Lethal effect of mononuclear cells derived from human umbilical cord blood differentiating into dendritic cells after in vitro induction of cytokines on neuroblastoma cells

BACKGROUND: Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor (rhG-MCSF) and recombinant human interleukin-4(rhIL-4) can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJECTIVE: To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN: Open experiment. SETTING: Department of Pediatrics, the Medical School Hospital of Qingdao University. MATERIALS: The study was carried out in the Shandong Provincial Key Laboratory (Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University) during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments: type 3111 CO2 incubator (Forma Scientific, USA), type 550 ELISA Reader (Bio-Rad, USA). Main reagents: neuroblastoma cell line SK-N-SH (Shanghai Institute of Life Science, Chinese Academy of Sciences), RPMI-1640 culture fluid and fetal bovine serum (Hyclone), rhIL-4 (Promega, USA), rhG-MCSF (Harbin Pharmaceutic Group Bioengineering Co.Ltd), rat anti-human CD1a monoclonal antibody and FITC-labeled rabbit anti-rat IgG (Xiehe Stem cell Gene Engineering Co.Ltd). METHODS: ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhIL-4. ② Different concentrations of dendritic cells[ dendritic cells: neuroblastoma cells=20∶1,50∶1,100∶1（2×108 L-1，5×108 L-1，1×109 L-1）], 1×109 L-1 T cells and 1×107 L-1 neuroblastoma cells were added in the experimental group. 1×109 L-1 T cells and 1×107 L-1 neuroblastoma cells were added in the control group. ③ Main surface marker CD1a molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a+ cells. ④ Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect(%)=（1-A experimental well-A effector cell well/A target cell well）×100%.⑤The experimental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES: ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a+ cellular expression rate. ③Lethal effect of dendritic cells on neuroblastoma cells. RESULTS: ①Morphological characters of dendritic cells in the process of induction and differentiation: On the 15th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhIL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a+ cells was （43.12±5.83）%. ③Lethal effect of dendritic cells on neuroblastoma cells: Lethal effect of dendritic cells stimulated T cells in each experimental group ( dendritic cells: neuroblastoma cells=100∶1,50∶1,20∶1 respectively) on neuroblastoma cells was significantly higher than that in control group[（31.00 ±4.41）%，（30.92±5.27）%，(33.57±5.35)%,(26.23±5.20)%, t=3.51，2.98，4.24, P < 0.01）; But the lethal effect of dendritic cells on neuroblastoma was significantly lower when their ratio was 100∶1 and 50∶1 in comparison with 20:1 （t=2.01，2.36, P < 0.05）, and no significant difference in lethal effect existed between the ratio at 100∶1 and 50∶1（t=0.06,P > 0.05）. CONCLUSION: Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.     

D2-43病毒E蛋白在酵母细胞表面的展示

目的：在酵母细胞表面展示登革2型病毒43株(D2—43)的E基因，探索利用酵母表面展示系统建立DNA改组筛选平台的可行性。方法：通过RT-PCR扩增获得D2-43的E基因，将该基因亚克隆至T载体后，再克隆至酵母表面展示载体pYDI，于酿酒酵母EBY100中利用半乳糖进行诱导表达。表达产物采用间接免疫荧光法和FACS进行检测。结果：酵母表面展示产物可与D2-43的腹水抗体特异性地结合；在半乳糖诱导后24h，展示E蛋白的酵母细胞百分数达22．07％。结论：本研究为建立基于酵母表面展示系统的DNA改组筛选平台奠定了基础。     

UB-I粘结剂粘结面的微形态观察

目的：研究UB-Ⅰ粘结剂的粘结界面和粘结表面的微形态特点。方法：用扫描电子显微镜观察UB-Ⅰ和牙本质的粘结界面和粘结表面。结果：UB-Ⅰ粘结剂在牙本质小管内可聚合形成树脂突，在牙本质小管内的树脂突表面有树脂侧枝呈树枝状横行排列，并且和相邻的树脂突相连接，在牙本质和复合树脂之间有杂合层形成。结论：UB-Ⅰ粘合剂具有良好的渗透性，与牙本质之间可以形成良好的机械嵌合。     

