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1.
树突状细胞 (DC)是免疫调节的关键细胞 ,为唯一能提呈抗原给初始型T细胞并诱导其活化和功能分化的细胞 ,可作为治疗恶性肿瘤或感染等疾病的细胞疫苗。随着基于DC的免疫治疗进入临床研究 ,稳定地产生足量有效的DC是技术关键。获得DC的方法除直接分离纯化外 ,还可用细胞因子(CK)体外培养。前一方法产量很低 ,后一方法也有其不尽如人意之处 ,如培养时间较长以及有的急性髓系白血病 (AML)细胞不能被CK诱导分化为DC。虽然有报道乙酸豆蔻佛波酯 (PMA)也能诱导CD34+细胞分化为DC ,但由于PMA的促癌作用 ,不能用于临床。…  相似文献   

2.
目的:研究lncRNA12753在β-葡聚糖诱导小鼠骨髓来源树突状细胞(DC)成熟和免疫应答中的作用及可能机制.方法:通过转录组测序和实时定量PCR,研究经β-葡聚糖诱导成熟的DC中差异表达的lncRNA;转染小干扰RNA(siRNA)敲低lncRNA12753的表达;流式细胞术检测DC表面主要组织相容性复合体Ⅱ(MH...  相似文献   

3.
小鼠脾脏树突状细胞免疫功能的体外研究   总被引:3,自引:0,他引:3  
本实验通过对小鼠脾脏分离到的树突状细胞免疫功能的研究表明,在ConA,PHA刺激的淋巴细胞增殖反应和混合淋巴细胞反应(MLR)中树突状细胞具有明显强于巨噬细胞的抗原递呈作用(P<0.001或P<0.005)。树突状细胞的这种作用可被特异性的单克隆抗体所抑制(抑制率>95%)。少量的巨噬细胞可促进其作用。  相似文献   

4.
小鼠脾脏树突状细胞的制备与鉴定   总被引:2,自引:0,他引:2  
本实验主要根据树突状细胞(DC)的半粘附和缺乏Fc受体的特性,进行DC的制备和鉴定。实验证明,脾脏单个核细胞径2h培养后,洗去非粘附细胞,继续培养16h,收集脱落DC后,再经Ig包被板的重复贴壁纯化,除去FcR~ 的巨噬细胞,即可获得较纯化的DC。DC在混合性淋巴细胞反应中起着重要的辅佐作用。  相似文献   

5.
目的 制备稳定表达FasL蛋白的小鼠骨髓源树突状细胞(dendritic cell,DC)并探讨其诱导异基因小鼠脾脏T细胞凋亡的机理.方法 采用培养基选择法体外培养小鼠骨髓源DC,脂质体法转染FasL基因至小鼠成熟DC,实时定量PCR检测转染前后FasL mRNA的表达,流式细胞仪和免疫蛋白印迹检测转染前后FasL蛋白的表达.异系小鼠静脉分别输注未转染DC、转染空质粒DC和转染FasL的DC,7 d后TdT介导的原位末端标记法(TUNEL)和流式细胞仪检测脾脏中T淋巴细胞凋亡.结果 体外培养可获得成熟的小鼠骨髓源DC,转染FasL基因的DC较未转染DC FasL mRNA和FasL蛋白表达明显升高.对脾脏中T淋巴细胞凋亡的检测发现,转染FasL的DC组凋亡指数(11.67±1.53)明显高于未转染DC组(2.67±0.58)和转染空质粒组(3.33±0.58),P<0.01.结论 培养基选择法可收获大量骨髓源DC,脂质体转染FasL基因至小鼠骨髓源DC,可以使DC高表达FasL蛋白.转染FasL的DC输注能明显诱导异系小鼠脾脏T淋巴细胞凋亡.  相似文献   

