树突状细胞与尤文肉瘤细胞融合诱导抗肿瘤免疫应答的体外研究

目的检测尤文肉瘤细胞A673和树突细胞(DC)融合构建的肿瘤疫苗对尤文肉瘤细胞株A673的杀伤作用。方法应用促融合剂PEG对尤文肉瘤A673细胞和从外周血单个核细胞(PBMC)诱生的树突细胞进行融合。利用细胞因子hGMCSF和hIL4从PBMC诱生DC,并对其表型进行流式细胞仪(FCM)分析,红色荧光染料PKH26标记A673细胞,绿色荧光染料PKH67标记DC,应用促融合剂PEG融合后光镜及电镜观察DC细胞和融合细胞形态,FCM检测融合效率,通过同种混合淋巴细胞反应检测其免疫刺激活性。通过IFNγELISA法产生细胞毒性T淋巴细胞(CTL)的量,通过51Cr细胞杀伤试验检测融合细胞对A673细胞的杀伤作用。结果FCM检测出从PBMC成功诱生出CD83、CD80、CD86及HLADR高表达的成熟DC。DCs/A673融合细胞的融合效率达到23%,同种混合淋巴细胞反应显示DCs/A673融合细胞有很强的免疫刺激活性。IFNγ分泌检测显示DCs/A673融合细胞组较对照组产生CTL水平显著增高。51Cr释放法检测融合细胞体外诱导抗原特异的CTL,融合细胞激活的CTL对肿瘤细胞系A673细胞的杀伤作用强于DCA673混合组、DC组、A673组的CTL(P<0.05),作用较对照各组显著增强。结论DC与A673细胞融合体外致敏自体T淋巴细胞能生成抗原特异CTL对尤文肉瘤细胞A673有一定的杀伤作用。     

树突状细胞与尤文肉瘤细胞融合诱导肿瘤免疫应答的体外研究

目的检测尤文肉瘤细胞A673和树突细胞(DC)融合构建的肿瘤疫苗对尤文肉瘤细胞株A673 的杀伤作用.方法应用促融合剂PEG对尤文肉瘤A673细胞和从外周血单个核细胞(PBMC)诱生的树突细胞进行融合.利用细胞因子hGM-CSF和hIL-4从 PBMC诱生DC,并对其表型进行流式细胞仪(FCM) 分析,红色荧光染料PKH-26标记A673细胞,绿色荧光染料PKH-67标记DC,应用促融合剂PEG融合后光镜及电镜观察DC细胞和融合细胞形态,FCM检测融合效率,通过同种混合淋巴细胞反应检测其免疫刺激活性.通过IFN-γ ELISA法产生细胞毒性T淋巴细胞(CTL)的量,通过51Cr细胞杀伤试验检测融合细胞对A673 细胞的杀伤作用.结果 FCM检测出从PBMC 成功诱生出CD83、CD80、CD86 及HLA-DR 高表达的成熟DC.DCs/A673融合细胞的融合效率达到23%,同种混合淋巴细胞反应显示DCs/A673融合细胞有很强的免疫刺激活性.IFN-γ分泌检测显示DCs/A673融合细胞组较对照组产生CTL水平显著增高.51Cr释放法检测融合细胞体外诱导抗原特异的CTL, 融合细胞激活的CTL对肿瘤细胞系A673细胞的杀伤作用强于DC-A673混合组、DC组、A673组的CTL(P<0.05),作用较对照各组显著增强.结论 DC与A673细胞融合体外致敏自体T 淋巴细胞能生成抗原特异CTL 对尤文肉瘤细胞A673 有一定的杀伤作用.     

树突状细胞融合瘤诱导抗结肠转移癌免疫应答

目的 检测结肠转移癌细胞SW620和树突状细胞（DC）融合构建的肿瘤疫苗对结肠转移癌细胞SW620及其同源结肠癌细胞SW480的杀伤作用。方法 应用促融合剂50％聚乙二醇（PEG）对SW620细胞和从外周血单个核细胞（PBMC）诱生的DC进行融合,用流式细胞仪（FCM）分析其表型并检测融合效率,电镜及免疫细胞化学观察DC、SW620和融合细胞（DC／SW620）形态;^51Cr释放法检测DC／SW620致敏CTL对SW620及SW480细胞的杀伤作用。结果 从PBMC成功诱生出高表达HLA-ABC、HLA-DR、CD80、CD86、CD83的成熟DC,DC／SW620的融合效率达到27．12％。融合细胞兼具DC与肿瘤细胞的结构特点及免疫表型。^51Cr释放法检测DC／SW620激活的CTL对SW620及SW480的杀伤作用强于各对照组（P〈0．01）。结论 DC与SW620细胞融合体外致敏自体T淋巴细胞能生成抗原特异CTL,对结肠转移癌细胞SW620及同源结肠癌细胞SW480有一定的杀伤作用。     

