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。     

树突状细胞与肝癌的免疫治疗

近20年来通过对树突状细胞(dendritic cells,DC)的研究,学者们逐渐认识到DC是目前发现的功能最强的抗原提呈细胞,DC是启动、调控、并维持免疫应答的中心环节。成熟活化的DC除了与初始型T细胞相互作用诱导抗原特异性细胞毒性T淋巴细胞外,还可以通过直接或间接方式影响B细胞的增殖,活化体液免疫应答;     

MAGE-1修饰的树突状细胞体外诱导杀伤人肝癌细胞

目的通过观察肿瘤相关抗原基因MAGE-1转导的树突状细胞(dendritic     

肝癌患者外周血树突状细胞免疫功能低下

目的研究肝癌患者外周血树突状细胞（ＤＣ）表面免疫分子ＨＬＡＤＲ及Ｂ７表达水平及其与ＤＣ免疫功能的相关性．方法以健康成人（ｎ＝１０）作为对照，检测临床确诊中晚期肝癌（ＨＣＣ）患者（ｎ＝１０）外周血ＤＣ表面免疫分子ＨＬＡＤＲ及Ｂ７表达水平及ＤＣ免疫诱导Ｔ淋巴细胞增殖的能力．结果ＨＣＣ患者ＤＣ表面ＨＬＡＤＲ及Ｂ７表达水平（ＶＯＦ）为６７±１６及６１±１１，明显低于对照组（１０７±１４及９６±１２，Ｐ＜００５）．其ＤＣ体外诱导Ｔ细胞增殖能力（ｍｉｎ－１）为３１００±１２０，亦明显低于对照组（６２００±９０，Ｐ＜００１）．结论．ＨＣＣ患者ＤＣ表面ＨＬＡＤＲ及Ｂ７表达水平下降，并与ＤＣ免疫功能低下密切相关．     

树突状细胞疫苗在肝病治疗中的研究进展

树突状细胞(dendritic cell,DC)疫苗疗法是很有前景的一种生物疗法.目前对DC疫苗用于黑色素瘤、恶性淋巴瘤和肝癌等恶性肿瘤已进行了大量的临床研究,其中一些研究结果令人鼓舞.此外,对DC疫苗用于慢性HBV和HIV感染也进行了初步临床研究,并显示出一定的疗效.本文对DC疫苗在肝癌和慢性乙型肝炎治疗中的研究进展...     

肝癌细胞放射敏感性与survivin蛋白表达的关系

目的 探讨肝癌细胞放射敏感性与survivin表达的关系.方法 肝癌细胞HepG2和SMMC-7721在接受不同剂量γ射线照射后,分别采用克隆形成法、免疫细胞化学法、流式细胞术、比色法等检测细胞存活率、survivin蛋白表达、细胞周期变化和Caspase-3活性.结果 在2Gy照射下HepG2和SMMC-7721细胞的存活分数分别为0.43±0.01与0.70±0.02,SMMC-7721较HepG2放射抗拒.γ射线对SMMC-7721细胞的G2/M期阻滞时间较HepG2细胞长(48 h对24 h),在阻滞峰各剂量点SMMC-7721细胞的G2/M期比例也更高.γ射线可上调两株肝癌细胞survivin蛋白的表达,照射后48～72 h,SMMC-7721细胞的survivin蛋白表达水平显著高于HepG2细胞(t值为2.81～5.20,P值均＜0.05).而Caspase-3的活化水平在放射敏感的HepG2细胞中更高(t值为6.05～6.72,P值均＜0.01).结论 射线诱导的survivin表达上调及survivin对Caspase-3的负调控可能是SMMC-7721细胞较HepG2细胞放射抗拒的原因之一.     

