轮状病毒感染患者外周血单个核细胞的转录组学分析

轮状病毒(Rotavirus,RV)是引起急性肠胃炎的主要病原体,分析RV感染患者的人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)中差异表达基因(Differentially expressed genes,DEGs)有利于探讨人PBMC在清除RV中的作用。为此,本研究采集2019年2月-2019年6月长春儿童医院中RV感染患者和健康儿童血液,分离PBMC,通过转录组测序(RNA sequencing,RNA-seq)技术比较RV感染患者与健康儿童之间的RNA表达图谱,借助基因本体论(Gene Ontology,GO)数据库功能富集分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)、Reactome通路富集分析DEGs,使用实时荧光定量PCR(Real-time quantitative PCR,qPCR)技术进行验证。结果显示,与健康对照组相比,RV感染轻症患者PBMC中有1619个DEGs;重症患者PBMC中有2816个DEGs,主要与干扰素(Interferon,IFN)反应、中性粒细胞、溶酶体、核小体、染色质等相关。qPCR验证轻症患者干扰素刺激基因(IFN-stimulated genes,ISGs)15表达上调,白介素(Interleukin,IL)1β表达下调;重症患者IL15、ISG15表达上调,IL1β表达下调,与转录组结果相一致。本研究提示,RV感染可能激活人I型和II型IFNs反应抵御病毒感染,但也会抑制溶酶体相关基因,对细胞自噬过程产生影响。     

Cytokines Released by Human Peripheral Blood Mononuclear Cells Inhibit the Production of Early and Late Cytomegalovirus Proteins

Cytomegalovirus-infected human fibroblasts are susceptible to lysis by natural killer cells and cytotoxic T cells. The purpose of this study was to determine whether non-lytic mechanisms might also contribute to the control of cytomegalovirus infection. The appearance of cytomegalovirus proteins in infected fibroblasts was determined by flow cytometry. Infected fibroblasts incubated with peripheral blood mononuclear cells for 3 days expressed less early and late proteins than fibroblasts incubated without peripheral blood mononuclear cells. Supernatants generated by the cocultivation of peripheral blood mononuclear cells with cytomegalovirus-infected fibroblasts inhibited the production of cytomegalovirus early and late proteins. The soluble factors in supernatants which contributed to the inhibitory effect were identified as interferons α, β and γ, and tumor necrosis factors α and β. The ability of supernatants to inhibit the production of cytomegalovirus early protein was mimicked by combinations of corresponding recombinant cytokines. The inhibition of cytomegalovirus protein production by cytokines produced by peripheral blood mononuclear cells may contribute to early containment of cytomegalovirus infection.     

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.     

从实验感染OPPV的山羊外周血单核细胞中分离病毒的研究

用绵羊胎肺细胞与接种绵羊进行性肺炎病毒（OPPV）的山羊外周血单核细胞共同培养的方法可以分离到病毒,这说明OPPV可以感染山羊。用细胞病变观察、间接荧光抗体试验、电镜切片观察和聚合酶链式反应对分离毒进行了鉴定,进一步证实了分离毒为OPPV。分离结果表明这是一种较为敏感的分离方法。绵羊胎肺细胞可传到40多代,且每一代次的细胞都可用于病毒的分离,因此这是一种非常实用的分离OPPV的方法。     

伏马菌素B1对人外周血单个核细胞抗原加工相关转运子表达的影响

探讨伏马菌素B1(FB1)对体外培养的人外周血单个核细胞(hPBMC)抗原加工相关转运子(TAP-1)表达的影响。采用流式细胞定量检测(FCM)、免疫印迹(Western印迹)及半定量RT-PCR方法,研究不同浓度FB1(0,10和50μmol/L)处理后人外周血单个核细胞TAP-1在mRNA和蛋白质水平表达的影响。RT-PCR检测结果表明,10和50μmol/L FB1处理24h后,处理组细胞TAP-1mRNA明显低于对照组。在蛋白质水平,FCM定量分析表明,两个处理组细胞表面TAP1的平均荧光强度均较对照组降低,以50μmol/LFB1处理组降低显著(P<0.05)。免疫印迹结果亦表明,FB1处理组TAP-1的表达均较对照组降低。10和50μmol/LFB1可抑制体外培养的hPBMCTAP-1mRNA和蛋白质表达。     

