化.反复冻融法制备胃癌可溶性抗原,将其与GM-CSF基因修饰的DC共同培养,制备DC疫苗,四甲基偶氮唑蓝(MTT)法检测活化的T淋巴细胞在体外对小鼠前胃癌细胞(MFC)的杀伤作用,ELISA法检测干扰素γ(INF-γ)的分泌情况.结果 CCL3注射后,外周血中B220- CD11c+细胞明显增加,48 h达到高峰[占外周血单个核细胞的(13.88±1.10)%].AdGM-CSF转染后,培养液上清中GM-CSF浓度升高,当感染复数(MOI)为1:100时达到高峰[(130.00±12.61)pg/m1].经GM-CSF.基因修饰的DC在形态上更趋成熟,MHCⅡ类分子、CD80、CD86等细胞表型明显上调,具有更强的刺激T细胞增殖的能力.荷载胃癌抗原的DC激活的T淋巴细胞对MFC细胞具有特异性杀伤作用,并产生高水平的INF-γ[(1245.00±13.75)pg/ml].结论 GM-CSF转染DC后,能大量表达GM-CSF,DC形态及细胞表型更趋成熟,刺激T细胞增殖能力明显增强.GM-CSF基因修饰的DC在体外可诱导出针对靶肿瘤细胞的特异性杀伤作用.     

人外周血树突状细胞的体外扩增及鉴定

树突状细胞(DC)作为抗原提呈细胞,在激发T细胞免疫应答及T细胞依赖性抗体生成中起重要作用,骨髓及外周血的CD34造血前体细胞在GM-CSF和TNF-α的作用下,可在体外分化发育,生成树突状细胞.本研究从正常人外周血分离获得单核细胞,加入100ng/ml hGM-CSF、500U/ml hIL-4,体外培养1周后,获得大量高纯度的树突状细胞,其高表达MHC-Ⅰ、MHC-Ⅱ类分子及共刺激分子B7-1和CD40,能强烈激发同种异体T淋巴细胞增殖,培养条件以应用自体血清或胎牛血清为最佳;单独应用hGM-CSF只能生成巨噬细胞;在培养末期加入TNFα可促进DC进一步成熟.本研究为DC的深入研究与临床应用奠定了基础.     

rhIL-17对小鼠造血前体细胞和人脐血CD34+干细胞分化发育的影响 *

目的:探讨重组人白细胞介素-17(Interleukin-17,IL-17)对小鼠骨髓造血前体细胞和人脐血来源的CD34~ 干细胞生长发育的影响.方法:采用常规方法采集小鼠造血前体细胞;采用Mini-MACS分离技术,从正常人脐血分离人CD34~ 干细胞.体外加入IL-17和/或GM-CSF、IL-4培养分离的前体细胞,应用流式细胞仪检测其表型,采用ELISA法检测了其分泌的IL-12水平,通过[~3H]-TdR掺入法测定其刺激同种异体T淋巴细胞增殖的能力.结果:IL-17促进了小鼠骨髓来源的未成熟DC表达Ia,B7-2等免疫分子,促使其分泌较高水平的IL-12,该细胞也能刺激同种异体T细胞有效增殖,表现出了成熟DC的特征.IL-17单独培养9d促使人脐血CD34~ 干细胞扩增了2倍,部分细胞高表达CD1a及B7-2,低表达HLA-DR,未检测到CD83的表达.该细胞能促使同种异体T细胞增殖,但作用较弱;而rhIL-17与GM-CSF联合培养后扩增了14倍,培养细胞中CD1a、B7-2阳性细胞的比例明显升高,且此细胞刺激同种异体T细胞增殖的能力较强.结论:IL-17体外可促进小鼠骨髓造血前体细胞来源的DC成熟;与GM-CSF联合培养既能促进CD34~ 干细胞增殖,又能使之获得DC特征,初步提示IL-17与GM-CSF联合作用可促进CD34~ 干细胞向DC分化.     