树突状细胞诱导的肿瘤特异性细胞毒性T淋巴细胞对裸鼠人肝癌转移的预防和治疗

目的探讨肿瘤抗原特异性细胞毒性T淋巴细胞(CTL)对裸鼠人肝癌转移模型LCI-D20的治疗作用。方法从健康人外周血单个核细胞中诱导树突状细胞,用重组人粒细胞-巨噬细胞集落刺激因子和白细胞介素-4刺激活化,经人肝癌细胞株MHCC97-H肿瘤抗原致敏后,诱导肿瘤抗原特异性CTL,经腹腔注射,以自然杀伤样T淋巴细胞(CIK)和磷酸盐缓冲液为对照,研究其对LCI-D20肝癌治疗和预防转移作用。结果肿瘤抗原特异性CTL组、CIK 组和对照组肝癌肿块重量、血清甲胎蛋白含量、肝癌肝内转移率和存活期依次为(1．11±0．63)g、(1．12±0．36)g 和(2．68±0．53)g;(52．1±9．7)μg／L、(48．6±5．2)μg／L和(82．2±7．2)μg/L;16．7％、16．7％和58．3％; (79．0±5．0)d、(73．3±7．0)d和(52．3±5．2)d。对照组与前两组比较差异均有统计学意义(P值均<0．01)。结论肿瘤抗原特异性CTL可以预防LCI-D20模型肝癌发生转移,延长动物存活时间。     

肺癌细胞裂解物负载对树突状细胞分泌的exosome诱导抗肿瘤作用的影响

目的为制备高效的胞外体（exosome）肿瘤疫苗提供理论依据。方法用细胞因子诱导培养树突状细胞（DC）,将肺癌细胞裂解物负载DC,提取exosome用exosome活化T细胞（负载组）,以未负载DC的exosome（未负载组）及肺癌细胞裂解物负载DC（DC组）活化的T细胞为对照,MTr法检测三组肺癌细胞的杀伤率。结果exosome中有HSP70、HLA及CEA表达。活化T细胞／肺癌细胞为25：1、10：1、5：1时负载组杀伤率均明显高于未负载组及DC组（P均〈0．05）。结论肺癌细胞裂解物负载能增强DC分泌的exosome诱导的抗肿瘤作用;本研究为制备高效的exosome肿瘤疫苗提供了理论依据。     

4种甲胎蛋白抗原表位肽段致敏树突状细胞诱导细胞毒性T淋巴细胞对肝癌细胞的杀伤效应比较

目的 通过体外杀伤试验,比较4种人肝癌特异性甲胎蛋白抗原表位肽段PLFQVEPV[hAFP(137～145)],FMNKFIYEI[hAFP(158～166)]、GLSPNLNRFL[hAFP(325-334)]和GVAL,QTMKQ[hAFP(542～550)]修饰树突状细胞(DC)后诱导的细胞毒性T淋巴细胞(CTL)对人肝癌细胞的特异性杀伤效应. 方法 选取健康人外周血单个核细胞,体外诱导成熟DC;合成4种hAFP抗原表位肽段分别修饰和诱导DC,体外刺激CTL,通过流式细胞仪法和细胞毒性检测法检测该修饰后的DC所诱导的CTL在体外对肝癌细胞株SMMC-7721细胞的杀伤作用.组间比较应用t检验进行统计学分析. 结果 4种人肝癌特异性甲胎蛋白片段均能在体外修饰和诱导DC细胞的增殖和成熟;这些成熟DC在体外均能诱导特异性CTL,CTL对SMMC-7721细胞均可产生杀伤效应.其中FMNKFIYEI[hAFP(158～166)]肽段诱导的CTL在效靶比分别为80:1、40:1及10:1时对肿瘤细胞的杀伤效应分别达到了78.1%±9.8%、43.9%±5.9%和28.2%±4.9%,比其他3种肽段的杀伤效应高(P<0.05). 结论 短肽段具有单独体外诱导特异CTL的作用,其诱导效果和杀伤效果均优于完整hAFP蛋白,其诱导的CTL有特异地杀伤SMM C-7721细胞的作用.     

