快速获取55型腺病毒基因组序列的方法

【目的】建立快速获取55型腺病毒的全长基因组序列的方法。【方法】根据55型腺病毒的基因组特点,设计覆盖55型腺病毒基因组序列的12对引物,分别以55型腺病毒DNA为模板,扩增得到12个PCR产物,通过对12个PCR产物测序及序列拼接,获得55型腺病毒的全长基因组序列。【结果】从本院急性上呼吸道感染者咽拭子标本中分离得到一株55型腺病毒毒株SF04/SC/2016,以其DNA为模板扩增成功获得12个PCR产物,对其进行测序,并对12段序列进行拼接得到55型腺病毒的全长基因组序列,与已报到的各型腺病毒序列进行比对,采用邻位相连法构建系统发育进化树,所得序列与55型腺病毒处于同一分支,进一步确认该病原体为55型腺病毒。【结论】研究公布的序列和方法,能够实现更方便对腺病毒的快速测序,为揭示55型腺病毒的进化特点及制订疾病防控策略提供了有效手段。     

病毒感染复数影响腺病毒感染T淋巴细胞效率的初步研究

以往研究发现,增加感染复数(multiplicity of infection,MOI)可以有效提高5型腺病毒(Ad5)感染T细胞的效率,但其具体机制并未十分清楚。选取T淋巴瘤细胞为靶细胞,通过荧光定量PCR及透射电镜观察等方法探讨不同MOI对重组腺病毒Ad5-GFP复制周期中病毒结合及病毒进入两个环节的影响,发现高MOI条件下不会影响T细胞结合的腺病毒数量,却可显著增加经胞吞进入T细胞的腺病毒数量。这些结果表明,增加MOI有利于腺病毒经胞吞进入T细胞,从而可提高腺病毒感染效率。这为进一步研究腺病毒感染T淋巴细胞的机制提供了理论基础。     

腺病毒55型抗原表位分析及评价

目的:筛选腺病毒55型(Ad55)抗原表位,为研制腺病毒免疫诊断试剂提供基础。方法:采用生物信息学方法预测Ad55六邻体、纤突的B细胞抗原表位;利用大肠杆菌优势密码子获得相应基因,插入载体pBVIL-1克隆表达后获得重组Ad55表位抗原;用临床疑似腺病毒呼吸道感染患者血清对重组Ad55表位抗原活性进行检测,用ROC曲线分析重组Ad55抗原的诊断意义;采用ClustalX软件进行多序列比较。结果:Ad55六邻体含有6个主要抗原表位,纤突含有2个主要抗原表位;采用退火延伸法获得上述8种表位基因,片段长度为90~180 bp;获得上述8种重组基因工程抗原,相对分子质量为18×103~21×103;血清学检测的ROC曲线分析显示,270~320、410~460和135~165氨基酸残基抗原表位的AUC面积超过0.75,具有一定的诊断意义(P0.05);序列比较结果显示,上述3种抗原表位与腺病毒11型序列高度同源,与3型存在较大差异。结论:获得了3个Ad55主要抗原,对研制通用性呼吸道传播腺病毒免疫诊断试剂具有一定的意义。     

人腺病毒36型与肥胖发生相互关系的研究进展

病毒感染与肥胖发生之间关系的研究在近年来受到关注,尤其是人腺病毒36型（Ad-36）感染。Ad-36抗体的存在与肥胖者体重指数增加具有相关性。体外动物模型和细胞模型实验也支持Ad-36诱导肥胖发生。在肥胖发生机制方面,E4 orf-1基因、脂肪细胞分化的细胞通路受到关注。磷脂酰肌醇3激酶（PI3K）途径被认为在Ad-36引发降血糖作用中发挥重要作用。目前,Ad-36的研究数据仍然有限,其与肥胖发生之间关系的确认仍需更多的流行病学数据和更深入的研究。     

