RSV毛细支气管炎患儿外周血CD4+ CD25+调节性T细胞与Th17细胞功能变化及意义

目的观察RSV毛细支气管患儿外周血CD4+CD25+调节性T细胞与Th17细胞及其分泌因子IL-10、TGF-β、IL-17水平并探讨与RSV毛细支气管炎发病的关系。方法采集33例RSV阳性毛细支气管炎患儿外周血,采用流式细胞仪检测外周血CD4+CD25+调节性T细胞、Th17细胞百分率,ELISA法检测血浆IL-10、TGF-β、IL-17的水平。28例非RSV感染的普通肺炎患儿作为阳性对照,26例健康儿童为正常对照组。结果 RSV毛细支气管炎患儿外周血CD4+CD25+调节性T细胞、IL-10、TGF-β水平低于肺炎组及正常对照组(P<0.05),毛支组Th17、IL-17水平高于肺炎组与正常对照组(P<0.05)。结论外周血CD4+CD25+调节性T细胞与Th1细胞7表达失衡,可能是RSV毛细支气管炎发病机制之一。     

类风湿关节炎滑膜液对体外诱导树突细胞的影响

目的了解树突细胞(DCs)在类风湿关节炎(RA)发病机制中的作用,对RA滑膜液是否能影响DCs的活化状态进行了研究。方法体外诱导外周血单个核细胞转化成为非活化的DCs,用此诱导的DCs再经RA滑膜液培养48h,然后用流式细胞仪对细胞表面表达的人类白细胞抗原(HLA)-DR和共刺激分子CD80进行分析。结果经滑膜液培养的DCs表达CD80为(45±13)%高于对照组的(26±6)%,差异有统计学意义;经滑膜液培养的DCs表达HLA-DR为(67±16)%高于对照组的(60±4)%,差异无统计学意义。结论 RA滑膜液在体外能诱导DCs活化。     

胃癌患者外周血树突状细胞的体外扩增、鉴定及内吞能力测定

目的 探讨胃癌患者外周血诱导扩增树突状细胞(DCs)的方法,观察其形态、表型、内吞及递呈抗原能力.方法 从胃癌患者外周血中分离单个核细胞(PBMNCs),在重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、白细胞介素4(IL-4)和肿瘤坏死因子α(TNF-α)的诱导下培养扩增树突状细胞.光学显微镜观察其体外培养过程中的形态特征,流式细胞仪(FACS)分析其表型特征,混合淋巴细胞反应(MLR)检测其功能,辣根过氧化酶(HRP)内吞实验检测其群体内吞能力.结果 PBMNCs经过rhGM-CSF、IL-4和TNF-α诱导培养9 d后,具典型的DCs形态,细胞表型为主要组织相容性复合体-2(MHCⅡ)类分子(HLA-DR)(69.70±6.12)%、CD80(65.32±5.19)%、CD86(71.65±7.61)%、CD1a(58.65±3.28)%和CD14(16.82±1.25)%,具有极强的激发MLR的能力,显示成熟DCs的特征.DCs群体的内吞能力约在培养第6天达高峰,之后有明显下降.结论 该方法是一种可靠的诱导、扩增DCs的方法,在其具有活跃的内吞能力时,肿瘤抗原致敏可作为胃癌生物治疗的手段之一.     

呼吸道合胞病毒、肺炎链球菌肺炎患儿免疫功能分析

目的 分析呼吸道合胞病毒(RSV)肺炎、肺炎链球菌(SP)肺炎患儿外周血T淋巴细胞亚群、B淋巴细胞以及NK细胞的变化.方法 对入院儿童行多病原联合检测,包括直接免疫荧光法检测7种病毒抗原、痰细菌培养、PCR检测痰肺炎支原体和肺炎衣原体,明确RSV、SP感染,并用流式细胞仪检测部分患儿外周血T淋巴细胞亚群、B淋巴细胞以及NK细胞.结果 RSV肺炎患儿外周血CD3+T细胞、CD4+T细胞、CD8+T细胞以及CD19+B细胞百分比与对照组相仿;但CD4+/CD8+比值和NK细胞比对照组低.SP肺炎患儿外周血T细胞亚群以及CD4+/CD8+比值与对照组相仿;但CD19+B细胞较正常组为高,而NK细胞比对照组为低.两感染组比较,PSV组CD4+Tlymphocyte升高;而SP组B lymphocyte升高.结论 RSV感染后刺激机体产生细胞免疫为主的反应,而SP感染后则产生体液免疫为主的反应.     