慢性乙型肝炎患者肠源性内毒素血症与树突状细胞表型及功能的关系

目的 探讨慢性乙型肝炎(CHB)患者树突状细胞(DC)与肠源性内毒素血症(IETM)的关系.方法 CHB患者80例,健康对照者21例,采集外周血,测定血浆内毒素含量、ALT、TBil.根据血浆内毒素水平,将患者分为内毒素阳性组和阴性组.同时用重组人粒细胞巨噬细胞集落刺激因子、重组人白细胞介素-4、酪氨酸激酶受体3配体和TNF-a体外诱导、培养CHB患者DC,采用流式细胞仪检测DC表型,混合淋巴细胞反应检测DC刺激T淋巴细胞的能力,用ELISA检测DC分泌细胞因子的水平.多组间比较采用单因素方差分析.结果 CHB患者DC表达CD83、CD80、CD86和人类白细胞抗原(HLA)-DR分子的水平及诱导同种异体混合T淋巴细胞增殖的能力均明显低于健康对照组.内毒素阳性组患者表达CD83、CD80、CD86、HLA-DR水平及诱导T淋巴细胞增殖的能力分别为(8.25±3.63)%、(10.63±4.52)%、(36.61±16.16)%、(61.65±14.33)%、0.812±0.311,明显低于内毒素阴性组的(11.39±4.35)%、(13.56±5.13)%、(45.90±15.35)%、(70.35±18.89)%、1.153±0.324(F=5.123、4.213、3.714、3.323、3.125,均P＜0.05).培养至第9天,CHB患者DC分泌IL-12和IFN-γ分别为(16.99±6.74)pg/mL和(10.52±4.19)pg/mL,明显低于健康者的(44.51±14.56)pg/mL和(17.94±5.86)pg/mL.内毒素阳性组患者IL-12水平为(13.14±5.71)pg/mL,明显低于内毒素阴性组的(20.98±9.03)pg/mL(F=3.225,P=0.016).IFN-γ水平在内毒素阳性组为(9.46±3.24)pg/mL,与阴性组的(11.54±5.20)pg/mL比较,差异无统计学意义(F=2.003,P=0.076).结论 IETM是导致CHB患者体内DC功能异常的原因之一.

Abstract：

Objective To investigate the relationship between dendritic cell (DC)and intestinal endotoxemia in patients with chronic hepatitis B (CHB).Methods Peripheral blood were collected from CHB patients (n = 80)and healthy controls (n = 21 ).Plasma endotoxin (ET)levels,liver function (alanine transaminase,total bilirubin)were detected.According to plasma ET concentration,all CHB patients were divided into two groups:ET positive and ET negative.The peripheral blood mononuclear cells (PBMCs)were isolated and then cultured with recombinant human granulocyte-macrophage colony-stimulating factor ( rhGM-CSF),recombinant human interleukin-4 ( rhIL-4 ),FMS-related tyrosine kinase 3 ligand (Flt3L)and tumor necrosis factor-alpha (TNF-α)to derive DC.The phenotypic patterns were characterized by flow cytometry.The proliferation of T lymphocytes was evaluated with mixed leukocytes reaction (MLR)and the levels of IL-12 and interferon-γ (IFN-γ)produced by DC were analyzed with enzyme-linked immunosorbent assay (ELISA).Comparisons among the two groups and healthy control group were done by single factor analysis of variance.Results Compared to healthy controls,the expressions of CD83,CD80,CD86,human leucocyte antigen (HLA)-DR and the proliferation of allogeneic T lymphocytes by DC were all significantly reduced in CHB patient groups.The expressions of CD83,CD80,CD86,HLA-DR and the activation of proliferation in ET positive subjects were lower than those in ET negative subjects [CD83 (8.25±3.63)% vs(11.39±4.35)% ,CD80 (10.63±4.52)% vs (13.56±5.13)%,CD86 (36.61±16.16)% vs (45.90±15.35)%,HLA-DR (61.65±14.33)% vs (70.35±18.89)%,the activation of proliferation0.812±0.311 vs 1.153±0.324; F=5.123,4.213,3.714,3.323 and 3.125,respectively; all P＜0.05].After cultured for 9 days,the secretions of IL-12 and IFN-γ by DC were significantly lower in CHB patients than in healthy controls [IL-12 (16.99± 6.74)pg/mL vs (44.51±14.56)pg/mL,IFN-γ (10.52±4.19)pg/mL vs (17.94±5.86)pg/mL].The level of IL-12 in the ET positive group was significantly lower than that ET negative group [( 13.14 ±5.71)pg/mL vs (20.98 ± 9.03)pg/mL; F= 3.225,P = 0.016].The level of IFN-γ was not different between two groups [(9.46 ± 3.24)pg/mL vs (11.54 ± 5.20)pg/mL; F = 2.003,P =0.076].Conclusion The intestinal endotoxemia may play a role in DC dysfunction in CHB patients.     

