外周血单个核细胞中乙型肝炎病毒核酸与细胞因子含量变化

目的：探讨慢性乙型肝炎患者外周血单个核细胞中乙型肝炎病毒核酸与血清细胞因子变化关系。方法：采用荧光实时PCR技术定量检测76例慢性乙型肝炎患者和15例健康者外周血单个核细胞中HBVDNA含量，用ELISA法定量检测血浆细胞因子(IFN-γ、TNF—α、IL-4、IL-6、TGF-β1、sIL-2R)水平。结果：①慢性乙型病毒性肝炎中细胞因子(IFN-γ、TNF-α、IL-4、IL-6、TGF-β1、sIL-2R)水平明显高于健康对照组；②单个核细胞HBVDNA阳性组IFN-γ、TNF-α含量明显低于阴性组，而sIL-2R含量则高于阴性组但无统计学差异，IL-4、IL-6、TGF-β1含量无差异；③随着PBMCs中HBVDNA含量升高，TNF—α含量逐渐降低，IL-4和sIL-2R含量逐渐升高，而IFN-γ，IL-6、TGF-β1含量无显著性变化。结论：外周血单个核细胞HBVDNA持续存在及含量变化与细胞因子相对异常有关，进而导致肝细胞损伤。     

外周血单个核细胞中乙型肝炎病毒感染的研究进展

乙型肝炎病毒（ＨＢＶ）感染乙肝（ＨＢ）患者外周血单个核细胞（ＰＢＭＣｓ），ＨＢＶ－ＤＮＡ以游离和整合型两种形式存在于ＰＢＭＣｓ内。在ＰＢＭＣｓ内复制与表达。研究ＰＢＭＣｓ内ＨＢＶ感染对于探讨ＨＢ的临床进程、治愈和指导治疗等均具有重要的意义。最近提出了一种与上述相反的观点，ＰＢＭＣｓ内有关的ＨＢＶ－ＤＮＡ和ＲＮＡ是吸附的结果，而非病毒的复制。     

2型糖尿病肾病患者血清及尿液PEDF水平及意义

目的：本研究通过观察色素上皮衍生因子( pigment epithelium derived factor,PEDF)在2型糖尿病肾病不同时期患者血清及尿液中的表达水平,探讨PEDF与糖尿病肾病(diabetic nephropathy,DN)病情进展的关联,进一步研究血清及尿液PEDF检测的临床意义。方法：选择研究对象共120例,分为健康对照组(n=30)、单纯糖尿病组(2型糖尿病、非糖尿病肾病组n=30),糖尿病肾病Ⅲ期(微量蛋白尿组、n=30)、Ⅳ期患者(临床蛋白尿组、n=30),采用酶联免疫(enzyme-linked immuno sorbent assay,ELISA)法检测空腹血清PEDF,尿液PEDF水平,静脉空腹血糖(fasting plasma glucose,FPG)、三酰甘油(triacylglycerol,TG)、总胆固醇(total cholesterol,TC)、低密度脂蛋白-胆固醇(low density lipoprotein,LDL-C)、糖化血红蛋白(glycosylated hemoglobin,HbA1c)、血肌酐(serum creatinine, sCr)、尿素氮(blood urea nitrogen,BUN)、24小时尿白蛋白(24 h-urinary albumin excretion,24hUAlb),计算尿白蛋白排泄率(urinary albumin excretion ratio,UAER)。结果：健康对照组、单纯糖尿病组、微量蛋白尿组、临床蛋白尿组患者血清中PEDF水平逐渐增高(5.51±0.44、6.27±0.52、8.85±0.68、12.44±0.87μg/mL),各组间有显著性差异(F=13.07,P<0.05)。各组尿液PEDF水平逐渐增高(4.98±0.21、5.20±0.18、6.29±0.46、8.63±0.85μg/mL),各组间差异有统计学意义(F=20.52, P<0.01)。尿白蛋白排泄率与血清PEDF含量成正相关(r=0.880,P<0.01);与尿液PEDF含量成正相关(r=0.809,P<0.01)。结论：本研究观察到不同时期糖尿病肾病患者血清及尿液PEDF水平随着病情进展逐渐升高,提示PEDF在DN的发生发展中可能起着重要作用,联合检测血清PEDF和尿液PEDF水平对糖尿病肾病早期诊断可能有重要意义。     