6.
为观察疟原虫感染早期卡介苗(BCG)接种对小鼠树突状细胞(DC)活化状态的影响,用Plasmodium berghei ANKA感染C57BL/6小鼠,3天后接种BCG(P.b-3-B实验组),同时以未接种BCG的感染小鼠为对照.通过动态观察两组感染鼠的原虫血症水平和生存率,流式细胞术检测感染后0、5和8天脾细胞中CD11c+CD11b+DC、CD11c+B220+DC、CD11c+CD80+DC和CD11c+MHCⅡ+ DC百分率.结果显示,P.b-3-B实验组的原虫血症从感染后第8天开始逐渐增高,66.7%的小鼠存活至第20天死亡,而对照组小鼠多于感染后8~10天死于脑型疟疾;感染后第5和8天,P.b-3-B实验组小鼠CD11c+CD11b+DC、CD11c+CD80+DC和CD11c+MHCⅡ+DC百分率均显著低于对照组(P〈0.05或P〈0.01),感染后第8天,CD11c+B220+DC百分率也明显低于对照组(P〈0.01).这些结果表明,疟原虫感染早期接种BCG可下调DC亚群百分率和表面分子表达水平,并以此影响疟疾的感染进程.  相似文献   

7.
小鼠脾脏树突状细胞的分离与鉴定   总被引:12,自引:1,他引:12  
本文根据Steinman方法并稍加改良从小鼠脾脏分离树突状细胞获得成功。经抗小鼠树突状细胞单克隆抗体(33D_1)作间接免疫荧光染色、微量细胞毒试验及EA花环形成试验等方法鉴定,其纯度大于80%。且经透射和扫描电镜观察,其形态结构明显不同于巨噬细胞。  相似文献   

8.
灵芝多糖对小鼠脾脏树突状细胞的增殖作用   总被引:5,自引:0,他引:5  
目的 观察灵芝多糖(GP)对小鼠脾脏树突状细(dendrirtie cells,DCs)的增殖作用。方法采用MTT法,以细胞因子(GM-CSF+IL-4)作比较,观察不同质量浓度GP以及细胞因子+不同浓度的GP对小鼠脾脏DCs的增殖作用。结果GP(5-80μg/mL)可明显刺激小鼠脾脏DCs增殖,与细胞因子组相比,其较高质量浓度组(20、40、80μg/mL)作用明显;GP+细胞因子,与对照组相比均有显著的增殖作用,且明显高于细胞因子组。结论GP不仅能促进小鼠脾脏DCs的增殖,而且与细胞因子有显著的协同作用。具有类生长因子和协同生长因子的作用。  相似文献   

9.
目的 观察NF-κB在黄芪多糖(APS)诱导脐血单核细胞向树突状细胞(DCs)分化过程的作用,探讨APS诱生DCs过程中的信号传导通路.方法 无菌条件下采集脐血,密度梯度离心法获得脐血单核细胞分为3组.对照组:在无药物的RPMI 1640完全培养液中培养;APS组:在含有黄芪多糖100mg/L的RPMI 1640完全培养液中培养;PDTC组:用NF-κB的抑制剂吡咯烷二硫氨基甲酸酯(PDTC)10μmol/L处理脐血单核细胞30 min后,加入含有黄芪多糖100 mg/L的RPMI 1640完全培养液中培养.培养过程中用倒置光学显微镜和透射电镜观察细胞形态变化;收集培养第12天的细胞,FCM检测细胞免疫表型;免疫荧光显微镜下观察细胞内NF-κB的激活、迁移情况.结果 (1)对照组细胞无成簇生长,培养至第12天细胞呈梭形巨噬细胞形态;黄芪多糖组细胞成簇生长,形态学由圆形逐渐变为典型的树突状细胞形态;抑制剂组细胞生长缓慢,未出现聚集现象,细胞形态学未见明显变化.(2)APS组高表达DCs特异性抗原CD80、CD83和CD86,与对照组、PDTC组比较差异有统计学意义(P<0.01),对照组与PDTC组表达相似,二者差异无统计学意义(P>0.05).(3)免疫荧光显微镜下观察NF-κB可见APS组伴有大量NF-κB荧光进入细胞核,尤以72 h显著,NF-κB激活率为(75.20±7.37)%,而对照组、PDTC组NF-κB激活率分别为(13.20±3.46)%、(8.20±1.92)%,与APS组相比,差异有统计学意义(P<0.01).结论 黄芪多糖能够诱导脐血单核细胞定向分化为DCs,NF-κB是黄芪多糖诱导脐血单核细胞向DCs分化的信号传导通路中的关键元件.
Abstract:
Objective To investigatethe role of NF-κB played in the process of the cord blood monocytes differentiating into dendritic cells(DCs)induced by astragalus polysaccharide(APS)and to explore the signal transduction pathway involved in this process.Methods Umbilica]cord blood was collected in aseptic conditions.The cord blood monocytes were obtained by density gradient centrifugation and were divided into three groups afterwards.In the control group.cells were cultivated in the RPMI 1640 complete medium.In the APS group.cells were cultivated in the RPMI 1640 complete medium containing 100 mg/L APS.In the PDTC group:cells were treated with 10 μmol/L disulfide carbamate(PDTC).NF-κB inhibitor in 30 min followed by cultivalion in the RPMI 1640 complete medium containing 100 mg/L APS.,The morphological changes were observed during the process of cultivation by the optical microscope and transmission electron microscopy.Cells were collected 12 d later and the cellular immunophenotyping was assayed by FCM.,The activation and migration of NF-κB fluorescence in the cells was examined by the immunoflouresce microscopy.Results (1)Cells in the control group grown up without cluster forformation and were found fusiform and macrophage-like in 12 d.Cells in the APS group grown up in clnstem,and morphological changes were found from the circular shape to a typical dendritic cells-like shape.Cells in the inhibitor group grown up slowly and without cluster formation,and cell morphdogy had no significant change.(2)The expression of DCs-specific antigen CD80,CD83 and CD86 in the APS group was higher than that in the control group and inhibitom group(P<0.01).The expression of those antigen in the control group and PDTC group was similar and had no statistically significance(P>0.05).(3)NF-κB fluorescence in the nuclei was examined by the immunoflourescence microscopy and was much higher in the APS group than that in khe other groups,especially in 72 h with the activation rate of NF-κB (75.20±7.37)%,while(13.20±3.46)% of PDTC group and(8.20 ±1.92)%,respectively(P<0.01).Conclusion Astragalus polysaccharide can induce the differentiation of umbilical cord blood cells into DCs,and NF-κB is the key component of the signal transduction pathway involved in this process.  相似文献   