重组腺病毒介导白细胞介素-18和甲胎蛋白基因修饰的树突状细胞体外诱导抗肝癌免疫活性的研究

目的 探讨甲胎蛋白(AFP)、白细胞介素(IL)-18基冈修饰树突状细胞(DC)较以往AFP基因修饰DC作为肝癌免疫治疗瘤苗是否更具优势.方法 分离外周血单个核细胞定向诱导为DC,以Ad-IL-18和Ad-AFP共感染DC,感染后细胞通过噻唑蓝(MTT)比色法检测其激活的T细胞对HepG2的杀伤活性.结果 检测结果显示目的 基因能够表达于DC细胞中.流氏细胞仪(FCM)结果显示共感染的DC细胞均高表达CD1a(73.4%)、CD11c(84.3%)、CD80(89.5%)、CD86(87.9%)和HLA-DR(91.7%).CTL结果显示IL-18/AFP-DC-T对HepG2的杀伤率(67.49±3.24)%明显高于对SMMC7721细胞(27.32±1.75)%和K562细胞(17.31±1.56)%的杀伤率,另外共感染IL-18/AFP-DC-T组对HepG2的杀伤率与AFP-DC-T、IL-18-DC-T组比较,其差异有统计学意义.结论 AFP和IL-18基因修饰DC,能够在体外诱导特异性细胞毒性T淋巴细胞(CTL)效应,而_且对HepG2细胞杀伤率大于AFP转染DC组.     

白细胞介素-12基因修饰对树突状细胞表面分子表达及细胞因子分泌的影响

目的 观察白细胞介素(IL)-12基因修饰对树突状细胞(DC)表面分子及细胞因子分泌的影响.方法 采用重组逆转录病毒介导IL-12基因修饰人外周血单个核细胞(PBMC)来源的Dc;ELISA法检测各组DCs和各组T细胞上清中IL-12、IL-10、IFN-γ因子的分泌水平;流式细胞仪(FACS)分析各组DC表面CD83、CD86的表达;MTT法检测DC刺激同源T淋巴细胞增殖的能力;统计学分析比较各组间的差异.结果 IL-12基因修饰使得DC高表达CD83和CD86分子,分泌高水平IL-12及IFN-γ,但对IL-10因子的分泌无明显影响,刺激同源T淋巴细胞增殖明显,诱导激活的T细胞上清中IFN-γ水平显著增高、IL-10分泌水平显著降低.结论 经IL-12基因修饰后的DC表型成熟,分泌IL-12及IFN-γ的能力增强,对IL-10因子的分泌无影响,能抑制T细胞分泌IL-10因子,优化抗原提呈的微环境.     

X-盒结合蛋白1和热休克蛋白靶向的树突状细胞疫苗对肾癌肿瘤免疫治疗作用的比较研究

目的:应用树突状细胞(DC)与X-盒结合蛋白1(XBP1)共培养制备XBP1-DC疫苗,通过与热休克蛋白70(HSP70)-DC疫苗对GRC2细胞的免疫杀伤作用进行比较,初步探讨应用XBP1蛋白与DC共培养制备融合瘤疫苗的可行性。方法:从肾癌患者的外周血中分离出外周血单核细胞(PBMC),经体外培养诱导为DC。以HSP70和XBP1与DC共培养制备HSP70-DC和XBP1-DC融合瘤疫苗。用HSP70-DC和XBP1-DC疫苗分别刺激患者外周血分离的T淋巴细胞,采用ELISA法检测所产生的CTL反应。以经HSP70-DC和XBP1-DC刺激形成的CTL作为效应细胞,以正常细胞、GRC2为靶细胞,测不同效靶比(10:1、20:1)下,效应细胞杀伤靶细胞的能力。结果:PBMC经细胞因子诱导后,CD1a、CD86、CD83、HLA-DR表达水平均显著高于诱导前表达水平(P0.05)。与DC组比较,XBP1-DC疫苗致敏后,CTL细胞均释放INF-γ显著增加,差异有统计学意义(P0.05),而与HSP70-DC疫苗致敏的CTL细胞比较,XBP1-DC释放的INF-γ差异无统计学意义(P0.05)。随着效靶比的升高,各组CTL细胞对GRC2细胞和RCC细胞的杀伤率均相应提高,差异均有统计学意义(P0.05)。在不同效靶比时,HSP-DC和XBP1-DC免疫的CTL对GRC2和GCC的杀伤作用差异均无统计学意义(P0.05)。结论:XBP1与DC共培养后,能增强DC的抗原呈递能力,增强T淋巴细胞分泌细胞因子的能力,从而特异性杀伤肾癌GRC2细胞系,且杀伤作用与HSP70-DC融合瘤疫苗一致,在抗肿瘤杀伤作用中具有一定的可行性。     

