树突状细胞联合细胞因子诱导的杀伤细胞体外抗肺癌的实验研究

目的探讨树突状细胞（DC）联合细胞因子诱导的杀伤细胞（CIK）在体内外能否有效的抗肺癌。方法正常人外周血单个核细胞经不同细胞因子作用后培养成DC和CIK细胞,单个核细胞经贴附塑料平皿获得单核细胞,加入GM—CSF,IL-4及TNF—a诱导获得DC,悬浮细胞加入IFN—g,24h后IL-1a,IL-2及CD3McAb诱导获得CIK,将A549制备成肿瘤细胞冻融物．作为肿瘤抗原刺激DC,并将DC与CIK细胞联合培养（DC＋CIK,负载抗原的DC＋CIK）,以CIK细胞单独培养作为对照。CytotoxicityTOX96体外杀伤实验测定体外细胞毒活性。结果在培养的第15d．与负载抗原的DC共同培养的CIK与CIK细胞单独培养细胞相比,增殖速率明显提高[（23．4±2．3）倍vs（16．7±2．7）倍,P〈0．05],CD3＋CD56＋细胞表达水平也明显提高,[（64．3±3．6）％vs（43．9±2．1）％,P〈0．05],同时负载抗原的DC的CIK对A549细胞的体外下细胞毒活性增强。结论DC与CIK细胞共培养后可提高CIK细胞的增殖速率,提高CIK细胞表型的表达水平,增强CIK抗肺癌的活性．将来可作为一种临床有效的过继免疫治疗策略。     

小鼠树突状细胞的体外扩增培养及形态观察

目的:建立体外诱导和扩增小鼠骨髓来源树突状细胞(DC)的方法,并进行形态学观察.方法:将健康小鼠股骨骨髓细胞分离,去除悬浮细胞,贴壁细胞中加入细胞因子(IL-4和GM-CSF)继续进行培养,2周后,诱导的DC经相差显微镜和透射电镜进行观察.结果:培养的DC表面有几个大而长的突起,具有DC的典型形态学特征.结论:小鼠骨髓细胞经贴壁处理后,加入细胞因子(IL-4和GM-CSF)进行培养是可行的,采用这种方法能获得大量较高纯度的具有典型形态特征的DC.     

人外周血单核细胞体外诱导成熟和激活的树突状细胞

目的　建立从人外周血单核细胞体外诱导培养成熟和激活的树突状细胞 (dendritic cell,DC)的方法。方法　从健康成人外周血分离单核细胞 (PBM) ,加入粒单系集落刺激因子 (GM-CSF) 10 0 μg/L+重组白细胞介素 -4 (rh IL -4 ) 5 0 0 k U /L体外培养 14 d,并于培养结束前 1d加入或不加入肿瘤坏死因子α (TNF -α ) (10 0μg/L ) ,流式细胞仪测定树突状细胞 (DC)的主要组织相容性复合体 (MHC) - 类分子、粘附分子和协同刺激分子 ,分析其成熟度和激活度。结果　 PBM经 GM-CSF+ rh IL-4诱导培养 14 d后 ,细胞成簇 ,表型为 CD83 2 2 .6%、CD865 5 .5 %、CD11c 3 6.1%、CD64 3 .2 %、人类白细胞抗原 (HLA) -DR 13 .4% ;培养结束前 1d加入 TNF -α诱导后 ,细胞表型为 CD83 81.5 %、CD8699.3 %、CD11c 98.8%、CD64 3 .4%、HLA-DR 88.3 %。结论　 GM-CSF +rh IL-4诱导 PB-M14 d,可获得大量不成熟的 DC,该体系有利于 DC扩增 ;培养结束前 1d加入 TNF-α,DC成熟度高 ,激活性好 ,适合于肿瘤免疫治疗     