慢性乙型肝炎患者外周血单个核细胞基因表达谱的研究

目的 分析慢性乙型肝炎患者外周血单个核细胞差异表达基因，探索慢性乙型肝炎形成的分子机制。方法 应用含14000条人体cDNA的微阵列芯片和来自外周血单个核细胞的标记cDNA，分析了10例慢性乙型肝炎患者和10例健康人基因表达谱。通过应用GenePix 4000B扫描仪和ImaGene3．0分析软件比较cy5标记的慢性乙型肝炎来源cDNA与Cy3标记的健康人来源cDNA的杂交结果，获得个体基因的相对表达比值。结果 在分析的14000条基因中，差异表达的基因有92条，占0．66％。其中51条基因表达水平显著上调，41条基因表达水平显著下调。这些差异表达的基因主要为细胞信号转导，细胞周期和代谢，凋亡及炎症相关类基因。结论 在乙型肝炎病毒致慢性乙型肝炎过程中，涉及到了众多基因的差异表达，为进一步阐明慢性乙型肝炎形成的分子机制提供基础。     

系统性红斑狼疮患者外周血单个核细胞中急性期蛋白反应因子的表达

目的　探讨系统性红斑狼疮 (SLE)患者外周血单个核细胞 (PBMC)中急性期蛋白反应因子 (APRF)的活性水平 ,以及IL 6和IL 10对APRF表达的影响。方法　采用凝胶阻滞电泳 (EMSA)的方法检测 4 0例SLE患者及 2 0例正常对照组PBMC中DNA结合蛋白APRF的表达水平。结果　所有活动期SLE患者均出现APRF电泳条带 ,17例非活动期SLE患者中有 10例出现APRF条带 ,而正常人对照组无 1例出现。 7例未出现APRF电泳条带的非活动期SLE患者PBMC加IL 10处理后均出现不同程度的APRF表达 ,而加IL 6处理时仍未出现APRF电泳条带。结论　SLE患者存在APRF的异常表达。在SLE中 ,IL 10信号转导途径中的某些调控机制 (如蛋白激酶 )可能发生改变 ,从而使得核内的APRF激活转录 ,提示IL 10很可能是通过APRF在SLE的发病机制中起作用 ,相反IL 6在SLE发病的作用机制很可能与APRF无关。     

外周血单个核细胞与HepG2.215细胞共培养体系的研究

目的建立外周血单个核细胞与HepG2.215细胞共培养体系,探讨不同培养基及效靶比对共培养体系的影响。方法分离正常人外周血单个核细胞,加入植物血凝素（PHA）,置于不同的培养基（DMEM、RPMI1640）中进行培养,采用不同效靶比（5∶1、10∶1、20∶1、40∶1）构建PBMCs与HepG2.215细胞共培养体系。用倒置显微镜观察细胞的形态及生长情况,台盼蓝拒染法检测PBMCs与HepG2.215细胞的细胞活力,cck-8法检测HepG2.215细胞的增殖活性。结果 DMEM培养基培养的HepG2.215细胞的细胞活力比RPMI1640培养基培养的细胞高;而两种培养基培养的PBMCs的细胞活力无明显变化。共培养条件下,PBMCs对HepG2.215细胞增殖活性的抑制作用随效靶比的不同而有所差别,效靶比为20∶1时抑制作用最强。结论在PBMCs与HepG2.215细胞共培养体系中,细胞培养基和效靶比对HepG2.215细胞的生长有影响。     

