乙型肝炎患者外周血单个核细胞中乙肝病毒前S/S基因片段的检测

1. 资料与方法：(1)研究对象：19例慢性乙型肝炎患者均符合1995年第6次全国病毒性肝炎会议诊断标准,为瑞金医院1998年门诊及住院患者,年龄12～46岁,平均32.79岁,女4例,男15例。同时收集PBMC和配对血清,并记录病程及ALT浓度。(2)分离PBMC及提取DNA：抽取肝素抗凝血7ml,用比重1.077的聚蔗糖－泛影葡胺液分离淋巴细胞,2500r/min,15min,吸出淋巴细胞,用HanKs液将细胞清洗5次,以去除可能残留的血浆DNA,分别收集第5次洗涤液（500μl）及细胞,提取PBMC DNA,DNA溶于500μl TE缓冲液。(3)血清及洗液中DNA的抽提：从500μl PBMC洗液及100μl血清中提取DNA后,分别溶于30μl及20μl TE缓冲液。     

一种改进的大鼠肝星状细胞分离方法

目的 改进大鼠肝星状细胞的分离方法,提高肝星状细胞分离的纯度和活力。方法 SD大鼠在静脉注射1 ml含二氯亚甲基二磷酸盐的脂质体3 d后,用含100 U/ml肝素的D-Hanks液灌注肝脏10～15 min,改用含0.05％胶原酶溶液的D-Hanks液灌注25～30 min,将肝脏细胞悬液置于0.025％胶原酶、0.005％DNase Ⅰ的溶液中振荡消化30 min,细胞悬液过200目筛网,50×g离心2 min去除肝细胞,300×g离心10 min得到肝脏非实质细胞沉淀,将细胞悬浮于Nycodenz使终浓度为11.5％,1400×g离心17 min,吸取Nycodenz上层的细胞即为肝星状细胞、台盼蓝拒染实验鉴定细胞活力,自发荧光、Desmin免疫细胞化学染色鉴定细胞纯度,内源性过氧化物酶染色检测库普弗细胞。结果 肝星状细胞得率每只大鼠约3×10~7个,细胞活力在95％以上,初分离的肝星状细胞在328 nm激发光下自发蓝绿色荧光,Desmin免疫细胞化学染色鉴定纯度达到90％,未检测到内源性过氧化物酶阳性细胞。结论 建立了一种无库普弗细胞混杂的大鼠肝星状细胞分离方法,可以用于原代肝易状细胞的生物学行为的研究。     

离心淘洗技术在分离大鼠肝非实质细胞亚群中的应用

目的：采用离心淘洗技术分离纯化大鼠肝非实质细胞亚群。方法：通过原粒肝脏酶灌注,准备肝非实质细胞悬液,离心淘洗,收集不同流速(20和40ml／min)的细胞群。结果：经培养鉴定,流速21ml／min获得的为肝窦内皮细胞,40ml／min的为库普弗细胞,细胞纯度均大于95％。结论:在本实验室,离心淘洗技术已能成功应用于肝脏细胞分离过程。     

高效液相色谱法测定血清卡马西平的优化方案

目的 建立以高效液相色谱法测定人血清CBZ的实验方法优化方案.方法 以600μL乙酸乙酯为萃取溶剂,样品用量0.2 mL血清,萃取时间3min,以3 500 r/min速度离心4 min,取400μL萃取剂于75℃水浴中挥发至干;用1.0mL流动相溶解残留物,10 000 r/min离心20 min后进样分析.分析条件：柱温30℃,流动相（甲醇∶水约为60∶40）,波长254 nm.结果 在优化条件下,CBZ在1.52～48.8 mg/L范围内呈线性,相关系数不低于0.999 1,检出限为0.4 mg/L,样品的平均加标回收率为103.7,相对标准偏差小于5％.结论 成功建立了以高效液相色谱法测定人血清CBZ浓度的优化方案,该方案确定了最佳样品预处理条件,优化了色谱分离及检测条件,灵敏、准确,能够满足血清中CBZ的监测要求.     