溶瘤腺病毒ZD55对类肝癌干细胞的抑制效应

溶瘤腺病毒能够靶向和杀死癌症干细胞,被认为是一种很有前景的抗癌药物.已有研究表明,溶瘤腺病毒ZD55能够靶向肝癌,并且表现出明显的细胞毒性效应.然而,其对肝癌干细胞是否具有同样地杀伤效力仍需进一步探讨.利用悬浮培养富集类肝癌干细胞,并验证其肝癌干细胞的特征.进一步通过MTT、结晶紫染色、Hoechst染色、Western blot和流式细胞术等检测ZD55对类肝癌干细胞的细胞存活率、凋亡诱导和病理效应等.结果发现,悬浮培养的类肝癌干细胞具有自我更新和分化能力、高表达干细胞相关转录因子(如NANOG和OCT4)、处于静息状态和具有耐药性等特性,溶瘤腺病毒处理后表现出明显的细胞毒性效应和杀伤特性,类肝癌干细胞的最低生存率仅为26.7%.ZD55能够非常明显地诱导类肝癌干细胞凋亡,其凋亡率最高达到60%.因此,ZD55可能会成为靶向肝癌干细胞的一种很有前景的治疗药物,对肝癌的临床治疗具有一定的应用价值.     

腺病毒E1B 55kD癌蛋白与hDaxx的相互作用

为了探讨腺病毒(adenovirus,Ad)E1B 55kD癌蛋白(Ad E1B 55kD)打破hDaxx和PML共定 位细胞核的作用机制,本文利用体内外共免疫沉淀反应研究Ad E1B 55kD与hDaxx的结合反应 ,并通过酵母双杂交体系测定两种蛋白质的相互作用及其作用的氨基酸残基序列.结果显示 Ad2 E1B 55kD通过C端58个氨基酸(aa)与hDaxx结合并发生相互作用.Ad12 E1B 5 5kD与hDaxx结合需全序列aa及其构象.共免疫沉淀反应和Western blot结果证实Ad2/5或Ad1 2 E1B 55kD能在体内外与hDaxx直接结合.     

腺病毒E1B 55KD癌蛋白与hDaxx的相互作用

为了探讨腺病毒 (adenovirus,Ad)E1B 5 5kD癌蛋白 (AdE1B 5 5kD)打破hDaxx和PML共定位细胞核的作用机制 ,本文利用体内外共免疫沉淀反应研究AdE1B 5 5kD与hDaxx的结合反应 ,并通过酵母双杂交体系测定两种蛋白质的相互作用及其作用的氨基酸残基序列。结果显示 :Ad2E1B 5 5kD通过C端 5 8个氨基酸 (aa)与hDaxx结合并发生相互作用。Ad12E1B 5 5kD与hDaxx结合需全序列aa及其构象。共免疫沉淀反应和Westernblot结果证实Ad2 / 5或Ad12E1B 5 5kD能在体内外与hDaxx直接结合     

腺病毒E1B55ku癌蛋白打破hDaxx与PML在细胞核的共定位

利用间接免疫荧光试验,通过Confocal激光扫描生物荧光显微镜和图像软件分析腺病毒(adenovirus,Ad)E1B 55 ku癌蛋白(Ad E1B 55 ku)与人Daxx(human Daxx,hDaxx)在细胞核的定位关系,研究它对早幼粒细胞性白血病蛋白(promyelocytic leukemia protein,PML)与hDaxx在细胞核定位关系的影响.实验结果表明,Ad E1B 55 ku与hDaxx共定位细胞核,并打破hDaxx与PML共定位于细胞核PML致癌结构域(PML oncogenic domams,PODs).     

Desmoglein 2 (DSG2) Is A Receptor of Human Adenovirus Type 55 Causing Adult Severe Community-Acquired Pneumonia

Virologica Sinica - Human adenovirus type 55 (HAdV-B55) is a re-emergent acute respiratory disease pathogen that causes adult community-acquired pneumonia (CAP). Previous studies have shown that...     