哮喘患者外周血单个核细胞IL-5、IL-3 mRNA表达的研究

嗜酸细胞（ＥＯＳ）是哮喘慢性炎症中的关键效应细胞。ＩＬ５、ＩＬ３能促进ＥＯＳ在骨髓的分化、成熟,使机体的ＥＯＳ产生增多,并促使ＥＯＳ在支气管肺组织的聚集及活化。哮喘患者外周血、支气管粘膜、支气管肺泡灌洗液（ＢＡＬＦ）中ＩＬ５明显升高［１］。ＩＬ３对ＥＯＳ的作用较ＩＬ５弱,它在哮喘发病机理中的作用仍有争议。本文通过对哮喘患者ＰＢＭＣＩＬ５ｍＲＮＡ、ＩＬ３ｍＲＮＡ表达水平的研究,探讨其在哮喘发病中的作用。１　材料与方法１．１　研究对象　哮喘组：哮喘患者１３例,男８例,女５例,平均年龄３…     

乳癌患者外周血及肿瘤局部单个核细胞亚群分析

对13例女性乳癌患者外周血MNC(半个核细胞)与15名正常女性比较时发现,乳癌患者血中Leul~+细胞及DR~+细胞较低(P<0.05,P<0.01),Leu2a~+细胞较高(P<0.01),Leu3a/Leu2a比值下降(P<0.001),从数量上反映患者机体细胞免疫功能紊乱,并发现可能存在的Leu3a~+、Leu2a~+双标记细胞增多的现象,也可能与免疫抑制有关。DR~+细胞的减少由何引起有待于进一步研究。配对资料分析表明:癌周间质MNC亚群与血中MNC各亚群水平相近(Leu7、Leullb细胞除外),而癌间间质则显示较低的MNC水平。     

Graves''甲亢患者外周血单个核细胞糖皮质激素受体表达类型的变化

目的：为检测Graves‘甲亢病人外周血单个核细胞hGRα/GRβ表达及影响因素，探讨糖皮质激素及其受体在该疾病中的作用。方法：本研究用半定量RT－PCR方法检测正常人及Graves‘甲亢病人外周血单个核细hGRα/GRβ，同时测定了晨8：00皮质醇、甲功水平，用多元回归分析法探讨了它们之间的关系。结果：该研究提示Graves甲亢组的hGRα/GRβmRNA比值与TT3、TT4相关（P＜0．05），相关系数分别为：0．61和 0．54。Graves‘甲亢组的血皮质醇明显高于正常组。TT3和TT4与皮质醇不相关。皮质醇与hGRα/GRβmRNA比值不相关。结论：Graves‘甲亢病人外周血单个核细胞hGRα/GRβ表达增高。     

2型糖尿病合并颈动脉粥样硬化患者外周血单个核细胞凋亡相关斑点样蛋白mRNA的表达

目的:分析2型糖尿病(T2DM)合并颈动脉粥样硬化(CAS)患者外周血单个核细胞(PBMCs)中衔接蛋白凋亡相关斑点样蛋白(ASC)mRNA的表达及作用。方法:根据华盛顿大学CAS斑块超声分级标准将107例T2DM患者分为单纯T2DM组(n=26)、T2DM轻度斑块组(n=32)、T2DM中度斑块组(n=38)和T2DM重度斑块组(n=11)。另选非T2DM的CAS患者作为单纯CAS组(n=35),以及体检健康者作为正常对照组(n=35)。采用实时荧光定量聚合酶链反应(RT-FQ-PCR)技术检测各组PBMCs中ASC mRNA表达水平。统计分析各组差异及ASC mRNA水平与CAS斑块严重程度的关系。结果:各病例组ASC mRNA表达水平显著高于正常对照组(P0.05),T2DM合并CAS组ASC mRNA表达水平显著高于单纯CAS组(P0.01);ASC mRNA表达水平轻度斑块组中度斑块组重度斑块组,即ASC mRNA表达水平与T2DM患者CAS斑块程度呈明显负相关(r=-0.43,P0.05)。结论:ASC mRNA表达增加可能是T2DM合并AS的发病因素和CAS斑块活跃性的指征。     

