负载卵巢癌抗原的树突状细胞抗卵巢癌作用的实验研究

卵巢上皮性癌（OEC）是妇科常见且致死率最高的恶性肿瘤，目前主要采用手术切除或联合化疗的方法治疗，但复发率较高，晚期OEC患者的5年生存率仅为28％-35％。近年来，免疫治疗在OEC的治疗中取得了较好的效果，且随着技术和方法的不断改进，免疫治疗在OEC治疗中的作用愈加明显。本研究以树突状细胞（dendritic cells，DC）为效应细胞。将用卵巢癌细胞系（SK-OV-3）提取的抗原负载到DC，检测其对卵巢癌细胞的特异性杀伤作用，为OEC的免疫治疗提供实验数据。     

HSP90多肽复合物对树突状细胞CD86、CD11c及IL-12p70表达的影响

目的研究HSP90多肽复合物对树突状细胞（dendritic cells,DC）功能的影响,并探讨HSP90多肽复合物修饰的DC疫苗对肿瘤的抑制作用。方法 HSP90多肽复合物刺激DC前后,ELISA法检测IL-12P70的表达水平,流式细胞仪检测CD86及CD11c的表达。建立C57BL/6小鼠Lewis肺癌模型,观察不同组别DC疫苗对肿瘤生长的抑制作用。结果 DC经HSP90多肽复合物刺激后,CD86及CD11c的表达上调（94.49±1.49及81.87±1.64）%,IL-12p70分泌量（97.11±5.26）pg/ml,均高于肿瘤粗提取物刺激DC组及未刺激空白DC组（P〈0.05）。HSP90多肽复合物修饰的DC疫苗可明显抑制肿瘤生长,抑制率为57.56%,凋亡率为26.35%,均明显高于对照组（P〈0.05）。结论 HSP90多肽复合物能够诱导DC成熟,经其修饰的DC能抑制肿瘤生长,可能与其能诱导肿瘤细胞凋亡有关。     

肿瘤细胞裂解物致敏的树突状细胞用于大鼠卵巢上皮癌免疫预防的体内研究

目的:通过负载肿瘤冻融抗原的树突状细胞(DC)预防大鼠卵巢上皮癌发生的体内试验,进行DC疫苗用于卵巢癌防治的可行性研究。方法:取大鼠骨髓单个核细胞(bone marrow mononuclear cells,BMMNC)体外培养获得成熟DC,诱导BMMNC细胞分化的第3天加入大鼠卵巢癌细胞株NuTu-19的冻融抗原,获得负载肿瘤抗原(TAA)的成熟DC;将致敏的DC制成DC疫苗经尾静脉注射大鼠体内,后于大鼠皮下接种NuTu-19细胞,观察大鼠皮下肿瘤的生长情况;注射生理盐水、NuTu-19冻融抗原、未致敏DC作为对照组。结果:大鼠骨髓来源的BMMNC在相关细胞因子的作用下可以培养出成熟的DC。TAA致敏的DC疫苗体内试验中肿瘤发生率较低,成瘤时间较晚,肿瘤生长速度缓慢,与对照组有显著性差别(P<0.01)。结论:大鼠骨髓来源的BMMNC可以培养出成熟的DC。DC可负载并提呈肿瘤抗原,体内试验证明负载癌抗原的DC可以杀伤肿瘤细胞,DC疫苗能够对肿瘤的发生起到主动免疫预防作用,在今后的肿瘤免疫防治方面有着广阔的前景。     

热休克蛋白—抗原肽复合物对移植黑色素瘤B16细胞小鼠的？…

制备黑色素瘤Ｂ１６细胞热休克蛋白－抗原肽复合物及其粗提物,并进行其抑瘤效应的研究。结果表明应用凝胶过滤制备的ＨＡＣ－ＣＥ３、ＨＡＣ－ＣＥ４和ＨＡＣ－ＣＥＳ及应用亲和层析纯化的ＨＡＣ６０、ＨＡＣ７５和ＨＡＣ９７均不同程度地降低肿瘤发生率、延迟肿瘤发生时间和减少Ｃ５７ＢＬ／６Ｊ小鼠死亡率。提示,６０－９７ｋＤＨＡＣｓ具有抑瘤效应,而且为制备肿瘤疫苗提供了重要的实验依据。     

