树突状细胞在榄香烯复合瘤苗主动免疫效应中的作用

本实验以HCa F或L6 1 5榄香烯复合瘤苗 (H TCV或L TCV )、H TCV溶解物 (TH )、短小棒状菌 (CP )免疫小鼠 ,分离制备其脾脏DC ,在体外分别用相应瘤细胞溶解物 (H或L )和瘤苗溶解物 (TH或TL )冲激后 ,以MTT法检测其诱导同系小鼠脾不粘附细胞增殖的能力。结果表明用榄香烯复合瘤苗或其溶解物免疫DC供鼠和体外冲激DC ,可增强其诱导同系小鼠脾不粘附细胞增殖的作用 ,给DC供鼠注射CP可进一步增强DC的这一作用     

树突状细胞在榄香烯复合瘤苗主动免疫保护效应中的作用

目的 :树突状细胞 (ＤＣ)是体内抗原提呈功能最强的抗原提呈细胞 (ＡＰＣ) ,是唯一能提呈抗原给初始型Ｔ细胞使之活化扩增为效应细胞的专职ＡＰＣ。ＤＣ在榄香烯复合瘤苗主动免疫保护效应中的作用如何 ,怎样提高其作用水平值得探讨。方法 :以ＨＣａ -Ｆ或Ｌ61 5榄香烯复合瘤苗 (Ｈ -ＴＣＶ或Ｌ -ＴＣＶ)、Ｈ -ＴＣＶ可溶性上清 (ＴＨ)、ＣＰ免疫小鼠 ,分离制备其脾脏ＤＣ ,在体外分别用相应瘤细胞上清 (Ｈ或Ｌ)和瘤苗上清 (ＴＨ或ＴＬ)冲激后 ,以ＭＴＴ法检测其诱导同系小鼠脾不粘附细胞增殖的能力。结果 :1 正常小鼠ＤＣ在体外可诱导同系…     

榄香烯复合瘤苗对小鼠腹腔巨噬细胞功能影响的分析

目的 :本室已证明榄香烯复合瘤苗对多种小鼠肿瘤 (如Ｌ61 5白血病、Ｈ2 2 肝癌等 )有明显的主动免疫保护作用。巨噬细胞在抗肿瘤免疫中不仅作为抗原递呈细胞 ,而且也是参与肿瘤杀伤的效应细胞。本实验检测了榄香烯复合瘤苗对小鼠腹腔巨噬细胞 (ＰＥＭΦ)的吞噬、杀瘤及一氧化氮 (ｎｉｔｒｉｃｏｘｉｄｅ ,ＮＯ)产生等功能的影响 ,以期从这一方面研究瘤苗免疫的机制。方法 :ＭＴＴ法检测不同浓度的榄香烯及榄香烯复合瘤苗体外刺激正常小鼠ＰＥＭΦ的活性变化 ;通过ＰＥＭΦ的吞噬功能 (血红蛋白释放法 )、对肿瘤靶细胞(ＨＣａ-Ｆ、Ｌ92 9)的…     

HTA-HSP70BCG对小鼠骨髓树突状细胞的冲击作用

目的 :用榄香烯复合瘤苗抗原 卡介苗热休克蛋白复合物 (HTA HSP70 BCG)冲击正常小鼠骨髓树突状细胞 ,检测其抗原递呈功能。方法 :用HTA HSP70 BCG刺激 ,并与GM CSM和IL 4联合培养的骨髓树突状细胞 ,用MTT法测增殖活性 ,用透射电镜检查形态学改变 ,用流式细胞仪检测FITC labeleddextran的摄取。结果 :与单用HTA或HSP70 BCG刺激的树突状细胞相比 ,HTA HSP70 BCG刺激的树突状细胞增殖指数明显增大 (2 10 7± 0 0 13) ,对脾不粘附细胞的激活作用明显增强 (增殖指数为 1 92 7±0 0 73) ,FITC labeleddextran摄取率明显增高 (达 5 8 6 1% ) ,形态上更趋于成熟。结论 :HTA HSP70 BCG 对树突状细胞具有较强的冲激作用 ,增强其抗原递呈功能。     