色素上皮衍生因子和血管内皮生长因子在糖尿病大鼠肾脏的不平衡表达

目的检测色素上皮衍生因子（PEDF）和血管内皮生长因子（VEGF）在糖尿病大鼠肾脏的表达，探讨其在糖尿病肾病（DN）发生发展中的作用。方法40只雄性Wistar大鼠随机分为：正常对照组（NC）、糖尿病模型组（DM）。4、8周末，检测血糖、糖化血红蛋白、肾重指数、尿白蛋白排泄率，免疫组织化学法检测肾脏PEDF、VEGF的表达。结果4、8周末，DM组大鼠肾脏PEDF的表达较NC组明显减少（P〈0．01）；VEGF的表达较NC组明显增多（P〈0．01）；肾脏PEDF的表达与VEGF明显负相关（r=-0．823，P〈0．01）。结论PEDF和VEGF在肾脏的不平衡表达可能参与了DN的发病。     

2型糖尿病视网膜病变患者血清炎症因子和脂联素水平的变化

目的: 观察2型糖尿病合并视网膜病变(DR)患者血清炎症因子和脂联素的变化。方法: 110例糖尿病患者分为3组:糖尿病无视网膜病变组(DM)35例、糖尿病伴非增殖期视网膜病变组(NPDR)45例和糖尿病伴增殖期视网膜病变组(PDR)30例,并与40名正常人对照(NC组)。观察患者的体检指标,并检测空腹血糖(FPG)、糖化血红蛋白(HbA1c)、空腹胰岛素(FINS)、餐后2 h血糖(2hPG),总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、脂联素、血清细胞间黏附分子-1(ICAM-1)、肿瘤坏死因子-α(TNF-α)和高敏C-反应蛋白(hs-CRP)。计算稳态模型评估的胰岛素抵抗指数(HOMA-IR)。结果: DM组、NPDR组和PDR组患者的收缩压、体重指数、腰臀比、血清TG、LDL-C、FPG、2hPG、 HbA1c、ICAM-1、TNF-α、hs-CRP水平和HOMA-IR均高于NC组(P<0.05),而NPDR组和PDR组的收缩压、血清ICAM-1、TNF-α、hs-CRP水平和HOMA-IR均高于DM组(P<0.05)。DM组、NPDR组及PDR组的患者血清脂联素水平均低于NC组(P<0.05),而NPDR组及PDR组的患者血清脂联素水平低于DM组患者(P<0.05)。血清脂联素水平与ICAM-1、TNF-α、hs-CRP和HOMA-IR之间呈负相关(r值分别为-0.735、-0.781、-0.768、-0.752,均P<0.01),HOMA-IR与ICAM-1、TNF-α和hs-CRP之间呈正相关(r值分别为0.857、0.906、0.888,均P<0.01)。结论: 炎症因子和脂联素参与了DR的发生和发展,而脂联素可能通过拮抗炎症反应减轻胰岛素抵抗,对DR有一定的改善作用。     

增生型糖尿病视网膜病变患者血清及玻璃体中IL-19和VEGF表达水平的研究

目的探讨增生型糖尿病视网膜病变(proliferative diabetic retinopathy,PDR)患者血清及玻璃体中白细胞介素-19(interleukin-19,IL-19)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达及临床意义。方法选取37例PDR患者和40例孔源性视网膜脱离(rhegmatogenous retinal detachment,RRD)患者分别作为实验组和对照组,采用酶联免疫吸附试验(ELISA)检测2组患者血清和玻璃体液中IL-19、VEGF的含量,PDR组患者检测空腹血糖(fasting blood glucose,FBG)、糖化血红蛋白(glycosylatedhemoglobin,Hb Alc)水平。结果 PDR组患者血清和玻璃体液中的IL-19和VEGF水平均显著高于对照组,差异均有统计学意义(t=8.747、16.437;t=10.417、14.978,均P<0.01),且两者之间呈正相关关系(r=0.394,P<0.05;r=0.574,P<0.01)。PDR组患者玻璃体液中IL-19和VEGF水平均高于血清水平,差异均有统计学意义(均P<0.05)。PDR组玻璃体中VEGF表达水平与FBG、Hb Alc均呈正相关性(r=0.361、0.672,均P<0.05);PDR组患者IL-19表达水平与糖尿病病程、FBG、Hb Alc均无明显相关性(均P>0.05)。结论 IL-19和VEGF在PDR的发病机制中扮演着重要角色;IL-19与VEGF正相关,可能参与了PDR新生血管病变的发生发展,有望为PDR的治疗带来更多的靶点。     

