大鼠IP3R1 miRNA慢病毒表达载体的构建及其沉默效应鉴定

目的 构建大鼠IP3R1 miRNA慢病毒表达载体,并利用PC12细胞株观察其对IP3R1基因的沉默效应.方法 根据已公布的IP3R1基因序列(GenBank No. NM_001007235),设计并合成4对miRNA的Oligo DNA(miRNA1、miRNA2、miRNA3和miRNA4),退火形成双链DNA后,与载体pcDNATM 6.2-GW/EmGFP-miR连接,构建pcDNATM 6.2-GW/EmGFP-miR-IP3R1表达载体;用Gateway技术将该表达载体先后与中间载体pDONRTM221、慢病毒载体pLenti6/V5-DEST连接,构建慢病毒表达载体pLenti6/V5-DEST-IP3R1,包装产生慢病毒,收集上清,感染PC12细胞株,用Real-time PCR和Western blotting技术检测慢病毒表达载体对PC12细胞内IP3R1表达的沉默效应.结果 测序结果表明,4对pcDNATM6.2-GW/EmGFP-miR-IP3R1表达载体序列与参考序列一致,将重组获得的慢病毒表达载体pLenti6/V5-DEST-IP3R1转染至PC12细胞株,48h后IP3R1的mRNA和蛋白表达下调,以miRNA2和miRNA3的沉默效应最佳.结论 成功构建了大鼠IP3R1慢病毒表达载体pLenti6/V5-DEST-IP3R1,其可使大鼠PC12细胞株IP3R1表达下调,为利用RNA干扰技术进一步研究细胞内钙释放通道的功能奠定了基础.     

人miRNA-埃博拉病毒相互作用的生物信息学研究

目的：初步探讨人微小RNA（ miRNA）与埃博拉病毒基因组5′尾标（ trailer）序列相互作用,为防治埃博拉病毒提供可能的靶向miRNA。方法运用Pita和RNAhybrid软件预测与埃博拉病毒5′尾标序列相互作用的人miRNA,并对其进行注释和分析。结果与结论发现人miRNA可能与埃博拉病毒5′尾标序列存在复杂的相互作用。根据以前关于宿主miRNA与病毒基因组相互作用的报道,我们认为,人miRNA与埃博拉病毒基因组5′尾标的相互作用可能会影响埃博拉病毒在人体内的复制以及人体细胞的正常功能。该研究将为埃博拉病毒的防治提供新的思考。     

HCMV gp52基因的克隆与表达

目的：克隆表达人巨细胞病毒(human cytomegalovirus，HCMV)gp52基因，制备特异性抗原，用于HCMV早期诊断。方法：从感染的HCMV细胞上清液中提取病毒的DNA，用PCR扩增gp52蛋白IgM抗原决定簇编码区DNA片段，并将其克隆到pGEM-Teasy载体测序，然后将其克隆到含有谷胱甘肽转移酶(GST)融合蛋白表达载体pGEX-5x-3中表达，表达产物经GST亲和层析柱纯化后，采用ELIA和免疫印迹(Westem blotting)检测重组蛋白的抗原性。结果：经序列测定gp52基因片段的序列正确，并在pGDX-5x-3表达载体中得到了高效表达。表达的融合蛋白用亲和层析柱纯化后经免疫印迹和ELISA分析具有良好的抗原性，能够与HCMV IgM阳性血清呈特异性反应。结论：通过基因工程制备的重组gp52蛋白可作为抗原用于HCMV感染者的早期诊断。     

