OPTIMIZATIONS FOR 5-AMINOLEVULINIC ACID BASED PHOTODYNAMIC THERAPY IN PURGING LEUKEMIA CELL HL60

Photodynamictherapy(PDT),whichinvolves administrationofatumorlocalizing photosensiti zingagentthatmayrequiremetabolizingsynthesis(i.e.,aprodrug),followedbyactivationofthea gentbylightofaspecificwavelength.Thistherapy resultsinasequenceofphotochemicalandph…     

CpG寡核苷酸对人外周血单个核细胞的免疫刺激作用

目的　观察CpGODN、GM CSF、CD40 L对正常人外周血单个核细胞 (PBMC)的免疫刺激作用 ,以期筛选出一种高效的疫苗佐剂。方法　用CpGODN 2 0 0 6、GM CSF、CD40 L刺激正常人PBMC ,48h后采用ELISA法检测培养上清液中IFN γ、IL 12及IL 10水平 ;应用免疫荧光法检测PBMC上CD40、CD86、HLA DR的表达。结果　CpGODN 2 0 0 6能诱导正常人PBMC分泌较高水平的IL 12、IFN γ ,抑制IL 10的分泌 ;上调PBMCCD40、CD86、HLA DR表达的作用最强。结论　CpGODN 2 0 0 6是一种免疫刺激作用强大的新型佐剂 ,可用于多种疫苗的制备。     

Methylation status of the interferon-gamma gene promoter in chronic hepatitis B

Objective To evaluate the methylation status at CpG site -55 in the interferon-gamma (IFN-7) gene promoter and its effect on IFN-7 expression in chronic hepatitis B. Method The authors recruited 30 patients with UBeAg-positive chronic hepatitis B (CHB), 30 HBeAg-negative CHB patients, and 30 healthy blood donors. Pyrosequeneing was used to determine the methylation status at CpG site -55 in the IFN-γ gene promoter following bisulfite treatment of DNA in peripheral blood mononuclear cells (PBMCs). The expression of IFN-γ was analyzed by real-time RT-PCR and ELISA. HBV DNA in PBMCs was detected by nested PCR. Results The methylation level at CpG site -55 in the IFN-γ gene promoter was significantly increased, resulting in subsequent down-regulation of the expression of this cytoldne in CHB. The methylation level at CpG site -55 was significantly higher in HBeAg-positive patients than in HBeAg-negative ones (P<0.01) and was also significantly higher in PBMCs from HBV DNA-positive patients than from HBV DNA-negative ones (P<0.01) ; the methylation level at CpG site -55 was positively correlated with the amount of HBV DNA in serum (P<0.01). Oonclusion IFN-γ gene expression appears to be regulated by methylation of the IFN-γ gene promoter in CHB; the methylation level at CpG site -55 is associated with HBV infection.     

胎盘、脐血和骨髓来源的贴壁细胞的生物学特性的比较

目的探讨人胎盘、脐血和骨髓来源的贴壁细胞的分离培养和生物学特性,为间充质干细胞(MSC)的选择和应用提供依据。方法采用酶消化法分离人胎盘组织,60 g/L羟乙基淀粉和密度梯度离心两步法分离脐血单个核细胞,密度梯度离心法分离骨髓单个核细胞,分别进行贴壁培养,观察不同来源MSC的生长、增殖和表面标志的表达并做比较。结果胎盘来源的贴壁细胞为一种混合性细胞,脐血单个核细胞体外培养可以获得形态不均一的贴壁细胞,二者在生长规律和形态学特征上与骨髓基质相比有一定差异性,CD106、CD44在三种细胞均有表达。结论胎盘和脐血来源的贴壁细胞具备间充质干细胞的基本特征。     