Antitumor effects of vaccine consisting of dendritic cells pulsed with tumor RNA from gastric cancer

AIM: To investigate the immunotherapeutic potential of vaccine consisting of dendritic cells (DCs) pulsed with total RNA from MFC gastric cancer cells. METHODS: DCs were prepared from the spleens of strain 615 mice by magnetic cell sorting (MACS). After culture for 24 h, DCs were pulsed with total RNA from MFC gastric cancer cells. Mice of one group were immunized with tumor RNA pulsed DC (RNA/DC) at the dosage of 1X10(6) on d 14 and 7 by s c inoculation before tumor implantation. Mice of another group were immunized with unpulsed DC (UDC) at the same dosage on days as the RNA/DC group. The third group of control mice was untreated. On d 0, all the mice were challenged with s c injections of 5X10(5) MFC gastric cancer cells. After inoculation, the mice were monitored closely with respect to tumor growth. Activities of NK cells in PBL and splenocytes and CTL were tested. RESULTS: On d 21 after tumor cell inoculation, the mice of control group manifested the largest tumors with volume at a mean of 2.6323+/-1.1435 cm(3), followed by the UDC and RNA/DC groups with mean volumes at 0.7536+/-0.3659 cm(3) and 0.3688+/-0.6571 cm(3), respectively. The activities of NK cells in PBL and splenocytes in RNA/DC group were 66.2% and 65.4%, respectively, higher than that in the control group. The tumor specific CTL activity in RNA/DC group was 49.5%, higher than that in the control group. CONCLUSION: The tumor vaccine with DCs pulsed with total RNA from gastric cancer cells possesses the ability to stimulate tumor specific CTL activity and to establish anti-tumor immunity when administered in vivo.     

树突状细胞致敏的肿瘤疫苗对胰腺癌细胞的杀伤效应

目的:研究胰腺癌细胞冻融物致敏树突状细胞(DC)诱导的细胞毒性T细胞(CTL)对原代培养的自体胰腺癌细胞的杀伤作用．方法:从6例手术切除的胰腺癌组织中分离胰腺癌细胞,反复冻融获得肿瘤抗原;以该肿瘤抗原致敏外周血DC,诱导T细胞转变为CTL;采用Cr51释放法观察CTL对原代培养的自身胰腺癌细胞的杀伤活性,分别以来源于胰腺癌细胞株Pancl的肿瘤抗原致敏DC和未致敏DC刺激的CTL作为抗原对照和阴性对照．结果:实验组CTL对自身细胞的杀伤活性为69．05%±15．79%→88．05%±15．34％,抗原对照组CTL的杀伤活性为43．08％±6．92％→67．30％±8．91％,两组CTL杀伤率均显著高于阴性对照组(P<0．01);而实验组与抗原对照组相比,前者的杀伤活性显著高于后者者(P<0．05)．结论:胰腺癌细胞冻融物致敏的DC疫苗可以诱导T细胞产生高效的针对自体癌细胞的细胞毒效应;新鲜肿瘤组织来源的胰腺癌细胞比传代的Pancl细胞具有更好的抗原性．     

Bone marrow-derived dendritic cells pulsed with tumor lysates induce anti-tumor immunity against gastric cancer ex vivo

AIM： To investigate whether bone marrow-derived denritic cells pulsed with tumor lysates induce immunity against gastric cancer ex vivo. METHODS： c-kit＋ hematopoietic progenitor cells were magnetically isolated with a MiniMACS separator from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFα to induce their maturation. They were analysed by morphological observation, phenotype analysis, and mixed lymphocyte reaction （MLR）. Bone marrowderived DCs （BM-DCs） were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1：3 DC：tumor cell ratio. Finally, cytotoxic T lymphocyte （CTL） activity and interferon gamma （IFNγ） secretion was evaluated ex vivo. RESULTS： c-kit^＋ hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 d showed the character of typical mature DCs.Morphologically, observed by light microscope, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed.high levels of la, DEC-205, CD11b, CD80 and CD86 antigen, moderate levels of CD40, and negative for F4/80. Functionally, these cells gained the capacity to stimulate allogeneic T cells in MLR assay. However, immature DCs cultured with cytokines for 5 d did not have typical DCs phenotypic markers and could not stimulate allogeneic T cells. Ex vivo primed T cells with SGC-7901 tumor cell lysate-pulsed （TP） DCs were able to induce effective CTL activity against SGC-7901 tumor cells （E：T = 100：1, 69.55% ± 6.05% specific lysis）, but not B16 tumor cells, and produced higher levels of IFNγ, when stimulated with SGC-7901 tumor cells but not when stimulated with B16 tumor cells （1575.31 ± 60.25 pg/mL in SGC-7901 group vs 164.11± 18.52 pg/mL in B16 group, P 〈 0.01）. CONCLUSION： BM-derived DCs pulsed with tumor lysates Can induce anti-tumor immunity specific to gastric cancer ex vivo.     