腺病毒-甲病毒杂合载体的构建

目的将腺病毒Ad5/F11p造血细胞靶向和甲病毒复制子载体目的基因高效转录的特点相结合,构建腺病毒-甲病毒杂合载体,以提高目的基因在造血细胞的表达水平。方法基本实验过程包括骨架质粒构建,穿梭质粒构建,同源重组产生杂合病毒基因组,杂合病毒在包装细胞的拯救和扩增,以及杂合病毒对造血细胞基因转移效率的初步评价。具体步骤如下：利用重叠延伸PCR技术将pFiber5/11p质粒上Ad5的E4区部分删除产生骨架质粒pAdEasy/F11pDE4orf3。在pShuttle质粒的多克隆位点处分别插入造血细胞特异性启动子（CD11a promoter,CD11Ap）、甲病毒载体元件（nsP）、目的基因（GFP）、PolyA加尾信号构建杂合穿梭质粒pSh-SFV-GFP。骨架质粒与穿梭质粒在大肠杆菌BJ5183菌株中同源重组产生杂合病毒质粒pAd5/F11p-SFV-GFP,经与辅助病毒质粒Ad5/f11p-HV共转染293细胞后拯救出杂合病毒Ad5/F11p-SFV-GFP,经CsCl密度梯度离心法纯化,得到的病毒滴度为3×1010pfu/ml。与对照病毒Ad5-GFP相同感染复数（100MOI）分别感染U937细胞,利用流式细胞术检测GFP＋细胞比例。结果pAdEasy-1/F11p-E4orf3和pSh-SFV-GFP经相应酶切和测序鉴定,与预期设计一致;CsCl密度梯度离心纯化得到杂合病毒;100MOI Ad5/F11p-SFV-GFP感染U937细胞2 d后,GFP＋细胞比例为55.79%,而对照病毒Ad5-GFP的感染效率为2.42%。结论成功构建了腺病毒-甲病毒杂合载体,为进一步评价其对造血细胞的基因转移效率奠定了基础。     

感染人巨细胞病毒后涎腺导管上皮细胞的病理形态学改变

目的：探讨涎腺导管上皮细胞感染人巨细胞病毒（HCMV）后的病理形态学改变。方法：应用免疫组化法标记11例腮腺巨细胞包涵体病尸检蜡块中HCMV感染细胞;应用HE染色、倒置相差显微镜和透射电镜从体内外观察HCMV感染细胞的形态学改变。结果：组织内HCMV感染细胞从未感染时的柱状变为圆形或椭圆形,体积增大,出现包涵体,近管腔处胞浆内见少量颗粒状物。体外培养的涎腺导管上皮细胞感染HCMV后,先是胞体皱缩变为球形,核略增大,以后细胞逐渐从培养瓶底部脱落。与体内感染相比,体外HCMV感染可导致宿主细胞在大小、形状、与周围细胞的连接和包涵体等方面出现差别。结论：体内外HCMV感染涎腺导管上皮细胞导致的形态学改变不同。     

X射线诱导非小细胞肺癌A549细胞凋亡的适应性反应及相关miRNA筛选的研究

目的 研究X射线辐射诱导非小细胞肺癌（NSCLC）A549细胞凋亡的适应性反应,并筛选适应性反应相关的微RNA（miRNA）。 方法 将NSCLC A549细胞分为6组,包括 50 mGy+20 Gy、200 mGy+20 Gy、20 Gy、50 mGy、200 mGy照射组及对照组（0 Gy）,前2组细胞分别用50、200 mGy初始剂量进行照射,培养6 h后用20 Gy的效应剂量进行照射,20 Gy、50 mGy、200 mGy照射组同时进行照射。培养24 h后使用流式细胞仪检测细胞凋亡情况。利用小RNA测序技术筛选差异表达miRNA,并对其靶基因进行基因本体（GO）及京都基因与基因组百科全书（KEGG）通路的功能富集分析。采用实时荧光定量PCR（qRT-PCR）对部分差异表达miRNA进行验证。2组间数据的比较采用Welch t检验。 结果 50 mGy+20 Gy照射组和200 mGy+20 Gy照射组的A549细胞早期凋亡率分别为（1.81±0.11）%和（2.17±0.19）%,低于20 Gy照射组的（4.54±0.23）%,且差异均有统计学意义（t=10.680、8.006,均P<0.01）。与20 Gy照射组相比,50 mGy+20 Gy照射组和200 mGy+20 Gy照射组共同差异表达趋势miRNA有1个上调（miR-3662）、15个下调（miR-185-3p、miR-1908-5p、miR-1307-5p、miR-182-3p、miR-92a-3p、miR-582-5p、miR-501-3p、miR-138-5P、miR-1260b、miR-484、miR-378d、miR-193b-3P、miR-127-3p、miR-1303及miR-654-5p）。GO富集分析结果显示,差异表达miRNA调控靶基因功能显著富集于细胞通讯调节、代谢过程的正向调节、代谢信号的调节、酶结合及催化活性等过程。KEGG富集分析结果显示,靶基因相关信号通路显著富集于溶酶体、丝裂原活化蛋白激酶、Ras和内吞作用等信号通路。qRT-PCR检测结果显示,miRNA表达情况与基因芯片结果趋势一致（10个miRNA表达水平得到验证）。 结论 X射线50、200 mGy照射剂量均能诱导NSCLC A549细胞凋亡的适应性反应,并筛选到一组共同差异表达的miRNA,可能在X射线辐射诱导细胞凋亡的适应性反应中发挥了重要作用,有可能成为调节电离辐射生物效应的潜在靶点。     