低氧对人外周血单个核细胞迁移活性的影响及其临床意义

目的探讨低氧对机体外周血单个核细胞（PBMC）迁移的影响及其临床意义。方法分离外周血单个核细胞，采用Transwell小室实验检测常氧和低氧条件下PBMC迁移能力的变化，流式细胞术（FCM）检测常氧和低氧条件下T、B淋巴细胞，NK细胞及单核细胞迁移的变化。结果低氧环境下PBMC的迁移率降低31％。对其细胞亚群的分析结果显示，NK细胞的迁移率降低31．7％，单核细胞的迁移率降低63．1％，T、B淋巴细胞的迁移率无显著变化。结论低氧能显著影响单核细胞及NK细胞的迁移功能；检测低氧条件下PBMC的迁移能力，可为以免疫细胞迁移功能为靶点的炎症等疾病提供新的治疗对策。     

一种简易分离大鼠肝脏库普弗细胞的方法

目的建立一种经济、简单、稳定的大鼠肝脏库普弗细胞(KCs)分离方法。方法采用前灌流液(D-Hanks)和胰酶消化液两步法原位灌注大鼠肝脏,低速离心去除肝细胞,Percoll分离液不连续密度梯度离心法和选择性贴壁法纯化KCs,将培养7 d的KCs采用吞噬latex-beads实验及ED1免疫细胞化学法鉴定并检测其纯度。结果每只大鼠肝脏KCs的得率为(1.2～1.8)×107个,细胞存活率达95%,KCs纯度近100%。结论此新方法分离获得的KCs细胞得率和纯度较高且较稳定,同时较传统方法胶原酶的使用相比,此方法更经济实用。     

老年人外周血树突状细胞的体外培养及功能研究

目的 探讨≥80岁老年人外周血单核细胞体外诱导培养生成树突状细胞(dendriticcells,DCs)的情况及其功能特点.方法 取30例≥80岁老年人及30例60～70岁老年人外周血,用淋巴细胞分离液离心获得单个核细胞,贴壁培养2h后获得单核细胞,用含有rhGM-CSF和rhIL-4的培养基培养1周,观察2组DCs的生长、活力、存活时间、同种T淋巴细胞的刺激反应,并进行比较.结果 ≥80岁组DCs培养过程中细胞逐渐死亡,培养至第7天大部分细胞死亡,获得的DCs数量较少,其中有8例未能培养出DCs;较60～70岁组对同种T淋巴细胞的刺激能力也显著下降,而60～70岁组培养细胞的活力保持较好,至第7天均可获得数量较多的DCs.结论 较60～70岁组,≥80岁组外周血培养的DCs体外的生长活力、T细胞刺激能力明显降低,可能是≥80岁老年人免疫功能降低的重要原因.     

离心淘洗技术分离纯化大鼠肝库普弗细胞

目的通过制备肝非实质细胞悬液并离心淘洗的方法,分离纯化大鼠肝库普弗细胞.方法采用原位肝脏酶灌注、肝非实质细胞悬液的准备、离心淘洗和原代培养等方法进行分离.结果在经过胶原酶和链酶蛋白酶消化、Nycodenz分离和离心淘洗后,获得大鼠肝库普弗细胞产量约为(28±6)×106/鼠肝,细胞纯度为95%,细胞活力大于90%.结论应用离心淘洗技术能有效地分离纯化大鼠肝库普弗细胞,为体外进一步研究提供了高纯度和活力的库普弗细胞群.     

体外灌注法分离大鼠肝脏Kupffer细胞及原代培养

目的:探讨一种简便、高效的大鼠离体肝脏分离Kupffer细胞(KCs)及原代培养的方法.方法:采用大鼠肝脏离体Ⅳ型胶原酶灌注,剪碎肝脏,37℃消化30 min,低速离心去除肝细胞,Pcrcoll不连续密度梯度离心和选择性贴壁的方法分离KCs.通过墨汁吞噬实验和ED2免疫细胞化学来鉴定KCs.结果:分离的KCs的得率在细胞贴壁前(2.1±0.3)×106/g肝脏、贴壁后(1.5±0.1)×106/g肝脏,用0.4%台盼蓝染色,活率大于92%,ED2染色阳性的细胞达90%以上,分离的细胞培养后功能完整并延伸呈不规则型.结论:本实验建立的大鼠肝脏KCs的分离培养方法简便、高效、稳定,培养的细胞具有良好的生物学性状,为进一步研究提供基础.     