树突状细胞联合细胞因子诱导的杀伤细胞体内外抗肺癌的实验效应研究

目的探讨树突状细胞（DC）联合细胞因子诱导的杀伤细胞（CIK）在体内外对肺癌的影响。方法正常人外周血单个核细胞经不同细胞因子作用后培养成DC和CIK细胞,单个核细胞经贴附塑料平皿获得单核细胞,加入GM-CSF,IL-4及TNF-a诱导获得DC,悬浮细胞加入IFN-g,24h后IL-1a,IL-2及CD3McAb诱导获得CIK,将A549制备成肿瘤细胞冻融物,作为肿瘤抗原刺激DC,并将DC与CIK细胞联合培养（DC＋CIK,负载抗原的DC＋CIK）,以CIK细胞单独培养作为对照。用流式细胞仪分析细胞表型,自体混合淋巴细胞反应和异体混合淋巴细胞反应检测其刺激T细胞的的增殖活性。Cytotoxicity TOX96体外杀伤实验测定体外细胞毒活性,同时用A549细胞系建立荷瘤裸鼠模型研究其体内抗肺癌活性。结果在培养的第15d,与负载抗原的DC共同培养的CIK与CIK细胞单独培养细胞相比,增殖速率明显提高[（23.4±2.3）倍vs（16.7±2.7）倍,P〈0.05],CD3＋CD56＋细胞表达水平也明显提高,[（64.3±3.6）%vs（43.9±2.1）%,P〈0.05],同时负载抗原的DC的CIK对A549细胞的体外下细胞毒活性增强,体内实验显示,与单独培养的CIK细胞相比,与负载抗原的DC共同培养的CIK,可明显抑制接种瘤细胞的裸鼠成瘤率,DC＋CIK与负载抗原的DC＋CIK在抗六效应上没有明显差异。结论 DC与CIK细胞共培养后可提高CIK细胞的增殖速率,提高CIK细胞表型的表达水平,增强CIK体内抗肺癌的活性。将来可作为一种临床有效的过继免疫治疗策略。     

小鼠髓源性树突状细胞分离培养及鉴定

目的 建立小鼠髓源性树突状细胞分离培养及鉴定方法。方法 取小鼠股骨和胫骨骨髓细胞,使用重组粒细胞-巨噬细胞集落刺激因子(recombinant granulocyte-macrophage colony-stimulating factor, rmGM-CSF)20 ng·mL^-1联合白细胞介素-4(interleukin 4,IL-4)10 ng·mL^-1诱导小鼠骨髓前体细胞分化为树突状细胞(dendritic cell)。体外培养10 d,使用流式细胞术鉴定细胞表面树突细胞特异性标志物CD11c、MHCⅡ、CD80、CD86、CD40及CCR7的表达。进一步使用OVA_257-264多肽处理细胞48 h,流式细胞术分析该多肽与MHC I形成的复合物的表达,验证该细胞的抗原提呈功能。结果 依据本文方法,培养第10天每只小鼠可获得1.5×10⁷～2×10⁷个细胞。其中,38.4%细胞表达CD11c, CD11c阳性的细胞中有15.2%细胞表达MHCⅡ、32.3%细胞表达CD80、...     

细胞因子IL-4对小鼠骨髓来源树突状细胞培养的影响

目的:建立从小鼠骨髓中分离、纯化、培养、扩增树突状细胞(DC)前体的方法,研究细胞因子IL-4对小鼠DC体外增殖、分化成熟的影响.方法:将健康小鼠股骨骨髓细胞分离,去除悬浮细胞,将贴壁细胞分为2组,第1组加入重组细胞/巨噬细胞集落刺激因子(GM-CSF)培养;第2组加入GM-CSF和白细胞介素-4(IL-4)联合培养.诱导的DC经相差显微镜观察.结果:分离的DC前体经8～9d的培养,镜下随机取8个视野进行细胞计数,第1组细胞数量是85.38±3.42;第2组细胞数量是119.25±3.96,两组间DC数量有显著差异(t=3.19,P<0.05).且细胞纯度达90%以上,具有典型的树枝状或裙褶状突起.结论:用GM-CSF和IL- 4联合作用更能促进DC的体外扩增及分化.     

不同细胞因子诱导树突状细胞体外抗肿瘤效应研究

目的研究不同细胞因子诱导的树突状细胞(DC)体外抗肿瘤效应。方法常规分离健康人外周血单核细胞,分别采用粒巨噬集落刺激因子(GM-CSF),白细胞介素(IL)-4,肿瘤坏死因子(TNF),GM-CSF、IL-4、α干扰素(IFN-α)细胞因子组合将其诱导为DC,并分别负载SMMC7721细胞冻融抗原;用流式细胞仪检测第9天时细胞表面HLA-DR、CD1α、CD80、CD83的表达;采用四甲基偶氮唑盐比色(MTT)法检测各组刺激自体淋巴细胞增殖能力,并检测各组DC诱导的细胞毒性T淋巴细胞(CTL)对SMMC7721细胞株的杀伤率。结果第9天显微镜下观察各组细胞,其细胞形态无明显差异,但流式细胞仪显示IFN-α组DC其膜表面标志CD80、CD83、CD1a表达上调,在刺激同种T细胞增殖能力方面,较TNF-α组DC效果明显。且IL-12分泌明显增加。在肝癌细胞株SMMC7221杀伤实验中,培养液中加入IFN-α后杀伤效应明显。结论与TNF-α相比,IFN-α诱导DC一定程度上能增强其特异性杀伤能力。     