CB6F1小鼠树突状细胞诱导细胞毒性T淋巴细胞的杀伤作用

[目的]探讨CB6F1小鼠脾树突状细胞(DC)的培养及其诱导针对小鼠路易斯肺癌(LLC)的细胞毒性T淋巴细胞(CTL)对肿瘤的杀伤效应.[方法]应用CB6F1小鼠脾细胞在GM-CSF、IL-4等细胞因子作用下培养出DC,反复冻融法制备LLC抗原致敏DC,与淋巴细胞及IL-2混合培养诱导出肿瘤特异性CTL,利用乳酸脱氢酶法检测CTL的杀伤活性.[结果]用GM-CSF、IL4联合培养小鼠脾细胞第4d,可见细胞形态发生改变,培养第8d,可见典型的刺突样DC,通过流式细胞术检测了DC表型高表达CD80占76.5％,CD86占60.0％,MHCⅡ占67.4％,CD11C占80.6％.[结论]应用GM-CSF、IL-4、LPS等细胞因子培养CB6F1小鼠脾细胞经过肿瘤抗原冲击,可以培养出成熟DC,并且DC可以诱导出具有杀伤活性的肿瘤特异性CTL.     

抗原致敏树突状细胞诱导CIK对胃癌细胞杀伤作用的研究

[目的]探讨抗原致敏的树突状细胞（DC）诱导CIK（cytokine induced killer）的杀伤作用。[方法]联合应用粒/巨细胞集落刺激因子（GM-CSF）及白介素-4（IL-4）从外周血单个核细胞中培养DC，用人胃癌细胞SGC提取肿瘤抗原致敏DC，流式细胞仪检测致敏前后DC表型的变化，用人胃癌细胞SGC提取肿瘤抗原致敏DC诱导CIK，用MTT法检测淋巴细胞的增殖及原致敏DC诱导CIK对SGC的杀伤效应。[结果]①利用GM-CSF及IL-4可从外周血单个核细胞中获取DC，肿瘤抗原可促进DC的成熟，肿瘤抗原致敏可促进DC成熟，细胞表面分子HLA-DR、CD86、CD86升高，CD14下降。②混合淋巴细胞反应提示成熟的DC可促进CIK细胞增殖。③SGC肿瘤抗原致敏DC诱导CIK对胃癌细胞SGC有特异性的杀伤作用，随着效靶比的升高，杀伤效应随之增强。[结论]抗原致敏的DC可通过诱导特异性CIK细胞及促进CIK细胞增殖两方面显著提高CIK细胞的杀瘤效应。     

脐血来源树突状细胞体外诱导抗卵巢癌免疫特异性

[目的]研究脐血来源树突状细胞(DC)体外诱导特异性抗卵巢癌细胞的免疫效应.[方法]①从脐血中分离单个核细胞(MNCs)后,获得单核细胞(Mo).粒单集落刺激因子(GM-CSF)和白介素4(IL-4)诱导分化,培养7天后应用流式细胞仪进行细胞表型分析.②诱导单核细胞分化的第3天加入人卵巢癌细胞株3AO的冻融抗原,共培养4天后获得负载肿瘤抗原的成熟DC;将致敏DC与从脐血中分离的同种异体T淋巴细胞共培养3天,获得细胞毒T淋巴细胞(CTL);四甲基偶氮唑蓝(MTT)法检测CTL及上清对人卵巢癌细胞株3AO、人胚肾细胞株293T(对照细胞)、人肝癌细胞株HCCC-9810的细胞毒作用.[结果]①脐血来源单核细胞(Mo)在GM-CSF和IL-4作用下,7天后可分化生成成熟的DC,高表达DC特异性抗原CDla、CD80(B7-1)、CD86(B7-2)、HLA-DR、CD83.②DC可负载并递呈肿瘤抗原,激活同种异体T淋巴细胞,诱导肿瘤特异性CTL产生.不同浓度CTL及上清对卵巢癌细胞3AO有特异性杀伤、抑制作用(P＜0.05).[结论]脐血中单核细胞可体外分化扩增为成熟的功能性DC,并诱导出特异性杀伤卵巢癌细胞的免疫效应.     