L-选择蛋白在小鼠肝癌细胞高低转移株HCa-F、HCa-P的表达及与肿瘤转移潜能的相关性

目的　探讨经淋巴道转移的小鼠肝癌细胞高低转移株HCa F、HCa P淋巴道转移能力及与淋巴细胞归巢受体L 选择蛋白的相关性。方法　应用免疫印迹分析、RT PCR及流式细胞术检测小鼠肝癌HCa F、HCa P细胞表面L 选择蛋白的表达情况 ,再通过E 、P 、L 选择蛋白的抗体对细胞粘附实验的影响检测HCa F、HCa P细胞淋巴道转移潜能与L 选择蛋白的相关性。结果　L 选择蛋白在HCa F、HCa P细胞表面均有表达 ,且HCa F细胞的表达量明显高于HCa P细胞 (P <0 .0 1) ,HCa F细胞与淋巴结之间的粘附可被抗L 选择蛋白的抗体所阻断。结论　小鼠肝癌细胞高低转移株HCa F、HCa P细胞均表达L 选择蛋白 ,其程度与该细胞的淋巴道转移潜能正相关     

β-榄香烯对胃癌耐药细胞裸鼠移植瘤生长的影响

目的观察中药β-榄香烯对胃癌耐药细胞株SGC-7901/Adr裸鼠成瘤能力的影响,探讨β-榄香烯对胃癌耐药细胞的作用及机制。方法利用MTT实验验证SGC7901/Adr细胞的耐药性及β-榄香烯的毒性作用,利用裸鼠移植瘤模型分析β-榄香烯对胃癌耐药细胞裸鼠移植瘤生长的影响,利用免疫组织化学方法检测各组移植瘤组织中Caspase-3蛋白的表达水平。结果相对于SGC7901细胞,SGC7901/Adr细胞对ADR具有耐药性;β-榄香烯对SGC7901/Adr细胞具有增殖抑制作用,且具有浓度依赖性。β-榄香烯能够较好地抑制胃癌耐药细胞裸鼠移植瘤的生长,能够明显缩小肿瘤体积。β-榄香烯处理后,移植瘤组织中Caspase-3蛋白表达有上升趋势。结论β-榄香烯促进肿瘤细胞凋亡、抑制胃癌耐药细胞裸鼠移植瘤的生长与上调Caspase-3蛋白表达水平相关。     

瘤苗免疫治疗对带瘤小鼠脾细胞IL-10和IL-12产生的影响

小鼠ＴＨ1 /ＴＨ2亚群的发现为人的ＴＨ1 /ＴＨ2免疫偏斜研究提供了依据 ,细胞因子对ＴＨ1 /ＴＨ2的漂移起着重要作用 ,其中ＩＬ - 1 2、ＩＬ - 1 0是调节ＴＨ0→ＴＨ1 /ＴＨ2分化两个最重要的细胞因子。目的 :以小鼠腹水型肝癌高淋巴道转移亚系ＨＣａ -Ｆ为模型 ,探讨榄香烯复合瘤苗对带瘤小鼠的免疫治疗作用及脾细胞ＩＬ - 1 0、ＩＬ - 1 2产生的影响 ,观察榄香烯复合瘤苗及免疫化学疗法的抗瘤效果、分析其机制。方法 :Ｂａｌｂ/Ｃ小鼠按体重随机分为 5组 ,正常组、攻击对照组、瘤苗治疗组、环磷酰胺组及瘤苗 环磷酰胺联合治疗组。在ＨＣ…     

榄香烯复合瘤苗HSP70与卡介苗HSP70免疫效应机制的对比研究

分别从榄香烯复合瘤苗及卡介苗中提取纯化HSP70，并以榄香烯复合瘤作为阳性对照，通过测定免疫小鼠全脾细胞的细胞增殖周期、细胞表型、细胞毒活性，研究两者在免疫效应及机制上的不同之处。     

B16F10/ESAT-6-GPI-IL-21瘤苗安全性及抗肿瘤效应研究

目的:评估B16F10/ESAT-6-GPI-IL-21瘤苗用于C57BL/6小鼠的安全性及其诱导的抗肿瘤免疫效应。方法:应用免疫荧光、FCM检测瘤苗细胞ESAT-6-GPI的表达情况;以Western blot方法检测瘤苗细胞IL-21的表达情况;动物实验检测瘤苗体内应用的安全性;制备荷瘤鼠术后免疫模型,评价瘤苗免疫效果。结果:B16F10/ESAT-6-GPI-IL-21瘤苗细胞表面有靶抗原ESAT-6-GPI表达,并分泌IL-21。于C57BL/6小鼠皮下接种2×105个B16F10/ESAT-6-GPI-IL-21瘤苗细胞,60天内未见致瘤,但能够诱导小鼠产生有效的抗肿瘤免疫效应。结论:B16F10/ESAT-6-GPI-IL-21瘤苗致瘤性明显下降,低剂量应用具有安全性,能够诱导小鼠产生有效的抗肿瘤免疫效应。     

