树突状细胞与肿瘤疫苗

细胞生物学、分子生物学和免疫学等学科的飞速发展,以及新的实验体系和新的技术方法的不断涌现,促进了肿瘤递呈性免疫治疗的日新月异,树突状细胞作为体内功能最强的抗原递呈细胞在以T细胞识别肿瘤抗原为核心的肿瘤生物治疗研究中展现了良好的应用前景。本文通过对树突状细胞在肿瘤疫苗中设计策略的综述,有助于更加准确地看待当今的肿瘤生物治疗。     

胃癌-树突状细胞融合细胞疫苗制备及其形态学观察

目的 利用细胞融合技术，获取胃癌细胞．树突状细胞融合细胞疫苗，并对其形态学特点进行观察，为基于树突状细胞（DC）的融合疫苗研究及应用提供实验基础。方法 利用聚乙二醇（PEG）诱导胃癌细胞、DC融合，HAT、HT筛选培养系统对融合细胞进行筛选培养，获取纯净融合疫苗；利用光镜、电镜对其形态学特点进行观察。结果 胃癌-DC细胞融合后，体积明显增大，悬浮生长，细胞表面树枝状突起较亲代Dc明显短小、稀少。结论 胃癌-DC融合细胞保持类似于亲代DC的悬浮生长特性，但数量减少、体积明显增大，具备与两种亲代细胞均不相同的形态学特性。     

树突状细胞肿瘤疫苗的研究进展

树突状细胞(DC)是目前发现的功能最强大的专职抗原递呈细胞(APC),能激活静息T细胞并使其增殖,产生抗原特异性细胞毒性T淋巴细胞(CTL),从而发挥抗肿瘤免疫效应.近年来,人们在肿瘤疫苗的研究中,采用多种策略制备DC肿瘤疫苗,尝试通过改变机体免疫系统对肿瘤的免疫状态来加强机体的免疫功能,进而清除肿瘤细胞,而对正常组织几乎无伤害,因此有很好的临床应用前景.     

RNA转染的树突状细胞疫苗及其在肿瘤治疗中的研究进展

树突状细胞（DC）是目前发现的功能最强的专职抗原提呈细胞（APC）,是机体免疫应答的主要启动者,在免疫应答的诱导中发挥关键作用,已成为肿瘤免疫治疗的新载体。近年来,人们发现转染肿瘤细胞的RNA制备的DC疫苗具有很多独特的优点,在肿瘤的治疗中占有重要的地位。     

树突状细胞肿瘤疫苗的研究进展

在抗肿瘤免疫应答中,关键的一步是抗原呈递细胞将肿瘤抗原呈递给T淋巴细胞。树突状细胞是专职抗原呈递细胞,它能够有效的将肿瘤抗原呈递给T细胞,引发机体产生抗肿瘤的免疫应答。因此,利用树突状细胞制备肿瘤疫苗可望提供一种有效的肿瘤免疫治疗方法。目前,体外实验、动物实验和初期的临床实验都已经证明了树突状细胞肿瘤疫苗的抗肿瘤作用。本文主要介绍树突状细胞的生物学特征和树突状细胞肿瘤疫苗在肿瘤免疫治疗中的研究进展。     

肺癌细胞总RNA转染的DC疫苗体外诱导肺癌患者特异性抗肿瘤免疫的实验研究

目的：为了探讨肺癌细胞总RNA转染的DC疫苗体外诱导特异性抗肿瘤免疫的能力。方法：采用分离肺癌患者外周血单核细胞体外诱导DC细胞，Trizol法提取肺癌细胞系Calu-6总RNA，用脂质体包裹总RNA转染DC并诱导特异性CTL的扩增，LDH法和ELISA法检测CTL的杀伤活性和IFN-γ分泌。结果：经肺癌细胞总RNA转染的DC特异性表面标志及功能相关分子表达均上调，转染后的DC可显著刺激异体／自体T淋巴细胞增殖，诱导的特异性CTL对携带Calu-6抗原的靶细胞的杀伤率显著高于LAK细胞，再次接触相同抗原时其IFN-γ分泌量显著增高。结论：肺癌细胞总RNA转染的DC疫苗可在体外诱导出特异性抗肿瘤免疫。     