转存活素基因树突状细胞抗消化道肿瘤的免疫效应

目的研究转染存活素基因对树突状细胞(DC)免疫功能的影响,观察修饰后的DC在体外诱导的抗消化道肿瘤免疫效应。方法脂质体介导存活素基因转染入DC,用蛋白印迹法检测培养上清存活素的表达,检测转存活素基因DC分泌细胞因子白细胞介素12(IL12)、肿瘤坏死因子α(TNFα)的功能,以及经流式细胞仪检测DC表面CD1a、CD83、MHCⅡ、CD80、CD86表达的高低,用噻唑蓝(MTT)法诱导人特异的细胞毒性T淋巴细胞(CTL)的能力。结果培养上清中均可以检测到存活素表达;转存活素基因DC的上清IL12、TNFα含量分别为(2652±327)pg/ml和(4371±835)pg/ml,比单纯DC组高(P<005);CD1a、CD83、MHCⅡ、CD80、CD86等在单纯DC表面低表达,在转基因DC表面高表达;MTT法检测,经转染存活素基因的DC提呈的细胞对胃癌细胞、结肠癌细胞、胆管癌细胞杀伤率分别为65%、77%、85%,而单纯DC杀伤作用较低。结论存活素基因转染修饰的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癌免疫效应。     

IκBα突变体基因修饰树突状细胞降低同种T细胞的反应性

目的观察IκBα突变体基因修饰的树突状细胞(IκBαM-DC)对同种T细胞的反应性。方法利用腺病毒载体将IκBαM基因转染WF大鼠骨髓树突状细胞(DC),Western-blot法检测DC中IκBα、IκBαM基因的表达;用流式细胞仪检测DC中共刺激分子MHCⅡ、CD80、CD86、CD40的表达;酶联免疫法(ELISA)分析DC分泌IL-12的含量。通过混合淋巴细胞反应(MLR)分析Lewis大鼠T细胞对IκBαM-DC刺激的增殖能力,二次MLR检测IκBαM-DC诱导的T细胞抗原特异性的低反应性。结果IκBαM抑制DC共刺激分子MHCⅡ、CD80、CD86、CD40的表达及IL-12分泌。同种T细胞对IκBαM-DC刺激的增殖能力较未转染的DC反应明显降低;T细胞低反应具有抗原特异性。结论表达IκBα突变体基因的DC能降低同种T细胞的反应性。     

转Survivin基因树突状细胞抗消化道肿瘤的免疫效应研究

目的　研究转染Survivin的树突状细胞 (DC)在体外诱导高效而特异的抗消化道肿瘤免疫效应。方法　用脂质体作为介质 ,将Survivin基因转染入DC ,用Westernblot法检测培养上清Survivin的表达 ,检测这种DC分泌细胞因子白介素 (IL 12 )、肿瘤坏死因子 (TNF) α的功能 ,以及表面分子CD1a、CD83、MHcⅡ、CD80、CD86表达的高低 ,用MTT法诱导人特异的细胞毒性T淋巴细胞 (CTLs)的能力。结果　培养上清中均可以检测到Survivin表达 ;转基因DC的上清IL 12、TNF α两种细胞因子含量为 (2 65 .2± 3 2 .7)ng/L和(4 3 7.1± 83 .5 )ng/L明显比单纯DC组高(P <0 .0 5 ) ;转基因DC表面高表达CD1a、CD83、MHCⅡ、CD80、CD86;转基因的DC提呈的T细胞对胃癌细胞、结肠癌细胞、胆管癌细胞杀伤率分别为 :65 %、77%、85 % ,而未修饰的单纯DC杀伤作用较低。结论　Survivin基因转染修饰的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癌免疫效应。     