脱氧雪腐镰刀菌烯醇对人外周血单个核细胞 HLA-I分子表达的影响

研究探讨了脱氧雪腐镰刀菌烯醇(DON)对人外周血单个核细胞HLA-I(human leucocyte cyte antigen I)分子表达影响.采用流式细胞术(FCM)和免疫印迹方法研究了不同剂量DON对体外培养人外周血单个核细胞表面HLA-I分子表达的影响及其量效关系.FCM定量检测结果表明,不同浓度DON处理均可一定程度降低人外周血单个核细胞表面HLA-I分子的表达,DON 50ng/mL、100ng/mL、1000 ng/mL和2000 ng/mL组HLA-I类分子的平均表达量分别为6.92±0.68、6.64±0.69、5.95±0.48和5.48±0.77,在50～2000ng/mL范围内随着DON浓度增加,外周血单个核细胞HLA-I分子表达降低,两者呈显著负相关(r=0.737,P＜0.01).Western印迹结果显示,大剂量DON(1000ng/mL和2000ng/mL)组人外周血单个核细胞HLA-I分子表达明显减弱.研究结果表明脱氧雪腐镰刀菌烯醇可剂量依赖地抑制体外培养的人外周血单个核细胞HLA-I分子的表达.     

脱氧雪腐镰刀菌烯醇抑制体外培养人外周血单个核细胞低分子量蛋白酶体-2表达

探讨脱氧雪腐镰刀菌烯醇(DON)对人外周血单个核细胞参与抗原呈递的低分子量蛋白酶体-2(LMP-2)表达的影响。采用流式细胞术(FCM)和半定量RT-PCR方法从蛋白质和mRNA水平分析了不同剂量DON对体外培养人外周血单个核细胞LMP-2分子表达的影响及其量效关系。FCM定量检测结果表明,不同浓度DON处理均可一定程度抑制人外周血单个核细胞LMP-2的表达,50ng/mlDON组、100ng/mlDON组、1000ng/mlDON组和2000ng/mlDON组LMP-2平均荧光强度分别为6.99±0.72、6.21±0.55、5.34±0.56和5.03±0.43,在50～2000ng/mL范围内随着DON浓度增加,外周血单个核细胞LMP-2表达降低,与DON浓度呈显著负相关(r=0.824,P<0.01)。半定量RT-PCR结果显示,不同浓度DON处理均可抑制人外周血单个核细胞LMP-2mRNA表达。DON在蛋白质和mRNA水平可剂量依赖地抑制体外培养的人外周血单个核细胞LMP-2的表达。     

系统性红斑狼疮患者外周血单个核细胞Toll样受体9的表达及血清白介素-10水平的研究

目的:研究Toll样受体9(TLR-9)在系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)上的表达水平及SLE患者血清白介素-10水平,探讨发病机制。方法:从23例活动期、19例缓解期SLE患者和20例正常对照组中分离PBMCs,利用反转录-聚合酶链反应(RT-PCR)法检测PBMCs中TLR9 mRNA的表达水平,利用酶联免疫吸附试验法检测其血清白介素-10水平。结果:活动期SLE患者PBMCs的TLR-9mRNA表达高于缓解组(P<0.01)及正常对照(P<0.01),缓解期和正常对照组相比,差异无统计学意义(P>0.05)。SLE活动期患者血清IL-10水平显著高于缓解期患者(P<0.01),并均高于正常对照组(P<0.01)。结论:活动期SLE患者PBMC的TLR9 mRNA的表达水平增高;并且活动期及缓解期SLE患者血清IL-10水平升高可能与TLR9 mRNA表达的上调相关。     

Cytokine Quantification in the Supernatant of Mononuclear Cell Cultures and in Blood Serum from Patients with Jorge Lobo's Disease