热休克蛋白-抗原肽复合物对移植黑色素瘤B16细胞小鼠的抑瘤效应

制备黑色素瘤B16细胞热休克蛋白-抗原肽复合物(HACs)及其粗提物(HAC-CEs),并进行其抑瘤效应的研究.结果表明应用凝胶过滤制备的HAC-CE3、HAC-CE4和HAC-CE5及应用亲和层析纯化的HAC60、HAC75和HAC97均不同程度地降低肿瘤发生率、延迟肿瘤发生时间和减少C57BL/6J小鼠死亡率.提示,60～97kD HACs具有抑瘤效应,而且为制备肿瘤疫苗提供了重要的实验依据.     

肿瘤抗原致敏的树突状细胞抗肿瘤作用的实验研究

目的研究肿瘤细胞裂解物体外致敏的树突状细胞的抗肿瘤效应.方法经粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)协同诱导小鼠骨髓树突状细胞(DC)前体为DC反复冻融制备肿瘤细胞可溶性抗原,将肿瘤细胞可溶性抗原与DC共育,获得肿瘤抗原致敏的DC,将其直接作为瘤苗对荷瘤小鼠进行免疫治疗实验.结果从每只小鼠股骨中分离的DC前体细胞经GM-CSF和IL-4协同诱导,可获得(3～5)×106个树突状细胞;用肿瘤细胞可溶性抗原致敏的DC瘤苗治疗的荷瘤小鼠,其脾淋巴细胞转化率明显高于其它实验组及对照组,其瘤体生长减缓,生存时间明显延长.结论肿瘤抗原体外致敏的DC对荷瘤小鼠具有明显的免疫治疗作用,可望成为肿瘤免疫治疗的新途径.     

抗原负载树突状细胞对胃癌患者自身肿瘤细胞体外杀伤作用研究

目的探讨用冻融的自身胃癌抗原冲击树突状细胞(DC),体外观察其对自身细胞因子诱导的杀伤细胞(CIK)对自身肿瘤细胞杀伤作用的研究。方法从胃癌手术切除肿瘤周围转移引流淋巴结,提取单个核细胞,将贴壁生长的细胞用细胞因子基因重组粒巨集落和生长因子(rhGM-CSF)、基因重组白介素4(rhIL-4)、以及基因重组α-肿瘤坏死因子(rhTNF-α)联合诱导培养DC,然后用经冻融制备的自身肿瘤细胞抗原冲击DC并作用CIK细胞,最后检测CIK细胞体外杀伤三种靶细胞(自身胃癌细胞、K562和SGC-7901细胞)的活性。结果经细胞因子联合诱导培养后,DC含量明显增高,CD1a/CD14和CD80/CD86阳性细胞比刚分离的单个核细胞明显增多。DC和CIK细胞共同培养后,CIK细胞高表达CD3+CD56+。负载胃癌抗原冲击的引流淋巴结DC活化的CIK细胞,杀伤自身肿瘤细胞活性达78.6%,而单纯CIK细胞为35.2%,两者比较有显著性差异p<0.01;而对K562和SGC-7901细胞杀伤活性二者间无明显差异。结论肿瘤周围转移引流淋巴结贴壁细胞在体外能定向诱导扩增出大量DC;胃癌抗原负载的DC和CIK细胞共同培养,可明显提高CIK细胞对自身肿瘤细胞的杀伤活性。     

树突状细胞激活效应细胞作用的初步研究

目的探讨树突状细胞（DC）对肿瘤浸润淋巴细胞（TIL）和小鼠脾淋巴细胞（小鼠脾LC）的最佳激活作用比例.方法联合应用GM-CSF、IL-4和小鼠H22肝癌细胞全细胞性抗原致敏从荷瘤小鼠四肢长骨提取的DC,依据不同的激活比例用致敏DC体外激活TIL和小鼠脾LC,采用乳酸脱氢酶（LDH）4小时释放法测定、观察被不同程度激活的TIL和小鼠脾LC对小鼠H22肝癌细胞的杀伤活性.结果当E/T为1：400和1：200时,所激活的TIL或小鼠脾LC的杀伤活性较弱,线性关系不明显,P＞0.05;当E/T为1：100时,杀伤活性有较明显的提高,与前两者比较,P＜0.05;当E/T=1：50时,杀伤活性徒增,与前三者比较均有显著性差异;而当E/T=1：25,1：12.5和1：6.25时,杀伤活性与E/T=1：50时基本没变化,P＞0.05.结论当E/T=1：50时,DC能使小鼠脾LC和TIL充分激活,显著提高其对小鼠H22肝癌细胞的体外杀伤活性.     