还原型辅酶Ⅰ(NADH)对PC12细胞鱼藤酮损伤的分子调控

为探讨NADH对PC12细胞线粒体鱼藤酮损伤的修复机制，采用细胞毒实验、免疫荧光和流式细胞仪检测细胞在鱼藤酮损伤前后细胞增殖基因（c-myc、c-erbB-2）、抗凋亡基因（bcl－2）、抑癌基因（p53）、细胞快反应基因（c-fos）相关蛋白和细胞增殖核抗原（PCNA）的表达。结果表明，鱼藤酮能明显抑制PC12细胞增殖，下调c-erbB-2、c-myc、bcl-2和p53基因的表达；NADH可以抑制鱼藤酮对PC12细胞线粒体的毒性作用，上调细胞bcl-2c、c-myc、c-erbB－2基因和PCR的表达，提示鱼藤酮可能通过控线粒体磷酸化过程和下调细胞增殖基因（c-rebB-2、c－myc）、抗凋亡基因（bcl-2），上调快反应基因(c-fos）表达致使细胞损伤，NADH抑制鱼藤酮对PC12细胞的损伤可能与bcl-2、c-myc、c-erbB-2和p53表达调控有关。     

WJBC株盖塔病毒基因组序列测定及与其他甲病毒的系统进化关系

目的对WJBC株盖塔病毒（GETV）进行基因组序列测定,阐明其与已报道的GETV及其他甲病毒的关系。方法将GETV基因组编码区分12段分别进行RT-PCR扩增,将扩增产物直接进行测序,非编码区采用RACE法进行扩增,扩增产物纯化后连入pGEM-T easy载体,经转化DH5α感受态细胞,挑取阳性克隆鉴定后测序,应用DNAStar软件将测序结果拼接获得基因组序列。结果与结论 GETV基因组核苷酸共11 696 nt,编码3721个氨基酸,非结构基因编码2468个氨基酸,结构基因编码1253个氨基酸,结构基因和非结构基因之间有44 nt的连接区为非翻译区。病毒基因组5＇端和3＇端分别有78、411 nt的非编码区。序列同源性分析结果表明,此株病毒与M1株盖塔病毒同源性最高,两者核苷酸序列同源性为99.8%,氨基酸序列同源性为99.2%。     

逆转录病毒介导的基因转移方法的建立

将人促红细胞生成素（ｈＥＰＯ）、人粒细胞集落刺激因子（ｈＧ－ＣＳＦ）、细胞程序死亡抑制基因（ＢＣＬ－２）重组逆转录病毒质粒ＤＮＡ转染病毒包装细胞ＰＡ３１７后，经Ｇ４１８抗性筛选，其单克隆细胞株培养上清液均具有感染ＮＩＨ３Ｔ３及大鼠原代成纤维细胞、成肌细胞并形成Ｇ４１８抗性克隆的能力。其感染细胞的染色体ＤＮＡＰＣＲ鉴定结果均为阳性，表明其染色体中均成功地整合了目的基因；感染的靶细胞均能表达外源基因编码蛋白，表明已成功地建立了逆转录病毒介导的基因转移技术。     

人巨细胞病毒包膜糖蛋白B线性B细胞抗原表位的预测

目的：通过生物信息学方法分析人巨细胞病毒（ human cytomegalovirus ,HCMV）包膜糖蛋白B （ glycopro－tein B,gB）的结构及理化特性,预测gB的线性B细胞抗原表位。方法基于HCMV gB的序列,利用两种在线B细胞表位预测程序及DNAstar软件对gB进行预测;利用SWISS－MODEL服务器同源构建gB三级结构模型,进行进一步验证。结果与结论综合多项分析,预测得到HCMVgB的多个线性B细胞表位,为后续验证其优势中和表位,建立富含高效价中和抗体的原料血浆的筛选方法奠定了基础。     