自体外周血纯化CD34+细胞移植对系统性红斑狼疮患者外周血CD4+CD25+T细胞表达的影响

目的研究自体外周血纯化造血干细胞移植对系统性红斑狼疮(SLE)患者CD4 CD25 T细胞表达的影响。方法15例接受自体外周血纯化造血干细胞移植的SLE患者列入研究,应用流式细胞技术动态监测移植前及移植后3、6、12、24个月的CD4 CD25 T细胞表达水平。结果自体外周血造血干细胞移植治疗前SLE患者外周血的CD4 CD25 T细胞表达明显低下,移植后3个月出现增高,移植6、12、24个月后达正常水平。复发者外周血CD4 CD25 T细胞的表达水平明显低于非复发者,差异有统计学意义(P<0.01)。结论自体外周血纯化CD34 细胞移植可通过促进CD4 CD25 T细胞的表达发挥治疗作用。     

Toll样受体4在佐剂性关节炎模型大鼠外周血单核细胞中的表达及意义

目的研究佛氏完全佐剂性关节炎(AIA)大鼠外周血单核细胞(PBMC)对Toll样受体4(Toll-like receptor 4,TLR4)、TLR4 mRNA的表达及其与关节炎评分的关系。方法流式细胞仪测定CD14 TLR4 细胞占CD14 细胞的比例和TLR4-PE的平均荧光强度(MIF);Northern blot法测定PBMC TLR4 mRNA的表达量。比较造模后15 d与正常对照组和造模后25 d时CD14 TLR4 /CD14 、MIF及其TLR4 mRNA表达量的差异,并用Mann-WhiteyU检验分析关节炎评分与TLR4 MIF的相关性。结果在造模后15 d,CD14 TLR4 /CD14 、TLR4 MIF和PB-MC TLR4 mRNA的表达较正常对照组和造模后25 d明显增高(P<0.01),而且TLR4 MIF与关节炎的评分有明显的相关性(15 d:r=0.826,P<0.01;25 d:r=0.688,P<0.05)。但造模后25 d,CD14 TLR4 /CD14 、TLR4 MIF和PBMC TLR4 mRNA的表达与正常对照组和造模前相比无统计学意义(P>0.05)。结论TLR4与AIA发病有密切关系,提示固有免疫反应在类风湿性关节炎(RA)及其他自身免疫性疾病的发病中有重要的作用。     

乙型肝炎病毒对T细胞亚群的侵犯及对其功能的影响

以单克隆抗体补作溶解法分离T4、T8细胞亚群，PCR检测65例各型乙肝患者T细胞亚群和PBMCs中的HBVDNA，结合细胞免疫功能改变探讨HBV侵犯免疫细胞对其功能的影响。结果表明：PBMCs中HBVDNA阳性24例（24／65，36．92％），不同类型乙肝患者阳性率差异较大；T4、T8、细胞亚群中HBVDNA阳性分别为16例和21例，HBVDNA阳性组IL2R表达明显降低，抑制细胞功能有不同程度的下降；提示HBV对T8细胞可能更具亲嗜性，HBV对免疫细胞的侵犯是乙肝患者细胞免疫功能紊乱的原因之一。     

硒对慢性肝病患者外周血单个核细胞膜流动性的影响

目的　探讨硒对慢性肝病患者外周血单个核细胞 (PBMC)膜流动性的影响。方法　分离健康人和慢性肝病患者的PBMC ,分别体外培养 ,对比观察了预加硒 ( 1.15 6× 10 -7mol·L-1)和不同剂量脂质过氧化诱导剂叔丁基过氧化氢 (tBHP)作用后各组PBMC膜流动性和培养液中过氧化脂质 (MDA)含量变化。结果　健康人PBMC膜流动性随tBHP剂量的增加而降低 ,培养液中MDA含量呈相反变化 ;慢性肝病患者PBMC膜流动性明显下降 ,培养液中MDA含量增加。而经硒预保护作用 6h后两组细胞上述指标均有改善。结论　脂质过氧化反应可影响人PBMC的膜流动性 ,硒对此具有保护作用。     