脐血分离、冷冻的实验研究

1　材料和方法1标本来源:47份脐血广州市妇婴医院提供,产妇为健康、足月分娩。标本采集方法按文献〔1〕,采用50ml以上脐血,<50ml的标本弃置不用。2分离方法:按1∶5加入6%羟乙基淀粉到脐血袋中,以500r/min倒置离心5min,垂挂挤出红细胞部分。混匀后正置1200r/min离心13min,以分浆夹去除血浆部分。3血液分析:应用美国CoulterJT-血细胞计数仪检测分离前后的白细胞数及白细胞分类计数。4台盼蓝拒染法测定细胞活力。5祖细胞分析:CFU-GM,BFU-E,CFU-GEMM混合培养体系按文献〔2〕配制,包含粒系刺激因子(G-CSF,100ng/ml,Kirin公司),红细胞生…     

急性心肌梗死患者外周血液单核细胞中血管内皮生长因子的表达

目的　探讨急性心肌梗死 ( AMI)后外周血单核细胞中血管内皮生长因子 ( VEGF)的表达及其与 AMI左心室收缩功能的关系。方法　分别抽取 2 5例行急诊经皮冠状动脉介入 ( PCI)治疗的 AMI患者发病后第 1、5、10和15 d的外周静脉血 ,分离、培养单核细胞 ,用酶联免疫吸附法 ( ELISA)测定 VEGF浓度。结果　单核细胞分泌的VEGF在 AMI后第 5 d即达高峰 ,显著高于对照组 ( 3 43 .2± 82 .5 pg/ml vs 14 3 .3± 2 4.2 pg/m l,P<0 .0 5 )。单核细胞分泌的 VEGF峰植与肌酸磷酸激酶 ( CK)峰值无显著相关。 PCI术后左心室收缩功能改善的 AMI患者 ,其单核细胞分泌的 VEGF水平显著高于左心室收缩功能未改善者 ( 867.6± 113 .1pg/ml vs2 3 4.8± 82 .4pg/ml,P<0 .0 5 )。结论　AMI患者 PCI术后单核细胞产生的 VEGF与左心室收缩功能改善有关     

Interactions of IgG from SLE patients with peripheral blood mononuclear cells and adherent cell populations

Summary Peripheral blood mononuclear cells (PBMCs) from normal control subjects were studied for their interactions with IgG isolated from normal or active systemic lupus erythematosus (SLE) sera. Preincubation of PBMCs with SLE IgG at 0.5–1.0 mg for 24 h followed by washing and subsequent cell culture for 7 days resulted in marked relative increase in cell supernatant IgG. These findings were noted with and without inclusion of cyclosporin A or indomethacin in cultures. Experiments using isolated normal adherent cell populations showed that SLE IgG but not normal IgG, when preincubated with adherent cell macrophage/monocyte populations, was capable of inducing the latter to produce suppressor factors capable of down modulating IgG production or release from normal non-adherent cells cultured with pokeweed mitogen. These findings suggest that IgG from active SLE patients may interact with both IgG-producing PBMC populations as well as adherent-cell populations to influence IgG production or release from subsequently cultured cells.     

Activation-induced cell death in peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis B may be related to abnormal production of interleukin 12 and 10

Peripheral blood mononuclear cells (PBMCs) from 70 patients with chronic hepatitis B and 32 normal healthy persons were isolated and cultured with or without Staphylococcus aureus enterotoxin B (SEB; 0.2 mg l^–1) and recombinant HBcAg (rHBcAg; 1.0 mg l^–1) for 48 h in vitro . After incubation, the cells were harvested by centrifugation and apoptosis of the PBMCs was studied by staining with fluorescent dyes YOPRO-1 and Hoechst 33342. The levels of IL-12 and IL-10 in the serum and the supernatants of cultured PBMCs were assayed by ELISA. The levels of IL-12 heterodimer in the serum and the supernatants of PBMCs cultured with SEB or rHBcAg were lower in patients than controls. The levels of IL-10 in both the serum and supernatants were higher in patients than controls. In addition, the percentage of apoptotic cells in PBMCs from the infected patients was significantly greater than from normal persons in the presence or absence of SEB and rHBcAg. Patients seropositive for HBeAg had much greater percentage of apoptotic cells in the PBMCs cultured with rHBcAg than patients seronegative for HBeAg, reaching 24.08%. We speculate that activation-induced cell death of PBMCs in the patients with hepatitis B may be related to abnormal expression of IL-12 heterodimer and IL-10, which may lead to persistent infection in the patients.     