Few studies are available about the participation of the immune response in the control or the development of Jorge Lobo's disease. Thus, the objective of the present study was to quantify macrophage and lymphocyte cytokines in the supernatant of cell cultures and in blood serum from patients with this disease. The study was conducted on 15 patients with the mycosis and on 15 healthy adult individuals (control group). Blood samples were collected in order to obtain serum and mononuclear cells. Monocytes were cultured for 24 h in the presence or absence of LPS and L. loboi, and lymphocytes were cultured for 48 h in the presence or absence of PHA and L. loboi. Cytokines IL-1beta, TNF-alpha and IL-6 were quantified by ELISA in the supernatants of monocyte cultures and in serum. Cytokines IL-2, IFN-gamma, IL-4 and IL-10 were quantified by FLISA in the supernatants of lymphocyte cultures and in serum. The quantification of the cytokines in the culture supernatant revealed a greater IL-4 and IL-6 production and lower IL-2 levels in patients compared to control. The production of IL-1beta, TNF-alpha, IL-10 and INF-gamma was similar in patients and controls. The mononuclear cells from patients with the non-localized form of the disease produced higher INF-gamma levels than those of patients with the localized form. The results suggest that patients with Jorge Lobo's disease show altered cytokine profiles represented by a predominance of the Th2 profile. However, further studies are needed to assess the participation of cytokines in the cell-fungus interaction in situ.     

Downregulation of Tumour Necrosis Factor α Gene Expression in Peripheral Blood Mononuclear Cells Cultured in the Presence of Tofacitinib Prior to Therapy Is Associated with Clinical Remission in Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by pain, synovial hyperplasia, mononuclear cell infiltration, bone erosion and joint destruction. Efficacy of personalized therapy in RA is associated with correct choice of therapeutic agent and a possibility to predict its effect prior to treatment. Our objective was to examine the association of baseline expression of metalloproteinase (MMP)-9 and cathepsin K, which are involved in cartilage and bone degradation, as well as proinflammatory cytokines tumour necrosis factor (TNF)α and interleukin (IL)-1β in the peripheral blood mononuclear cells (PBMCs) obtained from patients with RA cultured with tofacitinib (TFCN) and remission achievement. We examined 12 tofacitinib-naïve patients with RA, with a median age of 51 years and disease duration of 37.6 months. After three months of TFCN therapy, six of these patients reached clinical remission criteria while others preserved high and moderate disease activity. PBMCs were tested prior to therapy followed by their isolation in Ficoll density gradient and cultured with 100 nM TFCN for 48 h. Gene expression analysis for MMP-9, cathepsin K, IL-1β, and TNFα was performed with quantitative real-time RT-PCR using total RNA isolated from and cultured with TFCN PBMCs compared with untreated cells. Expression of all the examined genes was significantly upregulated in those cultured with TFCN PBMCs from patients who maintained high and moderate disease activity after TFCN therapy while TNFα gene expression was significantly downregulated in patients who gained remission compared with untreated counterparts. Downregulation of TNFα gene expression in PBMCs from TFCN-naïve patients with RA cultured with TFCN prior to therapy compared with untreated counterparts might serve a prognostic biomarker for remission attainment in response to tofacitinib therapy.     

人外周血单个核细胞与脐带间充质干细胞共培养的AFM研究

采用原子力显微镜与倒置显微镜在细胞层次上观察了人外周单个核细胞(PBMCs)与同种异源脐带间充质干细胞(hUC-MSCs)共培养的过程,并在单细胞水平上分析了共培养前后人外周单个核细胞的形貌和生物物理性质。结果发现:共培养后贴壁人外周单个核细胞的形态发生了很大的改变,并且表面分布着大小不一的颗粒状聚合物。利用AFM高空间分辨的力位移曲线测量系统,发现共培养72h后培养上清中人外周单个核细胞、贴壁的人外周单个核细胞的粘滞力分别是单纯培养72h的人外周单个核细胞的2倍、5倍,而细胞的硬度分别是单纯培养人外周单个核细胞的1.5倍、2倍。CCK-8检测提示,共培养过程中,干细胞的生长与外周血单个核细胞的生长出现了竞争作用。通过AFM探测人外周单个核细胞与脐带间充质干细胞共培养的可视化数据,有助于更好地了解间充质干细胞与外周血单个核细胞的相互作用。     