抗人CD3单链抗体四聚体基因在HeLa细胞中的表达和活性鉴定

目的　将抗人CD3单链抗体 (scFv) /人 p5 3四聚功能域融合基因转染到HeLa细胞 ,进行真核表达和活性测定。方法　将抗人CD3scFv /人 p5 3四聚功能域融合基因克隆入真核分泌表达载体 pSecTag2 B中 ,转染HeLa细胞进行表达 ,表达产物纯化后利用流式细胞仪进行活性测定。结果　表达产物经SDS PAGE和Westernblot证实 ,为Mr约35 0 0 0的特异蛋白条带。纯化后经流式细胞仪检测 ,可特异性地结合人外周血单个核细胞 (PBMCs) ,亲和力高于scFv。结论　获得了可与PBMCs特异性结合的抗人CD3scFv四聚体 ,为进一步临床应用奠定了基础     

蛋白印迹法与免疫荧光法检测白血病患者Survivin表达

目的　比较蛋白印迹法和免疫荧光法检测Survivin表达结果,以探寻临床上切实可行的检测方法。方法　应用蛋白印迹(Western blots)和免疫荧光法检测18例白血病患者和10例正常对照组外周血中单个核细胞Survivin的表达。结果　Western blots 法检测Survivin 表达阳性13 例(72. 2%);免疫荧光法检测Survivin 表达阳性11 例(61.1%)。两种方法检测10例正常对照组外周血单个核细胞中Survivin的表达均为阴性。两种检测方法的符合率为88.89%。结论　Survivin是否表达可能成为白血病临床诊断、预后判断的有效指标。两种检测方法具有良好的一致性,免疫荧光法操作比较简单,适于在临床上应用。     

白血病患者FasL的表达及功能研究

目的　观察白血病细胞表面FasL的表达水平及其是否具有功能性 ,探讨FasL是否在白血病的免疫逃逸中发挥作用。方法　通过链酶亲和素 碱性磷酸酶免疫组织化学法检测 10例正常人及 4 8例白血病患者外周血单个核细胞FasL的表达水平 ;将白血病细胞与Jurkat细胞混合培养 2 4h后 ,用DNA电泳法及MTT法从定性和定量两方面检测Jurkat细胞发生凋亡的情况。结果　FasL在白血病细胞膜中较正常人高表达 ,AML及ALL耐药未缓解者FasL表达水平明显高于缓解及部分缓解者 ,随着CML病情进展 ,白血病细胞膜FasL表达率升高。三种不同比例的效应细胞与靶细胞混合培养 ,效应细胞数越高 ,对靶细胞杀伤越明显。结论　FasL在白血病细胞表面高表达且对Fas阳性的敏感细胞具有杀伤功能 ,推测Fas/FasL途径可能在白血病的免疫逃逸中发挥作用。     

PCR法检测乙型肝炎患者外周血单个核细胞中HBV DNA

采用ＰＣＲ法检测了７７例乙肝患者外周血单个核细胞（ＰＢＭＣ）中ＨＢＶＤＮＡ，２７例阳性，总阳性率为３５．１％。慢性活动性肝炎（９／２１，４２．９％），肝炎后肝硬化（９／１９，４７．４％）及重型肝炎（８／１６，５０．０％）患者ＰＢＭＣＨＢＶＤＮＡ阳性率明显高于慢迁肝（１／１４，７．１％）和急肝（０／７）患者。结果提示ＰＢＭＣ中ＨＢＶＤＮＡ的存在在乙肝发病机理中具有一定意义。     

STUDY ON DIFFERENTIATION OF RATS EMBRYONIC STEM CELLS CULTURED IN BRL-CM INTO NEURAL PRECURSOR CELLS

Embryonicstem (ES)cellsrefertothosesepa ratedfromtheinteriorcellmassthathasnotyetim bedded .Thesetotipotentialcellsareinhighundif ferentiatedstate ,canbecultivatedandproliferateinvitro[1] .Underinductioninvitro ,EScellscanbedifferentiatedintocellsthatbelongtothreeembry oniclayers[2 ] ,suchasmyocardiumcells ,bloodcellsandneuralcells .GeneticoperationscanbedonewithoutlosingfeaturesoftheEScells.Thereforeithasbecomethemaintoolintheup to datedcelltherapy .Becausetheyarelikelytoformteratomawhentr…     