IL-15对树突状细胞激活的T淋巴细胞免疫反应的作用观察

  目的  观察IL-15和IL-2对树突状细胞疫苗(dendritic cells, DC)激活的淋巴细胞免疫反应的作用。  方法  取健康小鼠的脾淋巴细胞, 在体外采用肿瘤抗原负载的DC疫苗与脾淋巴细胞混合后, 分别加入IL-15或IL-2培养7d。采用流式细胞术检测淋巴细胞免疫表型的变化; 使用酶联免疫斑点实验(ELISPOT)检测分泌干扰素(interferon, IFN)-γ的细胞数量; 利用51Cr释放法检测淋巴细胞对肿瘤细胞的杀伤活性。  结果  IL-15联合DC疫苗增强了T细胞反应, 包括淋巴细胞免疫表型和功能的改变。  结论  与IL-2相比, IL-15能更好的增强DC疫苗诱发的免疫反应, 为IL-15与DC疫苗的联合应用提供了一定的依据。      

人外周血树突状细胞的体外诱导和鉴定

目的：建立从人外周血体外诱导扩增树突状细胞(DC)的方法：方法：从正常人外周血F1细胞分离单个核细胞，经三种细胞因子——重组人粒细胞一巨噬细胞集落刺激因子(rh GM—CSF)、重组人白细胞介素4(rh IL-4)和重组人肿瘤坏死因子(rh TNFα)联合诱导培养，10天后收获细胞，利用光学显微镜、透射电镜观察其外部形态和细胞超做结构，流式细胞仪检测其细胞表面抗原，自体混合淋巴细胞反应检测其刺激T细胞的增殖活性：结果：培养收获了高纯度的DC，这些细胞表面有大量的不规则突起，核也呈不规则状，核仁小，核膜异染色质聚集，含有丰富的细胞器，如核糖体、内质网、溶酶体等；细胞表面高水平表达HLA—DR、CD1α、CD80、CD83和CD86；自体混合淋巴细胞反应表明诱导所得的DC具有很强的激发同种T细胞增殖的能力。结论：人外周血单个核细胞经细胞因子诱导培养可以得到大量的成熟DC。     

重组人白介素2梯度胶的肽图分析

白细胞介素17(IL-17)是一新近发现的由活化T淋巴细胞产生的细胞因子,初步研究表明它可能参与T淋巴细胞与造血系统的相互作用,由于IL-17的研究工作刚刚起步,对其确切功能并不完全清楚.为了进一步了解IL-17的生物学功能及可能机制,我们从活化的人外周血单个核细胞中克隆了人IL-17基因的编码序列,将其克隆至本室构建的克隆、表达、测序一体化载体 pLCM182中,经序列测定结果与报道基因序列一致后在大肠杆菌中进行表达,结果发现经诱导后其表达量可达30%以上.IL-17的表达产物在体外能刺激原代培养的人成纤维细胞分泌IL-6和GM-CSF,证明所表达的IL-17具有生物学活性.     

CONSTRUCTION AND IDENTIFICATION OF RETROVIRAL VECTOR EXPRESSING HUMAN INTERLEUKIN-17 GENE

ＣＯＮＳＴＲＵＣＴＩＯＮＡＮＤＩＤＥＮＴＩＦＩＣＡＴＩＯＮＯＦＲＥＴＲＯＶＩＲＡＬＶＥＣＴＯＲＥＸＰＲＥＳＳＩＮＧＨＵＭＡＮＩＮＴＥＲＬＥＵＫＩＮ１７ＧＥＮＥＣａｏＸｕｅｔａｏ曹雪涛ＨｕａｎｇＸｉｎ黄欣ＷａｎＴａｏ万涛ＺｈａｏＺｈｏｎｇｌｉａ...     