小鼠端粒酶蛋白亚单位转染树突状细胞体外诱导特异性抗肝癌细胞免疫应答

目的 观察携带小鼠端粒酶蛋白亚单位(mTERT)基因蕈组腺病毒载体(AdmTERT)转染树突状细胞(DC)后诱发免疫效应细胞产生特异性抗肝癌细胞免疫应答的研究.方法 用流式细胞仪分析及电镜观察培养6 d DC的细胞表型及形态,Ad-mTERT重组腺病毒转染体外培养的小鼠DC,Western blot检测mTERT融合蛋白表达;用负载mTERT的DC刺激同型淋巴细胞,免疫磁珠分选CD8~+T细胞做为效应细胞,小鼠肝癌细胞株(H22)及小鼠结肠癌细胞(CT26)作为靶细胞,用酶联免疫吸附试验(ELISA)及酶联免疫斑点法(ELISPOT)检测干扰索(IFN)-γ分泌量和释放抗原特异性IFN-γ的T细胞数,~(51)Cr释放法检测细胞毒性T淋巴细胞(CTL)对肝癌细胞的杀伤活性.结果 细胞表型及形态观察证实小鼠骨髓来源的DC为成熟的树突状细胞;AdmTERT转染DC后能正确表达mTERT融合蛋白,用AdmTERT转染DC致敏的淋巴细胞IFN-γ分泌量(208.6μg/L)和分泌IFN-γ的特异性T细胞的数量(341/10~6脾细胞)都高于Ad-GFP转染的DC组(14.2μg/L,33/10~6脾细胞)和单纯DC组(12.1μg/L,19/10~6脾细胞,P＜0.05).AdmTERT修饰DC刺激产生的效应T细胞在效靶比为90:1时,对H22细胞的杀伤率(54.2%)明显高于AdGFP致敏组(8.2%)和未致敏DC组(4.5%,P＜0.05),而对CT26细胞无明显杀伤作用.结论 AdmTERT修饰的DC体外能够诱导出针对mTERT抗原特异性的CTL效应,可特异性杀伤mTERT阳性的肝癌细胞.     

一个与小鼠精子发生相关的基因—rjs 基因

应用改良的mRNA差异显示技术,对不同发育期小鼠（分别为1周龄至4周龄）睾丸曲细精管进行了差异表达的研究,并对其中一个在3周龄小鼠中具有高表达的差异表达片段进行了克隆和测序分析,其序列与Genebank中rjs基因具有高度同源性,表明rjs基因可能与小鼠精子发生相关。     

一个与小鼠精子发生相关的基因——ris基因

应用改良的mRNA差异显示技术,对不同发育期小鼠(分别为1周龄至4周龄)睾丸曲细精管进行了差异表达的研究,并对其中一个在3周龄小鼠中具有高表达的差异表达片段进行了克隆和测序分析,其序列与Genebank中ris基因具有高度同源性,表明ris基因可能与小鼠精子发生相关。     

基因敲除技术在精子发生相关基因功能研究中的应用

从基因水平研究男性不育症发病机理将有助于发现男性不育治疗的新途径。基因敲除技术是目前研究基因功能的主流方法。筛选鉴定精子发生过程中相关基因,探索其特性和功能,对了解睾丸功能、探索男科疾病新的治疗靶点具有重要意义。本文概述基因敲除技术在精子发生相关基因功能研究中的应用。     

基因芯片在膀胱癌基因研究中的应用

膀胱癌是泌尿系统最常见的恶性肿瘤,以空间上的多中心与时间上的反复发作为其生物学特点,其发生由多个基因控制、多步骤进行.基因芯片技术是一种高通量的基因分析平台,现已广泛用于疾病机制的研究、疾病的分类和诊断、疾病的预测和治疗,并用于膀胱癌发生、发展相关基因的筛选、分子治疗靶点的筛选、寻找膀胱癌亚型的分子标记以及肿瘤预后分类的研究.     