负载乳腺癌干细胞RNA树突状细胞疫苗抗肿瘤免疫机制的研究

目的:通过制备负载乳腺癌干细胞RNA的树突状细胞疫苗,研究其抗肿瘤免疫机制.方法制备乳腺癌干细胞RNA树突状细胞疫苗的采用细胞毒性试剂盒检测2种乳腺癌干细胞疫苗[(A组,CD44+CD24-+MCF-7-CTLs)、(B组,MCF-7-CTLs)]和(C组,DC-CTLs)的体外细胞杀伤能力.取24只小鼠均分为四组:A1组皮下接种活化的A组疫苗+MCF-7乳腺癌细胞;B1组接种活化的B组疫苗+MCF-7乳腺癌细胞;C1组皮下注射活化的DCs-CTLs+MCF-7乳腺癌细胞;D1组单用MCF-7乳腺癌细胞作为对照.分析裸鼠接种后肿瘤在体内的生长情况.结果:体外对CD44+CD24-+MCF-7乳腺癌细胞杀伤能力强度:A组>B组>C组(P<0.05).体外对MCF-7乳腺癌细胞杀伤能力强度:B组>A组>C组(P<0.05).在体成瘤实验显示,B1组和A1组的成瘤时间分别为第7周和第6周,明显长于D1组(第1周)和C1组(第2周)(P<0.05).结论:CD44+CD24-乳腺癌干细胞RNA树突状细胞疫苗诱导的特异性细胞毒性T淋巴细胞能够产生特异性免疫应答,从而发挥抗肿瘤作用.     

食道癌细胞RNA致敏树突状细胞诱导特异性抗肿瘤免疫的实验研究

目的观察人食道癌细胞株RNA致敏的树突状细胞(DC)所诱导的特异性抗肿瘤免疫效应,探讨肿瘤细胞RNA直接致敏DC进行食道癌生物治疗的可能性.方法食道癌细胞株TTn体外培养,提取RNA;正常人外周血,进行体外DC的培养扩增;食道癌RNA直接致敏DC,MTT法检测食道癌RNA活化DC所诱生的淋巴细胞对食道癌TTn、视网膜母细胞瘤SoRb-70的体外杀伤效应.结果正常人外周血来源的DC,经食道癌TTn细胞株RNA直接致敏后,成功诱导特异性抗肿瘤免疫反应,TTn RNA组在CTL在靶效比为20;1时对TTn、SoRb-70的杀伤率分别在84.54%、1.53%,而对照组对这两种细胞的杀伤率分别为1.34%和1.70%.结论肿瘤RNA致敏DC可以诱导特异性抗肿瘤免疫,是一种有前景的食道癌治疗手段,有进一步研究的价值.     

Heat shock protein 70 from Trichinella spiralis induces protective immunity in BALB/c mice by activating dendritic cells

Trichinella spiralis heat shock protein 70 (Ts-Hsp70) is a protective antigen that induces partial protective immunity against T. spiralis infection in mice. To determine whether dendritic cells are involved in the mechanism responsible for the protection induced by Ts-Hsp70, mouse bone marrow-derived dendritic cells (DCs) were incubated with recombinant Ts-Hsp70 (rTs-Hsp70), and the DC-secreted cytokines and expressed surface markers were measured. The results demonstrated that rTs-Hsp70 activated DC maturation that was characterized by the secretion of IL-1β, IL-12p70, TNF-α, and IL-6 and the increased surface expression of CD11c, MHC II, CD40, CD80, and CD86. The rTs-Hsp70-activated DCs enabled the stimulation, proliferation and secretion of Th1/2 cytokines (i.e., INF-γ, IL-2, IL-4 and IL-6) in CD4⁺ T cells from T. spiralis-infected mice. The mice that received rTs-Hsp70-activated DCs exhibited a 38.4% reduction in muscle larvae upon larval challenge with T. spiralis compared to the group that received PBS-incubated DCs. This partial protection was correlated with Th1 and Th2 mixed anti-Ts-Hsp70-specific immune responses that included high titers of total IgG, IgG1 and IgG2a and increased levels of Th1/2 cytokines (i.e., IFN-γ, IL-2, IL-4, IL-6). These results indicate that the rTs-Hsp70-induced protective immunity was mediated by the activation of the DCs and that rTs-Hsp70-loaded DCs could be an alternative vaccine approach against trichinellosis.     