Inoculation of human interleukin-17 gene-transfected Meth-A fibrosarcoma cells induces T cell-dependent tumor-specific immunity in mice

OBJECTIVE: The biological activities of interleukin-17 (IL-17), a newly cloned cytokine, have not been fully elucidated. The present study was designed to assess the in vitro and in vivo effect of transfecting the IL-17 gene into tumor cells. METHODS: A complementary DNA (cDNA) encoding human IL-17 (hIL-17) was obtained by polymerase chain reaction amplification from the human CD4+ T cell cDNA library and inserted into the plasmid pRc/cytomegalovirus to construct an expression vector for the hIL-17 gene. Murine Meth-A fibrosarcoma cells were transfected with the hIL-17 gene using the lipofectin method. The hIL-17 gene-expressing clone (Meth-A/IL-17) was selected and analyzed for cytokine expression by Northern blot. RESULTS: There was no significant difference in the in vitro proliferation rate among parent Meth-A, cells transfected with vector alone and Meth-A/IL-17 cells. When the tumor cells were transplanted subcutaneously into BALB/c nude (nu+/nu+) mice, there was no difference in in vivo growth rates among the three cell lines. Challenge with tumor cells in conventional BALB/c mice, however, resulted in the rejection of Meth-A/IL-17 cells, but the other two lines did grow. After immunization with Meth-A/IL-17 cells, the mice were rechallenged by parent Meth-A or syngeneic MOPC-104E plasmacytoma cells; the immunized mice rejected the Meth-A cells, but not the MOPC-104E cells. Injecting the anti-thy 1,2 (CD90), anti-CD4 or anti-CD8 monoclonal antibody into conventional BALB/c mice resulted in the resumption of in vivo growth of Meth-A/IL-17 cells, but injecting the anti-asialo GM1 antibody did not. Furthermore, flow cytometric analysis demonstrated a significant increase in the expression of major histocompatibility complex (MHC) class I and class II antigens and lymphocyte function-associated antigen-1 on Meth-A/IL-17 cells. CONCLUSION: Meth-A cells transfected with the hIL-17 gene can induce tumor-specific antitumor immunity by augmenting the expression of MHC class I and II antigens, and both CD4+ and CD8+ T cells may play important roles in inducing antitumor immunity, suggesting the possibility of developing a tumor vaccine incorporating IL-17-transfected tumor cells.     

Human and viral interleukin-10 in Hodgkin's disease,and its influence on CD4+ and CD8+ T lymphocytes

The Epstein-Barr virus (EBV) is closely related to Hodgkin's disease (HD), while the BCRF-I (viral [v] IL-10) gene of the EBV is highly homologous to the human interleukin-10 (h IL-10) gene. To investigate the relationship of IL-10 and HD, we performed both immunostaining and in situ hybridization (ISH) in 30 cases of HD. The presence of EBV in Hodgkin (H) and Reed-Sternberg (RS) cells was seen in 16 of the 30 cases, by ISH of the EBV EBER-I region and/or immunostaining of latent membrane protein (LMP-I). Of the 16 EBV-positive cases, 12 also showed IL-10 antigen (Ag) in H and RS cells by immunostaining, 5 of the 16 demonstrated hIL-10 RNA by ISH and 14 of the 16 showed vIL-10 RNA. But only 2 of the 14 EBV-negative cases showed IL-10 Ag, and one of them showed hIL-10 RNA, while none demonstrated vIL-10 RNA. The T cells in the HD-involved tissues were found to be mainly CD4-positive T cells, and had no association with EBV infection. However, the lymphocytes surrounding H and RS cells were more frequently CD4 cells and rarely CD8 cells in the EBV-positive cases, in contrast with the EBV-negative cases. The above results indicate that an EBV infection influenced both cytokine synthesis and the response of T cells in HD. © 1995 Wiley-Liss Inc.     