人巨细胞病毒^32P标记PCR产物探针敏感度

＾３２Ｐ探针杂交是病原微生物基因诊断应用最广泛的方法之一，在建立了人巨细胞病毒ＰＣＲ、套式ＰＣＲ的基础上，我们用６１３ｂｐ ＰＣＲ外引物扩增产物的制备了杂交探针，并与３００ｂｐ内引物扩增产物进行转膜杂交，可将单纯琼脂糖凝胶电泳１７ｎｇ的检测水平提高到０．５ｎｇ，释释梯度相当于提高２个数量级。因此，利用ＰＣＲ结合＾３２Ｐ杂交就可以检测出不到１个病毒的基因拷贝（１００ａｇ），为临床人巨细胞病毒潜伏感染     

Comprehensive circRNA-microRNA-mRNA network analysis revealed the novel regulatory mechanism of Trichosporon asahii infection

Background:Invasive Trichosporon asahii (T.asahii) infection frequently occurs with a high mortality in immunodeficient hosts,but the pathogenesis of T.asahii infection remains elusive.Circular RNAs (circRNAs) are a type of endogenous noncoding RNA that participate in various disease processes.However,the mechanism of circRNAs in T.asahii infection remains completely unknown.Methods:RNA sequencing (RNA-seq) was performed to analyze the expression profiles of circRNAs,microRNAs(miRNAs),and mRNAs in THP-1 cells infected with T.asahii or uninfected samples.Some of the RNA-seq results were verified by RT-qPCR.Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG)pathway analyses were used to analyze the differentially expressed mRNAs.A circRNA-miRNA-mRNA network was constructed and verified by dual-luciferase reporter assay and overexpression experiments.Results:A total of 46 circRNAs,412 mRNAs and 47 miRNAs were differentially expressed at 12 h after T.asahii infection.GO and KEGG analyses showed that the differentially expressed mRNAs were primarily linked to the leukocyte migration involved in the inflammatory response,the Toll-like receptor signaling pathway,and the TNF signaling pathway.A competing endogenous RNA (ceRNA) network was constructed with 5 differentially expressed circRNAs,5 differentially expressed miRNAs and 42 differentially expressed mRNAs.Among them,hsa circ 0065336 was found to indirectly regulate PTPN11 expression by sponging miR-505-3p.Conclusions:These data revealed a comprehensive circRNA-associated ceRNA network during T.asahii infection,thus providing new insights into the pathogenesis of the T.asahii-host interactions.     

肝肾移植术后受者人巨细胞病毒两种检测方法的对比研究

目的:探讨巢式PCR(Nest PCR)检测器官移植术后受体人巨细胞病毒(HCMV)DNA,并与传统ELISA法作方法学对照。方法:巢式PCR针对HCMV AD169株IEA基因设计内外两对引物,对59例肝、肾移植术后受体(肝移植27例、肾移植32例)血、尿标本进行HCMV DNA检测。分离血清作EIASA检测IgG、IgM。结果:血液Nest PCR阳性率肝移植62.9％,肾移植46.8％;ELISA法阳性率:IgC35.5％,IgM27.1％,IgG+IgM18.6％,IgM阳性标本(16例)DNA检测皆为阳性,6例IgG、IgM皆阴性标本DNA检测仍为阳性。两法检测,P<0.05,证明两法存在显著差异,结论:巢式PCR是一种敏感特异、简便快速诊断HCMV感染的方法,灵敏度及特异性高于传统ELISA,能弥补ELISA的假阴性,更适用于临床检测器官移植术后HCMV感染。     