10.
目的对粒-单集落刺激因子(GM-CSF)/白介素4(IL-4)或脱氧氟胸腺嘧啶配体(Flt3-L)体外诱导的小鼠骨髓来源树突状细胞(BMDC)亚群和正常小鼠脾脏DC亚群进行比较,探索诱生DC的特性。方法分离Balb/c小鼠骨髓细胞,分别加入含GM-CSF/IL-4或Flt3-L的培养液,体外培养7d,倒置显微镜观察细胞形态,并用流式细胞术检测CD11c、MHCⅡ、CD4、CD8α、CD45RA及Sirp-α分子;利用免疫磁珠从小鼠脾脏细胞中分离DC。结果两组细胞因子均可在体外诱导小鼠骨髓细胞分化发育为未成熟的DC;倒置显微镜下观察可见BMDCs表面有树突状突起,具有典型的DC形态学特点,与Flt3-L诱导的BMDC相比GM-CSF/IL-4诱导的DC体积大,树突长;但是,流式细胞术检测发现Flt3-L体外诱导的BMDCs与小鼠脾脏DC亚群更为相似。结论 GM-CSF/IL-4及Flt3-L均可在体外诱导小鼠骨髓细胞分化发育为DC,且Flt3-LDC亚群与脾脏DC亚群相似,有可能成为体外研究脾脏来源DC的一种方法。  相似文献   

11.
目的: 分离纯化小鼠胎肝间质干细胞(flMSCs)并探讨其分化潜能。方法: 通过改良贴壁法分离BABL/c小鼠胎肝间质干细胞;流式细胞术测定细胞周期和表型;体外诱导贴壁细胞向脂肪、软骨、骨组织以及神经细胞分化;化学染色、RT-PCR和免疫细胞化学染色鉴定分化。结果: 改进贴壁法分离的flMSCs呈均一梭形,(83.76±2.88)%处于G0/G1期,表达CD44、CD29,不表达造血细胞表面标志CD45、CD11b,诱导后能向脂肪、软骨、骨以及神经方向分化。结论: 贴壁法可以分离纯化小鼠flMSCs,小鼠flMSCs具有较强的分化潜能和“可塑性”,可用于干细胞治疗疾病的进一步研究。  相似文献   