雪莲黄酮总甙对人外周血单个核细胞功能的影响

研究了雪莲黄酮总甙对人外用血单个核细胞（PBMC）功能的影响，结果发现雪莲黄酮单独应用可诱导PBMC细胞毒活性，其浓度在0．6μg／ml时，作用最明显（p＜0．05）。且可显著增强亚适剂量HrIL－2诱导的PBMC的细胞毒活性，而对大剂量IL－2的诱导作用没有影响。     

Increased expression of the 20S proteasome in peripheral blood mononuclear cells of type 2 diabetic patients

Objective To investigate the dynamic expression of the 20S proteasome in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic patients without vascular complications. Methods PBMCs were prepared from 30 type 2 diabetic patients and 30 nondiabetic controls. The general indexes including weight, height and blood pressure were recorded. Fasting plasma glucose, fasting plasma insulin and glycosylated hemoglobin were measured. The protein level of the 20S proteasome was measured by Western blotting. The mRNA expression levels of the 20S proteasome β1, β2 and β5 subunits were detected by real-time PCR. Results Compared with that in the nondiabetic controls, the protein level of the 20S proteasome was significantly increased in the diabetic patients and was positively associated with glycosylated hemoglobin. Conclusion Type 2 diabetic patients without vascular complications have an increased 20S proteasome expression, the significance of which needs to be explored by further study.     

Foxp3在自身免疫性甲状腺病患者外周血和病变组织中的表达

目的检测自身免疫性甲状腺病(AITD)患者Foxp3的表达,探讨调节性T细胞(Treg)与AITD发病的关系。方法采用Real-time PCR检测AITD患者及健康对照者外周血及甲状腺组织中Foxp3mRNA的表达;免疫组织化学方法检测甲状腺内Foxp3蛋白的表达;Western blot方法检测外周血单个核细胞Foxp3蛋白的表达。结果与对照组相比,AITD患者外周血单核细胞Foxp3mRNA表达显著降低(P<0.05);桥本甲状腺炎(HT)患者外周血单个核细胞Foxp3蛋白表达下降(P<0.05),Graves病(GD)患者外周血单个核细胞Foxp3蛋白表达与对照组相比无差异(P>0.05)。与对照组相比,HT患者甲状腺组织Foxp3mRNA和蛋白表达均升高(P<0.05),而GD患者甲状腺组织Foxp3mRNA和蛋白表达水平与对照组相比无显著性差异(P>0.05)。结论 CD4+CD25+Foxp3+Tregs的减少和/或功能的减退可能是诱导AITD尤其是HT发生的因素。     

丹红注射液对U937细胞SR-AⅠ及ABCA1 mRNA表达的影响

目的 以人单核细胞株U937细胞为研究对象,观察不同浓度丹红注射液对U937细胞清道夫受体AⅠ(SR-AⅠ)和ATP结合盒转运体A1( ABCA1)mRNA表达的影响,探讨其抗动脉粥样硬化的机制.方法 以100 nmol/L佛波酯诱导U937细胞48 h使其分化为巨噬细胞,再以50 mg/Lox-L DL(氧化低密度脂蛋白)孵育细胞的同时加入不同浓度丹红注射液或其他干预因素处理24 h,用实时荧光定量PCR方法检测其SR-A Ⅰ和ABCA1 mRNA的表达量.结果 与50 mg/L ox-LDL组比较,LXR-a(肝X受体激动剂)(T-090137)干预组U937细胞SR-A ⅠmRNA表达下降、ABCAl mRNA表达升高,但SR-A Ⅰ mRNA的表达无统计学意义(P＞0.05),而ABCAl mRNA的表达有统计学意义(P＜0.01);3.0 mL/L丹红注射液干预组U937细胞SR-A Ⅰ mRNA的表达升高,10.0、30.0、60.0 mL/L丹红注射液于预组U937细胞SR-A Ⅰ mRNA的表达逐渐下降,但各浓度组均无统计学意义(P＞0.05);丹红注射液干预组ABCAl mRNA的表达则呈浓度依赖性升高,但于60.0mL/L浓度时出现表达下降,3.0、10.0、30.0 mL/L干预组有统计学意义(P＜0.05).与LXR-a(肝X受体激动剂)(T-090137)干预组比较,丹红注射液各浓度干预组ABCAl mRNA表达降低,10.0、30.0 mL/L丹红注射液干预组ABCA1 mRNA的表达无统计学意义(P＞0.05).结论 丹红注射液各浓度组对50 mg/Lox-LDL干预的U937细胞SR-A Ⅰ mRNA的表达无影响,而明显增加U937细胞ABCA1 mRNA表达,尤其在浓度10.0、30.0 mL/L时增加ABCA1 mRNA表达明显.增加U937细胞ABCA1 mRNA表达可能是其抗动脉粥样硬化的重要机制.     