慢性丙型肝炎患者外周血单个核细胞HCV RNA检测及其与机体免疫功能的相关性研究

目的观察慢性丙型肝炎患者外周血单个核细胞(PBMC)HCVRNA含量及其对T淋巴细胞亚群的影响,以探讨HCV感染者PBMC中HCVRNA水平及其与机体免疫功能的关系。方法采用荧光定量PCR(FQPCR)技术对128例丙型肝炎患者血清、外周血单个核细胞的HCVRNA含量进行了检测,同时检测CD3+、CD4+、CD8+、CD4+/CD8+。结果PBMC内HCVRNA阳性组与HCVRNA阴性组比较,前者CD3+、CD4+水平降低、CD8+水平增高,CD4+/CD8+比值下降大于后者,差异有显著性(P<0.05)。结论丙型肝炎病毒侵染PBMC后可加重患者的细胞免疫功能紊乱。     

Effect of interleukin-10 on the production of tumor necrosis factor-alpha by peripheral blood mononuclear cells from patients with chronic heart failure

Chronic heart failure (HF) is a state of inflammatory immune activation characterized by elevated circulating levels of tumor necrosis factor-alpha (TNF-alpha). Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine that inhibits TNF-alpha production and lessens endotoxin bioactivity. It is not known whether IL-10 reduces lipopolysaccharide (LPS) stimulated TNF-alpha production of peripheral blood mononuclear cells (PBMCs) from patients with chronic HF. PBMCs were isolated from 15 patients with chronic HF (New York Heart Association functional class 3.0 +/- 0.2, left ventricular ejection fraction 30 +/- 2%, peak oxygen consumption 18.1 +/- 0.8 ml/kg/min) and 15 healthy control subjects and stimulated with 1 and 10 ng/ml LPS for 24 hours with or without prior addition of IL-10 (10 ng/ml). TNF-alpha was quantified in cell-free supernatants by an enzyme-linked immunosorbent assay. TNF-alpha, soluble TNF receptors, IL-10, and LPS were quantified in plasma. LPS stimulated TNF-alpha production was highest in those patients in New York Heart Association class II (p <0.01 vs New York Heart Association class III and IV, p <0.001 vs control subjects). IL-10 reduced PBMC TNF-alpha production in all stimulated samples at 1 and 10 ng/ml LPS (mean reduction 43% at 1 ng/ml, p <0.01 and 55% at 10 ng/ml, p <0.0001). The percentage reduction in TNF-alpha release did not differ significantly between patients and control subjects or with respect to severity of chronic HF or baseline immune parameters. Independently of clinical severity, IL-10 profoundly inhibits TNF-alpha release from PBMCs isolated from patients with chronic HF. IL-10 is, therefore, a potential therapy for use in chronic HF associated with inflammatory immune activation.     

Spontaneous generation and survival of blood dendritic cells in mononuclear cell culture without exogenous cytokines

Studies on purified blood dendritic cells (DCs) are hampered by poor viability in tissue culture. We, therefore, attempted to study some of the interactions/relationships between DCs and other blood cells by culturing unseparated peripheral blood mononuclear cell (PBMC) preparations in vitro. Flow cytometric techniques were used to undertake a phenotypic and functional analysis of DCs within the cultured PBMC population. We discovered that both the CD11c(+) and CD11c(-) CD123(hi) DC subsets maintained their viability throughout the 3-day culture period, without the addition of exogenous cytokines. This viability was accompanied by progressive up-regulation of the surface costimulatory (CD40, CD80, CD86) and activation (CMRF-44, CMRF-56, CD83) molecules. The survival and apparent production of DCs in PBMC culture (without exogenous cytokines) and that of sorted DCs (with cytokines) were evaluated and compared by using TruCOUNT analysis. Absolute DC counts increased (for CD123(hi) and CD11c(+) subsets) after overnight culture of PBMCs. Single-cell lineage depletion experiments demonstrated the rapid and spontaneous emergence of "new" in vitro generated DCs from CD14(+)/CD16(+) PBMC radioresistant precursors, additional to the preexisting ex vivo DC population. Unlike monocyte-derived DCs, blood DCs increased dextran uptake with culture and activation. Finally, DCs obtained after culture of PBMCs for 3 days were as effective as freshly isolated DCs in stimulating an allogeneic mixed leukocyte reaction.     