树突状细胞疫苗在肿瘤免疫治疗领域的研究现状

树突状细胞 (DendriticCells ,DC)是目前所知的功能最强的、也是唯一能激活初始性T细胞 (NativeTcell)的专职抗原提呈细胞 (AntigenPresentingCell,APC) ,具有强大的激活CD8+ CTL及CD4 + T辅助细胞的能力 ,控制着体内免疫反应的过程 ,在免疫应答中处于中心地位 ,因而成为肿瘤免疫反应的中心环节。DC可以在体外培养后用于体内免疫治疗 ,也可辅以细胞因子或其它因子如Flt 3等在体内扩增[1] 。用DC疫苗进行抗肿瘤治疗已受到重视[2 ,3 ] ,且成为当今肿瘤生物治疗领域备受关注的热点课题 ,对树突状细胞疫苗的研究日趋深入而广泛。1　…     

以树突状细胞为基础的肿瘤疫苗研究进展

树突状细胞 (DC)是具有最强抗原提呈能力的专职性抗原提呈细胞。近年来 ,以DC为基础的肿瘤疫苗研究已成为肿瘤免疫治疗的热点之一。综述DC的功能研究、各种DC肿瘤疫苗的制备和研究策略 ,以及在体内外抗肿瘤排斥反应中的作用与临床应用前景。     

树突状细胞疫苗在妇科肿瘤免疫治疗中的研究进展

树突状细胞(DC)具有摄取、加工、处理和呈递抗原以及激活初始T淋巴细胞的功能,是体内功能最强的专职抗原递呈细胞,也是免疫应答的关键细胞.近年来研究发现,体外经肿瘤抗原刺激的DC回输到病人体内,能诱导机体产生细胞毒性T淋巴细胞,从而发挥特异性肿瘤杀伤作用.根据这一发现研制的DC疫苗目前在妇科肿瘤免疫治疗中已取得重要进展,...     

HPV树突状细胞疫苗研究进展

人乳头瘤病毒(HPV)感染在人群中非常普遍，尚无有效预防和治疗办法。本文概述了以树突状细胞(DC)为基础的HPV疫苗研究现状，包括病毒蛋白及肽段负载的DC疫苗、HPV-DNA、RNA、病毒样颗粒刺激的DC疫苗及肿瘤细胞与DC融合疫苗等，并讨论了其在尖锐湿疣防治中的可能应用。     

Genetically modified dendritic cells for cancer immunotherapy

The ability to grow and differentiate dendritic cells (DC) ex vivo has allowed their genetic manipulation to enhance immune activation against tumor antigens. Gene engineering of DC can be achieved with a variety of physical methods and using different viral vectors. RNA or DNA transfection, either alone (naked), coated with liposomes or using electroporation or gene guns leads to T cell activation while transgene expression is frequently undetectable. Adenoviral and retroviral vectors have proven to be highly efficient in DC genetic modification, and have been widely used in preclinical models. Other vectors like lentivirus, poxvirus, herpes virus and adeno-associated virus (AAV) can also lead to foreign transgene expression in DC leading to immune cell activation. DC have been genetically engineered to provide constitutive and high level of tumor antigen expression or to introduce genes that further enhance their immune stimulatory ability. The promising results from preclinical animal models and from in vitro human immune cell culture systems have provided a strong rationale to initiate pilot clinical trials. Recently published or communicated clinical experiences and ongoing trials have used defined tumor antigen RNA transfection for prostate carcinoma and melanoma, liposome-encoated DNA transfection for breast or pancreatic cancer, adenoviral vector tumor antigen gene modification for melanoma and small cell lung cancer, and poxvirus-mediated expression of costimulatory molecules for colon carcinoma. These preliminary experiences suggest that genetically modified DC can safely induce T cell responses but few clinical responses.     

转染自体胃癌细胞总RNA的树突状细胞诱导个体化免疫治疗的体外实验

目的探讨转染自体胃癌细胞总RNA的树突状细胞(DC)体外介导抗胃癌的免疫效应。方法制备短期培养的原代胃癌细胞。用rhGM-CSF、rhIL-4和TNF-α体外诱导胃癌患者外周血单个核细胞(PBMC)中DC的发育和成熟,并转染自体肿瘤细胞总RNA,激活自体T细胞产生CTL,用CCK-8试剂盒检测CTL的杀伤活性。应用流式细胞术及混合淋巴细胞培养技术检测DC的免疫功能状态。用ELISA法测定IL-12和INF-γ的水平。结果转染自体肿瘤细胞总RNA的成熟DC,不仅可高表达MHC-I、II类分子及CD80、CD83和CD86协同刺激分子,并可获得高效刺激自体或异体T细胞增殖的能力。转染RNA的成熟DC,分泌IL-12的水平及其刺激产生的CTL培养上清液中INF-γ的水平显著高于单纯成熟DC及未成熟DC;且CTL对自体胃癌细胞的杀伤率显著高于异体组。结论转染自体胃癌细胞总RNA的成熟DC能够体外诱导产生对自体肿瘤细胞具有高度抗原特异性杀伤活性的CTL。     

