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.     

胸腰椎骨折术后内固定松动、断裂的原因及预防

目的 探讨椎弓根螺钉系统用于胸腰椎骨折术后出现松动、断裂的原因，并提出相应的预防对策。方法 12例胸腰椎骨折术后内固定失效病例，采用RF系统3例，AF系统9例。均未行植骨融合，AF系统中未按要求安装横连结3例，术后伤椎前缘高度平均恢复至正常的85．2％。结果 术后8—21个月出现棒断裂4例，螺钉断裂3例，钉帽松动5例。结论 内固定物的自身问题、术者的经验欠缺及操作不规范是胸腰椎骨折术后椎弓根螺钉系统松动、断裂的原因，但伤椎骨结构未能得到良好重建及术后椎间盘退变可能是其更重要的因素。因此重视内固定物的选择、加强规范操作，强调对损伤椎体及椎间盘的全面处理是预防内固定失效的有效措施。     

The upregulation of glial glutamate transporter-1 participates in the induction of brain ischemic tolerance in rats.

Glial glutamate transporter-1 (GLT-1) plays an essential role in removing glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. To explore whether GLT-1 plays a role in the acquisition of brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIP), the present study was undertaken to observe in vivo changes in the expression of GLT-1 and glial fibrillary acidic protein (GFAP) in the CA1 hippocampus during the induction of BIT, and the effect of dihydrokainate (DHK), an inhibitor of GLT-1, on the acquisition of BIT in rats. Immunohistochemistry for GFAP showed that the processes of astrocytes were prolonged after a CIP 2 days before the lethal ischemic insult, which could protect pyramidal neurons in the CA1 hippocampus against delayed neuronal death induced normally by lethal ischemic insult. The prolonged processes extended into the area between the pyramidal neurons and tightly surrounded them. These changes made the pyramidal layer look like a 'shape grid'. Simultaneously, the prolonged and extended processes showed a great deal of GLT-1. Western blotting analysis showed significant upregulation of GLT-1 expression after the CIP, especially when it was administered 2 days before the subsequent lethal ischemic insult. Neuropathological evaluation by thionin staining showed that DHK dose-dependently blocked the protective role of CIP against delayed neuronal death induced normally by lethal brain ischemia. It might be concluded that the surrounding of pyramidal neurons by astrocytes and upregulation of GLT-1 induced by CIP played an important role in the acquisition of the BIT induced by CIP.     

Keratin 13基因在喉癌发生中的作用

探讨keratin 13基因在喉癌发生中的作用。方法在keratin 13基因内部及附近选择5个微卫星引物进行LOH分析，于DNA水平间接检测100例喉癌患者中该基因的缺失情况。结果5个STR位点均存在LOH，其中D17S1964E、D17S2092、D17S791、D17S1665及D17S808位点的LOH频率分别为30．48％、26．02％、21．62％、37．66%和21．51％，以D17S1665位点的LOH频率最高，杂合性丢失与临床分期无显著相关。结论Keratin13基因在喉癌的发生中具有重要作用，具体机制有待进一步研究。     