猪外周血内皮祖细胞的分离培养和鉴定

从猪外周血分离出单个核细胞,置于EGM-2培养基中培养,通过挑选细胞集落并对之进行免疫组织化学染色和荧光染色来鉴定内皮祖细胞。结果显示猪的内皮祖细胞为长梭形或纺锤形并呈集落生长,能够吞噬已酰化低密度脂蛋(ac-LDL)并结合凝结素BS-1,同时具有内皮细胞标志CD31、flk-1和von willebrand factor(vWF)。这些结果表明能够从猪的外周血中分离培养出内皮祖细胞,为自体内皮祖细胞移植促进猪慢性心肌缺血模型血管新生的研究打下了基础。     

Effect of Vitamin D on Peripheral Blood Mononuclear Cells from Patients with Psoriasis Vulgaris and Psoriatic Arthritis

BackgroundPsoriasis, a chronic skin disease with or without joint inflammation, has increased circulating proinflammatory cytokine levels. Vitamin D is involved in calcium homeostasis, bone formation, osteoclastogenesis and osteoclast activity, as well as regulation of immune response. We aimed to study osteoclast differentiation and cytokine secretion of peripheral blood mononuclear cells (PBMCs) from patients with psoriasis vulgaris and psoriatic arthritis, in response to 1,25(OH)₂D₃.MethodsSerum levels of bone turnover markers were measured by ELISA in patients with psoriasis vulgaris and psoriatic arthritis, and healthy controls. PBMCs were isolated and cultured with or without RANKL/M-CSF and 1,25(OH)₂D₃. Osteoclast differentiation and cytokine secretion were assessed.ResultsPsoriatic arthritis patients had lower osteocalcin, as well as higher C-telopeptide of type I collagen and cathepsin K serum levels compared with psoriasis vulgaris patients and controls. RANKL/M-CSF-stimulated PBMCs from psoriatic arthritis patients produced higher proinflammatory cytokine levels and had a differential secretion profile in response to 1,25(OH)₂D₃, compared with psoriasis vulgaris and control PBMCs.ConclusionsOur data confirmed altered bone turnover in psoriatic arthritis patients, and demonstrated increased osteoclastogenic potential and proinflammatory cytokine secretion capacity of these PBMCs compared with psoriasis vulgaris and controls. 1,25(OH)₂D₃ abrogated these effects.     

Herpesvirus DNA in Peripheral Blood Mononuclear Cells of Some Patients with Meniere's Disease

Herpes simplex virus‐1 (HSV) or varicella zoster virus (VZV) DNA was detected by nested polymerase chain reaction in peripheral blood mononuclear cells of patients with Meniere's disease (one of 28 patients for HSV‐1,2 of 28 patients for VZV) during acute illness (within 5 days after onset). On the other hand, neither HSV‐1 DNA or VZV DNA was detected in PBMCs of 50 age‐ and sex‐matched healthy individuals and 50 pregnant women. These findings may imply that reactivation of HSV‐1 or VZV may be associated with the development of some cases of Meniere's disease.     

外周血单核细胞HLA-DR/CD14分析的临床意义及其与降钙素原、C-反应蛋白、白细胞计数的相关性

目的:探讨外周血单核细胞HLA-DR/CD14表达在感染性疾病中的临床意义,及其与降钙素原(PCT)、C-反应蛋白(CRP)、白细胞(WBC)计数等指标的相关性。方法:收集2012-01~2013-07住院患者75例,包括脓毒血症患者47例,其中重度感染患者25例设为观察1组、中度感染患者22例设为观察2组;非感染炎症患者10例,设为观察3组;其他疾病患者18例,设为观察4组;以及本院健康体检者21例,设为正常对照组。用流式细胞术分析外周血单核细胞HLA-DR/CD14,同步定量检测各组的PCT、CRP、WBC指标,用SPSS13.0软件对各组间数据进行方差分析及相关性比较。结果:观察1、2组各项指标与正常对照组间均存在显著性差异,1、2组间除WBC外其他各项指标间均存在显著性差异,3、4组与1、2组比较各项指标均存在显著性差异,P值均0.01;观察1、2组内HLA-DR/CD14与PCT、CRP存在显著负相关关系,而与WBC不存在显著性相关关系。结论:HLA-DR/CD14在感染性疾病中具有重要意义,联合检测HLA-DR/CD14和PCT、CRP、WBC,有助于感染性疾病的临床评估、诊断治疗及疗效观察。     