系统性红斑狼疮患者血清IL-6对造血干细胞分化发育为树突状细胞的影响

目的 观察系统性红斑狼疮(SLE)患者血清中的白细胞介素6(IL-6)对脐血CD34 造血干细胞(HPC)体外诱导分化为树突状细胞(DCs)的影响.方法 淋巴细胞分离液分离获得脐血单个核细胞,免疫磁珠阳性分选CD34 HPC,采用粒-巨噬细胞集落刺激因子(GM-CSF) IL- 4 肿瘤坏死因子α(TNF-α)方案体外诱导分化形成DCs,加入高IL-6水平的SLE患者血清观察对细胞分化的影响.在培养的7、10和14d收集细胞,流式细胞术检测表型,细胞增殖/毒性(CCK-8)试剂盒分析DCs刺激同种异体淋巴细胞增殖的能力.结果 在高IL-6水平SLE血清的作用下,由HPC分化而来的DCs表达CD80、CD86上升,刺激同种异体淋巴细胞增殖的能力增强;IL-6中和抗体能够降低这种DCs的CD80、CD86表达水平和对同种异体淋巴细胞增殖的刺激能力.结论 SLE血清中的IL-6对造血干细胞向DCs分化、发育及DCs功能的活化可能起促进作用.     

α干扰素对慢性乙型肝炎患者树突状细胞表型的影响

目的观察α干扰素治疗对慢性乙型肝炎患者树突状细胞(Dendriticcells,DCs)表型的影响,进一步探讨α干扰素治疗慢性乙型肝炎的具体机制。方法分离32例慢性乙型肝炎患者及10名健康人外周血中的单个核细胞(Peripheralbloodmononuclearcells,PBMCs)进行体外诱导培养,使之发育成DCs,流式细胞仪测定其表面共刺激分子的表达。对32例慢性乙型肝炎患者进行α干扰素治疗3个月后,再次检测DCs表面分子的表达水平,并将治疗前后的数据进行分析、比较。结果与正常对照组相比较,慢性乙型肝炎患者组PBMCs来源的DCs表面共刺激分子CD83、CD86的表达水平明显降低(P<0.05)。在α干扰素治疗3个月后,慢性乙肝患者组其DCs表面CD83、CD86的表达水平与治疗前相比均明显升高(P<0.05),与正常对照组相比无明显差异(P>0.05)。结论α干扰素可以上调DCs表面共刺激分子的表达水平,促进DCs的成熟,这可能是α干扰素治疗慢性乙肝的重要机制之一。     

呼吸道合胞病毒下呼吸道感染病儿外周血微小 RNA-145表达与 T淋巴细胞亚群的关系

目的 探讨呼吸道合胞病毒(RSV)下呼吸道感染肺炎病儿外周血血浆中微小RNA-145(mir-145)表达与外周血单个核细胞(PBMC)中T淋巴细胞亚群的关系.方法 选取2017年1月至2018年12月海南现代妇女儿童医院收治的RSV下呼吸道感染肺炎病儿126例作为RSV肺炎组,同期在本院进行体检的健康儿童130例作为健康对照组.RSV肺炎组根据病情严重程度分为轻度组(n=54)、重度组(n=72),并依据重度组是否给予机械通气分为非机械通气组(n=38)、机械通气组(n=34).利用实时荧光定量PCR(qRT-PCR)检测外周血血浆中mir-145表达水平,使用流式细胞仪及配套试剂盒检测PBMC中T淋巴细胞亚群CD3+、CD4+、CD8+水平,并用MRFlow流式细胞分析软件自动行CD3+、CD4+、CD8+绝对计数,同时计算CD4+/CD8+值.结果 RSV肺炎组病儿血浆mir-145表达[(1.74±0.48)比(0.99±0.26)]及PBMC中CD8+水平[(30.75±8.39)％比(21.64±5.92)％]均显著高于健康对照组(P<0.05),PBMC中CD3+[(59.04±16.12)％比(68.77±18.35)％]、CD4+水平[(31.27±8.01)％比(46.90±12.84)％]及CD4+/CD8+值[(1.02±0.28)比(2.17±0.63)]均明显低于健康对照组(P<0.05).重度组病儿血浆mir-145表达[(1.87±0.55)比(1.52±0.40)]及PBMC中CD8+水平[(33.96±9.10)％比(25.34±7.02)％]均明显高于轻度组病儿(P<0.05),PBMC中CD3+[(57.05±15.83)％比(63.28±17.21)％]、CD4+水平[(23.78±6.62)％比(38.09±10.14)％]及CD4+/CD8+值[(0.70±0.21)比(1.50±0.39)]均显著低于轻度组病儿(P<0.05).重度RSV肺炎机械通气组病儿血浆mir-145表达[(1.95±0.58)比(1.68±0.45)]及PBMC中CD8+水平[(34.92±9.18)％比(28.17±7.49)％]均显著高于非机械通气组病儿(P<0.05),PBMC中CD3+[(54.72±13.53)％比(61.83±16.04)％]、CD4+水平[(20.45±5.79)％比(34.03±8.85)％]及CD4+/CD8+值[(0.59±0.17)比(1.21±0.36)]均明显低于非机械通气组病儿(P<0.05).RSV肺炎病儿血浆mir-145表达与PBMC中CD3+水平呈负相关(P<0.05),与PBMC中CD4+水平呈负相关(P<0.05),与PBMC中CD8+水平呈正相关(P<0.05),与CD4+/CD8+值呈负相关(P<0.05).结论RSV下呼吸道感染肺炎病儿外周血血浆中mir-145表达水平升高,其高表达可能与T淋巴细胞亚群异常有关.     