Intracellular levels of hepatitis B virus DNA and pregenomic RNA in peripheral blood mononuclear cells of chronically infected patients

Summary.  It remains uncertain whether hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and pregenomic RNA (pgRNA) can be detected in the serum or peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B (CHB) infection. We examined HBV cccDNA and pgRNA in the serum and PBMC, and investigated the effect of lamivudine therapy on the viral loads in the PBMC of CHB patients. Paired serum and PBMC samples from 50 treatment-naïve CHB patients [25 hepatitis B e antigen (HBeAg) positive and 25 HBeAg negative] were quantified for total HBV DNA, cccDNA and pgRNA by real time polymerase chain reaction. HBV cccDNA and pgRNA were below the lower detection limit in all serum samples, and in 84% of PBMC. HBV DNA ( r  = 0.889, P  <   0.001) and pgRNA ( r  = 0.696, P  <   0.001) in PBMC correlated with the HBV DNA in serum. In the longitudinal study, 30 patients treated with lamivudine therapy for a median duration of 34 weeks (range 12–48 weeks) were examined. The median HBV DNA reduction in PBMC before and after treatment was 1.318 (range −0.471 to 3.846) log units, which was significantly lower than serum HBV DNA reduction [3.371 (range −0.883 to 9.454) log units, P  <   0.05]. HBV cccDNA and pgRNA were undetectable in the serum of CHB patients. HBV viral loads in PBMC correlated with serum HBV DNA. Lamivudine therapy had less effect on the HBV viral loads in PBMC compared with the serum viral loads.     

实时荧光定量RT-PCR检测血清和单个核细胞中HCV RNA的意义

目的探讨实时荧光定量聚合酶链反应(FQ-RT-PCR)检测血清和外周血单个核细胞(PBMCs)中HCV RNA含量及其临床价值。方法采集可疑丙型病毒性肝炎病人血,用FQ-RT-PCR分别检测血清和PBMCs中HCV RNA含量。血清或PBMCS HCV RNA阳性者都视为HCV RNA阳性。结果检测可疑患者180例,HCV RNA阳性106例。其中血清HCV RNA阳性84例,占79.25%;PBMCs HCV RNA阳性63例,占59.43%。HCV RNA阳性比率血清高于PBMCs(χ2=9.78,P<0.01)。单独血清HCV RNA阳性43例,占40.57%;单独PBMCs HCV RNA阳性22例,占20.75%;血清和PBMCs中HCV RNA同时阳性41例,占38.68%。血清和PBMCs HCV RNA含量差异无显著性。结论同时检测血清和PBMCs HCV RNA可提高HCV感染诊断的阳性率,检测PBMCs HCV RNA对抗病毒治疗的疗效评价及治疗时间有重要意义。     

类风湿关节炎患者外周血高迁移率族蛋白1表达

目的通过检测类风湿关节炎（RA）患者外周血单个核细胞（PBMC）、血浆中高迁移率族蛋白1（HMGB1）表达,为寻找治疗RA的新靶点提供依据。方法采集38例活动期RA患者、24例相对稳定期RA患者和20例健康对照者外周血。RT．PCR检测PBMC HMGB1mRNA表达,Western blot检测PBMC、血浆HMGB1蛋白表达。结果与相对稳定期RA患者、健康对照者相比,活动期RA患者PBMC HMGB1mRNA表达水平差异无统计学意义（F=1．23,P〉0．05）,而HMGB1蛋白表达水平下降（F=70．91,P〈0．01）,血浆HMGB1水平显著增高（P〈0．001）。相对稳定期RA患者与健康对照者之间差异无统计学意义（P〉0．05）。活动期RA患者血浆HMGB1水平与ESR（r．=0．478。P〈0．001）、C-反应蛋白（rs=0．574,P〈0．05）呈正相关。结论HMGB1与RA发病密切相关,并可能成为新的治疗靶点。