胃癌单克隆细胞球和CD44＋及CD133＋亚群成瘤能力的比较

目的研究胃癌干细胞亚群的筛选方法,探讨建立胃癌干细胞稳定亚群的可行性。方法利用流式细胞仪从MKN45、MKN25、SGC7901细胞株和人胃癌组织中分选出不同CD44和CD133表型的亚群,比较不同亚群和无血清培养基培养的单克隆细胞球细胞在裸鼠移植瘤模型中的成瘤能力。结果不同细胞的CD44＋和CD133＋亚群在低数量级时几乎不具有裸鼠皮下成瘤能力,在高数量级接种时成瘤能力与母系细胞差异无统计学意义;单克隆细胞球细胞在各数量级下的成瘤能力、瘤体积和生瘤速度均显著高于母系细胞。结论与CD44和CD133等细胞表面标志物筛选法相比,单克隆细胞球细胞可更好的作为胃癌干细胞研究的细胞学基础。     

HSP70多肽复合物修饰树突细胞抗胰腺癌研究

目的：研究肿瘤热休克蛋白70（HSP70）多肽复合物修饰树突状细胞激活淋巴细胞治疗胰腺癌的策略和方法。方法：采用低渗裂解，ConA—Sepharose亲和层析柱及ADP—Agarose亲和层析柱，从小鼠胰腺癌（MPC83）瘤块中纯化HSP70多肽复合物；纯化出的70KD蛋白修饰小鼠骨髓来源诱导树突细胞（DC）并制备树突细胞HSP70多肽肿瘤疫苗，MTT法检测修饰后DC增殖活性；用修饰后DC激活小鼠脾淋巴细胞，MTT法检测激活淋巴细胞在不同效靶比下对MPC83的体外杀伤活性。结果：获得较高纯度分子量为70kD左右的蛋白质；50～100ng HSP70多肽复合物可修饰10^4树突细胞，每克瘤块能获取HSP70多肽复合物约100μg；来自MPC83细胞瘤块HSP70多肽复合物激活的淋巴细胞能特异性杀伤MPC83细胞。结论：采用低压亲和层析柱可从胰腺癌瘤块中获得较高纯度HSP70多肽复合物，HSP多肽复合物DC疫苗用于胰腺癌细胞免疫治疗能获得体外杀伤效果，为临床胰腺癌生物免疫治疗奠定基础。     

树突状细胞在胃癌中的研究进展

树突状细胞为目前功能最强的抗原呈递细胞,是抗原捕获和抗原递呈途径中的一个关键的细胞群.树突状细胞不仅与胃癌的发生、进展及预后有关,其在胃癌治疗方面的应用也得到了肯定.     

预输注RelB shRNA慢病毒修饰树突细胞诱导大鼠肝移植免疫耐受

背景：肝移植后的排斥反应是威胁患者和移植物长期存活的主要原因。诱导受者产生特异性免疫耐受是解决排斥反应的理想措施。 目的：探讨RNAi RelB树突状细胞预输注诱导大鼠肝移植特异性免疫耐受的可能性。 方法：将近交系雄性清洁级DA(RT1a)大鼠和近交系雄性SPF级Lewis(RT11)大鼠分别作为供、受体,行原位肝移植手术。术前随机配对分为4组：①对照组,受体鼠移植前不做预输注。②治疗组：受体鼠移植前7 d静脉输注供体大鼠RNAi RelB树突状细胞(5×106)。③未成熟树突状细胞组：受体鼠移植前7 d静脉输注供体大鼠未成熟树突状细胞(5×106)。④成熟树突状细胞组：受体鼠移植前7 d静脉输注供体大鼠成熟树突状细胞(5×106)。 结果与结论：与对照组、未成熟树突状细胞组以及成熟树突状细胞组相比,治疗组移植肝的平均生存时间显著延长。移植后第7天,治疗组天冬氨酸转氨酶,总胆红素水平低于对照组、成熟树突状细胞组、未成熟树突状细胞组(P < 0.01),移植后第14天治疗组、未成熟树突状细胞组天冬氨酸转氨酶,总胆红素均有轻微下降,两组比较差异仍有显著性意义(P < 0.01)。移植后第7天,与治疗组比较,对照组、成熟树突状细胞组、未成熟树突状细胞组γ-干扰素、白细胞介素2水平升高(P < 0.01),而白细胞介素4、白细胞介素10下降(P < 0.01);移植后第14天治疗组、未成熟树突状细胞组γ-干扰素、白细胞介素2水平均有下降,白细胞介素4、白细胞介素10水平均有升高,两组比较差异仍有显著性意义(P < 0.01)。对照组、成熟树突状细胞组、未成熟树突状细胞组移植后第7天排斥活动指数为8.0-9.0。未成熟树突状细胞组第14天肝细胞、内皮细胞坏死及汇管区炎性细胞浸润进一步增多。治疗组移植后第7天排斥活动指数为6.0-8.0,第14天时排斥活动指数为4.0-5.0。结果提示RNAi RelB树突状细胞预输注可以减轻移植肝排斥程度,延长移植肝生存时间,这是通过间接途径实现的,其机制可能与T细胞的调节和无能有关。     