Expression of BAFF and BR3 in patients with systemic lupus erythematosus

The objective of this study was to examine the relationship between the expression ofB cell activating factor (BAFF) and BAFF receptor in patients with disease activityof systemic lupus erythematosus (SLE). Real-time RT-PCR was used to examine BAFF mRNAexpression in peripheral blood monocytes of active and stable SLE patients andhealthy controls. The percentage of BAFF receptor 3 (BR3) on B lymphocytes wasmeasured by flow cytometry. Soluble BAFF levels in serum were assayed by ELISA.Microalbumin levels were assayed by an automatic immune analysis machine. BAFF mRNAand soluble BAFF levels were highest in the active SLE group, followed by the stableSLE group, and controls (P<0.01). The percentage of BR3 on B lymphocytes wasdownregulated in the active SLE group compared with the stable SLE group and controls(P<0.01). BAFF mRNA levels and soluble BAFF levels were higher in patients whowere positive for proteinuria than in those who were negative (P<0.01). Thepercentage of BR3 on B lymphocytes was lower in patients who were positive forproteinuria than in those who were negative (P<0.01). The BAFF/BR3 axis may beover-activated in SLE patients. BAFF and BR3 levels may be useful parameters forevaluating treatment.     

Urinary 6-sulfatoxymelatonin level in diabetic retinopathy patients with type 2 diabetes

Melatonin is a powerful antioxidant. Decreased melatonin excretion has been reported to be associated with several oxidative stress-related diseases. The urinary metabolite of melatonin, 6-sulfatoxymelatonin (aMT6s), has proved to be a very reliable index of melatonin production. The present study aims to evaluate the level of urinary aMT6s in patients with type 2 diabetes mellitus and diabetic retinopathy. Urine samples were collected from 10 patients with diabetes and no diabetic retinopathy (NDR), 19 patients with nonproliferative diabetic retinopathy (NPDR), 38 patients with proliferative diabetic retinopathy (PDR), and 16 subjects without diabetes mellitus, who served as controls. The level of aMT6s in specimens was assayed by a commercial aMT6s ELISA kit, creatinine levels were also measured for each sample to get urinary aMT6s/creatinine ratio. Creatinine-adjusted urinary aMT6s values were compared among four groups. The urinary aMT6s (mean ± SD) levels were 9.95 ± 2.42, 9.90 ± 2.28, 8.40 ± 1.84 and 5.58 ± 1.33 ng/mg creatinine in the controls and in patients with NDR, NPDR, or PDR, respectively. The urinary aMT6s level of the PDR group was significantly lower than that of the control, NDR and DR groups. No significant difference was found among the control, NDR and DR groups. After adjustment for various factors (age, smoking, cancer, and coronary heart disease) that may influence the aMT6s level, the odds-ratio of urinary aMT6s comparing PDR patients to controls was 0.246 (95% confidence interval = 0.108-0.558, P = 0.001). Therefore, the urinary aMT6s level is significantly decreased in diabetic patients with PDR but not in diabetic patients without PDR, which indicates that decreased urinary aMT6s level may be associated with the pathogenesis of PDR.     

糖尿病视网膜病变早期血管内皮生长因子作用机制的研究

目的:通过链脲佐菌素(streptozocin,STZ)糖尿病大鼠视网膜的石蜡切片及血管铺片,研究糖尿病大鼠视网膜病变时血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)作用的分子病理机制。方法:建立糖尿病大鼠模型,随机分为正常对照组(C),糖尿病1月组(M₁)、3月组(M₃)、5月组(M₅)。分别在视网膜石蜡切片及血管铺片上行VEGF原位杂交和免疫组化。结果:①石蜡切片:原位杂交仅M₅表达为67%;免疫组化M₃表达为34%,M₅表达为89%。②视网膜血管铺片:原位杂交仅M₅表达为34%;免疫组化仅M₅表达为56%。结论:①除内皮细胞外,周细胞及Müller细胞也可产生VEGF;②糖尿病视网膜病变早期VEGF可能是来源于旁分泌途径。     