干扰素抗病毒应答与慢性乙型肝炎患者外周血树突状细胞功能的关系

目的:探讨干扰素抗病毒应答与慢性乙型肝炎患者外周血树突状细胞(DCs)功能的关系。方法:分别采集23例慢性乙型肝炎患者干扰素治疗前和治疗满4mo时的抗凝外周静脉血,分离外周血单个核细胞(PBMCs),在重组人白细胞介素4和重组人粒细胞-巨噬细胞集落刺激因子的作用下培养7 d使DCs增殖、成熟。以间接免疫荧光流式细胞技术检测DCs的表型;以ELISA法检测DCs单独培养上清液中IL-12的水平;用DCs与HBsAg共同孵育,丝裂霉素C处理后,再与自体PBMCs共同培养,加入H-TDR,收集细胞测定cpm值。实验中以8例正常健康人作为对照。结果:干扰素完全应答组DCs表面CD,HLA-DR和ICAM-1的表达较治疗前均显著增加(均为P<0.05),且CD,CD,HLA-DR和ICAM-1的增高与无应答组相比具有显著性意义(分别为P<0.05、P<0.01、P<0.01和P<0.05);完全应答组DCs分泌IL-12的水平在治疗后显著增加(P<0.01);DCs的抗原提呈作用在治疗前、后,完全应答组比无应答组均显著增强(P<0.01和P<0.001),而部分应答与无应答组之间无显著性差异(P>0.05)。结论:慢性乙肝患者干扰素的抗病毒应答与外周血DCs的功能状态有关,干扰素能显著促进患者DCs功能的改善,进而可能增加患者对治疗的应答。     

小鼠树突状细胞体外培养的实验研究

目的研究体外大量扩增小鼠树突状细胞(DCs)的实验方法。方法用细胞因子[肿瘤坏死因子-α(TNF-α)、白介素-4(IL-4)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)]组合体外诱导培养DCs,混合淋巴细胞实验检测DCs的免疫刺激活性。结果培养6-8 d,细胞表面有大量毛刺状突起生长,即DCs;该DCs具有很强的刺激T淋巴细胞增殖的能力,且在T细胞与DCs比值为10∶1时最强。结论 TNF-α、IL-4、GM-CSF组合是诱导体外扩增DCs的较佳方法。     

小鼠骨髓来源树突状细胞对T细胞的活化作用

目的 从小鼠骨髓中体外诱导、扩增树突状细胞(DCs),检测DCs对T细胞的活化作用.方法 从小鼠骨髓中分离单个核细胞(MNCs),在重组鼠粒细胞-巨噬细胞集落因子(rmGM-CSF)、白细胞介素4(IL-4)和肿瘤坏死因子α(TNF-α)的诱导下培养扩增DCs.光学显微镜观察其形态特征,流式细胞仪分析其表型特征,混合淋巴细胞反应(MLR)检测DCs刺激同种异体T细胞增殖的能力.结果 MNCs经过rmGM-CSF、IL-4和TNF-α诱导培养12 d后,具有典型的DCs形态,高表达MHCⅡ类分子、CD11c、CD80、和CD86抗原.具有极强的激发刺激同种异体T细胞增殖的能力.结论 体外诱导、扩增DCs具有极强的激发刺激同种异体T细胞增殖的能力.     