细胞因子诱导外周血单个核细胞的转录和蛋白表达研究

采用干扰素-γ、抗CD3单克隆抗体和IL-2体外诱导扩增外周血单个核细胞成为cIK细胞,并于诱导培养前及培养第15d时分别收集细胞样本。在对培养前后细胞的增殖、形态及表面标志变化检测的同时。提取总蛋白进行定量、双向电泳和银染。利用ImageMasterTM软件对培养前后表达相同和不同的蛋白质点进行分析,并选择其中24个蛋白质点进行质谱鉴定。对于部分培养前后具有代表性的蛋白,进一步采用qPCR技术分析其的转录情况。结果表明,培养前后细胞的蛋白质组学特征是完全不同的,相同表达蛋白点主要与基因的转录因子和细胞骨架相关,诱导后特异表达蛋白主要与细胞生长、增殖相关。虽然在转录与蛋白水平上呈现出部分负相关现象,由于蛋白质组才是基因表达的最终形式,结合蛋白差异研究结果提示,经细胞因子诱导后,CIK细胞的大量扩增与细胞内蛋白表达改变相关。     

Haptoglobin Proved a Prognostic Biomarker in Peripheral Blood of Patients with Personalized Peptide Vaccinations for Advanced Castration-Resistant Prostate Cancer

The fopA gene encoding a fructooligosaccharide-producing β-fructofuranosidase was isolated from Aspergillus niger ATCC 20611. The primary structure deduced from the nucleotide sequence showed considerable similarity to those of two other β-fructofuranosidases from A. niger, but the fopA gene product had several amino acid insertions and an extra C-terminal polypeptide consisting of 38 amino acids that could not be found in the two others. We could successfully express the fopA gene in S. cerevisiae and the fopA gene product obtained from the culture supernatant of the S. cerevisiae transformant had similar characteristics to the β-fructofuranosidase purified from A. niger ATCC 20611. However, we could not detect any β-fructofuranosidase activity in either the culture supernatant or cell lysate when the C-terminal truncated fopA gene product by 38 amino acids was used to transform S. cerevisiae. In western analysis of those samples, there was no protein product that is cross-reacted with anti-β-fructofuranosidase antibody. These results suggested that the C-terminal region of the fopA gene product consisting of 38 amino acids was essential for the enzyme production.     

Focused Microarray Analysis of Peripheral Mononuclear Blood Cells from Churg-Strauss Syndrome Patients

DNA diagnostics are useful but are hampered by difficult ethicalissues. Moreover, it cannot provide enough information on theenvironmental factors that are important for pathogenesis ofcertain diseases. However, this is not a problem for RNA diagnostics,which evaluate the expression of the gene in question. We herereport a novel RNA diagnostics tool that can be employed withperipheral blood mononuclear cells (PBMCs). To establish thistool, we identified 290 genes that are highly expressed in normalPBMCs but not in TIG-1, a normal human fibroblast cell. Thesegenes were entitled PREP after predominantly expressed in PBMCand included 50 uncharacterized genes. We then conducted PREPgene-focused microarray analysis on PBMCs from seven cases ofChurg–Strauss syndrome (CSS), which is a small-vesselnecrotizing vasculitis. We found that PREP135 (coactosin-likeprotein), PREP77 (prosaposin), PREP191 (cathepsin D), PREP234(c-fgr), and PREP136 (lysozyme) were very highly up-regulatedin all seven CSS patients. Another 28 genes were also up-regulated,albeit more moderately, and three were down-regulated in allCSS patients. The nature of these up- and down-regulated genessuggest that the immune systems of the patients are activatedin response to invading microorganisms. These observations indicatethat focused microarray analysis of PBMCs may be a practical,useful, and low-cost bedside diagnostics tool.