树突状细胞负载肝癌相关抗原后成熟调控的研究

目的 研究树突状细胞（DC）负载肝癌相关抗原后的成熟调控。方法 用SDS－PAGE制备电泳纯化牛结核分枝杆菌热休克蛋白70，用其诱导DC的分化与成熟。结果 DC负载肝癌可溶性抗原后，10％2细胞失去了DC特征，同时其表面CD54（90．0％），CD83（78．0％），CD86（85．0％）分子表达下降；牛分枝杆菌卡介苗－热休克蛋白70（BCG HSP70）的活化有利于负载肝癌可溶性抗原后的DC维持其特异性标志，同时DC表面CD54（92．0％），CD83（90．0％），CD86（91．0％）分子表达增加，DC诱导同种异体淋巴细胞转化的能力增加，淋巴细胞增殖加快，从而促进DC成熟，增加其抗原呈递能力。结论 预示BCG HSP70有可能成为促进DC活化和成熟的另一重要分子。     

The use of dendritic cells in cancer immunotherapy

Cancer immunotherapy aims at eliciting an immune response directed against tumor antigens to help fight off residual tumor cells and thereby improve survival and quality of life of cancer patients. Different immunotherapeutic approaches share the use of dendritic cells (DCs) to present tumor-associated antigens to T-lymphocytes. Ex vivo generated DCs can be loaded with antigens and re-infused to the patients, or they can be used for ex vivo expansion of antitumor lymphocytes. Alternatively, methods exist to target antigens to DCs in vivo without need for ex vivo cell manipulations. The clinical studies have shown that DC administration to patients is safe and induces antigen-specific immunity. However, it seldom elicits objective clinical responses in patients with advanced-stage malignancies. Novel insights into DC and lymphocyte regulation are expected to lead to more effective vaccines in the near future. Meanwhile, efforts are directed at identifying the most appropriate clinical targets for active specific immunotherapies. Data suggests that vaccinations may indeed be beneficial when given in the adjuvant setting rather than to treat metastatic cancers. These issues are discussed here together with an overview of the DC-based antitumor immunotherapy studies.     

Apoptotic Plasmodium-infected hepatocytes provide antigens to liver dendritic cells

Malaria starts with infection of the host liver by Plasmodium sporozoites. Inoculation with radiation-attenuated Plasmodium sporozoites induces complete protection against malaria. Protection is mediated by dendritic cells (DCs) and CD8(+) T cells, but the source of parasite antigens mediating this response remains unclear. Here, we show that hepatocytes infected with irradiated Plasmodium sporozoites undergo apoptosis shortly after infection. Infection with irradiated sporozoites induces the recruitment of DCs to the liver, where they phagocytose apoptotic infected hepatocytes containing parasite antigens. We propose that apoptotic Plasmodium-infected hepatocytes provide a source of parasite antigens for the initiation of the protective immune response.     

骨髓瘤独特型抗原负载树突细胞介导的抗肿瘤效应研究

目的:研究骨髓瘤独特型抗原(Idiotype,Id)负载树突细胞(DC)对同源细胞因子诱导的杀伤细胞(CIK)体外抗瘤活性的影响。方法:采集健康供者外周血单个核细胞(PBMNC)用常规方法诱导DC和CIK细胞,将骨髓瘤OPM-2细胞培养上清提取的Id冲击或未冲击的DC与CIK细胞共培养(CIK、DC加CIK、Id-DC加CIK),用流式细胞术分析细胞表型,MTT法检测体外效应细胞杀伤活性。结果:在(5～20):1效靶比范围内, CIK细胞对OPM-2和K562细胞的杀伤率分别为(24．47±3．00)％～(40．64±1．62)％和(23．36±1．51)％～(42．52±2．06)％。DC加CIK及Id—DC加CIK对OPM-2和K562细胞的杀伤活性均高于CIK组,差异有统计学意义(P<0．05);而在相同效靶比之下,Id-DC加CIK对OPM-2细胞的杀伤活性最强,差异有统计学意义(P <0．05)。结论:CIK细胞对骨髓瘤细胞有强的杀伤活性,经Id负载的DC与CIK细胞共培养能进一步增强其特异性杀伤活性,对骨髓瘤可能有免疫治疗作用。     

规范肺功能测定技术提高肺功能测定水平

国外最早开始测量肺功能是在17世纪80年代。1979年美国胸科学会制定了肺功能测定操作规范，8年后，即1987年又对该操作规范进行了修订和补充。