喉癌患者树突状细胞体内诱导高效而特异的抗喉癌免疫

目的 探讨健康人外周血树突状细胞（DCs）在体内能否诱导出高效而特异的抗喉癌免疫应答。方法 从喉癌患者外周血诱导DCs回输荷瘤鼠，或DCs免疫后用癌细胞攻击。流式细胞术检测两组DCs的表面分子表达，MTT法检测其细胞毒作用．观察其免疫治疗及免疫保护作用。结果 （1）健康人外周血贴壁PBMCs经细胞因子诱导后可获大量DCs-纯度达70％以上。流式细胞术分析显示：两组DCs均高表达CD86、CD40、CD83、HLA—DR、CD54．负载抗原后的Db表达稍有下降．但两者差异无统计学意义（P〉0．05）。（2）负载抗原的DCs能诱导同种异体T淋巴细胞增殖分化为CTLs．经MTT检测实验组的OD值为（0．57±0．12），与对照组相比．差异有统计学意义（t=12．55．P〈0．01）。经过负载抗原的DCs诱导的CTLs可特异性抑制和杀伤Hep-2细胞，差异有统计学意义（P〈0．01）。（3）免疫保护实验：与对照组、未负载抗原D氏组、抗原粗提物组小鼠相比，负载抗原DCs组的成瘤率低（84％）、潜伏期长[（14±3）d，P〈0．01]、肿瘤体积及重量小（P〈0．01）。（4）免疫治疗实验：与其他三组相比．负载抗原DCs组的肿瘤体积及重量较小（P〈0．01）。结论 用GM—CSF及IL-4从人外周血诱导的DCs能有效提呈Hep-2喉癌抗原给T淋巴细胞，并且在体内诱导出高效而特异的抗肿瘤免疫，有望成为有效的肿瘤特异性免疫治疗新途径。     

脂肪干细胞诱导树突状细胞发生耐受性和抑制Th1极化的研究

目的观察脂肪干细胞（ADSCs）与异同种异体树突状细胞（DC）共培养后，DC细胞发生的耐受和Th1的极化情况。方法ADSCs与同种异体的DC共同培养3d后，通过流式细胞仪对共刺激分子的表达进行检测，用ELISA法对细胞因子的产生白细胞介素12（IL-12）和肿瘤坏死因子α（TNF—α）做进一步地分析。结果ADSCs和DC共同培养前CD80、CD83、CD86分别为（92．1±2．1）％、（90．4±8．3）％、（87．9±4．5）％，共同培养后分别为（76．2±5．1）％、（67．4±3．3）％、（57．1±3．2）％，差异均有统计学意义（均p〈0．05）；共同培养前IL-12和TNF．仅分别为（750．3±89．2）pg／L和TNF—α（282．5±15．3）ng／L，共同培养后分别为（480．6±79．5）pg／L和（192．6±22．3）ng／L，差异均有统计学意义（均P〈0．05）；吲哚-2，3-双加氧酶（IDO）表达量增高50％。共同培养后的DC可抑制CD4＋T细胞活化和幼稚T细胞向Th1细胞极化。结论脂肪干细胞（ADSCs）可能产生免疫力的负调节以及诱导免疫耐受，这对于移植或自身免疫性疾病来说无疑是提供了-个很有前途的有效方法。     