脾切断流术围术期患者外周血淋巴细胞的变化及免疫功能的研究

目的:本实验旨在探讨脾切除门奇静脉断流术后患者外周血T淋巴细胞、B淋巴细胞、NK细胞的变化,评估患者的抗肿瘤免疫能力是否有所影响,及机体的免疫系统有何变化.方法:选择择期行脾切除门奇静脉断流术的肝硬化患者20名,给予全凭静脉麻醉.分别于麻醉前(T0)、切皮前(T1)、脾切除即刻(T2)、脾切除后1h(T3)、手术完毕(T4)、手术后1d(T5)、手术后7d(T6)时抽取病人外周静脉血2mL,采用流式细胞仪测定CD3+、CD4+、CD8+、CD19+B细胞、CD3-56+(NK细胞)绝对数量.结果:与麻醉前相比,切皮前CD3+T细胞、CD3+CD4+T细胞、CD3+CD8+T细胞、CD19+B细胞、NK细胞均明显的降低,而在脾脏切除即刻各系细胞又明显恢复,基本与麻醉前水平相当.然而随着手术继续,在脾脏切除后1h,仅B细胞低于术前,一直持续到手术完毕,但是,此时B细胞与麻醉前比已没有统计学差异.手术完毕时T、B细胞和NK细胞再次降低,但仍明显高于切皮前水平.手术后1d时,CD4+T细胞与NK细胞仍然低于麻醉前,CD3T细胞、CD3+CD8+T细胞和B细胞已经恢复到麻醉前水平.术后7d时,CD3+T细胞、CD3+CD4+T细胞、CD3+CD8+T细胞及B细胞不仅得到恢复,而且还比麻醉前明显升高,但是NK细胞仍与麻醉前的水平相当.结论:异丙酚联合瑞芬太尼麻醉对门脉高压患者行脾切除门奇静脉断流术患者的T、B淋巴细胞和NK细胞有快速、短期的降低作用,术后7d人体淋巴细胞数量不仅得到恢复,并且反馈性地升高,提示脾脏切除手术能够有效提升患者的免疫细胞数量.     

肺癌患者外周血T淋巴细胞分型与抗核抗体之间的关系研究

摘要 目的：研究肺癌患者外周血T淋巴细胞分型与抗核抗体之间的关系。方法：选择2019年1月到2021年6月在我院接受治疗的肺癌患者81例作为研究组,并选择同期健康志愿者81例作为对照组,检测并比较两组患者外周血CD4⁺、CD8⁺和CD4⁺/CD8⁺淋巴细胞比例,以及抗核抗体血清滴度。比较不同抗核抗体、年龄、性别、TNM分期、肿瘤分化程度以及病理类型肺癌患者外周血CD4⁺、CD8⁺和CD4⁺/CD8⁺淋巴细胞比例。结果：（1）肺癌患者外周血CD4⁺和CD4⁺/CD8⁺淋巴细胞比例显著低于对照组,而CD8⁺淋巴细胞比例显著高于对照组（P<0.05）;（2）III+IV肺癌患者外周血CD4⁺、和CD4⁺/CD8⁺淋巴细胞比例均显著低于I+II肺癌患者,而CD8⁺淋巴细胞比例均显著高于I+II肺癌患者（P<0.05）;（3）小细胞肺癌患者外周血CD4⁺、和CD4⁺/CD8⁺淋巴细胞比例均显著低于非小肺癌患者,而CD8⁺淋巴细胞比例均显著高于非小肺癌患者（P<0.05）;（4）肺癌患者抗核抗体血清滴度显著高于对照组（P<0.05）;（5）抗核抗体阳性患者CD4⁺和CD4⁺/CD8⁺淋巴细胞亚群比例均显著低于抗核抗体阴性患者,而CD8⁺淋巴细胞亚群比例显著高于抗核抗体阴性患者（P<0.05）。结论：肺癌患者外周血T淋巴细胞亚群表达异常,并且其表达水平可能与抗核抗体滴度有关。     

人类外周血淋巴细胞中“自发”和诱发的SCE频数分布的比较

“自发”SCE频数在细胞间的分布,服从Poisson分布;而以CSC诱发的SCE频数则服从正态颁。分布类型的改变,可能是由于平均数的增加所致。平均数的增加,以A组和C组染色体为甚。     

Transport of the Immunosuppressant 15-Deoxyspergualin in Human Peripheral Blood Lymphocytes

15-deoxyspergualin (DSG) is a potent immunosuppressive compound currently in clinical trials. In this study, we have characterized the uptake and intracellular localization of DSG in human peripheral blood lymphocytes (PBL′s). DSG is transported into human PBL′s and reaches an estimated maximum concentration of approximately 500μM in 6 hours. The majority of the [³H]-DSG remains in the cytoplasm of cells and that which is associated with the nucleus is only loosely associated. DSG was transported by HeLa cells, as well, suggesting uptake is not specific for hematopoietic cells. Positively charged amino acids and polyamines, which are structurally similar to DSG, were unable to compete for DSG transport suggesting that DSG is transported into cells via a pathway distinct from amino acids or polyamines.     