12.
Dipeptidyl peptidase 2 (DPP2) is an N‐terminal dipeptidase, required for maintaining lymphocytes in a resting state. Mutant mice with T‐cell‐specific knock‐down (kd) of DPP2 (lck‐DPP2 kd) were generated and analyzed for their phenotype. Normal thymocyte development and a modest increase in the proportions of peripheral T cells were observed in these mice compared with littermate controls. Interestingly, the peripheral T cells were hyperactive upon TCR stimulation in vitro, although they did not express any activation markers. Furthermore, CD3‐crosslinking in the naïve CD4+ and CD8+ T cells of lck‐DPP2 kd mice resulted mainly in IL‐17 production. Similarly, the mutant T cells secreted primarily IL‐17 after in vivo priming and in vitro antigen‐specific restimulation. These data suggest that IL‐17 production is the default program for T‐cell differentiation in the absence of DPP2. Thus, DPP2 seems to impose a threshold for quiescent T cells, preventing them from drifting into cell cycle.  相似文献   

13.
《Seminars in immunology》2014,26(6):512-517
The Bacille Calmette–Guerin (BCG) vaccine is the only vaccine proved to be effective against tuberculosis and it remains the most commonly used vaccine worldwide. In addition to its effects on mycobacterial diseases, an increasing body of epidemiological evidence accumulated since its introduction in 1921 shows that BCG also exerts beneficial non-specific effects ranging from protection against non-mycobacterial diseases, decreased incidence of allergic diseases, and treatment of certain malignancies. The biological substrate of these effects is mediated partly by heterologous effects on adaptive immunity, but also on the potentiation of innate immune responses through epigenetic mechanisms, a process termed ‘trained immunity. The process of trained immunity may also play a role in the beneficial effects of BCG against tuberculosis and Mycobacterium tuberculosis infection, and this could have important consequences for our quest for improving vaccination strategies.  相似文献   

14.
 目的: 观察钙敏感受体(calcium sensing receptor,CaSR)对淫羊藿苷(ICA)诱导小鼠胚胎干细胞向心肌细胞分化的影响。方法: 129小鼠ES-D3细胞经直接悬浮法形成拟胚体(EBs),应用ICA定向诱导,透射电镜观察分化细胞的超微结构;免疫荧光和Western blot分别检测细胞有无α-辅肌动蛋白(α-actinin)和肌钙蛋白I(cTnI)的表达;流式细胞术检测细胞分化率;Western blot检测心肌特异转录因子NKx2.5、GATA-4和CaSR的蛋白表达。结果: ICA诱导2 d后,可见自发性收缩的细胞簇;随着诱导分化时间的延长,α-actinin和cTnI蛋白的表达逐渐增多;CaSR、NKx2.5和GATA-4蛋白在分化早期表达最多,持续表达至晚期;电镜观察分化细胞中可见连接结构、肌丝;CaSR激动剂新霉素能够增加早期EBs中CaSR、NKx2.5和GATA-4的表达,CaSR抑制剂NPS2390能够阻断上述作用。结论: CaSR在胚胎干细胞分化的心肌细胞中有表达,CaSR活化可通过增加NKx2.5和GATA-4的表达促进心肌细胞的分化。  相似文献   

15.
Abstract

Memory is no longer a privilege of adaptive immunity. Innate immune cells can exhibit a long-term immune activation after infection or vaccination, which is called “trained immunity.” In addition to defense against mycobacterial infection, BCG-induced trained immunity can also exert nonspecific protection, which is regulated by metabolic rewiring and epigenetic reprograming. Enhanced glycolysis and glutamine-driven tricarboxylic acid cycle have been proven to be important metabolic pathways for trained immunity induced by BCG, which is dependent on Akt/mTOR pathway.  相似文献   