M受体机制在粉防己碱保护心脏缺血再灌注损伤作用中的影响

目的　探讨M受体对粉防己碱抗心脏缺血再灌注损伤作用的影响。方法　以离体豚鼠工作心脏缺血再灌注为心肌损伤模型 ,观察乙酰甲胆碱 (Mch ,0 1μmol·L-1) ,粉防己碱 (Tet,0 3μmol·L-1)的心肌保护作用。结果　对照组在缺血复灌后 2 0min ,主动脉压 (AP)、左室收缩压(LVSP)、左室压力上升最大速率 (+dp/dtmax) ,左室压力下降最大速率 (-dp/dtmax)仅分别恢复至缺血前的 6 5 6 %±3 6 9%、6 7 9%± 9 0 9%、5 8 8%± 7 6 4%、48 6 %±8 38%。Mch、Tet使AP恢复至缺血前的 80 7%±11 70 %、77 2 %± 6 96 % (P <0 0 1vscontrol) ,LVSP恢复至 97 9%± 6 32 %、93 9%± 10 1% (P <0 0 1vscon trol) ,+dp/dtmax 恢复至 91 2 %± 11 99%、80 0 3%±9 2 4% (P <0 0 1vscontrol) ,-dp/dtmax恢复至 92 13%±6 89%、79 43%± 8 83% (P <0 0 1) ;对照组复灌后心输出量 (CO)、冠脉流量 (CF)均降低 ,恢复率不足 5 0 % ;Mch、Tet能使CO、CF维持于缺血前水平的 90 %、80 %以上。给予阿托品 0 1μmol·L-1阻断心脏M受体可见Mch、Tet保护心功能的作用明显受到抑制 ,心功能各项指标恢复程度与对照组差异无显著性。结论　调节心脏M受体能影响Tet的心肌保护作用。     

IL-12 suppression,enhanced endocytosis and up-regulation of MHC-II and CD80 in dendritic cells during experimental endotoxin tolerance

Aim:

The aim of this study was to investigate endocytosis, MHC-II expression and co-stimulatory molecule expression, as well as interleukin-12 (IL-12) production, in bone marrow dendritic cells (DCs) derived from endotoxin tolerant mice.

Methods:

Endotoxin tolerance was induced in C57BL/10J mice through four consecutive daily intraperitoneal injections of 1.0 mg/kg of 055:B5 Escherichia coli lipopolysaccharide (LPS). Bone marrow DCs were isolated in the presence of GM-CSF and IL-4 and purified by anti-CD11c Micro beads. FITC–dextran uptake by DCs was tested by flow cytometric analysis and the percentage of dextran-containing cells was calculated using a fluorescence microscope. The expression of surface MHC-II, CD40, CD80, and CD86 was also detected by flow cytometric analysis. An ELISA was used for the measurement of IL-12 production by DCs with or without LPS stimulation.

Results:

Endotoxin tolerance was successfully induced in C57BL/10J mice, evidenced by an attenuated elevation of systemic TNF-α. DCs from endotoxin tolerant mice possessed enhanced dextran endocytosis ability. The expression of surface MHC-II and CD80 was higher in DCs from endotoxin tolerant mice than in DCs from control mice, whereas the expression of CD40 and CD86 was not altered. Compared with DCs from normal control mice, DCs from endotoxin tolerant mice produced less IL-12 after subsequent in vitro stimulation with LPS.

Conclusion:

These data suggest enhanced endocytosis, selective up-regulation of MHC-II and CD80 and IL-12 suppression in DCs during in vivo induction of endotoxin tolerance.     

Modulation of costimulatory molecules CD80/CD86 on B cells and macrophages by stress proteins GroEL, GroES and DnaK

The aim of this study was to evaluate the effect of the Heat Shock Proteins GroES, GroEL and DnaK on the expression of the costimulatory molecules CD80/CD86 in B cells and macrophages. The interactions among these molecules are able to highly influence the immune response through the regulation of cytokine liberation which, on their own, are able to regulate the immunological response by a feedback mechanism. Our results showed that, on B cells, GroES and GroEL stimulated the expression of CD86 but did not induce the increase of the CD80 expression. CD86 peak expression showed a peak after 24-48 h of culture and decreased 60h after the stimulation. GroES and GroEL also stimulated the expression of CD80 and CD86 on macrophages. The same HSPs did not modify the expression of CD80 and CD86 on cells having characteristics of activated macrophages, the A-THP-1 cell line. DnaK did not induce any increase in the expression of CD80 and CD86 on lymphocytes or macrophages.     