巨细胞病毒体外感染对人精子存活率及其运动参数与超微结构的影响

目的探讨人巨细胞病毒（HCMV）感染对人精子存活率、精子各运动参数及其超微结构的影响。方法将HCMV与10例经过上游法优化的正常人精子体外共同孵育，于0、1、2、3、4h分别取样，检测精子存活率，并应用计算机辅助精子分析系统（CASA）测定精子直线运动速度（VSL）、曲线运动速度（VCL）、平均路径速度（VAP）、头部侧摆幅度（ALH）和前向运动百分率（a＋b级精子百分率）；同时，电镜观察HCMV感染不同时间人精子超微病理结构的改变。结果与空白、阴性对照组比较，人精子存活率在高浓度HCMV感染后1、3h时明显降低，差异显著（P〈0．05），4h时差异非常显著（P〈0．001）。a＋b级精子百分率仅在高浓度HCMV感染4h时明显降低，差异显著（P〈0．05）。精子VSL在各时间点各组比较差异均无统计学意义（P〉0．05）。精子VCL在高浓度HCMV感染3h时明显下降，差异显著（P〈0．05），4h时显著下降，差异非常显著（P〈0．001）。而中浓度HCMV感染4h时，精子VCL才下降，差异显著（P〈0．05）。精子VAP在高、中浓度HCMV感染2、3、4h时明显降低，差异显著（P〈0．05）。精子ALH在高浓度HCMV感染3、4h时明显下降，差异显著（P〈0．05）。电镜结果显示：人精子超微结构的异常改变在高浓度HCMV感染时常见，4h时严重；主要表现有精子核解聚、空泡增多、顶体破裂、质膜破损、线粒体肿胀及其排列紊乱。结论HCMV体外感染3～4h可造成人精子存活率与运动参数及精子超微结构的异常，且这种改变与HCMV浓度存有一定的量效关系，高浓度HCMV感染的影响结果显著。     

Screening and confirmation of microRNA markers for distinguishing between menstrual and peripheral blood

The identification of menstrual blood (MB) and peripheral blood (PB) left at a crime scene is crucial for crime reconstruction, especially in sexual assaults. MicroRNAs (miRNAs), a class of non-protein coding molecules, have been demonstrated to be a viable tool for body fluid identification in forensic casework. Several groups have searched for miRNAs that are specific to different body fluids. Blood has been studied the most extensively. However, menstrual blood was only involved in five studies, and the results confirming the presence of specific miRNAs could not be reproduced in other studies. In this study, we attempted to screen new markers that can differentiate between menstrual blood and peripheral blood by using Exiqon’s miRCURY™ LNA Array. Five miRNAs were selected based on the microarray results, namely, miR-141-3p, miR-373-3p, miR-497-5p, miR-143-5p, and miR-136-5p, whose expression levels exhibited 27.95-, 17.96-, 16.74-, 10.14-, and 9.21-fold changes, respectively, compared to the level in peripheral blood. Two classic quantitative methods, TaqMan hydrolysis probes (TaqMan for short) and SYBR Green fluorochrome (SYBR Green for short), were applied in the confirmation step to study the impact of different quantitative methods on the results. Three miRNAs (miR-141-3p, miR-497-5p, and miR-143-5p) were confirmed by TaqMan and one (miR-141-3p) by SYBR Green. Furthermore, bioinformatic methods were applied to interpret the candidate miRNAs. Our results established a multi-step procedure for body fluid identification and showed that the choice of quantitative method is important when miRNAs are used to identify the origin of blood samples.     

Massively parallel sequencing of microRNA in bloodstains and evaluation of environmental influences on miRNA candidates using realtime polymerase chain reaction

MicroRNAs (miRNA) are small (22–24 nucleotides) non-coding RNAs with potential application in forensic science because of their anti-degradation property and tissue specificity. Recent studies on the use of miRNA in forensic applications have mainly focused on body fluid identification using realtime polymerase chain reaction or microarray analysis. However, the exploration of miRNA in bloodstains, which are the most valuable source of biological evidence during case investigations, is currently lacking, particularly for aged and environmentally compromised forensic samples. Recent developments in massively parallel sequencing (MPS) technology provide the opportunity to establish a whole-genome miRNA profile with high throughput and efficiency. However, MPS analysis of genome-wide miRNA profiles from bloodstains has not been reported to date. In this study, the whole-genome miRNA profiles of bloodstains were examined using MPS, revealing 633 known miRNAs and 266 novel miRNAs. To further explore the stability of miRNAs in bloodstains under various circumstances, the expression levels of six miRNAs (miR-16-5p, miR-20a-5p, miR-486-5p, miR-148a-3p, miR-151a-3p, and miR-451a) that were abundant in blood/bloodstains were examined. The results showed that freezing/thawing and a high concentration of oxidant solution affects the absolute expression of miRNA significantly, while storage for up to 5 months and a temperature of 37 °C did not have any observed effects. This study not only provides a novel method to explore miRNA profiles in bloodstains using MPS, but also points to the circumstantial influences on miRNA expression, which are an important consideration for practical application. Collectively, our work may shed light on MPS-based approaches with miRNA analysis of bloodstains in forensics.     