小鼠肝癌细胞特异性淋巴道转移与其基质金属蛋白酶分泌的关系

目的　探讨肿瘤细胞基质金属蛋白酶 (MMPs)分泌与特异性淋巴结转移的关系。方法　将自建高、低转移小鼠肝癌细胞系HCa F(高转移 )和HCa P(低转移 )在培养时 ,分别加入淋巴结、肝脏或脾脏匀浆 ,离心收集培养上清 ,用明胶酶谱法检测两株癌细胞在不同培养环境下分泌的MMPs酶谱 ,分析两者的差异。结果　在单纯RPMI16 40培养基中HCa F和HCa P细胞仅能分泌少量MMP 9(12 5 6± 15 7,2 6 42± 385 ) ,加入淋巴结匀浆后HCa F细胞分泌MMP 9显著增强 (12 40 3± 894) ,且分泌MMP 9的活性型和MMP 2 ;HCa P细胞也分泌MMP 9(90 6 8± 6 86 )及其活性型 (3 914± 12 5 3)和MMP 2(2 997± 1990 ) ,但量均明显低于HCa F细胞 ;加入肝脏或脾脏匀浆后 ,HCa F和HCa P细胞均不分泌MMPs。结论　HCa F细胞强于HCa P细胞的转移能力可能与HCa F细胞分泌MMPs的能力强于HCa P细胞相关 ,而HCa F和HCa P细胞仅在淋巴结成分诱导下才能分泌较多的MMPs,这可能与它们具有特异性地向淋巴结转移的功能密切相关。     

Experimental study on the effect and mechanism of action of active immunization with elemene combo-tumor cell vaccine

Experimental study on the effect and mechanism of action of active immunization with elemene combo-tumor cell vaccine     

榄香烯复合瘤苗的HSP70表达及其主动免疫预防效应

为进一步阐明本室专利榄香烯复合瘤苗(EC-TCV)的主动免疫效应与其HSP70-肿瘤肽之间的关系,本文以42℃热休克(H)为对照,首先检测了榄香烯(E)、丝裂霉素C(MMC)、戊二醛(G)单独或复合处理时对小鼠HCa-F肝癌、L615白血病细胞膜HSP(HSP70、HSP90)表达的影响,发现只有膜HSP70的表达在两种榄香烯复合瘤苗中均升高,随后以E-MMC复合处理的HCa-F细胞中提纯的HSP70-肿瘤肽(HHSP70)为免疫原,以小鼠HCa-F肝癌细胞为模型,研究了HHSP70的主动免疫预防效应及其特异性。结果表明,E、MMC、G同H相似,也具有应激原作用,能诱导应激蛋白HSP70的表达;而且HSP70在HCa-F和L615两种肿瘤细胞均以E-MMC-G三者复合处理时为最高;HHSP70能诱发机体产生主动抗瘤效应,且这种效应有一定的肿瘤特异性。     

树突细胞对Lewis肺癌抗瘤作用的研究

目的：研究经体外骨髓细胞衍生的DC在Lewis肺癌中的抗瘤作用。方法：将肿瘤抗原MUT－1加载的DC（DC－MUT－1）经尾静脉免疫C57BL／6小鼠后，随后将肿瘤细胞接于小鼠皮下，观察DC能否诱发机体的免疫保护。同时检测DC－MUT－1免疫后小鼠脾脏T细胞的CTL活性。结果：经DC－MUT－1免疫后能诱发机体产生对相应肿瘤细胞的持久和特异性的免疫保护作用，体外检测CTL活性显示，免疫小鼠脾脏T细胞对相应肿瘤具有 特异性杀伤。结论：经DC－MUT－1免疫小鼠能诱发机体产生特异性抗肿瘤细胞免疫从而使宿主对随后的肿瘤细胞攻击产生特异性的免疫保护作用。     