吗啡依赖猴T细胞亚群CD4的变化

目的··:考察吗啡依赖猴及正常猴外周血T细胞亚群CD4表达的变化 ,以深入了解吗啡依赖对机体免疫功能的破坏程度及免疫功能自然恢复的进度和状态。方法··:应用流式细胞术和标记单抗法测定猴外周血T细胞亚群CD4表达的阳性率。结果·· :正常猴CD4阳性率为36.83 %±s1.44 % ;吗啡依赖性猴CD4阳性率为9.02 %±s0.12 % ;吗啡依赖猴戒断后50d ,80d ,120d和150d后CD4阳性率分别为22.68 %±s2.05 %,25.89%±s0.52%,29.55 %±s1.08 %,32.08 %±s1.88% ,至360d其CD4阳性率为35.26 %±s1.41 %。结论··:吗啡依赖可使T细胞亚群CD4表达下降 ,吗啡戒断体征消失后其CD4表达仍低下 ,自然条件下恢复至正常将需要1a的时间     

Chromium (VI)-induced immunotoxicity and intracellular accumulation in human primary dendritic cells

Chromium compounds, besides being occupational carcinogens, can also induce allergic contact dermatitis (ACD) and other immunomodulatory effects. In this study we investigate cell viability, uptake and intracellular distribution of chromium in human primary dendritic cells (DCs), either immature (iDCs) or driven to differentiate by a specific maturation stimulus (LPS) (mature DCs, mDCs), when exposed for 48 h to concentrations of soluble radiolabelled Na251CrO4 ranging from 5 to 0.5 microM. The modulation of the expression of membrane markers (CD80, CD86, MHC class II) correlated with the immunological functions of DCs was also measured. After 48 h of exposure the mean IC50 values in 4 donors were 36 and 31 microM in iDCs and mDC respectively, as detected by propidium iodide incorporation. Cellular uptake of chromium was nearly linear with increasing doses. At 48 h post-exposure chromium was accumulated preferentially in the nuclear and cytosolic fractions (44.1 to 66% and 13.1 to 31% of total cellular chromium, respectively). Although a high inter-individual variability was observed, an increase in the expression of CD86 and, to a lower extent, CD80 and MHC class II membrane markers was found in mDCs of single donors. These results highlight the relevance of searching for the biodistribution of trace metals in primary cells of the immune system. Moreover, they suggest that DCs differentiation markers can help in measuring the immunotoxicity of metal compounds with sensitisation potential.     

Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT.

Recent regulatory changes have placed a major emphasis on in vitro safety testing and alternative models. In regard to skin sensitization tests, dendritic cells (DCs) derived from human peripheral blood have been considered in the development of new in vitro alternatives. Human cell lines have been also reported recently. In our previous study, we suggested that measuring CD86 and/or CD54 expression on THP-1 cells (human monocytic leukemia cell line) could be used as an in vitro skin sensitization method. An inter-laboratory study among two laboratories was undertaken in Japan in order to further develop an in vitro skin sensitization model. In the present study, we used two human cell lines: THP-1 and U-937 (human histiocytic lymphoma cell line). First we optimized our test protocol (refer to the related paper entitled "optimization of the h-CLAT protocol" within this journal) and then we did an inter-laboratory validation with nine chemicals using the optimized protocol. We measured the expression of CD86 and CD54 on the above cells using flow cytometry after a 24h and 48h exposure to six known allergens (e.g., DNCB, pPD, NiSO(4)) and three non-allergens (e.g., SLS, tween 80). For the sample test concentration, four doses (0.1x, 0.5x, 1x, and 2x of the 50% inhibitory concentration (IC(50))) were evaluated. IC(50) was calculated using MTT assay. We found that allergens/non-allergens were better predicted using THP-1 cells compared to U-937 cells following a 24 h and a 48 h exposure. We also found that the 24h treatment time tended to have a better accuracy than the 48 h treatment time for THP-1 cells. Expression of CD86 and CD54 were good predictive markers for THP-1 cells, but for U-937 cells, expression of CD86 was a better predictor than CD54, at the 24h and the 48 h treatment time. The accuracy also improved when both markers (CD86 and CD54) were used as compared with a single marker for THP-1 cells. Both laboratories gave a good prediction of allergen/non-allergen, especially using THP-1 cells. These results suggest that our method, human Cell Line Activation Test (h-CLAT), using human cell lines THP-1 and U-937, but especially THP-1 cells at 24h treatment, may be a useful in vitro skin sensitization model to predict various contact allergens.     