Study of the mechanism on the apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase

Objective To investigate the mechanisms of apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase. Methods Human leukemia cell line K562 were exposed to indole-3-acetic acid （IAA） at 20, 40, 60, 80 or 100 mol/L and horseradish peroxidase（HRP） at 1.2 g/mL for varying times. MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect the arrest of cell cycle. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay was used to measure apoptosis. 2, 7-dichlorofluorescin diacetate （DCFH-DA） uptake was measured to determine free radical by confocal microscope. Content of malondiadehyde （MDA） and activity of superoxide dismutase （SOD） were measured by biochemical methods. Results IAA/HRP initiated growth inhibition of K562 cells in a dose- and time-dependent manner. Flow cytometry revealed that cell cycle arrested at G1/G0 after 24 hours treatment. After 72 hours treatment, apoptotic rate of 100 mol/L IAA group increased to 43.9 %, which was 5 times that of control（P〈0.01）. Content of MDA and activity of SOD increased respectively in treatments compared with control. Meanwhile, IAA/HRP stimulated the formation of free radical, which was increased by IAA concentration-dependently. Conclusion The combination of IAAand HRP can inhibit the growth of Human leukemia cell line K562 in vitro by inducing apoptosis which is associated with the increase of free radical. The combination of IAA and HRP might be a promising chemopreventive and chemotherapeutic agent against human leukemia.     

Identification and culture of neural stem cells isolated from adult rat subventricular zone following fluid percussion brain injury

Objective To analyze proliferation and differentiation of glial fibrillary acid protein (GFAP)- and nestin-positive (GFAP+/nestin+) cells isolated from the subventricular zone following fluid percussion brain injury to determine whether GFAP+/nestin+ cells exhibit characteristics of neural stem cells. Methods Male Sprague-Dawley rats, aged 12 weeks and weighing 200-250 g, were randomly and evenly assigned to normal control group and model group. In the model group, a rat model of fluid percussion brain injury was established. Five days later, subventricular zone tissue was resected from each group and made into single cell suspension. After serum-free neural stem cell medium culture and subsequent serum-induced differentiation, cell type, proliferation and differentiation capacities were determined by immunofluorescence staining and flow cytometry. Results At 3-7 days after fluid percussion brain injury, nestin+/GFAP+ cells in the single cell suspension from the model group significantly outnumbered those from the normal control group (P<0.01). In the model group, an increased number of small neurospheres with smooth cell edge and bulged center formed after primary culture, and were clearly visible with the increase of culture time and medium replacement. After several passages, many clonal spheres were obtained, suggesting strong self-proliferatiing capacity. Neurospheres from the model group differentiated into astrocytes, neurons and oligodendrocytes. Conclusion GFAP+/nestin+ cells isolated from the adult rat subventricular zone after fluid percussion brain injury are thought to be neural stem cells because of their self-renewal and multi-differentiation capacities.     

白细胞介素2诱生和检测的实验条件分析

本文对人外周血单个核细胞(PBMC)诱生白细胞介素2(IL-2)及用ConA 激活的小鼠脾细胞检测IL-2活性过程中的几个实验条件进行了摸索,发现1×10~6 PBMC/ml 经10ug ConA/ml 刺激48h 为诱生IL-2的最佳条件,检测IL-2活性的最佳条件是:2.5×10~4每孔检测细胞/0.1ml/与等量样品共同培养24h.作者认为,小鼠脾细胞经10ug ConA/ml 剌激48h 后.大部分转化为淋巴母细胞,适用于IL-2活性的检测,并具有一定的特异性和重复性。