Nonviral gene transfer for cancer gene therapy

Although the pre‐clinical and clinical results of gene therapy have shown promise for some cancers, cancer gene therapy is still at an early stage of clinical development. Due to the complexity of targeted vector delivery to the tumor, our strategy for gene therapy is focussed on the development of local non‐viral gene transfer to treat tumors. The local application of non‐viral gene therapy is of particular value in the context of pre‐ or intraoperative application of therapeutic genes. This ensures accessibility of targeted tumor areas and will contribute to better local control of the disease. In this regard, applicable transfer technologies are needed in gene therapy. Different physical procedures, such as in vivo electroporation, sonoporation, ballistic transfer etc. are employed to deliver naked DNA into the target cells or tissues in vitro and in vivo. Among the various non‐viral gene delivery technologies jet‐injection is gaining increasing acceptance, since this technique allows gene transfer into different tissues with deep penetration of naked DNA. The jet‐injection technology is based on low‐volume jets of high‐velocity to penetrate skin and deeper tissues associated with efficient transfection of the affected area. For non‐viral in vivo gene transfer a jet‐injector prototype was created and tested. The beta‐galactosidase (LacZ), green fluorescence protein reporter gene constructs were successfully jet‐injected into different syngeneic mouse and patient‐derived xenotransplanted human tumor models of colon‐ or mammary carcinoma and malignant melanoma. Qualitative and quantitative expression analysis of jet‐injected tumor tissues revealed the efficient expression of these genes. Therapeutic in vivo experiments using the jet‐injection transfer of the cytosine deaminase suicide gene in tumors demonstrated antitumor effects with significant growth inhibition of the jet‐injected xenotransplanted colon carcinomas. Furthermore, jet‐injection was also successfully used for the application of a heat‐inducible TNF‐α expressing vector system leading to efficient in vivo tumor growth inhibition in the combined non‐viral TNF‐α gene transfer and hyperthermia approach. Based on our pre‐clinical experiments for non‐viral gene transfer, a phase I clinical trial has been conducted at the Clinic for Surgery and Surgical Oncology, Charité, Berlin to evaluate the feasibility, efficiency, and safety of jet‐injection aided LacZ‐reporter gene transfer in patients with cutaneous metastases from breast cancer and malignant melanoma. In this study naked GMP‐plasmid DNA was applied intratumorally by jet‐injection. The jet‐injection was well tolerated by all patients and no side effects have been experienced. The study clearly demonstrated that the single application of plasmid‐DNA is safe and leads to the expression of the LacZ‐reporter gene in the tumor tissue, as shown at mRNA‐ and at protein level.     

Evaluation of gene promoters for liver expression by hydrodynamic gene transfer

OBJECTIVE: Gene therapy represents a promising treatment for hepatic disease. Most approaches today use viral methods to target tissues. While nonviral gene therapy is less prominent, hydrodynamic gene delivery represents a promising approach to direct gene expression to the liver. The purpose of the present study was to evaluate promoters for efficient gene expression in hepatocytes in vivo by hydrodynamic delivery and to test the findings in a model of hemophilia A. MATERIALS AND METHODS: Human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), elongation factor-1 alpha (EF1alpha), and phosphoglycerokinase (PGK) promoters were subcloned into plasmids with a luciferase reporter gene. In vitro calcium phosphate-mediated transfection of 2 x 10(5) HEK 293 cells was followed by in vivo whole animal bioluminescence and luminometry after hydrodynamic tail vein injection of plasmid DNA. Six-month-old FVB factor VIII (FVIII)-deficient mice were similarly injected with CBA- or EF1alpha-promoted constructs containing the FVIII heavy and light chains and expression was examined. RESULTS: In vitro transfection demonstrated a hierarchy of expression: hCMV-intron>CAG>EF1alpha>hCMV>PGK. In vivo luminometry demonstrated that the CAG construct produced 2.6x, 3.0x, 3.4x, and >1000x the expression of the hCMV-intron, EF1alpha, hCMV, and PGK constructs respectively. FVIII plasmid injected hemophilic mice demonstrated higher levels of FVIII expression with CAG versus EF1alpha, confirming the reporter gene studies. All FVIII-deficient mice injected with EF1alpha-FVIII or CAG-FVIII plasmids survived after tail clipping. CONCLUSIONS: The CAG promoter/enhancer combination is an excellent alternative to the human CMV promoter for hydrodynamic gene delivery to the liver.     

Multispecies comparison of the casein gene loci and evolution of casein gene family

Caseins, the major milk proteins, are present in a genomic cluster spanning 250–350 kb. The divergence at the coding level between human, rodent, and cattle sequences is rather extensive for most of the genes in this region. Nevertheless, comparative analysis of genomic sequences harboring the casein gene cluster region of these species (with equal evolutionary distances 79–88 Myr) shows that the organization and orientation of the genes is highly conserved. The conserved gene structure indicates that the molecular diversity of the casein genes is achieved through variable use of exons in different species and high evolutionary divergence. Comparative analysis also revealed the presence within two species of uncharacterized casein family members and ruled out the previously held notion that another gene family, located in this region, is primate-specific. Several other new genes as well as conserved noncoding sequences with potential regulatory functions were identified. All genes identified in this region are, or are predicted to be, secreted proteins involved in mineral homeostasis, nutrition, and/or host defense, and are mostly expressed in the mammary and/or salivary glands. These observations suggest a possible common ancestry for the genes in this region.