腺病毒载体携带HSV1-TK报告基因感染BMSCs后摄取~(131)I-FIAU的实验研究

用内部核糖体进入序列(IRES)将报告基因单纯疱疹I型病毒胸苷激酶(HSV1-TK)和治疗基因脑源性神经营养因子(BDNF)连接,双启动子法将TK-IRES-BDNF与增强绿色荧光蛋白(EGFP)包装到重组腺病毒载体中,得到Ad5-TK-IRES-BDNF-EGFP,扩增、纯化、测定病毒滴度;以感染复数(MOI)为0、50、100、150、200、250感染体外培养的骨髓间充质干细胞(BMSCs),以重组腺病毒Ad5-EGFP为无效对照病毒。荧光显微镜下观察感染细胞的绿色荧光细胞阳性率。四甲基偶氮唑蓝(MTT)比色法检测感染细胞增殖能力。碱性成纤维细胞生长因子(bFGF)和表皮生长因子(EGF)诱导感染细胞向神经元细胞分化,显微镜下观察细胞形态变化。实时定量聚合酶链反应(RQ-PCR)和2-△△CT法分析两目的基因的相对表达量(CT)。观察感染细胞对131I-FIAU的摄取情况。重组腺病毒Ad5-TK-IRES-BDNF-EGFP在MOI为150可高效感染BMSCs,感染细胞存活率98%,且保持良好的神经元细胞诱导分化能力。RQ-PCR检测两目的基因随着腺病毒MOI增加表达增强,且TK和BNDF两基因的表达有良好的线性相关(r=0.973,P0.05,n=3)。在前3h可见感染目的基因组细胞对131I-FIAU的摄取程度与时间呈正相关,3h感染目的基因组对131I-FIAU摄取率可达(31.42±0.46)%(n=3),随后增加不明显。各点感染目的基因组摄取率均显著高于对照组(t=23.06–173.83,P0.05,n=3)。重组腺病毒载体能高效、低毒感染BMSCs,IRES介导两目的基因表达有良好的线性相关。本研究表明感染目的基因细胞可有效介导HSV1-TK摄取131I-FIAU,为后续放射性核素报告基因活体显像示踪转基因BMSCs治疗脑梗死提供了细胞水平依据。     

Progresses of mycobacteriophage-based <i>Mycobacterium tuberculosis</i> detection

Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide, particularly in developing countries. A rapid and efficient method for TB diagnosis is indispensable to check the trend of tuberculosis expansion. The emergence of drug-resistant bacteria has increased the challenge of rapid drug resistance tests. Due to its high specificity and sensitivity, bacteriophage-based diagnosis is intensively pursued. In this review, we mainly described mycobacteriophage-based diagnosis in TB detection, especially two prevalent approaches: fluorescent reporter phage and phage amplified biologically assay (PhaB). The rationale of reporter phage is that phage carrying fluorescent genes can infect host bacteria specifically. Phage amplified biological assay based on the principle that phages can infect the live Mycobacterium tuberculosis< in the specimen under suitable conditions and produce plaques. Other phage-based diagnostic methods, such as a combination of the amplified biologically assay and nucleic acid amplification or lateral flow assays, are also actively explored. This review will help us improve the understanding of mycobacteriophages in TB detection and better promote the development of the rapid diagnosis of M. tuberculosis.     

Novel reporter cell line to analyze cytokine-mediated expression regulation of c-myc gene

Growth-promoting cytokines induce expression of nuclear proto-oncogenes that play critical roles in the regulation of cell proliferation. c-myc gene is one of those nuclear proto-oncogenes, whose regulation mechanisms of cytokine-mediated expression are not fully understood. Here, I generated a green fluorescent protein (GFP) reporter system that faithfully reflects interleukin-3 (IL-3)-induced c-myc gene expression. Flowcytometric analysis revealed cytokine-specific expression of reporter GFP. Kinetics of GFP mRNA expression was similar to that of endogenous c-myc mRNA. The reporter cell line will be a useful tool for studies of cell proliferation regulation through analysis of cytokine-induced c-myc gene expression.     

PowerBuilder中动态报表的设计

报表的设计一向是数据库应用系统的重要过程，所以灵活适用的报表无疑是非常重要的。通过实例介绍了在PowerBuilder中动态生成报表的两种设计方法，在这个设计实现过程中，首先对数据的来源（DataStore）以及ListView对象进行了说明，然后分析了实现的方法并提供了该实例的实现过程。     

On existence of reporter strands in DNA-based graph structures

Through self-assembly of branched junction molecules many different DNA structures (graphs) can be assembled. We show that every multigraph can be assembled by DNA such that there is a single strand that traces each edge in the graph at least once. This strand corresponds to a boundary component of a two-dimensional orientable surface that has the given graph as a deformation retract. This boundary component traverses every edge at least once, and it defines a circular path in the graph that “preserves the graph structure” and traverses each edge.     

人miR-34a靶基因的生物信息学分析及载体构建

目的对人miR-34a(hsa-miR-34a)下游靶基因进行预测,并构建含自噬相关基因靶结合序列的GFP报告基因载体。方法使用TargetScan Release 5.1和RNA22软件分析hsa-miR-34a可能的靶基因,并利用Gominer软件对靶基因功能进行分析。根据预测结果,合成含自噬相关基因hsa-miR-34a靶序列的寡核苷酸,以pEGFPC2为载体构建系列质粒,并检测各载体的真核表达情况。结果预测结果显示hsa-miR-34a共有2904个下游靶基因,功能涵盖生物过程、细胞组分、分子功能三大类,其中与自噬作用直接相关的靶基因有beclin 1和sestrin 2。构建含beclin 1和sestrin 2作用位点的GFP载体共5个,质粒转染Hela细胞后在荧光显微镜下可见绿色荧光。结论成功构建hsa-miR-34a下游自噬相关基因靶位点序列GFP报告基因载体,为进一步研究hsa-miR-34a对自噬的调控作用奠定基础。