枸橼酸减轻草酸钙晶体对大鼠肾小管上皮细胞损伤作用的实验研究

目的 通过研究枸橼酸能否减轻一水草酸钙（calcium oxalate monohydrate，COM）晶体对体外培养的Wistar大鼠肾小管上皮细胞造成的损伤，以探讨枸橼酸在尿路结石防治中的作用及其机制。方法分离、培养正常雄性Wistar大鼠的肾小管上皮细胞，在原代培养第5天时，将细胞分成3组（空白对照组、COM组及枸橼酸组），分别于各组培养液中培养2h后用比色法测定各组细胞培养基中丙二醛（malonaldehyde，MDA）的含量及细胞的钙镁ATP酶（Ca`2＋-Mg`2＋-ATPase）和钠钾ATP酶（Na`2＋K`＋-ATPase）的活性。结果①COM组和枸橼酸组细胞Ca2＋-Mg`2＋ATPase和Na`＋＋K`＋-ATPase活性均明显低于对照组（P〈0．05）；②与枸橼酸组相比，COM组细胞Ca`2＋-Mg`2＋-ATPase和a`＋＋K`＋-ATPase活性下降更加明显（P〈O．05）；③COM组和枸橼酸组细胞培养基中MDA的含量均明显高于对照组（P〈O．05）；④与枸橼酸组相比，COM组细胞培养基中MDA的含量更高（P〈O．05）。结论外源性枸橼酸能够减轻一水草酸钙晶体对体外培养的Wistar大鼠肾小管上皮细胞造成的脂质过氧化损伤。     

脾原发性淋巴瘤超微结构观察与流式细胞分析

目的 探讨诊断电镜（DEM）对脾原发性淋巴瘤的病理分型的超微结构特征。流式细胞术（FCM）测定瘤细胞DNA倍性与恶性程度的关系。方法 应用透射电镜和流式细胞术观察分析41例脾原发性淋巴瘤的超微结构和瘤细胞DNA倍性。结果41例脾原发性淋巴瘤中霍奇金淋巴瘤5例,电镜下R-S细胞呈双叶不规则核和对称致密大核仁,核内常染色质明显,胞质内见中等量线粒体,较长的粗面内质网。非霍奇金B细胞性淋巴瘤21例,裂细胞性5例,细胞核大,核伴有较深的裂沟,胞浆少,胞质见少数肿胀线粒体,无核裂细胞性7例,细胞核规则,呈圆或卵圆型,异染色质多呈条块状,核仁中等大,胞质少,见少量线粒体及长粗面内质网。混合细胞性,瘤细胞大小悬殊,大畸形核较多,核扭曲,不规则深沟及不对称分叶,形成多叶核。非霍奇金T免疫母细胞淋巴瘤11例,瘤细胞较大,核小胞质相对多,胞质透明,核多形性,有的呈圆或卵圆形,核膜薄,有深浅不一的凹陷,可见小核仁。淋巴母细胞性5例,淋巴母曲核性淋巴瘤,细胞为中小形,呈锯齿状核和扭曲状核,异染色质沿核膜聚集,核仁小。毛细胞性淋巴瘤1例,毛细胞的绒毛宽基底,核仁大,胞浆无核糖体板层复合物。组织细胞性淋巴瘤3例,细胞浆丰富,核圆或椭圆形,偶见核膜呈锯齿状或折叠,线粒体和溶酶体丰富,无细胞连接和Birbeck颗粒。FCM     

The CLDN5 locus may be involved in the vulnerability to schizophrenia.

The present study was designed to detect three single nucleotide polymorphisms (SNPs) located on 22q11 that was thought as being of particularly importance for genetic research into schizophrenia. We recruited a total of 176 Chinese family trios of Han descent, consisting of mothers, fathers and affected offspring with schizophrenia for the genetic analysis. The transmission disequilibrium test (TDT) showed that of three SNPs, rs10314 in the 3'-untranslated region of the CLDN5 locus was associated with schizophrenia (chi(2) = 4.75, P = 0.029). The other two SNPs, rs1548359 present in the CDC45L locus centromeric of rs10314 and rs739371 in the 5'-flanking region of the CLDN5 locus, did not show such an association. The global chi-square (chi(2)) test showed that the 3-SNP haplotype system was not associated with schizophrenia although the 1-df test for individual haplotypes showed that the rs1548359(C)-rs10314(G)-rs739371(C) haplotype was excessively non-transmitted (chi(2) = 5.32, P = 0.02). Because the claudin proteins are a major component for barrier-forming tight junctions that could play a crucial role in response to changing natural, physiological and pathological conditions, the CLDN5 association with schizophrenia may be an important clue leading to look into a meeting point of genetic and environmental factors.