Enhanced anti-tumor effect of dendritic cells modified by CIITA gene in hepatocellular carcinoma-bearing mice]

AIM: To study the effect of MHC class II transactivator(CIITA ) on the expression of MHC class I/II molecules, CD80 and CD86 molecules on dendritic cells(DCs), and explor the use of DCs modified by CIITA gene in the biotherapy of tumors. METHODS: We transfected the mouse CIITA gene by lipofectamine into DCs drived from mouse bone marrow. The expression of MHC class I/II, CD80, and CD86 molecules was detected by flow cytometry. 40 mice bearing hepatcellular carcinoma were divided into 4 groups: group 1 were treated with a para-tumor injection of PBS, group 2 DCs, group 3 modified DCs, and group 4 modified DCs pulsed with H22 tumor antigen. Then the anti-tumor effect of DCs modified by mCIITA was evaluated by measuring the tumor size. RESULTS: After transfection of mCIITA, the expression rate of MHC class I/II molecules of DCs increased from 74.2%/66.7% to 93.6%/91.4%, and that of CD80/CD86 increased from 52.3%/60.5% to 89.7%/91.5%. After immunization, the growth of H22 tumors in group 4 mice was significantly inhibited compared with that in other three groups (P<0.05 at 3, 4, 5, 6, and 7 weeks versus groups 1, 2; P<0.05 at 5, 6, 7 weeks versus group 3). CONCLUSION: The expression of MHC I/II, CD80 and CD86 on DCs was regulated markedly by transfection of mCIITA. mCIITA transfection also enhanced the anti-tumor effect of DCs. Our results provided a new method for biotherapy of tumors with DCs.     

Vaccination with plasmacytoid dendritic cells induces protection against infection with Leishmania major in mice

DC-based vaccination against Leishmania major induces a parasite-specific Th1 response and long-lasting protective immunity in susceptible mice. Since distinct DC subsets have been proposed to direct the predominant development of either Th1 or Th2 cells, we analyzed the capability of plasmacytoid DC (pDC) to induce protection and elicit a Th1 response against L. major. Pulsing with L. major lysate induced the activation and maturation of semi-mature murine pDC that had been isolated from the spleen, as indicated by up-regulation of the co-stimulatory molecules CD86 and CD80, but did not enhance the level of IFN-alpha secretion by pDC. Vaccination of susceptible mice with L. major lysate-pulsed pDC induced highly effective T cell-mediated immunity against subsequent infection with L. major parasites. Surprisingly, the protection was not accompanied by a polarized Th1 cytokine profile. Co-activation of pDC with CpG-containing oligodeoxynucleotides, which has been shown to be critical for activating the protective potential of myeloid DC, was not required for the protective effect of L. major antigen-pulsed pDC. These findings demonstrate that antigen-loaded pDC are able to induce T cell-mediated protection against a parasite disease and that experimental leishmaniasis is a suitable model to elucidate the mechanisms underlying DC-based vaccination against infections.     

H+ production by isolated cells from human gastric mucosa

Gastric mucosal cells were isolated from human mucosa obtained at surgery. H⁺ production was indirectly estimated by¹⁴C aminopyrine (AP) uptake. The maximal response to histamine occurred after 30 min of incubation whereas intrinsic factor (IF) secretion was maximal after only 7.5 to 15 min. According to the concentration response curve 10^–4 mol/l histamine proved to be the most effective concentration, the response to which was completely inhibited by ranitidine. Carbachol, dibutyryl cAMP and IMX also enhanced AP uptake, IMX being even more powerful than histamine. Carbachol and IMX failed to potentiate the response to histamine. Parietal cell fractions potentiate the response to histamine. Parietal cell fractions enriched by a Percoll^® density gradient revealed a pronounced background stimulation so that additional stimulation by test agents was less effective than in non-fractionized cells.