冻融抗原负载树突状细胞诱导细胞毒性T淋巴细胞特异性杀伤急性白血病细胞的实验研究

我们应用基因重组人白介素 4 (rhIL 4 ) ,基因重组人粒 /单细胞集落刺激因子 (rhGM CSF)联合培养体系自健康志愿者骨髓单个核细胞诱导产生DC ,使其负载急性髓系白血病 (AML)细胞冻融抗原 ,体外诱导细胞毒性T细胞 (CTL) ,观察负载AML细胞冻融抗原后DC的状态对所激发的T淋巴细胞表型及功能的影响及CTL对AML细胞特异性杀伤活性。我们发现负载AML冻融抗原后DC的CD1a、CD83、CD86、CD11C、HLA DR表达率为较培养前明显增高 (P <0 .0 1) ,且负载AML冻融抗原的DC诱导的CTL中CD3 CD8 T细胞比例 ,较诱导前明显增高 (P <0 .0 1) ,而负载AML细胞冻融抗原的DC诱导CTL对AML细胞有较强的杀伤作用 ,明显强于加或不加IL 2培养的T细胞对照组 (P <0 .0 1) ,而对K5 6 2细胞无明显的杀伤活性。通过以上实验我们认为经rhGM CSF ,rhIL 4培养产生的DC为CD14CD1a DC ,能诱导CTL对AML细胞产生明显的特异性杀伤作用 ,另外 ,负载AML细胞冻融抗原DC诱导的CTL中CD3 CD8 T细胞比例明显增高 ,提示CD8 T细胞在抗肿瘤免疫中具有极其重要的作用。     

The fraction 1 and V protein antigens of Yersinia pestis activate dendritic cells to induce primary T cell responses

The F1 and V antigens of Yersinia pestis, despite acting as virulence factors secreted by the organism during infection, also combine to produce an effective recombinant vaccine against plague, currently in clinical trial. The protective mechanisms induced by rF1 + rV probably involve interactions with dendritic cells (DC) as antigen uptake, processing and presenting cells. To study such interactions, naive ex vivo DC from bone marrow, spleen and lymph node were cultured with rF1, rV or combined antigens and demonstrated to secrete interleukin (IL)-4 and IL-12 into the culture supernatant. Cytokine production in response to pulsing was dependent on the maturity of the bone marrow-derived DC culture, so that pulsed 8-day-old cultures had accumulated significantly more intracellular IL-4 and IL-12 than unpulsed cells. DC, pulsed with rF1 + rV for 2-24 h, were able to prime naive autologous lymph node T cells to proliferate in an antigen dose-dependent manner, with an order of potency of 3d bone marrow-derived DC (BMDC) > 7d BMDC > splenic DC. Significantly, cell-free supernatants from rF1 + rV-pulsed BMDC and splenic DC were also able to induce specific primary responses effectively in naive T cells, suggesting that these supernatants contained stimulatory factor(s). This study suggests an important role for DC, or factors secreted by them, in the induction of protective immunity to plague by the rF1 and rV antigens.     

Protection against pulmonary infection with Pseudomonas aeruginosa following immunization with P. aeruginosa-pulsed dendritic cells

To develop a Pseudomonas aeruginosa vaccine that allows the host immune system to select the antigens, we hypothesized that dendritic cells (DC) pulsed with P. aeruginosa would induce protective immunity against pulmonary infections with P. aeruginosa. Incubation of murine bone marrow-derived DC with P. aeruginosa in vitro led to uptake of P. aeruginosa and activation of the DC. Spleen-derived CD4(+) cells from mice immunized with P. aeruginosa-pulsed DC showed increased proliferation, demonstrating that DC pulsed with P. aeruginosa were capable of eliciting a P. aeruginosa-specific immune response. To evaluate if P. aeruginosa-pulsed DC can induce protective immunity against P. aeruginosa pulmonary infection, DC incubated with P. aeruginosa in vitro were administered systemically to syngeneic mice, and the mice were then challenged by intrapulmonary infection with P. aeruginosa (5 x 10(4) CFU/mouse) 13 days later. Unimmunized control mice and mice who had previously received naive DC or DC stimulated with lipopolysaccharide or Escherichia coli died within 72 h. In contrast, 45% of mice receiving P. aeruginosa-pulsed DC demonstrated prolonged survival (>14 days). Finally, DC-pulsed with heat-inactivated P. aeruginosa protected CD8(-/-) but not CD4(-/-) mice, demonstrating that CD4(+) T cells were required for the DC pulsed with P. aeruginosa to induce protective immunity.     