Determination of foveal avascular zone in diabetic retinopathy digital fundus images

Monitoring FAZ area enlargement enables physicians to monitor progression of the DR. At present, it is difficult to discern the FAZ area and to measure its enlargement in an objective manner using digital fundus images. A semi-automated approach for determination of FAZ using color images has been developed. Here, a binary map of retinal blood vessels is computer generated from the digital fundus image to determine vessel ends and pathologies surrounding FAZ for area analysis. The proposed method is found to achieve accuracies from 66.67% to 98.69% compared to accuracies of 18.13-95.07% obtained by manual segmentation of FAZ regions from digital fundus images.     

Increased protein carbonylation of red blood cell membrane in diabetic retinopathy

We investigated the protein carbonylation of red blood cell (RBC) membrane in type 2 diabetic patients and the potential implication of carbonyl/oxidative stress in reflecting disease severity. Sixty-four diabetic patients with or without retinopathy of variable clinical severity (Groups DR and DM, respectively) and 20 healthy controls were included in the study. Protein carbonyls were determined in RBC membranes by immunoblotting. Compared to healthy volunteers, the RBC membranes of diabetic patients were characterized by significantly increased levels of carbonylated proteins. The carbonylation of Group DR was higher compared to that of Group DM. The subgroup of patients with proliferative retinopathy exhibited a trend towards a significant increase in protein carbonyls, compared to both free-of-retinopathy diabetic cases and non-proliferative diabetic retinopathy cases. The correlation between the chemical modifications of the erythrocyte membrane proteins and the clinical severity of diabetic retinopathy suggests a potential utility of membrane carbonylation as a marker and risk factor in the development of retinopathy.     

Astaxanthin inhibits oxidative stress and apoptosis in diabetic retinopathy

BackgroundThe pathophysiology of diabetic retinopathy (DR) is thought to be influenced by oxidative stress. Astaxanthin (ASX) is a natural product with antioxidant effect, but it is not clear whether its mechanism of inhibiting the development of DR is related to anti-oxidation.MethodsRats were intraperitoneally injected with streptozotocin (60 mg/kg) to create DR rat models followed by ASX (20 mg/kg) for 45 days. Retinal tissue was examined by Hematoxylin and Eosin staining. By using Enzyme-linked immunosorbent assay (ELISA), 2,7-Dichlorodrhydrofluorescein diace (DCFH-DA) probes, immunohistochemistry and western blot, it was feasible to evaluate the contents of inflammation-related factors (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and macrophage inhibitory cytokine-1 (MIC-1)), oxidative stress-related indicators (glutathione (GSH), malonic dialdehyde (MDA), glutathione peroxidase (GPx), reactive oxygen species (ROS) and Total antioxidant capacity (T-AOC)), antioxidant enzymes (hemoxgenase-1(HO-1) and Quinone Oxidoreductase 1 (NQO1)), and apoptosis-related proteins (Bcl-2, Bcl2 Associated X Protein (BAX), and cleaved-caspase-3). Additionally, antioxidant proteins downstream of the nuclear factor E2 related factors (Nrf-2) pathway, expression levels of Nrf2/ Kelch-like ECH-associated protein 1(Keap 1) pathway-associated proteins, and nuclear and cytoplasmic levels of Nrf2 were assessed using immunohistochemistry, western blot, or quantitative real-time polymerase chain reaction (qRT-PCR).ResultsASX alleviated retinal tissue damage by increasing overall retina thickness and ganglion cell layer (GCL) cell numbers and exerted the anti-inflammatory, anti-oxidative stress, and anti-apoptosis effects in DR rats. Additionally, ASX could inhibit the expression of Keap1, promote the transport of Nrf2 from cytoplasm to nucleus and facilitate the expressions of HO-1, NQO1, γ-glutamylcysteine synthetase, (γ-GCS) and GPx.ConclusionASX exerted antioxidant effects through Nrf2/keap1 pathway, thereby alleviating apoptosis, inflammation, and oxidative stress in retinal tissues of DR rats.