布鲁氏菌病患者外周血树突状细胞、Th1／Th2细胞因子含量的检测及意义

目的：观察布鲁氏菌病患者外周血树突状细胞表型、Th1／Th2细胞含量的检测及意义。方法选取诊治的布鲁氏菌病患者50例为病例组,另选取同期进行健康体检的正常人50例作为正常组。采用Real time-PCR测定2组Th1相关转录因子T细胞表达的T盒（T-bet）、GATA连接蛋白3（GATA-3）、维A酸相关核孤儿受体γt（RORγt）、叉头蛋白3（Foxp3）及Th1／Th2细胞因子肿瘤坏死因子α（TNF-α）、干扰素γ（IFN-γ）、白介素6（IL-6）、白介素10（IL-10）mRNA含量。酶联免疫吸附实验（ELISA）测定TNF-α、IFN-γ、IL-6、IL-10蛋白表达及补体C3、C4含量。流式细胞术测定树突状细胞表型、Th1、Th2及T淋巴细胞亚群（ CD4＋T细胞、CD8＋T细胞、NK细胞）含量。结果病例组外周血Th1细胞相关转录因子T-bet、RORγt、Foxp3及Th1细胞、Th1／Th2、TNF-α、IFN-γ含量较对照组显著降低,Th2细胞及IL-6、IL-10含量较对照组显著升高,差异有统计学意义（ P ＜0．05）;病例组CD8＋3、CD8＋0、CD8＋6阳性的树突状细胞比例、CD4＋T细胞、NK细胞及补体C3、C4含量较对照组显著降低,CD8＋T细胞含量较对照组显著升高,差异有统计学意义（ P ＜0．05）;TNF-α、IFN-γ含量与CD4＋T细胞、NK细胞、C3、C4含量呈正相关性（ r值分别为2．879、3．214、3．255和2．978, P ＜0．05）,与CD8＋T细胞含量呈负相关性（ r值分别为－3．146和－3．011, P ＜0．05）。 IL-6、IL-10含量与CD4＋T细胞、NK细胞、C3、C4含量呈负相关性（ r值分别为－2．124、－2．343、－3．423、－2．789、－2．993、－2．566、－3．758, P ＜0．05）,与CD8＋T细胞含量呈正相关性（ r值分别为3．465、3．129, P ＜0．05）。结论布鲁氏菌病患者外周血成熟树突状细胞数目减少,同时Th1／Th2细胞及相关细胞因子失衡,且与机体天然免疫和细胞免疫功能降低有关,这可能在布鲁氏菌病发生发展过程中发挥重要作用。     

Dendritic cells and T cells in immunotherapy

Autologous cellular immunotherapies have been used experimentally in humans to treat many types of cancer. These therapies are divided into two principal types: active cellular immunotherapies that rely on autologous dendritic cells or other antigen presenting cells; and adoptive T-cell therapies, in which large numbers of antigen-specific T lymphocytes are propagated ex vivo and then infused back into the patient. With the FDA approval of the antigen presenting cell vaccine sipuleucel-T for prostate cancer, active immunization has become an accepted approach for the treatment of established cancer.     

Stem cells in myelotoxicity

Myelotoxicity describes bone marrow failure due to adverse effect of xenobiotic on hematopoiesis. Hematopoiesis is a complex system in which pluripotent hematopoietic stem cells (PHSCs) differentiate into many highly specialized circulating blood cells involving the interaction of many cell types as well as the interaction of local and systemic growth factors. With respect to blood cell formation, two functional systems must be considered: the hematopoietic stem cells (PHSCs) and the progenitor cells, on one hand, and the stromal cells, which constitute the hematopoietic environment niche, on the other hand. There are three types of assays for hematopoietic progenitor clonogenic assays useable in myelotoxicology: CFU-GM assay for Colony Forming Unit Granulocyte and Macrophage, BFU-E assay for Burst Forming Unit Erythroid, and CFU-MK assay for Colony Forming Unit Megakaryocyte from several species as well as from murine as from mammalian and human.Clonogenic assays have been used to detect myelotoxicity induced by chemicals, drug, food and environmental contaminants. Designs and applications are described in this review.     

Proteomics in living cells

The study of protein dynamics in vivo is central to understanding the chemical machinery that makes up the process of life: in determining how, when, where and under what conditions proteins are created, form complexes, are modified and disintegrate. Until recently, facile methods to study these processes in intact living cells did not exist. This review describes promising experimental strategies that have been devised to visualize the dynamic nature of proteins and protein complex formation occurring in living cells. The approaches described are based largely on genetically encoded fusions to proteins of interest combined with labelling strategies. The feasibility of employing such strategies in genome-wide biochemical pathway mapping efforts is discussed.     

Excitotoxicity in glial cells

Excitotoxicity results from prolonged activation of glutamate receptors expressed by cells in the central nervous system (CNS). This cell death mechanism was first discovered in retinal ganglion cells and subsequently in brain neurons. In addition, it has been recently observed that CNS glial cells can also undergo excitotoxicity. Among them, oligodendrocytes are highly vulnerable to glutamate signals and alterations in glutamate homeostasis may contribute to demyelinating disorders. We review here the available information on excitotoxity in CNS glial cells and its putative relevance to glio-pathologies.     

Reversion in virus-transformed cells

The hamster cell line BHK21 can be transformed by the Schmidt-Ruppin strain of Rous sarcoma (RSV-SR) and by polyoma viruses. Revertant cells that have apparently lost the virus genes can be obtained in both cases. These occur spontaneously in the case of RSV-SR transformed cells and following the induction of chromosome loss from near tetraploid BHK21 hybrid cells transformed by polyoma. In both cases the cells can be retransformed by the same virus that transformed them.     