人外周血淋巴细胞体外诱导培养前后细胞表型与细胞毒活性相关性研究

为了分析体外细胞因子诱导培养CIK细胞过程中细胞表型的变化与其杀瘤活性的相关性及为临床过继免疫治疗提供实验依据,本研究采用体外诱导方法扩增培养正常人外周血淋巴细胞及单个核细胞,应用流式细胞术测定培养前、培养第7天和第14天的CD3~+等15种不同表型细胞百分率的变化,用CCK-8试剂检测第7天和第14天的细胞毒活性。结果显示,扩增培养后T细胞活化表型的表达和细胞毒活性在第7天最强,与其细胞表型CD3~+CD25~+、CD3~+CD28~+、CD3~+CD25~+CD28~+、CD3~+CD4~+呈正相关(P<0.05),与CD3~+CD45RA~+CD45RO~+呈负相关(P<0.05)。本研究表明测定培养细胞活化相关表型可以间接监测其杀瘤能力,为临床CIK细胞过继免疫治疗的应用提供实验依据。     

Evaluation of Costimulatory Molecules in Peripheral Blood Lymphocytes of Canine Patients with Histiocytic Sarcoma

Histiocytic sarcoma is a rapidly progressive and fatal neoplastic disease in dogs. It is unclear whether costimulatory molecules, including CD28, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and programmed death-1 (PD-1), are expressed on peripheral blood lymphocytes (PBLs) of canine patients with histiocytic sarcoma. The objective of this study was to evaluate the expression of CD28, CTLA-4, and PD-1 molecules on PBLs of patients with histiocytic sarcoma, patients with other tumors, and healthy controls. Twenty-six dogs were included in the study, with eight, ten, and eight dogs in the histiocytic sarcoma, other tumor, and healthy control groups, respectively. PBLs and serum were prospectively obtained from patients diagnosed histopathologically with histiocytic sarcoma, other tumors and healthy controls. The surface expression of CTLA-4, CD28, and PD-1 on T lymphocytes was examined using flow cytometric analysis. Serum samples were frozen at −30°C until serum interferon-γ (IFN-γ) was measured by enzyme-linked immunosorbent assay. The expression level of CTLA-4 on CD4+ lymphocytes was significantly higher in the histiocytic sarcoma group than in the control group. The expression of CTLA-4 on CD8+ lymphocytes was significantly higher in the histiocytic sarcoma group than in the other two groups. In addition, the expression of PD-1 on CD8+ lymphocytes was significantly higher in the histiocytic sarcoma group than in the control group. However, no significant differences in CD28 expressions and serum IFN-γ levels were observed. The present results provided evidence showing that the expression levels of CTLA-4 on both CD4+ and CD8+ lymphocytes and PD-1 on CD8+ lymphocytes in peripheral blood obtained from dogs with histiocytic sarcoma were upregulated. The overexpressions of CTLA 4 and PD-1 suggested that antitumor immunity may be suppressed in dogs with histiocytic sarcoma.     

β-N-Acetylhexosaminidase in Peripheral Blood Lymphocytes and Monocytes in the Different Forms and Stages of Multiple Sclerosis

Abstract: The activity of the acidic glycohydrolase β- N -acetylhexosaminidase, an enzyme system normally participating in the stepwise degradation of glycoproteins, glycolipids, and proteoglycans, appears to be modulated in lymphocytes and monocytes from peripheral blood of patients affected by multiple sclerosis during different stages of the disease. In particular, a significant decrease in this enzyme activity, compared with healthy subjects, was observed in patients affected by the relapsing-remitting form both in a stable clinical status and during a relapse as well as in patients with the progressive form. The decrease in total intracellular hexosaminidase activity in lymphomonocytes of multiple sclerosis patients was accompanied by an enrichment of this activity associated with the plasma membrane fraction as demonstrated by experiments of subcellular fractionation. The analysis carried out using two synthetic substrates, 4-methylumbelliferyl N -acetyl-β- d -glucosaminide and its sulfate derivative, enables us to demonstrate that this accumulation is mainly due to isoenzymes with a ββ structure, whereas lysosomal fractions confirmed the classical presence of both αβ and ββ forms (hexosaminidases A and B, respectively). This was particularly evident in the plasma membrane fraction from mononuclear cells of patients with a clinical exacerbation of the disease. Considered together, these observations provide additional insight into the abnormality of peripheral blood immune cells in multiple sclerosis and may contribute to the understanding of the basic mechanisms underlying the pathological events resulting in the demyelinating process.