Recombinant heat shock protein 65 carrying PADRE and HBV epitopes activates dendritic cells and elicits HBV-specific CTL responses

BCG Hsp65 and PADRE have been shown to be potent to enhance antigen specific immunity. In order to explore the possibility to utilize them for the development of HBV therapeutic vaccine, a chimeric protein, Hsp65-HBV, was created by fusing PADRE and epitopes from HBV to the carboxyl-terminus of BCG Hsp65 and expressed in E. coli. We evaluated its effects on human dendritic cell maturation and specific CTL induction in vitro. Results showed that Hsp65-HBV could activate human dendritic cells by up-regulating the expressions of HLA-A2, HLA-DR and CD86, companioning with high level of IL-12 secretion. Furthermore, Hsp65-HBV matured DCs could significantly stimulate human autologous CD8⁺ T cell proliferation and induce HBV-specific CTLs. Hsp65-HBV was also shown to generate HBsAg-specific CTLs in vivo in mice. These results indicated that Hsp65-HBV might be a candidate for the treatment of chronic HBV infection.     

乳腺癌患者外周血淋巴细胞热休克蛋白70表达水平及其意义

目的:探讨乳腺癌患者外周血淋巴细胞中热休克蛋白70(HSP70)表达水平及其临床意义。方法:用Westernblot方法检测45例乳腺癌患者、43例乳腺良性病变患者及43例正常对照组外周血淋巴细胞中HSP70的表达水平。结果:乳腺癌组、乳腺良性病变组外周血淋巴细胞中HSP70表达水平均高于正常对照组,差异有统计学意义(t=2.173,P<0.05);HSP70表达水平在肿瘤直径>2 cm组高于≤2 cm组,差异有统计学意义(t=-2.781,P<0.01);HSP70与乳腺癌患者年龄、组织学分级、临床分期、是否有淋巴结转移及月经状况无关。结论:术前外周血淋巴细胞中HSP70的检测有一定意义,其表达水平有可能成为判断预后的参考指标。     

L-Threonine induces heat shock protein expression and decreases apoptosis in heat-stressed intestinal epithelial cells

ObjectivesOsmotically acting amino acids can be cytoprotective following injury. As threonine (THR) induces osmotic cell swelling, our aim was to investigate the potential for THR to induce cellular protection in intestinal epithelial cells and evaluate possible mechanisms of protection.MethodsCells treated with a range of THR doses were evaluated following heat stress (HS) injury. Alpha-aminoisobutyric acid (AIB), a non-metabolizable amino acid analog, was used as an osmotic control. MTS assays were used to assess cell survival. Heat shock protein (HSP) expression and cleaved caspase-3 (CC3) were evaluated via Western blot. Cell morphology and cell size were analyzed via microscopy.ResultFollowing HS, THR treatment increased cell viability in a dose dependent manner vs. non-THR treated cells (CT). The non-metabolized amino acid analogue, AIB, also increased cell survival in heat-stressed cells versus HS controls. HSP70 and HSP25 expression increased with THR and AIB treatment versus HS controls. THR also increased HSP25 in non-stressed cells. Microscopic evaluation revealed both THR and AIB preserved the structural integrity of the actin cytoskeleton in heat-stressed cells versus HS controls. THR, but not AIB, enhanced nuclear translocation of HSP25 during HS. This nuclear translocation was associated with a 60% decrease in apoptosis in heat-stressed cells with THR. No antiapoptotic effect was observed with AIB.ConclusionsThis is the first demonstration that THR increases HSP70 and HSP 25 and protects cells from HS. THR's mechanism of protection may involve cytoskeletal stabilization, HSP up-regulation and nuclear translocation, and decreased apoptosis. THR's protection appears to involve both cell-swelling–dependent and –independent processes.     