粒细胞集落刺激因子动员的外周血干细胞悬液诱导培养树突状细胞的研究

目的　研究粒细胞集落刺激因子 (G CSF)动员的外周血干细胞 (PBSC)悬液中单核细胞和造血干/祖细胞分别诱导培养的树突状细胞 (DC)的特性 ,探讨临床应用PBSC悬液诱导DC的前景。方法　健康供者经G CSF动员后采集PBSC ,分两种方法诱导培养树突状细胞 :①贴壁细胞 (单核细胞 )经粒—单核细胞集落刺激因子 (GM CSF) +白细胞介素 4 (IL 4 )培养 2周 ,培养结束前 4 8、2 4h分别加入肿瘤坏死因子 (TNF α)、布雷菲德菌素 (brefeldinA ,BFA ) ;②非贴壁细胞 (含CD34+细胞 ) ,加入Flt3Ligand(FL) +干细胞因子 (SCF) +GM CSF +IL 4培养 1周 ,再按前一种方法继续培养 2周。培养结束后 ,流式细胞仪分析细胞表型和胞质 (c)IL 10 +(PE标记 )、cIL 12 (P4 0 ) +(TC标记 )细胞。结果　新鲜标本含CD14 +细胞 ( 18 4± 8 6 ) % ,CD34+细胞 ( 0 9± 0 4 ) %。以PBSC悬液中的 2× 10 6单个核细胞为起始细胞 ,前一种培养方法获得 ( 1 6± 0 6 )× 10 5细胞 ,细胞表型 :CD11c+( 97 3± 5 2 ) % ,CD86 +( 88 2± 6 8) % ,CD83+( 5 9 6± 8 4 ) % ,HLA DR+( 96 5± 7 1) % ,CD1a+( 36 6±7 5 ) % ,cIL 12 (P4 0 ) +( 15 9± 5 1) % ,cIL 10 +( 1 2± 0 4 ) % ;后一种培养方法获得 ( 1 2± 0 4 )× 10 6细胞 ,细胞表型     

腺苷通过内质网应激途径诱导HepG2细胞凋亡的研究

目的探讨腺苷(ADO)诱导人类肝癌HepG2细胞凋亡的分子机制。方法将不同浓度的ADO(0～6mmol.L-1)作用于HepG2细胞36h,采用MTT法测定ADO抑制细胞增殖效应。将HepG2细胞暴露于不同浓度的ADO(0～4mmol.L-1)作用36h或2mmol.L-1ADO作用不同时间(0～48h),观察细胞核的形态学改变;观察2mmol.L-1ADO处理12h和24h后细胞周期的变化;观察2mmol.L-1ADO作用前后Caspase-3和CHOP的亚细胞定位的变化;用West-ernblot检测不同浓度ADO作用后HepG2细胞Caspase-3,Caspase-4,CHOP,JNK的蛋白表达变化。结果ADO对HepG2细胞生长有明显的抑制作用,不同浓度ADO(0.5,1,2,4,6mmol.L-1)处理HepG2细胞36h后,与对照组相比,相对细胞存活数分别下降13.48%±0.12%,27.92%±0.25%,35.21%±0.42%,51.46%±0.24%,71.42%±0.58%,呈现剂量依赖性;不同浓度ADO作用36h或2mmol.L-1ADO作用不同时间(0～48h)后,随着ADO浓度的增加或作用时间的延长,HepG2细胞核发生典型核固缩、核碎裂、核分解等凋亡形态学改变;2mmol.L-1ADO作用12h或24h后,细胞周期分析出现亚二倍体峰,提示细胞发生凋亡,对照组、ADD处理12h及24h组细胞凋亡率分别为1.55%±0.12%、10.96%±0.07%和21.04%±0.26%;2mmol.L-1ADO诱导Caspase-3和CHOP表达增加,并从胞质易位进入胞核内;随着ADO浓度的升高,Caspase-4,Caspase-3,CHOP的表达均升高,均呈现剂量依赖性;而JNK的表达则没有变化。结论腺苷诱导HepG2细胞凋亡与内质网应激途径有关。     

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