Significant anti-tumour activity of adoptively transferred T cells elicited by intratumoral dendritic cell vaccine injection through enhancing the ratio of CD8+ T cell/regulatory T cells in tumour

We have shown that immunization with dendritic cells (DCs) pulsed with hepatitis B virus core antigen virus‐like particles (HBc‐VLP) packaging with cytosine–guanine dinucleotide (CpG) (HBc‐VLP/CpG) alone were able to delay melanoma growth but not able to eradicate the established tumour in mice. We tested whether, by modulating the vaccination approaches and injection times, the anti‐tumour activity could be enhanced. We used a B16‐HBc melanoma murine model not only to compare the efficacy of DC vaccine immunized via footpads, intravenously or via intratumoral injections in treating melanoma and priming tumour‐specific immune responses, but also to observe how DC vaccination could improve the efficacy of adoptively transferred T cells to induce an enhanced anti‐tumour immune response. Our results indicate that, although all vaccination approaches were able to protect mice from developing melanoma, only three intratumoral injections of DCs could induce a significant anti‐tumour response. Furthermore, the combination of intratumoral DC vaccination and adoptive T cell transfer led to a more robust anti‐tumour response than the use of each treatment individually by increasing CD8⁺ T cells or the ratio of CD8⁺ T cell/regulatory T cells in the tumour site. Moreover, the combination vaccination induced tumour‐specific immune responses that led to tumour regression and protected surviving mice from tumour rechallenge, which is attributed to an increase in CD127‐expressing and interferon‐γ‐producing CD8⁺ T cells. Taken together, these results indicate that repeated intratumoral DC vaccination not only induces expansion of antigen‐specific T cells against tumour‐associated antigens in tumour sites, but also leads to elimination of pre‐established tumours, supporting this combined approach as a potent strategy for DC‐based cancer immunotherapy.     

HTA-HSP70BCG冲激的小鼠骨髓树突状细胞的生物学活性变化

利用HTA、HSP70BCG、HTA-HSP70BCG体外冲激小鼠骨髓树突状细胞,观察树突状细胞的抗原捕获能力及抗原提呈能力的变化。用流式细胞仪检测不同冲激组树突状细胞对FITC-Dextran的摄取率;用MTT法检测不同冲激组树突状细胞对HCa-F的增殖抑制作用、对脾不黏附细胞的刺激增殖活性及活化后的脾不黏附细胞对HCa-F的杀伤活性;分别用ELISA法和一氧化氮硝酸还原酶法检测树突状细胞培养上清中IL-12和NO的浓度。结果显示,冲激后的树突状细胞对FITC-Dextran的摄取率降低,对HCa-F的增殖抑制率、脾不黏附细胞的刺激增殖指数、活化后的脾不黏附细胞对HCa-F的杀伤率及树突状细胞培养上清中IL-12和NO的浓度均较对照组树突状细胞增高(P<0.05)。结果表明HTA-HSP70BCG及单独HTA、HSP70BCG均能促进小鼠骨髓树突状细胞的成熟,成熟后的树突状细胞抗原捕获能力下降,而抗原提呈能力增强。     

MHC I类抗原多肽致敏IL-2基因修饰的树突状细胞对小鼠 引流淋巴结T细胞亚群的影响及其免疫保护作用

目的探讨MHCI类分子限制性肿瘤抗原多肽Mutl体外冲击白细胞介素2(Interleukin-2,IL-2)基因修饰的树突状细胞(Dendrticcells,DC)对小鼠体内特异性免疫的活化机制。方法用腺病毒作为载体介导小鼠IL-2基因修饰DC,用小鼠Lewis肺癌3LL细胞株MHCI类分子限制性八肽Mutl冲击IL-2基因修饰的DC(DC-IL-2-Mutl)免疫小鼠,用流式细胞术(FACS)分析免疫保护小鼠接受3LL细胞再攻击后引流淋巴结内T细胞亚群的比例变化。结果用Mutl冲击的DC免疫的小鼠抵抗3LL细胞再攻击时,引流淋巴结内CD8