Mouse Homologue of the Human SART3 Gene Encoding Tumor-rejection Antigen

We recently isolated a human SART3 ( hSART3 ) gene encoding a tumor-rejection antigen recognized by HLA-A2402-restricted cytotoxic T lymphocytes (CTLs). The hSART3 was also found to exist as an RNA-binding nuclear protein of unknown biological function. In this study, we cloned and analyzed the homologous mouse SART3 ( mSART3 ) gene in order to understand better the function of hSART3, and to aid in establishing animal models of specific immunotherapy. The cloned 3586-bp cDNA encoded a 962-amino acid polypeptide with high homology to hSART3 (80% or 86% identity at the nucleotide or protein level, respectively). Nonapeptides recognized by the HLA-A2402-restricted CTLs and all of the RNA-binding motifs were conserved between hSART3 and mSART3. The mSART3 mRNA was ubiquitously expressed in normal tissues, with low level expression in the liver, heart, and skeletal muscle. It was widely expressed in various organs from as early as day 7 of gestation. mSART3 was mapped to chromosome 5, a syntenic region for human chromosome 12q23–24, and its genomic DNA extended over 28-kb and consisted of 19 exons. This information should be important for studies of the biological functions of the SART3 protein and for the establishment of animal models of specific cancer immunotherapy.     

Mouse homologue of the human SART3 gene encoding tumor-rejection antigen.

We recently isolated a human SART3 (hSART3) gene encoding a tumor-rejection antigen recognized by HLA-A2402-restricted cytotoxic T lymphocytes (CTLs). The hSART3 was also found to exist as an RNA-binding nuclear protein of unknown biological function. In this study, we cloned and analyzed the homologous mouse SART3 (mSART3) gene in order to understand better the function of hSART3, and to aid in establishing animal models of specific immunotherapy. The cloned 3586-bp cDNA encoded a 962-amino acid polypeptide with high homology to hSART3 (80% or 86% identity at the nucleotide or protein level, respectively). Nonapeptides recognized by the HLA-A2402-restricted CTLs and all of the RNA-binding motifs were conserved between hSART3 and mSART3. The mSART3 mRNA was ubiquitously expressed in normal tissues, with low level expression in the liver, heart, and skeletal muscle. It was widely expressed in various organs from as early as day 7 of gestation. mSART3 was mapped to chromosome 5, a syntenic region for human chromosome 12q23-24, and its genomic DNA extended over 28-kb and consisted of 19 exons. This information should be important for studies of the biological functions of the SART3 protein and for the establishment of animal models of specific cancer immunotherapy.     

pIRES-PML-RARα-hIL-2重组质粒的构建和表达研究

目的构建PML-RARα融合基因与人白介素-2（hIL-2）基因真核双表达质粒。方法利用RT-PCR技术从NB4细胞的RNA中扩增出PML-RARα融合点附近的部分基因片段,通过RT-PCR扩增Jurkat细胞中的hIL-2基因,并分别将两基因片段连接到pIRES质粒的多克隆位点（MCS）A和B中,构建真核双表达质粒。利用酶切和序列分析方法验证所构建质粒的正确性。将构建成功的重组质粒转染A549细胞,通过RT-PCR检测重组质粒在真核细胞中的转录情况,利用点杂交、ELISA检测目的蛋白的表达情况。结果Nhe I／Mlu I和Sal I／Not I双酶切证明重组质粒中含有相应大小的PML-RARa基因片段及hIL-2基因,序列分析证明重组质粒中插入片段的碱基序列均完全正确。将所构建的pIRES-PML-RARα-hIL-2质粒转染A549细胞后经RT-PCR鉴定表明,插入到重组质粒中的PML—RARα和hIL-2基因能够在真核细胞中进行正常转录。经点杂交和ELISA检测显示重组质粒在真核细胞中可表达hIL-2蛋白。结论成功构建了pIRES-PML-RARα-hIL-2质粒,该重组质粒能够在真核细胞中正常转录并表达PML-RARα和hIL-2蛋白。