静脉血管平滑肌细胞中的多倍体细胞分析

目的探讨流式细胞术分析静脉血管平滑肌细胞多倍体细胞的方法和意义。方法选取普通级健康成年日本大耳白兔2只,雄性,8月龄,大约3 kg;普通级SD大鼠2只,雄性,8周龄,大约200 g。人大隐静脉为2例心脏冠状动脉旁路移植术中修补血管所得。在麻醉日本大耳白兔和大鼠后开腹腔获取下腔静脉,采用多种酶组合的消化液消化兔、大鼠以及人的静脉血管得到适合流式细胞检测的静脉血管平滑肌单细胞悬液,用碘化丙啶标记细胞,利用细胞流式仪分析兔、大鼠以及人的静脉血管平滑肌单细胞悬液各5 000个细胞,检测细胞DNA含量,DNA含量翻倍的细胞为多倍体细胞。并利用荧光原位杂交技术（FISH）验证阳性对照HEK293细胞系中的多倍体的存在。结果流式结果显示分析的5 000个细胞中,阳性对照HEK293细胞的多倍体细胞为1 355个（27.1%）,从2例患者中取得的大隐静脉多倍体细胞为310个（6.2%）和250个（5.0%）;2只大鼠的下腔静脉平滑肌细胞中多倍体细胞为360个（7.2%）和450个（9%）;2只兔的下腔静脉平滑肌细胞中多倍体细胞分别为270个（5.4%）和305个（6.1%）。结论成年的静脉血管包括人大隐静脉的平滑肌细胞中普遍存在一定比例的多倍体细胞。     

Interactions between dendritic cells and epithelial cells in allergic disease

Dendritic cells (DCs) play a crucial role in the sensitisation process. Upon encounter with an allergen, DCs require interactions with other cells and factors for triggering a primary or secondary immune response. Epithelial cells (ECs) express features of accessory cells, such as expression of HLA-DR, co-stimulatory molecules, functional FcgammaR, molecules of the antigen-processing machinery, and display an ability to internalise antigen. These features may authorize them to function as immunomodulators (e.g. amplification of memory T cells during secondary immune responses). ECs may increase chemokine (e.g. CCL20) secretion thereby attracting DCs. Epithelial human TSLP activates DC, which allow them to prime naive T cells for the production of pro-inflammatory cytokines, while down-regulating IFN-gamma and IL-10. ECs may also influence the local polarization of types l and 2 antigen-presenting cells via PGE(2) by impairing the ability of maturing DC to produce bioactive IL-12 p70. PGE(2) is synergistic with IL-1beta and TNF-alpha in the induction of functional and phenotypic maturation of DC and induce IL12 p40 production. Sensitisation via the respiratory route may be Th(2) skewed, possibly because the antigen recognition by DC occurs in an environment rich of airway EC-product such as PGE(2).     

Calcium-transporters in myocardial cells

This is a concise review of important calcium-transporters on the sarcolemma and organellar membranes of myocardial cells, and their functional roles in cell physiology. It briefly addresses L and T type calcium channels, store-operated calcium channel (SOC), sodium-calcium exchanger (NCX), and the plasma membrane calcium ATPase (PMCA) on the sarcolemma, ryanodine receptor (RyR), IP3 receptor (IP3R) and the sarcoplasmic reticulum (SR) calcium ATPase (SAERCA) on the SR membrane and their contributions to contraction and rhythm-generation. Several agonists and blockers for every transporter that are commonly used in research, and those with therapeutic applications have also been discussed.     

体外定向诱导脐血间充质干细胞向类肝细胞分化的研究

目的 探讨诱导人脐血间充质干细胞（UBCMSCs）向类肝细胞定向分化的可能性。方法 采用密度梯度离心法与直接贴壁法相结合，从人脐血中分离UBCMSCs；通过流式细胞仪检测细胞表面抗原CD34、CD44，确定UCBMSCs的比例。在UCBMSCs培养体系中，加入人肝细胞株L-02细胞培养上清液或诱导因子（联用HGF和FGF-4）共培养，检测诱导后第7天、14天细胞内细胞角蛋白18（CK18）、白蛋白及糖原的表达。结果 从脐血中分离获得均一贴壁的UBCMSCs；在UBCMSCs培养体系中，无论是添加L-02细胞培养上清液，还是采用诱导因子HGF和FGF-4，均可诱导UCBMSCs细胞内表达中晚期成熟肝细胞表面标志物CK8＆18、白蛋白及糖原。结论 密度梯度离心法和直接贴壁法相结合能够有效分离UCBMSCs；L-02人肝细胞培养上清液或诱导因子HGF、FGF-4均具有诱导UCBMSCs向类肝细胞定向分化的作用。