16.
目的: 探讨丁酸钠、激活素A (activin A)和地塞米松诱导小鼠胚胎干细胞(ES细胞)分化为胰腺外分泌细胞的可行性,并对诱导作用进行比较。方法: 小鼠 ES细胞悬浮培养为拟胚体后,以不同浓度的丁酸钠(1 mmol/L,2 mmol/L,3 mmol/L)诱导分化,通过RT-PCR检测不同时点胰腺特异性外分泌基因的表达水平,确定丁酸钠诱导ES细胞向胰腺外分泌细胞分化的最佳浓度和作用时间。进一步单独或联合应用丁酸钠、activin A、地塞米松诱导ES细胞分化,并通过细胞形态学变化、RT-PCR和免疫荧光检测观察不同诱导方案对胰腺外分泌基因和蛋白表达的影响,确定最佳诱导方案。结果:1 mmol/L丁酸钠能明显促进胰腺外分泌基因amylase、chymotrypsinogen、elastase1、elastase2和carboxypeptidase的表达,随着丁酸钠浓度的增加,丁酸钠的诱导作用逐渐减弱;1 mmol/L丁酸钠诱导第3 d后可检测到amylase、chymotrypsinogen、elastase1、elastase2和carboxypeptidase的表达,在第5 d外分泌基因mRNA表达水平达到高峰,随后逐渐下降。与自发对照组相比,单独应用丁酸钠、activin A、地塞米松诱导ES细胞分化,均能提高amylase、chymotrypsinogen、elastase1、elastase2和carboxypeptidase的表达水平。但联合应用丁酸钠、activin A、地塞米松诱导后,ES细胞形态更为均一,上述胰腺外分泌基因的表达进一步增强;免疫荧光结果显示amylase表达为阳性。结论: 低浓度的丁酸钠、activin A以及地塞米松均可以诱导小鼠ES细胞胰腺外分泌基因的表达,多种诱导因子的联合作用能明显提高胰腺外分泌细胞的诱导效率。  相似文献   

17.
目的: 研究nestin(巢蛋白)在人尿萃取物细胞分化剂CDA-2(又名尿多酸肽)诱导SWO-38人胶质瘤细胞分化中表达的变化及其意义。方法: 采用光镜观察和鉴定CDA-2对SWO-38细胞的分化作用;RT-PCR、细胞免疫荧光、Western blotting分析CDA-2诱导前后SWO-38细胞nestin表达的变化。 结果: 光镜观察发现经CDA-2诱导后SWO-38细胞胞体变大、核浆比减小、突起增多,表现出向成熟的星形细胞分化的现象;Nestin表达在细胞浆,呈丝状着色;Nestin在CDA-2诱导SWO-38细胞分化中表达下调。结论: Nestin在CDA-2诱导SWO-38细胞分化中表达下调,进一步验证nestin与细胞分化的关系,nestin有可能成为胶质瘤诱导分化治疗领域新的标志物。  相似文献   

18.
Adaptive features of innate immunity, also termed ‘trained immunity’, have recently been shown to characterize monocytes of BCG vaccinated healthy volunteers. Trained immunity leads to increased cytokine production in response to non-related pathogens via epigenetic reprogramming of monocytes. Recently, memory-like properties were also observed in NK cells during viral infections, but it is unknown if memory properties of NK cells contribute to trained immunity due to BCG vaccination.  相似文献   

19.
Following i.v. BCG infection, a new population of macrophages are recruited in the rabbit lung. These macrophages, known as activated macrophages, substitute the resident macrophages and can play a key role in the defence against mycobacteria. We report here that BCG-activated alveolar macrophages are equipped with a more active hexose monophosphate pathway, which can maintain an optimal intracellular concentration of NADPH and GSH, and allow to produce mycobactericidal free radicals and to become resistant to mycobacterium-induced programmed cell death. These findings suggest that sustaining the anti-oxidant properties of macrophages could represent a candidate process to be considered as a good therapeutic target in fighting Mycobacterium spp infections.  相似文献   

20.
目的:探讨蛇六谷提取物(TuAKe)抑制人慢性髓系白血病K562细胞增殖和诱导分化的作用机制。方法:不同浓度的TuAKe处理K562细胞,MTT比色法和半固体集落形成实验检测K562细胞的增殖能力;瑞氏-姬姆萨染色观察细胞形态;流式细胞术检测分化相关抗原CD11b、CD14和CD42b的阳性表达率;Western blot法检测细胞周期蛋白依赖性激酶2(CDK2)、细胞周期蛋白E1(cyclin E1)和红系分化核转录因子GATA-1的蛋白表达水平。结果:TuAKe能够抑制K562细胞增殖,改变细胞形态,抑制K562细胞进入S期,并阻滞在G_2/M期,提高分化相关抗原CD11b、CD14和CD42b阳性表达率,上调GATA-1蛋白表达,同时下调CDK2和cyclin E1蛋白表达(P0.05)。结论:TuAKe可能通过调控细胞周期抑制K562细胞增殖,同时诱导K562细胞多向分化。  相似文献   

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