Effects of Systemic or Topical Administration of Sodium Selenite on Early Radiation Effects in Mouse Oral Mucosa

PURPOSE: To quantify the effect of sodium selenite (selenium) on radiation-induced oral mucositis (mouse) after subcutaneous or topical administration. MATERIAL AND METHODS: Mucosal ulceration of the lower epithelium of mouse tongue was analyzed. Selenium (5 mug) was applied subcutaneously (s.c.) or locally, 60 min or 30 min prior to irradiation, respectively. In combination with single-dose irradiation, a single selenium application was given. With daily fractionated irradiation (3 Gy/fraction) for 1 week (days 0-4), selenium was administered at all 5 days of irradiation. With ten fractions over 2 weeks, selenium was applied in week 1, week 2, or both. All fractionation protocols were terminated by graded test doses to generate full dose-effect curves. RESULTS: In a single-dose control experiment, the ED(50) (dose after which ulcer induction is expected in 50% of the mice) was 12.9 +/- 1.6 Gy. Selenium increased the ED(50) to 17.7 +/- 2.6 Gy (s.c.; p = 0.0003) and 16.3 +/- 3.0 Gy (local; p = 0.0104). The ED(50) for test irradiation after 5 x 3 Gy was 7.4 +/- 2.2 Gy. Subcutaneous administration of selenium resulted in an ED(50) of 11.5 +/- 2.0 Gy (p = 0.0015), local application yielded an ED(50) of 10.0 +/- 2.1 Gy (p = 0.0284). The ED(50) for test irradiation after 10 x 3 Gy/2 weeks was 8.0 +/- 1.7 Gy. Subcutaneous or local administration of selenium in week 1 yielded a significant increase in ED(50) to 10.5 +/- 1.0 Gy (p = 0.0069) and 10.7 +/- 1.0 Gy (p = 0.0039), respectively. By clear contrast, selenium administration in week 2 had no significant effect. Administration in both weeks resulted in an ED(50) of 9.1 +/- 2.0 Gy (s.c.; p = 0.2747) and 9.7 +/- 1.4 Gy (local; p = 0.0541). CONCLUSION: Administration of sodium selenite during clinically relevant fractionated irradiation protocols has a significant effect during the initial treatment phase, i.e., week 1 in the mouse. Therefore, in clinical radiotherapy, the latent time to manifestation of confluent mucositis may be significantly prolonged, and hence the burden for the patient clearly reduced by selenium.     

Decreased chronotropic drive as an adaptation to chronic exercise; possible mechanisms.

Cardiovascular function was determined at rest and during exercise in twenty-eight healthy, elite distance runners. Maximum heart rate was 184 +/- 6 b x min(-1), which was more than one SD lower than the age predicted value (p < 0.001), and an inverse correlation was observed between maximum heart rate and VO2max (r = - 0.82, p < 0.001). The most aerobically trained athlete, a 27 year old male, presented with a maximum heart rate of 139b x min(-1). Echo-cardiac ultrasound revealed unremarkable intra-cardiac dimensions and flow characteristics relative to other endurance-trained athletes. A decreased chronotropic drive may represent a favourable physiological adaptation to endurance exercise which increases cardiovascular efficiency.     

EB病毒潜伏膜蛋白在胆管癌中的表达

目的 了解ＥＢ病毒感染与胆管癌的发生发展是否有关及ＥＢ病毒（ＥＢＶ）编码的潜伏膜蛋白（ＬＭＰ－１）在胆管癌组织中的表达情况。方法 采用免疫组化技术,检测３０例不同分化类型胆管癌组织和１５例胆总管囊肿组织中ＥＢＶ ＬＭＰ－１的表达。结果 ＥＢＶ ＬＭＰ－１在胆管高、中、低分化癌中均有一定比例的阳性,低分中中的表达明显高于高、中分化癌的表达。高分化癌与中分化癌间的表达无明显差异,与对照组相比差异有显著