锌对热应激大鼠热休克蛋白70mRNA表达的影响

目的：锌对热应激大鼠热休克蛋白７０ ｍ Ｒ Ｎ Ａ 表达的影响。方法：分别以高、中、低锌饲料（锌含量分别为９２２０、４５６１、２１７０ ｍ ｇ／ｋｇ 饲料）喂养３ 组大鼠,热暴露（干球４１５ ° Ｃ、湿球３２ ° Ｃ）３０ ｍ ｉｎ 时断头处死,应用 Ｎｏｒｔｈｅｒｎ 印迹杂交和斑点杂交检测热应激大鼠肝脏热休克蛋白７０ ｍ Ｒ Ｎ Ａ 的表达情况。结果：不经受热暴露的３ 组动物仅高锌组能检测到热休克蛋白７０ ｍ Ｒ Ｎ Ａ,而热暴露３０ ｍ ｉｎ 的３ 组动物均有热休克蛋白７０ ｍ Ｒ Ｎ Ａ 条带,且依锌水平不同,其表达强弱依次为高锌组强于中锌组,中锌组强于低锌组。结论：热应激时热休克蛋白７０ 基因的转录水平提高;锌通过活化热休克蛋白７０ 基因的转录而诱发热应激大鼠热休克蛋白７０ 的合成,从而使机体对热暴露产生更强的耐受力。     

热休克蛋白Hsp70在抗原呈递过程中的作用

目的研究热休克蛋白Hsp70家族在抗原呈递中的作用，探讨其与肿瘤免疫反应的关系。方法利用反义RNA技术，将反义Hsp70、反义Hsc70、B7表达质粒导入肿瘤细胞B16中，24h后检测细胞中热休克蛋白表达水平。利用混合淋巴细胞培养，检测T淋巴细胞增殖能力。结果在黑色素瘤细胞B16中转染pLXSN—antihsp70、pLXSN—antihsc70及2者共转染，转染反义质粒能够抑制B16细胞中Hsp70／Hsc70的表达，导致Hsp70表达下调。其刺激T淋巴细胞增殖能力均大幅下降。而将pLXSNmB7转染入B16细胞中，其刺激T细胞增殖的能力均有所上升。转染pLXSNmB7后，B16细胞中Hsp70／Hsc70的表达量有所提高。结论抑制各热休克蛋白Hsp70家族的表达能够导致T细胞增值数降低。在肿瘤细胞中导入B7分子能够提高Hsp70的表达水平，从而提高其抗原呈递的能力。     

Protection against pneumococcal infection elicited by immunization with multiple pneumococcal heat shock proteins

Heat shock proteins (HSPs) play important roles in the pathogenesis of pneumococcal infection, and they are considered as potential protein vaccine antigens. In this study, we investigated the efficacy of immunization with pneumococcal HSPs, including ClpP (hsp100/Clp peptidase subunit), DnaJ (hsp40) and GroEL (hsp60), to protect against pneumococcal carriage, lung colonization and sepsis in mouse models using different serotypes of Streptococcus pneumoniae. In a nasopharyngeal colonization model by serotype 6B or 14 and in a lung colonization model by serotype 19F, immunization with pneumococcal HSPs could elicit effective protection. Likewise, vaccination with ClpP, DnaJ or GroEL allowed significantly longer mouse survival times after lethal intranasal challenge with serotype pneumococcal 2, 3 or 4. Interestingly, combinations of these HSPs could consistently enhance the protection against nasopharynx carriage, lung colonization as well as invasive infection caused by different pneumococcal serotypes. In an in vitro killing assay, anti-sera against ClpP, DnaJ or GroEL could kill S. pneumoniae by polymorphonuclear leukocytes in a complement-dependent way, and combinations of multiple anti-sera against these HSPs could increase the killing ability compared with single anti-sera. Finally, passive immunization studies with anti-sera against pneumococcal HSPs also demonstrated that an additive effect could be achieved by using multiple anti-sera when compared with single anti-sera. Thus, inclusion of multiple pneumococcal HSPs is important for the development of protein-based pneumococcal vaccines.