苯乙酸对结直肠癌细胞中C-myc基因表达的影响

目的　观察诱导分化剂苯乙酸 (Phenylacetate,PA)抑制结直肠癌细胞HCT 8增殖过程中C myc基因表达的变化。方法　体外细胞培养 ,运用流式细胞技术观察苯乙酸对HCT 8细胞凋亡的影响。应用原位分子杂交方法观察应用PA后对HCT 8细胞C mycmRNA表达的情况。结果　PA组HCT 8细胞G1期比例从 32 3%增加到 6 1 0 % ,S期比例从 5 9 6 %下降到 2 9 7%。PA治疗后HCT 8细胞C mycmRNA表达阳性表达率为 (12 0 5± 7 92 ) % ,显著低于非治疗组中的阳性率 (5 5 15± 2 1 6 4 ) % ,P <0 0 1。通过细胞周期的检测发现 ,PA抑制大肠癌细胞HCT 8增殖的方式为抑制细胞周期G1期比例。结论　PA通过下调结直肠癌细胞C mycmRNA的生成以及细胞生长G1期阻滞在肿瘤诱导分化治疗方面发挥作用     

成功救治产科DIC 38例

报道成功救治38例产科DIC的临产资料,发病诱因以妊高征、死胎,胎盘早剥多见。治疗上以病因治疗与肝素抗凝治疗为主,主张早期、小量,快速的肝素疗法,每日剂量不得超过50mg。积极抗休克,辅以输血及抗纤溶综合治疗措施,不失时机地切除子宫。     

肾细胞癌伴静脉癌栓15例临床分析

１９８５～１９９４年治疗肾细胞癌伴静脉癌栓１５例。按癌栓水平分为肾型１０例，肝下型４例，肝上型１例。Ｂ超和ＣＴ检查总确诊率７３％。手术１４例均完整取出癌栓，术后１３例接受５－ＦＵ加ＭＭＣ方案化疗。随访３个月～５年，１例肝下型和２例肾型无瘤存活分别３６、４３、５２个月，余均在术后２年内死亡。认为Ｂ超与ＣＴ互补应用可基本确诊静脉癌栓，除肝上型和已有血管壁浸润者外大部分癌栓可采用松解游离同时渐渐拉出的方式取出，癌栓水平除肝上型外对预后影响不大。     

临床输液监控系统的设计

本文针对临床输液过程中出现的漏、停、输液速度改变等各种问题，讨论了一由单台微机和多个单片机通讯实现的多床输液自动监控、自动反应、自动报答系统的可行性，并给出了主要部分的硬件、软件设计方法。     

白细胞介素-10诱导的大鼠树突状细胞体外免疫功能的研究

目的　研究白细胞介素 10 (IL 10 )诱导的大鼠未成熟树突状细胞 (imDCs)体外诱导免疫耐受的可行性。方法　在经典诱导方案的基础上 ,应用IL 10 ( 10 μg/L)抑制大鼠骨髓来源DCs的成熟 (IL 10组 ,10例 ) ,并设对照组 (IL 4组 ,10例 )。培养期间观察DCs形态 ,检测DCs表型、摄取抗原能力、体外免疫功能及培养上清细胞因子水平。结果　与IL 4组比较 ,IL 10组DCs细胞表面CD80 、CD86及OX6低度表达 ( 2 5 .3 %、42 .4%、3 2 .3 % ) ,吞噬能力较强 ( 81.9) ,刺激同种异体淋巴细胞增殖能力下降 ,该淋巴细胞具有抗原特异性低反应性 ;培养上清中IL 12水平 ( 4 0 6.5pg/L)及初次MLR培养上清IL 2水平 ( 2 45 .4ng/L)均较低 ,差异有非常显著性 (P <0 .0 1)。 结论　IL 10作用的大鼠imDCs具有诱导免疫耐受的应用价值。     

扩散法被动式甲醛个体监测器（Ⅱ型）的研制

介绍了一种基于气体分子扩散原理的被动式甲醛个体采样器，它是在Ⅰ型采样器的基础上完成的。采用甘油（２０％）／偏重亚硫酸钠（０．２５％）浸渍的定量滤纸（φ４２ｍｍ）作为吸收层，空气中甲醛扩散到吸收层上，形成稳定的化合物。采样一定时间后，取出吸收层，洗脱后，用ＡＨＭＴ化学比色法测定所采集到的甲醛。这种采样器的平均采样速率为８３ｍｌ／ｍｉｎ，标准偏差为７．２ｍｌ／ｍｉｎ，与有动力吸收管采样的方法相比较，总不确定度小于±２５％。